WO2023245032A2 - Methods and compositions for treating pediatric autoimmune neuropsychiatric disorders - Google Patents

Methods and compositions for treating pediatric autoimmune neuropsychiatric disorders Download PDF

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WO2023245032A2
WO2023245032A2 PCT/US2023/068399 US2023068399W WO2023245032A2 WO 2023245032 A2 WO2023245032 A2 WO 2023245032A2 US 2023068399 W US2023068399 W US 2023068399W WO 2023245032 A2 WO2023245032 A2 WO 2023245032A2
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pans
cytokines
chemokines
gas
subject
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WO2023245032A3 (en
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Dritan Agalliu
Charlotte WAYNE
Tyler CUTFORTH
Wendy Sulina VARGAS
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The Trustees Of Columbia University In The City Of New York
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • the present invention relates to methods of diagnosing and treating pediatric autoimmune neuropsychiatric disorders such as Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS).
  • PANDAS Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections
  • the psychiatric syndrome is classified as Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS), which is defined by several diagnostic criteria: (i) abrupt “overnight” obsessive compulsive disorder (OCD), or dramatic, disabling tics, (ii) an episodic course, (iii) young age at onset (average 6-7 years), (iv) presence of neurological abnormalities and (v) temporal associate between symptom onset and GAS infection. https://www.pandasppn.org/what-are-pans-pandas/; Swedo et al., Am. J. Psychiatry 155:2, 264- 271 (1998)).
  • OCD obsessive compulsive disorder
  • PANDAS are part of a larger group of neuropsychiatric disorders termed Pediatric Acute- Onset Ncuropsychiatric Syndrome, or PANS, characterized by a sudden onset of OCD or eating restrictions, together with acute behavioral deterioration in at least two areas, anxiety, sensory amplification, or motor abnormalities, behavioral regression, deterioration in school performance, mood disorder, urinary symptoms or sleep disturbances. Swedo et al., Clinical Presentation of Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections in Research and Community Settings, J. Child Adolesc. Psychopharmacol. 2015, 25(1): 26-30.
  • the behavioral changes observed with PANDAS or PANS may be the result of an aberrant anti-pathogen immune response targeting the central nervous system (CNS) (Cunningham (2019). Microbiol. Spectr. 7. 10.1128/microbiolspec.GPP3-0045-2018).
  • the present disclosure provides for a method of treating a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in a subject in need thereof.
  • the method may comprise administering to the subject an inhibitor of IL- 17 A or its receptor.
  • the PANS may be Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).
  • PANDAS may be associated with Group A Streptococcus infection.
  • the PANS may be associated with an ongoing or prior infection.
  • the inhibitor may comprise an antibody or a fragment thereof that binds to IL- 17 A or its receptor.
  • the antibody comprises secukinumab (SEC), ixekizumab (IXE), brodalumab (BROD), bimekizumab, or combinations thereof.
  • the inhibitor may comprise a small molecule.
  • the inhibitor may be administered by inhalation, intranasally, intrathecally, orally, intravenously, subcutaneously or intramuscularly. In one embodiment, the inhibitor is administered intranasally. In certain embodiments, the inhibitor may be administered using an intranasal spray device, an atomizer, a nebulizer, a metered dose inhaler (MDI), a pressurized dose inhaler, an insufflator, an intranasal inhaler, a nasal spray bottle, a unit dose container, a pump, a dropper, a squeeze bottle, or a bi-directional device.
  • MDI metered dose inhaler
  • the present disclosure provides for a method for detecting or diagnosing a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in a subject, or assessing the subject’s risk of developing a pediatric autoimmune neuropsychiatric disorder such as PANS.
  • a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in a subject, or assessing the subject’s risk of developing a pediatric autoimmune neuropsychiatric disorder such as PANS.
  • PANS Pediatric Acute-onset Neuropsychiatric Syndrome
  • the method may comprise: (a) obtaining a sample from the subject; (b) determining levels of one or more cytokines, chemokines and/or growth factors in the sample, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; (c) comparing the levels obtained in step (b) with the levels of the one or more cytokines, chemokines and/or growth factors in a control sample; and (d) diagnosing that the subject has a pediatric autoimmune neuropsychiatric disorder (such as PANS) or an increased risk of developing a pediatric autoimmune neuro
  • Also encompassed by the present disclosure is a method of treating a subject with a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) or an increased risk of developing a pediatric autoimmune neuropsychiatric disorder such as PANS.
  • a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) or an increased risk of developing a pediatric autoimmune neuropsychiatric disorder such as PANS.
  • the method may comprise: (a) obtaining, or having obtained, a sample from the subject; (b) determining, or having determined, levels of one or more cytokines, chemokines and/or growth factors in the sample, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; (c) comparing, or having compared, the levels obtained in step (b) with the levels of the one or more cytokines, chemokines and/or growth factors in a control sample; and (d) treating the subject for PANS or an increased risk of developing PANS, if the level
  • the one or more cytokines, chemokines and/or growth factors may be selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, and TNFa.
  • the PANS may be Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).
  • PANDAS may be associated with Group A Streptococcus infection.
  • the PANS may be associated with an ongoing or prior infection.
  • the sample may be a plasma, serum or blood sample.
  • the control sample may be from a healthy subject or a plurality of healthy subjects.
  • the control sample may be from a subject without a pediatric autoimmune neuropsychiatric disorder such as PANS, or a plurality of subjects without a pediatric autoimmune neuropsychiatric disorder such as PANS.
  • the subject may be a mammal such as a human.
  • the subject’s existing PANS treatment regimen may be modified or maintained.
  • the level of the one or more cytokines, chemokines and/or growth factors may be determined by mass spectrometry (MS), or by enzyme-linked immunosorbent assay (ELISA).
  • kits comprising: (a) antibodies or fragments thereof that specifically bind to one or more cytokines, chemokines and/or growth factors in a blood, plasma or serum sample from a subject, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL- 23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; and (b) instructions for measuring the one or more cytokines, chemokines and/or growth factors for diagnosing Pediatric Acuteonset Neuropsychiatric Syndrome (PANS) in the subject or assessing the subject’s risk of developing PANS.
  • PANS Pediatric Acuteonset Neuropsychiatric Syndrome
  • Figures 1A-1I Microglia and CNS endothelial cells display major transcriptional shifts after multiple GAS infections.
  • Figure 1A Schematic diagram of the workflow for singlecell RNA sequencing (scRNAseq) and MERFISH experiments.
  • Figures 1B-1C Dimensionality reduction plot of scRNAseq from the olfactory bulbs (OBs) of GAS-infected and PBS control mice without cell type-specific enrichment (Figure IB), and with enrichment by sorting for CD31 + (endothelial) and CDllb + (myeloid) cells (Figure 1C).
  • Figure ID Dimensionality reduction plot of sample identity for OB cell types isolated from PBS and GAS conditions.
  • Figure IE Cell type abundance from OBs from scRNAseq (with and without enrichment) and MERFISH experiments.
  • the labeled bars represent the fraction of endothelial cells (ECs) and microglia among all isolated cells.
  • Figure IF Representative MERFISH coordinate plot.
  • Figure 1G /-SNE plot for all MERFISH samples.
  • Figures 1H-1I Plots of the numbers of differentially expressed genes (DEGs) by cluster, displaying upregulated and downregulated DEGs.
  • FIGS 2A-2H CNS endothelial cells upregulate inflammatory signatures and lose BBB marker expression following GAS infections.
  • Figure 2A r-SNE plot of the sample identity for ECs isolated from the OBs of PBS and GAS-infected mice.
  • Figure 2B Gene set enrichment analysis (GSEA) for genes differentially expressed in ECs following GAS infection. Bars indicate significance of enrichment (-logiiDR) and dots indicate the number of genes significantly enriched in each gene set.
  • GSEA Gene set enrichment analysis
  • Bars indicate significance of enrichment (-logiiDR) and dots indicate the number of genes significantly enriched in each gene set.
  • the inflammatory signatures are upregulated, whereas blood-brain barrier (BBB) gene signatures are downregulated in ECs from GAS- infected mice.
  • BBB blood-brain barrier
  • FIG. 2C Heat map of genes related to the endothelial response to systemic LPS in ECs from the OB of PBS and GAS-infected mice.
  • Figures 2D-2F Expression changes for three BBB transcripts (Itm2a, Itih5 and Mfsd2d) in ECs by scRNAseq (left). Statistical comparisons of scRNAseq by Wilcoxon Rank Sum test (* p-adj ⁇ 0.05).
  • FIG. 2G On the left, correlation of log2 fold changes in CNS EC genes identified from GAS versus PBS (x-axis) with those identified from either acute experimental autoimmune encephalomyelitis (EAE) versus Complete Freund Adjuvant (CFA) control (top panels, y-axis) and chronic EAE versus CFA (bottom paneled; y-axis). Correlations on the right display only BBB-associated genes. The correlation coefficient is displayed in the top left comer of each plot. Gray dotted lines mark the line of identity; black dashed line indicates the line of best fit.
  • EAE acute experimental autoimmune encephalomyelitis
  • CFA Complete Freund Adjuvant
  • FIGS 3A-3O Microglia respond to recurrent GAS infections by upregulating inflammatory gene signatures.
  • Figure 3A t-SNE plot showing the sample identity of microglia from the OBs of PBS and GAS-infected conditions.
  • Figure 3B GSEA for genes upregulated by microglia after GAS infection. Bars indicate significance of enrichment (-logirDR) and dots indicate number of genes significantly enriched in each gene set.
  • Figure 3C Heat map of genes related to antigen presentation, cytokines and chemokines, and interferon response in PBS and GAS samples.
  • Figure 3D Representative coordinate plot of microglia showing different OB layers in the MERFISH.
  • Figure 3E Representative coordinate plot of microglial distance to the nearest T cell in micrometers (pm) in each OB layer in PBS (left) and GAS-infcctcd (right) conditions from the MERFISH analysis, shaded by proximity to the nearest T cell. Microglia in the glomerular layer of the OB are closest to T cells.
  • Figure 3F-3J Expression changes of Cd74, Tnf, Ccl5, Cx3crl, and P2ryl2 in microglia by scRNAseq (left). Statistical comparisons of scRNAseq were performed with Wilcoxon Rank Sum test (* p-adj ⁇ 0.05).
  • Figures 4A-4Q Macrophages in the NALT/OE secrete similar chemokines & cytokines to those infiltrating the OB after multiple GAS infections.
  • Figure 4A t-SNE plot of microglia isolated from the OBs of all conditions examined in this study reveals six major clusters: two homeostatic microglia (hMG-1 and -2) and four Streptococcus-responsive, microglia (srMGl- 4) subtypes.
  • Figure 4B t-SNE plot of microglia isolated from the OBs by inoculate (PBS versus GAS) shows that hMG microglia are predominantly from PBS mice and srMGl-4 from GAS- infected mice.
  • Figure 4C Feature plot of the expression of selected homeostatic, disease- associated, antigen-presentation, and interferon-response genes across microglial populations. The scale indicates average level of expression.
  • Figure 4D Feature plot of the expression of key chemokine and cytokine genes across microglial populations. The scale indicates level of expression.
  • Figures 4E-4G Representative fluorescence images of perivascular (Figure 4E) and meningeal (Figure 4F) macrophages, and microglia (Figure 4G) in the OB from GAS-infected CX3CRl GFP /TMEM119 tdTomato reporter mice.
  • Figure 4J t- SNE plot of cell types isolated from NALT/OE based on scRNAseq data.
  • Figure 4K t-SNE plot of sample identity for NALT/OE-isolated cells from PBS and GAS mice.
  • Figure 4L Expression levels for select cytokines, chemokines and growth factors in NALT/OE-derived macrophages and OB-derived macrophages and microglia in PBS and GAS-infected conditions. The size of each dot indicates the percent of the population expressing each marker; the scale indicates average level of expression. The cell population analyzed is shown in the schematic diagram on the left.
  • FIG. 4M Dotted bar graph of the normalized mean fluorescence intensity of albumin- Alexa 594 localized inside primary human umbilical vein endothelial cells (HUVECs) exposed to cytokines and growth factors that are present in high concentrations in sera from PANDAS/PANS patients. Incubation with selected cytokines and growth factors for 48 hours upregulates albumin uptake in HUVEC cells. The comparisons were performed by one-way ANOVA (* p ⁇ 0.05; *** p ⁇ 0.001; **** p ⁇ 0.0001).
  • Figures 4O-4Q Identification of NALT and OE cell types and measurement of transcriptional shifts in perivascular macrophages after intranasal GAS infections.
  • Figure 40 Molecular markers used to assign cell identity in the nose associated lymphoid tissue (NALT) and olfactory epithelium (OE) in scRNAseq experiments.
  • the dot size indicates the percentage of the population expressing each marker; the scale indicates the average level of expression.
  • Figure 4P Heat map of genes related to antigen presentation, cytokines and chemokines, and interferon response, as well as the most upregulated gene (Saa3) in perivascular macrophages from the OB.
  • Figure 4Q Expression of chemokine receptors in immune cell clusters isolated from the OB (left) and NALT/OE (right).
  • the dot size indicates the percent of population expressing each marker; the color scale indicates the average level of expression.
  • the purple color in the grid indicates receptor-ligand binding pairs for the relevant GAS-induced chemokines in mouse microglia / macrophages.
  • Figures 5A-5V Lack of Thl7 cells or inhibition of IL-17A function rescues aberrant transcrip tome signatures in CNS endothelial cells and microglia associated with recurrent GAS infections.
  • Figure 5A Microglial surface expression of antigen-presentation markers by flow cytometry in wild-type PBS, wild-type GAS -infected and RORyt ' GAS- infected mice. Comparisons by one-way ANOVA. (ns, p > 0.05; * p ⁇ 0.05; ** p ⁇ 0.01).
  • Figure 5B Representative plots of CD74 and MHC II (I-A/I-E) expression in wild-type and RORyf 7- mutant microglia after GAS infections.
  • Figure 5C Quantification of IFNy concentrations in whole OBs from wildtype PBS, wild-type GAS and RORyt 7 ’ GAS mice after two and five GAS infections. Comparisons by one-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05; * p ⁇ 0.05).
  • Figure 5D Timeline of GAS infections and administration of an a-IL-17A- neutralizing antibody or isotype control.
  • FIGS. 5E-5F Dotted bar graphs show the quantification of endogenous IgG leakage (relative fluorescence intensity) in the glomerular ( Figure 5E) and granular ( Figure 5F) layers of the OB in PBS and GAS conditions treated with either an a-IL-17A-neutralizing antibody or isotype control. IgG intensity is significantly reduced in IL-17A mAb-treated mice relative to isotype mAb in the granular layer.
  • Figure 5G Representative images of IgG leakage in the granular layer of the OB.
  • FIG. 5H-5M Violin plots of expression changes in ECs and microglia from GAS -infected mice treated with either an a-IL-17A-neutralizing antibody or isotype control. Comparisons by Wilcoxon Rank Sum test (* p-adj ⁇ 0.05).
  • Figures 5N-5V Elimination of Thl7 cells or blockade of IL- 17A rescues BBB transcriptome changes in CNS ECs, and reduces expression of chemokine and interferon response genes by microglia in GAS-infected mice.
  • Figures 5N-5S Gene expression changes in endothelial cells (Figure 5N) and microglia ( Figures 5O-5S) isolated from the OBs of wildtype PBS, wild-type GAS-infected and RORyf /_ GAS-infected mice.
  • the displayed comparisons between PBS and GAS and WT GAS and RORyf /_ GAS were significant by Wilcoxon Rank Sum test with Bonferroni correction (* p-adj ⁇ 0.05).
  • the comparisons between PBS and RORyt 7 ’ GAS are not shown.
  • FIG. 5T Ridge plots showing expression of key major histocompatibility complex (MHC) class I genes for antigen presentation by various OB cell types in wild-type PBS, wild-type GAS-infected mice, RORyt 7 GAS-infected mice, isotype control antibody treated GAS-infected mice, and a-IL-17A mAb-treated GAS infected mice.
  • Figure 5U Quantification of the number of CD4 + T cells in the OBs of isotypecontrol and a-IL-17A mAb-treated GAS infected mice (bregmas 4.28, 3.8 and 3.2).
  • MHC major histocompatibility complex
  • Figures 6A-6R Although IFNy + IL-17A + and GM-CSF + CD4 T cells accumulate in the brain after multiple GAS infections, GM-CSF is primarily critical to induce GAS-related microglial transcription changes.
  • Figure 6A Dotted bar graph of the progressive increase in the proportion of IFNy + IL-17A + CD4 T cells in the brain with increasing numbers of GAS infections. The proportion of IFNy + IL- 17A + in mice infected twice over a five-week interval (li...2i) was not significantly different from that seen with two consecutive infections (2i). Comparisons by one-way ANOVA with Dunnett’s T3 multiple comparisons test.
  • FIG. 6B Dotted bar graph of the progressive increase in the proportion of GM-CSF + CD4 T cells in the brain with increasing numbers of GAS infections. Comparisons by two-way ANOVA with Tukey’s multiple comparisons test (* p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001; **** p ⁇ 0.0001; each dot represents one mouse).
  • Figure 6E Timeline of 4-OH- tamoxifen administration and GAS infections in Csf2 fl/fl and Csf2 ACD4 mice.
  • Figures 6F-6K Violin plots of expression changes in ECs and microglia from Csf2 ACD4 mutant and Csf2 fl/fl control GAS-infcctcd mice. Comparisons by Wilcoxon Rank Sum test (* p-adj ⁇ 0.05).
  • Figure 6L A model summarizing three key findings from the study. Left: scRNAseq of mouse OB reveals that microglia upregulate antigen presentation, cytokine and chemokine, and interferon-response genes after multiple GAS infections, whereas CNS ECs lose BBB transcriptome properties.
  • cytokines A small number of inflammatory cytokines, chemokines and growth factors, produced primarily by microglia and macrophages after GAS infections, are elevated in sera from acute PANDAS/PANS patients. These cytokines upregulate transcytosis in human EC in vitro suggesting a direct impact on EC function.
  • Th 17 cell-derived cytokines IFNy, IL-17A and GM-CSF play distinct roles on BBB dysfunction and microglial activation in post-infectious CNS sequelae after repeated GAS infections.
  • Figure 6M Ridge plot showing expression of Trem2 in PBS, wild- type GAS, RORyt 7 ’ GAS, isotype control antibody GAS, a-IL-17A mAb-treated GAS conditions, Csf2 fl/fl GAS and Csf2 ACD4 GAS mice.
  • Figure 6N Quantification and
  • Figure 60 representative images of Ibal + CD68 + myeloid cells in the glomerular layer of the OB (dashed outline) in PBS and GAS -infected Csf2 fl/fl and Csf2 ACD4 mice.
  • Figures 6P-6R Volcano plots showing significant upregulated (right) and downregulated (left) genes by bulk RNA sequencing in cultured primary brain microglia after 24 hours of treatment with IFNy (Figure 6P), IL-17A ( Figure 6Q), or GM-CSF ( Figure 6R) cytokines.
  • Figures 7A-7E Assessment of barrier properties of the human blood-brain barrier in an in vitro 3D human NVU/BBB microfluidic system using the MIMETAS 3-Lane-40 plates.
  • Figure 7A The schematic diagram of the MIMETAS 3-Lane-40 plates and chips.
  • Figure 7D BMECs are plated on the top channel and form tubules by making contact via cell junctions labelled with ZO-1.
  • the present disclosure relates to using inhibitors of interleukin 17A (IL- 17A) or its receptor for the treatment, prophylaxis or alleviation of Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) including Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).
  • IL- 17A interleukin 17A
  • PANS Pediatric Acute-onset Neuropsychiatric Syndrome
  • PANDAS Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus
  • the disclosure also provides diagnostic methods for PANS (such as PANDAS) where the methods assay the levels of one or more cytokines, chemokines and/or growth factors in a sample (e.g., a blood, plasma or serum sample) taken from a subject.
  • a sample e.g., a blood, plasma or serum sample
  • the present disclosure provides for a method of treating, or treating prophylactically, a pediatric autoimmune neuropsychiatric disorder such as PANS including PANDAS in a subject.
  • PANS may include a broad spectrum of neuropsychiatric conditions.
  • the syndrome of PANS may be associated with a variety of disease mechanisms and have multiple etiologies, ranging from psychological trauma or underlying neurological, endocrine, and metabolic disorders to post-infectious autoimmune and neuroinflammatory disorders, such as PANDAS, cerebral vasculitis, ncuropsychiatric lupus, and others.
  • PANS may be associated with infections, metabolic disturbances, and/or other inflammatory reactions.
  • PANS may be associated with bacterial infections or viral infections.
  • PANS may be associated with infections in the upper respiratory tract, including rhinitis, sinusitis, and pharyngitis.
  • the infection may be caused by group A Streptococcus (GAS), Mycoplasma pneumoniae, Staphylococcus bacteria, influenza viruses, and other viruses.
  • Infections that may be associated with PANS include the infections caused by, or associated with, Epstein Barr virus (EBV), herpes simplex virus (HSV), varicella, upper respiratory infections (including rhinosinusitis, pharyngitis, or bronchitis), and Borrelia burgdorferi (Lyme disease).
  • Other infections that may be associated with PANS include infection caused by babesia, bartonella, and coxsackie virus.
  • PANDAS may refer to Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus, or Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcal infections.
  • PANDAS may be associated with a Group A Streptococcus infection, or a Group A beta- hemolytic Streptococcus infection.
  • PANS or PANDAS may be associated with an ongoing infection or a prior infection.
  • the symptoms of PANS or PANDAS may start after, or at the time of, an infection (e.g., a Streptococcus infection).
  • the symptoms of PANDAS or PANS may start about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or longer, after an infection (e.g., a Streptococcus infection).
  • the symptoms of PANDAS or PANS may become more intense within about 24-48 hours after the onset of the infection (e.g., the Group A Streptococcus infection).
  • the term “inhibitor” refers to agents capable of down-regulating or otherwise decreasing or suppressing the level/amount and/or activity of IL- 17 A or its receptor.
  • the inhibitor can block the ability of IL- 17 A to bind to its receptor.
  • the mechanism of inhibition may be at the genetic level (e.g., interference with or inhibit expression, transcription or translation, etc.) or at the protein level (e.g., binding, competition, etc.).
  • the present inhibitor may inhibit IL-17A or its receptor through any mechanism, including, but not limited to, inhibiting/reducing IL- 17 A (or its receptor) activity, inhibiting/reducing IL- 17 A (or its receptor) level, and/or inhibiting/reducing IL-17A (or its receptor) gene expression.
  • the inhibitor may block an IL-17A-mediated immune response in the subject.
  • the present inhibitors may be a small molecule, a polynucleotide, a polypeptide, or an antibody or antigen-binding fragment thereof.
  • the inhibitor may comprise an antibody or a fragment thereof that binds to IL-17A or its receptor, such as secukinumab, ixekizumab, brodalumab, bimekizumab, or combinations thereof.
  • the polynucleotide is a small interfering RNA (siRNA) or an antisense molecule (e.g., antisense RNA), microRNA (miRNA), ribozymes, triple stranded DNA, etc.
  • the inhibitor is a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system specific for IL-17A or its receptor.
  • inhibitors By “inhibition”, “down-regulation” or “reduction” is meant any negative effect on the condition being studied; this may be total or partial.
  • the present inhibitor/compo sition is capable of reducing, ameliorating, or abolishing the level or activity of the protein (e.g., IL-17A or its receptor).
  • the inhibition or down-regulation of the level or activity of the protein achieved by the present agent may be at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to the level or activity of the protein (e.g., IL-17A or its receptor) in the absence of the present inhibitor/composition.
  • the protein e.g., IL-17A or its receptor
  • composition comprising the present inhibitor.
  • the composition further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
  • the present methods may further comprise administering a second therapeutic agent such as an immunosuppressant, a tumor necrosis factor antagonist (a TNF-antagonist), a CTLA4-antagonist, an anti-IL-6 receptor antibody, an anti-CD20 antibody, or a combination of any of the foregoing.
  • a second therapeutic agent such as an immunosuppressant, a tumor necrosis factor antagonist (a TNF-antagonist), a CTLA4-antagonist, an anti-IL-6 receptor antibody, an anti-CD20 antibody, or a combination of any of the foregoing.
  • the present inhibitor/composition can be administered alone, or may be co-administered together with antibiotics, anti-inflammatory or immune modulating therapies, and/or psychiatric medications and behavioral interventions.
  • Treatments may be sequential, with the present inhibitor/composition being administered before or after the other therapies. Agents/therapies may be administered concurrently.
  • the route of administration may vary, and can include, inhalation, intranasal, oral, transdermal, intravenous, subcutaneous or intramuscular injection.
  • the present disclosure provides for a method of treating, or treating prophylactically, a pediatric autoimmune neuropsychiatric disorder, such as PANS including PANDAS, in a subject.
  • the method may comprise administering to the subject an effective amount (e.g., a therapeutically effective amount) of an inhibitor of IL-17A or its receptor (e.g., interleukin 17 receptor A, or IL- 17RA).
  • an effective amount e.g., a therapeutically effective amount
  • an inhibitor of IL-17A or its receptor e.g., interleukin 17 receptor A, or IL- 17RA.
  • the method may comprise: (a) administering an inhibitor of IL-17A or its receptor to the subject during an induction regimen, where the induction regimen comprises a loading regimen, where the loading regimen comprises administering to the subject five doses of about 150 mg to about 300 mg of the inhibitor, each of the five doses being delivered weekly, beginning on week zero; and (b) thereafter administering the inhibitor to the subject during a maintenance regimen.
  • a “therapeutic regimen” encompasses treatment of an illness, such as PANDAS or PANS.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the patient can be given both an induction regimen and a maintenance regimen of the inhibitor of IL-17A or its receptor.
  • An “induction regimen” refers to a treatment regimen (or the portion of a treatment regimen) used for the initial treatment of a disease. The goal of an induction regimen is to provide a high level of the inhibitor in the system of a patient during the induction period.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the inhibitor than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the inhibitor during a maintenance regimen, or both.
  • the present inhibitor during an induction regimen may be delivered via a subcutaneous route, e.g., delivery of dosages of about 10 mg - about 300 mg s.c, via an intravenous route, e.g., delivery of dosages of about 0.1 mg/kg, - about 50 mg/kg i.v.
  • any other route of administration e.g., intramuscular, i.m.
  • any other route of administration e.g., intramuscular, i.m.
  • Maintenance regimen refers to the treatment of a subject over a longer period of time, days, months, years, during treatment of an illness in order to keep the patient in remission for longer periods of time (months or years). This time frame is referred to as a "maintenance period".
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined.
  • the inhibitor during a maintenance regimen may be delivered via a subcutaneous route, e.g., delivery of dosages of about 10 mg - about 300 mg s.c, via an intravenous route, e.g., delivery of dosages of about 0.1 mg/kg, - about 50 mg/kg i.v. (e.g., about, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, etc.), or any other route of administration (e.g., intramuscular, i.m.).
  • a subcutaneous route e.g., delivery of dosages of about 10 mg - about 300 mg s.c
  • an intravenous route e.g., delivery of dosages of about 0.1 mg/kg, - about 50 mg/kg i.v.
  • a subcutaneous route e.g., delivery of dosages of about 0.1 mg/kg, - about
  • the present inhibitor can be an antibody or antigen-binding fragment thereof that is specific to IL- 17 A or its receptor.
  • the antibody or antigen-binding fragment thereof may be the following: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab')2; and (e) a disulfide linked Fv.
  • the antibodies can be full-length or can include a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab, F(ab')2, Fab’, F(ab)’, Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment [c.g., Ward ct al., Nature, 341:544-546 (1989)], an isolated CDR, diabodics, triabodics, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • the antibody or antigen-binding fragment thereof may be monoclonal, polyclonal, chimeric and humanized.
  • the antibodies may be murine, rabbit or human antibodies.
  • Such antibodies include, but are not limited to, a fully human monoclonal antibody that targets IL-17A (secukinumab), a humanized monoclonal antibody specific for IL-17A (ixekizumab), and a fully human antibody that targets the IL-17 receptor A (brodalumab) [Adamas et al. Front. Immunol. 11: 1894 (2020)].
  • the humanized antibody of the present disclosure may be an antibody from a non-human species where the amino acid sequences in the non-antigen binding regions (and/or the antigenbinding regions) have been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • a chimeric antibody may be a molecule in which different portions are derived from different animal species.
  • an antibody may contain a variable region derived from a murine antibody and a human immunoglobulin constant region.
  • antibodies or antigen-binding fragments thereof in which specific amino acids have been substituted, deleted or added.
  • the present antibodies or antigen-binding fragments thereof may be variants, analogs, orthologs, homologs or derivatives of antibodies of antigen-binding fragments thereof disclosed herein, e.g., with less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 1% amino acid residues substituted or deleted but retain essentially the same or similar immunological properties including, but not limited to, binding to IL-17A or its receptor.
  • IgG e.g., IgGl, IgG2, IgG3, IgG4
  • IgM IgA (IgAl, IgA2)
  • IgD IgE.
  • the antibodies or antigen-binding fragments thereof of the present disclosure may be monospecific, bi-specific or multi- specific.
  • the present antibodies or fragments thereof can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein.
  • small molecules encompasses molecules other than proteins or nucleic acids without strict regard to size.
  • Non-limiting examples of small molecules that may be used according to the present methods and compositions include, small organic molecules, peptide-like molecules, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
  • Non-limiting examples of inhibitors of IL-17A or its receptor include S011806, LY3509754, LEO153339, and the compounds in Zhang et al., Small molecule modulators of IL- 17A/IL-17RA: a patent review (2013-2021), Expert Opinion on Therapeutic Patents, 2022, 32(11): 1161-1173 (which is incorporated by reference herein in its entirety).
  • the inhibitor used in the present methods and compositions is a polynucleotide that reduces expression of IL-17A or its receptor.
  • the nucleic acid target of the polynucleotides may be any location within the gene or transcript of IL- 17 A or its receptor.
  • SiRNAs small interfering RNAs
  • shRNA small-hairpin RNA
  • SiRNAs may have 16-30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • the siRNAs may have fewer than 16 or more than 30 nucleotides.
  • the polynucleotides of the invention include both unmodified siRNAs and modified siRNAs such as siRNA derivatives etc.
  • SiRNAs can be delivered into cells in vitro or in vivo by methods known in the art, including cationic liposome transfection and electroporation. SiRNAs and shRNA molecules can be delivered to cells using viruses or DNA vectors.
  • the polynucleotide is an antisense nucleic acid sequence that is complementary to a target region within the mRNA of TL- 17A or its receptor.
  • the antisense polynucleotide may bind to the target region and inhibit translation.
  • the antisense oligonucleotide may be DNA or RNA, or comprise synthetic analogs of ribo-deoxynucleotides. Thus, the antisense oligonucleotide inhibits or decreases expression of IL-17A or its receptor.
  • An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • antisense nucleic acid molecules of the invention may be administered to a subject, or generated in situ such that they hybridize with or bind to the mRNA of IL-17A or its receptor.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using viruses or DNA vectors.
  • the polynucleotide is a ribozyme that inhibits expression of the gene of IL- 17 A or its receptor.
  • Ribozymes can be chemically synthesized and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme encoding nucleotide sequences can be introduced into host cells through gene-delivery mechanisms known in the art.
  • vectors e.g., viral vectors, expression cassettes, plasmids
  • polynucleotides that act as inhibitors e.g., siRNA, antisense nucleic acids, and ribozymes
  • host cells genetically modified with polynucleotides or vectors.
  • the present inhibitors can also be a polypeptide exhibiting inhibitory activity toward IL- 17 A or its receptor.
  • Various means for delivering polypeptides to a cell can be utilized to carry out the methods of the subject invention.
  • protein transduction domains PTDs
  • PTDs protein transduction domains
  • the PTDs can be fused to the polypeptide, producing a fusion polypeptide, in which the PTDs are capable of transducing the polypeptide cargo across the plasma membrane (Wadia, J. S. and Dowdy, S. F., Curr. Opin. Biotechnol., 2002, 13(1)52-56).
  • Recombinant cells can be administered to a patient, wherein the recombinant cells have been genetically modified to express a nucleotide sequence encoding an inhibitory polypeptide.
  • composition comprising the present inhibitor.
  • the pharmaceutical composition may be administered intrathecally, subdurally, orally, intravenously, intramuscularly, topically, arterially, or subcutaneously.
  • Other routes of administration of pharmaceutical compositions include oral, intravenous, subcutaneous, intramuscular, inhalation, or intranasal administration.
  • the pharmaceutical compositions may be administered by any route, including, without limitation, oral, transdermal, ocular, intraperitoneal, intravenous, Intracerebventricular, intracisternal injection or infusion, subcutaneous, implant, sublingual, subcutaneous, intramuscular, intravenous, rectal, mucosal, ophthalmic, intrathecal, intra- articular, intra-arterial, sub-arachinoid, bronchial and lymphatic administration.
  • the pharmaceutical composition may be administered parenterally or systemically.
  • Specifically targeted delivery of the present composition could be delivered by targeted liposome, nanoparticle or other suitable means.
  • composition may be administered by bolus injection or chronic infusion.
  • claimed composition may be administered at or near the site of the disease, disorder or injury, in a therapeutically effective amount.
  • compositions of the present invention can be, e.g., in a solid, semisolid, or liquid formulation.
  • Intranasal formulation can be delivered as a spray or in a drop; inhalation formulation can be delivered using a nebulizer or similar device; topical formulation may be in the form of gel, ointment, paste, lotion, cream, poultice, cataplasm, plaster, dermal patch aerosol, etc.; transdermal formulation may be administered via a transdermal patch or iontophoresis.
  • Pharmaceutical compositions can also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, emulsions, suspensions, elixirs, aerosols, chewing bars or any other appropriate compositions.
  • the pharmaceutical composition may be administered locally via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed release bolus, or continuous administration.
  • compositions encompass any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions can additionally contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like.
  • Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • Liquid carriers particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols.
  • carriers, stabilizers, preservatives and adjuvants see Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
  • the pharmaceutically acceptable excipient may be selected from the group consisting of fillers, e.g. sugars and/or sugar alcohols, e.g. lactose, sorbitol, mannitol, maltodextrin, etc.; surfactants, e.g. sodium lauryle sulfate, Brij 96 or Tween 80; disintegrants, e.g. sodium starch glycolate, maize starch or derivatives thereof; binder, e.g. povidone, crosspovidone, polyvinylalcohols, hydroxypropylmethylcellulose; lubricants, e.g. stearic acid or its salts; flowability enhancers, e.g.
  • fillers e.g. sugars and/or sugar alcohols, e.g. lactose, sorbitol, mannitol, maltodextrin, etc.
  • surfactants e.g. sodium lauryle sulfate
  • silicium dioxide e.g. aspartame
  • sweeteners e.g. aspartame
  • colorants e.g., colorants.
  • Pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • Oral dosage forms may be tablets, capsules, bars, sachets, granules, syrups and aqueous or oily suspensions. Tablets may be formed form a mixture of the active agents with fillers, for example calcium phosphate; disintegrating agents, for example maize starch, lubricating agents, for example magnesium stearate; binders, for example microcry stallinc cellulose or polyvinylpyrrolidone and other optional ingredients known in the art to permit tabletting the mixture by known methods.
  • capsules for example hard or soft gelatin capsules, containing the active agents, may be prepared by known methods. The contents of the capsule may be formulated using known methods so as to give sustained release of the active agents.
  • dosage forms for oral administration include, for example, aqueous suspensions containing the active agents in an aqueous medium in the presence of a non-toxic suspending agent such as sodium carboxymethylcellulose, and oily suspensions containing the active agents in a suitable vegetable oil, for example arachis oil.
  • the active agents may be formulated into granules with or without additional excipients.
  • the granules may be ingested directly by the patient or they may be added to a suitable liquid carrier (e.g. water) before ingestion.
  • the granules may contain disintegrants, e.g. an effervescent pair formed from an acid and a carbonate or bicarbonate salt to facilitate dispersion in the liquid medium.
  • Intravenous forms include, but are not limited to, bolus and drip injections.
  • Examples of intravenous dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles including, but not limited to. Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water- miscible vehicles including, but not limited to, ethyl alcohol, polyethylene glycol and polypropylene glycol; and non-aqueous vehicles including, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.
  • Additional pharmaceutical compositions include formulations in sustained or controlled delivery, such as using liposome or micelle carriers, bioerodible microparticles or porous beads and depot injections.
  • the inhibitor or pharmaceutical composition may be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter.
  • the pharmaceutical composition can be prepared in single unit dosage forms.
  • Appropriate frequency of administration can be determined by one of skill in the art and can be administered once or several times per day (e.g., twice, three, four or five times daily).
  • the compositions may also be administered once each day or once every other day.
  • the compositions may also be given twice weekly, weekly, monthly, or semi-annually. Tn the case of acute administration, treatment is typically carried out for periods of hours or days, while chronic treatment can be carried out for weeks, months, or even years.
  • compositions can be carried out using any of several standard methods including, but not limited to, continuous infusion, bolus injection, intermittent infusion, inhalation, or combinations of these methods.
  • continuous infusion bolus injection
  • intermittent infusion inhalation
  • one mode of administration that can be used involves continuous intravenous infusion.
  • the infusion of the compositions of the invention can, if desired, be preceded by a bolus injection.
  • Methods of determining the most effective means and dosage of administration can vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject or patient being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific peptide, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • a daily dosage such as any of the exemplary dosages described above, is administered once, twice, three times, or four times a day for at least three, four, five, six, seven, eight, nine, or ten days.
  • a shorter treatment time e.g., up to five days
  • a longer treatment time e.g., ten or more days, or weeks, or a month, or longer
  • a once- or twice-daily dosage is administered every other day.
  • the present disclosure also provides for methods of assaying the levels of one or more cytokines, chemokines and/or growth factors in a sample (e.g., a blood, plasma or serum sample) taken from a subject. Based on the levels of the cytokine(s), chemokine(s) and/or growth factor(s), PANS (such as PANDAS) may be diagnosed or predicted (a risk of developing PANS (such as PANDAS)), and then the subject may be treated.
  • a sample e.g., a blood, plasma or serum sample
  • the present methods may determine/detect the presence, and/or severity of PANS (such as PANDAS).
  • PANS such as PANDAS
  • the levels of cytokines, chemokines and/or growth factors in the sample can be used for assessing the onset or severity of PANS (such as PANDAS), or as an indicator of the efficacy of a therapeutic intervention for treating PANS (such as PANDAS).
  • a plurality of cytokines, chemokines and/or growth factors may be measured.
  • PANS (such as PANDAS) may be diagnosed or predicted, and then the subject may be treated.
  • the therapeutic intervention may be continued when it is effective, or altered if ineffective or insufficient.
  • the method may also identify a subject at risk for PANS (such as PANDAS).
  • PANS such as PANDAS
  • the methods of the present disclosure can impact the way the subject is treated. For example, patients identified as having a high risk of PANS (such as PANDAS) can be treated more aggressively. Patients identified as low risk may be treated less aggressively.
  • the present methods can diagnose or predict PANS (such as PANDAS) in a subject who has an ongoing or prior infection (e.g., a streptococcal infection).
  • PANS such as PANDAS
  • an ongoing or prior infection e.g., a streptococcal infection
  • the method comprises the following steps: (a) obtaining a sample (e.g., a blood, plasma or serum sample, or other samples as discussed herein) from the subject; (b) assaying the level of one or more cytokines, chemokines and/or growth factors in the sample; and (c) comparing the level obtained in step (b) with the level of the one or more cytokines, chemokines and/or growth factors in a control sample.
  • a sample e.g., a blood, plasma or serum sample, or other samples as discussed herein
  • the subject is diagnosed to have PANS (such as PANDAS) (or diagnosed to have an increased risk of developing PANS (such as PANDAS)), if the level of at least one cytokine, chemokine and/or growth factor obtained in step (b) increases by at least 5% compared to its level in the control sample.
  • PANS such as PANDAS
  • PANDAS diagnosed to have an increased risk of developing PANS
  • the present methods may treat a subject with PANS (such as PANDAS) or an increased risk of PANS (such as PANDAS).
  • PANS such as PANDAS
  • PANDAS an increased risk of PANS
  • the subject may be treated with an inhibitor of IL- 17 A or its receptor.
  • PANS such as PANDAS
  • PANDAS predicted (or when an increased risk of PANS (such as PANDAS) is diagnosed)
  • the subject may be treated with an inhibitor of IL-17A or its receptor.
  • the method comprises the following steps: (a) obtaining a sample (e.g., a plasma or serum sample, or other samples as discussed herein) from the subject;
  • a sample e.g., a plasma or serum sample, or other samples as discussed herein
  • step (c) comparing the level obtained in step (b) with the level of the one or more cytokines, chemokines and/or growth factors in a control sample; and (d) treating the subject for PANS (such as PANDAS) or an increased risk of PANS (such as PANDAS), if the level of at least one cytokine, chcmokinc and/or growth factor obtained in step (b) increases by at least 5% compared to its level in the control sample.
  • PANS such as PANDAS
  • PANDAS an increased risk of PANS
  • the present method determines/detects the level of one or more cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, VEGFD, and combinations thereof.
  • the present method determines/detects the level of one or more cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, and combinations thereof.
  • the present method determines/detects the level of one or more cytokines, chemokines and/or growth factors selected from those listed in Table 1, and combinations thereof.
  • Table 1 provides an exemplary list of cytokines, chemokines and/or growth factors whose levels may be determined/detected by the present method.
  • cytokines There may be a number of different isoforms for each of these cytokines, chemokines and/or growth factors.
  • the method comprises the following steps: (a) obtaining a sample from the subject; (b) determining (detecting) in the sample a level of one or more cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, wherein an increase by at least 5% in the level of the one or more polypeptides relative to a control sample indicates that the subject has PANS (such as PANDAS) or have an increased risk of PANS (such as PANDAS).
  • PANS such as PANDAS
  • PANDAS PANDAS
  • the level of at least 1 or 1, at least 2 or 2, at least 3 or 3, at least 4 or 4, at least 5 or 5, at least 6 or 6, at least 7 or 7, at least 8 or 8, at least 9 or 9, at least 10 or 10, at least 11 or 11, at least 12 or 12, at least 13 or 13, at least 14 or 14, at least 15 or 15, at least 16 or 16, at least 17 or 17, at least 18 or 18, at least 19 or 19, at least 20 or 20, at least 21 or 21, at least 22 or 22, at least 23 or 23, at least 24 or 24, or at least 25 or 25, cytokines, chemokines and/or growth factors in the sample may increase by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 30%, at least or about 40%, at
  • the control sample may be from a patient who does not have PANS (such as PANDAS) or a plurality of patients who do not have PANS (such as PANDAS).
  • the control sample may be from a healthy subject or a plurality of healthy subjects.
  • the levels of a plurality of cytokines, chemokines and/or growth factors in the sample may be assayed, which comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, cytokines, chemokines and/or growth factors.
  • the samples may include, but are not limited to, serum, plasma, blood, whole blood and derivatives thereof, bone marrow, urine, cerebrospinal fluid (CSF), endothelium, skin, hair, hair follicles, saliva, oral mucus, vaginal mucus, sweat, tears, epithelial tissues, semen, seminal plasma, prostatic fluid, excreta, ascites, lymph, bile, as well as other samples or biopsies.
  • the biological sample is plasma or serum.
  • the level or amount of a cytokine, chemokine and/or growth factor in a patient sample can be compared to a reference level or amount of the cytokine, chemokine and/or growth factor present in a control sample.
  • Reference levels for a cytokine, chemokine and/or growth factor can be determined by determining the level of a cytokine, chemokine and/or growth factor in a sufficiently large number of samples obtained from normal, healthy control subjects to obtain a pre-determined reference or threshold value.
  • Reference (or calibrator) level information and methods for determining reference levels can be obtained from publicly available databases, as well as other sources.
  • the present diagnostic/treatment methods may be combined with an evaluation of the subject’s medical and psychiatric history, throat swabs, physical examination, viral/bacterial testing, laboratory testing of blood, laboratory testing cerebrospinal fluid (CSF), and selected paraclinical evaluations, such as magnetic resonance imaging (MRI), electrocardiogram/echocardiography, electroencephalography, positron emission tomography (PET), computerized tomography (CT), and polysomnography.
  • the blood testing may include testing for IgA, IgM, IgG (subclass 1, 2, 3, 4), CBC, ANA, ferritin (iron), B-12, vitamin D, and combinations thereof.
  • Viral/bacterial testing may include testing for strep throat culture, antistreptolysin O (ASO), group A strep, anti-DNase B, streptozyme, Mycoplasma Pneumoniae IgA & IgM, pneumococcal antibody titers, Lyme disease and co-inf ections, Epstein Barr virus panel, Coxsackie A & B titers, HHV-6 titers, or combinations thereof. Additional testing may include Cunningham Panel - autoimmune autoantibody levels (Dopamine D 1 receptor, Dopamine D2L receptor, Lysoganglioside GM1, Tubulin and CaM Kinase II); GAD65 antibody testing, or combinations thereof.
  • ASO antistreptolysin O
  • group A strep anti-DNase B
  • streptozyme Mycoplasma Pneumoniae IgA & IgM
  • the present diagnostic/treatment methods may be combined with other assessment/evidence, including, but not limited to, prior streptococcal infection; a rise in the antibody level(s) (e.g., anti-streptolysin O (ASO), anti-DNAse B (ADB), and/or anti- streptococcal carbohydrate (anti-CHO)); acute pharyngitis with a positive GAS throat culture, with or without a rising antibody level; pharyngitis with characteristic palatal petechiae; pharyngitis with a characteristic scarlatinaform rash; pharyngitis without a throat swab or serology, but intimate (usually household) exposure to a proven GAS case; asymptomatic pharyngeal colonization documented after an intimate exposure; asymptomatic pharyngeal colonization after a negative throat swab documented within the prior 3-4 months; single ASO or ADB antibody level within 6 months after the initial onset of neuropsychi
  • the present disclosure provides for methods of evaluating and/or monitoring the efficacy of a therapeutic intervention for treating PANS (such as PANDAS). These methods can include the step of measuring the level of at least one cytokine, chemokine and/or growth factor, or a panel of cytokines, chemokines and/or growth factors, in a biological sample from a subject. In some embodiments, the level of the at least one cytokine, chemokine and/or growth factor in the biological sample is compared to a reference level, or the level of the at least one cytokine, chemokine and/or growth factor in a control sample.
  • the control sample may be taken from the patient at a different time point after the start of the treatment, or from the patient before initiation of the therapeutic intervention, or from the patient at a different time point after initiation of the therapeutic intervention.
  • the measured level of the at least one cytokine, chemokine and/or growth factor is indicative of the therapeutic efficacy of the therapeutic intervention.
  • an increase or decrease in the level of the cytokine, chemokine and/or growth factor is indicative of the efficacy of the therapeutic intervention.
  • a change in the measured level of the at least one cytokine, chemokine and/or growth factor relative to a sample from the patient taken prior to treatment or earlier during the treatment regimen is indicative of the therapeutic efficacy of the therapeutic intervention.
  • An effective therapy may be continued, or discontinued if the patient’s condition has improved and is no longer in need of treatment.
  • An ineffective treatment may be altered or modified, or replaced with other treatment.
  • the method comprises detecting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cytokines, chemokines and/or growth factors described herein.
  • the patient sample may be classified as indicative of effective or non-effective intervention on the basis of a classifier algorithm.
  • samples may be classified on the basis of threshold values as described, or based upon mean and/or median cytokine, chemokine and/or growth factor levels in one population or versus another (e.g., a population of healthy controls or a population of patients without PANS (such as PANDAS), or levels based on effective versus ineffective therapy).
  • threshold values as described, or based upon mean and/or median cytokine, chemokine and/or growth factor levels in one population or versus another (e.g., a population of healthy controls or a population of patients without PANS (such as PANDAS), or levels based on effective versus ineffective therapy).
  • the present invention also provides methods for modifying a treatment regimen comprising detecting the level of at least one cytokine, chemokine and/or growth factor in a biological sample from a patient receiving the therapeutic intervention and modifying the treatment regimen based on an increase or decrease in the level of the at least one cytokine, chemokine and/or growth factor in the biological sample.
  • the methods for modifying the treatment regimen of a therapeutic intervention may comprise the steps of: (a) detecting the level of at least one cytokine, chemokine and/or growth factor in a biological sample from a patient receiving the therapeutic intervention; and (b) modifying the treatment regimen based on an increase or decrease in the level of the at least one cytokine, chemokine and/or growth factor in the biological sample.
  • the method comprises detecting 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cytokines, chemokines and/or growth factors described herein. In certain embodiments, the levels of less than 30, less than 25, less than 20, less than 10, less than 8, or less than 7, cytokines, chemokines and/or growth factors are detected.
  • Modifying the treatment regimen can include, but is not limited to, changing and/or modifying the type of therapeutic intervention, the dosage at which the therapeutic intervention is administered, the frequency of administration of the therapeutic intervention, the route of administration of the therapeutic intervention, as well as any other parameters that would be well known by a physician to change and/or modify. For example, where one or more cytokines, chemokines and/or growth factors decrease (or increase) during therapy or match reference levels, the therapeutic intervention is continued. In embodiments where one or more cytokines, chemokines and/or growth factors do not decrease (or increase) during therapy or match reference levels, the therapeutic intervention is modified.
  • the information regarding the increase or decrease in the level of at least one cytokine, chemokine and/or growth factor can be used to determine the treatment efficacy, as well as to tailor the treatment regimens of therapeutic interventions.
  • the present methods can include the steps of measuring the level of at least one cytokine, chcmokinc and/or growth factor in a sample from a patient receiving a therapeutic intervention, and comparing the measured level to a reference level or the level of at least one cytokine, chemokine and/or growth factor in a control sample.
  • the measured level of the at least one cytokine, chemokine and/or growth factor is indicative of the therapeutic efficacy of the therapeutic intervention.
  • therapy may be continued or altered, e.g., by change of dose or dosing frequency, or by addition of other active agents, or change of therapeutic regimen altogether.
  • the present invention also encompasses a method of predicting or assessing the level of severity of PANS (such as PANDAS) in a patient.
  • the method comprises measuring the level of at least one cytokine, chemokine and/or growth factor in a biological sample from a patient; and comparing the measured level to a reference level or the level of the at least one cytokine, chemokine and/or growth factor in a control sample, wherein the measured level of the at least one cytokine, chemokine and/or growth factor is indicative of the level of severity of PANS (such as PANDAS) in the patient.
  • an increase as described herein
  • in the level of the cytokines, chemokines and/or growth factors is indicative of the level of severity of PANS (such as PANDAS) in the patient.
  • the expression profile of the cytokines, chemokines and/or growth factors in a subject may be determined/detected.
  • the expression profile of the cytokines, chemokines and/or growth factors of the subject may be compared with a reference value, where the reference value is based on a set of cytokine, chemokine and/or growth factor expression profiles of a subject without PANS (such as PANDAS), and/or based on a set of cytokine, chemokine and/or growth factor expression profiles in an unaffected individual or unaffected individuals, and/or based on a set of cytokine, chemokine and/or growth factor expression profiles in the patient before, after and/or during therapy.
  • the changes in cytokine, chemokine and/or growth factor expression may be used to alter or direct therapy, including, but not limited to, initiating, altering or stopping therapy.
  • the sample is a body fluid.
  • the body fluid can include, but are not limited to, serum, plasma, blood, whole blood and derivatives thereof, urine, tears, saliva, sweat, cerebrospinal fluid (CSF), oral mucus, vaginal mucus, seminal plasma, semen, prostatic fluid, excreta, ascites, lymph, bile, and amniotic fluid.
  • the biological sample is plasma or scrum.
  • samples can include, but are not limited to, bone marrow, endothelium, skin, hair, hair follicles, epithelial tissues, as well as other samples or biopsies.
  • cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD, or selected from the cytokines, chemokines and/or growth factors in Table 1, and combinations thereof, are measured.
  • a panel of no greater than 25, no greater than 20, no greater than 15, no greater than 10, or no greater than 5 cytokines, chemokines and/or growth factors is tested, the panel including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cytokines, chemokines and/or growth factors as described herein.
  • the level or amount of cytokine, chemokine and/or growth factor in a patient sample can be compared to a reference level or amount of the cytokine, chemokine and/or growth factor present in a control sample.
  • the control sample may be from a patient who does not have PANS (such as PANDAS) or a plurality of patients who do not have PANS (such as PANDAS).
  • the control sample may be from a healthy subject or a plurality of healthy subjects.
  • a control sample is taken from a patient prior to treatment with a therapeutic intervention or a sample taken from an untreated patient.
  • Reference levels for a cytokine, chemokine and/or growth factor can be determined by determining the level of a cytokine, chemokine and/or growth factor in a sufficiently large number of samples obtained from a patient or patients who do not have PANS (such as PANDAS), or normal, healthy control subjects to obtain a pre-determined reference or threshold value.
  • a reference level can also be determined by determining the level of the cytokine, chemokine and/or growth factor in a sample from a patient prior to treatment with the therapeutic intervention.
  • the level of a cytokine, chemokine and/or growth factor can be detected and/or quantified by any of a number of methods well known to those of skill in the art.
  • the cytokines, chemokines and/or growth factors/proteins may be detected by, for example, mass spectrometry (e.g., LC-MS/MS) and Western blot
  • the methods may include various immunoassays such as cnzymc-linkcd immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), immunohistochemistry, antibody sandwich capture assay, immunofluorescent assay, Western blot, enzyme-linked immunospot assay (EliSpot assay), precipitation reactions (in a fluid or gel), immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), competitive binding protein assays, chemiluminescent assays, and the like.
  • ELISA cnzymc-linkcd immunosorbent as
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, liquid chromatography-tandem mass spectrometry, and the like.
  • HPLC high-performance liquid chromatography
  • TLC thin layer chromatography
  • hyperdiffusion chromatography liquid chromatography-tandem mass spectrometry, and the like.
  • the level of a cytokine, chemokine and/or growth factor may be detected by using molecules (e.g., polypeptides, etc.) that bind to the cytokine, chemokine and/or growth factor.
  • the binding polypeptide may be an antibody or antibody fragment, such as an Fab, F(ab)2, F(ab’)2, Fd, or Fv fragment of an antibody.
  • Any of the various types of antibodies can be used for this purpose, including, but not limited to, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies (e.g., generated using transgenic mice, etc.), single chain antibodies (e.g., single chain Fv (scFv) antibodies), heavy chain antibodies and chimeric antibodies.
  • the antibodies can be from various species, such as rabbits, mice, rats, goats, chickens, guinea pigs, hamsters, horses, sheep, llamas etc.
  • ELISA is used to detect and/or quantify one or more cytokines, chemokines and/or growth factors in a sample.
  • the ELISA can be any suitable methods, including, but not limited to, direct ELISA, sandwich ELISA, and competitive ELISA.
  • Western blot is used to detect and quantify one or more cytokines, chemokines and/or growth factors in a sample.
  • the technique may comprise separating sample proteins by gel electrophoresis, transferring the separated proteins to a suitable solid support, and incubating the sample with the antibodies that specifically bind the one or more cytokines, chemokines and/or growth factors.
  • the disclosure further includes protein microarrays (including antibody arrays) for the analysis of levels of a plurality of cytokines, chemokines and/or growth factors.
  • Protein microarray technology which is also known as protein chip technology and solid-phase protein array technology, is well known to those of ordinary skill in the art. Protein microarray may be based on, but not limited to, obtaining an array of identified peptides or proteins on a fixed substrate, binding target molecules or biological constituents to the peptides, and evaluating such binding. See, e.g., MacBeath et al., Printing Proteins as Microarrays for High-Throughput Function Determination, Science 289(5485): 1760-1763, 2000.
  • one or more control peptide or protein molecules are attached to the substrate.
  • the polypeptides that may be used to assay the level of a cytokine, chemokine and/or growth factor may be derived also from sources other than antibody technology.
  • binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties.
  • the cytokine, chemokine and/or growth factor can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the cytokine, chemokine and/or growth factor.
  • Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the cytokine, chemokine and/or growth factor.
  • the present methods may also assay the presence of or quantity the gene encoding a cytokine, chemokine and/or growth factor or the gene product.
  • Gene products include nucleic acids (e.g., mRNAs) derived from the gene.
  • the level of the DNA or RNA (e.g., mRNA) molecules may be determined/detected using routine methods known to those of ordinary skill in the art.
  • the measurement result may be an absolute value or may be relative (e.g., relative to a reference oligonucleotide, relative to a reference mRNA, etc.).
  • the level of the nucleic acid molecule may be determined/detected by nucleic acid hybridization using a nucleic acid probe, or by nucleic acid amplification using one or more nucleic acid primers.
  • Nucleic acid hybridization can be performed using Southern blots, Northern blots, nucleic acid microarrays, etc.
  • the DNA encoding a cytokine, chemokine and/or growth factor in a sample may be evaluated by a Southern blot.
  • a Northern blot may be used to detect a cytokine, chemokine and/or growth factor mRNA.
  • mRNA is isolated from a given sample, and then clcctrophorcscd to separate the mRNA species. The mRNA is transferred from the gel to a solid support. Labeled probes are used to identify or quantity the cytokine, chemokine and/or growth factor nucleic acids.
  • labeled nucleic acids are used to detect hybridization.
  • Complementary nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. One method of detection is the use of autoradiography.
  • Other labels include ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • Nucleic acid microarray technology which is also known as DNA chip technology, gene chip technology, and solid-phase nucleic acid array technology, may be based on, but not limited to, obtaining an array of identified nucleic acid probes on a fixed substrate, labeling target molecules with reporter molecules (e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP, etc.), hybridizing target nucleic acids to the probes, and evaluating target-probe hybridization.
  • reporter molecules e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP, etc.
  • the sensitivity of the assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected.
  • Nucleic acid amplification assays include, but are not limited to, the polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, quantitative RT-PCR, etc.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • real-time RT-PCR real-time RT-PCR
  • quantitative RT-PCR quantitative RT-PCR
  • Measuring or detecting the amount or level of mRNA in a sample can be performed in any manner known to one skilled in the art and such techniques for measuring or detecting the level of an mRNA are well known and can be readily employed.
  • a variety of methods for detecting mRNAs have been described and may include, Northern blotting, microarrays, realtime PCR, RT-PCR, targeted RT-PCR, in situ hybridization, deep-sequencing, single-molecule direct RNA sequencing (RNAseq), bioluminescent methods, bioluminescent protein reassembly, BRET (bioluminescence resonance energy transfer)-based methods, fluorescence correlation spectroscopy and surface-enhanced Raman spectroscopy (Cissell, K. A. and Deo, S. K. (2009) Anal. Bioanal. Chem., 394: 1109-1116).
  • the present methods may include the step of reverse transcribing RNA when assaying the level or amount of an mRNA.
  • cytokines, chemokines and/or growth factors may include the use of a label(s).
  • the labels can be any material having a detectable physical or chemical property.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Such labels may include, but are not limited to, a fluorescent label, a radiolabel, a chemiluminescent label, an enzyme, a metallic label, a bioluminescent label, a chromophore, biotin etc.
  • a fluorescently labeled or radiolabeled antibody that selectively binds to a cytokine, chemokine and/or growth factor may be contacted with a tissue or cell to visualize the cytokine, chemokine and/or growth factor.
  • a label may be a combination of the foregoing molecule types.
  • the level, amount, abundance or concentration of one or more cytokines, chemokines and/or growth factors may be measured.
  • the measurement result may be an absolute value or may be relative (e.g., relative to a reference cytokine, chemokine and/or growth factor, etc.)
  • a difference e.g., an increase or decrease
  • the measured level of the cytokine, chemokine and/or growth factor relative to the level of the cytokine, chemokine and/or growth factor in the control sample e.g., a sample in at least one healthy individual, in the patient prior to treatment, at a different time point during treatment, or an untreated patient
  • a pre-determined reference value is indicative of the therapeutic efficacy of the therapeutic intervention.
  • an increase or decrease in the measured level of the cytokine, chemokine and/or growth factor relative to the level of the cytokine, chemokine and/or growth factor in the control sample or pre-determined reference value is indicative of the therapeutic efficacy of the therapeutic intervention.
  • the increase (or decrease) is indicative of therapeutic efficacy of the therapeutic intervention.
  • a reduction or decrease in the measured level of the cytokine, chemokine and/or growth factor relative to the level of the cytokine, chemokine and/or growth factor in the control sample (e.g., a sample in the patient prior to treatment or an untreated patient) or pre-determined reference value can be indicative of the therapeutic efficacy of the therapeutic intervention.
  • the control sample e.g., a sample in the patient prior to treatment or an untreated patient
  • pre-determined reference value can be indicative of the therapeutic efficacy of the therapeutic intervention.
  • the level of one or more cytokines, chemokines and/or growth factors is decreased (or increased) when compared to the level in a control sample or pre-determined reference value in response to a therapeutic intervention, the decrease (or increase) is indicative of therapeutic efficacy of the therapeutic intervention.
  • cytokines, chemokines and/or growth factors Patients showing different (elevated or reduced) levels of one or more cytokines, chemokines and/or growth factors can be identified.
  • the expression profile of these cytokines, chemokines and/or growth factors may be used to calculate a score for the combined or individual cytokine, chemokine and/or growth factor expression.
  • the scores of these patients will be compared to the score of unaffected individuals (e.g., patients without PANS (such as PANDAS)).
  • the clinical condition of these patients may be correlated with the cytokine, chemokine and/or growth factor expression profiles.
  • the scores may be used to identify groups of patients having PANS (such as PANDAS) responsive to treatment.
  • the subject may be treated by an inhibitor of IL-17A or its receptor.
  • the subject may be treated with 1, 2 or 3 of the following three modes of intervention:
  • the subject may be treated by an inhibitor of IL-17A or its receptor, in addition to 1, 2 or 3 of the above three modes of intervention.
  • the subject may (further) be treated with antibiotics, anti-inflammatory or immune modulating therapies, and/or psychiatric medications and behavioral interventions.
  • the subject may (further) be treated by an antibiotic, such as penicillin, amoxicillin, cephalosporin, azithromycin, cefdinir, cephalexin, cefadroxil, clindamycin, or clarithromycin etc.
  • the subject may (further) be treated by intravenous immune globulin (TVTG).
  • the subject may (further) be treated by plasmapheresis (which can remove harmful auto-antibodies from the blood to reduce PANDAS symptoms).
  • the subject may (further) be treated with other therapies, including tonsil removal, NSAIDs (e.g., ibuprofen), steroids (e.g., prednisone), and psychological interventions such as cognitive- behavioral therapy or exposure and response therapy (ERP).
  • NSAIDs e.g., ibuprofen
  • steroids e.g., prednisone
  • psychological interventions such as cognitive- behavioral therapy or exposure and response therapy (ERP).
  • kits containing a reagent for measuring at least one cytokine, chemokine and/or growth factor in a biological sample, instructions for measuring at least one cytokine, chemokine and/or growth factor, and instructions for evaluating or monitoring PANS (such as PANDAS) in a patient based on the level of the at least one cytokine, chemokine and/or growth factor.
  • the kit contains reagents for measuring from 1 to about 20 human cytokines, chemokines and/or growth factors, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more cytokines, chemokines and/or growth factors.
  • kits for assessing or predicting the severity or progression of PANS such as PANDAS
  • the kit may comprise a reagent for measuring at least one cytokine, chemokine and/or growth factor in a biological sample, and instructions for assessing severity or progression of PANS (such as PANDAS) based on the level of the at least one cytokine, chemokine and/or growth factor.
  • the kit comprises antibodies specific to one or more cytokines, chemokines and/or growth factors.
  • the kit comprises primers and/or probe for reverse transcribing, amplifying, and/or hybridizing to one or more mRNAs of one or more cytokines, chemokines and/or growth factors.
  • kits can further comprise one or more normalization controls and/or a TaqMan probe specific for each mRNA.
  • the kit may comprise one or more biochips to assay the levels of a plurality cytokines, chemokines and/or growth factors.
  • Biochips may contain a microarray of molecules (e.g., antibodies, peptides etc. as described herein) which are capable of binding to the cytokines, chemokines and/or growth factors described herein.
  • the term “effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician.
  • the term "therapeutically effective amount” is an amount sufficient to treat a specified disorder or disease or alternatively to obtain a pharmacological response treating a disorder or disease.
  • the term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
  • the term also includes within its scope amounts effective to enhance normal physiological function.
  • the subject may be a mammal.
  • Mammals may refer to human subjects and non-human subjects, such as dogs, cats, rats, mice, rabbits, monkeys, etc. (e.g., for veterinary purposes). Mammals include humans (infants, children, adolescents and/or adults), and animals such as dogs and cats, farm animals such as cows, pigs, sheep, horses, goats and the like, and laboratory animals (e.g., rats, mice, guinea pigs, and the like). In certain embodiments, the subjects are infants, children, and/or adolescents.
  • numeric value refers to ⁇ 10% of the stated numeric value. In other words, the numeric value can be in a range of 90% of the stated value to 110% of the stated value.
  • Thl7 effector cytokines IL-17A and GM-CSF, differentially promote BBB dysfunction and microglial expression of interferon-response and chemokine genes in a mouse model of intranasal GAS infections.
  • Antibody blockade of the signature Th 17 effector cytokine, IL- 17 A, in wild-type mice is sufficient to phenocopy the transcriptome rescue in both microglial and endothelial cells after GAS infections, suggesting that IL-17A is a major driver of the CNS pathology after GAS infections.
  • IL-17A blockade partially rescues BBB permeability to serum IgG, indicating that it can be used to treat disorders involving anti-neuronal autoantibodies which have been postulated to underlie the CNS pathology in PANDAS/PANS.
  • Microglia and CNS endothelial cells show major transcriptional shifts after multiple GAS infections
  • CNS endothelial cells upregulate inflammatory signatures and downregulate BBB- specific transcripts after GAS infections
  • microglia isolated from GAS samples showed downregulation of homeostatic genes Tmemll9, Cx3crl, P2ryl2, and Gpr34) and upregulation of disease-associated microglia (DAM) genes (Cst7, Axl, Lpl, Sppl, and Apoe).
  • DAM disease-associated microglia
  • Ccl2 and Ccl3 were expressed primarily by microglia and brain macrophages, whereas Ccl4 and Ccl5 were also expressed by infiltrating peripheral immune cells (Figure 3C).
  • microglial expression of Ccl3 and Ccl4 was not attributable to enzymatic dissociation [Marsh et al. (2022). Nat Neurosci 25, 306-316], since neither correlate with expression of ex vivo activation signature genes.
  • the clusters could not be distinguished by expression of single genes, but rather by gradients of up- or downregulated gene expression.
  • Ccl3 and Ccl4 were expressed by all srMG clusters, whereas Ccl2 levels were the highest in srMG2 and srMG4 ( Figure 4D).
  • Ccl5 and Lllb were expressed by a smaller subset of srMG cells.
  • Cluster srMG4 showed the highest expression of interferon-response genes (Irf7, Isgl5, lfi30, Ifi207, Statl, CxcllO Figures 4C, 4D), despite no detectable upregulation of IFNa or IFNp, either transcriptionally or by multiplex cytokine immunoassay, in the OB after the fifth infection.
  • interferon-response genes Irf7, Isgl5, lfi30, Ifi207, Statl, CxcllO Figures 4C, 4D
  • Streptococcus -responsive microglia are enriched in the glomerular OB layer in close proximity to T cells
  • Microglia which are more numerous throughout the OBs of GAS-infected mice (Figure 3N), had a significantly shorter distance to the nearest T cell in the glomerular and external plexiform layers of the OB compared to the granular layer as quantified by MERFISH ( Figures 3D, 3E, and Figure 30).
  • Brain-derived macrophages are restricted to perivascular and meningeal sites after GAS infections and resemble transcriptionally nasal-derived macrophages
  • the GFP + IdTomalo cells were restricted to perivascular and meningeal regions in both PBS and GAS OBs, with few to none within the brain parenchyma ( Figures 4E-4G), although the number of macrophages and microglia was increased in the OBs of GAS compared to PBS mice ( Figures 4H, 41). Therefore, unlike other models of neuroinflammation, peripheral macrophages do not penetrate the brain after multiple GAS infections.
  • perivascular macrophages which express Cdl63, Cd207 and Lyvel by scRNAseq, revealed upregulation of similar pathways to those found in microglia after GAS infections, including cytokine/chemokine expression, antigen presentation and interferon response.
  • Saa3, encoding serum amyloid A (SAA) protein 3 was the most upregulated gene in perivascular macrophages.
  • SAA3 is produced by myeloid cells and has been shown to sustain Thl7 responses and inflammation in EAE [Lee et al. (2020). Cell 180, 79-91 el6. 10.1016/j.cell.2019.11.026].
  • NALT/OE macrophages showed a similar response to brain macrophages after GAS infections at the molecular level by upregulating several cytokines and chemokines, particularly Ccl5, Ccl6, Ccl9, CxcllO and III b ( Figure 4L).
  • the cytokine/chemokine expression patterns of OB macrophages resembled more closely to NALT/OE macrophages than OB microglia ( Figures 3C, 4L) suggesting a shared origin.
  • PANDAS/PANS patients have elevated serum levels of inflammatory cytokines and growth factors expressed by microglia/macrophages
  • cytokines and growth factors present in patient sera may act on ECs
  • HUVECs human umbilical vein endothelial cells
  • Table 1 we quantified the uptake of fluorescently labeled albumin in HUVECs as a measure of caveolar-mediated uptake and transport [Lutz et al. , S.E., Smith, J.R., Kim, D.H., Olson, C.V.L., Ellefsen, K., Bates, J.M., Vogel, S.P., and Agalliu, D. (2017).
  • ND not detectable.
  • Thl7 cells rescues BBB dysfunction, microglial Activation, and olfactory circuitry deficits in the OB after multiple GAS infections (Platt et al. (2020). Proc Natl Acad Sci U S A 777, 6708-6716)
  • Thl7 fate-specifying transcription factor RORyt (Ivanov et al. (2006). Cell 126, 1121 -1133. 10.1016/j.cell.2006.07.035).
  • ECs from GAS-infected RORyt 7- mice showed a partial restoration of several BBB transcripts (c.g., Mfsd a, ltm2a and Ilih5) and dampened expression of transcripts related to inflammation and LPS response (e.g., Lcn2) compared to wild-type (WT) GAS mice by scRNAseq (Figure 5N).
  • microglia from GAS-infected RORyt 7- mice showed higher expression of homeostatic genes (P2ryl2, Tmemll9, Cx3crl, Gpr34) and lower expression of DAM genes (Cst7, Lpl, Ctsl, Sppl) compared to wild-type GAS-infected microglia by scRNAseq ( Figures 50, 5P).
  • Expression of chemokines and cytokines Ccl2, Ccl3, Ccl4, Tnf
  • interferon-response Irf7, lfitm3, lfi207, lsg!5 genes was also reduced relative to wild-type GAS microglia ( Figures 5Q, 5R).
  • MHC class I markers B2m, H2-D1 and H2-K1 were significantly elevated in astrocytes, OECs, and to a lesser extent in neurons, ECs and microglia from GAS-infected RORyt 7- mice ( Figure 5T).
  • a potential mechanism for this effect could be increased IFNy in RORyt 7- mice, since IL-17A negatively regulates IFNy and Thl cell identity (O'Connor et al. (2009). Nat Immunol 10, 603-609. 10.1038/ni.l736; Ajendra et al. (2020). Mucosal Immunol 13, 958-968.
  • IL-17A is required for BBB dysfunction and microglial activation in vivo following GAS infections
  • Thl7 cells contribute to neurovascular and microglial dysfunction
  • two inflammatory cytokines produced by Thl7 cells IL-17A and GM- CSF.
  • IL-17A-neutralizing monoclonal antibody mAb
  • isotype control antibody administered twice weekly starting 24 hours before the first GAS infection to parse the contribution of IL-17A in the RORyt-dcpcndcnt pathology ( Figure 5D).
  • IL- 17A blockade did not impact CD4 T cell infiltration into the anterior brain since their number was similar between the two conditions (Figure 5U).
  • mice treated with the IL-17A- neutralizing antibody had a significantly higher mortality due to sepsis (Figure 5V), reflective of a key role for IL-17A in controlling infection.
  • IL-17A blockade partially rescued BBB leakage after GAS infections, since there was a two-fold reduction in serum IgG extravasation in the granular layer of the OB in IL-17A blocking condition compared to isotype controls ( Figures 5E-5G), an effect similar to that seen in RORyt /_ mutant mice.
  • IL-17A blockade did not rescue expression of Mfsd a, Itiih5 and Itm2a (Figure 5H), it restored expression of other BBB genes including cell-junction (e.g., Cldn5, Ocln. Cngll, Ctnnal, Tjpl , Tjp2), and transporter genes (e.g., Bsg, Slc7a5, Ap2bl, Abcbla, Slcolcl).
  • IL-17A blockade reduced EC expression of some inflammation and LPS response genes including Lcn2 (Figure 5H).
  • IL- 17A contributes to BBB dysfunction after GAS infections, both at a molecular and functional level.
  • microglia from the IL-17A mAb-treated condition showed rescue of many homeostatic transcripts and decreased expression of DAM signature, chemokines/cytokines, and interferon-response transcripts ( Figures 5I-5L).
  • IL-17A blockade exacerbated upregulation of antigen-presentation genes (Figure 5M).
  • Genes related to antigen presentation by MHC class I were also upregulated in other OB cell types, including OECs, astrocytes and ECs, in IL-17A mAb-treated mice compared to isotype controls (Figure 5T). Therefore, IL-17A promotes several microglial-related neuroinflammatory responses after GAS infections.
  • GM-CSF does not drive BBB breakdown but negatively regulates microglial abundance after multiple GAS infections
  • GM-CSF is another cytokine downstream of RORyt in Thl7 cells with the potential to drive neuroinflammation and vascular dysfunction following GAS infections.
  • GM-CSF plays a pathogenic role in some autoimmune paradigms, such as EAE, but is protective in others 51 .
  • Streptococcus pneumoniae infections GM-CSF is expressed by T cells only in chronic inflammation [Kara et al. (2015). Nat Commun 6, 8644. 10.1038/ncomms9644]. Tn addition, GM- CSF was among the scrum proteins significantly elevated in PANDAS/PANS patients (Table 1).
  • mice lacking GM-CSF in CD4 T cells Csf2 ACD4
  • CD4-Cre ERT2 53 CD4-Cre ERT2 53
  • Csf2 ⁇ 54 mice mice lacking GM-CSF in CD4 T cells
  • 4-OH-tamoxifen 4-OH-tamoxifen between P16 and P20 prior to beginning GAS infections at P28.
  • flow cytometry we confirmed GM-CSF knockdown in CD4 T cells isolated from the OB of GAS-infected mice after 4-OH-tamoxifen administration.
  • Trem2 was even further elevated in Csf2 ACD4 compared to Csf2 fl/fl microglia after GAS infections ( Figure 6M).
  • Loss of GM-CSF expression by CD4 T cells caused a significant increase in Ibal + CD68 + cell number in the glomerular layer of the OB ( Figures 6N, 60). This was not reflected in expression of proliferation genes since Csf2 ACD4 microglia downregulated several cell cycle-related transcripts (e.g. Top2' a. Mcm2, Mcm5 and Mki67) relative to Csf2 fl/fl microglia after GAS infections by scRNAseq.
  • IL-17A and GM-CSF have distinct roles in BBB dysfunction and neuroinflammatory microglial responses after GAS infections in vivo.
  • mBECs mouse brain capillary endothelial cells
  • IFNy which is produced by infiltrating Thl and IFNy + Th 17 cells after multiple GAS infections
  • microglia responded robustly to GM-CSF in culture by upregulating several genes including Cd300f, a gene downregulated in vivo in RORyt /_ , IL-17A mAb-treated and Csf2 ACD4 microglia.
  • Cd300f Cd300f
  • IL-17A mAb-treated Csf2 ACD4 microglia.
  • minimal changes were observed in glial cell with IL-17A treatment, although this is difficult to interpret since cultured microglia expressed low levels of the IL-17A receptor transcript Ill7ra [Timmerman et al. Cell Neurosci 12, 242. 10.3389/fncel.2018.00242].
  • T cell effector cytokines induce distinct gene expression profiles in microglia responsible for neuroinflammatory changes after repeated GAS infections.
  • the endothelial response to GAS includes upregulating antigen presentation and inflammation genes, and downregulating BBB-associated transcripts.
  • BBB-enriched transcripts such as Itm2a, Itih5, Mfsd2a, cell junction regulators (e.g. Cldn5, Ocln, Tjp2, Amot, Magi3, Cgnll ), and ECM proteins and receptors (e.g. Col4a3, Lamc3, Itgal), critical for BBB formation and maintenance, were significantly downregulated after GAS infections.
  • Mfsd2a Decreased expression of Mfsd2a could explain, in part, the upregulation in EC transcellular transport since it inhibits caveolae formation [Ben-Zvi et al., Nature (2014) 509, 507-511]; however, breakdown of cell junctions or increased bulk transcytosis or macropinocytosis can also promote transport across the BBB.
  • Lcn2 The gene most strongly induced in ECs after GAS infections, Lcn2, is involved in the innate immune response with pleiotropic roles in the CNS, and has also been shown to depend on IL- 17 A signaling [Karlsen et al., J Biol Chem (2010) 285, 14088-14100; Shen et al., J Biol Chem (2006) 281, 24138-24148], and the inflammatory cytokine response to LPS administration is exacerbated in Lcn2-deficient brains [Kang et al., Mol Psychiatry (2016) 23, 344-350]. Therefore, Lcn2 may regulate this response by ECs in GAS infections.
  • Microglia also undergoes major transcriptional changes after GAS infections reflected in induction of DAMs, cytokine and chcmokinc, antigen-presentation and intcrfcron-rcsponsc genes, and suppression of homeostatic genes.
  • the MERFISH analysis revealed that expression of Streptococcus-responsive genes is higher in microglia in the OB areas with more infiltrating T cells, suggesting that the proximity to T cells is a key factor driving the shift to disease-associated state in microglia.
  • Trem2 which drives expression of DAM genes in other disease models, was downregulated in microglia after GAS infections.
  • TREM2 may be required for development of “stage 2” DAM signatures, including upregulation of genes such as Lpl, Cst7, Axl, Itgcix and Sppl.
  • T cells that infiltrate the brain after GAS infections secrete IFNy (type II interferon); however, microglial transcription appeared skewed toward a type I IFN response, with increased expression of genes like Irf7, Isgl5, Ifitl, Ifit2, lfit3, Rsad2 and Ms4a4c, which are preferentially induced by IFNa.
  • IFNy type II interferon
  • genes like Irf7, Isgl5, Ifitl, Ifit2, lfit3, Rsad2 and Ms4a4c which are preferentially induced by IFNa.
  • the third class of genes elevated in treptococcus-responsive microglia and macrophages were cytokines and chemokines including Ccl2, Ccl3, Ccl4, Ccl5, CxcllO, Cxcll2, Illb, and Tnf. These cytokines are also upregulated in other models of neuroinflammation and neurodegeneration, although with distinct combinatorial patterns.
  • the microglial cytokine expression profile more closely resembles that of OB than NALT/OE macrophages; however, the proportion of microglia expressing cytokines was higher than that of OB macrophages.
  • chemokines by microglia and macrophages could contribute to recruitment of peripheral immune cells, particularly T cells and infiltrating macrophages, into the CNS.
  • peripheral immune cells particularly T cells and infiltrating macrophages
  • CCL2-CCR2 signaling is required for Thl7 path cell recruitment to the CNS in EAE and S. pneumoniae intranasal infection.
  • Thl7 cells are critical to induce BBB dysfunction, microglial activation, and olfactory circuitry deficits in the OB after multiple GAS infections [Platt et al. (2020). Proc Natl Acad Sci U S A 117, 6708-6716].
  • the molecular transcriptome analysis of microglia from RORyt 1 ' mice supports the rescue of the pathological phenotype (i.e., microglia activation) after GAS infections. Id.
  • Our molecular analysis of microglia shows a rescue in most Streptococcus- responsive pathways in RORyl " mice, particularly in chemokine/cytokine and interferon- response genes.
  • BBB transcripts e.g., Mfsd2a, Itm2a and Iiih5
  • Lcn2 transcripts related to inflammation and LPS response
  • the decrease in EC transcytosis seen in RORyt 1 ' mice after GAS infections is not paralleled by a significant rescue of transcytosis genes (e.g., Mfsd2a or Cavl)
  • the rescue in either cell-junction e.g., Cdh5, Jam2, Tjpl
  • receptor-mediated endocytosis transcripts may explain BBB functional rescue in the absence of Thl7 cells.
  • expression of genes involved in antigen presentation was elevated further in GAS-infected RORyt /_ CNS cells possibly due to increased expression of IFNy at two, but not five, GAS infections.
  • Thl7 effector cytokines IL-17A and GM-CSF
  • IL-17A Thl7 effector cytokine
  • GM-CSF GM-CSF
  • IL-17A blockade partially rescues BBB permeability to serum IgG, indicating that it may have therapeutic potential in disorders involving anti-neuronal autoantibodies which have been postulated to underlie the CNS pathology in PANDAS/PANS [Xu et al. (2021). Am J Psychiatry 178, 48-64. Frick et al. (2016). Brain Behav Immun 69, 304-311. Kirvan et al. (2006). J Neuroimmunol 179, 173-179].
  • GM-CSF an alternate Thl7 effector cytokine
  • CD4 T cells only after multiple GAS infections, which is interesting since PANDAS is thought to result from repeated GAS exposures.
  • Genetic ablation of GM-CSF in T cells failed to rescue BBB leakage to scrum IgG after GAS infections, although at a molecular level some BBB transcripts (e.g., Itih.5, Ilm2a) were increased and inflammatory genes (e.g., Lcn2) were decreased in Csf2 ACD4 mutant ECs after GAS infections.
  • BBB transcripts e.g., Itih.5, Ilm2a
  • inflammatory genes e.g., Lcn2
  • mice were approved by Columbia University’s Institutional Animal Care and Use Committees. Mice were bred in the CUIMC vivarium, under 12-hour light/12-hour dark, pathogen-free conditions. Female mice were used for all experiments, except the time course analysis of Thl7 cell subtypes by flow cytometry and Csf2 recombination confirmation flow cytometry, which used even numbers of males and females.
  • the RORyf GFF mice [Platt et al. (2020), Proc Natl Acad Sci U S A 117, 6708-6716; Ivanov et al. (2006).
  • B6.129P2(Cg)-Rorctm2Litt/J, strain 007572 were obtained from the Jackson Laboratory.
  • the Csf2 fl/fl mouse strain Louis et al. (2020). J Exp Med 217. 10.1084/jem.20191421) was provided by Bogoljub Ciric (Thomas Jefferson University, Philadelphia, PA).
  • CD4-CreER T2 transgenic mice B6(129Xl)-Tg(Cd4-cre/ERT2)llGnri/J, strain 022356) (Aghajani et al. (2012). Genesis 50, 908- 913) were obtained from the Jackson Laboratory and crossed to Csf2 fl/fl mice for two generations.
  • CD4-CreER T2+/_ Csf2 fl/fl males were mated to Csf2 fl/fl females to generate CD4-CreER T2+/ “; Csf2 fl/fl experimental mice and Csf2 fl;fl littermate controls.
  • P16 pups were intraperitoneally injected daily with 100 pg of (Z)-4-Hydroxytamoxifen (Millipore Sigma, H7904), dissolved in 50 pL of com oil (Millipore Sigma, C8267) for 5 days (P16-P20).
  • TMEM119 tdTon,ato (Ruan et al. (2020). Brain Behav Immun 83, 180-191) and CX3CR1 GFP (Jung et al. (2000). Mol Cell Biol 20, 4106-4114) reporter mouse lines were generously provided by Wassim Elyaman (CUIMC).
  • mice received weekly intranasal inoculations with either a suspension of Streptococcus pyogenes [Group A Streptococcus (GAS)], or phosphate -buffered saline (PBS) control, starting at P21-P28.
  • GAS Streptococcus pyogenes
  • PBS phosphate -buffered saline
  • mice were provided with nutritional supplements (ClearH2O, 72-27-5022). Neutralizing antibody treatment
  • mice were injected intraperitoneally twice weekly with 500 pg of either InVivoMAb anti-mouse IL-17A monoclonal antibody, clone 17F3 (Bio X Cell catalog, BE0173), or mouse IgGl isotype control monoclonal antibody, clone MOPC-21 (Bio X Cell, catalog BE0083), in 100 pL of dilution buffer (Bio X Cell catalog IP0070 and IP0065, respectively).
  • InVivoMAb anti-mouse IL-17A monoclonal antibody clone 17F3
  • mouse IgGl isotype control monoclonal antibody clone MOPC-21
  • mice were anesthetized with isoflurane and perfused intracardially with PBS for 3 minutes.
  • Nasal associated lymphoid tissue (NALT), olfactory epithelium (OE), or olfactory bulb (OB) were dissected and placed in Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ and cut up with a sterile scalpel blade.
  • HBSS Balanced Salt Solution
  • Two or three animals were pooled per sample. Tissue was then placed in C Tubes (Miltenyi Biotec, 130-093-237), along with dissociation reagents from the MACS Neural Tissue Dissociation Kit (P) (Miltenyi Biotcc, 130-092-628).
  • nucleated cells (DRAQ5+ PIlo) were sorted on a FACSAria II (BD), equipped with 355 nm, 405 nm, 488 nm, 561 nm and 640 nm lasers and a 130 pm nozzle.
  • BD FACSAria II
  • CD31+ and CDl lb-i- populations were collected to enrich for cell types of interest. Sequencing was performed by the Columbia Single Cell Core using 10X Genomics Chromium Single Cell 3’ technology, with reads aligned to the mml0-2020-A transcriptome.
  • mice were anesthetized with isoflurane and perfused intracardially with PBS for 4 minutes, followed by 4% paraformaldehyde (PFA) for 6 minutes. Brains were extracted and post-fixed in 4% PFA for 4-6 hours, then washed three times in PBS, incubated overnight in 30% sucrose, embedded in Tissue-Plus O.C.T. compound (Fisher, 4585) and stored at -80°C. Coronal sections (12 pm) were cut on a Leica CM3O5O S Cryostat and stored at -80"C.
  • PFA paraformaldehyde
  • Plasmids were obtained from Transomic Technologies and the antisense mRNAs were synthesized using the Digoxigenin RNA Labeling Kit (SP6/T7; Roche, 11175025910). DIG RNA in situ hybridization (ISH) and fluorescent in situ hybridization (FISH) experiments were performed as previously described (Biswas et al., Development, 2022; 149(17):dev200610). Mice used for ISH or FISH were perfused for 4 minutes with PBS and brains were dissected out and embedded in O.C.T.
  • ISH Digoxigenin RNA Labeling Kit
  • FISH fluorescent in situ hybridization
  • mice were anesthetized with isoflurane and intracardially perfused with cold RNAse-free PBS for 4 minutes. Brains were dissected out and immediately embedded in Tissue-Plus O.C.T compound and stored at -80"C until samples could be shipped overnight on dry ice to UMass Chan Medical School. Samples were prepared according to the Vizgen Fresh Frozen Tissue Sample Preparation protocol. Tissue was sectioned in 10 pm slices onto a functionalized coverslip covered with fluorescent beads. Each coverslip contained a section from one PBS sample and a section from one GAS sample. Tissue on coverslips was fixed for 15 minutes at room temperature in 4% paraformaldehyde in PBS, followed by three washes with PBS.
  • Tissue was then permeabilized in 70% ethanol for 24 hours, washed with PBS and incubated with blocking solution for 1 hour, followed by 1 hour of incubation with the primary antibody against vessel marker CD31 (BioLegend, 102502), diluted 1:20 in blocking solution (Vizgen).
  • the tissue was then washed three times with PBS and incubated for 1 hour with an oligo-conjugated secondary antibody diluted 1:1000 in blocking solution.
  • the sample was washed three times with PBS and fixed for 15 minutes at room temperature in 4% paraformaldehyde in PBS, followed by three washes with PBS.
  • MERFISH imaging was performed as previously described97 with parameter files provided by Vizgen. Briefly, the sample was loaded into a flow chamber connected to the Vizgen Alpha Instrument. First, a low-resolution mosaic image was acquired (405 nm channel) with a low magnification objective (lOx). Then the objective was switched to a high magnification objective (60x) and seven 1.5-pm z-stack images of each field of view position were generated in 749 nm, 638 nm and 546 nm channels. A single image of the fiducial beads on the surface of the coverslip was acquired and used as a spatial reference (477 nm channel).
  • Probes for the following transcripts were used: Abca7, Abcc3, Abcg2, Abliml, Actb,Acvrll , AdamlO, Adaml7, AdgrfS, Adgrl4, Adoral, Aff3, Ago4, Agt, Ahr, Akapl2, Aldoc, Anxal, Ap2ml, Aqp4, Arc, Argl, Arhgap29, Arll5, Arpc2, Atmin, AtplOa, Axl, Baiap2ll, Bardl, Binl, Birc5, Bmp6, Brcal, Btk, Clqa, Clqb, Clqbp, Clqc, C3, C3arl, C4a, C5arl, Caldl, Casp7, Casp8, Cass4, Ccl2, Ccl22, Ccl3, Ccr2, Cdl4, Cdl63, Cd27, Cd33, Cd3e, Cd4, Cd47, Cd68, Cd72
  • mice were anesthetized with isoflurane and intracardially perfused with PBS for 4 minutes. Brain, as well as combined nasal associated lymphoid tissue/olfactory epithelium (NALT/OE), were dissected out, placed in cold Dulbecco’s Modified Eagle’s Medium (DMEM) (Genesee, 25-500), and pressed through a cell strainer with the end of a sterile syringe. Samples were collected in 10 mL of a 30% Percoll (Cytiva, GE17-0891-01) suspension in DMEM, and underlaid with 1 mL of 70% Percoll.
  • DMEM Modified Eagle’s Medium
  • Spleen samples were suspended in 3 mL of Red Blood Cell Lysis Buffer (155 mM NH4C1, 10 mM KHC03, 0.1 mM EDTA) at room temperature for 10 minutes. Samples were centrifuged at 4°C at 1300 ref for 30 minutes, then immune cells were collected at the interface.
  • Red Blood Cell Lysis Buffer 155 mM NH4C1, 10 mM KHC03, 0.1 mM EDTA
  • Intracellular stains were diluted in IX permeabilization buffer and incubated for 1 hour, then washed in permeabilization buffer and resuspended in FACS buffer. Samples were analyzed in the Columbia Stem Cell Initiative Flow Cytometry Core. Time course experiments were analyzed using a ZE-5 analyzer (Bio-Rad, Hercules, CA) equipped with 355 nm, 405 nm, 488 nm, 561 nm, and 640 nm lasers. Compensation controls used splenocytes for most cell surface markers and compensation beads (BD Biosciences, 552844 for rat hosts; ThermoFisher, 01-3333- 41 for mouse or Armenian hamster hosts) for cytokines.
  • mBECs Primary mouse brain endothelial cells (mBECs; Cell Biologies, C57-6023) were cultured as monolayers at 37°C with 5% CO2 and used to evaluate the effect of cytokines in vitro.
  • Cells were grown to confluence on collagen IV-coated (Corning, CB-40233) dishes in endothelial cell media (Cell Biologies, M1168) supplemented with 10% FBS (Cytiva, SH30071.03) and supplements recommended by the supplier.
  • FBS Cytiva, SH30071.03
  • TEER Trans-endothelial electrical resistance
  • ECIS electric ccll-substratc impedance sensing
  • Cytokines or vehicle were added at a concentration of 50 ng/mL, except IL-1 (R&D Systems, 401-ML) and TNF (R&D Systems, 410-MT), which were added at lOng/mL. Resistance was monitored over 24 hours from the start of cytokine treatment. The area under the curve (AUC) was calculated for each condition and normalized to AUC of vehicle-treated cells, with values for each independent experiment compared by one-way ANOVA. Albumin transcytosis was measured in vitro by culturing mBECs on collagen IV-coated 3.0-pm PET membrane inserts for 24-well plates (Corning, 353096).
  • Cytokines or vehicle were added at a concentration of 100 ng/mL, with 500 ng/mL lipopolysaccharide (LPS) used as a positive control.
  • LPS lipopolysaccharide
  • Cells were switched to endothelial cell media with 2% FBS and without phenol.
  • Media added to wells contained bovine serum albumin at 400 pg/mL, while media added to inside of transwell inserts contained albumin conjugated to AlexaFluor 647 (ThermoFisher, A34785) at 400 pg/mL. Cells were incubated at 37°C with flow-through samples collected from bottom wells at 30 minutes, 1, 2, 4 and 6 hours).
  • microglia were obtained using a shake off protocol and cultured in serum-free media containing neurobasal solution (ThermoFisher, 211039049), insulin (Sigma, 16634, 1: 100), sodium pyruvate (Invitrogen, 11360-070, 1: 100), pen/strep (Life Technologies, 15140-122, 1:100), SATO (containing transferrin, BSA, putrescine, progesterone, and sodium selenite; 1:100), thyroxine, GlutaMAX (Life Technologies, 35050-061, 1: 100), B27 (ThermoFisher, 17504- 044, 1:50), N-acetyl cysteine (1: 1000), and mouse M-CSF (Shenandoah Biotechnology, 200-08- 10, 1: 1000). Cultures were treated with 100 ng/mL of either IFNy, IL-17A or GM-CSF for 24 hours, then washed and collected into TRIzol for RNA extraction and sequencing
  • Serum protein concentrations were measured by the Irving Institute for Clinical and Translational Research Biomarkers Core Laboratory using a 45- Plex Luminex assay (Invitrogen, EPX260-26088-901). Samples were run in duplicate, and standard curves were run for each analyte. Undetectable values were replaced with half of the lower detection limit for statistical comparison.
  • RNA sequencing data was analyzed using Seurat package v4.0.298 in RStudio. Upon data import, genes detected in fewer than three cells, and cells with fewer than 200 genes were excluded. Cells were removed from the merged data set if they had fewer than 1 ,000 or more than 50,000 molecules detected, or greater than 20% mitochondrial reads. Data was normalized and highly variable features identified using default parameters, then scaled, followed by linear dimensional reduction using PCA. Dimensionality of the data was selected using the Elbow plot method with 50 dimensions, and cells were clustered with a resolution of 1 for OB, 0.4 for endothelial cells and microglia, and 2 for NALT/OE. Dimensionality reduction for visualization was performed with /-distributed stochastic neighbor embedding (t-SNE). The Harmony package vl.099 was used for batch correction.
  • t-SNE stochastic neighbor embedding
  • Cluster identity was assigned using the following cell type markers: neurons (Map2, Snap25), astrocytes (Gfap, Aqp4). olfactory ensheathing cells (Frzb), oligodendrocytes/oligodendrocyte precursor cells (Pdgfra), endothelial cells (Cldn5.
  • Pecaml pericytes (Pdgfrb, Atpl3a5), fibroblasts (Coll al, Fblnl), microglia (Tmemll9, P2ryl2), macrophages (Aifl, Plac8), neutrophils (Ly 6g, Camp), dendritic cells (Xcrl, Ccr9, Cd209 ), B cells (Cdl9), CD4 T cells (Cd4), CD8 T cells (Cd8a), NK cells (Klrblc), and 78 T cells (Cdl63ll).
  • FindMarkers was used to compare GAS to PBS conditions with minimum percent of 0.2, fold-change threshold of 0.5 and p-value cutoff of less than 0.05.
  • Expression of ex vivo activation genes Duspl, Fos, Histlhld, Histlh2ac, Jun, Nfkbid, Nfkbiz) were added using AddModuleScore and plotted against Ccl3 and Ccl4 using FeatureScatter. Additional analysis was performed with BB Browser 3 (BioTuring) software.
  • Signature scores were plotted in BBrowser3 using the following pathway markers: Antigen presentation (B2m, Cd74, H2-Aa, H2-Abl, H2-D1 , H2-Ebl , H2-K1 , H2-Q4, H2- Q6, H2-Q7, Tapi, Tap2), disease-associated microglia (Apoe, Axl, Cd9, Csfl, Cst7, Itgax, Lpl, Sppl, Tyrobp), homeostatic microglia (Cd33, Cst3, Cx3crl, Feris, Gpr34, 0lfml3, P2ryl2, P2ryl3, Salll, Tmeml 19), and interferon signaling (Ifi30, lfi204, lfi211, Ifitl, Ifitm3, Irfl, Irf7, Isgl5, Oasla, Stall, Stat2).
  • Antigen presentation B2m, Cd74, H2-Aa, H
  • GSEA31 Gene set enrichment analysis (GSEA)31 was performed using curated and database derived gene lists for blood-brain barrier, response to LPS, inflammation, extracellular matrix, interferon response, antigen presentation, chemokine and cytokine signaling, endothelial cell proliferation, endothelial cell migration, disease-associated microglia, apoptosis, leukocyte chemotaxis and phagocytosis. Analysis was run using the GSEA desktop tool (Broad Institute, v4.1.0), using preranked weighted settings. Stacked violin plots were generated using scripts by Dr. Ming Tang (divingintogeneticsandgenomics.rbind.io).
  • MERFISH data was analyzed in RStudio using Seurat 4.1.0.9005, R 4.0.0 and custom- made scripts as previously describedlOO. Cell segmentations with volume ⁇ 50pm3 or ⁇ 10 unique transcripts were first excluded. Cell gene expression data of each cell was then normalized to that cell’s volume and the total transcript count of that cell, then scaled. To correct for global differences in total transcript counts between coverslips (each containing one GAS sample and one PBS sample), we performed ComBatlOl batch correction (sva 3.38.0). To identify individual cell types, we performed principal component analysis was performed using the entire probe library (391 transcripts) as the variable features, followed by linear dimensional reduction.
  • t-SNE stochastic neighbor embedding
  • Raw counts were normalized to the PBS condition for each batch and used for gene expression analysis. Endothelial cell gene expression was compared on log2 fold change values using a one-sample t test. Microglia gene expression between the glomerular and granular layers used a ratio t test. Nearest neighbor analysis of microglial distance to T cells was calculated based the x,y coordinates of the centers of the cell segmentations using a custom python script.
  • Thl7 effector cytokines differentially promote microglial and blood-brain barrier inflammatory responses during post-infectious encephalitis, bioRxiv, doi: https://doi.org/10.1101/2023.03.10.532135, which is incorporated by reference herein in its entirety.
  • This 3D system includes flow (shear stress is crucial for BMEC maturity and function) and uses a glass bottom plate with 3 microfluidic channels separated by middle-channel phase guides (Figure 7A).
  • Intact BEC tubule formation is confirmed by dual tracer barrier integrity assays (BI), TEER measurements and key cell-type marker IF staining (Figure 7B).
  • BI tracer barrier integrity assay
  • TEER measurements and key cell-type marker IF staining
  • Figure 7B We have established the 3D NVU system for 10 days prior to exposure to sera from PANDAS/PANS patients and from healthy controls (described in Example 1).
  • sera from PANDAS/PANS patients, but not controls enhance permeability of the BBB and promote migration of tracer.
  • vGluT2 Vesicular glutamate transporter 2
  • GAD67 glutamate decarboxylase 67
  • Blockade of IL-17A with antibodies are expected to restore vGluT2 expression within glomeruli by 48 hours after the final GAS infection.
  • Electrophysiology To determine whether there are persistent deficits in olfactory processing, we will record odor-evoked spiking activity in populations of mitral/tufted (M/T) neurons within the OB in awake, head-fixed GAS-infected mice around the 48-hour time point after the last GAS infection (Bolding et al., (2017) Elife 6:e22630; Bolding et al., (2016), Science 361:6407). Extracellular unit recordings and analyses of odor-evoked spiking activity in the mitral/tufted (M/T) neurons within the OB will be performed in awake, head-fixed mice as published (Platt et al., (2020) Proc Natl Acad Sci U S A, 117 (12) 6708-6716).
  • mice After four GAS infections, mice will be surgically fitted with a head plate to allow for awake, head-fixed recording from the OB. Recordings will be performed in a ten-channel olfactometer 48 hours after the final infection. A 32-site polytrode acute probe will be placed into the posterior OB near the ventromedial M/T neuronal layer. Signals will be collected through an A32-OM32 adaptor connected to a Cereplex digital head stage. Six monomolecular odors will be used: methyl tiglate, y-terpinene, 2-hexanone, isoamyl acetate, ethyl butyrate and valeraldehyde.
  • M/T neurons are expected to be either activated or suppressed by different monomolecular odorants as they relay the information to various regions of the olfactory cortex.
  • Single M/T neurons are expected to show robust odor responses in PBS control mice.
  • GAS-infected mice will show a striking absence of odor responses in M/T neurons
  • GAS- infected mice treated with IL- 17 A blocking antibody may show either a partial or complete rescue in single M/T neuronal responses, indicating that physiological deficits are partially regulated by an IL-17A-dependent mechanism.
  • mice Animal behavior: The transient loss of vGluT2 expression in OB glomeruli raises the question whether olfactory function is impaired persistently in GAS-infected mice.
  • We will use a habituation-dishabituation behavioral paradigm, which leverages a mouse’s curiosity for new olfactory stimuli. Mice are presented with an unscented Q-tip, which they first investigate by sniffing but then rapidly lose interest at subsequent presentations of the same stimulus (habituation). However, if the Q-tip is impregnated with an odor, mice initially investigate the novel odor (dishabituation) indicating odor detection, but they rapidly habituate to subsequent presentations of the same odor.
  • mice will re-investigate if a different odor is presented, measuring odor discrimination.
  • Each odorant (almond extract, vanilla extract, ethyl butyrate, and 2- phenylethanol) will be presented three times, with water presented first as described (Platt et al., (2020) Proc Natl Acad Sci U S A, 117 (12) 6708-6716).
  • mice Normal mice will actively investigate an unscented Q-tip, but then rapidly lose interest (Root et al., (2014) Nature 515, 269-273) and perform normally the other behavior tasks. In contrast, GAS-infected mice will show both impaired odor detection and odor discrimination at 6 h after the last infection, but show normal spontaneous motor activity in the OF test. Finally, GAS mice treated with an IL-17A blocking antibody will show restoration of odor detection.

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Abstract

The present disclosure relates to using inhibitors of interleukin 17A (IL-17A) or its receptor for the treatment, prophylaxis or alleviation of Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) including Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). Also provided are diagnostic methods for PANS (such as PANDAS) where the methods assay the levels of one or more cytokines, chemokines and/or growth factors in a sample taken from a subject.

Description

METHODS AND COMPOSITIONS FOR TREATING PEDIATRIC AUTOIMMUNE NEUROPSYCHIATRIC DISORDERS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Patent Application Nos. 63/352,195 (filed on June 14, 2022) and 63/352,201 (filed on June 14, 2022), which are incorporated herein by reference in their entirety.
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under MH 112849 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
The present invention relates to methods of diagnosing and treating pediatric autoimmune neuropsychiatric disorders such as Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS).
BACKGROUND
Even in the absence of brain infections, bacterial or viral infections may trigger neuropsychiatric and cognitive dysfunction [Blackburn et al. (2020) Ther. Adv. Neurol. Disord. 13, 1756286420952901]. Infections with Group A Streptococcus (GAS), can give rise to secondary sequelae in the patient, including movement abnormalities such as Sydenham’ s chorea, or disabling tics and psychiatric manifestations. Clinically, the psychiatric syndrome is classified as Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS), which is defined by several diagnostic criteria: (i) abrupt “overnight” obsessive compulsive disorder (OCD), or dramatic, disabling tics, (ii) an episodic course, (iii) young age at onset (average 6-7 years), (iv) presence of neurological abnormalities and (v) temporal associate between symptom onset and GAS infection. https://www.pandasppn.org/what-are-pans-pandas/; Swedo et al., Am. J. Psychiatry 155:2, 264- 271 (1998)). Swedo et al., Pediatr. Therapeut. 2012, 2:2. PANDAS are part of a larger group of neuropsychiatric disorders termed Pediatric Acute- Onset Ncuropsychiatric Syndrome, or PANS, characterized by a sudden onset of OCD or eating restrictions, together with acute behavioral deterioration in at least two areas, anxiety, sensory amplification, or motor abnormalities, behavioral regression, deterioration in school performance, mood disorder, urinary symptoms or sleep disturbances. Swedo et al., Clinical Presentation of Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections in Research and Community Settings, J. Child Adolesc. Psychopharmacol. 2015, 25(1): 26-30. Mechanistically, the behavioral changes observed with PANDAS or PANS may be the result of an aberrant anti-pathogen immune response targeting the central nervous system (CNS) (Cunningham (2019). Microbiol. Spectr. 7. 10.1128/microbiolspec.GPP3-0045-2018).
During the early stages of the bacterial infection, antibiotic therapy, even for an extended time period such several months, is recommended. Cooperstock et al., Clinical Management of Pediatric Acute-Onset Neuropsychiatric Syndrome: Part III — Treatment and Prevention of Infections, Journal of Child and Adolescent Psychopharmacology, 2017, 27(7): 594-606. Antibiotics, such as amoxicillin, penicillin, azithromycin, and cephalosporins, which are typically used to treat Streptococcus infection can be used and treatment often results in immediate improvement. Anti-inflammatory agents (including both steroids and non-steroidal anti-inflammatory drugs (NSAIDS)) may also be helpful. Although treatment with prednisone or corticosteroids has been used with some reports of improvements, in some cases tic conditions have actually worsened. Frankovich et al., Clinical Management of Pediatric Acute-Onset Neuropsychiatric Syndrome: Part II — Use of Immunomodulatory Therapies, Journal of Child and Adolescent Psychopharmacology, 2017, 27(7): 574-593. Due to possible long-term complications, such treatments, antibiotics or steroids, can only be used for short time periods. Even if patients show improvement after treatment, the symptoms, sometimes even worse, often return after treatment is stopped. U.S. Patent No. 10,228,376; https://www.pandasppn.org/symptom-severity/.
Thus, there is a continuing need to develop more effective methods for diagnosing and treating both PANDAS and PANS. SUMMARY
The present disclosure provides for a method of treating a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in a subject in need thereof. The method may comprise administering to the subject an inhibitor of IL- 17 A or its receptor.
The PANS may be Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). The PANDAS may be associated with Group A Streptococcus infection. The PANS may be associated with an ongoing or prior infection.
The inhibitor may comprise an antibody or a fragment thereof that binds to IL- 17 A or its receptor. In certain embodiments, the antibody comprises secukinumab (SEC), ixekizumab (IXE), brodalumab (BROD), bimekizumab, or combinations thereof.
The inhibitor may comprise a small molecule.
The inhibitor may be administered by inhalation, intranasally, intrathecally, orally, intravenously, subcutaneously or intramuscularly. In one embodiment, the inhibitor is administered intranasally. In certain embodiments, the inhibitor may be administered using an intranasal spray device, an atomizer, a nebulizer, a metered dose inhaler (MDI), a pressurized dose inhaler, an insufflator, an intranasal inhaler, a nasal spray bottle, a unit dose container, a pump, a dropper, a squeeze bottle, or a bi-directional device.
The present disclosure provides for a method for detecting or diagnosing a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in a subject, or assessing the subject’s risk of developing a pediatric autoimmune neuropsychiatric disorder such as PANS. The method may comprise: (a) obtaining a sample from the subject; (b) determining levels of one or more cytokines, chemokines and/or growth factors in the sample, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; (c) comparing the levels obtained in step (b) with the levels of the one or more cytokines, chemokines and/or growth factors in a control sample; and (d) diagnosing that the subject has a pediatric autoimmune neuropsychiatric disorder (such as PANS) or an increased risk of developing a pediatric autoimmune neuropsychiatric disorder (such as PANS), if the level of at least one cytokine, chemokine and/or growth factor obtained in step (b) increases by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, compared to its level in the control sample.
Also encompassed by the present disclosure is a method of treating a subject with a pediatric autoimmune neuropsychiatric disorder such as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) or an increased risk of developing a pediatric autoimmune neuropsychiatric disorder such as PANS. The method may comprise: (a) obtaining, or having obtained, a sample from the subject; (b) determining, or having determined, levels of one or more cytokines, chemokines and/or growth factors in the sample, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; (c) comparing, or having compared, the levels obtained in step (b) with the levels of the one or more cytokines, chemokines and/or growth factors in a control sample; and (d) treating the subject for PANS or an increased risk of developing PANS, if the level of at least one cytokine, chemokine and/or growth factor obtained in step (b) increases by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1- fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, compared to its level in the control sample.
In certain embodiments, the one or more cytokines, chemokines and/or growth factors may be selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, and TNFa.
The PANS may be Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). The PANDAS may be associated with Group A Streptococcus infection. The PANS may be associated with an ongoing or prior infection.
The sample may be a plasma, serum or blood sample.
The control sample may be from a healthy subject or a plurality of healthy subjects. The control sample may be from a subject without a pediatric autoimmune neuropsychiatric disorder such as PANS, or a plurality of subjects without a pediatric autoimmune neuropsychiatric disorder such as PANS. The subject may be a mammal such as a human.
The subject’s existing PANS treatment regimen may be modified or maintained.
The level of the one or more cytokines, chemokines and/or growth factors may be determined by mass spectrometry (MS), or by enzyme-linked immunosorbent assay (ELISA).
The present disclosure provides for a kit comprising: (a) antibodies or fragments thereof that specifically bind to one or more cytokines, chemokines and/or growth factors in a blood, plasma or serum sample from a subject, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL- 23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; and (b) instructions for measuring the one or more cytokines, chemokines and/or growth factors for diagnosing Pediatric Acuteonset Neuropsychiatric Syndrome (PANS) in the subject or assessing the subject’s risk of developing PANS.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1I: Microglia and CNS endothelial cells display major transcriptional shifts after multiple GAS infections. Figure 1A, Schematic diagram of the workflow for singlecell RNA sequencing (scRNAseq) and MERFISH experiments. Figures 1B-1C, Dimensionality reduction plot of scRNAseq from the olfactory bulbs (OBs) of GAS-infected and PBS control mice without cell type-specific enrichment (Figure IB), and with enrichment by sorting for CD31+ (endothelial) and CDllb+ (myeloid) cells (Figure 1C). Figure ID, Dimensionality reduction plot of sample identity for OB cell types isolated from PBS and GAS conditions. Figure IE, Cell type abundance from OBs from scRNAseq (with and without enrichment) and MERFISH experiments. The labeled bars represent the fraction of endothelial cells (ECs) and microglia among all isolated cells. Figure IF, Representative MERFISH coordinate plot. Figure 1G, /-SNE plot for all MERFISH samples. Figures 1H-1I: Plots of the numbers of differentially expressed genes (DEGs) by cluster, displaying upregulated and downregulated DEGs.
Figures 2A-2H: CNS endothelial cells upregulate inflammatory signatures and lose BBB marker expression following GAS infections. Figure 2A, r-SNE plot of the sample identity for ECs isolated from the OBs of PBS and GAS-infected mice. Figure 2B, Gene set enrichment analysis (GSEA) for genes differentially expressed in ECs following GAS infection. Bars indicate significance of enrichment (-logiiDR) and dots indicate the number of genes significantly enriched in each gene set. The inflammatory signatures are upregulated, whereas blood-brain barrier (BBB) gene signatures are downregulated in ECs from GAS- infected mice. Figure 2C, Heat map of genes related to the endothelial response to systemic LPS in ECs from the OB of PBS and GAS-infected mice. Figures 2D-2F, Expression changes for three BBB transcripts (Itm2a, Itih5 and Mfsd2d) in ECs by scRNAseq (left). Statistical comparisons of scRNAseq by Wilcoxon Rank Sum test (* p-adj < 0.05). Representative images (center) and quantification (right) of expression changes in the BBB transcripts by fluorescence in situ RNA hybridization (FISH) for the mRNA probes combined with immunofluorescence staining for EC marker Glutl (Figure 2D-2E) and immunofluorescence staining for Mfsd2a and EC marker CD31 (Figure 2F). Scale bars = 25 pm. Statistical comparisons were performed with unpaired t test with Welch’s correction (* p < 0.05; n = 7-9 per condition). Error bars represent mean with SEM. Figure 2G, On the left, correlation of log2 fold changes in CNS EC genes identified from GAS versus PBS (x-axis) with those identified from either acute experimental autoimmune encephalomyelitis (EAE) versus Complete Freund Adjuvant (CFA) control (top panels, y-axis) and chronic EAE versus CFA (bottom paneled; y-axis). Correlations on the right display only BBB-associated genes. The correlation coefficient is displayed in the top left comer of each plot. Gray dotted lines mark the line of identity; black dashed line indicates the line of best fit. Figure 2H: Downregulation of BBB-related genes Itm2a, Itgal and Itgbl by MERFISH (* p < 0.05; ** p < 0.01; *** p < 0.001; one sample t-test; n = 4 mice per group). Error bars represent mean with SEM.
Figures 3A-3O: Microglia respond to recurrent GAS infections by upregulating inflammatory gene signatures. Figure 3A, t-SNE plot showing the sample identity of microglia from the OBs of PBS and GAS-infected conditions. Figure 3B, GSEA for genes upregulated by microglia after GAS infection. Bars indicate significance of enrichment (-logirDR) and dots indicate number of genes significantly enriched in each gene set. Figure 3C, Heat map of genes related to antigen presentation, cytokines and chemokines, and interferon response in PBS and GAS samples. Figure 3D, Representative coordinate plot of microglia showing different OB layers in the MERFISH. Figure 3E, Representative coordinate plot of microglial distance to the nearest T cell in micrometers (pm) in each OB layer in PBS (left) and GAS-infcctcd (right) conditions from the MERFISH analysis, shaded by proximity to the nearest T cell. Microglia in the glomerular layer of the OB are closest to T cells. Figure 3F-3J, Expression changes of Cd74, Tnf, Ccl5, Cx3crl, and P2ryl2 in microglia by scRNAseq (left). Statistical comparisons of scRNAseq were performed with Wilcoxon Rank Sum test (* p-adj < 0.05). Representative plots (center) and quantification (right) of the normalized median fluorescence intensity (MFI) for each protein by flow cytometry. All comparisons were performed by unpaired t test (** p < 0.01; **** p < 0.0001). Figure 3K, Expression of P2ryl2, Gpr34, Cd.74, Ifi30, and Ax/ in representative spatial plots from PBS and GAS OBs in MERFISH samples (n = 4 mice per group). Figure 3L, Expression of homeostatic and GAS-responsive genes in microglia from the glomerular and granular layers of the OB. Data were normalized to the PBS glomerular value by batch. Comparisons by ratio t test (* p < 0.05; ** p < 0.01; **** p < 0.0001; n = 4 mice per group). Figure 3M: Dotted bar graphs for select cytokine levels (pg/mg) in whole OB by multiplex immunoassay. Comparisons between PBS and GAS-infected mice were done by student t-test [* p < 0.05; n = 5 (PBS) and n = 6 (GAS)]. Figures 3N-3O, The number of microglia (Figure 3N) and the distance from the nearest T cell (Figure 30) in the OB after GAS infections as assessed by MERFISH. Comparisons in I between PBS and GAS-infected mice were done by one-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001; n = 4 mice per group).
Figures 4A-4Q: Macrophages in the NALT/OE secrete similar chemokines & cytokines to those infiltrating the OB after multiple GAS infections. Figure 4A, t-SNE plot of microglia isolated from the OBs of all conditions examined in this study reveals six major clusters: two homeostatic microglia (hMG-1 and -2) and four Streptococcus-responsive, microglia (srMGl- 4) subtypes. Figure 4B, t-SNE plot of microglia isolated from the OBs by inoculate (PBS versus GAS) shows that hMG microglia are predominantly from PBS mice and srMGl-4 from GAS- infected mice. Figure 4C, Feature plot of the expression of selected homeostatic, disease- associated, antigen-presentation, and interferon-response genes across microglial populations. The scale indicates average level of expression. Figure 4D, Feature plot of the expression of key chemokine and cytokine genes across microglial populations. The scale indicates level of expression. Figures 4E-4G, Representative fluorescence images of perivascular (Figure 4E) and meningeal (Figure 4F) macrophages, and microglia (Figure 4G) in the OB from GAS-infected CX3CRlGFP/TMEM119tdTomato reporter mice. White arrows (Figure 4E, Figure 4F) indicate macrophages (CX3CR1GFP+ TMEM119tdTomato0 and arrowhead outlines (Figure 4G) indicate microglia (CX3CR1GFP+ TMEM119tdlomato+). Scale bars = 50 pm. Figures 4H-4I, Dotted bar graph of the number of macrophages (Figure 4H) and microglia (Figure 41) averaged for OB bregmas 4.5, 4.28 and 3.92 in PBS (n = 3) and GAS-infected (n = 3) CX3CR1GFP/TMEM1 ] 9td '’"i l'’ reporter mice. Comparisons were performed with unpaired t test (* p < 0.05; *** p < 0.001). Figure 4J, t- SNE plot of cell types isolated from NALT/OE based on scRNAseq data. Figure 4K, t-SNE plot of sample identity for NALT/OE-isolated cells from PBS and GAS mice. Figure 4L, Expression levels for select cytokines, chemokines and growth factors in NALT/OE-derived macrophages and OB-derived macrophages and microglia in PBS and GAS-infected conditions. The size of each dot indicates the percent of the population expressing each marker; the scale indicates average level of expression. The cell population analyzed is shown in the schematic diagram on the left. Figure 4M, Dotted bar graph of the normalized mean fluorescence intensity of albumin- Alexa 594 localized inside primary human umbilical vein endothelial cells (HUVECs) exposed to cytokines and growth factors that are present in high concentrations in sera from PANDAS/PANS patients. Incubation with selected cytokines and growth factors for 48 hours upregulates albumin uptake in HUVEC cells. The comparisons were performed by one-way ANOVA (* p < 0.05; *** p < 0.001; **** p < 0.0001). Figure 4N, Representative fluorescence images of the albumin-Alexa 594 uptake in HUVECs after 48-hour incubation with the following cytokines and concentrations: IL- ip/TNF at 20 ng/mL; CCL2 at 0.5 ng/mL; CCL5 at 4 ng/mL, and IL-IRA, PDGFBB and HGF at 10 ng/mL). Scale bar = 25 pm. Figures 4O-4Q: Identification of NALT and OE cell types and measurement of transcriptional shifts in perivascular macrophages after intranasal GAS infections. Figure 40, Molecular markers used to assign cell identity in the nose associated lymphoid tissue (NALT) and olfactory epithelium (OE) in scRNAseq experiments. The dot size indicates the percentage of the population expressing each marker; the scale indicates the average level of expression. Figure 4P, Heat map of genes related to antigen presentation, cytokines and chemokines, and interferon response, as well as the most upregulated gene (Saa3) in perivascular macrophages from the OB. Figure 4Q, Expression of chemokine receptors in immune cell clusters isolated from the OB (left) and NALT/OE (right). The dot size indicates the percent of population expressing each marker; the color scale indicates the average level of expression. The purple color in the grid indicates receptor-ligand binding pairs for the relevant GAS-induced chemokines in mouse microglia / macrophages.
Figures 5A-5V: Lack of Thl7 cells or inhibition of IL-17A function rescues aberrant transcrip tome signatures in CNS endothelial cells and microglia associated with recurrent GAS infections. Figure 5A, Microglial surface expression of antigen-presentation markers by flow cytometry in wild-type PBS, wild-type GAS -infected and RORyt ' GAS- infected mice. Comparisons by one-way ANOVA. (ns, p > 0.05; * p < 0.05; ** p < 0.01). Figure 5B, Representative plots of CD74 and MHC II (I-A/I-E) expression in wild-type and RORyf7- mutant microglia after GAS infections. Contour plots represent n = 3,640 and 4,657 cells, respectively. Figure 5C, Quantification of IFNy concentrations in whole OBs from wildtype PBS, wild-type GAS and RORyt 7’ GAS mice after two and five GAS infections. Comparisons by one-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05; * p < 0.05). Figure 5D, Timeline of GAS infections and administration of an a-IL-17A- neutralizing antibody or isotype control. Figures 5E-5F, Dotted bar graphs show the quantification of endogenous IgG leakage (relative fluorescence intensity) in the glomerular (Figure 5E) and granular (Figure 5F) layers of the OB in PBS and GAS conditions treated with either an a-IL-17A-neutralizing antibody or isotype control. IgG intensity is significantly reduced in IL-17A mAb-treated mice relative to isotype mAb in the granular layer. Statistical analyses by two-way ANOVA with Tukey’s multiple comparison test (* p < 0.05; *** p < 0.001; n = 3-7 mice per group). Figure 5G, Representative images of IgG leakage in the granular layer of the OB. The vasculature is marked with Caveolin 1. Figures 5H-5M, Violin plots of expression changes in ECs and microglia from GAS -infected mice treated with either an a-IL-17A-neutralizing antibody or isotype control. Comparisons by Wilcoxon Rank Sum test (* p-adj < 0.05). Figures 5N-5V: Elimination of Thl7 cells or blockade of IL- 17A rescues BBB transcriptome changes in CNS ECs, and reduces expression of chemokine and interferon response genes by microglia in GAS-infected mice. Figures 5N-5S, Gene expression changes in endothelial cells (Figure 5N) and microglia (Figures 5O-5S) isolated from the OBs of wildtype PBS, wild-type GAS-infected and RORyf/_ GAS-infected mice. The displayed comparisons between PBS and GAS and WT GAS and RORyf/_ GAS were significant by Wilcoxon Rank Sum test with Bonferroni correction (* p-adj < 0.05). The comparisons between PBS and RORyt 7’ GAS are not shown. Figure 5T, Ridge plots showing expression of key major histocompatibility complex (MHC) class I genes for antigen presentation by various OB cell types in wild-type PBS, wild-type GAS-infected mice, RORyt 7 GAS-infected mice, isotype control antibody treated GAS-infected mice, and a-IL-17A mAb-treated GAS infected mice. Figure 5U, Quantification of the number of CD4+ T cells in the OBs of isotypecontrol and a-IL-17A mAb-treated GAS infected mice (bregmas 4.28, 3.8 and 3.2). The comparison was done by mixed-effects analysis with Sidak’s multiple comparisons test (ns, p > 0.05; n = 4 isotype-control, n = 6 a-IL-17A mAb-treated mice). Figure 5V, a-IL-17A mAb- treated mice showed a significant increase in mortality after GAS infection relative to isotype controls by Mantel-Cox test (* p < 0.05; n = 4 PBS isotype-control; n = 3 PBS a-IL-17A mAb; n = 11 GAS isotype-control and n = 19 GAS a-IL-17A mAb mice).
Figures 6A-6R: Although IFNy+IL-17A+ and GM-CSF+ CD4 T cells accumulate in the brain after multiple GAS infections, GM-CSF is primarily critical to induce GAS-related microglial transcription changes. Figure 6A, Dotted bar graph of the progressive increase in the proportion of IFNy+IL-17A+ CD4 T cells in the brain with increasing numbers of GAS infections. The proportion of IFNy+IL- 17A+ in mice infected twice over a five-week interval (li...2i) was not significantly different from that seen with two consecutive infections (2i). Comparisons by one-way ANOVA with Dunnett’s T3 multiple comparisons test. Figure 6B, Dotted bar graph of the progressive increase in the proportion of GM-CSF+ CD4 T cells in the brain with increasing numbers of GAS infections. Comparisons by two-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; each dot represents one mouse). Figure 6C, Dotted bar graph of the proportion of GM-CSF+ CD4 T cells over the total CD4 T cells from the NALT/OE of wildtype (n = 5) and RORyl ' (n = 7) GAS-infected mice. Statistical analysis by unpaired t-test (** p < 0.01). Figure 6D, Dotted bar graph of the proportion of GM-CSF+ CD4 T cells from the NALT/OE that were Thl7 cells or IFNy+ Thl7 cells in wild-type (n = 4) and RORyt7- (n = 4) GAS-infected mice. Approximately 90% of GM-CSF+ CD4 T cells in the wild-type NALT/OE were Thl7 cells or IFNy+ Thl7 cells. Comparisons by two-way ANOVA with Tukey’s multiple comparisons test (*** p < 0.001; **** p < 0.0001). Figure 6E, Timeline of 4-OH- tamoxifen administration and GAS infections in Csf2fl/fl and Csf2ACD4 mice. Figures 6F-6K, Violin plots of expression changes in ECs and microglia from Csf2ACD4 mutant and Csf2fl/fl control GAS-infcctcd mice. Comparisons by Wilcoxon Rank Sum test (* p-adj < 0.05). Figure 6L, A model summarizing three key findings from the study. Left: scRNAseq of mouse OB reveals that microglia upregulate antigen presentation, cytokine and chemokine, and interferon-response genes after multiple GAS infections, whereas CNS ECs lose BBB transcriptome properties. Center: A small number of inflammatory cytokines, chemokines and growth factors, produced primarily by microglia and macrophages after GAS infections, are elevated in sera from acute PANDAS/PANS patients. These cytokines upregulate transcytosis in human EC in vitro suggesting a direct impact on EC function. Right: Th 17 cell-derived cytokines IFNy, IL-17A and GM-CSF play distinct roles on BBB dysfunction and microglial activation in post-infectious CNS sequelae after repeated GAS infections. Figure 6M, Ridge plot showing expression of Trem2 in PBS, wild- type GAS, RORyt7’ GAS, isotype control antibody GAS, a-IL-17A mAb-treated GAS conditions, Csf2fl/fl GAS and Csf2ACD4 GAS mice. (Figure 6N) Quantification and (Figure 60) representative images of Ibal+CD68+ myeloid cells in the glomerular layer of the OB (dashed outline) in PBS and GAS -infected Csf2fl/fl and Csf2ACD4 mice. Statistical analyses were done with two-way ANOVA with Tukey’s multiple comparison test (ns, p > 0.05; * p < 0.05, *** p < 0.001; **** p < 0.0001; n = 4-6 mice / group). All comparisons were done by one-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3-5 experiments per condition). Figures 6P-6R, Volcano plots showing significant upregulated (right) and downregulated (left) genes by bulk RNA sequencing in cultured primary brain microglia after 24 hours of treatment with IFNy (Figure 6P), IL-17A (Figure 6Q), or GM-CSF (Figure 6R) cytokines.
Figures 7A-7E: Assessment of barrier properties of the human blood-brain barrier in an in vitro 3D human NVU/BBB microfluidic system using the MIMETAS 3-Lane-40 plates. Figure 7A: The schematic diagram of the MIMETAS 3-Lane-40 plates and chips. Brightfield image (Figure 7B) and immunofluorescence staining (Figure 7C) of human primary brain microvascular endothelial cells (BMECs), pericytes (PCs), and astrocytes (ACs) in the MIMETAS chips. Figure 7D: BMECs are plated on the top channel and form tubules by making contact via cell junctions labelled with ZO-1. Pericytes and astrocytes are plated on the bottom chamber to allow assembly of the neurovascular unit (NVU). The plates incorporate bidirectional flow of fluids. Figure 7E: Analysis of tracer permeability (Dextran 70kDa) from the top to the middle and bottom channels in the human 3D NVU/BBB system cultured with media (circles), sera from healthy donors (N=3; squares), or sera from PANDAS patients (N=4; triangles) for 48 hours. Sera from PANDAS patients increase the permeability of the tracer from the top to the bottom channel of the MIMETAS platform indicative of a leaky human BBB (n=8 chips / patient; two-way ANOVA with Bonferroni correction; **p<0.01).
DETAILED DESCRIPTION
The present disclosure relates to using inhibitors of interleukin 17A (IL- 17A) or its receptor for the treatment, prophylaxis or alleviation of Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) including Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).
The disclosure also provides diagnostic methods for PANS (such as PANDAS) where the methods assay the levels of one or more cytokines, chemokines and/or growth factors in a sample (e.g., a blood, plasma or serum sample) taken from a subject.
The present disclosure provides for a method of treating, or treating prophylactically, a pediatric autoimmune neuropsychiatric disorder such as PANS including PANDAS in a subject.
PANS may include a broad spectrum of neuropsychiatric conditions. The syndrome of PANS may be associated with a variety of disease mechanisms and have multiple etiologies, ranging from psychological trauma or underlying neurological, endocrine, and metabolic disorders to post-infectious autoimmune and neuroinflammatory disorders, such as PANDAS, cerebral vasculitis, ncuropsychiatric lupus, and others.
PANS may be associated with infections, metabolic disturbances, and/or other inflammatory reactions. PANS may be associated with bacterial infections or viral infections. PANS may be associated with infections in the upper respiratory tract, including rhinitis, sinusitis, and pharyngitis. The infection may be caused by group A Streptococcus (GAS), Mycoplasma pneumoniae, Staphylococcus bacteria, influenza viruses, and other viruses. Infections that may be associated with PANS include the infections caused by, or associated with, Epstein Barr virus (EBV), herpes simplex virus (HSV), varicella, upper respiratory infections (including rhinosinusitis, pharyngitis, or bronchitis), and Borrelia burgdorferi (Lyme disease). Other infections that may be associated with PANS include infection caused by babesia, bartonella, and coxsackie virus.
The term “PANDAS” may refer to Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus, or Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcal infections.
PANDAS may be associated with a Group A Streptococcus infection, or a Group A beta- hemolytic Streptococcus infection. PANS or PANDAS may be associated with an ongoing infection or a prior infection.
The symptoms of PANS or PANDAS may start after, or at the time of, an infection (e.g., a Streptococcus infection). The symptoms of PANDAS or PANS may start about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or longer, after an infection (e.g., a Streptococcus infection). The symptoms of PANDAS or PANS may become more intense within about 24-48 hours after the onset of the infection (e.g., the Group A Streptococcus infection).
As used herein, the term "inhibitor" refers to agents capable of down-regulating or otherwise decreasing or suppressing the level/amount and/or activity of IL- 17 A or its receptor. For example, the inhibitor can block the ability of IL- 17 A to bind to its receptor.
The mechanism of inhibition may be at the genetic level (e.g., interference with or inhibit expression, transcription or translation, etc.) or at the protein level (e.g., binding, competition, etc.). The present inhibitor may inhibit IL-17A or its receptor through any mechanism, including, but not limited to, inhibiting/reducing IL- 17 A (or its receptor) activity, inhibiting/reducing IL- 17 A (or its receptor) level, and/or inhibiting/reducing IL-17A (or its receptor) gene expression. The inhibitor may block an IL-17A-mediated immune response in the subject.
The present inhibitors may be a small molecule, a polynucleotide, a polypeptide, or an antibody or antigen-binding fragment thereof. The inhibitor may comprise an antibody or a fragment thereof that binds to IL-17A or its receptor, such as secukinumab, ixekizumab, brodalumab, bimekizumab, or combinations thereof. In one embodiment, the polynucleotide is a small interfering RNA (siRNA) or an antisense molecule (e.g., antisense RNA), microRNA (miRNA), ribozymes, triple stranded DNA, etc. In one embodiment, the inhibitor is a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system specific for IL-17A or its receptor.
By “inhibition”, "down-regulation" or “reduction” is meant any negative effect on the condition being studied; this may be total or partial. Thus, where the level or activity of a protein (e.g., IL-17A or its receptor) is being detected, the present inhibitor/compo sition is capable of reducing, ameliorating, or abolishing the level or activity of the protein (e.g., IL-17A or its receptor). The inhibition or down-regulation of the level or activity of the protein achieved by the present agent may be at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to the level or activity of the protein (e.g., IL-17A or its receptor) in the absence of the present inhibitor/composition.
Also encompassed by the present disclosure is a pharmaceutical composition comprising the present inhibitor. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
In addition to the use of an inhibitor of IL-17A or its receptor, the present methods may further comprise administering a second therapeutic agent such as an immunosuppressant, a tumor necrosis factor antagonist (a TNF-antagonist), a CTLA4-antagonist, an anti-IL-6 receptor antibody, an anti-CD20 antibody, or a combination of any of the foregoing.
The present inhibitor/composition can be administered alone, or may be co-administered together with antibiotics, anti-inflammatory or immune modulating therapies, and/or psychiatric medications and behavioral interventions.
Treatments may be sequential, with the present inhibitor/composition being administered before or after the other therapies. Agents/therapies may be administered concurrently.
The route of administration may vary, and can include, inhalation, intranasal, oral, transdermal, intravenous, subcutaneous or intramuscular injection.
The present disclosure provides for a method of treating, or treating prophylactically, a pediatric autoimmune neuropsychiatric disorder, such as PANS including PANDAS, in a subject. The method may comprise administering to the subject an effective amount (e.g., a therapeutically effective amount) of an inhibitor of IL-17A or its receptor (e.g., interleukin 17 receptor A, or IL- 17RA).
The method may comprise: (a) administering an inhibitor of IL-17A or its receptor to the subject during an induction regimen, where the induction regimen comprises a loading regimen, where the loading regimen comprises administering to the subject five doses of about 150 mg to about 300 mg of the inhibitor, each of the five doses being delivered weekly, beginning on week zero; and (b) thereafter administering the inhibitor to the subject during a maintenance regimen.
A "therapeutic regimen" encompasses treatment of an illness, such as PANDAS or PANS. A therapeutic regimen may include an induction regimen and a maintenance regimen. The patient can be given both an induction regimen and a maintenance regimen of the inhibitor of IL-17A or its receptor. An "induction regimen" refers to a treatment regimen (or the portion of a treatment regimen) used for the initial treatment of a disease. The goal of an induction regimen is to provide a high level of the inhibitor in the system of a patient during the induction period. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the inhibitor than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the inhibitor during a maintenance regimen, or both. The present inhibitor during an induction regimen may be delivered via a subcutaneous route, e.g., delivery of dosages of about 10 mg - about 300 mg s.c, via an intravenous route, e.g., delivery of dosages of about 0.1 mg/kg, - about 50 mg/kg i.v. (e.g., about, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, etc.) or any other route of administration (e.g., intramuscular, i.m.).
“Maintenance regimen" refers to the treatment of a subject over a longer period of time, days, months, years, during treatment of an illness in order to keep the patient in remission for longer periods of time (months or years). This time frame is referred to as a "maintenance period". A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined. The inhibitor during a maintenance regimen may be delivered via a subcutaneous route, e.g., delivery of dosages of about 10 mg - about 300 mg s.c, via an intravenous route, e.g., delivery of dosages of about 0.1 mg/kg, - about 50 mg/kg i.v. (e.g., about, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, etc.), or any other route of administration (e.g., intramuscular, i.m.).
Treating PANS/PANDAS using inhibitors of IL-17A or its receptor
Antibodies
The present inhibitor can be an antibody or antigen-binding fragment thereof that is specific to IL- 17 A or its receptor.
The antibody or antigen-binding fragment thereof may be the following: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab')2; and (e) a disulfide linked Fv. The antibodies can be full-length or can include a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab, F(ab')2, Fab’, F(ab)’, Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment [c.g., Ward ct al., Nature, 341:544-546 (1989)], an isolated CDR, diabodics, triabodics, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present disclosure [Bird et al. Science, 1988, 242:423-426. Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883],
The antibody or antigen-binding fragment thereof may be monoclonal, polyclonal, chimeric and humanized. The antibodies may be murine, rabbit or human antibodies.
Examples of such antibodies include, but are not limited to, a fully human monoclonal antibody that targets IL-17A (secukinumab), a humanized monoclonal antibody specific for IL-17A (ixekizumab), and a fully human antibody that targets the IL-17 receptor A (brodalumab) [Adamas et al. Front. Immunol. 11: 1894 (2020)].
The humanized antibody of the present disclosure may be an antibody from a non-human species where the amino acid sequences in the non-antigen binding regions (and/or the antigenbinding regions) have been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
A chimeric antibody may be a molecule in which different portions are derived from different animal species. For example, an antibody may contain a variable region derived from a murine antibody and a human immunoglobulin constant region.
Also within the scope of the disclosure are antibodies or antigen-binding fragments thereof in which specific amino acids have been substituted, deleted or added. The present antibodies or antigen-binding fragments thereof may be variants, analogs, orthologs, homologs or derivatives of antibodies of antigen-binding fragments thereof disclosed herein, e.g., with less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 1% amino acid residues substituted or deleted but retain essentially the same or similar immunological properties including, but not limited to, binding to IL-17A or its receptor.
All antibody isotypes are encompassed by the present disclosure, including, but not limited to, IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgA (IgAl, IgA2), IgD or IgE.
The antibodies or antigen-binding fragments thereof of the present disclosure may be monospecific, bi-specific or multi- specific. The present antibodies or fragments thereof can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein.
Small Molecule Inhibitors
As used herein, the term "small molecules" encompasses molecules other than proteins or nucleic acids without strict regard to size. Non-limiting examples of small molecules that may be used according to the present methods and compositions include, small organic molecules, peptide-like molecules, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
Non-limiting examples of inhibitors of IL-17A or its receptor include S011806, LY3509754, LEO153339, and the compounds in Zhang et al., Small molecule modulators of IL- 17A/IL-17RA: a patent review (2013-2021), Expert Opinion on Therapeutic Patents, 2022, 32(11): 1161-1173 (which is incorporated by reference herein in its entirety).
In certain embodiments, the inhibitor used in the present methods and compositions is a polynucleotide that reduces expression of IL-17A or its receptor.
The nucleic acid target of the polynucleotides (e.g., siRNA, antisense oligonucleotides, and ribozymes) may be any location within the gene or transcript of IL- 17 A or its receptor.
RNA Interference
SiRNAs (small interfering RNAs) or small-hairpin RNA (shRNA) may be used to reduce the level of IL- 17 A or its receptor.
SiRNAs may have 16-30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The siRNAs may have fewer than 16 or more than 30 nucleotides. The polynucleotides of the invention include both unmodified siRNAs and modified siRNAs such as siRNA derivatives etc.
SiRNAs can be delivered into cells in vitro or in vivo by methods known in the art, including cationic liposome transfection and electroporation. SiRNAs and shRNA molecules can be delivered to cells using viruses or DNA vectors.
Antisense Polynucleotides
In other embodiments, the polynucleotide is an antisense nucleic acid sequence that is complementary to a target region within the mRNA of TL- 17A or its receptor. The antisense polynucleotide may bind to the target region and inhibit translation. The antisense oligonucleotide may be DNA or RNA, or comprise synthetic analogs of ribo-deoxynucleotides. Thus, the antisense oligonucleotide inhibits or decreases expression of IL-17A or its receptor.
An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
The antisense nucleic acid molecules of the invention may be administered to a subject, or generated in situ such that they hybridize with or bind to the mRNA of IL-17A or its receptor. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using viruses or DNA vectors.
Ribozyme
In other embodiments, the polynucleotide is a ribozyme that inhibits expression of the gene of IL- 17 A or its receptor.
Ribozymes can be chemically synthesized and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme encoding nucleotide sequences can be introduced into host cells through gene-delivery mechanisms known in the art.
Other aspects of the disclosure include vectors (e.g., viral vectors, expression cassettes, plasmids) comprising or encoding polynucleotides that act as inhibitors (e.g., siRNA, antisense nucleic acids, and ribozymes), and host cells genetically modified with polynucleotides or vectors.
Polypeptides
The present inhibitors can also be a polypeptide exhibiting inhibitory activity toward IL- 17 A or its receptor. Various means for delivering polypeptides to a cell can be utilized to carry out the methods of the subject invention. For example, protein transduction domains (PTDs) can be fused to the polypeptide, producing a fusion polypeptide, in which the PTDs are capable of transducing the polypeptide cargo across the plasma membrane (Wadia, J. S. and Dowdy, S. F., Curr. Opin. Biotechnol., 2002, 13(1)52-56).
Recombinant cells can be administered to a patient, wherein the recombinant cells have been genetically modified to express a nucleotide sequence encoding an inhibitory polypeptide.
Pharmaceutical Composition
Also encompassed by the present disclosure is a pharmaceutical composition comprising the present inhibitor.
The pharmaceutical composition may be administered intrathecally, subdurally, orally, intravenously, intramuscularly, topically, arterially, or subcutaneously. Other routes of administration of pharmaceutical compositions include oral, intravenous, subcutaneous, intramuscular, inhalation, or intranasal administration. The pharmaceutical compositions may be administered by any route, including, without limitation, oral, transdermal, ocular, intraperitoneal, intravenous, Intracerebventricular, intracisternal injection or infusion, subcutaneous, implant, sublingual, subcutaneous, intramuscular, intravenous, rectal, mucosal, ophthalmic, intrathecal, intra- articular, intra-arterial, sub-arachinoid, bronchial and lymphatic administration. The pharmaceutical composition may be administered parenterally or systemically.
Specifically targeted delivery of the present composition could be delivered by targeted liposome, nanoparticle or other suitable means.
The composition may be administered by bolus injection or chronic infusion. The claimed composition may be administered at or near the site of the disease, disorder or injury, in a therapeutically effective amount.
The pharmaceutical compositions of the present invention can be, e.g., in a solid, semisolid, or liquid formulation. Intranasal formulation can be delivered as a spray or in a drop; inhalation formulation can be delivered using a nebulizer or similar device; topical formulation may be in the form of gel, ointment, paste, lotion, cream, poultice, cataplasm, plaster, dermal patch aerosol, etc.; transdermal formulation may be administered via a transdermal patch or iontophoresis. Pharmaceutical compositions can also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, emulsions, suspensions, elixirs, aerosols, chewing bars or any other appropriate compositions.
The pharmaceutical composition may be administered locally via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed release bolus, or continuous administration.
Pharmaceutically acceptable carriers that can be used in the present compositions encompass any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions can additionally contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. For examples of carriers, stabilizers, preservatives and adjuvants, see Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
The pharmaceutically acceptable excipient may be selected from the group consisting of fillers, e.g. sugars and/or sugar alcohols, e.g. lactose, sorbitol, mannitol, maltodextrin, etc.; surfactants, e.g. sodium lauryle sulfate, Brij 96 or Tween 80; disintegrants, e.g. sodium starch glycolate, maize starch or derivatives thereof; binder, e.g. povidone, crosspovidone, polyvinylalcohols, hydroxypropylmethylcellulose; lubricants, e.g. stearic acid or its salts; flowability enhancers, e.g. silicium dioxide; sweeteners, e.g. aspartame; and/or colorants. Pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
Oral dosage forms may be tablets, capsules, bars, sachets, granules, syrups and aqueous or oily suspensions. Tablets may be formed form a mixture of the active agents with fillers, for example calcium phosphate; disintegrating agents, for example maize starch, lubricating agents, for example magnesium stearate; binders, for example microcry stallinc cellulose or polyvinylpyrrolidone and other optional ingredients known in the art to permit tabletting the mixture by known methods. Similarly, capsules, for example hard or soft gelatin capsules, containing the active agents, may be prepared by known methods. The contents of the capsule may be formulated using known methods so as to give sustained release of the active agents. Other dosage forms for oral administration include, for example, aqueous suspensions containing the active agents in an aqueous medium in the presence of a non-toxic suspending agent such as sodium carboxymethylcellulose, and oily suspensions containing the active agents in a suitable vegetable oil, for example arachis oil. The active agents may be formulated into granules with or without additional excipients. The granules may be ingested directly by the patient or they may be added to a suitable liquid carrier (e.g. water) before ingestion. The granules may contain disintegrants, e.g. an effervescent pair formed from an acid and a carbonate or bicarbonate salt to facilitate dispersion in the liquid medium.
Intravenous forms include, but are not limited to, bolus and drip injections. Examples of intravenous dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles including, but not limited to. Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water- miscible vehicles including, but not limited to, ethyl alcohol, polyethylene glycol and polypropylene glycol; and non-aqueous vehicles including, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.
Additional pharmaceutical compositions include formulations in sustained or controlled delivery, such as using liposome or micelle carriers, bioerodible microparticles or porous beads and depot injections.
The inhibitor or pharmaceutical composition may be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. The pharmaceutical composition can be prepared in single unit dosage forms.
Appropriate frequency of administration can be determined by one of skill in the art and can be administered once or several times per day (e.g., twice, three, four or five times daily). The compositions may also be administered once each day or once every other day. The compositions may also be given twice weekly, weekly, monthly, or semi-annually. Tn the case of acute administration, treatment is typically carried out for periods of hours or days, while chronic treatment can be carried out for weeks, months, or even years.
Administration of the compositions can be carried out using any of several standard methods including, but not limited to, continuous infusion, bolus injection, intermittent infusion, inhalation, or combinations of these methods. For example, one mode of administration that can be used involves continuous intravenous infusion. The infusion of the compositions of the invention can, if desired, be preceded by a bolus injection.
Methods of determining the most effective means and dosage of administration can vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject or patient being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. The specific dose level for any particular subject depends upon a variety of factors including the activity of the specific peptide, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Different dosage regimens may be used. For example, a daily dosage, such as any of the exemplary dosages described above, is administered once, twice, three times, or four times a day for at least three, four, five, six, seven, eight, nine, or ten days. Depending on the stage and severity of the the disease, a shorter treatment time (e.g., up to five days) may be employed along with a high dosage, or a longer treatment time (e.g., ten or more days, or weeks, or a month, or longer) may be employed along with a low dosage. In some embodiments, a once- or twice-daily dosage is administered every other day.
Cytokine Profiling for Diagnosing PANS
The present disclosure also provides for methods of assaying the levels of one or more cytokines, chemokines and/or growth factors in a sample (e.g., a blood, plasma or serum sample) taken from a subject. Based on the levels of the cytokine(s), chemokine(s) and/or growth factor(s), PANS (such as PANDAS) may be diagnosed or predicted (a risk of developing PANS (such as PANDAS)), and then the subject may be treated.
The present methods may determine/detect the presence, and/or severity of PANS (such as PANDAS). The levels of cytokines, chemokines and/or growth factors in the sample can be used for assessing the onset or severity of PANS (such as PANDAS), or as an indicator of the efficacy of a therapeutic intervention for treating PANS (such as PANDAS). A plurality of cytokines, chemokines and/or growth factors may be measured. Based on the levels of the cytokines, chemokines and/or growth factors, PANS (such as PANDAS) may be diagnosed or predicted, and then the subject may be treated. For patients under treatment for PANS (such as PANDAS), based on the cytokine, chemokine and/or growth factor levels, the therapeutic intervention may be continued when it is effective, or altered if ineffective or insufficient.
The method may also identify a subject at risk for PANS (such as PANDAS). As such, the methods of the present disclosure can impact the way the subject is treated. For example, patients identified as having a high risk of PANS (such as PANDAS) can be treated more aggressively. Patients identified as low risk may be treated less aggressively.
The present methods can diagnose or predict PANS (such as PANDAS) in a subject who has an ongoing or prior infection (e.g., a streptococcal infection).
In certain embodiments, the method comprises the following steps: (a) obtaining a sample (e.g., a blood, plasma or serum sample, or other samples as discussed herein) from the subject; (b) assaying the level of one or more cytokines, chemokines and/or growth factors in the sample; and (c) comparing the level obtained in step (b) with the level of the one or more cytokines, chemokines and/or growth factors in a control sample. The subject is diagnosed to have PANS (such as PANDAS) (or diagnosed to have an increased risk of developing PANS (such as PANDAS)), if the level of at least one cytokine, chemokine and/or growth factor obtained in step (b) increases by at least 5% compared to its level in the control sample.
The present methods may treat a subject with PANS (such as PANDAS) or an increased risk of PANS (such as PANDAS). When diagnosed with PANDAS, the subject may be treated with an inhibitor of IL- 17 A or its receptor. Alternatively, when PANS (such as PANDAS) is predicted (or when an increased risk of PANS (such as PANDAS) is diagnosed), the subject may be treated with an inhibitor of IL-17A or its receptor.
In certain embodiments, the method comprises the following steps: (a) obtaining a sample (e.g., a plasma or serum sample, or other samples as discussed herein) from the subject;
(b) assaying the level of one or more cytokines, chemokines and/or growth factors in the sample;
(c) comparing the level obtained in step (b) with the level of the one or more cytokines, chemokines and/or growth factors in a control sample; and (d) treating the subject for PANS (such as PANDAS) or an increased risk of PANS (such as PANDAS), if the level of at least one cytokine, chcmokinc and/or growth factor obtained in step (b) increases by at least 5% compared to its level in the control sample.
In certain embodiments, the present method determines/detects the level of one or more cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, VEGFD, and combinations thereof.
In certain embodiments, the present method determines/detects the level of one or more cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, and combinations thereof.
In certain embodiments, the present method determines/detects the level of one or more cytokines, chemokines and/or growth factors selected from those listed in Table 1, and combinations thereof. Table 1 provides an exemplary list of cytokines, chemokines and/or growth factors whose levels may be determined/detected by the present method.
There may be a number of different isoforms for each of these cytokines, chemokines and/or growth factors.
In certain embodiments, the method comprises the following steps: (a) obtaining a sample from the subject; (b) determining (detecting) in the sample a level of one or more cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, wherein an increase by at least 5% in the level of the one or more polypeptides relative to a control sample indicates that the subject has PANS (such as PANDAS) or have an increased risk of PANS (such as PANDAS).
The level of at least 1 or 1, at least 2 or 2, at least 3 or 3, at least 4 or 4, at least 5 or 5, at least 6 or 6, at least 7 or 7, at least 8 or 8, at least 9 or 9, at least 10 or 10, at least 11 or 11, at least 12 or 12, at least 13 or 13, at least 14 or 14, at least 15 or 15, at least 16 or 16, at least 17 or 17, at least 18 or 18, at least 19 or 19, at least 20 or 20, at least 21 or 21, at least 22 or 22, at least 23 or 23, at least 24 or 24, or at least 25 or 25, cytokines, chemokines and/or growth factors in the sample may increase by about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 1.1 fold, at least or about 1.2 fold, at least or about 1.3 fold, at least or about 1.4 fold, at least or about 1.5 fold, at least or about 1.6 fold, at least or about 1.7 fold, at least or about 1.8 fold, at least or about 1.9 fold, at least or about 2.5 fold, at least or about 3.5 fold, at least or about 11 fold, at least or about 12 fold, at least or about 13 fold, at least or about 14 fold, at least or about 15 fold, at least or about 16 fold, at least or about 17 fold, at least or about 18 fold, at least or about 19 fold, at least or about 20 fold, from about 10% to about 20 fold, from about 20% to about 20 fold, from about 30% to about 20 fold, from about 40% to about 20 fold, from about 50% to about 20 fold, from about 60% to about 20 fold, compared to the level(s) in the control sample.
The control sample may be from a patient who does not have PANS (such as PANDAS) or a plurality of patients who do not have PANS (such as PANDAS). The control sample may be from a healthy subject or a plurality of healthy subjects.
In certain embodiments, the levels of a plurality of cytokines, chemokines and/or growth factors in the sample may be assayed, which comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, cytokines, chemokines and/or growth factors.
The samples may include, but are not limited to, serum, plasma, blood, whole blood and derivatives thereof, bone marrow, urine, cerebrospinal fluid (CSF), endothelium, skin, hair, hair follicles, saliva, oral mucus, vaginal mucus, sweat, tears, epithelial tissues, semen, seminal plasma, prostatic fluid, excreta, ascites, lymph, bile, as well as other samples or biopsies. In one embodiment, the biological sample is plasma or serum.
The level or amount of a cytokine, chemokine and/or growth factor in a patient sample can be compared to a reference level or amount of the cytokine, chemokine and/or growth factor present in a control sample. Reference levels for a cytokine, chemokine and/or growth factor can be determined by determining the level of a cytokine, chemokine and/or growth factor in a sufficiently large number of samples obtained from normal, healthy control subjects to obtain a pre-determined reference or threshold value. Reference (or calibrator) level information and methods for determining reference levels can be obtained from publicly available databases, as well as other sources.
The present diagnostic/treatment methods may be combined with an evaluation of the subject’s medical and psychiatric history, throat swabs, physical examination, viral/bacterial testing, laboratory testing of blood, laboratory testing cerebrospinal fluid (CSF), and selected paraclinical evaluations, such as magnetic resonance imaging (MRI), electrocardiogram/echocardiography, electroencephalography, positron emission tomography (PET), computerized tomography (CT), and polysomnography. The blood testing may include testing for IgA, IgM, IgG (subclass 1, 2, 3, 4), CBC, ANA, ferritin (iron), B-12, vitamin D, and combinations thereof. Viral/bacterial testing may include testing for strep throat culture, antistreptolysin O (ASO), group A strep, anti-DNase B, streptozyme, Mycoplasma Pneumoniae IgA & IgM, pneumococcal antibody titers, Lyme disease and co-inf ections, Epstein Barr virus panel, Coxsackie A & B titers, HHV-6 titers, or combinations thereof. Additional testing may include Cunningham Panel - autoimmune autoantibody levels (Dopamine D 1 receptor, Dopamine D2L receptor, Lysoganglioside GM1, Tubulin and CaM Kinase II); GAD65 antibody testing, or combinations thereof.
The present diagnostic/treatment methods may be combined with other assessment/evidence, including, but not limited to, prior streptococcal infection; a rise in the antibody level(s) (e.g., anti-streptolysin O (ASO), anti-DNAse B (ADB), and/or anti- streptococcal carbohydrate (anti-CHO)); acute pharyngitis with a positive GAS throat culture, with or without a rising antibody level; pharyngitis with characteristic palatal petechiae; pharyngitis with a characteristic scarlatinaform rash; pharyngitis without a throat swab or serology, but intimate (usually household) exposure to a proven GAS case; asymptomatic pharyngeal colonization documented after an intimate exposure; asymptomatic pharyngeal colonization after a negative throat swab documented within the prior 3-4 months; single ASO or ADB antibody level within 6 months after the initial onset of neuropsychiatric symptoms may be accepted as positive if it is >95th percentile; both ASO and ADB are elevated at >80% percentile for age in the same serum sample within 6 months after the initial onset of neuropsychiatric symptoms; culture-documented streptococcal dermatitis; GAS cultured from a normal- appearing throat, without rising antibody titers; pharyngitis without a swab; either ASO or ADB, but not both, elevated at >80% percentile of age norms within 6 months after the initial onset of ncuropsychiatric symptoms, and culture negative or unavailable; household exposure to a proven GAS case without clinical pharyngitis or suggestive dermatitis, and without a diagnostic swab; intertrigo or perianal dermatitis without confirmatory culture, or combinations thereof.
Cooperstock et al., Clinical Management of Pediatric Acute-Onset Neuropsychiatric Syndrome: Part III — Treatment and Prevention of Infections, Journal of Child and Adolescent Psychopharmacology, 2017, 27(7): 594-606, which is incorporated by reference in its entirety.
The present disclosure provides for methods of evaluating and/or monitoring the efficacy of a therapeutic intervention for treating PANS (such as PANDAS). These methods can include the step of measuring the level of at least one cytokine, chemokine and/or growth factor, or a panel of cytokines, chemokines and/or growth factors, in a biological sample from a subject. In some embodiments, the level of the at least one cytokine, chemokine and/or growth factor in the biological sample is compared to a reference level, or the level of the at least one cytokine, chemokine and/or growth factor in a control sample. The control sample may be taken from the patient at a different time point after the start of the treatment, or from the patient before initiation of the therapeutic intervention, or from the patient at a different time point after initiation of the therapeutic intervention. The measured level of the at least one cytokine, chemokine and/or growth factor is indicative of the therapeutic efficacy of the therapeutic intervention. In some cases, an increase or decrease in the level of the cytokine, chemokine and/or growth factor is indicative of the efficacy of the therapeutic intervention. In some embodiments, a change in the measured level of the at least one cytokine, chemokine and/or growth factor relative to a sample from the patient taken prior to treatment or earlier during the treatment regimen is indicative of the therapeutic efficacy of the therapeutic intervention.
An effective therapy may be continued, or discontinued if the patient’s condition has improved and is no longer in need of treatment. An ineffective treatment may be altered or modified, or replaced with other treatment.
In certain embodiments, the method comprises detecting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cytokines, chemokines and/or growth factors described herein. When the levels of a panel of cytokines, chemokines and/or growth factors are determined/detected in the patient sample, the patient sample may be classified as indicative of effective or non-effective intervention on the basis of a classifier algorithm. For example, samples may be classified on the basis of threshold values as described, or based upon mean and/or median cytokine, chemokine and/or growth factor levels in one population or versus another (e.g., a population of healthy controls or a population of patients without PANS (such as PANDAS), or levels based on effective versus ineffective therapy).
The present invention also provides methods for modifying a treatment regimen comprising detecting the level of at least one cytokine, chemokine and/or growth factor in a biological sample from a patient receiving the therapeutic intervention and modifying the treatment regimen based on an increase or decrease in the level of the at least one cytokine, chemokine and/or growth factor in the biological sample. The methods for modifying the treatment regimen of a therapeutic intervention may comprise the steps of: (a) detecting the level of at least one cytokine, chemokine and/or growth factor in a biological sample from a patient receiving the therapeutic intervention; and (b) modifying the treatment regimen based on an increase or decrease in the level of the at least one cytokine, chemokine and/or growth factor in the biological sample. In some embodiments, the method comprises detecting 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cytokines, chemokines and/or growth factors described herein. In certain embodiments, the levels of less than 30, less than 25, less than 20, less than 10, less than 8, or less than 7, cytokines, chemokines and/or growth factors are detected.
Modifying the treatment regimen can include, but is not limited to, changing and/or modifying the type of therapeutic intervention, the dosage at which the therapeutic intervention is administered, the frequency of administration of the therapeutic intervention, the route of administration of the therapeutic intervention, as well as any other parameters that would be well known by a physician to change and/or modify. For example, where one or more cytokines, chemokines and/or growth factors decrease (or increase) during therapy or match reference levels, the therapeutic intervention is continued. In embodiments where one or more cytokines, chemokines and/or growth factors do not decrease (or increase) during therapy or match reference levels, the therapeutic intervention is modified. In another embodiment, the information regarding the increase or decrease in the level of at least one cytokine, chemokine and/or growth factor can be used to determine the treatment efficacy, as well as to tailor the treatment regimens of therapeutic interventions. The present methods can include the steps of measuring the level of at least one cytokine, chcmokinc and/or growth factor in a sample from a patient receiving a therapeutic intervention, and comparing the measured level to a reference level or the level of at least one cytokine, chemokine and/or growth factor in a control sample. The measured level of the at least one cytokine, chemokine and/or growth factor is indicative of the therapeutic efficacy of the therapeutic intervention.
Based on the measured cytokine, chemokine and/or growth factor levels, therapy may be continued or altered, e.g., by change of dose or dosing frequency, or by addition of other active agents, or change of therapeutic regimen altogether.
The present invention also encompasses a method of predicting or assessing the level of severity of PANS (such as PANDAS) in a patient. In one embodiment, the method comprises measuring the level of at least one cytokine, chemokine and/or growth factor in a biological sample from a patient; and comparing the measured level to a reference level or the level of the at least one cytokine, chemokine and/or growth factor in a control sample, wherein the measured level of the at least one cytokine, chemokine and/or growth factor is indicative of the level of severity of PANS (such as PANDAS) in the patient. In other embodiments, an increase (as described herein) in the level of the cytokines, chemokines and/or growth factors is indicative of the level of severity of PANS (such as PANDAS) in the patient.
The expression profile of the cytokines, chemokines and/or growth factors in a subject may be determined/detected. The expression profile of the cytokines, chemokines and/or growth factors of the subject may be compared with a reference value, where the reference value is based on a set of cytokine, chemokine and/or growth factor expression profiles of a subject without PANS (such as PANDAS), and/or based on a set of cytokine, chemokine and/or growth factor expression profiles in an unaffected individual or unaffected individuals, and/or based on a set of cytokine, chemokine and/or growth factor expression profiles in the patient before, after and/or during therapy. The changes in cytokine, chemokine and/or growth factor expression may be used to alter or direct therapy, including, but not limited to, initiating, altering or stopping therapy.
In certain embodiments, the sample is a body fluid. For example, the body fluid can include, but are not limited to, serum, plasma, blood, whole blood and derivatives thereof, urine, tears, saliva, sweat, cerebrospinal fluid (CSF), oral mucus, vaginal mucus, seminal plasma, semen, prostatic fluid, excreta, ascites, lymph, bile, and amniotic fluid. In certain embodiments, the biological sample is plasma or scrum.
In certain embodiments, samples can include, but are not limited to, bone marrow, endothelium, skin, hair, hair follicles, epithelial tissues, as well as other samples or biopsies.
In certain embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, cytokines, chemokines and/or growth factors selected from CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD, or selected from the cytokines, chemokines and/or growth factors in Table 1, and combinations thereof, are measured. In some embodiments, a panel of no greater than 25, no greater than 20, no greater than 15, no greater than 10, or no greater than 5 cytokines, chemokines and/or growth factors is tested, the panel including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cytokines, chemokines and/or growth factors as described herein.
The level or amount of cytokine, chemokine and/or growth factor in a patient sample can be compared to a reference level or amount of the cytokine, chemokine and/or growth factor present in a control sample. The control sample may be from a patient who does not have PANS (such as PANDAS) or a plurality of patients who do not have PANS (such as PANDAS). The control sample may be from a healthy subject or a plurality of healthy subjects. In other embodiments, a control sample is taken from a patient prior to treatment with a therapeutic intervention or a sample taken from an untreated patient. Reference levels for a cytokine, chemokine and/or growth factor can be determined by determining the level of a cytokine, chemokine and/or growth factor in a sufficiently large number of samples obtained from a patient or patients who do not have PANS (such as PANDAS), or normal, healthy control subjects to obtain a pre-determined reference or threshold value. A reference level can also be determined by determining the level of the cytokine, chemokine and/or growth factor in a sample from a patient prior to treatment with the therapeutic intervention.
Protein-based assays
The level of a cytokine, chemokine and/or growth factor can be detected and/or quantified by any of a number of methods well known to those of skill in the art. The cytokines, chemokines and/or growth factors/proteins may be detected by, for example, mass spectrometry (e.g., LC-MS/MS) and Western blot The methods may include various immunoassays such as cnzymc-linkcd immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), immunohistochemistry, antibody sandwich capture assay, immunofluorescent assay, Western blot, enzyme-linked immunospot assay (EliSpot assay), precipitation reactions (in a fluid or gel), immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), competitive binding protein assays, chemiluminescent assays, and the like. Also included are analytic biochemical methods such as electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, liquid chromatography-tandem mass spectrometry, and the like. U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168. Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991).
The level of a cytokine, chemokine and/or growth factor may be detected by using molecules (e.g., polypeptides, etc.) that bind to the cytokine, chemokine and/or growth factor. For example, the binding polypeptide may be an antibody or antibody fragment, such as an Fab, F(ab)2, F(ab’)2, Fd, or Fv fragment of an antibody. Any of the various types of antibodies can be used for this purpose, including, but not limited to, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies (e.g., generated using transgenic mice, etc.), single chain antibodies (e.g., single chain Fv (scFv) antibodies), heavy chain antibodies and chimeric antibodies. The antibodies can be from various species, such as rabbits, mice, rats, goats, chickens, guinea pigs, hamsters, horses, sheep, llamas etc.
In certain embodiments, ELISA is used to detect and/or quantify one or more cytokines, chemokines and/or growth factors in a sample. The ELISA can be any suitable methods, including, but not limited to, direct ELISA, sandwich ELISA, and competitive ELISA.
In certain embodiments, Western blot (immunoblot) is used to detect and quantify one or more cytokines, chemokines and/or growth factors in a sample. The technique may comprise separating sample proteins by gel electrophoresis, transferring the separated proteins to a suitable solid support, and incubating the sample with the antibodies that specifically bind the one or more cytokines, chemokines and/or growth factors.
The disclosure further includes protein microarrays (including antibody arrays) for the analysis of levels of a plurality of cytokines, chemokines and/or growth factors. Protein microarray technology, which is also known as protein chip technology and solid-phase protein array technology, is well known to those of ordinary skill in the art. Protein microarray may be based on, but not limited to, obtaining an array of identified peptides or proteins on a fixed substrate, binding target molecules or biological constituents to the peptides, and evaluating such binding. See, e.g., MacBeath et al., Printing Proteins as Microarrays for High-Throughput Function Determination, Science 289(5485): 1760-1763, 2000. In some embodiments, one or more control peptide or protein molecules are attached to the substrate.
The polypeptides that may be used to assay the level of a cytokine, chemokine and/or growth factor may be derived also from sources other than antibody technology. For example, such binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties. The cytokine, chemokine and/or growth factor can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the cytokine, chemokine and/or growth factor. Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the cytokine, chemokine and/or growth factor.
Nucleic Acid-based Assays
The present methods may also assay the presence of or quantity the gene encoding a cytokine, chemokine and/or growth factor or the gene product. Gene products include nucleic acids (e.g., mRNAs) derived from the gene.
The level of the DNA or RNA (e.g., mRNA) molecules may be determined/detected using routine methods known to those of ordinary skill in the art. The measurement result may be an absolute value or may be relative (e.g., relative to a reference oligonucleotide, relative to a reference mRNA, etc.). The level of the nucleic acid molecule may be determined/detected by nucleic acid hybridization using a nucleic acid probe, or by nucleic acid amplification using one or more nucleic acid primers.
Nucleic acid hybridization can be performed using Southern blots, Northern blots, nucleic acid microarrays, etc.
For example, the DNA encoding a cytokine, chemokine and/or growth factor in a sample may be evaluated by a Southern blot. Similarly, a Northern blot may be used to detect a cytokine, chemokine and/or growth factor mRNA. Tn one embodiment, mRNA is isolated from a given sample, and then clcctrophorcscd to separate the mRNA species. The mRNA is transferred from the gel to a solid support. Labeled probes are used to identify or quantity the cytokine, chemokine and/or growth factor nucleic acids.
In certain embodiments, labeled nucleic acids are used to detect hybridization. Complementary nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. One method of detection is the use of autoradiography. Other labels include ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
Nucleic acid microarray technology, which is also known as DNA chip technology, gene chip technology, and solid-phase nucleic acid array technology, may be based on, but not limited to, obtaining an array of identified nucleic acid probes on a fixed substrate, labeling target molecules with reporter molecules (e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP, etc.), hybridizing target nucleic acids to the probes, and evaluating target-probe hybridization. Jackson et al. (1996) Nature Biotechnology, 14: 1685- 1691. Chee et al. (1995) Science, 274: 610-613.
The sensitivity of the assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected.
Nucleic acid amplification assays include, but are not limited to, the polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, quantitative RT-PCR, etc.
Measuring or detecting the amount or level of mRNA in a sample can be performed in any manner known to one skilled in the art and such techniques for measuring or detecting the level of an mRNA are well known and can be readily employed. A variety of methods for detecting mRNAs have been described and may include, Northern blotting, microarrays, realtime PCR, RT-PCR, targeted RT-PCR, in situ hybridization, deep-sequencing, single-molecule direct RNA sequencing (RNAseq), bioluminescent methods, bioluminescent protein reassembly, BRET (bioluminescence resonance energy transfer)-based methods, fluorescence correlation spectroscopy and surface-enhanced Raman spectroscopy (Cissell, K. A. and Deo, S. K. (2009) Anal. Bioanal. Chem., 394: 1109-1116). The present methods may include the step of reverse transcribing RNA when assaying the level or amount of an mRNA.
These assays of determining/detecting the presence and/or level of one or more cytokines, chemokines and/or growth factors may include the use of a label(s). The labels can be any material having a detectable physical or chemical property. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels may include, but are not limited to, a fluorescent label, a radiolabel, a chemiluminescent label, an enzyme, a metallic label, a bioluminescent label, a chromophore, biotin etc. For example, a fluorescently labeled or radiolabeled antibody that selectively binds to a cytokine, chemokine and/or growth factor may be contacted with a tissue or cell to visualize the cytokine, chemokine and/or growth factor. In some aspects, a label may be a combination of the foregoing molecule types.
The level, amount, abundance or concentration of one or more cytokines, chemokines and/or growth factors may be measured. The measurement result may be an absolute value or may be relative (e.g., relative to a reference cytokine, chemokine and/or growth factor, etc.)
In one embodiment, a difference (e.g., an increase or decrease) in the measured level of the cytokine, chemokine and/or growth factor relative to the level of the cytokine, chemokine and/or growth factor in the control sample (e.g., a sample in at least one healthy individual, in the patient prior to treatment, at a different time point during treatment, or an untreated patient) or a pre-determined reference value is indicative of the therapeutic efficacy of the therapeutic intervention. In another embodiment, an increase or decrease in the measured level of the cytokine, chemokine and/or growth factor relative to the level of the cytokine, chemokine and/or growth factor in the control sample or pre-determined reference value is indicative of the therapeutic efficacy of the therapeutic intervention. For instance, in such embodiments, when the level of one or more cytokines, chemokines and/or growth factors is increased (or decreased) when compared to the level in a control sample or pre-determined reference value in response to a therapeutic intervention, the increase (or decrease) is indicative of therapeutic efficacy of the therapeutic intervention.
In certain embodiments, a reduction or decrease in the measured level of the cytokine, chemokine and/or growth factor relative to the level of the cytokine, chemokine and/or growth factor in the control sample (e.g., a sample in the patient prior to treatment or an untreated patient) or pre-determined reference value can be indicative of the therapeutic efficacy of the therapeutic intervention. For instance, in such embodiments, when the level of one or more cytokines, chemokines and/or growth factors is decreased (or increased) when compared to the level in a control sample or pre-determined reference value in response to a therapeutic intervention, the decrease (or increase) is indicative of therapeutic efficacy of the therapeutic intervention.
Patients showing different (elevated or reduced) levels of one or more cytokines, chemokines and/or growth factors can be identified. The expression profile of these cytokines, chemokines and/or growth factors may be used to calculate a score for the combined or individual cytokine, chemokine and/or growth factor expression. The scores of these patients will be compared to the score of unaffected individuals (e.g., patients without PANS (such as PANDAS)). The clinical condition of these patients may be correlated with the cytokine, chemokine and/or growth factor expression profiles. The scores may be used to identify groups of patients having PANS (such as PANDAS) responsive to treatment.
Therapeutic intervention/combination therapy
The subject may be treated by an inhibitor of IL-17A or its receptor.
The subject may be treated with 1, 2 or 3 of the following three modes of intervention:
1. Treating the symptoms with psychoactive medications, psychotherapies (e.g., cognitive behavioral therapy), and supportive interventions.
2. Removing the source of the inflammation with antimicrobial interventions.
3. Treating disturbances of the immune system with immunomodulatory and/or antiinflammatory therapies.
The subject may be treated by an inhibitor of IL-17A or its receptor, in addition to 1, 2 or 3 of the above three modes of intervention.
The subject may (further) be treated with antibiotics, anti-inflammatory or immune modulating therapies, and/or psychiatric medications and behavioral interventions.
The subject may (further) be treated by an antibiotic, such as penicillin, amoxicillin, cephalosporin, azithromycin, cefdinir, cephalexin, cefadroxil, clindamycin, or clarithromycin etc. The subject may (further) be treated by intravenous immune globulin (TVTG). The subject may (further) be treated by plasmapheresis (which can remove harmful auto-antibodies from the blood to reduce PANDAS symptoms).
The subject may (further) be treated with other therapies, including tonsil removal, NSAIDs (e.g., ibuprofen), steroids (e.g., prednisone), and psychological interventions such as cognitive- behavioral therapy or exposure and response therapy (ERP).
Kits
Another aspect of the disclosure is a kit containing a reagent for measuring at least one cytokine, chemokine and/or growth factor in a biological sample, instructions for measuring at least one cytokine, chemokine and/or growth factor, and instructions for evaluating or monitoring PANS (such as PANDAS) in a patient based on the level of the at least one cytokine, chemokine and/or growth factor. In some embodiments, the kit contains reagents for measuring from 1 to about 20 human cytokines, chemokines and/or growth factors, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more cytokines, chemokines and/or growth factors. Also encompassed by the disclosure are kits for assessing or predicting the severity or progression of PANS (such as PANDAS) in a subject. The kit may comprise a reagent for measuring at least one cytokine, chemokine and/or growth factor in a biological sample, and instructions for assessing severity or progression of PANS (such as PANDAS) based on the level of the at least one cytokine, chemokine and/or growth factor.
In certain embodiments, the kit comprises antibodies specific to one or more cytokines, chemokines and/or growth factors.
In certain embodiments, the kit comprises primers and/or probe for reverse transcribing, amplifying, and/or hybridizing to one or more mRNAs of one or more cytokines, chemokines and/or growth factors. Such kits can further comprise one or more normalization controls and/or a TaqMan probe specific for each mRNA.
The kit may comprise one or more biochips to assay the levels of a plurality cytokines, chemokines and/or growth factors. Biochips may contain a microarray of molecules (e.g., antibodies, peptides etc. as described herein) which are capable of binding to the cytokines, chemokines and/or growth factors described herein.
As used herein, the term “effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician.
As used herein, the term "therapeutically effective amount" is an amount sufficient to treat a specified disorder or disease or alternatively to obtain a pharmacological response treating a disorder or disease. The term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.
The subject may be a mammal. Mammals may refer to human subjects and non-human subjects, such as dogs, cats, rats, mice, rabbits, monkeys, etc. (e.g., for veterinary purposes). Mammals include humans (infants, children, adolescents and/or adults), and animals such as dogs and cats, farm animals such as cows, pigs, sheep, horses, goats and the like, and laboratory animals (e.g., rats, mice, guinea pigs, and the like). In certain embodiments, the subjects are infants, children, and/or adolescents.
The term “about” in reference to a numeric value refers to ±10% of the stated numeric value. In other words, the numeric value can be in a range of 90% of the stated value to 110% of the stated value.
The following are examples of the present invention and are not to be construed as limiting.
Example 1
Infections with S. pyogenes are a major cause of both short- and long-term morbidity in infants and adults worldwide [Ralph et al. Curr Top Microbiol Immunol 368, 1-27. 10.1007/82_2012_280], not only due to direct primary infections causing pharyngitis, but also due to secondary sequelae including neuropsychiatric disorders [Cunningham, M.W. (2019). Microbiol Spectr 7. 10.1128/microbiolspec.GPP3-0045-2018]. Although an aberrant humoral immune response to GAS infections has been proposed to underlie secondary CNS sequelae, the pathogenesis of CNS complications of GAS infections remains elusive from a molecular standpoint. Using single cell RNA sequencing (scRNAseq) and validation with a variety of approaches, here we provide a molecular atlas of transcriptomic changes that distinct CNS cell populations undergo after multiple peripheral GAS infections [Dileepan et al. (2016). I Clin Invest 72(5, 303-317],
Second, we show that cytokines or chcmokincs derived from myeloid cells arc highly elevated in sera from PANDAS/PANS patients at the acute phase of the disease and enhance transport across the endothelial barrier. These findings support a neuroinflammatory origin of CNS sequelae (SC and PANDAS) [Platt, et al. (2017) Front Immunol 8, 442].
Third, we demonstrate that two Thl7 effector cytokines, IL-17A and GM-CSF, differentially promote BBB dysfunction and microglial expression of interferon-response and chemokine genes in a mouse model of intranasal GAS infections.
Antibody blockade of the signature Th 17 effector cytokine, IL- 17 A, in wild-type mice is sufficient to phenocopy the transcriptome rescue in both microglial and endothelial cells after GAS infections, suggesting that IL-17A is a major driver of the CNS pathology after GAS infections. Moreover, IL-17A blockade partially rescues BBB permeability to serum IgG, indicating that it can be used to treat disorders involving anti-neuronal autoantibodies which have been postulated to underlie the CNS pathology in PANDAS/PANS. [Xu et al. (2021). Am J Psychiatry 178, 48-64; Frick et al. (2018). Brain Behav Immun 69, 304-311; Kirvan et al. (2006) J Neuroimmunol 179, 173-179].
Our findings in the mouse disease model indicate that these antibodies can be used to treat PANDAS/PANS in children.
Microglia and CNS endothelial cells show major transcriptional shifts after multiple GAS infections
We have previously shown that multiple intranasal GAS infections induce infiltration of CD4 T cells from the nose into the anterior brain (predominantly in the OB), BBB disruption, microglial activation, and degradation of excitatory synapses leading to aberrant odor-evoked neural circuitry responses [Dileepan et al. (2016). J Clin Invest 126, 303-317; Platt et al. (2020) Proc Natl Acad Sci U S A 117, 6708-6716].
To understand at the transcriptional level how CNS cell types respond to GAS infections, we isolated and profiled OB cells from P60 mice 18 hours after the fifth GAS infection using scRNAseq (Figure 1A). Inoculation with PBS was used as a control condition. Following quality filtering and batch correction, data from OB cells were represented using dimensionality reduction with /-distributed stochastic neighbor embedding (t-SNE) and cluster identities were assigned using established cell-type markers (Figure IB). A marked transcriptional shift was evident in microglia, which had the highest number of differentially expressed genes (DEGs) between PBS and GAS conditions, followed by olfactory ensheathing cells (OECs), a subtype of glia specific to the olfactory mucosa and OB (Figure 1H). Despite playing a key role in response to inflammation in other CNS diseases, astrocyte clusters showed relatively few DEGs (Figure II).
Since multiple GAS infections trigger BBB disruption and microglial activation in the mouse disease model, we focused on the transcriptional shifts in ECs and microglia by enriching for CD31+ and CDl lb+ cells, respectively, using fluorescence-activated cell sorting (FACS). Microglia, macrophages and ECs were present in greater abundance in FACS-enriched runs (Figure IE), and showed striking transcriptional shifts in gene expression between PBS and GAS conditions (Figures 1C, ID, and Figure II).
In addition to scRNAseq, we performed spatial transcriptomics to gain insight into the regional distribution of cell populations identified by scRNAseq after GAS infections. We used MERFISH [Chen et al. (2015) Science 348, aaa6090] on mouse OB sections and probed for 391 genes predominantly expressed by microglia and ECs given their large transcriptional shifts by scRNAseq (Figure 1A). Cluster identities were assigned using both cell-type markers and spatial localization. Since MERFISH does not enrich for any cell type, it better captured the abundance and heterogeneity of OB neuronal subtypes (Figures 1E-1G).
CNS endothelial cells upregulate inflammatory signatures and downregulate BBB- specific transcripts after GAS infections
To characterize endothelial-specific transcriptional changes after multiple GAS infections, we subsetted and re-clustered ECs from the scRNAseq data (Figure 2A) and performed gene set enrichment analysis (GSEA) [Subramanian et al. Proc Natl Acad Sci U S A 102, 15545-15550. 10.1073/pnas.0506580102] on DEGs using curated gene lists. There was a significant upregulation in genes related to interferon response, antigen presentation, inflammation and EC response to lipopolysaccharide (LPS) [Chen et al. (2020). Brain Endothelial Cells Are Exquisite Sensors of Age Related Circulatory Cues. Cell Rep 30, 4418-4432 e4414], as well as downregulation of BBB- associated genes [Daneman et al. (2010) PLoS One 5, el3741] in ECs isolated from GAS compared to PBS OBs (Figures 2B-2F). CNS specific EC genes and BBB transporters were the main clusters downregulated in the GAS condition. We validated decreased expression of two BBB specific genes, Itm2a and Itih5 [Daneman et al. (2010) PLoS One 5, el 3741 ] by FISH (Figures 2D, 2E) and MERFISH Itm2d). In OB sections from both conditions. In line with increased transport of serum IgG across the leaky BBB in brains from GAS-infected mice [Platt et al (2020). Proc Natl Acad Sci U S A 117, 6708-6716], the BBB transcytosis suppressor gene Mfsd2a [Ben-Zvi et al. (2014). Nature 509, 507-511] was downregulated at both the mRNA and protein level in CNS ECs (Figure 2F). Several genes that promote EC caveolar transport (e.g. Cavl, Cav2, Cavinl, Cavin2, Cavin3) were downregulated in CNS ECs from GAS-infected mice.
Pathway analysis of scRNAseq data also revealed significant downregulation of genes related to cell junctions (e.g., CldnS, Ocln, Tjp2, Amot, Magi3, Cgnll) and the extracellular matrix (ECM), including Col4a3, Lamc3, and Itgal, critical for BBB formation and maintenance [Biswas et al. (2020) Development 147. 10.1242/dev.182279]. We validated decreases in EC expression of Itgal and Itgbl, two integrin receptors that interact with ECM proteins, in GAS-infected OBs using MERFISH (Figure 2H).
Since BBB disruption occurs in other models of neuroinflammation (e.g., EAE), we investigate how the transcriptional shifts identified by scRNAseq in OB ECs from GAS versus PBS conditions compare to those identified by scRNAseq in spinal cord ECs isolated from either acute or chronic EAE versus Complete Freund Adjuvant controls (CFA) [Shahriar et al. (2022). bioRxiv, 2022.2011.2015.516660. 10.1101/2022.11.15.516660]. The correlation analysis performed on all EC genes revealed that log2 fold changes seen in ECs between GAS and PBS correlated more closely with acute (r2 = 0.302), than chronic (r2 = 0.1401) EAE (Figure 2G). The correlation coefficient was higher when we focused only on BBB-associated genes in both acute (r2 = 0.4282) and chronic EAE (r2 = 0.2313) (Figure 2G). These findings suggest the presence of a core EC transcriptional response module to neuroinflammation, regardless of the trigger. In contrast, however, the inflammatory mediator lipocalin 2 (Lcn2) was the most highly upregulated gene in GAS ECs - as validated by MERFISH (Figure 2C) - and was the most upregulated in ECs after LPS exposure [Chen et al. (2020). Cell Rep 30, 4418-4432 e4414], but was downregulated in ECs from EAE (Figure 2G). Overall, the scRNAseq data confirm at the molecular level our prior functional data that GAS infections trigger BBB breakdown [Dileepan et al. (2016) I Clin Invest 126, 303-317; Platt et al. (2020) Proc Natl Acad Sci U S A 117, 6708- 6716], Microglia increase expression of interferon-response, antigen-presentation and cytokine genes in response to GAS infections
The GSEA on DEGs by microglia in the scRNAseq data set from GAS compared to PBS OB samples (Figure 3A) showed enrichment for genes associated with interferon response, antigen-presentation, and cytokine production (Figure 3C). A large number of these genes were also elevated in ECs. Similar to other rodent models of neuroinflammation (e.g., EAE) and neurodegeneration (e.g., Alzheimer’s disease), microglia isolated from GAS samples showed downregulation of homeostatic genes Tmemll9, Cx3crl, P2ryl2, and Gpr34) and upregulation of disease-associated microglia (DAM) genes (Cst7, Axl, Lpl, Sppl, and Apoe). Trem2, which drives expression of select DAM genes in other disease models [Deczkowska et al. (2018) Cell 173, 1073-1081] was downregulated in microglia after GAS infections. Expression of Syk, another DAM regulatory gene (Ennerfelt et al. (2022). Cell 185, 4135-4152 e4122) was also significantly decreased in microglia after GAS infections. It is unclear whether DAM genes are upregulated in microglia independently of Trem2 after GAS infections. Alternatively, Trem2 mRNA may be upregulated earlier in the course of GAS infections, or at the protein level.
To validate some of the DEGs obtained from scRNAseq analysis of microglia following GAS infections, we confirmed by flow cytometry microglial upregulation of antigen-presentation marker CD74 and cytokines TNF and CCL5, as well as downregulation of homeostatic receptors CX3cRl and P2RY12 (Figures 3F-3J). In addition, we analyzed levels of CCL2, CCL4, CCL5, CXCL10 and TNF proteins in whole OB lysates using a multiplex immunoassay and found their levels were higher in OBs from GAS-infected mice (Figure 3M). At the mRNA level Ccl2 and Ccl3 were expressed primarily by microglia and brain macrophages, whereas Ccl4 and Ccl5 were also expressed by infiltrating peripheral immune cells (Figure 3C). Importantly, microglial expression of Ccl3 and Ccl4 was not attributable to enzymatic dissociation [Marsh et al. (2022). Nat Neurosci 25, 306-316], since neither correlate with expression of ex vivo activation signature genes.
To examine the heterogeneity within the microglial response to GAS using a larger cell population, we combined microglia from all scRNAseq experiments including those where either Thl7 cells were absent, or specific Thl7 effector cytokines were eliminated [Figure 4A; see below for more details). We could identify six distinct microglial clusters. Two clusters comprising nearly all cells from the PBS condition (clusters hMGl and hMG2) expressed homeostatic microglial genes (Figures 4B, 4C). Four “StoeptococcM.v-responsive” clusters (srMGl-4) showed upregulation of cytokinc/chcmokinc, disease-associated and antigen-presentation genes (Figure 4C). The clusters could not be distinguished by expression of single genes, but rather by gradients of up- or downregulated gene expression. For example, Ccl3 and Ccl4 were expressed by all srMG clusters, whereas Ccl2 levels were the highest in srMG2 and srMG4 (Figure 4D). In contrast, Ccl5 and Lllb were expressed by a smaller subset of srMG cells. Cluster srMG4 showed the highest expression of interferon-response genes (Irf7, Isgl5, lfi30, Ifi207, Statl, CxcllO Figures 4C, 4D), despite no detectable upregulation of IFNa or IFNp, either transcriptionally or by multiplex cytokine immunoassay, in the OB after the fifth infection.
Streptococcus -responsive microglia are enriched in the glomerular OB layer in close proximity to T cells
To gain insight into the spatial distribution of srMG cells, we performed spatial transcriptomics using MERFISH in the OB after intranasal inoculation with PBS or GAS. There was a distinct spatial distribution of genes upregulated in response to GAS infection. “Streptococcus-responsive” genes including Cd74, Ifi30 and Axl, were expressed at higher levels in microglia located in the glomerular layer of the OB (Figures 3D, 3K, and 3L). In contrast, expression of homeostatic genes P2ryl2 and Gpr34 was higher in the granular layer. A potential driver of this pattern could be the proximity of microglia to T cells, which are most abundant in the glomerular layer of the OB and meninges. Microglia, which are more numerous throughout the OBs of GAS-infected mice (Figure 3N), had a significantly shorter distance to the nearest T cell in the glomerular and external plexiform layers of the OB compared to the granular layer as quantified by MERFISH (Figures 3D, 3E, and Figure 30).
Brain-derived macrophages are restricted to perivascular and meningeal sites after GAS infections and resemble transcriptionally nasal-derived macrophages
Although scRNAseq data indicated that OB macrophages are enriched in GAS-infected mice (Figures 1B-1D), it is unclear whether macrophages infiltrate the brain parenchyma. CX3cRlgFP transgenic mice [Jung et al. (2000). Mol Cell Biol 20, 4106-4114] were crossed to TMEM119tdTomato reporters [Ruan et al. Brain Behav Immun 83, 180-191. 10.1016/j.bbi.2019.10.009] to distinguish between microglia (GFP+ tdTomato+) and macrophages (GFP+ IdTomalo ) in tissue sections. The GFP+ IdTomalo cells were restricted to perivascular and meningeal regions in both PBS and GAS OBs, with few to none within the brain parenchyma (Figures 4E-4G), although the number of macrophages and microglia was increased in the OBs of GAS compared to PBS mice (Figures 4H, 41). Therefore, unlike other models of neuroinflammation, peripheral macrophages do not penetrate the brain after multiple GAS infections.
Analysis of perivascular macrophages, which express Cdl63, Cd207 and Lyvel by scRNAseq, revealed upregulation of similar pathways to those found in microglia after GAS infections, including cytokine/chemokine expression, antigen presentation and interferon response. Saa3, encoding serum amyloid A (SAA) protein 3 was the most upregulated gene in perivascular macrophages. SAA3 is produced by myeloid cells and has been shown to sustain Thl7 responses and inflammation in EAE [Lee et al. (2020). Cell 180, 79-91 el6. 10.1016/j.cell.2019.11.026].
Since the olfactory axons may provide a route for immune cell entry into the CNS after repeated intranasal GAS infections, we performed scRNAseq of cells from the NALT/olfactory epithelium (OE) (Figures 4J, 4K). NALT/OE macrophages showed a similar response to brain macrophages after GAS infections at the molecular level by upregulating several cytokines and chemokines, particularly Ccl5, Ccl6, Ccl9, CxcllO and III b (Figure 4L). The cytokine/chemokine expression patterns of OB macrophages resembled more closely to NALT/OE macrophages than OB microglia (Figures 3C, 4L) suggesting a shared origin.
PANDAS/PANS patients have elevated serum levels of inflammatory cytokines and growth factors expressed by microglia/macrophages
Since distinct cytokines and chemokines derived from either T cells, microglia or macrophages are highly upregulated after GAS infections in the rodent model (Figures 3C, 4L), we wondered whether similar cytokines are also elevated in sera of PANDAS/PANS patients at the acute phase of the disease. We obtained serum samples from 10 PANDAS cases recruited for an IVIg clinical trial at the National Institute for Mental Health (NIMH) [Williams et al. J Am Acad Child Adolesc Psychiatry 55, 860-867 e862. 10.1016/j.jaac.2016.06.017] and 11 age-and sex-matched controls from NIMH, and recruited 13 PANDAS/PANS cases at Columbia University Irving Medical Center (CUIMC) and analyzed them for the presence of 45 serum proteins using a multiplex immunoassay. Serum from PANDAS/PANS patients showed a significant elevation in 13 of 45 tested cytokines, chcmokincs and growth factors compared to healthy controls (Table 1; p-adjusted < 0.05 for 24 significant comparisons). Among the elevated PANDAS/PANS sera proteins, six (CCL2, CCL3, CCL4, CCL5, CXCL10 and TNF) were also highly upregulated by mouse microglia or macrophages after GAS infections (Table 1). GM-CSF, a cytokine produced by pathogenic Thl7 cells (see below), was also significantly elevated in patient sera (Table 1). These data indicate that serum of PANDAS/PANS patients shows an inflammatory signature during the acute phase of the disease.
To understand how cytokines and growth factors present in patient sera may act on ECs, we treated human umbilical vein endothelial cells (HUVECs) with some of the cytokines that were upregulated in PANDAS/PANS patient sera (Table 1). After 24 hours of incubation with cytokines/growth factors, we quantified the uptake of fluorescently labeled albumin in HUVECs as a measure of caveolar-mediated uptake and transport [Lutz et al. , S.E., Smith, J.R., Kim, D.H., Olson, C.V.L., Ellefsen, K., Bates, J.M., Gandhi, S.P., and Agalliu, D. (2017). Caveolinl Is Required for Thl Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation. Cell Rep 21, 2104-2117. 10.1016/j.celrep.2017.10.094; Knowland et al. Neuron. 10.1016/j.neuron.2014.03.003], since albumin uptake is elevated in the mouse BBB after multiple GAS infections [Platt et al. (2020). Proc Natl Acad Sci U S A 777, 6708- 6716]. Several cytokines and growth factors, including CCL2, CCL5, IL-IRA, PDGFBB and HGF, potently upregulated transcytosis in HUVECs (Figures 4M, 4N), suggesting that these elevated serum proteins sera may induce endothelial barrier breakdown in PANDAS/PANS patients during the acute phase of the disease.
Table 1: Cytokines, chemokines and growth factors upregulated in sera from acute PANDAS/PANS patients
Figure imgf000046_0001
Figure imgf000047_0001
ND: not detectable.
Forty-five proteins encompassing cytokines, chemokines and growth factors were measured by multiplex immunoassay in sera from acute PANDAS/PANS patients (n = 23) and controls (n = 11). Twenty-four proteins were significantly elevated in PANDAS/PANS patients compared to controls. Serum concentrations are provided as median (with interquartile range). Statistical comparisons were performed using the two-sample Mann- Whitney test, p-values with Bonferroni correction and adjusted p values (p-adj) for 24 comparisons (ns, p > 0.05; * p < 0.05; ** p < 0.01).
GAS-induced transcriptome shifts in microglial and CNS endothelial cells are rescued in RORyt-deficient mice
We have previously shown that elimination of Thl7 cells rescues BBB dysfunction, microglial Activation, and olfactory circuitry deficits in the OB after multiple GAS infections (Platt et al. (2020). Proc Natl Acad Sci U S A 777, 6708-6716) To better understand how infiltrating Thl7 cells affect microglial and EC responses after GAS infections, we performed scRNAseq in mice lacking the Thl7 fate-specifying transcription factor RORyt (Ivanov et al. (2006). Cell 126, 1121 -1133. 10.1016/j.cell.2006.07.035). ECs from GAS-infected RORyt7- mice showed a partial restoration of several BBB transcripts (c.g., Mfsd a, ltm2a and Ilih5) and dampened expression of transcripts related to inflammation and LPS response (e.g., Lcn2) compared to wild-type (WT) GAS mice by scRNAseq (Figure 5N). Similarly, microglia from GAS-infected RORyt7- mice showed higher expression of homeostatic genes (P2ryl2, Tmemll9, Cx3crl, Gpr34) and lower expression of DAM genes (Cst7, Lpl, Ctsl, Sppl) compared to wild-type GAS-infected microglia by scRNAseq (Figures 50, 5P). Expression of chemokines and cytokines (Ccl2, Ccl3, Ccl4, Tnf) and interferon-response (Irf7, lfitm3, lfi207, lsg!5 genes was also reduced relative to wild-type GAS microglia (Figures 5Q, 5R). Intriguingly, expression of genes involved in antigen presentation (H2-Abl, H2-Ebl, Cd74, H2-D1 ) was elevated further in GAS-infected RORyt7- compared to wild-type mice (Figure 5S). Flow cytometry analysis confirmed significant elevation in microglial surface expression of CD74 and major histocompatibility complex (MHC) class II (I-A/I-E) proteins in GAS-infected RORyt7- compared to wild-type mice (Figures 5A, 5B). Thl7 cell-dependent suppression of antigen-presentation genes was not limited to antigen-presenting cells. MHC class I markers B2m, H2-D1 and H2-K1 were significantly elevated in astrocytes, OECs, and to a lesser extent in neurons, ECs and microglia from GAS-infected RORyt7- mice (Figure 5T). A potential mechanism for this effect could be increased IFNy in RORyt7- mice, since IL-17A negatively regulates IFNy and Thl cell identity (O'Connor et al. (2009). Nat Immunol 10, 603-609. 10.1038/ni.l736; Ajendra et al. (2020). Mucosal Immunol 13, 958-968. 10.1038/s41385- 020-0318-2), and IFNy upregulates MHC gene expression in myeloid cells (Gonalons et al. (1998). J Immunol 161, 1837-1843). To determine whether the overall concentration of IFNy is higher in RORyt7 mice, we measured IFNy from whole OB lysates of PBS wild-type, GAS wildtype and GAS RORyt7- mice 18 hours after either the second (2i) or fifth (5i) infection. We found a significant increase in IFNy concentration in RORyt7- compared to wild-type GAS mice at 2i, but not 5i (Figure 5C), indicating that IFNy may drive expression of antigen-presentation genes earlier in disease.
IL-17A is required for BBB dysfunction and microglial activation in vivo following GAS infections
To better understand how Thl7 cells contribute to neurovascular and microglial dysfunction, we focused on two inflammatory cytokines produced by Thl7 cells: IL-17A and GM- CSF. We treated mice with either an IL-17A-neutralizing monoclonal antibody (mAb), or an isotype control antibody, administered twice weekly starting 24 hours before the first GAS infection to parse the contribution of IL-17A in the RORyt-dcpcndcnt pathology (Figure 5D). IL- 17A blockade did not impact CD4 T cell infiltration into the anterior brain since their number was similar between the two conditions (Figure 5U). However, mice treated with the IL-17A- neutralizing antibody had a significantly higher mortality due to sepsis (Figure 5V), reflective of a key role for IL-17A in controlling infection. Importantly, IL-17A blockade partially rescued BBB leakage after GAS infections, since there was a two-fold reduction in serum IgG extravasation in the granular layer of the OB in IL-17A blocking condition compared to isotype controls (Figures 5E-5G), an effect similar to that seen in RORyt/_ mutant mice. We next performed scRNAseq on CD31- and CD 1 Ib-enriched OB cells from GAS-infected mice following treatment with the IL-17A-neutralizing antibody or isotype control. Although IL-17A blockade did not rescue expression of Mfsd a, Itiih5 and Itm2a (Figure 5H), it restored expression of other BBB genes including cell-junction (e.g., Cldn5, Ocln. Cngll, Ctnnal, Tjpl , Tjp2), and transporter genes (e.g., Bsg, Slc7a5, Ap2bl, Abcbla, Slcolcl). In addition, IL-17A blockade reduced EC expression of some inflammation and LPS response genes including Lcn2 (Figure 5H). Thus, IL- 17A contributes to BBB dysfunction after GAS infections, both at a molecular and functional level.
As was the case for RORyC mice (Figures 5O-5S), microglia from the IL-17A mAb- treated condition showed rescue of many homeostatic transcripts and decreased expression of DAM signature, chemokines/cytokines, and interferon-response transcripts (Figures 5I-5L). However, IL-17A blockade exacerbated upregulation of antigen-presentation genes (Figure 5M). Genes related to antigen presentation by MHC class I were also upregulated in other OB cell types, including OECs, astrocytes and ECs, in IL-17A mAb-treated mice compared to isotype controls (Figure 5T). Therefore, IL-17A promotes several microglial-related neuroinflammatory responses after GAS infections.
GM-CSF does not drive BBB breakdown but negatively regulates microglial abundance after multiple GAS infections
GM-CSF is another cytokine downstream of RORyt in Thl7 cells with the potential to drive neuroinflammation and vascular dysfunction following GAS infections. GM-CSF plays a pathogenic role in some autoimmune paradigms, such as EAE, but is protective in others51. In Streptococcus pneumoniae infections, GM-CSF is expressed by T cells only in chronic inflammation [Kara et al. (2015). Nat Commun 6, 8644. 10.1038/ncomms9644]. Tn addition, GM- CSF was among the scrum proteins significantly elevated in PANDAS/PANS patients (Table 1). To determine if GM-CSF+ CD4 T cells are present in the brains of GAS-infected mice, we used flow cytometry to track CD4 T cell subsets over the course of GAS infections. The proportions of GM-CSF+ CD4 T cells increased with repeated GAS infections, as did the number of IFNy+ Th 17 (IFNy+IL-17A+) cells in both the OB and NALT/OE (Figures 6A, 6B). Approximately 90% of GM-CSF+ CD4 T cells were either conventional Thl7s or IFNy+ Thl7 cells, and were significantly decreased in RORyt7’ OBs after repeated GAS infections (Figures 6C, 6D).
To determine the role of GM-CSF in CNS pathology after GAS infections, we generated mice lacking GM-CSF in CD4 T cells (Csf2ACD4) by crossing CD4-CreERT2 53 to Csf2^ 54 mice and administering 4-OH-tamoxifen between P16 and P20 prior to beginning GAS infections at P28 (Figure 6e). Using flow cytometry, we confirmed GM-CSF knockdown in CD4 T cells isolated from the OB of GAS-infected mice after 4-OH-tamoxifen administration. There was no significant difference in either CD4 T cell infiltration or serum IgG extravasation across the BBB and most BBB-related genes including tight junctions, transcytosis (e.g., Mfsd2d) or transporters were unchanged between Csf2ACD4 and Csf2fl/fl mice after GAS infections (Figure 6F). However, loss of GM-CSF in CD4 T cells rescued EC expression of some BBB-related (e.g., Itih5, Itm2a) or inflammation genes (e.g., Lcn2) (Figure 6F).
Examination of gene expression profiles in Csf2ACD4 microglia by scRNAseq indicated a slight rescue in some homeostatic and disease-associated transcripts (Figures 6G, 6H), and a significant reduction in interferon-response and antigen-presentation transcripts (Figures 6J, 6K) compared to Csf2fl/fl microglia after GAS infections. Unlike IL-17A blockade, elimination of GM- CSF in CD4 T cells did not reduce microglial expression of Ccl3 and Ccl4, although Ccl2 and Tnf were reduced in Csf2ACD4 microglia (Figure 61). Expression of Trem2 was even further elevated in Csf2ACD4 compared to Csf2fl/fl microglia after GAS infections (Figure 6M). Loss of GM-CSF expression by CD4 T cells caused a significant increase in Ibal+ CD68+ cell number in the glomerular layer of the OB (Figures 6N, 60). This was not reflected in expression of proliferation genes since Csf2ACD4 microglia downregulated several cell cycle-related transcripts (e.g. Top2' a. Mcm2, Mcm5 and Mki67) relative to Csf2fl/fl microglia after GAS infections by scRNAseq. Overall, IL-17A and GM-CSF have distinct roles in BBB dysfunction and neuroinflammatory microglial responses after GAS infections in vivo. To evaluate cell-autonomous effects of TL-17A and GM-CSF on ECs, we tested their ability to disrupt barrier properties of mouse brain capillary endothelial cells (mBECs) in vitro, along with IFNy, which is produced by infiltrating Thl and IFNy+ Th 17 cells after multiple GAS infections (Figure 6A). While treatment with positive controls IL-ip/TNF and IFNy significantly decreased trans-endothelial electrical resistance (TEER), a readout of paracellular EC barrier integrity, neither IL-17A nor GM-CSF had any effect on the TEER. IFNy, IL-17A and GM-CSF treatments failed to induce the transport of fluorescently labeled albumin, a readout of transcellular transport, in mBECs in an in vitro transwell assay. Since RORyt deficiency and blockade of IL- 17A rescued BBB transcytosis in vivo, these findings suggest that IL-17A likely exerts its effects on CNS ECs indirectly.
To evaluate the effect of T cell-derived cytokines on microglia in vitro, we performed bulk RNA sequencing on microglia cultures from postnatal brains after 24 hour incubation with IL- 17A, GM-CSF or IFNy. There was a dramatic shift in gene expression of cultured microglia upon treatment with IFNy, including upregulation of inflammatory and interferon-response genes (e.g., Irfl and CxcllO) that were also identified in microglia in vivo after GAS infections (Figure 3C). Similarly, primary microglia responded robustly to GM-CSF in culture by upregulating several genes including Cd300f, a gene downregulated in vivo in RORyt /_, IL-17A mAb-treated and Csf2ACD4 microglia. In contrast, minimal changes were observed in glial cell with IL-17A treatment, although this is difficult to interpret since cultured microglia expressed low levels of the IL-17A receptor transcript Ill7ra [Timmerman et al. Cell Neurosci 12, 242. 10.3389/fncel.2018.00242]. Nevertheless, T cell effector cytokines induce distinct gene expression profiles in microglia responsible for neuroinflammatory changes after repeated GAS infections.
Discussion
Using scRNAseq and validation with a variety of approaches, here we provide a molecular atlas of transcriptomic changes that distinct CNS cell populations undergo after multiple peripheral GAS infections [Dileepan et al. J Clin Invest (2016) 126, 303-317]. Second, we show that cytokines or chemokines derived from myeloid cells are highly elevated in sera from PANDAS/PANS patients at the acute phase of the disease, and enhance transport across the endothelial barrier. These findings support the hypothesis for a neuroinflammatory origin of CNS sequelae (SC and PANDAS) [Platt et al. (2017) Front Immunol 8, 442], Third, we demonstrate that two Th 17 effector cytokines, TL-17A and GM-CSF, differentially promote BBB dysfunction and microglial expression of intcrfcron-rcsponsc and chcmokinc genes in a mouse model of intranasal GAS infections. Below, we elaborate on these three key findings in relation to other neuroinflammatory/neuroinfectious diseases and their relevance for the diagnosis and treatment of CNS sequelae of GAS infections.
We have employed a naturalistic rodent model of intranasal infections to explore how recurrent GAS exposures trigger BBB breakdown and neuroinflammation at a molecular level, and how these molecular findings relate to the post-Streptococcal human CNS disorders PANDAS and SC. Our scRNAseq analysis of more than 100,000 cells from the mouse OB revealed that ECs and microglia are among the CNS cell types most transcriptionally altered after GAS infections.
The endothelial response to GAS includes upregulating antigen presentation and inflammation genes, and downregulating BBB-associated transcripts. Several BBB-enriched transcripts such as Itm2a, Itih5, Mfsd2a, cell junction regulators (e.g. Cldn5, Ocln, Tjp2, Amot, Magi3, Cgnll ), and ECM proteins and receptors (e.g. Col4a3, Lamc3, Itgal), critical for BBB formation and maintenance, were significantly downregulated after GAS infections. These molecular changes are consistent with our findings that BBB structure and function are impaired after GAS infections [Dileepan et al., J Clin Invest, (2016) 126, 303-317. Platt et al., Proc Natl Acad Sci U S A, (2020) 117, 6708-6716]. Although increased EC transcellular transport is one of the cell biological mechanisms by which the BBB becomes permeable to large proteins (e.g., IgG), our molecular data indicate that genes promoting formation of caveolae (e.g., Cavl, Cav2, Cavinl and Cavin2) were downregulated after GAS infections. Decreased expression of Mfsd2a could explain, in part, the upregulation in EC transcellular transport since it inhibits caveolae formation [Ben-Zvi et al., Nature (2014) 509, 507-511]; however, breakdown of cell junctions or increased bulk transcytosis or macropinocytosis can also promote transport across the BBB. The gene most strongly induced in ECs after GAS infections, Lcn2, is involved in the innate immune response with pleiotropic roles in the CNS, and has also been shown to depend on IL- 17 A signaling [Karlsen et al., J Biol Chem (2010) 285, 14088-14100; Shen et al., J Biol Chem (2006) 281, 24138-24148], and the inflammatory cytokine response to LPS administration is exacerbated in Lcn2-deficient brains [Kang et al., Mol Psychiatry (2018) 23, 344-350]. Therefore, Lcn2 may regulate this response by ECs in GAS infections. Microglia also undergoes major transcriptional changes after GAS infections reflected in induction of DAMs, cytokine and chcmokinc, antigen-presentation and intcrfcron-rcsponsc genes, and suppression of homeostatic genes. Importantly, the MERFISH analysis revealed that expression of Streptococcus-responsive genes is higher in microglia in the OB areas with more infiltrating T cells, suggesting that the proximity to T cells is a key factor driving the shift to disease-associated state in microglia. Surprisingly, Trem2, which drives expression of DAM genes in other disease models, was downregulated in microglia after GAS infections. Studies of the DAM response in neurodegenerative diseases (e.g., Alzheimer’s disease or amyotrophic lateral sclerosis) indicate that TREM2 may be required for development of “stage 2” DAM signatures, including upregulation of genes such as Lpl, Cst7, Axl, Itgcix and Sppl. [Deczkowska et al. (2018) Cell 173, 1073-1081.]
Although we could identify four distinct microglial “Streptococcus-responsive” clusters (srMGl-4) after GAS infections by scRNAseq, the expression of distinct srMG genes was graded, rather than discrete, across the four clusters. Interferon-response genes were an exception since they were strongly elevated in the srMG4 cluster. Both type I and type II interferon signaling have been implicated in defense against GAS infections. [Hyland et al. (2009). FEMS Immunol Med Microbiol 55, 422-431; Gratz et al. (2011). PLoS Pathog 7, el001345.] T cells that infiltrate the brain after GAS infections secrete IFNy (type II interferon); however, microglial transcription appeared skewed toward a type I IFN response, with increased expression of genes like Irf7, Isgl5, Ifitl, Ifit2, lfit3, Rsad2 and Ms4a4c, which are preferentially induced by IFNa. [Liu et al.. (2012). Proc Natl Acad Sci U S A 109, 4239-4244.] However, no upregulation of IFNa or - was detected, either transcriptionally or by multiplex cytokine immunoassay in the OB.
The third class of genes elevated in treptococcus-responsive microglia and macrophages were cytokines and chemokines including Ccl2, Ccl3, Ccl4, Ccl5, CxcllO, Cxcll2, Illb, and Tnf. These cytokines are also upregulated in other models of neuroinflammation and neurodegeneration, although with distinct combinatorial patterns. The microglial cytokine expression profile more closely resembles that of OB than NALT/OE macrophages; however, the proportion of microglia expressing cytokines was higher than that of OB macrophages. Upregulation of chemokines by microglia and macrophages could contribute to recruitment of peripheral immune cells, particularly T cells and infiltrating macrophages, into the CNS. For example, CCL2-CCR2 signaling is required for Thl7path cell recruitment to the CNS in EAE and S. pneumoniae intranasal infection. [Kara ct al. (2015). Nat Commun 6, 8644.]
Although the human studies have mainly focused on “pathological” antibodies, we have previously shown that Thl7 cells are critical to induce BBB dysfunction, microglial activation, and olfactory circuitry deficits in the OB after multiple GAS infections [Platt et al. (2020). Proc Natl Acad Sci U S A 117, 6708-6716]. The molecular transcriptome analysis of microglia from RORyt 1' mice supports the rescue of the pathological phenotype (i.e., microglia activation) after GAS infections. Id. Our molecular analysis of microglia shows a rescue in most Streptococcus- responsive pathways in RORyl " mice, particularly in chemokine/cytokine and interferon- response genes. Similarly, there is a partial restoration of some BBB transcripts (e.g., Mfsd2a, Itm2a and Iiih5) and reduced expression of transcripts related to inflammation and LPS response (e.g., Lcn2) in RORyf'~ compared to wild-type GAS conditions. Although the decrease in EC transcytosis seen in RORyt1' mice after GAS infections is not paralleled by a significant rescue of transcytosis genes (e.g., Mfsd2a or Cavl), the rescue in either cell-junction (e.g., Cdh5, Jam2, Tjpl). or receptor-mediated endocytosis transcripts may explain BBB functional rescue in the absence of Thl7 cells. Id. Unexpectedly, expression of genes involved in antigen presentation was elevated further in GAS-infected RORyt /_ CNS cells possibly due to increased expression of IFNy at two, but not five, GAS infections.
Our analysis of the distinct roles that two Thl7 effector cytokines, IL-17A and GM-CSF, play in post-Streptococcal neuropathology expands further our understanding of the role of Thl7 cells in GAS -mediated CNS sequelae. Antibody blockade of the signature Th 17 effector cytokine, IL- 17 A, in wild-type mice is sufficient to phenocopy the transcriptome rescue in both microglial and endothelial cells after GAS infections, suggesting that IL- 17 A is a major driver of the CNS pathology after GAS infections. Moreover, IL-17A blockade partially rescues BBB permeability to serum IgG, indicating that it may have therapeutic potential in disorders involving anti-neuronal autoantibodies which have been postulated to underlie the CNS pathology in PANDAS/PANS [Xu et al. (2021). Am J Psychiatry 178, 48-64. Frick et al. (2018). Brain Behav Immun 69, 304-311. Kirvan et al. (2006). J Neuroimmunol 179, 173-179].
We also examined the contributions of GM-CSF, an alternate Thl7 effector cytokine, in CNS pathology since it is significantly elevated in the serum of PANDAS/PANS patients. We found that GM-CSF is expressed by CD4 T cells only after multiple GAS infections, which is intriguing since PANDAS is thought to result from repeated GAS exposures. Genetic ablation of GM-CSF in T cells failed to rescue BBB leakage to scrum IgG after GAS infections, although at a molecular level some BBB transcripts (e.g., Itih.5, Ilm2a) were increased and inflammatory genes (e.g., Lcn2) were decreased in Csf2ACD4 mutant ECs after GAS infections. Similarly, the normalization of homeostatic and the reduction in disease-associated microglia transcripts is less significant in Csf2ACD4 compared to RORyt mutant and IL- 17 A mAb-treated wild-type mice. While Type I IFN response genes were strongly downregulated in Csf2ACD4 microglia similar to RORyt mutant and IL-17A mAb-treated conditions, the upregulation of some chemokine gene (e.g., Ccl3 and Ccl4) is not rescued in Csf2ACD4 microglia. Finally, antigen presentation genes were largely rescued in Csf2ACD4 microglia consistent with published reports that GM-CSF upregulates MHC II and CD74. Re et al. (2002). J Immunol 169, 2264-2273.
Currently it is unclear whether PANDAS/PANS have a neuroinflammatory origin, although higher neuroinflammation has been seen in the basal ganglia of a small number of PANDAS patients (Kumar et al. (2015). J Child Neurol 30, 749-756) using a PET ligand for TSPO that cannot distinguish between activated microglia and astrocytes [Nutma et al. (2021). Eur J Nucl Med Mol Imaging 49, 146-163]. Our mouse model supports microglial, rather than astrocytic reactivity, in response to repeated intranasal GAS exposures. The analysis of serum samples from 23 acute PANDAS/PANS patients and 11 age- and sex-matched healthy controls shows a significant elevation in 12 of the 45 cytokines, chcmokincs and growth factors. Among the elevated proteins were six that were also highly upregulated by mouse microglia after GAS infections (CCL2, CCL3, CCL4, CCL5, CXCL10 and TNF), further supporting a critical role for microglial activation as a driver of CNS pathology. Some of the elevated cytokines seen in PANDAS/PANS sera can be attributed to GAS infections since patients with invasive GAS diseases have elevated levels of IL-10, IL-6, IL-8, IL-10 and IL-18 compared to children with non- invasive GAS infections. Wang et al. (2008). Diagn Microbiol Infect Dis 61, 165-169. Moreover, stimulation of human peripheral blood mononuclear cells (PBMCs) with heat-killed lyophilized GAS for 24 hours evokes production of IL- 10, IL-2, IL-6, IL-8, IFNy, TNF and GM-CSF [Cavaillon et al. (1997). Adv Exp Med Biol 418, 869-879]. Among serum proteins significantly elevated in PANDAS/PANS, only IL-6 and CXCL10 were shown to be upregulated in acute S. pyogenes pharyngitis in adults [Anderson et al. (2022). Nat Commun 13, 769]. In the same study, CCL2, CCL4, CCL5, IL-7 and TNF - all significantly upregulated in PANDAS/PANS cases compared to controls - were unchanged or downregulated in serum following 8. pyogenes pharyngitis challenge. Overall, these studies suggest some specificity for the cytokine signature seen in PANDAS/PANS children’s sera. Importantly, several cytokines and growth factors including CCL2, CCL5, IL- IRA, PDGFBB and HGF potently upregulated transcytosis in ECs, suggesting that these elevated cytokines in PANDAS/PANS patient sera may induce endothelial barrier breakdown during the acute phase of the disease. These findings support a neuroinflammatory origin of CNS sequelae (SC/PANDAS) and provide some additional mechanisms that can drive the entry of pathological antibodies into the CNS in the acute phase of the disease [Platt et al. Front Immunol 8, 442].
Methods and Materials
Mice
Experiments involving mice were approved by Columbia University’s Institutional Animal Care and Use Committees. Mice were bred in the CUIMC vivarium, under 12-hour light/12-hour dark, pathogen-free conditions. Female mice were used for all experiments, except the time course analysis of Thl7 cell subtypes by flow cytometry and Csf2 recombination confirmation flow cytometry, which used even numbers of males and females. The RORyfGFF mice [Platt et al. (2020), Proc Natl Acad Sci U S A 117, 6708-6716; Ivanov et al. (2006). Cell 126, 1121-1133] B6.129P2(Cg)-Rorctm2Litt/J, strain 007572 were obtained from the Jackson Laboratory. The Csf2fl/fl mouse strain (Louis et al. (2020). J Exp Med 217. 10.1084/jem.20191421) was provided by Bogoljub Ciric (Thomas Jefferson University, Philadelphia, PA). CD4-CreERT2 transgenic mice (B6(129Xl)-Tg(Cd4-cre/ERT2)llGnri/J, strain 022356) (Aghajani et al. (2012). Genesis 50, 908- 913) were obtained from the Jackson Laboratory and crossed to Csf2fl/fl mice for two generations. CD4-CreERT2+/_ Csf2fl/fl males were mated to Csf2fl/fl females to generate CD4-CreERT2+/“; Csf2fl/fl experimental mice and Csf2fl;fl littermate controls. P16 pups were intraperitoneally injected daily with 100 pg of (Z)-4-Hydroxytamoxifen (Millipore Sigma, H7904), dissolved in 50 pL of com oil (Millipore Sigma, C8267) for 5 days (P16-P20). TMEM119tdTon,ato (Ruan et al. (2020). Brain Behav Immun 83, 180-191) and CX3CR1GFP (Jung et al. (2000). Mol Cell Biol 20, 4106-4114) reporter mouse lines were generously provided by Wassim Elyaman (CUIMC).
Human serum studies
The experiments with human sera were approved by Columbia University’s Institutional Review Board (TRB-AAAQ9999). The NIMH sera used in this study were analyzed in a previous publication (Swcdo ct al. (2015). J Child Adolcsc Psychopharmacol 25, 26-30. 10.1089/cap.2014.0073), and were obtained from the NIMH using an MTA agreement. Informed consent / assent was obtained from all subjects.
GAS intranasal infections
Mice received weekly intranasal inoculations with either a suspension of Streptococcus pyogenes [Group A Streptococcus (GAS)], or phosphate -buffered saline (PBS) control, starting at P21-P28. We used a recombinant GAS strain expressing a 2W epitope-tagged M protein as describedl8-20. GAS was streaked out on new blood agar plates each week. Culture media consisted of an autoclaved solution of 3% Todd-Hewitt Broth (Bacto, 90003-430) and 2% Neopeptone (Bacto, 90000-268). Several GAS colonies were used to inoculate 10 mL of culture medium and incubated overnight at 37° C in 5% CO2. The following day, the culture was diluted to an OD600 of 0.2 and grown to OD6000.6, centrifuged and washed in 1 mL PBS (without Ca2+ and Mg2+) and resuspended in 110 pL of PBS. The GAS suspension was kept briefly on ice prior to intranasal infections. All intranasal infections were performed in an ABSL2 vivarium facility. Mice were immobilized with light anesthesia and a P20 pipette was used to drip GAS suspension into nostrils. To reduce lethality due to sepsis, a smaller GAS dose was used during the first two weeks (the first GAS inoculation is 8 x 107 CFU per nostril, the second is 12 x 107 CFU per nostril, and the third, fourth and fifth are 2 x 108 CFU per nostril). During the first two weeks of GAS infections, mice were provided with nutritional supplements (ClearH2O, 72-27-5022). Neutralizing antibody treatment
Starting 24 hours prior to the first GAS infection, mice were injected intraperitoneally twice weekly with 500 pg of either InVivoMAb anti-mouse IL-17A monoclonal antibody, clone 17F3 (Bio X Cell catalog, BE0173), or mouse IgGl isotype control monoclonal antibody, clone MOPC-21 (Bio X Cell, catalog BE0083), in 100 pL of dilution buffer (Bio X Cell catalog IP0070 and IP0065, respectively).
Single-cell RNA sequencing
Mice were anesthetized with isoflurane and perfused intracardially with PBS for 3 minutes. Nasal associated lymphoid tissue (NALT), olfactory epithelium (OE), or olfactory bulb (OB) were dissected and placed in Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ and cut up with a sterile scalpel blade. Two or three animals were pooled per sample. Tissue was then placed in C Tubes (Miltenyi Biotec, 130-093-237), along with dissociation reagents from the MACS Neural Tissue Dissociation Kit (P) (Miltenyi Biotcc, 130-092-628). Samples were loaded onto a Gentle MACS Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427) and the 37C_NTDK_1 program was run. Following dissociation, samples were filtered through a 70 pm cell strainer, washed in HBSS and resuspended in MACS buffer with myelin removal beads (Miltenyi Biotec, 130-096-733), then purified with an LS column (Miltenyi Biotec, 130-042-401), according to manufacturer instructions. Eluent was washed twice, incubated with DRAQ5 (BioLegend, 424101, 1: 1000) and CD16/CD32 Fc block (BD Biosciences, 553141, 1:200) at room temperature for 15 minutes. Cells were washed and incubated with antibodies against CD31 (FITC, BD Biosciences, 561813, 1:200) and CDllb (BV421, BioLegend, 101235, 1: 100) for 30 minutes on ice. Cells were washed and resuspended in FACS buffer with propidium iodide (1: 10,000). Live, nucleated cells (DRAQ5+ PIlo) were sorted on a FACSAria II (BD), equipped with 355 nm, 405 nm, 488 nm, 561 nm and 640 nm lasers and a 130 pm nozzle. In a subset of experiments, CD31+ and CDl lb-i- populations were collected to enrich for cell types of interest. Sequencing was performed by the Columbia Single Cell Core using 10X Genomics Chromium Single Cell 3’ technology, with reads aligned to the mml0-2020-A transcriptome.
Immunofluorescence
Mice were anesthetized with isoflurane and perfused intracardially with PBS for 4 minutes, followed by 4% paraformaldehyde (PFA) for 6 minutes. Brains were extracted and post-fixed in 4% PFA for 4-6 hours, then washed three times in PBS, incubated overnight in 30% sucrose, embedded in Tissue-Plus O.C.T. compound (Fisher, 4585) and stored at -80°C. Coronal sections (12 pm) were cut on a Leica CM3O5O S Cryostat and stored at -80"C. For immunofluorescence staining, slides were washed in PBS for 10 minutes, incubated for 1 hour at room temperature in blocking buffer (10% BSA in IX PBS with 0.1% Triton-X-100), and with primary antibodies diluted in PBST (0.1% Triton-X-100 in IX PBS) with 1% BSA overnight at 4°C. The table below provides a complete list of immunofluorescence antibodies used for this study.
Figure imgf000058_0001
Figure imgf000059_0001
Slides were incubated in primary antibodies at 4°C in a humidified chamber overnight. After three 10-minute washes with PBST, slides were incubated for 2 hours at room temperature in secondary antibodies diluted in PBST with 1% BSA. These were conjugated to AlexaFluor 488 (1: 1000), AlexaFluor 594 (1: 1000), or AlexaFluor 647 (1:500). Following three washes with PBST and two with PBS, slides were cover slipped with Vectashield (Vector Labs, Burlingame, CA) containing the nuclear stain DAPI, then sealed with clear nail polish and stored at -20"C.
In situ hybridization
Plasmids were obtained from Transomic Technologies and the antisense mRNAs were synthesized using the Digoxigenin RNA Labeling Kit (SP6/T7; Roche, 11175025910). DIG RNA in situ hybridization (ISH) and fluorescent in situ hybridization (FISH) experiments were performed as previously described (Biswas et al., Development, 2022; 149(17):dev200610). Mice used for ISH or FISH were perfused for 4 minutes with PBS and brains were dissected out and embedded in O.C.T.
Multiplexed error-robust in situ hybridization (MERFISH}
Mice were anesthetized with isoflurane and intracardially perfused with cold RNAse-free PBS for 4 minutes. Brains were dissected out and immediately embedded in Tissue-Plus O.C.T compound and stored at -80"C until samples could be shipped overnight on dry ice to UMass Chan Medical School. Samples were prepared according to the Vizgen Fresh Frozen Tissue Sample Preparation protocol. Tissue was sectioned in 10 pm slices onto a functionalized coverslip covered with fluorescent beads. Each coverslip contained a section from one PBS sample and a section from one GAS sample. Tissue on coverslips was fixed for 15 minutes at room temperature in 4% paraformaldehyde in PBS, followed by three washes with PBS. Tissue was then permeabilized in 70% ethanol for 24 hours, washed with PBS and incubated with blocking solution for 1 hour, followed by 1 hour of incubation with the primary antibody against vessel marker CD31 (BioLegend, 102502), diluted 1:20 in blocking solution (Vizgen). The tissue was then washed three times with PBS and incubated for 1 hour with an oligo-conjugated secondary antibody diluted 1:1000 in blocking solution. The sample was washed three times with PBS and fixed for 15 minutes at room temperature in 4% paraformaldehyde in PBS, followed by three washes with PBS. After a 30-minute wash with Formamide Wash Buffer (30% formamide in 2X saline sodium citrate, or SSC) at 37°C, the MERFISH library mix was added and allowed to hybridize for 48 hours. Sample was then washed and incubated at 47°C with Formamide Wash Buffer twice for 30 minutes each and then the tissue was embedded in a polyacrylamide gel followed by incubation with tissue clearing solution (2X SSC, 2% SDS, 0.5% v/v Triton X-100, and proteinase K 1: 100) overnight at 37 °C. Then, tissue was washed and hybridized for 15 minutes with the first hybridization buffer containing DAPI, polyT and the readout probes associated with the first round of imaging. After washing, the coverslip was assembled into the imaging chamber and placed into the microscope for imaging. MERFISH imaging was performed as previously described97 with parameter files provided by Vizgen. Briefly, the sample was loaded into a flow chamber connected to the Vizgen Alpha Instrument. First, a low-resolution mosaic image was acquired (405 nm channel) with a low magnification objective (lOx). Then the objective was switched to a high magnification objective (60x) and seven 1.5-pm z-stack images of each field of view position were generated in 749 nm, 638 nm and 546 nm channels. A single image of the fiducial beads on the surface of the coverslip was acquired and used as a spatial reference (477 nm channel). After each round of imaging, the readout probes were extinguished, and the sample was hybridized with the next set of readout probes. This process was repeated until combinatorial FISH was completed. Raw data were decoded using the MERlin pipeline (Vizgen, vO.1.12) using the relevant library codebook. Cell boundaries were segmented in each FOV using a seeded watershed algorithm with DAPI signal as the seed and poly-T signal as the watershed channel. The volume, X position, and Y position of these cell boundaries, as well as the probe counts within each cell boundary, were output for further analysis. Probes for the following transcripts were used: Abca7, Abcc3, Abcg2, Abliml, Actb,Acvrll , AdamlO, Adaml7, AdgrfS, Adgrl4, Adoral, Aff3, Ago4, Agt, Ahr, Akapl2, Aldoc, Anxal, Ap2ml, Aqp4, Arc, Argl, Arhgap29, Arll5, Arpc2, Atmin, AtplOa, Axl, Baiap2ll, Bardl, Binl, Birc5, Bmp6, Brcal, Btk, Clqa, Clqb, Clqbp, Clqc, C3, C3arl, C4a, C5arl, Caldl, Casp7, Casp8, Cass4, Ccl2, Ccl22, Ccl3, Ccr2, Cdl4, Cdl63, Cd27, Cd33, Cd3e, Cd4, Cd47, Cd68, Cd72, Cd74, Cd79a, Cd84, Cd86, Cd8a, Cdh5, Cdh9, Cemip, Cenpa, Cgnll, Chek2, Chitl, Cldn5, Clec7a, CUc4, Clu, Cmtm8, Cobill, Col6a3, Colli, Criml, Csad, Csflr, Csf2, Csj2ra, Csf2rb, Cspg4, Cstb, Ctgf, Ctsb, Cx3crl, CxcllO, Cyr61, Dachl, Dapkl, Dclrela, Ddx58, Des, Did , Dna2, Dock2, Dock9, Duspl, E2fl, Ebfl, Ebi3, Ecel, Ednl, Edn3, Efnb2, Egfl7, Egfr, Egrl, Elovl7, Emcn, Empl, Eng, Enpp6, Entpdl, Epasl, Epb41l4a, Ephal, Erg, Esam, Esyt2, Fancd2, Fbrs, Fbxwl7, Fcerlg, Fcgrl, Fcgrt, Feris, Fgd2, Flcn, Flnb, Fill, Flt3, Flt4, Fnl, Fold, Fos, Foxjl , Foxpl , Ftsj3, Gad! , Gad2, Galntl8, Gbp2, Gfer, Gnal3, Gnal5, Gpil , Gprl83, Gpr34, Gpr84, GrblO, Gm, Gusb, H2-D1, Hcar2, Hegl, Hells, Hexb, Hmgb2, Hmoxl, Icaml, Ifi30, Ifihl, Igsf6, Illa, Illb, Illrl2, Illrn, H21r, H2rg, Il3ra, Impact, Inpp5d, Irak4, Irf7, Irf8, Itgal, Itga2, Itga6, Itgae, Itgal, Itgam, Itgax, Itgbl, Itgb3, Itgb5, Itm2a, Jcad, Jun, Kif26a, Kit, Laccl, Lairl, Lama2, Lcn2, Ldb2, Ldlrad3, Lefl, Lfng, Lgals3, Lgmn, Ligl, Liph, Lmnbl, Lrch3, Lrpl, Lrrc3, Ly6g, Ly9, Lyn, Lyvel, Lyz2, Mb21dl, Mcm2, Mcm5, Mcm6, Mctpl, Mecom, Mef2c, Meg3, Mertk, Mfsd6l, Mgatl, Mki67, Mkl2, Mmp2, Mmp9, Mpnd, Mrcl, Ms4al, Ms4a6d, Ms4a7, Msn, Mvp, Mybl2, Myrip, Nampt, Napsa, Ncaph, Ncfl, Nckapl I, Nebl, Nfib, Nlrp3, Nos3, Nostrin, Nrm, Ocln, Olfm2, Olfml3, Osm, Osmr, P2ryl2, Palmd, Pam, Pard3, Pena, PdelOa, Pde4d, Pdgfra, Pdgfrb, Pdlim5, Pdpn, Pdzm3, Pecaml, Picalm, Pik3cg, Pla2g4a, Plac8, Plcb4, Plcg2, Plekha6, Plekhgl, Plpl, Pippi, Pltp, Plxdc2, Plxna2, Podxl, Pole, Ppfibpl, Prdx5, Prickle2, Prkgl, Prosl, Psmb8, Ptk2b, Ptpn6, Ptprc, Ptprg, Ptprj, Ptprm, Pvalb, Qars, Rad23b, Rad51, Rael, Rapgef4, Rbms3, Rngtt, Rora, Rorb, Rrm2, Rsad2, Rundc3b, Salll, Sardh, Sashl, Sdf4, Sell, Serpina3n, Serpinel, Serpinfl, Siglecf, Slamf8, Slamf9, Slcl7a6, Slclal, Slc39al0, Slc40al, Slc4a4, Slc7al, Slco2bl, Slfn8, Smc3, Snx2, Sorbsl, Sorbs2, SoxlO, Sox2, Sox9, Spil, Sppl, Sptbnl, Srgn, Sst, St8sia6, Statl, Syk, Synel, Syne2, Taccl, Teadl, Tek, Tgfa, Tgfbi, Tgfbrl, Tgfbr2, Tgm2, Thsd4, Timeless, Timp2, Timp3, Tlr2, Tlr4, Tmemll9, Tmeml73, Tmtcl, Tnfrsflla, Tnfrsfla, Tnfsf1' 3b, Top2a, Treml, Trem2, Trim47, Tshz2, Tspan33, Ttlll2, Ttr, Txnrdl, Tyrobp, Uncl3b, Ung, Utrn, Vacl4, Vavl, Vcl, Vegfa, Vim, Vip, Vtn, Vwf, Was, Wwtrl, Xpol, Zbpl, Zbtb46.
Flow cytometry
Mice were anesthetized with isoflurane and intracardially perfused with PBS for 4 minutes. Brain, as well as combined nasal associated lymphoid tissue/olfactory epithelium (NALT/OE), were dissected out, placed in cold Dulbecco’s Modified Eagle’s Medium (DMEM) (Genesee, 25-500), and pressed through a cell strainer with the end of a sterile syringe. Samples were collected in 10 mL of a 30% Percoll (Cytiva, GE17-0891-01) suspension in DMEM, and underlaid with 1 mL of 70% Percoll. Spleen samples were suspended in 3 mL of Red Blood Cell Lysis Buffer (155 mM NH4C1, 10 mM KHC03, 0.1 mM EDTA) at room temperature for 10 minutes. Samples were centrifuged at 4°C at 1300 ref for 30 minutes, then immune cells were collected at the interface. All samples were then filtered through a cell strainer, washed with 2 mL DMEM, and centrifuged for 10 minutes at 800 ref, and resuspended in T cell media (RPMI with fetal bovine serum (FBS) 1: 10, penicillin/streptomycin 1: 100, MEMNEAA 1: 100, glutamine 1:100, 55 pM P- mercaptoethanol 1 : 1000) with 1X Cell Stimulation Cocktail (plus protein transport inhibitors) (cBioscicncc, 00-4975- 93). Samples were incubated at 37°C for 4 hours, washed with FACS buffer, and incubated in anti-CD16/CD32 Fc block (BD Biosciences, 553141, 1:200) for 15 minutes on ice. All subsequent steps were performed at 4°C. After washing with FACS buffer, samples were incubated in cell surface stains (a complete list of antibodies is included in table below) with either Fixable Viability Dye 780 (Invitrogen, 65086518, 1:4000) or Live/Dead Aqua (ThermoFisher, L34965, 1: 1000) in the dark for 1 hour, fixed for 30 minutes using the Intracellular Fixation & Permeabilization kit (eBioscience, 88-8824-00) according to manufacturer instructions. Intracellular stains were diluted in IX permeabilization buffer and incubated for 1 hour, then washed in permeabilization buffer and resuspended in FACS buffer. Samples were analyzed in the Columbia Stem Cell Initiative Flow Cytometry Core. Time course experiments were analyzed using a ZE-5 analyzer (Bio-Rad, Hercules, CA) equipped with 355 nm, 405 nm, 488 nm, 561 nm, and 640 nm lasers. Compensation controls used splenocytes for most cell surface markers and compensation beads (BD Biosciences, 552844 for rat hosts; ThermoFisher, 01-3333- 41 for mouse or Armenian hamster hosts) for cytokines. All other flow cytometry experiments were analyzed using a NovoCyte Penteon (Agilent, Santa Clara, CA) equipped with 349 nm, 405 nm, 488 nm, 561 nm and 637 nm lasers. Compensation controls used splenocytes for live/dead control and compensation beads for all other markers. All flow cytometry data analysis was performed using FlowJo 10.5 (FlowJo, LLC). Gates for forward and side scatter, singlets, live cells and CD4 T cells were set by eye, while all other gates were set using fluorescence minus one (FMO) controls, with a typical cutoff of <1% of the population.
Cell culture
Primary mouse brain endothelial cells (mBECs; Cell Biologies, C57-6023) were cultured as monolayers at 37°C with 5% CO2 and used to evaluate the effect of cytokines in vitro. Cells were grown to confluence on collagen IV-coated (Corning, CB-40233) dishes in endothelial cell media (Cell Biologies, M1168) supplemented with 10% FBS (Cytiva, SH30071.03) and supplements recommended by the supplier. One day prior to cytokine treatment, cells were switched to 2% FBS without growth factor supplements. Media containing either mouse IFNy (R&D Systems, 485-MI-100), mouse IL-17A (R&D Systems, 7956-ML-025), mouse GM-CSF (R&D Systems, 415- ML-010) or vehicle (PBS with Ca2+ and Mg2+) was applied to corresponding wells. Trans-endothelial electrical resistance (TEER) was measured in real time using an electric ccll-substratc impedance sensing (ECIS) instrument (Applied BioPhysics, ZThcta 96 Well Array Station) as previously described96. mBECs were plated on 96-well plates containing electrode arrays (Applied BioPhysics, 96W20idf). Cytokines or vehicle were added at a concentration of 50 ng/mL, except IL-1 (R&D Systems, 401-ML) and TNF (R&D Systems, 410-MT), which were added at lOng/mL. Resistance was monitored over 24 hours from the start of cytokine treatment. The area under the curve (AUC) was calculated for each condition and normalized to AUC of vehicle-treated cells, with values for each independent experiment compared by one-way ANOVA. Albumin transcytosis was measured in vitro by culturing mBECs on collagen IV-coated 3.0-pm PET membrane inserts for 24-well plates (Corning, 353096). Cytokines or vehicle were added at a concentration of 100 ng/mL, with 500 ng/mL lipopolysaccharide (LPS) used as a positive control. Cells were switched to endothelial cell media with 2% FBS and without phenol. Media added to wells contained bovine serum albumin at 400 pg/mL, while media added to inside of transwell inserts contained albumin conjugated to AlexaFluor 647 (ThermoFisher, A34785) at 400 pg/mL. Cells were incubated at 37°C with flow-through samples collected from bottom wells at 30 minutes, 1, 2, 4 and 6 hours). Absorbance of flow-through was quantified using the accuSkan FC plate reader (Fisher Scientific, 14-377-576). Background fluorescence was subtracted and AUC normalized to untreated for each experiment. Mixed glial cultures were generated from forebrains of P0-P2 C57BL/6J pups. After 7-14 days, microglia were obtained using a shake off protocol and cultured in serum-free media containing neurobasal solution (ThermoFisher, 211039049), insulin (Sigma, 16634, 1: 100), sodium pyruvate (Invitrogen, 11360-070, 1: 100), pen/strep (Life Technologies, 15140-122, 1:100), SATO (containing transferrin, BSA, putrescine, progesterone, and sodium selenite; 1:100), thyroxine, GlutaMAX (Life Technologies, 35050-061, 1: 100), B27 (ThermoFisher, 17504- 044, 1:50), N-acetyl cysteine (1: 1000), and mouse M-CSF (Shenandoah Biotechnology, 200-08- 10, 1: 1000). Cultures were treated with 100 ng/mL of either IFNy, IL-17A or GM-CSF for 24 hours, then washed and collected into TRIzol for RNA extraction and sequencing.
Multiplex immunoassays
Mouse olfactory bulb multiplex immunoassay. Twenty-four hours after the final GAS inoculation, pairs of OBs were dissected and flash frozen in liquid nitrogen, then stored at -80°C. Samples were pulverized on ice in cell lysis buffer (Abeam, abl52163) with protease inhibitor cocktail (ThermoFisher, 78440) and EDTA, using an electric pestle. To remove detergents that may interfere with downstream analysis, the resulting supernatant was dialyzed overnight against PBS with a 2 kDa cassette (ThermoFisher, 66205). After normalization to total protein concentration by Pierce bicinchoninic acid assay (ThermoFisher, 23225), analytes were measured by the Irving Institute for Clinical and Translational Research Biomarkers Core Laboratory using a custom mouse Luminex panel (ThermoFisher, PPX-12-MXEPUF3). Samples were run in duplicate, and standard curves were generated for each analyte. Undetectable values were replaced with half of the lower detection limit for purposes of statistical comparison.
Patient serum multiplex immunoassay: Serum protein concentrations were measured by the Irving Institute for Clinical and Translational Research Biomarkers Core Laboratory using a 45- Plex Luminex assay (Invitrogen, EPX260-26088-901). Samples were run in duplicate, and standard curves were run for each analyte. Undetectable values were replaced with half of the lower detection limit for statistical comparison.
QUANTIFICATION AND STATISTICAL ANALYSIS
Analysis of scRNAseq data
Single-cell RNA sequencing data was analyzed using Seurat package v4.0.298 in RStudio. Upon data import, genes detected in fewer than three cells, and cells with fewer than 200 genes were excluded. Cells were removed from the merged data set if they had fewer than 1 ,000 or more than 50,000 molecules detected, or greater than 20% mitochondrial reads. Data was normalized and highly variable features identified using default parameters, then scaled, followed by linear dimensional reduction using PCA. Dimensionality of the data was selected using the Elbow plot method with 50 dimensions, and cells were clustered with a resolution of 1 for OB, 0.4 for endothelial cells and microglia, and 2 for NALT/OE. Dimensionality reduction for visualization was performed with /-distributed stochastic neighbor embedding (t-SNE). The Harmony package vl.099 was used for batch correction.
Cluster identity was assigned using the following cell type markers: neurons (Map2, Snap25), astrocytes (Gfap, Aqp4). olfactory ensheathing cells (Frzb), oligodendrocytes/oligodendrocyte precursor cells (Pdgfra), endothelial cells (Cldn5. Pecaml), pericytes (Pdgfrb, Atpl3a5), fibroblasts (Coll al, Fblnl), microglia (Tmemll9, P2ryl2), macrophages (Aifl, Plac8), neutrophils (Ly 6g, Camp), dendritic cells (Xcrl, Ccr9, Cd209 ), B cells (Cdl9), CD4 T cells (Cd4), CD8 T cells (Cd8a), NK cells (Klrblc), and 78 T cells (Cdl63ll). To determine the number of differentially expressed genes for each cell type, FindMarkers was used to compare GAS to PBS conditions with minimum percent of 0.2, fold-change threshold of 0.5 and p-value cutoff of less than 0.05. Expression of ex vivo activation genes Duspl, Fos, Histlhld, Histlh2ac, Jun, Nfkbid, Nfkbiz) were added using AddModuleScore and plotted against Ccl3 and Ccl4 using FeatureScatter. Additional analysis was performed with BB Browser 3 (BioTuring) software. Signature scores were plotted in BBrowser3 using the following pathway markers: Antigen presentation (B2m, Cd74, H2-Aa, H2-Abl, H2-D1 , H2-Ebl , H2-K1 , H2-Q4, H2- Q6, H2-Q7, Tapi, Tap2), disease-associated microglia (Apoe, Axl, Cd9, Csfl, Cst7, Itgax, Lpl, Sppl, Tyrobp), homeostatic microglia (Cd33, Cst3, Cx3crl, Feris, Gpr34, 0lfml3, P2ryl2, P2ryl3, Salll, Tmeml 19), and interferon signaling (Ifi30, lfi204, lfi211, Ifitl, Ifitm3, Irfl, Irf7, Isgl5, Oasla, Stall, Stat2).
Gene set enrichment analysis (GSEA)31 was performed using curated and database derived gene lists for blood-brain barrier, response to LPS, inflammation, extracellular matrix, interferon response, antigen presentation, chemokine and cytokine signaling, endothelial cell proliferation, endothelial cell migration, disease-associated microglia, apoptosis, leukocyte chemotaxis and phagocytosis. Analysis was run using the GSEA desktop tool (Broad Institute, v4.1.0), using preranked weighted settings. Stacked violin plots were generated using scripts by Dr. Ming Tang (divingintogeneticsandgenomics.rbind.io).
Analysis of MERFISH data
MERFISH data was analyzed in RStudio using Seurat 4.1.0.9005, R 4.0.0 and custom- made scripts as previously describedlOO. Cell segmentations with volume < 50pm3 or < 10 unique transcripts were first excluded. Cell gene expression data of each cell was then normalized to that cell’s volume and the total transcript count of that cell, then scaled. To correct for global differences in total transcript counts between coverslips (each containing one GAS sample and one PBS sample), we performed ComBatlOl batch correction (sva 3.38.0). To identify individual cell types, we performed principal component analysis was performed using the entire probe library (391 transcripts) as the variable features, followed by linear dimensional reduction. Dimensionality of the data was selected using the jackstraw method with 28 dimensions, and cells were clustered with a resolution of 2.4. Dimensionality reduction was performed with /-distributed stochastic neighbor embedding (t-SNE). Clusters were manually annotated based on the spatial distribution of the cells in the tissue and the expression cell typespecific marker genes: neurons (Meg3, Gadl), astrocytes (Aqp4, Sox9), olfactory ensheathing cells (Plpl, Cldn5), oligodcndrocytcs/oligodcndrocytc precursor cells (Pdgfra, Sox 10). endothelial cells (Cldn5. Itm2a), pericytes Pdgfrb), fibroblasts (Cemip), microglia (Tmeml 19, P2ryl2), macrophages (Mrcl), neutrophils (Itgal, Mmp9), and T cells (Cd3e, Cd4, Cd8d).
Because of imperfections in cell boundary segmentation, a small fraction of cells expressed cell type markers for multiple cell types. Raw images of a subset of these cells were visually inspected using MERSCOPE Visualizer software (Vizgen, 2.L2589.1) to confirm that these clusters were due to cell segmentation errors (typified by two distinct clusters of cell-type specific transcripts within the same cell boundary). Clusters composed of these “hybrid” cells were removed from the analysis, and embedding and clustering analysis were iteratively repeated until all “hybrid” clusters were removed. The glomerular, external plexiform, and granular layers of the OB for each sample were outlined using MobileFish and coordinates recorded for point-in-polygon analysis and regional assignment of microglia. Raw counts were normalized to the PBS condition for each batch and used for gene expression analysis. Endothelial cell gene expression was compared on log2 fold change values using a one-sample t test. Microglia gene expression between the glomerular and granular layers used a ratio t test. Nearest neighbor analysis of microglial distance to T cells was calculated based the x,y coordinates of the centers of the cell segmentations using a custom python script.
Analysis of bulk RNAseq data
All sequencing and analysis of microglia cultures were performed by the Single Cell Analysis Core at the JP Sulzberger Columbia Genome Center. Following TRIzol RNA extraction, polyA libraries were prepared with the TruSeq Stranded mRNA kit (Illumina, San Diego, CA). Data processing used RTA (Illumina) for base calling, bcl2fastq2 (v2.19) for converting FASTQ and adaptor trimming, pseudoalignment from mouse transcriptomes GRCm38, kallisto (v0.44.0) for abundance quantification, and DESeq2 for differential expression analysis. Statistical analysis
Most statistical analyses were performed by GraphPad Prism 9. All tests were two-sided using a significance level a = 0.05. Outliers were identified and excluded using the ROUT method (Q = 1%). Significance was notated as ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent mean with SEM throughout. Differential expression analysis of scRNAseq data was performed using the FindMarkers function in Seurat Comparisons of more than two conditions underwent Bonfcrroni correction for multiple comparisons.
Immunohistochemistry Quantification
Quantification of microglial number: Three OB sections, corresponding to bregmas 4.5, 4.28 and 3.92, were imaged using a Zeiss Axiolmager microscope at magnification 20x for each animal. The number of Ibal+CD68+ cells (corresponding to activated microglia) in the glomerular layer was manually counted, and averaged across the three sections. Ibal+ cells (microglia) were considered CD68+ if the CD68 fluorescence occupied more than 50% of the cell surface area, as previously described18,19.
Quantification of BBB leakage: Bregma 4.28 sections were imaged using a Zeiss Axiolmager microscope at magnification lOx. Using ImageJ102, 20 small, rectangular regions of interest (ROIs) were placed around the glomerular layer of the OB, avoiding the vasculature, and average fluorescence quantified for each animal. The same process was repeated for the granular layer.
Quantification of EC marker expression: Tiled images of bregma 4.28 sections were taken at 40x using a Zeiss LSM700 confocal microscope, and maximum intensity projections were created. Using FIJI, ROIs across the whole OB were selected using Otsu thresholding on a vessel marker. Then average fluorescence intensity was measured within the ROIs. Due to neuronal expression of ltih5 in the glomerular layer, ROIs were restricted to the granular layer of the OB for quantification.
See also, Wayne et al., Distinct Thl7 effector cytokines differentially promote microglial and blood-brain barrier inflammatory responses during post-infectious encephalitis, bioRxiv, doi: https://doi.org/10.1101/2023.03.10.532135, which is incorporated by reference herein in its entirety.
Example 2 Assessment of barrier properties of the human blood-brain barrier in an in vitro 3D human NVU/BBB
We have used the Mimetas 40-chip, 3-Lane OrganoPlate microfluidic system (Figure 7A), which is suitable for endothelial cell tube formation and perfusion (models brain capillaries in size), angiogenesis and immune cell infiltration analysis. The platform allows for significant rigor and reproducibility. The low cells numbers that are required (2.500-40.000 pericytes, astrocytes, BMECs per chip) lend to feasibility. We have successfully generated 3D NVU (neurovascular unit) cultures using primary human brain microvascular endothelial cells (hBMECs) pericytes and astrocytes. This 3D system includes flow (shear stress is crucial for BMEC maturity and function) and uses a glass bottom plate with 3 microfluidic channels separated by middle-channel phase guides (Figure 7A). BMECs are seeded in the top channel, coated with Col IV/Fibronectin/Laminin, and are allowed to form an intact, perfused vessel against an extracellular matrix (Collagen I/IV, ratio = 80:20) gel (middle channel). Pericytes and astrocytes are added to the bottom channel (BMEC: astrocytes: pericytes ratio = 4:2: 1). Perfusion is established by placing the plate on the rocker platform. Intact BEC tubule formation is confirmed by dual tracer barrier integrity assays (BI), TEER measurements and key cell-type marker IF staining (Figure 7B). We have established the 3D NVU system for 10 days prior to exposure to sera from PANDAS/PANS patients and from healthy controls (described in Example 1). We assessed paracellular permeability of human BMECs in culture by applying a tracer (Dextran 70kDa) to the top chamber. We analyzed tracer diffusion from the top to the bottom chamber across the BMEC tubules in real time. We found that sera from PANDAS/PANS patients, but not controls, enhance permeability of the BBB and promote migration of tracer. This effect could be mediated by inflammatory cytokines present in PANDAS/PANS patient sera. We have observed that sera from 4 PANDAS cases were associated with a dramatic increase in barrier permeability of the in vitro human 3D NVU/BBB microfluidic system (Figures 7D-7E).
Example 3 Analysis of the Effects of IL-17A Blocking Antibody on Neuronal Function
Synaptic integrity: Olfactory neurons that provide sensory information to the olfactory bulb (OB) are particularly vulnerable to inflammatory injury after GAS infections, because their cell bodies reside within the nasal mucosa outside the CNS. Vesicular glutamate transporter 2 (vGluT2) is expressed at OSN terminals and glutamate decarboxylase 67 (GAD67) is an enzyme expressed by GABAergic local inhibitory interneurons in the OB glomeruli. To address whether reduction in vGluT2 expression in olfactory bulb (OB) glomeruli after multiple GAS infections (Dileepan et al., (2016) J. Clin. Invest. 126, 303-317) depends on IL-17A, we will perform immunofluorescence staining on brain (OB) sections for several synaptic markers (vGluT2, PSD95, GAD67) at 6-hour and 48-hour time points after the last GAS infection to quantify distribution and densities of excitatory (vGluT2+) and inhibitory (GAD67+) synapses. We will quantify as a positive signal, the areas that are 2 standard deviations brighter than the mean fluorescence intensity. We will compare the fraction of excitatory over inhibitory synapses with Prism as we have shown in the past (Dileepan et al., (2016) J. Clin. Invest. 126, 303-317; Platt et al., (2020) Proc Natl Acad Sci U S A, 117 (12) 6708-6716).
Blockade of IL-17A with antibodies are expected to restore vGluT2 expression within glomeruli by 48 hours after the final GAS infection.
Electrophysiology: To determine whether there are persistent deficits in olfactory processing, we will record odor-evoked spiking activity in populations of mitral/tufted (M/T) neurons within the OB in awake, head-fixed GAS-infected mice around the 48-hour time point after the last GAS infection (Bolding et al., (2017) Elife 6:e22630; Bolding et al., (2018), Science 361:6407). Extracellular unit recordings and analyses of odor-evoked spiking activity in the mitral/tufted (M/T) neurons within the OB will be performed in awake, head-fixed mice as published (Platt et al., (2020) Proc Natl Acad Sci U S A, 117 (12) 6708-6716). After four GAS infections, mice will be surgically fitted with a head plate to allow for awake, head-fixed recording from the OB. Recordings will be performed in a ten-channel olfactometer 48 hours after the final infection. A 32-site polytrode acute probe will be placed into the posterior OB near the ventromedial M/T neuronal layer. Signals will be collected through an A32-OM32 adaptor connected to a Cereplex digital head stage. Six monomolecular odors will be used: methyl tiglate, y-terpinene, 2-hexanone, isoamyl acetate, ethyl butyrate and valeraldehyde. These odors will be presented for one second using a custom flow-dilution olfactometer at 0.04%, 0.2% and 1% (vol/vol). Fifteen trials per odor concentration will be presented at 10-second intervals. Individual units will be automatically recorded using Spyking-Circus (https://github.com/spyking-circus) and quantified as described in the paper (Platt et al., (2020) Proc Natl Acad Sci U S A, 117 (12) 6708-6716).
Individual M/T neurons are expected to be either activated or suppressed by different monomolecular odorants as they relay the information to various regions of the olfactory cortex. Single M/T neurons are expected to show robust odor responses in PBS control mice. In contrast, GAS-infected mice will show a striking absence of odor responses in M/T neurons, whereas GAS- infected mice treated with IL- 17 A blocking antibody may show either a partial or complete rescue in single M/T neuronal responses, indicating that physiological deficits are partially regulated by an IL-17A-dependent mechanism. Regarding the M/T neuronal responses over a 25-fold range of odor concentration from 0.04% to 1% v/v odor dilution, PBS control mice will show a systematic, conccntration-dcpcndcnt increase in the fraction of responsive M/T neurons as they sense more odor. We anticipate that neuronal responses across all odor concentrations will be abolished (equivalent to mineral oil controls) in GAS-infected mice with no change in baseline firing rates among all groups. However, these responses may be rescued by treatment with an IL-17A blocking antibody.
Animal behavior: The transient loss of vGluT2 expression in OB glomeruli raises the question whether olfactory function is impaired persistently in GAS-infected mice. We will use a habituation-dishabituation behavioral paradigm, which leverages a mouse’s curiosity for new olfactory stimuli. Mice are presented with an unscented Q-tip, which they first investigate by sniffing but then rapidly lose interest at subsequent presentations of the same stimulus (habituation). However, if the Q-tip is impregnated with an odor, mice initially investigate the novel odor (dishabituation) indicating odor detection, but they rapidly habituate to subsequent presentations of the same odor. Mice will re-investigate if a different odor is presented, measuring odor discrimination. Each odorant (almond extract, vanilla extract, ethyl butyrate, and 2- phenylethanol) will be presented three times, with water presented first as described (Platt et al., (2020) Proc Natl Acad Sci U S A, 117 (12) 6708-6716).
We will use the open field ( OF) test to examine spontaneous motor activity. Mice will be placed in the center of an arena and tracked with AnyMaze to quantify time in central versus peripheral areas (anxiety-like behavior), total distance and speed (motor activity).
Normal mice will actively investigate an unscented Q-tip, but then rapidly lose interest (Root et al., (2014) Nature 515, 269-273) and perform normally the other behavior tasks. In contrast, GAS-infected mice will show both impaired odor detection and odor discrimination at 6 h after the last infection, but show normal spontaneous motor activity in the OF test. Finally, GAS mice treated with an IL-17A blocking antibody will show restoration of odor detection.
SEQUENCES
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000072_0002
Figure imgf000073_0001
Gene Target Primer sequence Products Size
Figure imgf000073_0002
The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there arc suitable alternatives to the depicted examples of materials, configurations, constructions and dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety. Variations, modifications and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. While certain embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention. The matter set forth in the foregoing description is offered by way of illustration only and not as a limitation.
1

Claims

Claims
1. A method of treating Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in a subject in need thereof, the method comprising administering to the subject an inhibitor of IL- 17 A or its receptor.
2. The method of claim 1, wherein the PANS is Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).
3. The method of claim 2, wherein the PANDAS is associated with Group A Streptococcus infection.
4. The method of any preceding claim, wherein the PANS is associated with an ongoing or prior infection.
5. The method of claim 1, wherein the inhibitor comprises an antibody or a fragment thereof that binds to IL- 17 A or its receptor.
6. The method of claim 5, wherein the antibody comprises secukinumab (SEC), ixekizumab (IXE), brodalumab (BROD), bimekizumab, or combinations thereof.
7. The method of claim 1, wherein the inhibitor comprises a small molecule.
8. The method of any preceding claim, wherein the inhibitor is administered by inhalation, intranasally, intra thee ally, orally, intravenously, subcutaneously or intramuscularly.
9. The method of any preceding claim, wherein the inhibitor is administered using an intranasal spray device, an atomizer, a nebulizer, a metered dose inhaler (MDI), a pressurized dose inhaler, an insufflator, an intranasal inhaler, a nasal spray bottle, a unit dose container, a pump, a dropper, a squeeze bottle, or a bi-directional device.
10. A method for detecting or diagnosing Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in a subject or assessing the subject’s risk of developing PANS, the method comprising: a) obtaining a sample from the subject; b) determining levels of one or more cytokines, chemokines and/or growth factors in the sample, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; c) comparing the levels obtained in step (b) with the levels of the one or more cytokines, chemokines and/or growth factors in a control sample; and d) diagnosing that the subject has PANS or an increased risk of developing PANS, if the level of at least one cytokine, chemokine and/or growth factor obtained in step (b) increases by at least 10% compared to its level in the control sample.
11. A method of treating a subject with Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) or an increased risk of developing PANS, the method comprising: a) obtaining or having obtained a sample from the subject; b) determining or having determined levels of one or more cytokines, chemokines and/or growth factors in the sample, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; c) comparing or having compared the levels obtained in step (b) with the levels of the one or more cytokines, chemokines and/or growth factors in a control sample; and d) treating the subject for PANS or an increased risk of developing PANS, if the level of at least one cytokine, chemokine and/or growth factor obtained in step (b) increases by at least 10% compared to its level in the control sample.
12. The method of claims 10 or 11, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, and TNFa.
13. The method of any of claims 10-12, wherein the PANS is Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).
14. The method of claim 13, wherein the PANDAS is associated with Group A Streptococcus infection.
15. The method of any of claims 10-14, wherein the increase in the level of the at least one cytokine, chemokine and/or growth factor is at least 30%.
16. The method of any of claims 10-15, wherein the increase in the level of the at least one cytokine, chemokine and/or growth factor is at least 50%.
17. The method of any of claims 10-16, wherein the increase in the level of the at least one cytokine, chemokine and/or growth factor is at least 1-fold.
18. The method of any of claims 10-17, wherein the increase in the level of the at least one cytokine, chemokine and/or growth factor is at least 2-fold.
19. The method of any of claims 10-18, wherein the sample is a plasma, serum or blood sample.
20. The method of any of claims 10-19, wherein the control sample is from a healthy subject or a plurality of healthy subjects.
21. The method of any of claims 10-19, wherein the control sample is from a subject without PANS or a plurality of subjects without PANS.
22. The method of any of the preceding claims, wherein the subject is human.
23. The method of any of the preceding claims, wherein the subject’s existing PANS treatment regimen is modified or maintained.
24. The method of any of claims 10-23, wherein the level of the one or more cytokines, chemokines and/or growth factors is determined by mass spectrometry (MS).
25. The method of any of claims 10-23, wherein the level of the one or more cytokines, chemokines and/or growth factors is determined by enzyme-linked immunosorbent assay (ELISA).
26. A kit comprising: a) antibodies or fragments thereof that specifically bind to one or more cytokines, chemokines and/or growth factors in a blood, plasma or serum sample from a subject, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM-CSF, TNFa, CXCL8, CXCL12, EGF, HGF, IL-IRA, IL-6, IL-7, IL-12a/B, IL-15, IL-22, IL-23, KITLG, LIF, PDGFB, PGF, TNF, VEGFA, and VEGFD; and b) instructions for measuring the one or more cytokines, chemokines and/or growth factors for diagnosing Pediatric Acute -onset Neuropsychiatric Syndrome (PANS) in the subject or assessing the subject’s risk of developing PANS.
27. The kit of claim 26, wherein the one or more cytokines, chemokines and/or growth factors are selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL10, GM- CSF, and TNFa.
28. The kit of claims 26 or 27, wherein the PANS is Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS).
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