WO2022032592A1 - Interleukine-17d et cd93 utilisés en tant que nouvelle paire cytokine-récepteur dans le système immunitaire - Google Patents

Interleukine-17d et cd93 utilisés en tant que nouvelle paire cytokine-récepteur dans le système immunitaire Download PDF

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WO2022032592A1
WO2022032592A1 PCT/CN2020/109014 CN2020109014W WO2022032592A1 WO 2022032592 A1 WO2022032592 A1 WO 2022032592A1 CN 2020109014 W CN2020109014 W CN 2020109014W WO 2022032592 A1 WO2022032592 A1 WO 2022032592A1
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medicament
ilc3
cells
activity
dysregulation
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Chen Dong
Jinling Huang
Xiaohu Wang
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • 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
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases

Definitions

  • the present invention relates to the field of medicinal technology, in particular, to the treatment or prevention of intestinal homeostasis related diseases in a subject.
  • IL-17A-F structurally related members
  • IL-17A and IL-17F two most homologous cytokines produced by lymphocytes including Th17, ILC3 and ⁇ 17 cells, signal through IL-17RA/IL-17RC and IL-17RA/IL-17RD complexes to promote the development of autoimmune diseases while playing important roles in host defense against bacterial and fungal infections.
  • Other members in this family, including IL-17E, IL-17B, and IL-17C are produced by epithelial cells and bind to receptor complexes on epithelial cells to regulate tissue inflammation, and also mediate differentiation and function of specific lymphocyte subsets.
  • IL-17 family members emerge as important mediators in mucosal immunity, and serve as important drug targets in inflammatory disases, IL-17D is least studied in this family. Although it was initially shown to promote IL-6, IL-8, and GM-CSF production in endothelial cells, and recently, studies showed that overexpression of IL-17D in poorly immunogenic cancer cells mediate tumor rejection.
  • ILCs Innate lymphoid cells
  • ILCs represent a family of heterogeneous lymphocytes that lack variable antigen receptors yet produce cytokines similar to CD4+ helper T cell subsets. Based on the expression of cytokines and transcription factors, ILCs are divided into three main groups: IFN- ⁇ -producing ILC1s that depend on T-bet; IL-5-and IL-13-producing ILC2s that express Gata-3; IL-17-and/or IL-22-producing ILC3s that require ROR ⁇ t.
  • ILCs typically reside at mucosal tissues and promptly produce effector cytokines in response to environmental stimuli, thus playing critical roles in host defense against pathogens, regulation of adaptive immune responses, deveopment of lymphoid architectures, as well as tissue remodeling.
  • ILC3s further subdividing into natural cytotoxicity receptor (NCR) -ILC3s, NCR+ ILC3s and LTi cells, were the first characterized ILC subset in tonsils in human and intestinal lamina intestinal in both mice and human.
  • NCR natural cytotoxicity receptor
  • ILC3 cells were the first characterized ILC subset in tonsils in human and intestinal lamina intestinal in both mice and human.
  • Previous studies have established the importance of ILC3 cells in intestinal homeostasis at the steady state and in models of intestinal inflammation; they produce IL-22 to trigger epithelial cells to produce antimicrobial peptides, such as RegIII ⁇ and RegIIII ⁇ , offering innate immunity to infection.
  • Il22-/-mice also exhibited accelerated tumorigenesis than wild-type mice in a colon cancer model.
  • ILC3 cells arise from hematopoietic progenitors in the bone marrow and ILC3 cells in peripheral tissues are functionally mature in expressing transcription factors ROR ⁇ t and AhR, which allow their rapid production of IL-22 upon IL-23 stimulation.
  • ILC3s act as “pre-primed” effector cells and respond rapidly to cytokine IL-23 to produce IL-22.
  • the initial lineage development of ILC3 precursor cells in the bone marrow has been characterized, how they are regulated in the peripheral tissues for their effector function and IL-22 production in peripheral tissues remains poorly understood.
  • IL-17D-CD93 axis thus exert a protective role at the barrier surfaces against intestinal inflammation.
  • reagent in preparing medicament wherein the reagent is used for regulating IL-17D-CD93 axis, and the medicament is used for treating or prevention of inflammatory or immune diseases, including dysregulation of intestinal homeostasis related diseases.
  • the inventors have found that IL-17D-CD93 axis exert a protective role during intestinal homeostasis, therefore, the medicament prepared with the reagent used for regulating IL-17D-CD93 axis can be used for regulation of intestinal homeostasis and treating or prevention of dysregulation of intestinal homeostasis related diseases effectively.
  • the dysregulation of intestinal homeostasis related diseases comprise at least one of the following: microbiota dysbiosis, colitis, colon cancer, inflammatory bowel diseases (IBD) , pathogen infection. crohn’s disease and ulcerative colitis .
  • IBD inflammatory bowel diseases
  • the reagent is used for activating or upregulating IL-17D and/or CD93 or enhancing the binding of IL-17D and CD93, and the medicament is used for treating or prevention of microbiota dysbiosis, colitis, colon cancer, inflammatory bowel diseases or pathogen infection.
  • the reagent comprises at least one of the following: IL-17D, Nrf2 agonists, nucleic acid used for overexpression of IL-17D and/or CD93.
  • nucleic acid used for overexpression of IL-17D and/or CD93 has a nucleotide sequence shown as any one of SEQ ID NO: 1 and 2.
  • the reagent is used for suppressing or downregulating IL-17D and/or CD93 or preventing the binding of IL-17D and CD93, and the medicament is used for treating or prevention of crohn’s disease and ulcerative colitis.
  • the reagent comprises at least one of the following: nucleic acid used for downregulating IL-17D and/or CD93, MMRN2 siRNA and MMRN2 inhibitor, wherein the MMRN2 siRNA or MMRN2 inhibitor can be used for downregulating CD93.
  • nucleic acid used for downregulating IL-17D and/or CD93 has a nucleotide sequence shown as any one of SEQ ID NO: 3 ⁇ 5.
  • gRNA sequence can be as following:
  • CD93-gRNA GCCCCAATGGGCTGTATAGC (SEQ ID NO: 3) .
  • sequence can be as following:
  • sequence can be as following:
  • ILC3 function as a double-edged sword in gut. ILC3 are responsible for intestinal homeostasis through moderate generation of IL-22 in the physiological state. However, ILC3 also contribute to the progression and aggravation of IBD (such as crohn’s disease and ulcerative colitis) while IL-22 is overexpressed by dysregulation of ILC3 in the pathological state.
  • IL-17D-CD93 axis in screening monoclonal antibodies or other proteins/peptides or small molecules that preventing the binding of IL-17D and CD93.
  • the method comprises: 1) using a chemical or biological activator to enhance the activity of IL-17D and/or CD93; 2) using a chemical or biological regent to enhance the amount of IL-17D and/or CD93; 3) using a chemical or biological regent to enhancing the binding of IL-17D and CD93.
  • the method comprises: 1) using a chemical or biological inhibitor to suppress the activity of IL-17D and/or CD93; 2) using a chemical or biological regent to reduce the amount of IL-17D and/or CD93; 3) using a chemical or biological regent to prevent the binding of IL-17D and CD93.
  • the method comprises: administrating a promoter for IL-17D expression and secretion by colonic epithelial cells; administrating an activator for enhancing the activity of IL-17D; administrating a promoter for CD93 expressing on ILC3 cells; administrating an activator for enhancing the activity of CD93; or administrating an enhancer for enhancing the binding of IL-17D and CD93.
  • the dysregulation of intestinal homeostasis related diseases comprise at least one of the following: microbiota dysbiosis, colitis, colon cancer, inflammatory bowel diseases, pathogen infection.
  • the method comprises: administrating an inhibitor for IL-17D expressing and secretion by colonic epithelial cells; administrating an inhibitor for suppressing the activity of IL-17D; administrating an inhibitor for CD93 expressing on ILC3 cells; administrating an inhibitor for suppressing the activity CD93; or administrating an inhibitor for preventing the binding of IL-17D and CD93.
  • the dysregulation of intestinal homeostasis related diseases comprise at least one of the following: crohn’s disease and ulcerative colitis.
  • the method comprises: contacting a candidate medicament with colonic epithelial cells, the elevation of the expression of IL-17D or the upregulating of IL-17D activity or the enhancing of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.
  • the method comprises: contacting a candidate medicament with ILC3 cells, the elevation of the expression of CD93 or the upregulating of CD93 activity or the enhancing of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.
  • the method comprises: contacting a candidate medicament with colonic epithelial cells, the downregulating of the expression of IL-17D or the IL-17D activity or the inhibition of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.
  • the method comprises: contacting a candidate medicament with ILC3 cells, the downregulating of the expression of CD93 or the CD93 activity or the weakening of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.
  • the methods described above can screen medicament used for treatment or prevention of homeostasis related diseases effectively.
  • Fig. 1 shows IL-17D protects against DSS-induced colitis
  • Fig. 2 shows IL-17D in non-hematopoietic cells is required for protection against colitis
  • Fig. 3 shows IL-17D administration partially rescued the disease symptoms of colitis in Il17d-/-mice
  • (A) Relative mRNA expression of indicated genes in colons of WT and Il17d-/-mice at day 9 of colitis (n 4) .
  • Fig. 4 shows IL-17D is essential for IL-22 production by ILC3,
  • LPLs Small intestinal lamina limbal lymphocytes
  • Fig. 5 shows Il17d-deficient mice show dysregulated fecal microbiota
  • Fig. 6 shows IL-17D binds to CD93
  • A Cultured RAW 264.7 cells were cross-linked with 30 ⁇ g/ml human IgG or mouse IL-17D-human IgG1 Fc fusion protein, followed by Western blot analysis probed with HRP conjugated anti-human IgG.
  • B to E Flow cytometry analysis of IL-17A or IL-17D binding to 293T cells transfected with empty, IL-17RA, or CD93 expression vector.
  • 293T cells were subsequently incubated 30 min with His-tagged proteins (B) , human IgG fusion proteins (C) , biotin labelled proteins (D) , or commercial proteins bought from R&D systems (E) , followed by staining with anti-His (B) , anti-human IgG (C) , streptavidin (E) , anti-IL-17A or anti-IL-17D antibodies (E) .
  • CTLD C-type lectin like domain
  • X domain of unknown structural function
  • EGF-like EGF-like repeats
  • Mucin mucin-like domain
  • TM transmembrane domain.
  • Fig. 7 shows CD93 plays a protective role in DSS-induced colitis and colitis-associated colon cancer (CAC) .
  • (A) Flow cytometric staining of CD93 expression in ILC3s isolated from the small intestine lamina propia in WT mice stimulated with IL-23 (Gated on live CD45+ CD3-CD90.2+ ROR ⁇ t+cells) .
  • (B) Flow cytometry staining of IL-17D-hIg binding to ILC3s isolated from the SI LPL of WT and Cd93-/-mice. Left: CD93 staining; Middle: hIg binding control; Right: IL-17D-hIg binding.
  • Data are a representative of three (A to C) or two (D, G, H) independent experiments, or pooled from two experiments (E and F) . Data are shown as mean ⁇ s.e.m. *p ⁇ 0.05, **p ⁇ 0.01, ****p ⁇ 0.0001 by unpaired t test (C to F, H) , two-way ANOVA (G) ; and
  • Fig. 8 shows CD93 regulates ILC3 function
  • (B, D) Analysis of the frequency of wild-type or CD93-knockout ILC3s and IL-22-producing ILC3s among total ILCs in the colon (B) and small intestine (D) of mice (n 5) .
  • IL-17D-CD93 axis refers to IL-17D in the intestine identifies its receptor, CD93.
  • IL-17D-CD93 axis is in the selective regulation of ILC3s function during intestinal homeostasis.
  • "treat” , “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In other embodiments, “treat” , “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom) , physiologically, (e.g., stabilization of a physical parameter) , or both. In other embodiment, “treat” , “treating” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.
  • the term “therapeutically effective amount” or “therapeutically effective dosage” refers to the amount of the regent of the invention which is capable of eliciting biological or medical response (Such as alleviating symptoms, slowing or delaying the development of the disease, or preventing diseases, etc. ) of an individual.
  • the term “therapeutically effective amount” refers to, when the regent of the present invention is administered to a subject.
  • the term “therapeutically effective amount” refers to, when administering the cell, or organ, or non-cellular biological material, or medium, an effective amount of the regent of the present invention, which can be at least used for regulating IL-17D-CD93 axis.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the reagent used for regulating IL-17D-CD93 axis.
  • the pharmaceutical composition can further comprise pharmaceutically acceptable excipient, carrier, adjuvant, solvent and a combination thereof.
  • the present invention provides a method of treating, preventing or ameliorating a disease or disorder, comprising administrating a safe and effective amount of a combination of drugs containing regents and one or more therapeutic active agents.
  • the combination of drugs comprises one or more additional drugs for treatment of dysregulation of intestinal homeostasis related disease.
  • Other drugs for treatment autoimmune diseases are not limited to: Aminosalicylates (5-ASA) , Antibiotics, Biologics, Corticosteroids, Immunomodulators, Small molecules.
  • the amount of the regent of the pharmaceutical composition disclosed herein refers to an amount which can be effectively regulating IL-17D-CD93 axis.
  • the dosage of active ingredient in the compositions of this invention may be varied, however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.
  • the active ingredient may be administered to patients (animals or human) in need of such treatment in dosage that will provide optimal pharmaceutical efficacy.
  • the selected dosage upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment.
  • the dosage will vary from patient to patient depending upon the nature and severity of disease, the patient's weight, special diet then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize.
  • the weight ratio of the regent of the present invention to the second active ingredient may be varied and depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used.
  • the weight ratio of the regent of the present invention to the other agent will generally range from about 1000: 1 to about 1: 1000, such as about 200: 1 to 1: 200.
  • Combinations of a regent of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
  • “Pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled, such that interactions which would substantially reduce the efficacy of the regent of the invention when administered to a patient and would result in pharmaceutically unacceptable compositions are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.
  • Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen.
  • suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition.
  • certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms.
  • Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms.
  • Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting the regent of the present invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body.
  • Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.
  • Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweetners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.
  • compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company) .
  • another aspect of the present invention is related to a method for preparing a pharmaceutical composition.
  • the pharmaceutical composition contains the regent disclosed herein and pharmaceutically acceptable excipient, carrier, adjuvant, vehicle or a combination thereof, the method comprises mixing various ingredients.
  • the pharmaceutical composition containing the regent disclosed herein can be prepared at for example environment temperature and under barometric pressure.
  • compositions provided herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.
  • compositions provided herein may be administered parenterally by injection, infusion, or implantation, for local or systemic administration.
  • Parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.
  • compositions provided herein may be formulated for single or multiple dosage administration.
  • the single dosage formulations are packaged in an ampoule, a vial, or a syringe.
  • the multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.
  • the pharmaceutical compositions are provided as ready-to-use sterile solution.
  • the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle prior to use.
  • the pharmaceutical compositions are provided as ready-to-use sterile suspensions.
  • the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use.
  • the pharmaceutical compositions are provided as ready-to-use sterile emulsions.
  • the regents of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agents.
  • the regents of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition .
  • ILC3 interleukin 17D
  • ILC3 interleukin 17D
  • ILC3 interleukin 17D
  • Il17d deficiency impaired IL-22 production by ILC3 cells, which resulted in microbiota dysbiosis, worsened severity in acute colitis and increased development of colon cancer.
  • IL-17D was found to interact with CD93, a glycoprotein expressed on ILC3 cells.
  • Mice lacking Cd93 also showed aggravated colonic inflammation in experimental colitis and CD93 expression in ILC3 cells was required for their function.
  • IL-17D-CD93 axis important in tissue-specific regulation of ILC3 cell function required for intestinal homeostasis.
  • mice used in this study were housed under specific-pathogen-free (SPF) condition and on the C57BL/6 background.
  • C57BL/6 and GF mice were obtained from animal facility of Tsinghua University.
  • Il17d-/-mice was purchased from MMRRC facility (032380-UCD-SPERM) as cryo-preserved spermatozoa.
  • Cd93-/-mice were generated at Tsinghua University as described below.
  • the pcDNA3.1-Cas9 plasmid was digested with XbaI to synthesis Cas9 mRNA using mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies) .
  • the pUC19-sgRNA plasmid encoding gRNA sequence was in vitro transcribed using MEGAshortscript T7 Transcription Kit (Life Technologies) .
  • Cas9 mRNA and gRNA were subsequently purified with MEGAclearTM Transctiption Clean-Up Kit (Life Technologies) and resuspended in RNase-free water.
  • the mixture of Cas9 mRNA and gRNA was injected into 0.5-day zygotes of C57BL/6 mice. CRISPR/Cas9 knockout founder mice were crossed with WT mice to generate F1 generation mice.
  • mice Age-matched male mice were given drinking water containing 2.5%DSS for 5 days, and then followed by regular water for 3-4 days. The DSS water was replaced daily.
  • mice were injected i.p. with 0.5 ⁇ g recombinant mouse IL-17D (R&D) or IL-22 (R&D) everyday starting from Day 3. Body weight was monitored throughout the experiments.
  • WT and Il17d-/-mice were injected intraperitoneally with AOM (10mg/kg) . 5 days later, 2.5%DSS was added to the drinking water for 5 consecutive days, followed by regular water for 14 days. This cycle was repeated three times.
  • Mice were sacrificed for analysis on day 80 of the experiments. Mice were orally gavaged with 2 ⁇ 109 CFU C. rodentium (ATCC 51459) and weighed daily after infection. Bacterial load was assessed by plating fecal homogenates overnight on MacConkey.
  • the intestine tissues were harvested from indicated mice, longitudinally opened and cut into small pieces.
  • the LPMCs were isolated as described previously. Briefly, the tissue pieces were incubated in pre-digestion media (RPMI 1640 containing 20mM HEPES, 5mM EDTA, 1%penicillin and streptomycin, 1mM DTT) for 30min at 37 °C. After washing, the pieces were incubated in digestion buffer (RPMI 1640 supplemented with 1%penicillin and streptomycin, 20mM HEPES, 0.5mg/ml Collagenase D, 1mg/ml Dispase, 0.5mg/ml DNase) for 30min at 37 °C.
  • pre-digestion media RPMI 1640 containing 20mM HEPES, 5mM EDTA, 1%penicillin and streptomycin, 1mM DTT
  • digestion buffer RPMI 1640 supplemented with 1%penicillin and streptomycin, 20mM HEPES, 0.5m
  • leukocytes were purified on Percoll density-gradient separation. Purified LPMCs were washed and stimulated with 5ng/ml IL-23 or 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of GolgiPlug (BD) for 4 hr prior to staining for flow cytometry analysis by LSR Fortessa (BD) .
  • BD GolgiPlug
  • Flurochrome-conjugated antibodies against CD45 (clone 30-F11) , CD3 (clone 500A2) , CD90.2 (clone 53-2.1) , Nkp46 (clone 29A1.4) , CD4 (clone RM4-5) , ROR ⁇ t (clone Q31-378) , IL-22 (clone IL22JOP) , CD45.1 (clone A20) , CD45.2 (clone 104) , CD93 (AA4.1) , TCR ⁇ (clone GL3) , IFN- ⁇ (clone XMG1.2) , IL-17A (clone eBio17B7) , Ki67 (BD, catalog no.
  • Il17d 5’ -TGAGTGCATAATTGTAGTGCTCAG-3’ and 5’ -ACTCAGGGACAGGGCACA-3’ ;
  • Il22 5’ -CATGCAGGAGGTGGTACCTT-3’ and 5’ -CAGACGCAAGCATTTCTCAG-3’ ;
  • Reg3b 5’ -CTCTCCTGCCTGATGCTCTT-3’ and 5’ -GTAGGAGCCATAAGCCTGGG-3’ ;
  • Reg3g 5’ -TCAGGTGCAAGGTGAAGTTG-3’ and 5’ -GGCCACTGTTACCACTGCTT-3’ ;
  • Il6 5’-GATGGATGCTACCAAACTGGAT-3’ and 5’ -CCAGGTAGCTATGGTACTCCAGA-3’ ;
  • Il1b 5’-TGTAATGAAAGACGGCACACC-3’ and 5’ -TCTTCTTTGGGTATTGCTTGG-3’ .
  • Bone marrow cells from WT and Il17d-/-donor mice were collected and flushed with 1x PBS.
  • Cell suspension containing 3 ⁇ 10 6 donor BM cells was intravenously injected into lethally irradiated WT and Il17d-/-recipient mice (1050 rads) .
  • To generate mixed bone marrow chimeras bone marrow cells were isolated from CD45.1/2 Cd93+/+ and CD45.2 Cd93-/-mice.
  • the expression vectors of IL-17D-hIg fusion protein or his-tagged IL-17D were first generated by cloning the Il17d gene sequence (Genbank accession number NM_145837.3) into pVRC vector and transfected into 293F cells.
  • the secreted IL-17D-hIg and IL-17D-his protein were purified with a protein A column and Metal Affinity Resins, respectively.
  • 293T cells were transfected with indicated vectors,
  • the IL-17D fusion proteins or recombinant proteins bought from R&D System were added to the transfected 293T cells for 30 min, followed by staining with indicated antibodies conjugated with proper flurochrome.
  • RAW264.7 cells plated on 15 cm dishes were incubated with 30 ⁇ g/ml IL-17D-hIg fusion protein or control human IgG for 30 min.
  • Cells were cross-linked by 0.1%formaldehyde and then stopped with 0.2M Glycine, and subsequently lysed with 2 ml of buffer containing 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10%glycerol, 0.5%NP-40, and 1 ⁇ protease inhibitor cocktail.
  • the cell lysates were precipitated with 250 ⁇ l of Protein A beads, washed with NETN buffer (20 mM Tris-HCl pH 7.8, 100 mM NaCl, 1 mM EDTA, 0.5%NP-40) , and eluted with 50 ⁇ l of non-5 reducing SDS-PAGE loading buffer. The samples were then subjected to 8%SDS-PAGE followed by western blot analysis probed with HRP conjugated anti-human IgG or coomassie blue staining. The indicated gels bands were cut and subjected to mass spectrometry by Centre for Protein Technology of Tsinghua University.
  • CD93-ECD-Fc (R&D, catalog no. 1696-CD-050) was immobilized to a CM5 sensorchip (GE Healthcare) to a level of ⁇ 750 response units (RUs) using a Biacore T200 (GE Healthcare) and a running buffer composed of 10 mM HEPES pH 7.2, 150 mM NaCl and 0.05%Tween 20. Serial dilutions of mIL-17D (R&D, catalog no. 2274-ML-025/CF) was flowed through with a concentration ranging from 150 to 9.375 nM. The resulting data were fit to a 1: 1 binding model using Biacore Evaluation Software (GE Healthcare) .
  • CD45 low CD3-CD90.2 high ILC3s were sorted from small intestine LPLs of indicated mice.
  • Total RNAs were isolated with TRIzol reagent (Invitrogen) and the RNA-seq library was built by the Beijing Genomics Institute. Sequence reads were obtained by BGISEQ-500 and the clean reads were mapped to mouse genome. Gene expression was indicated by RPKM (Reads Per Kilobases per Million reads) , and genes with at least 1.5 fold changes were used to select as differentially expressed genes.
  • Il17d expression in epithelial cells is required to control colonic inflammation
  • IL-17D we first assessed its mRNA expression in multiple mouse tissues by quantitative RT-PCR. High levels of Il17d expression was observed in the colon as well as lung, but not in thymus or bone mrrow of adult mice (Fig. 1A-B) , while small intestine and colon of newborn mice also expressed substantial levels of Il17d (Fig. 1C) . Il17d expression in intestine exhibited no difference between SPF and germ-free animals (Fig. 1D) , suggesting that it was not dependent on microbiota. Interestingly, the expression of Il17d was higher in colon than in small intestine (Fig. 1B-D) .
  • Il17d-/-mice did not exhibit any significant difference in their T cells, dendritic cells or macrophages from thymus, spleen, small intestine and colon compared to WT mice, and appeared to have normal mesenteric lymph node and Peyer’s patches (not shown) .
  • WT wild-type
  • DSS 2.5%dextran sulfate sodium
  • Il17d-deficient animals exhibited further decrease in body weights between days 6 and 9 (Fig. 1E) , with increased colon shortening than WT mice when they were sacrificed on day 9 (Fig. 1F) .
  • Fig. 1G-H there was stronger inflammation and destruction of intestinal epithelium structure in Il17d-/-mice (Fig. 1G-H) .
  • Fig. 1I we performed qPCR to analyze the expression of cytokine mRNA in colon tissue after DSS administration and found that ablation of Il17d resulted in significantly increased expression in pro-inflammatory cytokines IL-6 and IL-1 ⁇ (Fig. 1I) .
  • IL-17D plays an important protective role in DSS colitis model.
  • IL-17D is required for IL-22 production by ILC3s
  • IL-17A-and IFN- ⁇ -expressing T cells are important regulators in inflammatory diseases.
  • LPLs intestinal lamina limbal leukocytes
  • IFN- ⁇ +, IL-17A+ and IL-22+cells among CD4+ T and ⁇ T populations were similar in WT and Il17d-deficient mice during colitis, suggesting that the worsened colitis in Il17d-/-15 mice was unlikely to be related to pathogenic T cells.
  • IL-22 treatment improved severe colitis in DSS-treated Il17d-/-mice (Fig. 3H-I) .
  • IL-22 may be an important factor downstream of IL-17D signaling to protect mice against DSS-induce colitis.
  • ROR ⁇ t+ILC3 cells an important source of IL-22, are critical mediators in host defense against pathogens and in tissue repair, especially at the intestinal barrier.
  • Mice lacking IL-22 or ROR ⁇ t + ILC3s exhibited increased susceptibility to DSS-induced colitis similar to mice deficient in Il17d.
  • An IL-17D-hIg fusion protein was generated and was found to specifically bind to ILC3 but not T cells or CD3-CD90.2-cells in small intestine LPLs, suggesting that IL-17D might regulate ILC3s in the gut.
  • ILC3 cells in LPLs from small intestine and colon were analyzed.
  • ILC3-associated genes including Cd93, Ahr, Foxs1, Il17f, Il1r2 and Il22, were down-regulated, whereas some genes associated with ILC1 or ILC2 cells, including klrd1, Ifng, Gata3, Il4, Il17rb and Ccr8, were up-regulated in ILC3 cells from Il17d-deficient mice (Fig. 4H) .
  • IL-17D is required for reinforcing ILC3 transcriptional programs in the gut.
  • IL-22-producing ILC3 cells are critical for the maintenance of microbiota homeostasis, and microbiota dysbiosis has been reported to contribute to the pathogenesis of colitis.
  • 16S rRNA sequencing in feces from WT and Il17d-deficient mice.
  • fecal from Il17d-deficient mice showed a remarkable decrease of bacterial diversity (Fig. 5A-C) and changes in bacterial composition (Fig. 5D) , when compared to WT mice, consistent with reports on mice deficient in IL-22.
  • Verrucomicrobia and Proteobacteria two bacteria strains increased after DSS treatment shown in a previous study, were the most significantly upregulated phyla in Il17d-deficient mice at steady state and during colitis, respectively (Fig. 5E) .
  • Enterobacteriaceae which are susceptible to the antimicrobial Verrucomicrobiaceae and Enterobacteriaceae, belonging to the Verrucomicrobia and Proteobacteria phyla separately, were the most increased families in Il17d-deficient mice at steady state and colitis, respectively (Fig. 5F) . Therefore, our data indicate that Il17d deficiency results in microbiota dysbiosis, consistent with defective ILC3 function in these mice proteins and can be suppressed by IL-22, mediate DSS-induced colitis.
  • IL-17D binds to CD93
  • CD93 also known as C1qRp, is a cell-surface glycoprotein of 645 amino acids with its extracellular region composed of a C-type lectin-like domain (CTLD) , five epidermal growth factor (EGF) -like repeats and a mucin-like domain.
  • CTL C-type lectin-like domain
  • EGF epidermal growth factor
  • CD93 was originally proposed as a surface receptor involved in C1q-mediated phagocytosis.
  • contradictory results were reported subsequently showing no interaction between CD93 with C1q. Asking which part of CD93 was required for the binding to IL-17D, we generated several CD93 deletional mutants (Fig. 6F) , and transfected in 293T cells to test the binding of IL-17D.
  • CD93 protects against colon inflammation through IL-22 expression
  • CD93 was highly expressed by ROR ⁇ +ILCs from SI, but not from skin or lung (Fig. S7B) .
  • CD93 When gating on CD45+LPLs, CD93 were mainly expressed by CD3-and CD90.2+ population, and among CD3-CD90.2+ cells, CD93 was selectively expressed by three subsets of ROR ⁇ + ILC3s including CCR6+ CD4-ILC3, CCR6+ CD4+ ILC3 and CCR6-ILC3. Gating on CD45+ CD93+ revealed that 88%of these cells were ROR ⁇ t-expressing cells. Previous single-cell RNA-sequencing analysis also revealed ILC3s, but ILC1s or ILC2s, were enriched for expression of Cd93 transcript, consistent with an earlier report suggesting that CD93 is specifically expressed by ILC3 cells.
  • CD93 is a physiological receptor for IL-17D
  • Cd93 deletion diminished surface staining of CD93, as well as the binding of IL-17D, on ILC3 cells (Fig. 7B) . Therefore, our findings indicate that IL-17D interacts directly with ILC3 cells in a CD93-dependent manner.
  • CD93 appears to be a physiological receptor for IL-17D.
  • mice with Cd93 deficiency showed worsened colitis, including a significant decrease in body weight and shortened colon length (Fig. 7C) .
  • Fig. 7D AOM-DSS-induced tumor model
  • IL-17D regulates IL-22 production by ILC3 cells
  • mRNA expression of Il22, Reg3b, and Reg3g in total colon tissues of WT mice as well as Cd93-deficient mice at steady state.
  • Fig. 7E-F i.p. admisitration of IL-22 partially rescued phenotype of Cd93-/-5 mice (Fig. 7G-H) , indicative of IL-22 as a functional factor downstream CD93.

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Abstract

L'invention concerne l'utilisation d'un réactif dans la préparation d'un médicament, le réactif étant utilisé pour réguler l'axe IL-17D-CD93, et le médicament étant utilisé pour le traitement ou la prévention de maladies inflammatoires ou immunitaires, notamment un dérèglement de l'homéostasie intestinale.
PCT/CN2020/109014 2020-08-13 2020-08-13 Interleukine-17d et cd93 utilisés en tant que nouvelle paire cytokine-récepteur dans le système immunitaire WO2022032592A1 (fr)

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Citations (4)

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WO2011044563A2 (fr) * 2009-10-10 2011-04-14 The Board Of Trustees Of The Leland Stanford Junior University Compositions de cytokines de la famille il-17 et utilisations
EP2392352A2 (fr) * 2009-01-28 2011-12-07 Korea Research Institute of Bioscience and Biotechnology Utilisation de cd93 ou d'un fragment soluble de celui-ci
CN105073775A (zh) * 2013-02-08 2015-11-18 诺华股份有限公司 抗-il-17a抗体及其在治疗自身免疫性和炎性病症中的用途
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EP2392352A2 (fr) * 2009-01-28 2011-12-07 Korea Research Institute of Bioscience and Biotechnology Utilisation de cd93 ou d'un fragment soluble de celui-ci
WO2011044563A2 (fr) * 2009-10-10 2011-04-14 The Board Of Trustees Of The Leland Stanford Junior University Compositions de cytokines de la famille il-17 et utilisations
CN105073775A (zh) * 2013-02-08 2015-11-18 诺华股份有限公司 抗-il-17a抗体及其在治疗自身免疫性和炎性病症中的用途
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KLOSE CHRISTOPH S. N.; ARTIS DAVID: "Innate lymphoid cells control signaling circuits to regulate tissue-specific immunity", CELL RESEARCH, vol. 30, no. 6, 6 May 2020 (2020-05-06), Singapore , pages 475 - 491, XP037153246, ISSN: 1001-0602, DOI: 10.1038/s41422-020-0323-8 *
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