WO2015200094A1 - Methods and compositions for systemic lupus erythematosus (sle) therapy - Google Patents

Abstract

Embodiments concern methods and composition related to systemic lupus erythematosus (SLE) therapy, comprising administering certain pharmacentical compositions including a mitochondrial phosphodiesterase (PDE) inhibitor; a mitochondrial protein kinase A (PKA) enhancer; and/or a receptor for advanced glycation end products (RAGE) inhibitor. In certain embodiments, a method for treating a patien is provided. The method comprises administering to the patient a composition comprising an effective amount of: a) a mitochondrial phosphodiesterase (PDE) inhibitor; b) a mitochondrial protein kinase A (PKA) enhancer; and/or c) a receptor for advanced glycation end products (RAGE) inhibitor.

Description

DESCRIPTION

METHODS AND COMPOSITIONS FOR SYSTEMIC LUPUS ERYTHEMATOSUS

(SLE) THERAPY

BACKGROUND OF THE INVENTION [0001] This application claims the benefit of priority to U.S. Provisional Patent

Application Serial No. 62/015,266, filed June 20, 2014, hereby incorporated by reference in its entirety.

[0002] The invention was made with government support under Grant No. P50 AR054083-01 and U19 AI082715 awarded by the National Institutes of Health. The government has certain rights in the invention.

1. Field of the Invention

[0003] The present invention relates generally to the field of medicine. More particularly, it concerns compositions and methods related to Systemic Lupus Erythematosus (SLE) therapy. 2. Description of Related Art

[0004] Systemic Lupus Erythematosus (SLE) is a chronic automimmmune disorder in which patients suffer a number immunological abnormalities that is not specific to any one organ. SLE is manifested in various forms, including facial lesions, nephritis, endocarditis, hemolytic anemia and leukopenia. Specifically, SLE has been linked to disruption of complex T-cell mediated pathways, thus presenting a challenge to researchers attempting to elucidate the mechanism of the disease.

[0005] Many immunological phenomena are connected to SLE. In SLE patients, antibodies form against certain endogenous antigens, such as the basement membrane of the skin, against lymphocytes, erythrocytes and nuclear antigens. Antibodies may be directed against double- stranded DNA (ds-DNA) to form complexes, which are then deposited together on small blood vessels, resulting in vasculitis. These deposits are especially dangerous when they occur on the renal glomeruli, and may lead to glomerulonephritis and kidney failure. The incidence of clinically detectable involvement of the kidneys ranges from 50 to 80%. [0006] Current treatments involve preventive disease management (e.g, avoiding intense sun exposure) and drug therapies involving agents that suppress inflammation or interfere with immune functions (e.g., with NSAIDs, corticosteroids, antimalarials, methotrexate, diaminodiphenylsulfone, azathioprine, nitrogen mustard alkylating agents, danazol, and investigational therapies with cyclosporin A, immune globulin, plasma exchange and total lymphoid irradiation). Given the many combinations of organ system involvement and the need to address treatment of comorbid conditions accounting for considerable morbidity and mortality, it has remained desired to provide an effective treatment for SLE. SUMMARY OF THE INVENTION

[0007] In certain embodiments, a method for treating a patient is provided. The method comprises administering to the patient a composition comprising an effective amount of: a) a mitochondrial phosphodiesterase (PDE) inhibitor; b) a mitochondrial protein kinase A (PKA) enhancer; and/or c) a receptor for advanced glycation end products (RAGE) inhibitor. The patient may have symptoms of SLE, may have been diagnosed with SLE, and/or be at risk for SLE. In certain embodiments, the patient has been diagnosed with, or is having, suspected of having or at risk of SLE, any one or more of which patient group may be referred to as an "SLE patient." It is specifically contemplated that the patient may be a human. [0008] In certain embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial phosphodiesterase (PDE) inhibitor to the SLE patient. In further embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising mitochondrial protein kinase A (PKA) enhancer to the SLE patient. In still further embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising the RAGE inhibitor in dendritic cells of the patient.

[0009] In certain embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial PDE inhibitor and a mitochondrial PKA enhancer to the SLE patient or a patient having, suspected of having or at risk of SLE. In further embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial PDE inhibitor and a RAGE inhibitor to the SLE patient. In still further embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial PKA enhancer and a RAGE inhibitor to the SLE patient. In still further embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial PDE inhibitor, a mitochondrial PKA enhancer and a RAGE inhibitor to the SLE patient. In some embodiments, a patient is given the mitochondrial PDE inhibitor with the mitochondrial PKA enhancer, or the RAGE inhibitor, or both, in one or more doses together; in some embodiments, the mitochondrial PDE inhibitor is always given with the mitochondrial PKA enhancer, or the RAGE inhibitor, or both. In some embodiments, a patient is given the mitochondrial PKA enhancer with the mitochondrial PDE inhibitor, or the RAGE inhibitor, or both, in one or more doses together; in some embodiments, the mitochondrial PKA enhancer is always given with the mitochondrial PDE inhibitor, or the RAGE inhibitor, or both. In further embodiments, a patient is given the RAGE inhibitor with the mitochondrial PDE inhibitor, or the mitochondrial PKA inhibitor, or both, in one or more doses together; in some embodiments, the RAGE inhibitor is always given with the mitochondrial PDE inhibitor, or the mitochondrial PKA inhibitor, or both. In certain embodiments, the active ingredient(s) in a pharmaceutical composition are the mitochondrial PKA enhancer, the mitochondrial PDE inhibitor, and/or the RAGE inhibitor. In one or more embodiments, a composition or a method may be substituted with the term "consisting essentially of or "consisting of for the term "comprising." In addition, in certain embodiments, it is contemplated that only synthetic or manmade versions of the mitochondrial PKA enhancer, the mitochondrial PDE inhibitor, and/or the RAGE inhibitor are contemplated.

[0010] In some embodiments, the mitochondrial PDE inhibitor comprises IBMX. In certain embodiments, the mitochondrial PKA enhancer comprises 8-Br-cAMP. In further embodiments, the RAGE inhibitor comprises RAGE Fc Chimera.

[0011] Non-limiting examples of routes of administration include, but are not limited to the following: intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra- articular, intrasynovial, intrathecal, oral, topical, inhalation, or a combination of two or more recited routes.

[0012] In further aspects, administering a composition comprises targeting the pharmaceutical composition to mitochondria or neutrophils, or particularly, neutrophil mitochondria or dendritic cells, depending on the therapeutic agents delivered. For example, the PKA enhancer or the PDE inhibitor may be targeted to mitochondria or neutrophils, or particularly, neutrophil mitochondria. In other aspects, the RAGE inhibitor may be targeted or delivered to dendritic cells. [0013] In certain aspects, administering a composition involves delivering the pharmaceutical composition in a lipid vehicle. As used herein, the term "lipid" will be defined to include a substance that is a hydrophobic or amphiphilic small molecule that is characteristically insoluble in water and soluble in an organic solvent. This class of compounds is well known to those of skill in the art, and as the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds which contain long- chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, fatty acids, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, waxes, natural oils, lipids with ether and ester- linked fatty acids and polymerizable lipids, their derivatives, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods in accordance with certain embodiments of the present invention. [0014] In aspects wherein the active ingredient is associated with the lipid vehicle, the active ingredient may be conjugated to, fused to, or enclosed within the lipid vehicle. The active ingredient may also be associated within the lipid layer (either by conjugation or by non-chemical attraction) or attached to the surface of the lipid vehicle.

[0015] Further aspects of the disclosure relate to pharmaceutical compositions comprising the active ingredients described herein. Also disclosed are uses of the compositions for the preparation of a medicament for treating systemic lupus erythematosus (SLE) in patient having, suspected of a having or at risk of SLE. Further aspects relate to uses of the compositions for treating systemic lupus erythematosus (SLE) in patient having, suspected of a having or at risk of SLE. [0016] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the pharmaceutical composition may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0017] Methods may involve administering to the patient or subject at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a pharmaceutical composition or a composition described herein. A dose may be a composition comprising about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,

3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,

5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,

7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,

15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms (meg) or μg/ml or micrograms/ml or mM or μΜ (or any range derivable therein) of each therapeutic agent or compound or the total amount of a combination of therapeutic agents or compounds or the compositions. A treatment may comprise such a dosage regimen.

[0018] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0019] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more. [0020] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0021] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0023] FIG 1A-1F - Live neutrophils actively extrude mitochondria DNA/protein complexes. (A) Unstimulated healthy neutrophils spontaneously extrude mtDNA/protein complexes (mtC). Untreated or proteinase K-digested (PK) healthy neutrophil supernatants were visualized on agarose gels. (B) Neutrophils extrude matrix mitochondrial components. TFAM, MnSOD, TOMM20, Glyceraldehyde- 3 -phosphate dehydrogenase (GAPDH) and Histone H3 (H3) were quantified in concentrated neutrophil supernatants by Western blot. (Nec= necrotic neutrophil). (C) Neutrophils form a subset of SLE patients spontaneously extrude higher amounts of mtDNA. Supernatant mtDNA content was assessed by agarose gel electrophoresis (top panel) or by Picogreen dsDNA (bottom panel). (D) Extrusion of mtDNA is specific to neutrophils. Neutrophil and monocyte mitochondrial mass was measured with MitoTracker Green (upper panel) and the quantity of extruded mtDNA by Picogreen (lower panel). (E) Degradation of mtDNA by serum-resident DNAse-I is impaired in a subset of SLE patients. The integrity of extruded mtDNA was assessed by agarose gel. Neutrophils were cultured with 2% or 20% of healthy sera (upper panel) or with 20% of SLE sera (lower panel). (F) Synthetic TLR7/8 agonists increase mtC extrusion. R837 (TLR7/8 agonist) stimulation increases the extrusion of mtDNA by neutrophils (upper panel) without compromising the cell membrane integrity as assessed by LDH activity in the supernatants (lower panel). The percentage of cell lysis was calculated using the LDH activity in the total cell lysate (input). Data shown are representative of two or more experiments. ***p<0.001.

[0024] FIG 2A-2E - Extruded mtC derive from depolarized mitochondria. (A) MTDR(-) TFAM(+) mitochondrial vesicles are detected in the cytoplasm of neutrophils but not in monocytes. Neutrophils (left) and monocytes (right) were immunostained with MitoTracker DeepRed and anti-TFAM antibody. Four different sequential confocal sections (0.25 μιη) are shown. Arrowheads indicate a vesicle budding from respiring mitochondria. (B) TEM visualization of mitochondrial vesicles (yellow arrowhead) and neutrophil granules (arrowhead) (I). Vesicles are present both as independent structures (II) or joined to healthy mitochondria (III). (C) TEM (left) and flow cytometric analysis (right) of vesicle/plasma membrane fusion. TEM shows vesicles cargo extrusions. Histograms show selective translocation of the OMM protein TOMM20 onto the plasma membrane. (D) Mitochondrial vesicle-plasma membrane fusion leads to the appearance of DNA aggregates on the neutrophils surface. TEM (left) and IF analysis on non-permeabilized neutrophils (right). PanCadherin was used as a cell membrane marker. (E) MtDNA extrusion is calcium- dependent. Extruded mtDNA was quantified in the supernatnats of neutrophils cultured with or without BAPTA-AM. Data shown are representative of two of more independent experiments (mean +SD). **p<0.01. [0025] FIG 3A-3E - Neutrophils fail to undergo complete mitophagy in response to depolarization. (A) The protonophore CCCP, but not the mtROS generator Rotenone, induces neutrophil mitochondrial depolarization. (B) Depolarization, but not mtROS production, increases neutrophil mtDNA extrusion. (C) CCCP-treated neutrophils display reduced intracellular TFAM levels. Neutrophils or monocytes were treated with media or CCCP in the presence of the protein synthesis inhibitor cycloheximide. Proteins levels were analyzed by Western blot in the total cell lysate. (D) Neutrophils fail to fuse autophagosomes with lysosomes in response to CCCP-induced depolarization. Data shown are representative of two of more independent experiments (means +SD). **p<0.01. (E) Ingenuity pathway analysis of transcripts selectively unregulated in monocytes but not in neutrophils after 60 min exposure to CCCP. Raw microarray data are submitted to GEO.

[0026] FIG 4A-4F - MtDNA oxidation is required for extruded neutrophil mtC to activate pDCs. (A) The combination of IFNa and anti-Sm/RNP autoantibodies (IFN/aRNP) (lupus combination) alters the quality but not the quantity of extruded mtDNA. pDCs were incubated with supernatants from healthy neutrophils treated with IFNa and aRNP alone or in combination. IFNa leves (upper panel) and mtDNA quantification (lower panel) are shown. (B) Neutrophil mtROS scavenging reduces the interferogenic effect of the extruded mtDNA. Neutrophils were stimulated with IFN/aRNP in the absence of presence of MitoTempo (MT). The corresponding supernatants were then assessed for their interferogenic effect and mtDNA content. (C) Neutrophils activation with IFN/aRNP leads to the extrusion of Ox mtDNA. Dot blot analysis of the extruded mtDNA. Anti-dsDNA antibody was used as a loading control. Bars represent the relative quantification of 8-OHdG intensity. (D) Extrusion of interferogenic mtC is TLR7-dependent. Neutrophils were treated with IFN/aRNP in the presence of IRS661 (TLR7 inhibitor) or DVX42 (TLR8 inhibitor). The corresponding supernatants were assessed for their interferogenic effect. (E) Interferogenic mtC activate pDCs in a TFAM/RAGE-dependent manner. IFNa production was assessed after pDC incubation with supernatants form neutrophils activated with IFN/aRNP in the presence/absence of (upper panel) or recombinant RAGE-Fc chimera (lower panel). (F) Synthetic TLR7 agonists increase mitochondrial depolarization and mtDNA extrusion but do not induce oxidation. Mitochondrial depolarization was assessed with MitoTracker DeepRed (upper left). The amount of extruded mtDNA was quantified with Picogreen (upper right) and its oxidation status by 8-OHdG dot blot (lower left). Data shown are representative of two of more independent experiments (mean +SD). **p<0.01, ***p<0.001.

[0027] FIG 5A-5D - Under steady state conditions oxidized mtDNA is exported within cytosolic vesicles from mitochondria to lysosomes. (A) Detection by IF (left) and quantification (right) of cytoplasmic 8-OHdG(+) MDVs in neutrophils and monocytes. For IF, four different sequential confocal sections (0.25 μιη) are shown. Each dot in the graph represents the 8-OHdG MFI in a single cell. (B) 8-OHdG(+) MDVs include inner (Mitofilin) but not outer (TOMM20) mitochondrial membrane proteins. Arrowheads indicate the co- localization of 8-OHdG/Mitofilin. Each dot in the graph represents the percentage of 8- OHdG co-localizing with Mitofilin or TOMM20 in a single cell. (C) 8-OHdG(+) MDVs do not contain damaged nuclear DNA. The reactivity of anti-yH2A.X antibody was assessed on apoptotic neutrophils. (D) MtROS modulation correlates with the number of 8-OHdG(+) MDVs. Each dot in the graph represents the 8-OHdG MFI in a single cell. *p<0.05, **p<0.01, ***p<0.001.

[0028] FIG 6A-6C - IFN/aRNP activation of neutrophils blocks the detoxification of oxidized mtDNA. (A) Formation of large Ox mtDNA aggregates (left) and accumulation of intracellular Ox mtDNA (right) in neutrophils exposed to IFN/aRNP. (B) Ox mtDNA co- localizes with aggregated mitochondria. TOMM20 was used as a mitochondrial marker. (C) TEM confirms the mitochondrial swelling and aggregation (arrowheads) in neutrophils exposed to IFN/aRNP. **p<0.01, ***p<0.001.

[0029] FIG 7A-7D - IFN/aRNP inhibits oxidized mitochondrial nucleoids disassembly. (A) IFN/aRNP increases the amount of intracellular TFAM as assessed by Western blot on the total cell lysate. (B) Left panel: co-localization of Ox mtDNA and TFAM in neutrophils exposed to IFN/aRNP. Right panel: IP of TFAM (total cell lysate) confirms its association with Ox mtDNA. (C) IFN/aRNP block PKA-mediated TFAM phosphorylation. Cell lysates were subjected to IP with anti-TFAM antibody and the immunoprecipitates blotted with an antibody against PKA-phosphorylated proteins (a-PKA sub). The PKA inhibitor H89 was used as a positive control. (D) IFN/aRNP block Ox mtDNA detoxification through activation of PDEs. The 8-OHdG content of extruded mtDNA was assessed by dot blot. The effects of IFN/aRNP alone or in combination with 8Br-cAMP or IB MX are shown. Bars represent the relative quantification in each sample. Data shown are representative of two or more experiments.

[0030] FIG. 8A-8F - (A-B) Mitochondrial origin of extruded neutrophil DNA. (A) Real-time PCR amplification of the mitochondrial gene ND1. (B) IP of extruded mtC with anti-dsDNA antibody. Neutrophil supernatants (Orig Sup) were subjected to immunoprecipitation with antidsDNA antibody. The beads (IP Beads) and the resulting supernatants (IP Sup) were then analyzed by agarose gel (left) or by Western blot with anti- TFAM (upper right) or anti-H3 (lower right) antibodies. (C) MtDNA extrusion is not the result of cell lysis. LDH activity in live or necrotic (Nec) neutrophil supernatants. (D) TEM (upper panel) and immunofluorescence analysis (lower panel) show the tubular morphology of neutrophil mitochondria. Arrowheads indicate the mitochondria. (E) Extrusion of mtDNA is not a consequence of spontaneous neutrophil apoptosis. Picogreen quantification of extruded mtDNA in the presence of recombinant GM-CSF (right panel). Apoptosis progression was assessed by TUNEL assay (left panel). (F) MtDNA extrusion increases over time in culture in the absence of plasma membrane disruption. Supernatants from 6 h or 15 h cultured neutrophils were assessed for mtDNA content (upper panel) and LDH activity (lower panel). LDH activity in the total cell lysate (TCL) was used as a reference. Data shown are representative of two of more independent experiments (mean +SD).

[0031] FIG. 9A-9G - (A) Co-localization analysis of cells stained as in Figure 2A.(B) TEM shows that autophagosomes (I) or phagosomes (II) are morphologicallydifferent from mitochondrial vesicles. PM: plasma membrane. (C) Flow cytometric quantification of TOMM20 and LAMP1 translocation to the cellsurface as in Figure 2C. MFIs of permeabilized (Total) or non-permeabilized (Surface)cells were used to calculate the percentages of protein that translocate to the cell surface. (D) The specificity of surface dsDNA staining (Figure 2D) was assessed by adding DNAse-I to the cell cultures before IF staining. Three different sequential confocal sections (0.25 mm) are shown (left). Each dot in the chart represents the dsDNA antibody MFI in one single cell (right). (E) MtDNA extrusion is not ROS dependent. Picogreen quantification of extruded mtDNA in the presence of DPI (NADPH Oxidase Inhibitor) or MitoTempo (mitochondrial ROS scavenger). (F) The PARKIN/ubiquitin pathway is not involved in mitochondrial vesicle formation. Western blot analysis on total cell lysate shows expression of PARKIN in neutrophils. As a positive control for the anti-ubiquitin antibody (Ub), apoptotic neutrophils were included in these experiments. (G) Depolarized mitochondrial vesicles are not degraded in the lysosomal compartment. IF (left) and co-localization analysis (right) of TFAM and LAMP1. The percentage of TFAM co-localizing with MitoTracker Deep Red was used as reference. Each dot in the chart represents the percentage of co-localization in one single cell. Data shown are representative of two of more independent experiments (means +SD). **p<0.01, ***p<0.001. [0032] FIG. 10A-10D - (A) Autophagosome formation and cargo recognition in neutrophils and monocytes upon mitochondrial depolarization. Neutrophils and monocytes were treated with CCCP for 20 or 60 min and immunostained with anti-TOMM20 and anti- LC3B antibodies. The LC3B MFI of single cells is also shown. (B) Co-localization analysis of cells stained as in Figure 3D. (C) Fold change ratio of transcripts selectively upregulated in monocytes compared to neutrophils upon CCCP treatment. (D) CCCP fails to inhibit mTOR in neutrophils. mTOR activity was assessed by measuring the phosphorylation levels of the mTOR specific substrate 70s6K. **p<0.01, ***p<0.001.

[0033] FIG. 11A-11G - (A) Neutrophil activation with IFN/aRNP does not increase the amount of extruded TFAM as assessed by Western blot in the neutrophil supernatnats. (B) IFN/aRNP increase the amount of extruded Ox mtDNA. 8-OHdG levels were assessed by ELISA in the neutrophils supernatants. (C) Mitotempo treatment reduces the oxidation status of extruded mtDNA in IFN/aRNP activated neutrophils. Bars represent the relative quantification of 8-OHdG intensity. (D) IFNa production by pDCs stimulated with Non-Ox or Ox mtDNA alone or in combination with recombinant LL-37. (E) Uptake (upper panel) or intracellular stability (lower panel) of Non-Ox or Ox mtDNA in pDCs. Grey filled histogram represents cells incubated with medium. (F) Similar intracellular distribution of Non-Ox or Ox mtDNA in pDCs. Cy5 labeled Non-Ox or Ox mtDNA were assessed for their intracellular localization by IF with the early endosome marker TfR or the late endosome/lysosome marker LAMP-1. (G) MtC do not contain LL-37 or HMGBl. Upper panel: DNA from IFN/aRNP activated neutrophil supernatants was immunoprecipitated with anti-dsDNA antibody. The IP was analyzed by Western blot with anti-LL37 or anti-HMGBl antibodies. Lower panel: HMGBl neutralization with BoxA does not prevent pDC activation by IFN/aRNP activated neutrophil supernatants. Data shown are representative of two of more independent experiments (mean + SD).**p<0.01, ***p<0.001.

[0034] FIG. 12A-12D - (A) Four different sequential confocal sections (0.25 mm) (from Fig. 5B) are shown. (B) Upper panel: TUNEL assay shows the absence of fragmented DNA in the cytoplasm of live neutrophils. Apoptotic neutrophils were used as a positive control. Lower panel: cytoplasmic 8-OHdG(+) vesicles do not contain TFAM. (C) 8- OHdG(+) vesicles are delivered to the lysosomal compartment, as shown by their accumulation in the presence of Bafilomycin Al (upper panel) and their co-localization with LAMP-1 (lower panel; arrowhead). Each dot in the graph represents the 8-OHdG antibody MFI in a single cell. (D) Blocking MPTP formation with Cyclosporin A does not affect 8- OHdG(+) vesicle formation. Each dot in the graph represents the percentage of 8-OHdG co- localizing with TOMM20 in a single cell. ***p<0.001.

[0035] FIG. 13A-13C - (A) Six different sequential confocal sections (0.25 mm) (from Fig. 6B) are shown. (B) Induction of mtROS production with Rotenone recapitulates the effect of IFN/aRNP on neutrophil mitochondria. Three different sequential confocal sections (0.25 mm) are shown. (C) IFN/aRNP activation does not increase mtROS (assessed by MitoSox) or the total cellular ROS (assessed by CellRox) levels in neutrophils.

[0036] FIG. 14A-14E - (A) TFAM turnover is regulated by PKA and the Lon protease. Western blot analysis on total cell lysate shows accumulation of TFAM in neutrophils treated with the PKA inhibitor H89 or with the Lon protease inhibitor MG132. (B-C) Accumulation of TFAM in IFN/aRNP treated neutrophils is not a consequence of PGCla up-regulation (B) and/or enhanced mitogenesis (C). Total mitochondrial mass was assessed by MitoTracker Green. (D) Neutrophil lysates were subjected to IP with anti-TFAM antibody and the immunoprecipitates blotted with anti-phospho serine antibody (a-pSer). Bars represent the relative quantification in each sample. Data shown are representative of two or more experiments. (E) In healthy neutrophils, oxidized nucleoids are promptly removed from the mitochondria after a process that includes PKA-mediated TFAM phosphorylation/dissociation (1) and inclusion of Ox mtDNA inside MDVs. These microvesicles are transported to lysosomes for degradations (2). Upon depolarization QA* ), neutrophil mitochondria swell (3), fuse with the plasma membrane and release their content (nucleoids) into the extracellular space (4). The extruded nucleoids are immunological silent. In SLE, neutrophil TLR7 stimulation by anti-RNP/Sm autoantibodies leads to the activation of mitochondrial PDEs (5). This reduces the intramitochondrial cAMP pool and subsequently inactivates PKA. As a result, the detoxification of Ox mtDNA is blocked (6). This leads to the accumulation inside mitochondria of Ox nucleoids (7) that are eventually extruded. The extruded Ox nucleoids activate pDCs in a TF AM/RAGE dependent manner.

[0037] FIG. 15A-H - Live neutrophils extrude mitochondrial DNA/protein complexes. (A) Neutrophil supernatants from three healthy donors (HD) were run on agarose gels. High molecular weight complexes (mtC) yield a single DNA band of -16 Kb upon digestion with proteinase K (PK). (B) The abundance of mitochondrial and genomic DNA was assessed on DNA isolated from neutrophil supernatants by Real-Time PCR (mtDNA Copy Number; left) or by conventional PCR (right). (n=3). (C) Extrusion of mtDNA is not the consequence of neutrophil apoptosis. Apoptosis progression in untreated or GMCSF treated neutrophil was assessed by TUNEL assay (left). The corresponding supernatants were analyzed on agarose gel (right). (D) Extrusion of mtDNA is neutrophilspecific. Mitochondrial mass and extruded mtDNA were quantified with MitoTracker Green and Picogreen, respectively. (n=6). (E) TLR7, but not TLR9, stimulation increases the extrusion of mtDNA from neutrophils. (n=5). (F) TOMM20 (Outer Mitochondrial Membrane: OMM protein) and Mitofilin (Inner Mitochondrial Membrane: IMM protein) but not LAMPl or Rab7 (lysosomal markers) translocate to the neutrophil cell surface. Open grey histograms represent the isotype control. (G) TLR7 stimulation amplifies TOMM20 plasma membrane translocation. Δ MFI (Mean Fluorescence Intensity) = MFI antibody - MFI isotype control. (H) MtDNA extrusion requires autophagy. Quantification of extruded mtDNA in the presence of 3- Methyladenine (3MA) (upper) and co-localization of TOMM20 with LC3B on neutrophil plasma membrane (lower). (n=3). Scale bar: 10 μιη. Values represent mean + s.d. *p<0.05, **p<0.01, ***p<0.001.

[0038] FIG. 16A-H - (A) Left: DNA extracted from neutrophil supernatant and purified mitochondria yield a similar band of 16 Kb upon PK digestion. Right: Amplification of the mitochondrial gene ND1, but not the nuclear gene GAPDH, from DNA isolated from live neutrophil supernatant. Total DNA was used as control. (B) DNA from neutrophil supernatants is associated with TFAM but not H3. Neutrophil supernatant (Orig Sup) was immunoprecipitated with anti-dsDNA antibody. The beads (IP Beads) and the resulting supernatant (IP Sup) were analyzed by agarose gel (left) or by Western blot with anti-TFAM or anti-H3 antibodies (right). (C) MtDNA extrusion by live neutrophils is not associated with cell lysis. Supernatant LDH activity (upper) or Western blot analysis (lower). * indicates non- specific band. TLC = Total Cell Lysate. (n=3). (D) Quantification of extruded mtDNA in the presence of DPI (NADPH Inhibitor) or MitoTempo (mtROS scavenger). (n=5). (E) Upper: GM-CSF/LPS combination increases the extrusion of mtDNA. Lower: R837 treatment does not increase cell lysis. (n=3). (F) Neutrophils selectively extrude mitochondrial matrix components. The presence of TFAM, MnSOD and TOMM20 was assessed in live neutrophil supernatants by Western blot. (G) Δ MFI of FIG. 15F. (n=3). (H) Upper: reduction in LC3 puncta staining and LC3II/LC3I ratio in the presence of 3MA. Lower: 3MA treatment reduces TOMM20 translocation to the plasma membrane. (n=3). Scale bar: 10 μιη. Values represent mean + s.d. *p<0.05, **p<0.01. [0039] FIG. 17A-F - Neutrophils fail to complete mitophagy in response to mitochondrial depolarization. (A) The protonophore CCCP, but not the mitochondrial ROS generator Rotenone, amplifies neutrophil mitochondrial depolarization. (n=5). (B) Depolarization, but not mitochondrial ROS production, increases mtDNA extrusion from neutrophils but not from monocytes. (n=5). (C) CCCP-treated neutrophils selectively display reduced intracellular TFAM levels. Neutrophils or monocytes were treated with media or CCCP in the presence of 50 μΜ of the protein synthesis inhibitor cycloheximide. Total cell lysates were analyzed by western blot. (D) Similar autophagosome formation and cargo recognition in neutrophils and monocytes upon mitochondrial damage. Neutrophils and monocytes were treated with CCCP for 60 min and immunostained with anti-TOMM20 and anti-LC3B antibodies. Scale bar: 10 μιη. (E) Incomplete mitophagy in neutrophils. Neutrophils and monocytes were treated with CCCP for 60 min and immunostained with anti-TOMM20 and anti-LAMPl antibodies. Scale bar: 10 μιη. (F) Fold up or down regulation of mitophagy-related transcripts in monocytes and neutrophils after CCCP treatment. Raw microarray data were submitted to GEO under accession #. Values represent mean + s.d. **p<0.01, ***p<0.001.

[0040] FIG. 18A-E - (A) Early time point images of Fig. 17D. Neutrophils and monocytes were treated with CCCP for 20 min and immunostained with anti-TOMM20 and anti-LC3B antibodies. Scale bar: 10 μιη. (B) Co-localization analysis of Fig. 17E. (C) Bafilomycin Al (BafAl) dampens autophagosome/lysosome fusion in CCCP-treated monocytes (left, middle) but does not increase mtDNA extrusion in these cells (right). Scale bar: 10 μιη. (D) The combination Oligomycin (10 μΜ) and Antimycin (1 μΜ) (O+A) recapitulates the effect of CCCP on autophagosome-lysosome fusion. Mitochondria depolarization and mtDNA extrusion (left) and quantification of autophagosome/lysosome fusion (right) are shown. (n=3). Scale bar: 5 μιη. (E) CCCP fails to inhibit mTOR in neutrophils. mTOR activity was assessed by measuring phosphorylation levels of the mTOR specific substrate 70s6K by Western blot. Values represent mean + s.d. *p<0.05, **p<0.01, ***p<0.001.

[0041] FIG. 19A-F - mtDNA oxidation is required for pDC activation. (A) The combination of IFNa and anti-Sm/RNP autoantibodies (IFN/aRNP) alters the quality of extruded mtDNA. pDCs were incubated with supernatants from neutrophils treated with IFNa and/or aRNP. IFNa levels (upper) and mtDNA quantification (lower) are shown. (n=5). (B) MtDNA extruded upon IFN/aRNP treatment is highly oxidized. Dot blot analysis using anti-8-OHdG and anti-dsDNA (loading control) antibodies. Bars represent the quantification of 8-OHdG intensity. (n=5). (C) MtROS scavenging reduces the interferogenic capacity of extruded mtDNA. Neutrophils were stimulated with IFN/aRNP in the absence of presence of MitoTempo (MT). The corresponding supernatants were assessed for interferogenic effect on pDCs and for mtDNA content. (n=4). (D) Extrusion of interferogenic mtDNA is TLR7- dependent. Neutrophils were treated with IFN/aRNP in the presence of IRS661 (TLR7 inhibitor) or DVX42 (TLR8 inhibitor) and the corresponding supernatants were assessed for their interferogenic effect. (n=6). (E) Ox mtDNA activates pDCs in a TFAM/RAGE- dependent manner. IFNa production was assessed after pDC incubation with interferogenic neutrophil supernatants in the presence/absence of anti-TFAM antibodies (left; n=3) or recombinant RAGE-Fc chimera (right; n=6). (F) The synthetic TLR7 agonist R837 increases mitochondrial depolarization and mtDNA extrusion but does not increase oxidation. Mitochondrial depolarization was assessed with MitoTracker DeepRed, the amount of extruded mtDNA was quantified with Picogreen and its oxidation status by 8-OHdG dot blot. (n=4). Values represent mean + s.d. *p<0.05, **p<0.01, ***p<0.001.

[0042] FIG. 20A-I - (A) IFN/aRNP do not increase the amount of extruded mtDNA (top) or TFAM (bottom). (n=7). (B) MtDNA extruded upon exposure to IFN/aRNP is highly oxidized. 8-OHdG levels were assessed by ELISA. (C) MT treatment reduces the oxidation status of extruded mtDNA in IFN/aRNP activated neutrophils. Bars represent the relative quantification of 8-OHdG intensity. (D, E) The ROS inhibitor DPI (D) and the TLR7 inhibitor IRS661 (E) reduce the oxidation status of extruded mtDNA. (F) IFNa production by pDCs stimulated with LL-37 mixed with genomic, Non-Oxidized (Non-Ox) or Oxidized (Ox) mtDNA or with the supernatant from neutrophils treated with IFN and/or aRNP. (n=3). (G) Uptake (upper) and intracellular stability (lower) of Non-Ox or Ox mtDNA in pDCs. Grey filled histogram represents cells incubated with medium alone. (H) Non-Ox or Ox mtDNA were assessed for their intracellular localization by IF. Transferrin Receptor (TfR): early endosome marker. LAMP1: late endosome/lysosome marker. Scale bar: 10 μιη. (I) MtC does not contain LL-37 or HMGBl. Upper: DNA from IFN/aRNP activated neutrophil supernatants was immunoprecipitated with anti-dsDNA antibody. The IP was analyzed by Western blot with anti-LL37 or anti-HMGBl antibodies. Lower: HMGBl neutralization with BoxA does not prevent pDC activation by interferogenic neutrophil supernatants (n=3). Values represent mean + s.d. *p<0.05, **p<0.01, ***p<0.001. [0043] FIG. 21A-D - Oxidized mtDNA is exported from mitochondria to lysosomes under steady state conditions. (A) Cytoplasmic 8-OHdG(+) staining can be detected in neutrophils, but not in monocytes. 8-OHdG quantification is also shown. Scale bar: 10 μιη. (B) 8-OHdG staining does not co-localize with the damaged nuclear DNA marker γΗ2Α.Χ. Apoptotic neutrophils were used as a control. Scale bar: 5 μιη. (C) 8-OHdG staining is modulated according to mitochondrial ROS production (decreased with Mito tempo and increased with Rotenone). TOMM20 was used as mitochondrial marker. Scale bar: 10 μιη. (D) 8-OHdG(+) vesicles incorporate selective mitochondrial components. Supernatant (Sup) from mitochondrial budding assay was subjected to Western blot analysis or analyzed for mtDNA content by agarose gel. The mitochondrial fraction (Mitos) was loaded as a control. * indicates non-specific band. ***p<0.001.

[0044] FIG. 22A-E - (A) TUNEL assay demonstrates the absence of fragmented DNA in the cytoplasm of live neutrophils. Apoptotic neutrophils were used as control. Scale bar: 10 μπι. (B) 8-OHdG quantification of FIG. 21C. (C) 8-OHdG(+) vesicles are delivered to lysosomes for degradation. Neutrophils were treated with the lysosomal inhibitor Bafilomycin Al (BafAl) and stained with anti-8-OHdG and LAMP1 antibodies. 8-OHdG quantification and the percentage of LAMP1 that co-localizes with 8-OHdG (n = 30 cells) are also shown. Scale bar: 5 μιη. (D) The dynamin-related protein Drpl, but not the autophagic machinery, is involved in 8-OHdG(+) vesicle formation. Neutrophils were treated with the autophagy inhibitor 3MA or with the DRPl inhibitor MDrVT-l before staining with anti-8- OHdG and TOMM20 antibodies. The percentage of 8-OHdG that colocalizes with TOMM20 is also shown. Scale bar: 10 μιη. (E) MDIVT1 treatment blocks the translocation of DPR1 to mitochondria and induces their elongation. The percentage of DRPl that co-localizes with TOMM20 is also shown (n = 20 cells). Scale bar: 10 μιη. Values represent mean + s.d. ***p<0.001.

[0045] FIG. 23A-D - IFN/aRNP activation of neutrophils blocks the lysosomal exportation of oxidized mtDNA. (A) Appearance of large Ox mtDNA aggregates in neutrophils exposed to IFN and aRNP. 8-OHdG quantification is also shown. Scale bar: 10 μιη. (B) IF (left) and dot blot (right) analysis show Ox mtDNA accumulation within mitochondria in neutrophils exposed to IFN/aRNP. Scale bar: 10 μιη. (C, D) The combination of IFN and aRNP does not increase ROS production (C) and does not block DRP1 translocation to the mitochondria (D). Rotenone and MDIVI-1 were used as controls. ***p<0.001.

[0046] FIG. 24 - The percentage of TOMM20 that co-localizes with 8-OHdG (top) and six different sequential confocal sections (0.25 μιη each; bottom) of Fig. 23B are shown. Scale bar: 10 μιη. **p<0.01.

[0047] FIG. 25A-F - IFN/aRNP inhibit oxidized mitochondrial nucleoid disassembly. (A) The association of Ox mtDNA and TFAM increases in neutrophils exposed to IFN/aRNP as assessed by IF (left) or Co-IP (right). Scale bar: 5 μιη. (B) IFN/aRNP increases the amount of intracellular TFAM, as assessed by Western blot on the total cell lysate. (n=3). (C) IFN/aRNP decreases TFAM phosphorylation. Cell lysates were subjected to IP with anti-TFAM antibody and the immunoprecipitates were blotted with an antibody against PKA-phosphorylated peptides (a-PKA sub) or TFAM (loading control). The PKA inhibitor H89 was used as a control. (n=3). (D) The cAMP analog 8Br-cAMP or the PDE inhibition IBMX decrease the amount of extruded Ox mtDNA in IFN/aRNP treated neutrophils. Dot blot analysis of extruded mtDNA (left) and IFN production by pDCs (right) are shown. (n=3). (E) Accumulation of Ox DNA within mitochondria of neutrophils freshly isolated from SLE but not healthy control or JDM blood. Scale bar: 10 μιη. (F) Presence of anti-Ox mtDNA autoantibodies in SLE but not healthy control or JDM sera. Anti-8-OHdG antibody was used as a control. Values represent mean + s.d. *p<0.05, **p<0.01, ***p<0.001.

[0048] FIG. 26A-G - (A, B) Accumulation of TFAM in IFN/aRNP-treated neutrophils is not a consequence of PGCla up-regulation (A) and/or enhanced mitogenesis (B). Total mitochondrial mass was assessed by MitoTracker Green. (C) PK protection assay shows that PKA is present inside neutrophil mitochondria. * indicates non-specific band or an alternative isoforms. (D) TFAM turnover in neutrophils is regulated by PKA and Lon protease. Western blot analysis of total cell lysate shows accumulation of TFAM in neutrophils treated with the PKA inhibitor H89 or with the Lon protease inhibitor MG132. (E) Neutrophil lysates were subjected to IP with anti-TFAM antibody and the immunoprecipitates were blotted with anti-phospho serine (a-pSer) or anti-TFAM (loading control) antibody. Bars represent the relative quantification. (F) Mitochondrial cAMP levels are reduced in IFN/aRNP treated neutrophils. cAMP levels are normalized to the total protein content and expressed in Arbitrary Units (AU). (n=3). (G) Absence of cross-reactivity between anti-Ox and anti-Non Ox mtDNA autoantibodies. Values represent mean + s.d. *p<0.05.

[0049] FIG. 27 - Proposed effect of IFN/aRNP on neutrophil mitochondria. In healthy neutrophils, oxidized nucleoids are promptly removed from mitochondria upon PKA- mediated TFAM phosphorylation, which leads to (i) TFAM dissociation from mtDNA and (ii) TFAM degradation (1). Ox mtDNA is then sorted into vesicles that are directed to lysosomes for degradation (2). Upon depolarization (|ΔΨ), neutrophil mitochondria are not removed by mitophagy (3) but, instead, matrix components (including nucleoids) are released into the extracellular space (4). The extruded nucleoids are devoid of Ox DNA and therefore immunological silent. In SLE, neutrophil activation with TLR7-agonist autoantibodies decreases the mitochondrial cAMP pool (5) and leads to a reduction of matrix PKA activity. As a result, nucleoids are not disassembled. This leads to the intra-mitochondrial retention and eventual extrusion of Ox nucleoids (6). Extracellular Ox nucleoids activate pDCs in a TFAM/RAGE-dependent manner. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. INTRODUCTION

[0050] Certain embodiments are, in part, based on the finding of certain novel therapeutic targets for Systemic Lupus Erythematosus (SLE) in the pathway of oxidization of mitochondrial DNA. It was discovered that oxidized mitochondrial DNA (mtDNA) is responsible for induction of patient neutorphils to release interferogenic factors in SLE patients. Some aspects establish for the first time a link between extracellular Ox mtDNA and SLE pathogenesis.

II. SYSTEMIC LUPUS ERYTHEMATOSUS

[0051] Methods and compositions are provided for treating autoimmune diseases such as SLE. Lupus has long been considered a disease of adaptive immunity where altered lymphocyte signaling thresholds lead to breakdown of tolerance to self-antigens (Shlomchik et al., 2001). Genomic studies including genome-wide association (GWAS) and gene expression profiling have recently brought up the concept of interplay between innate and adaptive immunity at the core of human SLE pathogenesis. Thus, expression of type I interferon (IFN)-, neutrophil- and plasmablast-related transcripts correlate with disease activity, and common allelic variants within these pathways confer disease susceptibility (Moser et al., 2009; Pascual et al., 2006). More recently, identification of monogenic SLE cases due to mutations in genes involved in extracellular DNA degradation (i.e. DNaselL3 mutations) (Al-Mayouf et al., 2011), as well as intracellular nucleic acid degradation/sensing (i.e. Trexl) (Crow, 2011) support that proper disposal of nucleic acids is a fundamental and upstream event in SLE.

[0052] Clinical manifestations of SLE include: constitutional, arthritis, arthralgia, skin, mucous membranes, pleurisy, lung, pericarditis, myocarditis, Raynaud's, thrombophlebitis, vasculitis, renal, nephrotic syndrome, azotemia, CNS, cytoid bodies, gastrointestinal, pancreatitis, lymphadenopathy and myositis. The most common skin manifestation is the "butterfly" rash, commonly precipitated by exposure to sunlight. Subacute cutaneous lupus erythematosus is a relatively distinct cutaneous, lesion, nonfixed, nonscarring, exacerbating, and remitting, again correlated to sun exposure. Discoid lesions are chronic cutaneous lesions and may occur in the absence of systemic manifestations. Alopecia and mucous membrane lesions are other common features. The most common presentations of SLE include latent lupus (patients presenting one or two classification criteria over a period of years), drug-induced lupus (induced, e.g., by chlorpromazine, methyldopa, hydralazine, procainamide and isoniazid, with typically less severe clinical features), antiphospholipid antibody syndrome, and late stage lupus (typically involving mortality from complications that result from the disease itself or as a consequence of its therapy).

[0053] A characteristic feature of SLE is the presence of autoantibodies against dsDNA and RNA/protein complexes. In vitro, internalization of endogenous nucleic acids carried within SLE immune complexes (ICs) leads to endosomal TLR activation and type I IFN production by pDCs (Bave et al., 2003; Means et al., 2005). Recent evidence from us and others supports that neutrophils, which also internalize SLE ICs via FcRs, contribute to amplify pDC activation and IFN production. Thus, SLE neutrophils respond to activation with TLR7-agonistic (anti-Sm/RNP) autoantibodies with the extrusion of highly interferogenic DNA, while healthy neutrophils require priming with IFN in order to recapitulate this response (Garcia- Romo et al., 2011). TLR7 is IFN-inducible, and IFN priming might be necessary to increase the low baseline expression levels of this receptor in healthy neutrophils (Hayashi et al., 2003). Interestingly, however, high constitutive expression of TLR7 in pDCs is insufficient to render these cells directly responsive to the same TLR7-agonistic autoantibodies that trigger neutrophil activation (Garcia- Romo et al., 2011; Hagberg et al., 2011). As neutrophils significantly outnumber pDCs in vivo, pathogenic self-amplifying loops involving neutrophils might be highly relevant to disease.

[0054] The role of TLR7 is SLE pathogenesis is well accepted. Thus, TLR7 polymorphisms increase SLE risk in some ethnic groups (Shen et al., 2010), and TLR7 duplication in mice accelerates autoimmunity (Pisitkun et al., 2006). Furthermore, crossing SLE-prone mice with TLR7 but not TLR9 KO strains ameliorates disease (Wu and Peng, 2006). In addition, ssRNA-protein complexes are a common autoantigen in human SLE. Thus, autoantibody screening using a highly sensitive solution phase luciferase immunoprecipitation system (LIPS) recently found that >80 SLE patients express such specificities (Ching et al., 2012), which are associated with higher IFN signatures in patients (Niewold et al., 2007).

[0055] Additional SLE auto specificities trigger neutrophil activation and DNA release through the recognition of anti-microbial peptides displayed on their surface (Lande et al., 2011). Furthermore, a population of low-density neutrophils that is expanded in SLE blood (Pascual et al., 2010) seems to spontaneously release depolymerized chromatin DNA in the form of extracellular traps (NETs) (Brinkmann et al., 2004) that induce both endothelial cell death and pDCs activation (Villanueva et al., 2011).

[0056] Activated granulocytes release mitochondrial DNA (mtDNA) in culture in the absence of cell membrane disruption (Yousefi et al., 2009) (Yousefi et al., 2008) but the release mechanism and the potential role of mtDNA in IFN production and/or lupus pathogenesis have never been addressed. MtDNA contains hypomethylated CpG motifs that resemble bacterial DNA. When injected into mice joints, it causes inflammatory arthritis (Collins et al., 2004). In addition, mtDNA released by necrotic cells during severe trauma directly activates neutrophils through TLR9 engagement (Zhang et al., 2010). Moreover, cytoplasmic leakage of mtDNA, or lack of its appropriate disposal due to mitophagy defects, lead to cell autonomous activation of NALP3 inflammasome and TLR9, respectively (Nakahira et al., 2011; Oka et al., 2012).

[0057] It was previously described that activation of neutrophils under SLE-like conditions led to the extrusion of highly interferogenic DNA (Garcia-Romo et al., 2011). This phenomenon was interpreted in the context of chromatin depolarization and NET release (Brinkmann et al., 2004).

[0058] In certain aspects of the invention, it was discovered that live human neutrophils spontaneously extrude mitochondrial DNA-protein complexes (mtC) under steady state conditions. These mtC derive from depolarized mitochondria and lack the capacity to activate pDCs. Activation with type I IFN and TLR7-agonistic SLE-specific autoantibodies interferes with a novel neutrophil- specific pathway that routes oxidized mtDNA into lysosomes for degradation. As the result of this, extruded mtC become enriched in oxidized residues that explain their exceptional interferogenic effect and might be central to SLE pathogenesis.

III. PHARMACEUTICAL COMPOSITIONS

[0059] Methods and compositions may be provided for the treatment of SLE.

[0060] The term "treatment" or "treating" means any treatment of a disease in a mammal, including:

[0061] (i) preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease;

[0062] (ii) suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease;

[0063] (iii) inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; and/or

[0064] (iv) relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance.

[0065] The term "effective amount" means a dosage sufficient to provide treatment for the disease state being treated. This will vary depending on the patient, the disease and the treatment being effected. [0066] In certain embodiments, there may be provided methods and compositions involving pharmaceutical compositions that comprise one or more therapeutic agents as described herein.

[0067] For example, pan-PDE inhibitor or mitochondrial PDE inhibitors may be used. A phosphodiesterase (PDE) inhibitor is an agent, such as a drug or an inhibitory nucleic acid, that blocks or inhibits one or more subtypes of the enzyme phosphodiesterase (PDE), thereby preventing the inactivation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by the respective PDE subtype(s). PDE inhibitor may refer to any member of the class of compounds having an IC50 of 100 μΜ or lower concentration for a phosphodiesterase, for example, at least or at most or about 200, 100, 80, 50, 40, 20, 10, 5, 1 μΜ, 100, 10, 1 nM or lower concentration. When a PDE inhibitor is described herein as having activity against a particular type of PDE, the inhibitor may also have activity against other types, unless otherwise stated.

[0068] Non-limiting examples of pan-PDE or Nonselective PDE inhibitors include methylated xanthines and derivatives such as: caffeine; aminophylline; IB MX (3-isobutyl-l- methylxanthine; paraxanthine; pentoxifylline; theobromine; theophylline, a bronchodilator.

[0069] In other embodiments, a PDE inhibitor may be a molecule or composition that inhibits the expression of a target PDE, such as an antisense nucleotide (e.g., siRNA) that specifically hybridizes with the mitochodiral mRNA or DNA, or in some aspects, cellular mRNA and/or genomic DNA corresponding to the gene(s) of the target PDE so as to inhibit their transcription and/or translation, or a ribozyme that specifically cleaves the mRNA of a target PDE. Antisense nucleotides and ribozymes can be delivered directly to cells, or indirectly via an expression vector which produces the nucleotide when transcribed in the cell. Methods for designing and administering antisense oligonucleotides and ribozymes are known in the art, and are described, e.g., in Mautino et al., 2002; Pachori et al., 2002, herein incorporated by reference. Examples of antisense compositions against PDEs include, e.g., the anti-PDE4 compositions disclosed in US20030045490 and WO 00/40714 , and the anti- PDE1 and anti-PDE4 compositions disclosed in 5,885,834, all of which are herein incorporated by reference. [0070] Protein kinase A (PKA) is a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase (EC 2.7.11.11). Protein kinase A has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism. PKA enhancers may include any cAMP analogs that can enhance PKA activity or active PKA-mediate pathway such as 8-Br-cAMP, which is an analogue of the natural signal molecule cyclic AMP in which the hydrogen in position 8 of the heterocyclic nucleobase is replaced by bromine.

[0071] A PDE inhibitor or a PKA enhancer may be combined with mitochondrial targeting methods for mitochondria targeting.

[0072] Advanced glycosylation end product- specific receptor, also known as receptor for advanced glycosylation end products, AGER and RAGE, is a single-pass type I membrane protein and belongs to the immunoglobulin superfamily. AGER / RAGE contains two Ig-like C2-type (immunoglobulin-like) domains and one Ig-like V-type (immunoglobulin-like) domain. AGER / RAGE mediates interactions of advanced glycosylation end products (AGE). These are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. AGER / RAGE acts as a mediator of both acute and chronic vascular inflammation in conditions such as atherosclerosis and in particular as a complication of diabetes.

[0073] The term "anti-RAGE antibody" as used herein encompass to all types of antibodies which, preferably, specifically binds to RAGE and inhibits RAGE activity. Particularly the antibody may be a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody or any fragment or derivative of such antibodies being still capable of binding to RAGE and inhibiting at least one of its biological activities. Such fragments and derivatives comprised by the term antibody as used herein encompass a bispecific antibody, a synthetic antibody, an Fab, F(ab)2Fv or scFv fragment, or a chemically modified derivative of any of these antibodies. Specific binding as used in the context of the antibody of the present invention means that the antibody does not cross-react with other polypeptides. Specific binding can be tested by various well known techniques. Preferably, specific binding can be tested as described in the accompanying Examples. Antibodies or fragments thereof, in general, can be obtained by using methods which are described, e.g., in Harlow and Lane (1988). Monoclonal antibodies can be prepared by the techniques which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals and, preferably, immunized mice (Kohler (1975) and Galfre (1981)). Preferably, an immunogenic peptide having the extracellular domain of RAGE is applied to a mammal. The said peptide is, preferably, conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants encompass, preferably, Freund's adjuvant, mineral gels, e.g., aluminum hydroxide, and surface active substances, e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Monoclonal antibodies which specifically bind to RAGE can be subsequently prepared using the well known hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique. Specifically binding antibodies which affect at least one biological activity of RAGE can be identified by assays known in the art and described, e.g., in the accompanying Examples, below.

[0074] In a particular embodiment, the RAGE inhibitors may comprise a RAGE-Fc chimera comprising the human AGER isoform 1 ( NP_001127.1 ) extracellular domain ( Met 1 - Ala 344 ) fused with the Fc region of human IgGl at the C-terminus. [0075] WO 2008/137552 A2 (incorporated herein in its entirety) discloses certain monoclonal anti-RAGE antibodies binding to different domains of RAGE. Most of said antibodies inhibit the interaction of human RAGE and a complex of HMGB land CpG DNA. WO 2006/077101 (incorporated herein in its entirety) relates to the identification, functionality and use of peptides designated AGER-RME and AGER-CDP of RAGE. Said peptides are inter alia applicable for identifying and preparing RAGE binding ligands like anti-RAGE antibodies. WO 2009136382 (incorporated herein in its entirety) describes certain monoclonal antibodies that bind to the C-domains of RAGE and the specific interaction and competition with the binding of Αβ with monoclonal antibodies for the CI and C2-domain in RAGE. [0076] The compounds useful in the methods may be in the form of free acids, free bases, or pharmaceutically acceptable addition salts thereof. Such salts can be readily prepared by treating the compounds with an appropriate acid. Such acids include, by way of example and not limitation, inorganic acids such as hydrohalic acids (hydrochloric, hydrobromic, hydrofluoric, etc.), sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as acetic acid, propanoic acid, 2-hydroxyacetic acid, 2-hydroxypropanoic acid, 2- oxopropanoic acid, propandioic acid, and butandioic acid. Conversely, the salt can be converted into the free base form by treatment with alkali. [0077] Aqueous compositions in some aspects comprise an effective amount of the therapeutic compound, further dispersed in pharmaceutically acceptable carrier or aqueous medium. The phrases "pharmaceutically or pharmacologically acceptable" refer to compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.

[0078] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[0079] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0080] In certain embodiments, the compositions may be targeted to mitochondria by any methods known in the art, for example, by a mitochondrial targeting signal peptide. An example of the mitochondrial targeting signal is a 10-70 amino acid long peptide that directs a newly synthesized proteins to the mitochondria. It is found at the N-terminus and consists of an alternating pattern of hydrophobic and positively charged amino acids to form what is called an amphipathic helix. Mitochondrial targeting signals can contain additional signals that subsequently target the protein to different regions of the mitochondria, such as the mitochondrial matrix. Like signal peptides, mitochondrial targeting signals are cleaved once targeting is complete.

[0081] The pharmaceutical compositions may be advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. For instance, the composition may contain at least about, at most about, or about 1, 5, 10, 25, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0082] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti- oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0083] Administration of pharmaceutical compositions will be via any common route so long as the target tissue, cell or intracellular department is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration will be by orthotopic, intradermal subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. Volume of an aerosol is typically between about 0.01 mL and 0.5 mL.

[0084] Additional formulations may be suitable for oral administration. "Oral administration" as used herein refers to any form of delivery of an agent or composition thereof to a subject wherein the agent or composition is placed in the mouth of the subject, whether or not the agent or composition is swallowed. Thus, "oral administration" includes buccal and sublingual as well as esophageal administration. Absorption of the agent can occur in any part or parts of the gastrointestinal tract including the mouth, esophagus, stomach, duodenum, ileum and colon. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0085] In one embodiment, the oral formulation can comprise the therapeutic compounds and one or more bulking agents. Suitable bulking agents are any such agent that is compatible with the therapeutic compounds including, for example, lactose, microcrystalline cellulose, and non-reducing sugars, such as mannitol, xylitol, and sorbitol. One example of a suitable oral formulations includes spray-dried therapeutic compounds- containing polymer nanoparticles (e.g., spray-dried poly(lactide-co-glycolide)/amifostine nanoparticles having a mean diameter of between about 150 nm and 450 nm; see Pamujula, et al., 2004, which is here by incorporated by reference in its entirety). The nanoparticles can contain between about 20 and 50 w/w % therapeutic compounds for example, between about 25% and 50%.

[0086] In some embodiments, when the route is topical, the form may be a cream, ointment, salve or spray. Topical formulations may include solvents such as, but not limited to, dimethyl sulfoxide, water, Ν,Ν-dimethylformamide, propylene glycol, 2-pyrrolidone, methyl-2-pyrrolidone, and/or N-methylforamide. To enhance skin permeability, if necessary, the skin area to be treated can be pre-treated with dimethylsulf oxide; see Lamperti et al., 1990, which is hereby incorporated by reference in its entirety.

[0087] In other embodiments, the pharmaceutical compositions may be for subcutaneous administration (e.g., injection and/or implantation). For example, implantable forms may be useful for patients which are expected to undergo multiple CT scans over an extended period of time (e.g., one week, two weeks, one month, etc.). In one example, such subcutaneous forms can comprise the therapeutic compounds and a carrier, such as a polymer. The polymers may be suitable for immediate or extended release depending on the intended use. In one example, the therapeutic compounds can be combined with a biodegradable polymer (e.g., polylactide, polyglycolide, and/or a copolymers thereof). In another example, subcutaneous forms can comprise a microencapsulated form of the therapeutic compounds, see, e.g., Srinivasan et al., 2002, which is hereby incorporated by reference in its entirety. Such microencapsulated forms may comprise the therapeutic compounds and one or more surfactant and other excipients (e.g., lactose, sellulose, cholesterol, and phosphate- and/or stearate-based surfactants).

[0088] In a further embodiment, the therapeutic compounds or pharmaceutical compositions may be administered transdermally through the use of an adhesive patch that is placed on the skin to deliver the therapeutic compounds through the skin and into the bloodstream. An advantage of the transdermal drug delivery route relative to other delivery systems such as oral, topical, or intravenous is that the patch provides a controlled release of the therapeutic compound into the patient, usually through a porous membrane covering a reservoir of the therapeutic compound or through body heat melting thin layers of therapeutic compound embedded in the adhesive. In practicing certain aspects, any suitable transdermal patch system may be used including, without limitation, single-layer drug-in-adhesive, multilayer drug-in-adhesive, and reservoir.

[0089] The pharmaceutical compositions may optionally further comprise a second therapeutic agent. The second therapeutic agent can be an antioxidant. Examples of suitable antioxidants include, but are not limited to ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, β-carotene, lycopene, lutein, resveratrol, retinol (vitamin A), a-tocopherol (vitamin E), ubiquinol, selenium, and catalase. In certain embodiments, the second therapeutic agent is vitamin E, selenium or catalase. [0090] An effective amount of the pharmaceutical composition is determined based on the intended goal, such as treating SLE, reducing extrusion of oxidized DNA from neutrophil mitochondria, or leading to a decrease of mitochondria PDE activity, an increase of mitochondria PKA, or an decrease of RAGE of a cell. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen.

[0091] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect, for example, a decrease of mitochondria PDE activity, an increase of mitochondria PKA, or an decrease of RAGE of a cell. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these compounds. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg/kg, mg/kg, μg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.

[0092] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 μΜ to 150 μΜ. In another embodiment, the effective dose provides a blood level of about 4 μΜ to 100 μΜ.; or about 1 μΜ to 100 μΜ; or about 1 μΜ to 50 μΜ; or about 1 μΜ to 40 μΜ; or about 1 μΜ to 30 μΜ; or about 1 μΜ to 20 μΜ; or about 1 μΜ to 10 μΜ; or about 10 μΜ to 150 μΜ; or about 10 μΜ to 100 μΜ; or about 10 μΜ to 50 μΜ; or about 25 μΜ to 150 μΜ; or about 25 μΜ to 100 μΜ; or about 25 μΜ to 50 μΜ; or about 50 μΜ to 150 μΜ; or about 50 μΜ to 100 μΜ (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the compound that results from a therapeutic compound being administered to a subject: about, at least about, or at most about 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μΜ or any range derivable therein. In certain embodiments, the therapeutic compound that is administered to a subject is metabolized in the body to a metabolized therapeutic compound, in which case the blood levels may refer to the amount of that compound. Alternatively, to the extent the therapeutic compound is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic compound.

[0093] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0094] It will be understood by those skilled in the art and made aware of this invention that dosage units of μg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of μg/ml or mM (blood levels), such as 4 μΜ to 100 μΜ. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein. IV. EXAMPLES

[0095] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 - Live neutrophils spontaneously extrude mitochondrial DNA-protein complexes.

[0096] To characterize the composition of the interferogenic factors released by activated SLE neutrophils, we first collected the supernatants from non-activated healthy and SLE neutrophils after 6 h in culture. Electrophoresis of these supernatants revealed the presence of DNA-protein complexes that upon proteinase K digestion yielded a single DNA band with a MW of -16Kb, which corresponds to the size of the mitochondrial genome (Fig. 1A). The mitochondrial origin of this DNA was confirmed by amplification of the NADH Dehydrogenase 1 (ND1) gene (Fig. 8A) and by the presence of the mitochondrial DNA binding protein TFAM in both total supernatant (Fig. IB) as well as in immunoprecipitated DNA fractions (Fig. 8C). The absence of the nuclear DNA-binding protein H3 (Figs. IB and 8C) further supports the mitochondrial origin of the extruded complexes. This process appears to be selective for some mitochondrial components, as we could detect mitochondrial matrix proteins such as MnSOD (Fig. IB) but not inner (IMM) or outer (OMM) membrane proteins such as Mitofilin (not shown) and TOMM20 (Fig. IB). The absence of the cytoplasmic protein GAPDH and LDH enzymatic activity (Figs. IB and 8B) together with the inability of the cells to incorporate Sytox Orange (not shown), rule out non-specific membrane disruption and/or necrosis.

[0097] Neutrophils are short living cells that undergo spontaneous apoptosis in vitro and in vivo (Caielli et al., 2012). To further rule out that mitochondrial DNA extrusion is related to cell death, we inhibited apoptosis by exposing neutrophils to GM-CSF. This exposure increases neutrophil lifespan (as assessed by TUNEL assay) without affecting the amount of extruded DNA (Fig. 8E). Selective stimulation with the TLR7 agonist R837, but not with the TLR9 agonist ODN2216 or other TLR ligands (not shown), increased the amount of extracellular DNA, suggesting that this event is an active process (Fig. IF).

[0098] While all tested healthy neutrophils extrude 150-400 ng/ml of mtDNA in culture, neutrophils from -1/3 of SLE patients extrude up to double this amount (Fig. 1C). Neutrophils characteristically display low mitochondrial mass compared to monocytes, which do not extrude significant amounts of mtDNA in culture (Fig. ID). The mitochondrial morphology in these two cell types is also different (Fig. 8D), as neutrophil mitochondria show matrix condensation and expansion of the cristae space. This unusual morphology has been described in healthy and cancer cells upon switching from glycolysis to oxidative phosphorylation (Hackenbrock et al., 1971; Rossignol et al., 2004).

[0099] To determine the stability of the extruded mtDNA, we tested the effect of 2- 20% serum from healthy donors and SLE patients. At the higher concentration (20%) all tested healthy donor sera induced the degradation of mtDNA, while 50% of the tested SLE patient sera failed to do so (Fig. IE). The observed DNA degradation with high serum concentration is due to DNAse I activity as it is partially inhibited by G-actin (Fig. IE).

[00100] Thus, healthy and SLE neutrophils spontaneously release mitochondrial DNA/protein complexes (mtC) in the absence of overt cell death and/or membrane disruption. This extrusion is selectively enhanced by endosomal TLR7 ligation, suggesting that it might be an active process. EXAMPLE 2 - Extruded mtC derive from depolarized mitochondria.

[00101] We surmised that neutrophil mtC extrusion might be the result of mitochondrial damage such as depolarization. To track depolarization we used Mitotracker DeepRed (MTDR), which selectively accumulates inside respiring polarized mitochondria, together with antibodies against TFAM, a transcription factor that tightly coats mtDNA into compact aggregates known as nucleoids (Hallberg and Larsson, 2011). Confocal microscopy showed that monocyte nucleoids universally co-localize with respiring mitochondria, while 20-70% of neutrophil nucleoids do not (Figs. 2A and 9A). Furthermore, the depolarized neutrophil structures (TFAM+/MTDR-) are in some areas intimately associated with their polarized counterparts (TFAM+/MTDR+) (Fig. 2A, arrowheads), suggesting that they might be the result of a fission event. [00102] To better characterize these mitochondria-derived structures, we performed transmission electron microscopy. In addition to the classic neutrophil cytoplasmic electro-dense granules (Fig. 2BI; arrowhead), we observed large electro-lucent vesicles (Fig. 2BI; yellow arrowhead) that appear to differ from double-membrane autophagosomes (Fig. 9BI) and/or phagosomes (Fig. 9BII). These vesicles contain strands of electron-dense material that resembles depolymerized nucleic acid associated with proteins. They are found both as independent structures (Fig. 2BII) or joined to healthy mitochondria (Fig. 2BIII). In this case, it is possible to recognize the two condensed mitochondrial poles at the edges of the vesicles, resembling the final stage of a mitochondria fission event (Fig. 2BIII, arrowheads). [00103] Depolarized mitochondria can acquire a vesicular-like structure upon proteasome-mediated membrane rupture. This event requires the translocation of PARKIN to mitochondria and the subsequent ubiquitinilation of the outer mitochondrial membrane (OMM) (Yoshii et al., 2011). In our system however, immunofluorescence analysis showed neither significant PARKIN translocation nor membrane ubiquitinilation (Fig. 9F). No co- localization was found either with the lysosomal marker LAMPl (Fig 9G), thus ruling out that these vesicles are autolysosomes and/or lysosomes. Moreover, these vesicles are frequently found in the periphery of the cell, where they fuse with the plasma membrane (Fig. 2C) and release their cargo (Fig. 2D). Their mitochondrial origin was confirmed by detecting the translocation of the OMM protein TOMM20, but not the lysosomal trans- membrane protein LAMPl, on the plasma membrane (Figs. 2C and 9C). As a result of this fusion, DNAse-I sensitive aggregates of dsDNA could be visualized on the neutrophil surface (Fig. 2D). The translocation of TOMM20 into the plasma membrane is in line with the observed selective extrusion of mitochondrial matrix components (Fig. IB).

[00104] The docking and fusion of mitochondrial vesicles to the plasma membrane is reminiscent of synaptic vesicle exocytosis, a calcium dependent process (de Saint Basile et al., 2010; Reddy et al., 2001; Yogalingam et al., 2008). To assess whether calcium signals are involved in mitochondrial vesicle trafficking, intracellular calcium levels were quenched with BAPTA-AM, a widely used calcium chelator. As shown in Fig. 2E, calcium quenching significantly reduced the amount of extruded mtC. In contrast to the process of chromatin extrusion during NETosis (Brinkmann et al., 2004), extrusion of mtDNA appears to be ROS-independent, as neither DPI (NADPH inhibitor) nor MitoTempo (mtROS scavenger) could block it (Fig. 9E). [00105] Thus, under the steady state depolarized neutrophil mitochondria swell and acquire a vesicular structure. These vesicles traffic towards the plasma membrane and extrude their cargo that is enriched in mtDNA.

EXAMPLE 3 - Extrusion of mitochondrial DNA-protein complexes might represent a neutrophil alternative to mitophagy.

[00106] As damaged mitochondria are usually removed through mitophagy

(Ashrafi and Schwarz, 2013), we surmised that the formation of depolarized mitochondrial vesicles in neutrophils could result from defective mitophagy.

[00107] Since only a small fraction of mitochondria undergoes spontaneous depolarization under steady-state conditions, we amplified this phenomenon using the protonophore CCCP. As shown in Fig. 3A, this decoupling agent, which rapidly depolarizes the entire mitochondrial pool, increases the amount of extruded mtC, as assessed by increased mtDNA in the neutrophil supernatants (Fig. 3B) and decreased TFAM in the total cell lysates (Fig. 3C). This effect is specific to neutrophils, as monocytes exposed to CCCP do not extrude more mtC (Fig. 3B) despite reaching a similar degree of mitochondrial depolarization (not shown). To test if mtC extrusion is a universal response to mitochondrial injury in neutrophils, we used the complex I inhibitor rotenone, which increases mtROS without inducing depolarization (Chu et al., 2013). As shown in Fig. 3B, however, this compound does not increase mtC extrusion. [00108] The process of mitophagy includes three sequential steps: 1) autophagy machinery activation; 2) damaged cargo recognition/sequestration into the autophagosome; and 3) autophagosome fusion with the lysosomal compartment for degradation (Ashrafi and Schwarz, 2013). To elucidate the mitophagy step(s) that may be impaired in neutrophils, we used immunofluorescence to track CCCP-induced mitochondrial damage. Both in neutrophils and monocytes this compound is able to activate the autophagy machinery, as shown by the dramatic increase in LC3B puncta staining (Fig. 10A). The subsequent cargo recognition/sequestration step does not seem to be impaired in neutrophils either, as shown by the complete recruitment of LC3B membranes to damaged mitochondria (Fig. 10A). The last autophagy step, autophagosome-lysosome fusion, can be assessed by the degree of TOMM20 co-localization with the lysosomal marker LAMP1. While fusion of autophagosomes with lysosomes in monocytes was complete after 60 min of CCCP exposure, this process did not occur in neutrophils (Figs. 3D and 10B). [00109] To identify the molecular basis of this defect, we compared the gene expression profiles of neutrophils and monocytes upon 60 min exposure to CCCP. The analysis revealed that neutrophils downregulate a series of transcripts involved in different steps of autolysosome formation compared to monocytes (Figs. 3E and IOC). These steps include autophagy progression (ULK2, ULK3), autophagosome movement (Rab27a, Rab4a), autophagosome docking/fusion (NSF, NAPA, SNAP23, SNAP29, STX2, LAMP2) and lysosome acidification/activation (ATP6V0C, Presenilin-1). Over all, TOM1 came out as the most down-regulated transcript. This gene encodes an ESCRT-0 protein that plays a pivotal role in the maturation of the autophagosome (Rusten and Stenmark, 2009; Tumbarello et al., 2012). In addition, CCCP treatment of neutrophils fails to inhibit mTOR (Fig. 10D), a process recently associated with lysosome activation during the progression of autophagy (Zhou et al., 2013).

[00110] Thus, the steady-state extrusion of neutrophil mtC is linked to mitochondrial depolarization. This most likely represents an alternative pathway to eliminate damaged mitochondria due to the inability of neutrophils to undergo classic mitophagy.

EXAMPLE 4 - MtDNA oxidation is required for extruded neutrophil mtC to activate pDCs.

[00111] We reported that SLE neutrophils activated with anti-Sm/RNP antibodies extrude interferogenic DNA, an effect that can be reproduced in healthy neutrophils provided they are primed with IFNa (Garcia- Romo et al., 2011). To examine the role of IFN and anti-Sm/RNP antibodies in mtC extrusion, we quantified the amount of mtDNA and TFAM protein in the supernatants of neutrophils cultured with each of these activators alone or in combination. Similar concentrations of both mtDNA and TFAM were found regardless of the stimulus (Figs. 4A and 11A). As only the "lupus-like" combination induced the extrusion of interferogenic mtC (Fig. 4A), we surmised that unique DNA and/or protein modifications might be responsible for converting inert complexes into potent pDC activators.

[00112] The most common DNA modification associated with the acquisition of immuno stimulatory capacity is oxidation of purine and pyrimidine bases by ROS (Gehrke et al., 2013; Mathew et al., 2012; Shimada et al., 2012; Yoshida et al., 2011). mtDNA is more susceptible to oxidative damage than nuclear DNA due to its lack of association to histones, proximity to the respiratory chain and inefficient repair machinery (Yakes and Van Houten, 1997). Thus, we tested the impact of scavenging mitochondrial ROS on the generation/extrusion of immuno stimulatory mtC. Indeed, incubation of neutrophils with the mitochondrial-targeted MitoTEMPO (MT) abrogated the release of interferogenic mtC without affecting the total amount of extruded DNA (Fig. 4B). Conversely, high levels of 8- hydroxydeoxyguanosine (8-OHdG), the most common marker of DNA oxidative damage (Aruoma et al., 1989; Kasai and Nishimura, 1984), were only detected in the DNA extruded by neutrophils activated with both IFN and anti-Sm/RNP antibodies (Figs. 4C, 11B, 11C). This effect requires TLR7 engagement, as it can be reverted using TLR7- but not TLR8- specific antagonists (Fig. 4D). [00113] Synthetic TLR7 agonists such as the imidazole compound R837 trigger actually strong mitochondrial depolarization and subsequently mtDNA extrusion, but they do not increase its oxidation status (Fig 4F). Thus, different internalization routes leading to unique subcellular compartments or a differential ability to recruit adaptor molecules to TLR7 might be responsible for these differences. [00114] To further confirm that mtDNA oxidation is required to induce pDC activation, we used rt-PCR to generate oxidized (Ox) and non-oxidized (NonOx) mtDNA fragments. Neither of these DNA preparations alone activated pDCs in vitro. Addition of the neutrophil cationic peptide carrier LL37 led to the production of large amounts of IFNa only in the presence of Ox mtDNA (Fig. 11D). We next explored if the superior capacity to induce IFN could be due to better internalization rates and/or intracellular stability of Ox mtDNA- LL37 complexes in pDCs, but failed to detect any difference with their NonOx counterparts (Fig HE). Furthermore, they both localize to the same endosomal (TfR+) compartment and do not traffic to lysosomes (Fig. 11F).

[00115] While activated neutrophils secrete high levels of LL37 and HMGB1 (Garcia- Romo et al., 2011), neither protein is an integral part of the interferogenic mtC, as they do not co-immunoprecipitate with the mtDNA from neutrophil supernatants.

Furthermore, the HMGB1 antagonist BoxA does not block pDC activation (Fig. 11G).

Within mitochondria, however, DNA is tightly associated with TFAM, a cationic protein that facilitates the internalization of nucleic acids into pDCs through the engagement of RAGE (Julian et al., 2012). Indeed, blocking either TFAM or RAGE totally abrogated the interferogenicity of the extruded neutrophil mtC (Fig. 4E). [00116] Taken together, these data indicates that oxidation is essential for the conversion of inert self-DNA into a potent pDC activator.

EXAMPLE 5 - Oxidized mtDNA is constitutively exported within cytosolic vesicles from mitochondria to lysosomes.

[00117] Our data show that neutrophils steadily extrude non-oxidized mtDNA.

We next wondered how these cells manage to dispose of oxidized DNA that is constantly generated in the mitochondrial matrix in the absence of a repair DNA machinery and/or classic mitophagy. To visualize intracellular Ox mtDNA, we labeled neutrophils and monocytes with antibodies against 8-OHdG. These experiments revealed the presence of abundant 8-OHdG (+) vesicles only in the neutrophil cytosol (Fig. 5A).

[00118] Partially damaged mitochondria have developed distinct mechanisms to selectively remove their oxidized components (proteins or nucleic acids) without engaging the mitophagy process. These include chaperone-mediated extraction (Margineantu et al., 2007) and mitochondrial derived vesicle (MDV) formation (Neuspiel et al., 2008; Soubannier et al., 2012a; Soubannier et al., 2012b). In this latter case, the oxidized cargo is sorted inside micro-vesicles (70-100 nm) that can incorporate either inner or outer mitochondrial membrane proteins. Co-localization experiments in neutrophils demonstrate that 8-OHdG (+) vesicles incorporate the IMM protein Mitofilin but not the OMM protein TOMM20 (Figs. 5B and 12A). Furthermore, the fact that these vesicles do not include TFAM (Fig. 12A) supports that mtDNA dissociates from this nucleoid component before being sorted into them.

[00119] Upon budding from the mitochondria, MDV are delivered to peroxisomes and/or lysosomes for degradation (Neuspiel et al., 2008; Soubannier et al., 2012a). Neutrophil 8-OHdG (+) vesicles both i) accumulate in the cytosol in the presence of the lysosomal inhibitor Bafilomycin Al, and ii) co-localize with the lysosomal marker LAMP1, indicating their merging with the lysosomal compartment (Fig. 12B). The mechanism of MDV budding is not completely understood, but a potential role for the permeability transition pore (PTP) opening could be entertained. Nevertheless, culturing neutrophils in the presence of high concentrations of the PTP opening inhibitor CsA did not alter the number of 8-OHdG (+) vesicles (Fig. 12C). [00120] In addition to the complete co-localization of oxidized DNA with

Mitofilin (Fig. 5B), the mitochondrial origin of this cytosolic DNA was further confirmed by i) lack of co-localization with the marker of lethally damaged nuclear DNA γ.Η2Α.Χ (Fig. 5C), ii) down-modulation upon scavenging of mitochondrial ROS with MT, and iii) accumulation upon increasing mtROS production with Rotenone (Fig. 5D).

[00121] Taken together these data reveal the presence of a mytophagy- independent, MDV-like pathway that removes Ox DNA from healthy neutrophil mitochondria and routes it to lysosomes for degradation.

EXAMPLE 6 - Type I IFN and anti-Sm/RNP antibodies synergistically block the detoxification of oxidized mtDNA in neutrophils.

[00122] Type I IFN and anti-Sm/RNP antibodies induce the extrusion of Ox mtDNA by neutrophils, while normally this damaged DNA is diverted into lysosomes. Quantitative Immunofluorescence Analysis shows that the "lupus-like" combination considerably increases the amount of cytoplasmic Ox mtDNA fragments (Fig. 6A) that are neither incorporated within MDVs nor free in the cytoplasm. Instead, they form large aggregates inside the mitochondria, as shown by their complete co-localization with TOMM20 (Figs. 6B and 13A), and cluster in one pole of the cell (Figs. 6B and 6C). These "mito-aggregates" are reminiscent of those observed in CCCP-treated neurons, which represent a hallmark of mitochondrial stress/damage (Okatsu et al., 2010; Vives-Bauza et al., 2010).

[00123] Since this phenotype can be recapitulated by treating control neutrophils with the mtROS generator Rotenone (Fig. 13B), we asked whether the IFN /anti- Sm/RNP antibody effect would be mediated through the induction of ROS production. Lack of mitochondrial and/or total cellular ROS increase under these conditions ruled out, however, this possibility (Fig. 13B). An alternative explanation could be that "lupus-like" activation of neutrophils would interfere with the incorporation of oxidized cargo into MDVs.

[00124] Under steady-state conditions, cytoplasmic Ox mtDNA does not co- localize with TFAM. Due to the small dimensions of MDVs compared to nucleoids, damaged mtDNA might need to detach from TFAM before being incorporated into these vesicles. MtDNA/TFAM dissociation is normally prompted by TFAM phosphorylation through the mitochondrial resident Protein Kinase A (PKA) (Lu et al., 2013). This post-translational modification reduces the affinity of TFAM for mtDNA and subsequently promotes TFAM degradation by the AAA+ Lon protease (Fig. 14A). Our results show that lupus-like activation conditions lead to an accumulation of intracellular TFAM (Fig. 7A). This event reflects a decrease in TFAM degradation rather than an increase in its biosynthesis, as suggested by the lack of concomitant up-regulation of the TFAM master regulator PCGl (Fig. 14B) (Hock and Kralli, 2009). The increase in TFAM is not a consequence of mitogenesis either, as other mitochondrial proteins such as TOMM20 or Mitofilin, as well as the total mitochondrial mass did not change (Fig. 14C).

[00125] IFNa and anti-Sm/RNP antibodies not only slow-down TFAM degradation but also increase its association with oxidized mtDNA, as demonstrated by both IF and IP (Fig. 7B). Using antibodies to detect PKA-specific and non-specifically phosphorylated substrates, we found a significant reduction in phosphorylated TFAM in neutrophils activated under lupus conditions (Figs. 7C and 14D). This can be recapitulated by treating neutrophils with the selective PKA inhibitor H89 (Fig. 7C). However lupus-like activation does not directly inhibit PKA, as concomitant treatment with the non-hydrolysable reactive cAMP-analog 8Br-cAMP rescues the detoxification pathway (Figs. 7D). PKA activity is regulated by phosphodiesterases (PDE), which quickly convert cAMP to AMP to reduce the amount of cyclic nucleotides required for kinase activation. Mammalian cells contain different isoforms of PDE distributed along different subcellular compartments, including mitochondria (Acin-Perez et al., 2011; Acin-Perez et al., 2009). In fact, we show that the detoxification pathway originally blocked by IFNa and anti-Sm/RNP antibodies can be rescued by treatment with the pan-PDE inhibitor IB MX (Fig. 7D).

[00126] Thus, it was discovered that for IFN and SLE-specific TLR7 agonistic antibodies on the activation of mitochondrial-resident PDEs, which leads to the inhibition of mitochondrial nucleoid (TFAM/mtDNA) disassembly, intra-mitochondrial accumulation and subsequent release of oxidized DNA.

EXAMPLE 7 - Live neutrophils spontaneously extrude mitochondrial DNA-protein complexes

[00127] Short-term (6h) cell-free supernatants from non-activated,neutrophils from healthy individuals contain DNA-protein complexes that, upon digestion with proteinase K, yield a 16Kb DNA band, the size of mtDNA (FIGS. 15 A and 16A). This origin was confirmed by i) selective amplification of the mitochondrial-encoded gene ND1 (FIG. 16A) and ii) co-immunoprecipitation of DNA and the mitochondrial transcription factor TFAM (FIG. 16B).

[00128] Unlike NETotic or necrotic neutrophils that release gDNA and mtDNA, live neutrophils selectively extrude mtDNA (FIG. 15B). Absence of nuclear (H3) and cytoplasmic (GAPDH) proteins and of LDH activity in supernatants rules out cell membrane disruption (FIG. 15C). Furthermore, addition of GM-CSF, a pro- survival factor for neutrophils (Klein, J.B., et al. Journal of immunology 164, 4286-4291 (2000)), does not decrease the amount of extruded mtDNA (FIG. 15C), supporting that constitutive apoptosis does not drive this process. Extrusion of mtDNA is neutrophil-specific, since monocytes extrude insignificant amounts of mtDNA despite higher mitochondrial mass (FIG. 15D).

[00129] In contrast to chromatin extrusion during NETosis (Brinkmann, V., et al. Science 303, 1532-1535 (2004)), extrusion of mtDNA is ROS -independent as neither DPI nor MitoTempo (MT) blocks it (FIG. 16D).

[00130] As reported (Yousefi, S., et al. Cell death and differentiation 16, 1438- 1444 (2009)), stimulation with GM-CSF plus LPS amplifies mtDNA extrusion (FIG. 16E). Furthermore, TLR7- but not TLR9- synthetic agonists enhance mtDNA extrusion in the absence of cell lysis (FIGS. 15E and 16E).

[00131] Western Blot analysis of cell free supernatants from neutrophils reveals the presence of mitochondrial matrix (TFAM and MnSOD) but not membrane- associated (TOMM20) proteins (FIG. 16F). The translocation of outer and inner membrane proteins (TOMM20 and Mitofilin) to the neutrophil cell surface indicates that mitochondrial membranes fuse with the plasma membrane (FIGS. 15F and 16F); this translocation is enhanced by TLR7-agonists (FIG. 16G).

[00132] Recent studies indicate that autophagy is associated with exocytosis of intracellular components(Duran, J.M., et al. J Cell Biol 188, 527-536 (2010) and Manjithaya, R., et al. J Cell Biol 188, 537-546 (2010)). The inhibition of neutrophil mtDNA release and translocation of TOMM20 to the plasma by the PI3K inhibitor 3-methyladenine (3MA) indicate a possible role for autophagy (FIGS. 15H and 16H). Indeed, LC3B co-localizes with TOMM20 at the plasma membrane (FIG. 15H). Exocytosis of lysosomes(Yogalingam, G., et al. Dev Cell 15, 74-86 (2008)) or autophagosomes containing full mitochondria (Duran, J.M., et al. J Cell Biol 188, 527-536 (2010)) is ruled out by the lack of translocation of LAMP1 and Rab7 (FIG. 15F) and absence of TOMM20 in the supernatants (FIG. 16F).

[00133] Thus, neutrophils spontaneously release mitochondrial DNA/protein complexes (mtC) in the absence of overt cell death and/or membrane disruption in an autophagy-dependent manner.

EXAMPLE 8 - Extrusion of mitochondrial DNA-protein complexes as a neutrophil alternative to mitophagy

[00134] MtC extrusion could be the result of improper disposal of damaged mitochondria. To address this, Applicants amplified mitochondrial depolarization using CCCP or mtROS production using Rotenone (FIG. 17A). CCCP, but not Rotenone, increased mtDNA extrusion (FIG. 17B) and consequently decreased TFAM intracellular levels (FIG. 17C).

[00135] In most cells, damaged mitochondria are removed by mitophagy. This process requires i) autophagy activation; ii) sequestration of damaged mitochondria into autophagosomes; and iii) fusion of autophagosomes with lysosomes (Ashrafi, G. & Schwarz, T.L. Cell death and differentiation 20, 31-42 (2013)). Upon exposure to CCCP, autophagy activation (detected by increased LC3B puncta staining) and cargo sequestration (detected by TOMM20-LC3B co-localization) were similar in neutrophils and monocytes (FIGS. 17D and 18A). However, autophagosome-lysosome fusion (assessed by TOMM20-LAMP1 co- localization) was considerably lower in neutrophils (FIGS. 17E and 18B). Autophagy arrest with Bafilomycin Al (BafAl) reproduces this phenotype in monocytes (FIG. 18C) without increasing mtDNA extrusion, indicating that extrusion is neutrophil- specific (FIG. 18C). Amplification of mitochondrial depolarization with oligomycin/antimycin (O+A) yielded similar results as those obtained with CCCP (FIG. 18D). Of note, the mitochondrial protein content did not decrease in CCCP-treated monocytes, which undergo complete mitophagy, because CCCP blocks lysosomal function (Padman, B.S., et al.. Autophagy 9, 1862- 1875 (2013)) (FIG. 17C).

[00136] Gene expression profiling revealed that in response to CCCP monocytes, but not, neutrophils, upregulate transcripts related to autophagy activation (ULK2, ULK3), autophagosome trafficking (Rab27a, Rab4a) and fusion (NSF, NAPA, SNAP23, SNAP29, STX2, LAMP2), as well as lysosome activation (ATP6V0C, Presenilin- 1). TOM1, which participates in autophagosome maturation (Tumbarello, D.A., et al. Nature cell biology 14, 1024-1035 (2012)), was significantly downregulated in neutrophils (FIG. 17F). Interestingly, CCCP also failed to inhibit mTOR in neutrophils (FIG. 18F), a process associated with lysosome activation during autophagy (Zhou, J., et al. Cell Res 23, 508-523 (2013)).

[00137] Thus, extrusion of mtC might result from a constitutive defect of neutrophil mitophagy.

EXAMPLE 9 - Extruded neutrophil mtC activate pDCs only when enriched in oxidized mtDNA

[00138] SLE neutrophils activated with anti-Sm/RNP antibodies extrude interferogenic DNA while healthy neutrophils require IFNa priming to recapitulate this effect (Garcia-Romo, G.S., et al. Science translational medicine 3, 73ra20 (2011)). Yet, both SLE and healthy neutrophils extrude mtC in the steady state (FIG. 19A). Exposure to IFNa and anti-Sm/RNP antibodies must alter the quality of mtC, as similar concentrations of mtDNA and TFAM were found in supernatants regardless of activation (FIGS. 19A and 20 A). MtDNA is highly susceptible to oxidation (Yakes, F.M. & Van Houten, Proceedings of the National Academy of Sciences of the United States of America 94, 514-519 (1997)), a proinflammatory modification (Shimada, K., et al. Immunity 36, 401-414 (2012)). Indeed, high levels of 8 -hydroxydeoxy guano sine (8-OHdG), a marker of DNA oxidation (Kasai, H. & Nishimura, S. Nucleic acids research 12, 2137-2145 (1984)), were detected only in mtDNA extruded by IFN/aRNP treated neutrophils (FIGS. 19B and 20B). The role of oxidation is further supported by the loss of interferogenicity of extruded DNA upon reduction of 8- OHdG levels with MT (FIGS.19C and 20C) or DPI (FIG. 20D). Neutrophils require TLR7 engagement to release interferogenic mtDNA, as a specific TLR7 -antagonist reduced both the oxidation status and the interferogenicity of extruded mtDNA in response to anti-Sm/RNP antibodies (FIGS. 19D and 20E).

[00139] To further establish that only oxidized mtDNA is a powerful pDC activator, oxidized (Ox) and non-oxidized (Non Ox) mtDNA fragments were generated using rt-PCR. Addition of the cationic peptide LL-37 to facilitate their uptake (Lande, R., et al. Nature 449, 564-569 (2007)) resulted in large amounts of IFNa only in the presence of Ox mtDNA (FIG. 20F). This is not due to differential internalization rates, intracellular stability and/or intracellular localization of these two forms of mtDNA (FIGS. 19G and 20H). Importantly, the ability of gDNA to induce IFNcc production is much lower than that of Ox mtDNA (FIG. 20F).

[00140] While activated neutrophils secrete LL37 and HMGB1, neither protein is an integral part of mtC, as they do not co-immunoprecipitate with mtDNA. Furthermore, the HMGB1 antagonist BoxA does not block pDC activation (FIG. 201). In contrast, blocking either TFAM or its receptor RAGE abrogates the interferogenicity of extruded mtC (FIG. 19E).

[00141] As opposed to IFN/ccRNP antibodies, exposure of neutrophils to synthetic TLR7 ligands amplifies mitochondrial depolarization and the extrusion of Non Ox mtDNA (FIG. 19F). This different outcome might be due to differential compartmentalization of TLR7, as previously reported for CpGA- versus anti-dsDNA IC- activation of TLR9 in pDCs (Henault, J., et al. Immunity 37, 986-997 (2012)).

[00142] Taken together these data indicate that oxidation is essential for self- mtDNA to become a potent pDC activator. EXAMPLE 10 - Ox mtDNA is exported in steady state neutrophils from mitochondria to lysosomes within cytosolic vesicles

[00143] To address how neutrophils handle Ox mtDNA in the steady state,

Applicants stained neutrophils and monocytes with anti 8-OHdG antibodies. Cytosolic 8- OHdG was only detected in neutrophils (FIG. 21A). [00144] Two recent studies showed that damaged gDNA fragments are exported to lysosomes for degradation in cells other than neutrophils (Ivanov, A., et al. J Cell Biol 202, 129-143 (2013) and Lan, Y.Y., et al. Cell reports 9, 180-192 (2014)). Lack of cytosolic staining for both damaged (γ.Η2Α.Χ; FIG. 21B) and fragmented gDNA (TUNEL; FIG. 22A) rules out the nuclear origin of the 8-OHdG(+) cytosolic staining. In addition, its reduction upon mtROS scavenging and accumulation upon mtROS induction support its mitochondrial origin (FIGS. 21C and 22B).

[00145] Sorting of oxidized cargo to lysosomes through mitochondrial-derived vesicles (MDVs) has been described in HeLa cells as a mechanism whereby partially damaged mitochondria remove oxidized components without engaging mitophagy (Soubannier, V., et al. Curr Biol 22, 135-141 (2012)). Accordingly, cytosolic 8-OHdG(+) vesicles accumulate and merge with the LAMP1(+) compartment in the presence of BafAl (FIG. 22C).

[00146] The generation of MDVs does not require mitochondrial fission or autophagy, as it was shown that MDV number is not reduced after DRPl or ATG5 knockdown (Soubannier, V., et al. Curr Biol 22, 135-141 (2012)). Likewise, the generation of neutrophil 8-OHdG(+) vesicles is independent on autophagy (FIG. 22D). While blocking DRPl with MDIVI-1 (Cassidy-Stone, A., et al. Dev Cell 14, 193-204 (2008)) (FIG. 22E) induces intra-mitochondrial accumulation of Ox mtDNA (FIG. 22D), more studies are needed to establish its requirement in this pathway in neutrophils. [00147] To analyze the composition of 8-OHdG(+) vesicles, a mitochondrial cell-free budding assay (Soubannier, V., et al. PloS one 7, e52830 (2012)) was performed. Similar to lysosome-targeting MDVs, It was found that 8-OHdG(+) vesicles selectively incorporate IMM proteins, the matrix enzyme Pyruvate Dehydrogenase (PDH) and mtDNA (FIG. 21D). TFAM, however, is excluded (FIG. 21D), suggesting that Ox mtDNA dissociates from it before its exportation.

[00148] Taken together, these data reveal that Ox mtDNA dissociates from

TFAM and is steadily removed from neutrophil mitochondria and routed to lysosomes.

EXAMPLE 11 - IFN/aRNP block the routing of neutrophil oxidized mtDNA to lysosomes [00149] These studies show that the combination IFN/aRNP induces neutrophils to extrude Ox mtDNA, which in the steady state is diverted into lysosomes. This form of activation also increases the total amount of intracellular Ox mtDNA (FIG. 23A) and its retention inside mitochondria (FIGS. 23B and 24A). This phenotype is not due to increased ROS production or decreased DRPl mitochondrial translocation (FIGS. 23C and 23D).

[00150] While Ox mtDNA is not associated with TFAM in unstimulated neutrophils, Ox mtDNA/TFAM complexes can be easily detected in the presence of IFN/aRNP (FIG. 25A), suggesting an interference with nucleoid disassembly. This activation also increases intracellular TFAM levels (FIG. 25B), which reflects decreased TFAM degradation rather than increased biosynthesis, as supported by lack of up-regulation of the TFAM master regulator PGClcc (Hock, M.B. & Kralli, A. Annual review of physiology 71, 177-203 (2009)) and of increased mitogenesis (FIGS. 26A and 26B).

[00151] TFAM turnover requires dissociation from mtDNA and degradation by the Lon Protease. The dissociation step requires TFAM phosphorylation by Protein Kinase A (PKA) (Lu, B., et al. Mol Cell 49, 121-132 (2013)). As previously reported (Acin-Perez, R., et al. Cell Metab 9, 265-276 (2009) and Ryu, H., et al. Proceedings of the National Academy of Sciences of the United States of America 102, 13915-13920 (2005)), neutrophils express PKA within mitochondria (FIG. 26C). In addition, TFAM turnover in neutrophils is also regulated by PKA and Lon protease, as the inhibition of these two enzymes with H89 and MG132, respectively, increases intracellular TFAM (FIG. 26D). IFN/ccRNP significantly reduce TFAM phosphorylation (FIGS. 25C and 26E) without directly inhibiting PKA, as its activator 8Br-cAMP decreases the extrusion of Ox mtDNA (FIG. 25D). PKA is activated by cyclic-AMP (cAMP), which levels are modulated by adenylyl cyclase (AC) and phosphodiesterases (PDE). Both enzymes are present in the mitochondrial matrix. Indeed, IFN/ccRNP exposure reduces mitochondrial cAMP levels (FIG. 26F) and, as expected, the pan-PDE inhibitor IB MX decreases the oxidation status and interferogenicity of the extruded mtDNA (FIGS. 25D and 26F). Whether this is due to AC inhibition, PDE activation or mitochondrial ATP level reduction remains to be addressed.

[00152] Thus, these results link lupus- specific neutrophil activation with decreased mitochondrial cAMP levels, impaired PKA activation and lack of mtDNA/TFAM disassembly. This reduces the removal of Ox mtDNA from mitochondria and lead to its extrusion.

EXAMPLE 12 - SLE patients retain Ox mtDNA within their neutrophil mitochondria and develop anti-Ox mtDNA autoantibodies

[00153] Overexpression of IFN-inducible transcripts and development of TLR7 agonist autoantibodies are hallmarks of human SLE. We therefore analyzed whether patient blood neutrophils would recapitulate the mitochondrial alterations induced by IFN/ccRNP in vitro. IF analysis shows intramitochondrial accumulation of Ox mtDNA in 50% of SLE (12/24), but not in healthy control (0/50) or Juvenile Dermatomyositis (0/6) patient neutrophils (FIG. 25E). Furthermore, as shown in FIG. 25F (and FIG. 26G), anti-Ox mtDNA autoantibodies are detected in SLE sera (FIG. 25F. Thus, in addition to inducing pDC activation and IFN production, Ox mtDNA is an autoantigen in SLE. [00154] SLE neutrophils activated with anti-Sm/RNP antibodies extrude interferogenic DNA in an FcyR, TLR7 and ROS-dependent manner. This effect is recapitulated when healthy neutrophils are primed with IFNcc. While Applicants previously reported that interferogenic DNA originated from gDNA released during NETosis, subsequent analyses have led us to uncover that it consists of oxidized mtDNA. These studies also reveal that human neutrophils display a constitutive defect in mitophagy that is compensated by two complementary pathways: i) extrusion of inner mitochondrial components, including nucleoids devoid of oxidized residues, and ii) exportation of Ox mtDNA into lysosomes for degradation. Exposure to IFN/aRNP disrupts the disposal of oxidized mitochondrial products and leads to intramitochondrial accumulation and extracellular release of interferogenic Ox mtDNA.

[00155] Extrusion of mtDNA by granulocytes has been reported (Yousefi, S., et al. Cell death and differentiation 16, 1438-1444 (2009) and Yousefi, S., et al. Nature medicine 14, 949-953 (2008)) but the mechanism remained elusive. Here we demonstrate that this process requires autophagy. Absence of extrusion of outer mitochondrial components, however, suggests that it differs from "exophagy", whereby the autophagosome content is released into the extracellular space. Lack of LAMP1 and Rab7 translocation to the neutrophil surface also rules out exocytosis of lysosomes containing mitochondrial products. Instead, TOMM20 translocates to the cell surface where it co-localizes with LC3B. Thus, it is surmised that LC3B might be directly recruited to mitochondrial membranes independently of double-membrane autophagosomes, as previously reported for LAP (Sanjuan, M.A., et al. Nature 450, 1253-1257 (2007) and Florey, O., et al. Nature cell biology 13, 1335-1343 (2011)). Due to its highly fusogenic properties (Yang, A., et al. Chembiochem : a European journal of chemical biology 14, 1296-1300 (2013)), LC3B might favor the direct fusion of mitochondrial membranes with the plasmalemma.

[00156] Nucleoid extrusion seems to be linked to the inability of neutrophils to complete mitophagy. In particular, autophagosome-lysosome fusion seems to be constitutively impaired in these cells. The inability to transfect human neutrophils with appropriate reporter constructs, however, does not permit us to formally conclude. Autophagosome maturation is regulated by different proteins, including Rab7 (Jager, S., et al. Journal of cell science 117, 4837-4848 (2004)), presenilin-1 (Neely, K.M. & Green, K.N. Autophagy 7, 664-665 (2011)) and UVRAG (Liang, C, et al. Nature cell biology 10, 776-787 (2008)). In addition, TOM1, a constituent of the alternative endosomal sorting complex required for transport (ESCRT)-class 0, has been implicated in autophagosome/lysosome fusion. Accordingly, we observe that neutrophils fail to upregulate TOM1 expression upon induction of mitochondrial depolarization. Lack of mTOR inhibition under similar conditions could also contribute to this phenotype 24.

[00157] Mitochondria use MDVs to deliver their oxidized cargo to lysosomes for degradation. MDVs contain damaged respiratory chain subunits, but were never found to incorporate mtDNA. This data reveals, for the first time, that neutrophil mitochondria remove oxidized mtDNA through microvesicles. Like MDVs, these vesicles do not require autophagy for their generation and originate from EVIM. However, while MDV formation does not require DRP1 in cell lines, this GTPase might be necessary in neutrophils. Whether this is due to different requirements for MDV formation in neutrophils remains to be addressed.

[00158] IFN/ccRNP impairs the exportation of Ox mtDNA into lysosomes. This process requires nucleoid disassembly, as TFAM is never found associated with Ox mtDNA. Nucleoid disassembly relies on TFAM phosphorylation by Protein Kinase A (PKA). We show that IFN/ccRNP interfere with this step. The activity of PKA is directly regulated by the bioavailability of cAMP in the mitochondrial matrix. Accordingly, decreased availability of cAMP is responsible for the lupus phenotype.

[00159] Endosomal TLRs play a fundamental role in the IFN production and downstream immune alterations that characterize SLE (Rowland, S.L., et al. The Journal of experimental medicine 211, 1977-1991 (2014); Sisirak, V., et al. The Journal of experimental medicine 211, 1969-1976 (2014); and Guiducci, C, et al. Nature 465, 937-941 (2010)). The origin and nature of the NA ligands that trigger these sensors remain unclear. Here we show that oxidized mtDNA has a remarkable capacity to activate TLR9, but the mechanism responsible for the superior capacity of Ox mtDNA to activate this receptor remains an open question. A recent study demonstrated that cytosolic Ox gDNA is less susceptible to TREX1- mediated degradation and contribute to STING activation 48. In our hands, Ox and Non Ox mtDNA display similar susceptibility to degradation by extracellular deoxyribonucleases (not shown). Likewise, we did not observe differences in uptake, intracellular compartmentalization or stability of these two forms of mtDNA. Unique TLR9 binding affinities and/or recruitment of specific adaptors might explain these findings. [00160] Ox mtDNA needs to be in complex with TFAM, which binds RAGE on pDCs, to be internalized and activate TLR9. In healthy neutrophils, the steady state dissociation of Ox mtDNA from TFAM might take place to avoid the uncontrolled release of these interferogenic complexes under conditions that lead to plasma membrane rupture such as necrosis or NETosis. In the context of lupus, where NETosis rate is increased (Garcia- Romo, G.S., et al. Science translational medicine 3, 73ra20 (2011); Lande, R., et al. Science translational medicine 3, 73ral9 (2011); and Ivanov, A., et al. J Cell Biol 202, 129-143 (2013)) and dying cells might not be properly handled (Rothlin, C.V. & Lemke, G. Current opinion in immunology 22, 740-746 (2010)) this could be particularly detrimental. [00161] To address the relevance of our findings, we analyzed neutrophils isolated from SLE patients. In a large fraction of patients these neutrophils recapitulate the mitochondrial alterations observed in vitro, including aggregation, polarization and retention of Ox DNA. These alterations are not found in neutrophils from healthy children or JDM patients, who also over-express IFN-inducible transcripts but do not carry the same TLR7 agonist autospecificities (Robinson, A.B. & Reed, A.M. Nature reviews. Rheumatology 7, 664-675 (2011)). Furthermore, Ox mtDNA elicits an immune response in SLE, as autoantibodies are detected in patient sera. Future studies will address whether neutrophil mitochondrial changes and/or the presence of anti-Ox mtDNA autoantibodies can be used as biomarkers to stratify SLE patients with unique clinical phenotypes and/or disease course. [00162] This data supports that therapeutic efforts to increase extracellular and/or intracellular pathways involved in Ox mtDNA degradation should be explored in human SLE, a disease for which only one new drug has been approved in the past 50 years.

EXAMPLE 13 - Materials and Methods

[00163] Antibodies, recombinant proteins and chemicals: Mito tracker Green, Mitotracker DeepRed, MitoSox and CellRox are from Molecular Probes. Proteinase K, recombinant DNAse-I are from Roche. Recombinant human LL-37, R837, ODN-2216 are from Invivogen. Antibody against 8-OHdG (J-l) are from Santa Cruz Biotechnology. Antibodies against LL-37, Transferrin Receptor (TfR), TFAM (18G102B2E11), Histone H3, Mitofilin, γΗ2Α.Χ (3F2), TOMM20 (4F3), GADPH (6C5), dsDNA (HYB331-01), LAMP1, LC3B, Ubiquitin, Parkin are from Abeam. Antibody against MnSOD is from Millipore. Antibodies against P70s6K, HMGB1, PGCla, Phospho-(Ser), Phospho-(Ser/Thr) PKA Substrate are from Cell Signaling Technologies. IRS661 and DVX41 are from Dynavax Technologies Corporation. All chemicals are from Santa Cruz Biotechnology.

[00164] Anti-RNP/Sm autoantibody isolation: Serum samples from SLE patients were filtered through a 0.45-μιη polyvinylidene fluoride syringe. Anti-RNP/Sm and anti- dsDNA titer levels were measured using commercially available ELISA kits (GenWay Biotech). Samples positive for anti-RNP/Sm and negative for anti-dsDNA were selected and the total IgG fraction was then purified using HiTrap Protein G HP column (GE Healthcare). Once purified the total IgG fraction was desalted, dialyzed against PBS (Phosphate-Buffered Saline pH 7.4) and then quantified. [00165] Cells isolation and culture: Neutrophils were isolated from healthy donors or SLE patients peripheral blood following the dextran/NaCl protocol. All neutrophils cultures were carried out in complete RPMI supplemented with 2% FBS at cell density 1.25x106 cells/500 μΐ. For IFN priming neutrophils were pre-incubated with IFN α 2β (1000 U/ml; Schering Corp.) for 90 min at 37°C before stimulation. Neutrophils were made necrotic by freezing and thawing or apoptotic by UV irradiation. Monocytes were isolated from apheresis fraction 5 obtained from healthy donors. Monocytes were further enriched using negative selection by magnetic separation (Stem Cell Technology). Total dendritic cells fraction was obtained from healthy buffy coats by magnetic cell sorting with the pan-DC Enrichment Kit (Stem Cell Technology). Highly pure (>99%) plasmacytiod dendritic cells (Lin- HLADR+ CDl lc- CD123+) were then isolated from this fraction by FACS sorting as previously described (). pDCs were cultured (3 x 105 cells / 100 μΐ) with 40% of neutrophils- derived supernatants or with synthetic mtDNA pre-incubated (30 min at room temperature) with medium or LL-37 (50 μg/ml). After 18 hrs IFNa levels in the corresponding supernatants were measured by Flex Set Kit (BD Biosciences). For blocking experiments pDCs were pre-incubated with anti-TFAM (7 μg/ml; Cell Signaling Technology) or the corresponding isotype control or Recombinant Human RAGE-Fc Chimera (10 μg/ml; R&D System) or BoxA (10 μ^πύ; HMGBiotech).

[00166] Supernatant analysis: Neutrophils supernatants were centrifuged (1400 g/5 min) to remove cellular debris then digested with proteinase K (1 mg/ml) for 60 min at 60°C. 20 μΐ of digested supernatants were loaded on 1% agarose gel and DNA visualized with GelRed Nucleic Acid Stain (Phenix Research Products) or assessed for DNA quantity with Quant-iT Picogreen dsDNA Assay Kit (Invitrogen). For western blot analysis supernatants were concentrated 5 times with Concentrators PES Spin Columns (Pierce; MWCO 3K) before being subjected to SDS-PAGE/western blot analysis.

[00167] Electron microscopy - Immunofluorescence microscopy: Cells were settled on poly-L-lysine coated glass coverslips (BD Biocoat) for 3 hrs. Where specified Mitotracker DeepRed (25 μΜ) or recombinant DNAse-I (1 U/ml) were added the last 30 min of culture. Cells where then rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. For 8-OHdG detection after fixation, cells were incubated with 2 M HC1 (20 min at room temperature) and 0.1 M sodium borate, pH 8.5 (2 min at room temperature) before proceeding with antibody staining. Primary and secondary antibodies staining were carried out with blocking/staining buffer (1% goat serum 0.1% saponin, 1% BSA in PBS). Anti-mouse or anti-rabbit Alexa Fluor 488 or Alexa Fluor 568 were used as a secondary antibodies. Samples were embedded in ProLong Gold Antifade Reagent (Molecular Probes) and examined with a Leica TCS SP5 confocal laser scanning microscope equipped with a 63x/1.4 oil objective. ImageJ 1.47t was used to analyze confocal images. [00168] Flow cytometry: For mitochondrial mass assessment, cells were labeled with Mitotracker Green (25 μΜ) for 30 min at 37 °C and then analyzed immediately by flow cytometry. Apoptosis were assessed with the APO-BrdU TUNEL assay kit (Invitrogen). For surface TOMM20 or LAMP1 expression cells were cultured for 3 hrs, washed with FACS buffer (PBS + 1% FBS) and then stained with the corresponding antibodies or isotype controls. For total TOMM20 or LAMP1 expression cells were cultured for 3 hrs, washed with FACS buffer then fixed and permeabilized with BD cytofix/cytoperm (BD Biosciences) according to the manufacturer's instructions before proceed with antibody staining. For mitochondria depolarization cells were incubated for 3 hrs at 37 °C in 96 well plate (Corning Corporated). Mitotracker DeepRed (25 μΜ) was added the last 30 min of culture to label respiring mitochondria. Cells were then washed in PBS and analyzed immediately by flow cytometry. For mtDNA uptake and intracellular stability plasmacytoid DCs were incubated for 60 min at 37 °C in the presence of Cy5-labeled mtDNA (400 ng/ml) with or without LL- 37 (50 μg/ml). Cells were then washed in complete RPMI 10% FBS to remove unbound mtDNA and analyzed immediately or returned to the incubator for different time points before flow cytometry analysis. For ROS measurement cells were pre-loaded for 30 min at 37 °C with MitoSox (2.5 μΜ) or with CellRox (2.5 μΜ). Cells were then washed, cultured for 3 hrs as described and then subjected to flow cytometry analysis. [00169] LDH activity assay: LDH activity was measured in the cell-free supernatants using the Lactate Dehydrogenase Activity Assay Kit (Sigma) according to the manufacturer's instructions.

[00170] 8-OHdG ELISA: 8-OHdG ELISA was carried out with OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) according to the manufacturer's instructions.

[00171] Western blot: Cells were cultured as described, washed in PBS and then lysed in RIPA buffer in the presence of Halt Protease and Phosphates Inhibitor Cocktail (Pierce). Samples were incubated on ice for 30 min and then centrifuged (13,000 g for 10 min at 4°C). The supernatants containing the protein fraction, were collected and stored at -80°C. Proteins were estimated using the BCA kit (Pierce) following the manufacturer's instructions. 30 μg of proteins were resuspended in 5x reducing loading buffer (Pierce), boiled for 5 min at 100 °C and then subjected to electrophoresis with Mini-PROTEAN TGX Precast Gel (BIO- RAD). The proteins were then transferred to nitrocellulose membranes (BIO-RAD), blocked for 1 h 5% nonfat dry milk in TBST (Tris Buffered Saline containing 0.1% Tween20) and exposed overnight at 4° C to the primary antibodies. After washing in TBST, the membranes were incubated for 1 h at room temperature with Poly HRP-conjugated anti-rabbit or anti- mouse IgG (Pierce). ECL Plus reagents (Amersham) was used for detection.

[00172] Immunoprecipitation: For immunoprecipitation of mtDNA/protein complexes, 1 ml of crude neutrophil supernatants was pre-cleared with 20 μΐ of protein A/G plus agarose (Santa Cruz Biotechnology) for 1 hr at 4°C. Immunoprecipitation was carried out over night at 4°C with anti-dsDNA antibody (10 μg/ml) followed by addition of 20 μΐ of protein A/G plus agarose for another 4 hrs. Immunoprecipitates (IP beads) were collected and washed five times with PBS, resuspended in 5x reducing loading buffer (Pierce), boiled for 5 min at 100 °C and then being subjected to SDS-PAGE/western blot analysis. For intracellular TFAM/8-OHdG association or phosphor-TFAM detection cells were gently lysed in ice-cold IP Lysis/Wash Buffer (Pierce). Cell lysate (75 μg proteins) was incubated over night with anti-TFAM antibody (10 μg/ml). Subsequently 20 μΐ of protein A/G plus agarose were added for additional 6 hrs. Immunoprecipitates were then washed five times with 10 mM TrisHCl - 20 mM NaCl and the associated complexes eluted by boiling the beads for 5 min at 100 °C with 2% SDS. The corresponding supernatant was dot-blotted and UV cross-linked to a nitrocellulose membrane or subjected to SDS-PAGE/western blot analysis. To avoid interference of heavy and light antibody chains HRP-conjugated Clean-Blot IP Detection Reagent (Pierce) was used as a detection reagent.

[00173] Extruded mtDNA isolation and analysis: Neutrophils supernatants (500 μΐ) were digested with proteinase K (1 mg/ml) for 60 min at 60°C before DNA extraction with UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24: 1, v/v; Invitrogen). The DNA was then precipitated from the aqueous phase with Ammonium Acetate and 100% Ethanol. Glycogen (20 μg/ml; Santa Cruz Biotechnology) was added as a carrier. The DNA pellet was washed with 75% Ethanol, air-dried and then resuspended in TE Buffer pH 8. Real-Time PCR was carried out with 2.5 ng of isolated DNA, Power SYBR Green PCR Master Mix (Invitrogen) and 0.5 μΜ of the following primers: NADH dehydrogenase subunit 1 (ND1) (5 ' -GCATTCCTAATGCTTACCGAAC-3 ' (SEQ ID NO. 1) and 5'- AAGGGTGGAGAGGTTAAAGGAG-3 ' (SEQ ID NO. 2)). For Oxidized DNA Dot Blot assay 5ng of DNA was blotted on positively charged nylon membrane (Roche) and UV-cross linked. The membrane was then blocked with 5% nonfat dry milk in TBST for 2 h at room temperature before being exposed overnight to the primary antibodies at 4° C. After washing in TBST, the membranes were incubated for 1 h at room temperature with Poly HRP- conjugated anti-rabbit or anti-mouse IgG (Pierce). ECL Plus reagents (Amersham) was used for detection.

[00174] Synthetic mtDNA generation and labeling - Microarray analysis: Cells were cultured with medium or CCCP (25 μΜ) for 60 min and then lysed with mirVana Lysis Buffer (Invitrogen). Total RNA was isolated using the RNeasy kit (Qiagen), amplified and then labeled with Illumina TotalPrep RNA amplification kit (Applied Biosystems). Agilent 2100 Analyzer (Agilent Technologies) was used to assess RNA integrity. Biotinylated complementary RNA (cRNA) was hybridized to Illumina Human-6 Beadchip Array version 2 and scanned on Illumina Beadstation 500. Fluorescent hybridization signals were assessed with Beadstudio software (Illumina), and statistical analysis and hierarchical clustering were performed with GeneSpring 7.3.1 software (Agilent Technologies). Functions and interactions of differentially expressed genes were studied using Ingenuity Pathways Analysis (IPA 8.5). [00175] Statistics: To compare differences, the two-tailed Students i-test (at 95% confidence interval) was used to compare unpaired samples. A p value less than 0.05 was considered significant. [00176] Patient information: Blood samples were obtained from patients fulfilling the diagnosis of SLE according to the criteria established by the American College of Rheumatology and of JDM patients with biopsy-proven disease. Healthy pediatric controls were children visiting the clinic either for reasons not related to autoimmunity or for surgery not associated with any inflammatory diseases.

[00177] Antibodies, recombinant proteins and chemicals: Mito tracker Green, Mitotracker DeepRed, MitoSox and CellRox were purchased from Molecular Probes. Proteinase K was purchased from Roche. Recombinant human GM-CSF was purchased from BD Biosciences. Recombinant human LL-37, R837 and ODN-2216 were purchased from Invivogen. FcR Blocking Reagent was purchased from Miltenyi Biotech. Antibody against 8- OHdG (J-l; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology. Antibodies against LL-37, Transferrin Receptor (TfR), TFAM, Histone H3, Mitofilin, H2A.X, TOMM20, Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3-phosphate dehydrogenase (GADPH), dsDNA, LAMP1, LC3B, Rab7 and DRP-1 were purchased from Abeam. Antibodies against MnSOD and PKA (alpha isoform of the catalytic subunit) were purchased from Millipore (Billerica, MA, USA). Antibodies against P70s6K, HMGB 1, PGC1, Phospho-(Ser) and Phospho-(Ser/Thr) PKA Substrate were purchased from Cell Signaling Technologies. Genomic DNA was from BioChain (San Francisco, CA, USA). IRS661 and DVX41 were a gift from Dynavax Technologies Corporation (Berkeley, CA, USA).

[00178] Anti-RNP/Sm autoantibodies (aRNP) isolation: Serum samples from SLE patients were filtered through a 0.45-μιη polyvinylidene fluoride syringe. Anti- RNP/Sm and anti-dsDNA titers were measured using commercially available ELISA kits (GenWay Biotech). Samples positive for anti-RNP/Sm and negative for anti-dsDNA were selected and the total IgG fraction was purified using HiTrap Protein G HP column (GE Healthcare). Once purified, the total IgG fraction (aRNP) was desalted, dialyzed against PBS (Phosphate-Buffered Saline pH 7.4) and quantified.

[00179] Cell isolation and culture: Blood from healthy donors was collected in ACD tubes (BD Vacutainer). Neutrophils were then immediately isolated as previously described6. All neutrophil cultures were carried out in complete RPMI supplemented with 2% FBS at cell density 1.25 millions cells/500 μΐ. Were specified neutrophils were cultured in the presence of aRNP-IgG (50 μg/ml), recombinant human GM-CSF (50 ng/ml), 3- methyladenine (3MA; 1 mM), diphenylene iodonium (DPI; 10 μΜ), MitoTempo (MT; 10 μΜ), Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 25_μΜ), Rotenone (5 μΜ), Oligomycin (10 μΜ) plus Antimycin (1 μΜ) (O+A), IRS661 (TLR7 antagonist; 1 μΜ), DVX42 (TLR8 antagonits; 1 μΜ), Bafilomycin Al (BafAl; 1 μΜ), MDVI-1 (10 μΜ), 8BrcAMP (100 μΜ), IBMX (50 μΜ), H89 (1 μΜ) or MG132 (10 μΜ). For IFN priming, neutrophils were pre-incubated with IFNaP (2000 U/ml; Schering Corp.) for 90 min at 37°C before stimulation. To study the effect of TLR ligands on mtDNA extrusion neutrophils were cultured with R837 (TLR7 agonist; 1 μg/ml) alone or in combination with IRS661 (TLR7 antagonist, 1 μΜ) or with ODN2216 (TLR9 agonist, 1 μg/ml). Neutrophils were made necrotic by culturing the cells for 48 h. Cells were made apoptotic by UV irradiation or were made NETotic by PMA treatment (25 nM). Monocytes were isolated from apheresis fraction V obtained from healthy donors. Monocytes were further enriched using negative selection by magnetic separation (Stem Cell Technology). For pDCs isolation, the total dendritic cells fraction was obtained from healthy buffy coats by magnetic cell sorting with the pan-DC Enrichment Kit (Stem Cell Technology). Highly pure (>99 ) plasmacytoid dendritic cells (Lin- HLADR+ CDl lc- CD123+) were then isolated from this fraction by FACS sorting as previously described. PDCs were cultured (3x105 cells /100 μΐ) with 40% neutrophil supernatants. Alternatively, 400 ng/ml of synthetic oxidized or non-oxidized mtDNA or genomic DNA, all preincubated with LL-37 (50 μg/ml), were used to stimulate pDCs. After 18 h, IFNa levels in the corresponding supernatants were measured with the Human IFNa- Flex Set Kit (BD Biosciences). For blocking experiments, pDCs were preincubated, for 30 min at 37°C, with anti-TFAM (7 μg/ml; Cell Signaling Technology) or the corresponding isotype control or Recombinant Human RAGEFc Chimera (10 μg/ml; R&D System) or BoxA (10 μg/ml; HMGBiotech). [00180] Supernatant analysis: Neutrophil supernatants were collected after 6 h of culture, centrifuged for 10 min at 1400g to remove cellular debris and stored at - 80°C. For agarose gel electrophoresis crude supernatants where treated with proteinase K (PK; 1 mg/ml) for 60 min at 60°C and 20 μΐ of the digested supernatants were loaded on 1% agarose gel and DNA was visualized with GelRed Nucleic Acid Stain (Phenix Research Products; Candler, NC, USA). The amount of extruded mtDNA was measured with Quant-iT Picogreen dsDNA Assay Kit (Invitrogen) or by densitometric analysis of the mtDNA gel band and expressed in Arbitrary Units (AU). For Western blot analysis, crude supernatants were concentrated with Concentrators PES Spin Columns (MWCO 3K) (Pierce), boilded in 5x Lane Marker Reducing Sample Buffer (Pierce) for 5 min at lOOC before SDS-PAGE/Western blot analysis. For Total Cell Lysate (TCL) neutrophils were subjected to one cycle of freeze/thaw, centrifuged for 5 min at 13000g to remove debris and then concentrated as described above. [00181] Immunofluorescence microscopy (IF): Neutrophils or monocytes were settled on poly-L-lysine coated glass coverslips and cultured for 3 h or 60 min (for CCCP and O+A experiments). Cells where then rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilized with 0.05% TritonX-100 in PBS for 5 min at room temperature (for non-permeabilized cells staining this step was omitted) and then treated with Blocking Buffer (1% goat serum, 1% FcR Blocking Reagent and 1% BSA in PBS) for 30 min at room temperature. Primary and secondary antibody staining were carried out in Staining Buffer (1% BSA in PBS). Isotype specific anti- mouse or anti-rabbit AlexaFluor-488 or AlexaFluor-568 were used as secondary antibodies. Counterstaining of cell nuclei was performed with the Hoechst stains (Molecular Probes). Samples were embedded in Pro Long Gold Antifade Reagent (Molecular Probes) and examined with a Leica TCS SP5 confocal laser- scanning microscope equipped with a 63x/1.4 oil objective. ImageJ software (NIH; Bethesda MD - Version 1.47t) was used for analysis. For the TUNEL assay the Click-iT TUNEL Imaging Assay Kit (Molecular Probes) was used accordingly to the manufacturer's instructions. For mtDNA intracellular trafficking, pDCs were incubated for 60 min at 37°C in the presence of Cy5-labeled mtDNA (400 ng/ml) and LL-37 (50 μ^πύ). Cells were then chased in complete RPMI 10% FBS for 30 min before proceeding with the staining as described before. The percentage of co-localization was calculated from the Manders' Overlap Coefficient using the "Co- localization analysis" plugin (ImageJ; NIH; Bethesda MD - Version 1.47t). [00182] Flow cytometry: For mitochondrial mass assessment, cells were labeled with Mitotracker Green (25 nM) for 30 min at 37°C and then analyzed immediately by flow cytometry. Apoptosis progression was assessed with the APO-BrdU TUNEL assay kit (Molecular Probes) following the manufacturer's instructions. For TOMM20, Mitofilin, LAMPl or Rab7 surface expression, cells were cultured for 3 h, washed with FACS buffer (PBS + 1%FBS) and then stained with the corresponding antibodies or isotype controls. For total TOMM20, Mitofilin, LAMPl or Rab7 expression, cells were permeabilized with cytofix/cytoperm (BD Biosciences) before proceeding with antibody staining. For mitochondria depolarization, cells were incubated for 3 h at 37°C. Mitotracker DeepRed (25 nM) was added the last 30 min of culture. Cells were then washed in PBS and analyzed immediately by flow cytometry. For mtDNA uptake and intracellular stability, plasmacytoid DCs were incubated for 60 min at 37 °C in the presence of Cy5 -labeled mtDNA (400 ng/ml) with or without LL-37 (50 μg/ml). Cells were then washed in complete RPMI 10% FBS to remove unbound mtDNA and analyzed immediately or returned to the incubator for different time points before flow cytometry analysis. For ROS quantification, cells were loaded for 30 min at 37°C with MitoSox (2.5 μΜ) or CellRox (2.5 μΜ). Cells were then washed, cultured for 3 h as described and then subjected to flow cytometry analysis. [00183] LDH activity assay: LDH activity was measured in the cell-free supernatants using the Lactate Dehydrogenase Activity Assay Kit (Sigma- Aldrich) according to the manufacturer's instructions. Results are normalized to the total (intracellular) enzyme activity.

[00184] 8-OHdG ELISA: 8-OHdG ELISA was performed on isolated DNA with OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs; San Diego, CA, USA) according to the manufacturer's instructions.

[00185] SDS-PAGE and Western blot: Cultured cells were washed in PBS and then lysed in RIPA buffer in the presence of Halt Protease and Phosphates Inhibitor Cocktail (Pierce). Samples were incubated on ice for 30 min and then centrifuged (13000g for 10 min at 4°C). The supernatants containing the protein fraction were collected and stored at -80°C until further analysis. Proteins concentration was estimated using the BCA kit (Pierce) following the manufacturer's instructions. 40 μg of proteins were resuspended in 5x Lane Marker Reducing Sample Buffer (Pierce), boiled for 5 min at 100°C and then subjected to electrophoresis with Mini-PROTEAN TGX Precast Gel (BIO-RAD). The proteins were then transferred to nitrocellulose membranes, blocked for 1 h 5% nonfat dry milk in TBST (Tris Buffered Saline containing 0.1% Tween20) and incubated overnight at 4°C with the primary antibodies. After washing in TBST, the membranes were incubated for 1 h at room temperature with Poly HRP-conjugated anti-rabbit or anti-mouse IgG (Pierce). ECL Plus reagents (Amersham) were used for detection. Digital images were acquired with ChemiDoc MP System (BIO-RAD) and analyzed with Image Lab Software (BIO-RAD). [00186] Immunoprecipitation (IP) and Co-Immunoprecipitation (Co-IP):

For coimmunoprecipitation of mtDNA/protein complexes, 1 ml of crude neutrophil supernatants was pre-cleared with 20 μΐ of protein A/G plus agarose (Santa Cruz Biotechnology) for 1 h at 4°C. Immunoprecipitation was carried out over night at 4°C with anti-dsDNA antibody (10 μg/ml) followed by addition of 20 μΐ of protein A/G plus agarose for another 4 h. Immunoprecipitates (IP beads) were collected and washed five times with PBS, resuspended in 5x Lane Marker Reducing Sample Buffer, boiled for 5 min at 100°C and then subjected to SDSPAGE/western blot analysis. Alternatively washed IP beads were treated with PK (1 mg/ml) for 60 min at 60°C. The digested material was then loaded on 1% agarose gel and DNA was visualized with GelRed Nucleic Acid Stain. For intracellular for TFAM/8-OHdG complexes or phospho-TFAM detection cells were gently lysed in ice-cold IP Lysis/Wash Buffer (Pierce) supplemented with Halt Protease and Phosphatase Inhibitor Cocktails (Pierce). Cell lysate (75 μg proteins) was incubated over night with anti-TFAM antibody (10 μg/ml). Subsequently 20 μΐ of protein A/G plus agarose were added for additional 6 h. The beads were then washed five times with 10 mM TrisHCl - 20 mM NaCl (for TFAM/8-OHdG complexes) or with PBS (for phospho-TFAM detection) and the associated complexes/proteins were released from the immunocomplexes by incubation for 5 min at 100°C with 2% SDS (for TFAM/8-OHdG complexes) or with 5x Lane Marker Reducing Sample Buffer (for phospho-TFAM detection). The dissociated complexes/proteins were then collected by centrifugation and dot-blotted to a nitrocellulose membrane (for TFAM/8-OHdG complexes) or subjected to SDS-PAGE for western blot analysis (for phospho-TFAM detection). To avoid interference of heavy and light antibody chains HRP- conjugated Clean-Blot IP Detection Reagent (Pierce) was used as a detection reagent.

[00187] Extruded mtDNA isolation and analysis: Neutrophil supernatants (500 μΐ) were digested with PK (1 mg/ml) for 60 min at 60°C and then mixed with 1 volume of UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24: 1 v/v/v; Invitrogen). The DNA was then precipitated from the aqueous phase with Ammonium Acetate and 100% Ethanol. Glycogen (20 μg/ml) was added as a carrier. The DNA pellet was washed twice with 75% Ethanol, vacuum-dried and then resuspended in TE Buffer, pH 8. DNA concentration was assessed with Quant-iT Picogreen dsDNA Assay Kit (Invitrogen). PCR was carried out with 5 ng of isolated DNA, AmpliTaq Gold 360 (Invitrogen) and 0.5 μΜ of the following primers: mitochondrial DNA encoded NADH dehydrogenase subunit 1 (ND1) (5'- GCATTCCTAATGCTTACCGAAC-3 ' and 5 ' - A AGGGTGG AG AGGTT A A AGG AG- 3 ' ) ; genomic DNA encoded Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (5'- AGGCAACTAGGATGGTGTGG-3 ' and 5 ' -TTGATTTTGGAGGGATCTCG-3 ' ) . The PCR conditions were as follows: 95°C for 10 min; 30 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 60 sec with a final extension of 72°C for 7 min. PCR products were visualized on a 3% agarose gel. For mtDNA copy number 3 ng of isolated DNA were subjected to Real Time PCR with Power SYBR Green PCR Master Mix (Invitrogen) and 0.5 μΜ of each primer. MtDNA copy number was then calculated as described. For Dot Blot assay, 5 ng of DNA were blotted on a positively charged nylon membrane using the Bio-Dot Microfiltration System (BIORAD) and then cross-linked by UV irradiation. The membranes were blocked with 5% nonfat dry milk in TBST for 2 h at room temperature before overnight incubation, at 4°C, with the primary antibodies or with the patients' sera (1:200 in 1% nonfat dry milk in TBST). After washing in TBST, the membranes were incubated for 1 h at room temperature with Poly HRP-conjugated anti-rabbit or anti-mouse or anti-human IgG. ECL Plus Western Blotting Detection Reagent (Amersham) was used for detection. [00188] In vitro mtDNA generation and labeling: The mtDNA was amplified using two overlapping fragments each about 8.5 kb with primers previously reported. Amplification reaction was carried out against human genomic DNA using elongase enzyme mix (Invitrogen). Oxidized mtDNA was generated by performing PCR reaction in presence of 200 M 8-Oxo-2'-dGTP (TriLink; San Diego, CA, USA). Cy5 fluorescently labeled mtDNA and its oxidized form were generated by replacing half of the normal dCTP with Cy5-dCTP (GE Healthcare). Amplicons were purified from residual primers and dNTPs by MSB Spin PCRapace (B-Bridge International; Cupertino, CA, USA).

[00189] Microarray analysis: Cells were cultured with medium or CCCP (25 μΜ) for 60 min and then lysed with RLT Lysis Buffer (Qiagen). Total RNA was isolated using the RNeasy kit (Qiagen), amplified and then labeled with Illumina TotalPrep RNA amplification kit (Invitrogen). Agilent 2100 Analyzer (Agilent Technologies) was used to assess RNA integrity. Biotinylated complementary RNA (cRNA) was hybridized to Illumina Human-6 Beadchip Array version 2 and scanned on Illumina Beadstation 500. Fluorescent hybridization signals were assessed with Beadstudio software (Illumina), and statistical analysis and hierarchical clustering were performed with GeneSpring 7.3.1 software (Agilent Technologies). Function and interactions of differentially expressed genes were inferred using Ingenuity Pathways Analysis (IPA 8.5). [00190] Mitochondria isolation and Western/Dot Blot assay: Mitochondria were isolated from 10 millions of neutrophils using the Pierce Mitochondria Isolation Kit (Pierce) following the manufacturer's instructions. For western blot analysis the mitochondrial pellet was resuspended in 5x Lane Marker Reducing Sample Buffer (Pierce), boiled for 10 min at lOOC and then subjected to SDSPAGE/ Western blotting as described. For Oxidized DNA Dot Blot assay mitochondrial pellet was digested with PK (1 mg/ml) and 0.5% SDS for 60 min at 60°C. The mtDNA was then precipitated, quantified and subjected to Dot Blot assay as described before.

[00191] Mitochondrial protease protection assay: Purified neutrophils mitochondria were resuspended in digestion buffer (20 mM Hepes-KOH, pH 7.4, 250 mM sucrose, 80 mM KOAc) with or without 25 μg/ml of PK and incubated 30 min on ice. The reaction was then stopped by adding phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1 mM. Samples were centrifuged at 12000g for 5 min and the pellet was processed for SDS-PAGE/Western blotting as described. [00192] Reconstitution of MDV formation in vitro: Mitochondrial budding assay was as described. Briefly, purified mitochondria from 200 millions of neutrophils were incubated in 100 μΐ of an osmotically controlled, buffered environment including an energy regenerating system, where the final concentrations of the reagents were: 50 μΜ Antimycin, 220 mM mannitol, 68 mM sucrose, 80 mM KC1, 0.5 mM EGTA, 2 mM magnesium acetate, 20 mM Hepes pH 7.4, 1 mM ATP, 5 mM Succinate, 80 μΜ ADP, 2 mM K2HP04, pH 7.4. After 60 min at 37°C, the intact mitochondria were removed from the mixture by two sequential centrifugations at 7400g at 4°C. For western blot, the supernatants containing the MDVs fraction were treated with 0.5 mg/ml trypsin for 10 min at 4°C. Following trypsin treatment, loading buffer was added and the samples were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted. For mtDNA content, the supernatants containing the MDVs fraction were incubated 20 min at 4°C in the presence of 25 U/ml of DNAse I (Roche) to degrade unprotected mtDNA. Thereafter the supernatants were digested with 1 mg/ml PK and 0.5% SDS for 60 min at 60°C. MtDNA was then precipitated and subjected to agarose gel electrophoresis as described. [00193] Mitochondrial cAMP assay: Neutrophil-enriched mitochondrial fraction was obtained as previously described. cAMP levels were measured with the Cyclic AMP XP Assay Kit (Cell Signaling Technologies; Beverly, MA, USA) following the manufacturer's instructions and results were then normalized to the protein content.

[00194] Statistics: To compare differences, the two-tailed Students t-test (at

95% confidence interval) was used to compare unpaired samples. A p value less than 0.05 was considered significant.

* * *

[00195] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. A method for treating systemic lupus erythematosus (SLE), comprising administering to a patient having, suspected of having or at risk of SLE a pharmaceutical composition comprising an effective amount of: a) a mitochondrial phosphodiesterase (PDE) inhibitor; b) a mitochondrial protein kinase A (PKA) enhancer; and/or c) a receptor for advanced glycation end products (RAGE) inhibitor.

2. The method of claim 1, wherein the method comprises administering a pharmaceutical composition comprising an effective amount of a mitochondrial phosphodiesterase (PDE) inhibitor to the patient.

3. The method of claim 1 or 2, wherein the method comprises administering a pharmaceutical composition comprising an effective amount of mitochondrial protein kinase A (PKA) enhancer to the patient.

4. The method of any one of claims 1-3, wherein the method comprises administering a pharmaceutical composition comprising an effective amount of the RAGE inhibitor in dendritic cells of the patient.

5. The method of any one of claims 1-4, wherein the method comprises administering a pharmaceutical composition comprising an effective amount of a mitochondrial PDE inhibitor and a mitochondrial PKA enhancer to the patient.

6. The method of any one of claims 1-5, wherein the method comprises administering a pharmaceutical composition comprising an effective amount of a mitochondrial PDE inhibitor and a RAGE inhibitor to the patient.

7. The method of any one of claims 1-6, wherein the method comprises administering a pharmaceutical composition comprising an effective amount of a mitochondrial PKA enhancer and a RAGE inhibitor to the patient.

8. The method of any one of claims 1-7, wherein the method comprises administering a pharmaceutical composition comprising an effective amount of a mitochondrial PDE inhibitor, a mitochondrial PKA enhancer and a RAGE inhibitor to the patient.

9. The method of any one of claims 1-8, wherein the mitochondrial PDE inhibitor comprises IB MX.

10. The method of any one of claims 1-9, wherein the mitochondrial PKA enhancer comprises 8-Br-cAMP.

11. The method of any one of claims 1-10, wherein the RAGE inhibitor comprises RAGE Fc Chimera.

12. The method of any one of claims 1-11, wherein the administering is by a route selected from the group consisting of intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, inhalation, and a combination of two or more recited routes.

13. The method of any one of claims 1-12, wherein the administering comprises targeting the pharmaceutical composition to mitochondria.

14. The method of any one of claims 1-13, wherein the administering comprises delivering the pharmaceutical composition in a lipid vehicle.

15. A composition comprising a) a mitochondrial phosphodiesterase (PDE) inhibitor; b) a mitochondrial protein kinase A (PKA) enhancer; and/or c) a receptor for advanced glycation end products (RAGE) inhibitor.

16. The composition of claim 15, wherein the composition comprises an effective amount of a mitochondrial phosphodiesterase (PDE) inhibitor.

17. The composition of claim 15 or 16, wherein the composition comprises an effective amount of a mitochondrial protein kinase A (PKA) enhancer.

18. The composition of any one of claims 15-17, wherein the composition comprises an effective amount of the RAGE inhibitor.

19. The composition of any one of claims 15-18, wherein the composition comprises an effective amount of a mitochondrial PDE inhibitor and a mitochondrial PKA enhancer.

20. The composition of any one of claims 15-19, wherein the composition comprises an effective amount of a mitochondrial PDE inhibitor and a RAGE inhibitor.

21. The composition of any one of claims 15-20, wherein the composition comprises an effective amount of a mitochondrial PKA enhancer and a RAGE inhibitor.

22. The composition of any one of claims 15-21, wherein the composition comprises an effective amount of a mitochondrial PDE inhibitor, a mitochondrial PKA enhancer and a RAGE inhibitor.

23. The composition of any one of claims 15-22, wherein the mitochondrial PDE inhibitor comprises IB MX.

24. The composition of any one of claims 15-23, wherein the mitochondrial PKA enhancer comprises 8-Br-cAMP.

25. The composition of any one of claims 15-24, wherein the RAGE inhibitor comprises RAGE Fc Chimera.

26. The composition of any one of claims 15-25, wherein the composition comprises lipid vehicles.

27. The composition of claim 26, wherein the PDE inhibitor, PKA enhancer, and/or RAGE inhibitor are associated with the lipid vehicles.

28. Use of a composition according to any one of claims 15-27 in the preparation of a medicament for treating systemic lupus erythematosus (SLE) in patient having, suspected of a having or at risk of SLE.

29. Use of a composition according to any one of claims 15-27 for treating systemic lupus erythematosus (SLE) in patient having, suspected of a having or at risk of SLE.