WO2015200094A1 - Procédés et compositions pour le traitement du lupus érythémateux systémique (les) - Google Patents

Procédés et compositions pour le traitement du lupus érythémateux systémique (les) Download PDF

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Publication number
WO2015200094A1
WO2015200094A1 PCT/US2015/036459 US2015036459W WO2015200094A1 WO 2015200094 A1 WO2015200094 A1 WO 2015200094A1 US 2015036459 W US2015036459 W US 2015036459W WO 2015200094 A1 WO2015200094 A1 WO 2015200094A1
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mitochondrial
inhibitor
mtdna
neutrophils
composition
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PCT/US2015/036459
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English (en)
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Virginia Pascual
Simone CAIELLI
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Baylor Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the present invention relates generally to the field of medicine. More particularly, it concerns compositions and methods related to Systemic Lupus Erythematosus (SLE) therapy.
  • SLE Systemic Lupus Erythematosus
  • SLE 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.
  • a method for treating a patient 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.
  • PDE mitochondrial phosphodiesterase
  • PKA mitochondrial protein kinase A
  • RAGE receptor for advanced glycation end products
  • the patient may have symptoms of SLE, may have been diagnosed with SLE, and/or be at risk for SLE.
  • 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.
  • the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial phosphodiesterase (PDE) inhibitor to the SLE patient.
  • the method comprises administering an effective amount of a pharmaceutical composition comprising mitochondrial protein kinase A (PKA) enhancer to the SLE patient.
  • the method comprises administering an effective amount of a pharmaceutical composition comprising the RAGE inhibitor in dendritic cells of the patient.
  • PDE mitochondrial phosphodiesterase
  • PKA mitochondrial protein kinase A
  • 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.
  • the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial PDE inhibitor and a RAGE inhibitor to the SLE patient.
  • the method comprises administering an effective amount of a pharmaceutical composition comprising a mitochondrial PKA enhancer and a RAGE inhibitor to the SLE patient.
  • 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.
  • 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.
  • 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.
  • the active ingredient(s) in a pharmaceutical composition are the mitochondrial PKA enhancer, the mitochondrial PDE inhibitor, and/or the RAGE inhibitor.
  • composition or a method may be substituted with the term "consisting essentially of or “consisting of for the term “comprising.”
  • the mitochondrial PDE inhibitor comprises IBMX.
  • the mitochondrial PKA enhancer comprises 8-Br-cAMP.
  • the RAGE inhibitor comprises RAGE Fc Chimera.
  • 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.
  • 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.
  • the PKA enhancer or the PDE inhibitor may be targeted to mitochondria or neutrophils, or particularly, neutrophil mitochondria.
  • the RAGE inhibitor may be targeted or delivered to dendritic cells.
  • administering a composition involves delivering the pharmaceutical composition in a lipid vehicle.
  • 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.
  • lipid 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.
  • fatty acids neutral fats
  • phospholipids phosphoglycerides
  • steroids terpenes
  • lysolipids glycosphingolipids
  • glycolipids glycolipids
  • sulphatides waxes
  • waxes lipids with ether and ester- linked fatty acids and polymerizable lipids
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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 ⁇ ) 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.
  • 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.
  • FIG 4A-4F - MtDNA oxidation is required for extruded neutrophil mtC to activate pDCs.
  • IFN/aRNP anti-Sm/RNP autoantibodies
  • 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).
  • 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 ⁇ ) are shown. Each dot in the graph represents the 8-OHdG MFI in a single cell.
  • 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.
  • 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.
  • FIG 7A-7D IFN/aRNP inhibits oxidized mitochondrial nucleoids disassembly.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 12A-12D (A) Four different sequential confocal sections (0.25 mm) (from Fig. 5B) are shown.
  • Each dot in the graph represents the 8-OHdG antibody MFI in a single cell.
  • Each dot in the graph represents the percentage of 8-OHdG co- localizing with TOMM20 in a single cell. ***p ⁇ 0.001.
  • 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.
  • 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.
  • Ox nucleoids activate pDCs in a TF AM/RAGE dependent manner.
  • 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).
  • 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).
  • ⁇ MFI (Mean Fluorescence Intensity) MFI antibody - MFI isotype control.
  • 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).
  • FIG. 17A-F Neutrophils fail to complete mitophagy in response to mitochondrial depolarization.
  • C CCCP-treated neutrophils selectively display reduced intracellular TFAM levels. Neutrophils or monocytes were treated with media or CCCP in the presence of 50 ⁇ 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.
  • 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 ⁇ . (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 ⁇ .
  • FIG. 19A-F - mtDNA oxidation is required for pDC activation.
  • 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).
  • 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.
  • FIG. 21A-D Oxidized mtDNA is exported from mitochondria to lysosomes under steady state conditions.
  • FIG. 22A-E - TUNEL assay demonstrates the absence of fragmented DNA in the cytoplasm of live neutrophils. Apoptotic neutrophils were used as control. Scale bar: 10 ⁇ .
  • B 8-OHdG quantification of FIG. 21C.
  • 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 ⁇ .
  • 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 ⁇ .
  • B IF (left) and dot blot (right) analysis show Ox mtDNA accumulation within mitochondria in neutrophils exposed to IFN/aRNP. Scale bar: 10 ⁇ .
  • 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.
  • FIG. 24 The percentage of TOMM20 that co-localizes with 8-OHdG (top) and six different sequential confocal sections (0.25 ⁇ each; bottom) of Fig. 23B are shown. Scale bar: 10 ⁇ . **p ⁇ 0.01.
  • 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 ⁇ .
  • C IFN/aRNP decreases TFAM phosphorylation.
  • 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.
  • G Absence of cross-reactivity between anti-Ox and anti-Non Ox mtDNA autoantibodies. Values represent mean + s.d. *p ⁇ 0.05.
  • FIG. 27 Proposed effect of IFN/aRNP on neutrophil mitochondria.
  • 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).
  • 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.
  • 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.
  • SLE Systemic Lupus Erythematosus
  • 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.
  • GWAS genome-wide association
  • gene 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).
  • 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.
  • SLE SLE
  • 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 typically involving mortality from complications that result from the disease itself or as a consequence of its therapy.
  • a characteristic feature of SLE is the presence of autoantibodies against dsDNA and RNA/protein complexes.
  • SLE immune complexes ICs
  • 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).
  • neutrophils which also internalize SLE ICs via FcRs, contribute to amplify pDC activation and IFN production.
  • TLR7-agonistic anti-Sm/RNP
  • IFN priming might be necessary to increase the low baseline expression levels of this receptor in healthy neutrophils (Hayashi et al., 2003).
  • TLR7 SLE pathogenesis
  • TLR7 polymorphisms increase SLE risk in some ethnic groups (Shen et al., 2010), and TLR7 duplication in mice accelerates autoimmunity (Pisitkun et al., 2006).
  • crossing SLE-prone mice with TLR7 but not TLR9 KO strains ameliorates disease (Wu and Peng, 2006).
  • ssRNA-protein complexes are a common autoantigen in human SLE.
  • MtDNA mitochondrial DNA
  • 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).
  • mtC mitochondrial DNA-protein complexes
  • Methods and compositions may be provided for the treatment of SLE.
  • treatment means any treatment of a disease in a mammal, including:
  • 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.
  • 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 phosphodiesterase
  • PDE inhibitor may refer to any member of the class of compounds having an IC50 of 100 ⁇ or lower concentration for a phosphodiesterase, for example, at least or at most or about 200, 100, 80, 50, 40, 20, 10, 5, 1 ⁇ , 100, 10, 1 nM or lower concentration.
  • IC50 100 ⁇ or lower concentration for a phosphodiesterase
  • the inhibitor may also have activity against other types, unless otherwise stated.
  • 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.
  • 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.
  • an antisense nucleotide e.g., siRNA
  • 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.
  • PDA Protein kinase A
  • cAMP cyclic AMP
  • 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.
  • a PDE inhibitor or a PKA enhancer may be combined with mitochondrial targeting methods for mitochondria targeting.
  • Advanced glycosylation end product-specific receptor also known as receptor for advanced glycosylation end products, AGER and RAGE
  • 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.
  • anti-RAGE antibody encompass to all types of antibodies which, preferably, specifically binds to RAGE and inhibits RAGE activity.
  • 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.
  • 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).
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH).
  • various adjuvants can be used to increase the immunological response.
  • 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.
  • 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.
  • WO 2008/137552 A2 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 ⁇ with monoclonal antibodies for the CI and C2-domain in RAGE.
  • 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.
  • the salt can be converted into the free base form by treatment with alkali.
  • Aqueous compositions in some aspects comprise an effective amount of the therapeutic compound, further dispersed in pharmaceutically acceptable carrier or aqueous medium.
  • 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.
  • 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.
  • 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.
  • the compositions may be targeted to mitochondria by any methods known in the art, for example, by a mitochondrial targeting signal peptide.
  • a mitochondrial targeting signal peptide 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.
  • 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.
  • 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.
  • non-aqueous solvents examples 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.
  • 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.
  • oral administration 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.
  • 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.
  • 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.
  • 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%.
  • the form 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, ⁇ , ⁇ -dimethylformamide, propylene glycol, 2-pyrrolidone, methyl-2-pyrrolidone, and/or N-methylforamide.
  • solvents such as, but not limited to, dimethyl sulfoxide, water, ⁇ , ⁇ -dimethylformamide, propylene glycol, 2-pyrrolidone, methyl-2-pyrrolidone, and/or N-methylforamide.
  • 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.
  • the pharmaceutical compositions may be for subcutaneous administration (e.g., injection and/or implantation).
  • 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.).
  • 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.
  • the therapeutic compounds can be combined with a biodegradable polymer (e.g., polylactide, polyglycolide, and/or a copolymers thereof).
  • 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.
  • 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).
  • 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 adhesive patch that is placed on the skin to deliver the therapeutic compounds through the skin and into the bloodstream.
  • the pharmaceutical compositions may optionally further comprise a second therapeutic agent.
  • the second therapeutic agent can be an antioxidant.
  • suitable antioxidants include, but are not limited to ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, ⁇ -carotene, lycopene, lutein, resveratrol, retinol (vitamin A), a-tocopherol (vitamin E), ubiquinol, selenium, and catalase.
  • the second therapeutic agent is vitamin E, selenium or catalase.
  • 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.
  • 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.
  • the quantity to be administered 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.
  • doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these compounds.
  • 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 ⁇ g/kg, mg/kg, ⁇ g/day, or mg/day or any range derivable therein.
  • doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 ⁇ to 150 ⁇ .
  • the effective dose provides a blood level of about 4 ⁇ to 100 ⁇ .; or about 1 ⁇ to 100 ⁇ ; or about 1 ⁇ to 50 ⁇ ; or about 1 ⁇ to 40 ⁇ ; or about 1 ⁇ to 30 ⁇ ; or about 1 ⁇ to 20 ⁇ ; or about 1 ⁇ to 10 ⁇ ; or about 10 ⁇ to 150 ⁇ ; or about 10 ⁇ to 100 ⁇ ; or about 10 ⁇ to 50 ⁇ ; or about 25 ⁇ to 150 ⁇ ; or about 25 ⁇ to 100 ⁇ ; or about 25 ⁇ to 50 ⁇ ; or about 50 ⁇ to 150 ⁇ ; or about 50 ⁇ to 100 ⁇ (or any range derivable therein).
  • 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 ⁇ or any range derivable therein.
  • 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.
  • the blood levels discussed herein may refer to the unmetabolized therapeutic compound.
  • 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.
  • dosage units of ⁇ g/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of ⁇ g/ml or mM (blood levels), such as 4 ⁇ to 100 ⁇ .
  • 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
  • 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).
  • 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).
  • extrusion of mtDNA appears to be ROS-independent, as neither DPI (NADPH inhibitor) nor MitoTempo (mtROS scavenger) could block it (Fig. 9E).
  • DPI NADPH inhibitor
  • MitoTempo mtROS scavenger
  • EXAMPLE 3 Extrusion of mitochondrial DNA-protein complexes might represent a neutrophil alternative to mitophagy.
  • 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).
  • 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).
  • 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).
  • EXAMPLE 4 - MtDNA oxidation is required for extruded neutrophil mtC to activate pDCs.
  • 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.
  • 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.
  • HMGB1 antagonist BoxA does not block pDC activation (Fig. 11G).
  • 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.
  • 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.
  • MDV 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.
  • PTP permeability transition pore
  • EXAMPLE 6 Type I IFN and anti-Sm/RNP antibodies synergistically block the detoxification of oxidized mtDNA in neutrophils.
  • 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).
  • 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.
  • 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.
  • PKA activity is regulated by phosphodiesterases (PDE), which quickly convert cAMP to AMP to reduce the amount of cyclic nucleotides required for kinase activation.
  • PDE phosphodiesterases
  • Mammalian cells contain different isoforms of PDE distributed along different subcellular compartments, including mitochondria (Acin-Perez et al., 2011; Acin-Perez et al., 2009).
  • IB MX pan-PDE inhibitor
  • FIG. 15B 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).
  • neutrophils spontaneously release mitochondrial DNA/protein complexes (mtC) in the absence of overt cell death and/or membrane disruption in an autophagy-dependent manner.
  • mtC mitochondrial DNA/protein complexes
  • EXAMPLE 8 Extrusion of mitochondrial DNA-protein complexes as a neutrophil alternative to mitophagy
  • MtC extrusion could be the result of improper disposal of damaged mitochondria.
  • 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).
  • 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).
  • 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)).
  • extrusion of mtC might result from a constitutive defect of neutrophil mitophagy.
  • 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).
  • Ox mtDNA is a powerful pDC activator
  • Ox oxidized
  • Non Ox non-oxidized
  • mtDNA fragments were generated using rt-PCR.
  • Addition of the cationic peptide LL-37 to facilitate their uptake resulted in large amounts of IFNa only in the presence of Ox mtDNA (FIG. 20F).
  • the ability of gDNA to induce IFNcc production is much lower than that of Ox mtDNA (FIG. 20F).
  • 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).
  • 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).
  • 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)).
  • PKA Protein Kinase A
  • neutrophils express PKA within mitochondria (FIG. 26C).
  • 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.
  • 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.
  • EXAMPLE 12 SLE patients retain Ox mtDNA within their neutrophil mitochondria and develop anti-Ox mtDNA autoantibodies
  • Ox mtDNA is an autoantigen in SLE.
  • 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.
  • 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.
  • MDVs 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.
  • 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).
  • PKA Protein Kinase A
  • 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.
  • 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.
  • Ox mtDNA needs to be in complex with TFAM, which binds RAGE on pDCs, to be internalized and activate TLR9.
  • 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, ⁇ 2 ⁇ . ⁇ (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.
  • Anti-RNP/Sm autoantibody isolation Serum samples from SLE patients were filtered through a 0.45- ⁇ 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.
  • PBS Phosphate-Buffered Saline pH 7.4
  • 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 ⁇ ) with 40% of neutrophils- derived supernatants or with synthetic mtDNA pre-incubated (30 min at room temperature) with medium or LL-37 (50 ⁇ g/ml). After 18 hrs IFNa levels in the corresponding supernatants were measured by Flex Set Kit (BD Biosciences).
  • pDCs were pre-incubated with anti-TFAM (7 ⁇ g/ml; Cell Signaling Technology) or the corresponding isotype control or Recombinant Human RAGE-Fc Chimera (10 ⁇ g/ml; R&D System) or BoxA (10 ⁇ ; HMGBiotech).
  • Electron microscopy - Immunofluorescence microscopy Cells were settled on poly-L-lysine coated glass coverslips (BD Biocoat) for 3 hrs. Where specified Mitotracker DeepRed (25 ⁇ ) 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.
  • Apoptosis were assessed with the APO-BrdU TUNEL assay kit (Invitrogen).
  • FACS buffer PBS + 1% FBS
  • 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.
  • mitochondria depolarization cells were incubated for 3 hrs at 37 °C in 96 well plate (Corning Corporated).
  • Mitotracker DeepRed 25 ⁇ 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 ⁇ g/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.
  • 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.
  • 8-OHdG ELISA was carried out with OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) according to the manufacturer's instructions.
  • Immunoprecipitation For immunoprecipitation of mtDNA/protein complexes, 1 ml of crude neutrophil supernatants was pre-cleared with 20 ⁇ 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 ⁇ g/ml) followed by addition of 20 ⁇ of protein A/G plus agarose for another 4 hrs.
  • IP beads 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.
  • 5x reducing loading buffer Pierce
  • phosphor-TFAM detection cells were gently lysed in ice-cold IP Lysis/Wash Buffer (Pierce).
  • 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.
  • 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
  • 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).
  • Anti-RNP/Sm autoantibodies (aRNP) isolation Serum samples from SLE patients were filtered through a 0.45- ⁇ 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.
  • PBS Phosphate-Buffered Saline pH 7.4
  • neutrophils were pre-incubated with IFNaP (2000 U/ml; Schering Corp.) for 90 min at 37°C before stimulation.
  • IFNaP 2000 U/ml; Schering Corp.
  • TLR7 agonist 1 ⁇ g/ml
  • IRS661 TLR7 antagonist, 1 ⁇
  • ODN2216 TLR9 agonist, 1 ⁇ g/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 ⁇ ) with 40% neutrophil supernatants.
  • pDCs were preincubated, for 30 min at 37°C, with anti-TFAM (7 ⁇ g/ml; Cell Signaling Technology) or the corresponding isotype control or Recombinant Human RAGEFc Chimera (10 ⁇ g/ml; R&D System) or BoxA (10 ⁇ g/ml; HMGBiotech).
  • 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).
  • AU Arbitrary Units
  • 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.
  • TCL Total Cell Lysate neutrophils were subjected to one cycle of freeze/thaw, centrifuged for 5 min at 13000g to remove debris and then concentrated as described above.
  • IF Immunofluorescence microscopy
  • the Click-iT TUNEL Imaging Assay Kit (Molecular Probes) was used accordingly to the manufacturer's instructions.
  • pDCs were incubated for 60 min at 37°C in the presence of Cy5-labeled mtDNA (400 ng/ml) and LL-37 (50 ⁇ ). 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).
  • 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.
  • 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.
  • FACS buffer PBS + 1%FBS
  • 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 ⁇ g/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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g proteins) was incubated over night with anti-TFAM antibody (10 ⁇ g/ml). Subsequently 20 ⁇ 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).
  • HRP- conjugated Clean-Blot IP Detection Reagent Pieris
  • 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 ⁇ 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 ' ) .
  • ND1 mitochondrial DNA encoded NADH dehydrogenase subunit 1
  • GPDH genomic DNA encoded Glyceraldehyde 3-phosphate dehydrogenase
  • 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.
  • 3 ng of isolated DNA were subjected to Real Time PCR with Power SYBR Green PCR Master Mix (Invitrogen) and 0.5 ⁇ of each primer. MtDNA copy number was then calculated as described.
  • 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).
  • Microarray analysis Cells were cultured with medium or CCCP (25 ⁇ ) 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.
  • cRNA Biotinylated complementary RNA
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g/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.
  • PMSF phenylmethylsulfonyl fluoride
  • purified mitochondria from 200 millions of neutrophils were incubated in 100 ⁇ of an osmotically controlled, buffered environment including an energy regenerating system, where the final concentrations of the reagents were: 50 ⁇ 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 ⁇ 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.
  • 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.
  • 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.
  • 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.

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Abstract

Des modes de réalisation de l'invention concernent des procédés et une composition associés au traitement du lupus érythémateux systémique (LES), comprenant l'administration de certaines compositions pharmaceutiques comprenant un inhibiteur de phosphodiestérase (PDE) mitochondriale ; un activateur de protéine kinase A (PKA) mitochondriale ; et/ou un inhibiteur de récepteur pour les produits terminaux de glycation avancée (RAGE). Dans certains modes de réalisation, l'invention concerne un procédé de traitement d'un patient. Le procédé comprend l'administration au patient d'une composition comprenant une quantité efficace de : a) un inhibiteur de phosphodiestérase (PDE) mitochondriale ; b) un activateur de protéine kinase A (PKA) mitochondriale ; et/ou c) un inhibiteur de récepteur pour les produits terminaux de glycation avancée (RAGE).
PCT/US2015/036459 2014-06-20 2015-06-18 Procédés et compositions pour le traitement du lupus érythémateux systémique (les) WO2015200094A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040142391A1 (en) * 1998-10-05 2004-07-22 The Trustees Of Columbia University In The City Of New York Methods for determining whether a compound is capable of inhibiting the interaction of a peptide with RAGE
US20060233743A1 (en) * 2002-10-21 2006-10-19 Kelly Rodney W Compositions and methods of therapy
US20070213296A1 (en) * 2006-03-07 2007-09-13 Yanzhen Zhang Compositions and methods for the treatment of immunoinflammatory disorders
WO2012109569A1 (fr) * 2011-02-11 2012-08-16 The Trustees Of Columbia University In The City Of New York Récepteur pour produits terminaux avancés de glycation (rage) en tant que récepteur de l'acide lysophosphatidique (lpa)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040142391A1 (en) * 1998-10-05 2004-07-22 The Trustees Of Columbia University In The City Of New York Methods for determining whether a compound is capable of inhibiting the interaction of a peptide with RAGE
US20060233743A1 (en) * 2002-10-21 2006-10-19 Kelly Rodney W Compositions and methods of therapy
US20070213296A1 (en) * 2006-03-07 2007-09-13 Yanzhen Zhang Compositions and methods for the treatment of immunoinflammatory disorders
WO2012109569A1 (fr) * 2011-02-11 2012-08-16 The Trustees Of Columbia University In The City Of New York Récepteur pour produits terminaux avancés de glycation (rage) en tant que récepteur de l'acide lysophosphatidique (lpa)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARKOPOULOU ET AL.: "Small Molecules in the treatment of systemic lupus erythematosus", CLINICAL IMMUNOLOGY, vol. 148, no. Iss. 3, 2 October 2012 (2012-10-02), pages 359 - 368, XP028688848 *

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