Clq AND HMGB1 FUSION PROTEINS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 62/370,402, filed August 3, 2016, the contents of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers AR065506, AR057084, and OD012042 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Throughout this application various publications are referred to, including by number. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0004] The ability of the mammalian immune system to avoid reactivity to self relies on a fine balance of multiple, interrelated, signaling pathways, in which excitatory pathways are balanced by inhibitory pathways. Systemic Lupus Erythematosus (SLE) is a disease in which the inhibitory pathways are inadequate and autoreactivity and inflammation result (1). In particular, SLE is characterized by activation of cytosolic toll-like receptors (TLRs) leading to an immunogenic and inflammatory milieu (2). To date, therapeutic strategies for SLE have been largely palliative or rely on non-specific immunosuppressive drugs with serious toxicities. Developing a targeted therapeutic requires a better understanding of the molecular mechanisms through which the body achieves a natural program of quiescence and how this immune homeostasis is disrupted in SLE.

[0005] The present invention addresses this need for new therapeutics to treat SLE and other immune and inflammatory disorders. SUMMARY OF THE INVENTION

[0006] A polypeptide is provided comprising (i) a C lq dodecamer peptide, or a Clq nonamer peptide wherein the nonamer peptide comprises KGEQGEPGA (SEQ ID NO: 5), and (ii) a HMBGl A-box peptide or a HMBG1 B-box peptide.

[0007] Also provided is a method of treating an autoimmune inflammatory condition comprising administering an amount of a polypeptide as described herein effective to treat an autoimmune inflammatory condition.

[0008] Also provided is a method to quiesce a monocyte in a subject comprising administering an amount of the polypeptide as described herein effective to quiesce a monocyte in a subject

[0009] Also provided is a method to induce an M2 phenotype in a monocyte in a subject and/or reducing an adaptive immune activation in a subject comprising administering an amount of the polypeptide as described herein effective to induce M2 phenotype in a monocyte in a subject and/or reducing an adaptive immune activation in a subject.

[0010] Also provided is a method of reducing a hyper-activated innate immune response in a subject comprising administering an amount of the polypeptide as described herein effective to treat reduce a hyper-activated innate immune response.

[0011] Also provided is a polypeptide comprising (i) a C lq dodecamer peptide, or a Clq nonamer peptide wherein the nonamer peptide is KGEQGEPGA (SEQ ID NO:5), and (ii) a DWEYS peptide.

[0012] Also provided is a method of treating an autoimmune inflammatory condition comprising administering an amount of the polypeptide as described herein comprising the

DWEYS peptide effective to treat an autoimmune inflammatory condition.

[0013] Also provided is a method of treating an inflammatory condition in sepsis comprising administering an amount of the polypeptide as described herein comprising the

DWEYS peptide effective to treat an inflammatory condition in sepsis.

[0014] Also provided is a method of maintaining a systemic lupus erythematosus (SLE) remission state in a subj ect having had SLE but in remission, comprising administering an amount of the polypeptide as described herein comprising the DWEYS peptide effective to maintain a remission state in a subject having had systemic lupus erythematosus.

[0015] Also provided is a method of reducing a hyper-activated innate immune response in a subject comprising administering an amount of the polypeptide as described herein comprising the DWEYS peptide effective to treat reduce a hyper-activated innate immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. Clq inhibits expression of HMGBl -induced IFNa, MXl and inflammatory cytokines by human monocytes, (a) mRNA levels for IFNa, MXl, IL-6, TNFa and IL12p35 in monocytes stimulated with or without HMGBl (3 μg/ml) and various concentrations of Clq ml-1) for 6 h in serum free medium, (b) HMGBl (3 μg/ml) induced IL-6, TNFa and IL12p70 protein production by monocytes which was reduced in the presence of various concentrations of Clq, assessed at 24 h. (c) Increasing the concentration of HMGBl (10 μg ml-1) abolished the Clq-mediated inhibition of transcription as assessed by q-PCR and ELISA. (d) IFNa, MXl, IL-6, TNFa mRNA expression by monocytes transfected with control siRNA or LAIR-1 siRNA and treated with Clq (25 ug/ml), HMGBl (3 ug/ml) for 6 h. (e) MXl and TNFa mRNA expression in monocytes treated with Clq (25 ug/ml), LAIR-2 (20 ug/ml) and HMGBl (3 ug/ml) for 6 h. (f) IFNa, IL-6 and TNFa mRNA expression by the adherent cell fraction of peripheral blood mononuclear cells treated with Clq (25 g ml), HMGBl (3 μg/ ml) for 6 h. (g) LAIR-1 deficient monocytes exhibit no Clq-mediated inhibition for IFNa, MXl, IL-6 and TNFa mRNA expression. Data are expressed as fold induction relative to controls (mean ± s.d. of triplicates). R.E; relative expression. ELISA data reflect mean ± s.d. of duplicate samples. Differences, determined by the unpaired t-test and One-way ANOVA, are: not significant, ns; *P<0.05; **P<0.01; ***P<0 001. Data are representative of four independent experiments.

[0017] Figure 2. Clq inhibits HMGBl internalization by human monocytes. Human monocytes were treated with FITC-conjugated HMGBl (3 μg/ml) in the absence or presence of Clq (25 μg ml-1) for 15 min at 4°C (a) or 37°C (b). FITC-conjugated HMGBl (green) and DAPI (blue) were viewed using Axiovert 200M digital deconvolution microscope (63x; oil). Scale bar: 10 um. (c) Mean intensity per cell was calculated over 200 cells by Zen2 software. Similar results were obtained in four independent experiments, and representative images are shown. [0018] Figure 3. Clq, HMGB1 and soluble RAGE form a tri -molecular complex, (a) Clq inhibits HMGB1 -mediated IL-12a and MX1 transcription in wild type but not RAGE- deficient monocytes. mRNA expression in splenic monocytes of C57BL/6 wild type or RAGE deficient mice treated with HMGB1 (3 μg/ml) and co-incubated with or without Clq (25 g/ml) for 6 h. R.E; relative expression (mean ± s.d. of triplicates), not significant, ns; *P<0.05; **P<0.01. Data are representative of four independent experiments, (b) Surface plasmon resonance (SPR) assay of Clq and RAGE binding; KD=855 nM. (c) SPR assay of RAGE-Clq-HMGBl trimolecular complex. sRAGE was immobilized onto a CM5 chip and the first analyte (Clq, 200 nM) was added to saturation. HMGB1 (500 nM) was injected to the sRAGE-Clq complex in multiple pulses (left). HMGB1 was injected to immobilized sRAGE until the chip was saturated followed by Clq addition (right), (d) SPR assay for HMGB1 and Clq binding; KD=200 nM. (e) SPR assay of different redox states of HMGB1 and Clq binding. SPR experiments were repeated at three times; representative data are shown, (f) Clq-coated beads were incubated with saturating amounts of HMGB1, followed by 250 ng or 500 ng of RAGE. Complexes were analyzed by Western blot using anti-RAGE, anti-Cbp antibody for HMGB1 or IR-labeled streptavidin for biotinylated-Clq. Experiments were repeated three times, representative data are shown.

[0019] Figure 4. Clq and HMGB1 cross-link LAIR-1 and RAGE in lipid rafts, (a) Colocalization of LAIR-1 and RAGE on the plasma membrane was assessed by proximity ligation assay (PLA). Red dots (PLA positive), representing colocalization between RAGE and LAIR-1, are only seen in the presence of Clq, with or without HMGB1. Percent PLA positive cells over total cells were counted from different random field (>200 cells). One of four similar assays is shown, (b) Lipid raft fractions were concentrated and analyzed by Western blot for LAIR-1, RAGE, HMGB1, Clq or Flotillinl as a lipid raft marker. Data are representative of three independent experiments.

[0020] Figure 5. Clq dephosphorylates RAGE, recruits SHP-1 to LAIR-1 and inhibits the HMGBl-induced NF-κΒ signaling pathway, (a) Human monocytes were treated with Clq and/or HMGB1 and subjected to immunoprecipitation (IP) with antibodies to RAGE followed by immunoblotting with antibodies specific for phospho-serine (top) or RAGE (bottom). Numbers below the immunoblots indicate the signal intensity ratio. Data are representative of three independent experiments, (b) Total cell ly sates were subjected to immunophosphorylation array (R&D) to observe the phosphorylation of LAIR-1 ITIM motifs. Relative quantification for the phosphorylation of LAIR-1 was normalized to control spots. Data are representative of three independent experiments, (c) Human monocytes were treated with Clq and/or HMGB1 and subjected to immunoprecipitation with antibodies to LAIR-1 followed by immunoblotting with antibodies for SHP-1 (top) or LAIR-1 (bottom). Data are representative of four independent experiments, (d) Human monocytes were treated with Clq and/or HMGB1 and subjected to immunoblotting with antibodies for activated ΙΚΚα (Ρ-ΙΚΚα, top), p65 (P-p65, middle) or β-actin. Experiments were repeated four times and representative data are shown, (e) Nuclear translocation of NF-KB p65 was analyzed following HMGB1 or HMGB1 plus Clq stimulation for 1 h. Maximal fluorescent intensity was evaluated for DAPI (blue) and NF-κΒ p65 (red) across the red arrow The percentage of maximal fluorescent intensity along the red arrow traced in the merged image is displayed. Scale bar: 10 μιη. Data are representative of three independent experiments.

[0021] Figure 6. HMGB1 and Clq induce anti-inflammatory molecules and promote an M2-like phenotype. (a-e) Human monocytes treated with Clq (25 μg/ml) or Clq tail (53 μg/ml) and/or HMGB1 (3 μg/ml) for 24 h were processed for mRNA and protein, (a) Mer tyrosine kinase as assessed by q-PCR (reft) and flow cytometry (right). R.E; relative expression. Error bars indicate mean ± s.d. (b) Programmed Death-Ligand 1 (PDL-1) was measured by q-PCR (left) and flow cytometry (right), (c) IL-10 was measured by q-PCR (left) and ELISA of culture supernatant (right), (d) CD163 as assessed by q-PCR (left) was determined by flow cytometry (right), (e) In the presence of different concentration of Clq (10, 25, 50 or 75 μg/ ml), Mer, PDL-1, IL-10 and CD163 mRNA transcription was assessed after 24 h stimulation. Statistical analysis was performed by One-way ANOVA and t-test; ns, not significant; * PO.05, ** P <0.01 *** PO.001. All data are representative of four independent experiments

[0022] Figure 7. HMGB1 alters cellular metabolism, induces aerobic glycolysis, and is inhibited by Clq. HMGBl 's effect on cellular mitochondrial (a) and glycolytic (b) activity was evaluated using a SeaHorse metabolic analyzer. Monocytes were treated with HMGB1 and/or Clq for 24 h, assessed for basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), and then sequentially treated with listed reagents. One representative experiment of three is shown, (a) For evaluation of mitochondrial activity, cells were incubated with ohgomycm, FCCP, and rotenone plus antimycin A. Mitochondrial ATP production was calculated by subtracting oxygen consumption following oligomycin treatment from basal levels, (b) For evaluation of cellular glycolytic activity, cells were incubated in assay media lacking glucose or pyruvate, then treated sequentially with glucose, oligomycin, and 2-deoxy-glucose. Max glycolytic capacity following oligomycin treatment is shown. Statistical analysis was performed using One-way ANOVA with correction for multiple comparisons. * PO.05, **P <0.01 ***P<0.001. (c) HMGB1 treated cells showed a marked increase of glycolysis in comparison to oxidative phosphorylation (OCR/ECAR). Data represents sum of four independent experiments. Statistical analysis was performed by Kruskal-Wallis test/Dunn correction for subgroup analysis. *P<0.05.

[0023] Figure 8. HMGB1 and Clq terminate M2-like macrophage phenotypes. Phenotype analysis of DC differentiation induced by GM-CSF and IL-4 was performed by flow cytometry. Monocytes were treated with Clq and/or HMGB1 for 24 h (Day 0), then further cultured with GM-CSF and IL-4 for 2 days (Day 2). (a) High levels of CD 14 and LAIR-1 represent suppression of DC differentiation. Data are expressed as mean ± s.e.m. of triplicate samples. Transcription of Mer and CD 163 were measured by q-PCR. Data are expressed as mean ± s.d. of triplicate samples. Significant differences are indicated *P <0.05; **P <0.01 ; ***p <0.001 (t-test). Data are representative of three independent experiments, (b) Monocytes were exposed to HMGB1 (3 μg/ml), Clq (25 μg/ml) or both for 24 h washed, and further incubated 2 days in X-Vivo 15 medium. Cell Trace Violet-stained allogeneic primary human CD4 T cells were added (2: 1). After 4 days, the nonadherent cells were removed and assessed by flow cytometry. Live CD4+ T cells were analyzed. Significant differences are indicated *P <0.05; **P <0.01; ***P <0.001 (t-test). Data are representative of three independent experiments.

[0024] Figure 9. Model showing how Clq utilizes a natural pathway to dampen inflammation, (a) DNA/RNA binding HMGB1 is internalized and activates endosomal TLRs and induces Ml-like macrophages (b) In the presence of Clq without inflammation, Clq and LAIR-1 signaling prevents HMGB1 internalization, (c) However, in inflammation, Clq mediates M2 differentiation by cross-linking RAGE and LAIR-1 in lipid rafts to facilitate. SHP-1 binding to LAIR-1 via phosphorylated ITIMs, and induces differentiation of M2-like macrophage.

[0025] Fig. 10: Synthetic Clq peptide activates LAIR-1.

[0026] Fig. 11 : HMGBl-linker-Clq peptide mimics Clq; cross-links RAGE and LAIR-1. [0027] Fig. 12: Exemplary sequences of Clq A peptide (SEQ ID NO:3); HMGBl A- Box (22 aa) (SEQ ID NO: l); HMGBl A-Box linker Clq (SEQ ID NO: 10); HMGBl -B box linker Clq (SEQ ID NO:7); and DWEYS linker Clq (SEQ ID NO: 11).

[0028] Fig. 13A-B: 13A - Determination of bound LAIR-1 to Clq tail; 13B - Determination of bound P-LAIR-1 to Clq tail.

[0029] Fig. 14: 15 min PL A for RAGE and LAIR- 1 - showing HMGB 1 -B box linker Clq, DWEYS linker Clq, and control.

[0030] Fig. 15: B box fusion: HMGBl-B box linker Clq DWEYS fusion: DWEYS linker Clq. Left panel shows IL-6 levels after 4 hrs with each of the listed treatments. Right panel shows TNF-alpha levels after 4 hrs with each of the listed treatments.

[0031] Fig. 16: Fusion protein induces PDL-1.

[0032] Fig. 17: Fusion protein does not induce IL-10.

[0033] Fig. 18: Left panel shows MX-1 induction levels with each of the listed treatments. Right panel shows OAS1 induction levels in each of the listed conditions.

[0034] Fig. 19: DWEYS fusion: DWEYS linker Clqa. Left panel shows IL-6 levels after each of the listed treatments. Right panel shows TNF-alpha levels after each of the listed treatments.

[0035] Fig. 20: DWEYS fusion: DWEYS linker Clqa. Left panel shows PDL-1 levels after each of the listed treatments. Right panel shows IL-10 levels after each of the listed treatments.

[0036] Fig. 21 : LAIR- 1 -mediated inhibition of CI qA peptide.

DETAILED DESCRIPTION OF THE INVENTION

[0037] A polypeptide is provided comprising (i) a Clq dodecamer peptide, or a Clq nonamer peptide wherein the nonamer peptide comprises KGEQGEPGA (SEQ ID NO: 5), and (ii) a HMBGl A-box peptide or a HMBGl B-box peptide.

[0038] A Clq dodecamer peptide is a peptide comprising the sequence KGEQGEPGAPGI (SEQ ID NO: 3). A Clq nonamer peptide is a peptide comprising the sequence KGEQGEPGA (SEQ ID NO:5). A HMBG1 A-box peptide is a peptide comprising the sequence MGKGDPKKPRGKMSSYAFFVQT (SEQ ID NO: l). A HMBG1 B-box peptide is a peptide comprising the sequence KLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKK (SEQ ID NO:2). In an embodiment, the polypeptide is recombinantly produced. In an embodiment, the polypeptide is a recombinantly produced fusion protein. A nucleic acid, encoding the polypeptide comprising (i) a Clq dodecamer peptide, or a Clq nonamer peptide wherein the nonamer peptide comprises KGEQGEPGA (SEQ ID NO: 5), and (n) a HMBG1 A-box peptide or a HMBG1 B-box peptide, is also provided. In an embodiment, the nucleic acid is recombinantly produced. In an embodiment, the nucleic acid is a cDNA.

[0039] In an embodiment, the polypeptide comprising the Clq dodecamer peptide, or Clq nonamer peptide, and the HMBG1 A-box peptide or HMBG1 B-box peptide is up to 70 amino acids in length. In an embodiment, the polypeptide is up to 65 amino acids in length. In an embodiment, the polypeptide is up to 60 amino acids in length.

[0040] In an embodiment of the polypeptide, the carboxy terminal amino acid or the amino terminal amino acid residue of the Clq dodecamer or nonamer is bound to the amino terminal amino acid or the carboxy terminal amino acid residue, respectively, of the HMBG1 A-box or HMBG1 B-box peptide.

[0041] In an embodiment of the polypeptide, the Clq dodecamer or nonamer is bound directly by a peptide bond to the HMBG1 A-box or HMBG1 B-box peptide.

[0042] In an embodiment of the polypeptide, the Clq dodecamer or nonamer is bound by a peptide bond to a linker peptide which is bound by a peptide bond to the HMBG1 A- box or HMBG1 B-box peptide. In an embodiment of the polypeptide, the linker peptide comprises (GGGGS)n or A(EAAAK)nA (where n= 2, 3, 4, or 5). In an embodiment the linker comprises (Gh/ ₄ Ser)3. In an embodiment, the linker is rigid. In an embodiment the linker is cleavable. Non-limiting examples of cleavable linkers within the scope of the invention include disulfide links and protease cleavable linkers. In a preferred embodiment, the linker is a peptide linker.

[0043] In an embodiment the polypeptide further comprises a plasma half-life extending moiety. In an embodiment, the plasma half-life extending moiety is covalently attached to the polypeptide. Plasma half-life extending moieties are well known in the art, such as PEG molecules, fatty acids bound to peptide side chains, further polypeptides such as Fc, human serum albumin, XTEN and PAS. In an embodiment, the polypeptide further comprising an immunoglobulin Fc monomer or dimer. In an embodiment, the polypeptide does not further comprise a plasma half-life extending moiety. In an embodiment, the polypeptide does not further comprise an immunoglobulin Fc. In an embodiment, the polypeptide does further comprise an immunoglobulin Fc. In an embodiment, the immunoglobulin Fc is an immunoglobulin G Fc. In an embodiment, the immunoglobulin Fc has the sequence of a human immunoglobulin Fc. In an embodiment, the immunoglobulin Fc has the sequence of a human immunoglobulin IgGl Fc. Human immunoglobulin IgGl Fc are well known in the art and are readily and routinely identified by those of skill in the art. Automatic sequences can be used for such and widely-available alignment matching tools. In an embodiment of the Fc, the Fc is de-fucosylated of one or more N-linked oligosaccharides on the Fc region. In an embodiment, the polypeptide does not further comprise a plasma half-life extending entity.

[0044] In an embodiment, the polypeptide comprises the HMBG1 A-box. In an embodiment, the HMBG1 A-box comprises a 22-amino acid residue sequence. In an embodiment, the HMBG1 A-box comprises MGKGDPKKPRGKMSSYAFFVQT (SEQ ID NO: l). In an embodiment, the polypeptide comprises

MGKGDPKKPRGKMSSYAFFVQTGGGGSGGGGSGGGGSKGEQGEPGAPGI (SEQ ID NO: 10).

[0045] In an embodiment, the polypeptide comprises the HMBG1 B-box. In an embodiment, the HMBG1 B-box comprises a 34-amino acid residue sequence. In an embodiment, the HMBG1 B-box comprises

KLKEKYEKDIAAYRA GKPDAAKKGVVKAEKSKK (SEQ ID NO:2). In an embodiment, the polypeptide comprises the sequence

KLKEKYEKDIAAYRA GKPDAAKKGVVKAEKSKKGGGGSGGGGSGGGGSKGEQ GEPGAPGI (SEQ ID NO:7).

[0046] In an embodiment, the polypeptide comprises the Clq dodecamer peptide which has the sequence KGEQGEPGAPGI (SEQ ID N0 3).

[0047] In an embodiment, the polypeptide comprises the Clq nonamer peptide but not the Cl q dodecamer peptide which has the sequence KGEQGEPGAPGI (SEQ ID NO: 3).

[0048] In an embodiment, the polypeptide does not comprise a mouse Clq nonamer sequence.

[0049] Also provided is a method of treating an autoimmune inflammatory condition comprising administering an amount of a polypeptide as described herein effective to treat an autoimmune inflammatory condition.

[0050] In an embodiment, the autoimmune inflammatory condition is systemic lupus erythematosus (SLE). In an embodiment, the autoimmune inflammatory condition is rheumatoid arthritis. [0051] Also provided is a method to quiesce a monocyte in a subject comprising administering an amount of the polypeptide as described herein effective to quiesce a monocyte in a subject.

[0052] Also provided is a method to induce an M2 phenotype in a monocyte in a subject and/or reduce an adaptive immune activation in a subject comprising administering to the subject an amount of the polypeptide as described herein effective to induce M2 phenotype in a monocyte in a subject and/or reduce an adaptive immune activation in a subject.

[0053] Also provided is a method of reducing a hyper-activated innate immune response in a subject comprising administering an amount of the polypeptide as described herein effective to treat reduce a hyper-activated innate immune response.

[0054] Also provided is a polypeptide comprising (i) a Clq dodecamer peptide, or a Clq nonamer peptide wherein the nonamer peptide is KGEQGEPGA (SEQ ID NO:5), and (ii) a DWEYS peptide. In an embodiment, the DWEYS peptide consists of DWEYS (SEQ ID NO: 8).

[0055] In an embodiment, the Clq dodecamer or nonamer is bound by a peptide bond to a linker peptide which is bound by a peptide bond to the DWEYS peptide. In an embodiment, the linker peptide compnses (GGGGS)n or A(EAAAK)nA (where n= 2, 3, 4, or 5). In an embodiment, polypeptide has the sequence

D WE Y S GGGGS GGGGS GGGGS GEQ GEP GAP GI (SEQ ID NO: 11)

[0056] In an embodiment, the polypeptide further comprises a plasma half-life extending entity. In an embodiment, the plasma half-life extending entity is covalently attached to the polypeptide. Plasma half-life extending entities are well known in the art, such as PEG molecules, fatty acids bound to peptide side chains, further polypeptides such as Fc, human serum albumin, XTEN and PAS. In an embodiment, the polypeptide further comprising an immunoglobulin Fc monomer or dimer. In an embodiment, the polypeptide does not further comprise a plasma half-life extending entity. In an embodiment, the immunoglobulin Fc is an immunoglobulin G Fc. In an embodiment, the immunoglobulin Fc has the sequence of a human immunoglobulin Fc. In an embodiment, the immunoglobulin Fc has the sequence of a human immunoglobulin IgGl Fc. In an embodiment of the Fc, the Fc is de-fucosylated of one or more N-linked oligosaccharides on the Fc region. In an embodiment, the polypeptide does not further comprise a plasma half-life extending entity.

[0057] In an embodiment, the Clq dodecamer peptide has the sequence KGEQGEPGAPGI (SEQ ID NO:3). In an embodiment, the polypeptide comprises the Clq nonamer peptide but not the Clq dodecamer peptide having the sequence KGEQGEPGAPGI (SEQ ID NO: 3).

[0058] In an embodiment, the polypeptide does not comprise a mouse Clq nonamer sequence.

[0059] Also provided is a method of treating an autoimmune inflammatory condition comprising administering an amount of the polypeptide as described herein comprising the DWEYS peptide effective to treat an autoimmune inflammatory condition.

[0060] In an embodiment, the autoimmune inflammatory condition is systemic lupus erythematosus (SLE). In an embodiment, the autoimmune inflammatory condition is rheumatoid arthritis.

[0061] Also provided is a method of treating an inflammatory condition in sepsis comprising administering an amount of the polypeptide as described herein comprising the

DWEYS peptide effective to treat an inflammatory condition in sepsis.

[0062] Also provided is a method of maintaining a systemic lupus erythematosus (SLE) remission state in a subject having had SLE but in remission, comprising administering an amount of the polypeptide as described herein comprising the DWEYS peptide effective to maintain a remission state in a subject having had systemic lupus erythematosus.

[0063] Also provided is a method of reducing a hyper-activated innate immune response in a subject comprising administering an amount of the polypeptide as described herein comprising the DWEYS peptide effective to treat reduce a hyper-activated innate immune response.

[0064] In an embodiment of the methods described herein, the subject is a human.

[0065] In an embodiment of the methods and polypeptides described herein, the Clq peptide has a sequence identical to a portion of human Clq having the sequence:

MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPG APGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGT GSPGNIKDQ PRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQW EICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKG HIYQGSEADSVFSGFLIFPSA. (SEQ ID NO:4)

[0066] In an embodiment, the Clq peptide comprises the sequence KGEQGEPGA (SEQ ID NO:5).

[0067] In an embodiment, the Clq peptide comprises the sequence KGEQGEPGAPGI (SEQ ID NO:3). [0068] In an embodiment, the Clq peptide comprises the sequence KGEQGEPGA (SEQ ID NO:5) but not GEQGEPGAPGI (SEQ ID NO:3).

[0069] In an embodiment, the Clq peptide comprises the sequence KGEQGEPGA KGEQGEPGAPGI (SEQ ID NO: 6).

[0070] In an embodiment of the methods described herein, the polypeptide can be administered as an active ingredient in a pharmaceutical composition. In an embodiment, the polypeptide is the only pharmaceutically active ingredient in the pharmaceutical composition. In an embodiment, the pharmaceutical composition comprises a pharmaceutical carrier.

[0071] In the methods described herein, administration of the polypeptide, or of a pharmaceutical composition comprising the polypeptide, can be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravagmal, intragingival, mtraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topically, transdermal, transmucosal, transplacental, transtracheal, ureteral, uretheral, and vaginal.

[0072] As used herein, "treating" an autoimmune disease means that one or more symptoms of the disease, such as inflammation or other parameters by which the disease is characterized, are reduced, ameliorated, prevented, placed in a state of remission.

[0073] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0074] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS

[0075] This disclosure reveals interactions of two proteins that are dysregulated in SLE: High Mobility Group Box 1 (HMGBl) and the first component of complement (Clq). HMGBl is an evolutionary ancient DNA-binding nucleoprotein found in almost all mammalian cells (3,4). In addition to its role as a transcriptional regulator, HMGBl also functions as a Damage-Associated Molecular Pattern (DAMP) molecule when either released from necrotic cells (passive release) or secreted from activated leukocytes such as monocytes, macrophages and myeloid dendritic cells (active release) (5,6). HMGBl is significantly elevated in the serum from patients with SLE, where its role as a necessary co- factor for activation of endosomal TLRs is believed to be critical in lupus pathogenesis (4). HMGBl is a ligand for the Receptor for Advanced Gly cation Endproducts (RAGE), and functions to transport RNA and DNA to endosomal TLRs, leading to production of type 1 interferon (IFN), IFN-inducible genes and pro-inflammatory cytokines (6-8). Administration of antibodies against HMGBl confers significant protection against tissue injury in experimental models of autoimmune disease and inflammation (3). Selective deletion of HMGBl reduced survival of mice in a sepsis model (9). HMGBl has also been shown to suppress inflammation (10), promote the regeneration of skin grafts in mice (11) and enhance ATP production in pancreatic tumor cell lines (12,13). Although HMGBl is the subject of intense investigation, we are still learning about this protein and how it elicits both positive and negative immune responses.

[0076] Post-translational modification of HMGBl significantly influences the biological activity of this molecule (3). One post-translational modification that dramatically affects the activity of HMGBl is the redox state of three critical cysteine residues (4). Disulfide HMGBl, bearing a disulfide bond between C23 and C45, with a free cysteine at C106, binds the TLR4 co-receptor MD2 (14). Reduced HMGBl, which bears three fully reduced cysteine thiol residues, on the other hand, signals through CXCR4 to mediate chemokine-like activity. Finally, sulfonyl HMGBl, which contains a sulfonyl group on any of the cysteines, has no activity for cell migration or cytokine induction.

[0077] Since HMGBl is an evolutionarily conserved molecule which predates the development of adaptive immunity, other ancient proteins were investigated as possible HMGB1 regulatory factors within the scope of innate immunity. Clq is a 460 kDa protein formed by six homotrimeric subunits containing an N-terminal collagen-like sequence and a C-terminal globular region (15). In addition to its role in initiating the complement cascade, Clq has long been known to possess immunoregulatory properties (16,17). Clq binds to molecular partem molecules derived from pathogens and endogenous damage associated molecules, including antibody-antigen complexes, bacterial toxins, myelin, and β amyloid 18. Patients with active SLE have lower levels of Clq because the immune complex formation in SLE consumes complement components and because SLE patients produce antibodies that target and remove Clq (19). Although rare, Clq deficiency is the strongest genetic risk factor for developing SLE (20,21)

[0078] Recently, it was determined that Leukocyte-Associated Ig-like Receptor 1

(LAIR-1; CD305), a transmembrane protein Ig superfamily member, is a high-affinity receptor for Clq (22). Binding of Clq to LAIR-1 mediates inhibition of monocyte-to- dendritic cell (DC) differentiation and plasmacytoid DC (pDC) activation, functions that help explain the contribution of Clq deficiency to SLE pathogenesis.

[0079] It was investigated whether the mechanism by which Clq interacts with LAIR-1 to inhibit immune responses may provide insight into the activity of both HMGB1 and Clq in SLE. The results disclosed herein illuminate a specific previously unknown Clq- HMGB1 interaction: Clq binds to disulfide HMGB1, catalyzing formation of a multimeric protein complex comprising HMGB1, Clq, LAIR-1 and RAGE. This multimeric complex triggers monocytes to differentiate into an M2 phenotype, upregulating the expression of several anti -inflammatory molecules (e.g., Programmed Death Ligand-1 (PDL-1), Mer tyrosine-kinase (Mer), Interleukin-10 (IL-10), and effectively limits the differentiation of monocytes into dendritic cells (DCs), blocking the downstream adaptive immune response These findings identify a mechanism by which Clq levels modulate HMGBl 's inflammatory activity to achieve immune-regulation, a mechanism that is impaired in SLE due to genetic or acquired Clq deficiency.

[0080] Clq inhibits HMGB1 -induced monocyte activation: This laboratory has demonstrated that Clq inhibits the activation of monocytes and plasmacytoid dendritic cells by engaging LAIR-1 (22). It was investigated whether Clq might act through inhibiting the pro-inflammatory activity of HMGB1. To test this hypothesis, cultures of human monocytes isolated from peripheral blood of healthy volunteers were incubated in the presence or absence of HMGB1 with and without Clq and assessed downstream cytokine production. Cultures were performed in serum-free medium to avoid contamination by Clq in serum and permit accurate control of the concentration of Clq. As anticipated, the addition of HMGBl dramatically increased the transcription and secretion of type 1 IFN, IFN-inducible genes and NFi B-dependent proinflammatory cytokines, generating Ml -like macrophages, as has previously been reported (23) (Fig. la-b). While Clq did not alter transcription of these genes in the absence of HMGBl, the addition of human Clq to HMGBl counteracted the HMGBl -mediated cytokine transcription and protein induction in a dose-dependent manner (Fig. la-b). Physiologic levels of Clq effectively blocked monocyte activation while levels commonly present in SLE patients (-25-50 μg/ml) were less inhibitory. Increasing the levels of HMGBl abrogated the inhibitory effect of Clq (Fig lc), suspecting that the ratio of Clq to HMGBl is critical.

[0081] Next assessed was the involvement of LAIR- 1 in this Clq-mediated inhibition of HMGBl-induced monocyte activation. Consistent with the notion that Clq inhibits the activity of HMGBl by engaging LAIR-1, no effect was observed of Clq on HMGBl - mediated cytokine induction in monocytes treated with LAIR-1 -specific siRNA (Fig. Id). Clq failed to inhibit monocyte activation in the presence of soluble decoy receptor LAIR-2 (Fig. le). To study activation and inhibition of monocytes by HMGBl and Clq, respectively, when monocytes are present in mixed cell populations, peripheral blood mononuclear cells (PBMCs) were incubated with HMGBl and Clq and then the adherent cell population analyzed for cytokine induction. HMGBl induced IFN, IL-6 and TNFa; Clq inhibited this HMGBl -mediated activation (Fig. If). This finding in a mixed cell population confirmed the previous observation in isolated monocytes. LAIR-1 deficient monocytes purified from spleens of mice expressing Lysozyme-Cre and harboring a floxed LAIR-1 gene also showed no inhibitory effect of Clq (Fig. lg) Taken together, these results indicate that Clq can modulate immune homeostasis in blood by inhibiting HMGBl- induced monocyte activation through engaging LAIR-1.

[0082] Clq inhibits HMGBl internalization: HMGBl has an important role in potentiating the innate immune response to foreign (and endogenous) nucleic acids by transporting them into the cytoplasm of immune cells such as monocytes and DCs where they bind to endosomal TLRs. Since Clq inhibited HMGBl-induced cytokine secretion, it was asked whether this might result from a Clq-mediated inhibition of the internalization of HMGBl. FITC-labeled human HMGBl was incubated with freshly isolated monocytes in the presence or absence of Clq and performed immunofluorescence microscopy. When the monocytes were maintained at 4°C, HMGBl bound to surface receptors in the presence or absence of Clq (Fig. 2a). When monocytes were incubated at 37°C in the absence of Clq, labeled HMGBl accumulated in the cytoplasm. In contrast, cytosolic HMGBl was significantly decreased in monocytes co-incubated with labeled HMGBl and Clq. (Fig. 2b and 2c). To test whether internalization of TLR ligands, which rely on HMGBl for intracellular transport and trafficking, was also inhibited by Clq, the internalization of CpG oligonucleotides was assessed. FITC -labeled CpG oligonucleotides were internalized in the absence of Clq, but addition of Clq significantly reduced cellular internalization. Clq is known to block the effects of CpG (24), and the findings presented here provide a plausible molecular mechanism through which Clq accomplishes this previously described effect.

[0083] Clq's immunoregulatory function requires RAGE: It was previously reported that the internalization of HMGBl requires HMGBl binding to RAGE (25). Here it is investigated whether Clq was altering the interaction of HMGBl with RAGE. As a first step toward probing the potential interactions between Clq, HMGBl and RAGE, it was asked whether Clq could block HMGBl activation in RAGE-deficient cells. To ensure a complete absence of RAGE, murine monocytes genetically deficient in RAGE (26) were used. Monocytes from the spleens of both wild type and RAGE-deficient mice were incubated with HMGBl in the presence or absence of Clq. Consistent with previous studies (25), it was observed that RAGE-deficient monocytes failed to internalize HMGBl, with or without Clq (data not shown). HMGBl did, however, induce cytokine expression in both wild type and RAGE-deficient monocytes (Fig. 3a), in agreement with previous reports that disulfide HMGBl can signal through TLR4 and MD2 (26,27). As anticipated, co-incubation with Clq led to a reduction in HMGBl -induced proinflammatory cytokine gene transcription in wild type cells. In contrast, Clq mediated no change in HMGBl - induced gene transcription in RAGE-deficient cells (Fig. 3a), demonstrating that RAGE is required for Clq-mediated regulation of HMGBl.

[0084] To further probe the specificity of Clq-mediated monocyte inhibition, similar experiments were performed examining two TLR ligands, both of which can utilize HMGBl as a cofactor: CpG and LPS. CpG mimics bacterial DNA and, through cytosolic TLR9 engagement in endosomes, leads to activation of MyD88 and downstream pathways (28). In contrast, LPS (endotoxin) is a component of the outer membrane of Gram-negative bacteria and, through cell surface TLR4 engagement, leads to activation of a proinflammatory cytokine cascade (29). Incubating these ligands with human monocytes, it was determined that while Clq inhibits CpG-mediated induction of pro-inflammatory cytokines (e.g., TNF) and IFN inducible genes (the IFN signature) (e.g., MX1), it has no effect on LPS-induced cytokine transcription. These striking findings suggest that Clq does not inhibit all monocyte activation; Clq inhibits HMGBl -mediated TLR9 but not HMGBl - mediated TLR4 signaling.

[0085] Since Clq inhibited the activation of RAGE by HMGBl and since both Clq and HMGBl have been shown to bind RAGE directly (30,31), it was determined if Clq prevented the interaction of HMGBl with RAGE. For these experiments a surface plasmon resonance assay was employed and it was demonstrated that Clq binds RAGE in a dose- dependent manner (Kd=855nM, Fig. 3b). No binding of the Clq collagen-like tail to RAGE was detected, confirming an interaction of RAGE with the globular head of Clq (data not shown). It was next asked whether Clq prevented an interaction of HMGBl with RAGE, or whether a trimolecular complex of Clq, HMGBl and RAGE could form. For this experiment, soluble RAGE (sRAGE) was immobilized on the sensor chip and Clq was introduced until the chip was saturated; then HMGBl was added to form a sRAGE-Clq- HMGB1 complex (Fig. 3c, left). Since HMGBl is significantly smaller than Clq, displacement of Clq by HMGBl would have produced a lower signal after addition of HMGBl. This did not occur. Qualitatively similar results were observed when HMGBl was added to immobilized sRAGE followed by Clq addition (Fig 3c, right). In both cases Clq, HMGBl and sRAGE formed a trimolecular complex. Interestingly, Clq also bound directly to HMGBl in a concentration-dependent manner (Kd=200nM, Fig. 3d), preferentially binding to the disulfide form of HMGBl, the form that functions as a cytokine (Fig. 3e-g). The existence of a trimolecular complex was confirmed by immobilizing Clq on beads, incubating with saturating amounts of HMGBl, followed by increasing concentrations of sRAGE (Fig. 3f). sRAGE was bound to Clq beads even in the presence of saturating amounts of HMGBl (Fig. 3c). The data give evidence that Clq, RAGE and HMGBl all interact.

[0086] Clq bridges RAGE and LAIR-1 : It was previously demonstrated that Clq binds and activates the inhibitory receptor LAIR-1 (22) and it was shown above that this binding is critical to its inhibitory function. Since the Clq globular head binds RAGE while the Clq collagen tail binds LAIR-1, the next question was whether Clq might cross-link LAIR- 1 to RAGE on the surface of monocytes. For these experiments we used a proximity ligation assay to investigate the localization of RAGE and LAIR-1 in the absence and presence of Clq. Polymerase-amplified fluorescence, indicative of RAGE-LAIR- 1 binding, was only detected in the presence of Clq, with or without HMGBl (Fig. 4a). This interaction is specific because probe control or isotype controls showed little or no signal and co-incubating Clq with LAIR-2 (a soluble decoy receptor) negated the cross-linking of RAGE and LAIR-1 (data not shown). Since Clq binds both RAGE and HMGBl through its globular head, the fact that HMGBl does not alter the Clq-mediated cross-linking of LAIR-1 and RAGE suggests that RAGE and HMGBl bind Clq on different regions of the globular head. This agrees well with the surface plasmon resonance results which also demonstrate that HMGBl and Clq bind different epitopes on RAGE. In the presence of HMGBl and Clq, LAIR-1 migrated to lipid rafts where LAIR-1 colocalized with RAGE (Fig. 4b). Taken together, these results suggest that Clq can create a tetra-molecular complex of Clq, LAIR-1, HMGBl and RAGE, bringing these molecules into the lipid raft with RAGE.

[0087] Phosphorylation of RAGE and LAIR-1 differs in the presence of HMGBl and Clq In order to understand how the colocalization of RAGE and LAIR-1 might affect their downstream signaling pathways, the phosphorylation of several molecules was examined on primary human monocytes in the presence of HMGBl, Clq or both. A previous report that HMGBl enhances phosphorylation of RAGE, which others have shown is mediated by ΡΚ£ζ was confirmed (32), and it was demonstrated that the HMGBl - induced phosphorylation is diminished in the presence of Clq (Fig. 5a). It was also shown that Clq induces phosphorylation of LAIR-1 22, and observed that more SHP-1 is recruited to LAIR-1 when both HMGBl and Clq are present (Fig. 5b and c). Since phosphorylation on both ITIMs induces LAIR-1 to bind SHP-1, our data reveal that HMGBl and Clq together enhance the phosphorylation of both ITIM motifs. Finally, Clq inhibited the downstream activation and nuclear translocation of NF-κΒ induced by HMGBl (Fig. 5d and e). Diminished HMGBl -induced phosphorylation of IKKa and diminished nuclear localization of p65 was observed in the presence of Clq. Together, these results indicate that in the presence of both Clq and HMGBl, RAGE is dephosphorylated, SHP-1 is recruited to LAIR-1, and F-κΒ signaling pathway is inhibited.

[0088] Monocytes exposed to both HMGBl and Clq express a novel set of genes: To assess whether HMGBl and Clq might further alter the activity of monocytes, primary human monocytes were assessed for expression of immunomodulatory proteins after incubation alone or in the presence of HMGBl, Clq or both for 24 h. Exposure to both HMGB1 and Clq induced the transcription and protein expression of a variety of antiinflammatory factors, including Mer, a receptor tyrosine kinase important in the clearance of apoptotic debris, Fig. 6a); PDL-1, a molecule that suppresses T cell activation, Fig. 6b); transcription and secretion of IL-10, an anti-inflammatory cytokine (Fig. 6c); and transcription and expression of CD 163, a marker of M2 macrophages (Fig. 6d). Physiologic levels of Clq were more potent than levels often observed in patients with SLE (25-50 μg/ml) (Fig. 6e). Importantly, exposure to HMGB1 and Clq tail did not mediate similar effects (Fig. 6a), confirming that Clq must interact with RAGE as well as LAIR-1 to induce M2 polarization and that coincident but uncoordinated signaling through RAGE and LAIR- 1 is not sufficient. These findings indicate that when Clq cross-links LAIR-1 with RAGE and HMGB1 in lipid rafts it engages a program of monocyte differentiation into M2 macrophages.

[0089] Metabolic consequences of HMGB1 or HMGB1 and Clq exposure: The metabolic phenotype of immune cells has been previously shown to correlate with function. Classically activated (Ml) macrophages have been shown to mainly utilize aerobic glycolysis for energy, while alternatively activated (M2) macrophages rely on fatty acid oxidative phosphorylation or gluconeogenesis (33). This shift toward aerobic glycolysis is coupled with transcriptional induction of glycolytic enzymes such as PKM2, or gluconeogenesis enzymes such as FBPl, and is thought to be important for cell survival in during oxidative stress (34,35). Importantly, aerobic glycolysis drives further HMGB1 secretion. HMGB1 has recently been shown to increase ATP production in a number of cell lines, including Jurkat and HL-60, owing to RAGE mediated signaling (12,36). It was investigated whether HMGB1 might promote aerobic glycolysis, and whether Clq could regulate this metabolic shift.

[0090] For these experiments, human monocytes were treated with buffer, HMGB1,

Clq or HMGB1 with Clq for 24 hours and assessed the cellular bioenergetics by simultaneously measuring mitochondrial respiration and glycolysis using a SeaHorse XF analyzer. A more complete bioenergetic analysis was afforded by the sequential use of oligomycin (inhibits mitochondrial ATP synthase), FCCP (uncouples mitochondrial respiration from ATP synthesis) and rotenone plus antimycin A (blocks mitochondrial electron transport). While exposing monocytes to HMGB1 alone enhanced both baseline oxidative phosphorylation (as measured by oxygen consumption rate (OCR), Fig. 7a) and glycolytic metabolism (as measured by extracellular acidification rate (ECAR), Fig. 7b), the OCR/EC AR ratio was significantly decreased in HMGBl -exposed cells (Fig. 7c), demonstrating a shift to enhanced aerobic glycolysis, consistent with an Ml phenotype. In contrast, the inclusion of Clq together with HMGBl prevented the alteration in cellular metabolism, presumably due to blockade of RAGE mediated signaling. This bioenergetic analysis, therefore, demonstrated that HMGBl -activated monocytes exhibited an Ml macrophage profile while HMGBl /Clq-treated monocytes exhibited an M2 macrophage profile 36, confirming the Ml-like pro-inflammatory (HMGBl -mediated) vs. M2-like antiinflammatory (HMGBl /Clq-mediated) gene expression patterns.

[0091] Monocyte differentiation to DCs is prevented by HMGBl and Clq: As an early step toward an adaptive immune response, monocytes can differentiate into DCs and function in an antigen presenting role. In view of the M2-like state that Clq and HMGBl elicited in monocytes, we wondered whether monocytes previously exposed to HMGBl and Clq would lose their ability to differentiate into DCs. For these experiments, we induced monocyte differentiation to M2 macrophages followed by treatment with DC cytokines (GM-CSF and IL-4). Monocytes previously exposed to either Clq or HMGBl and Clq for 24 hours retained expression of CD14 and LAIR-1, but expression was significantly higher in monocytes that had been exposed to both HMGBl and Clq. In contrast, monocyte to DC differentiation occurred in previously untreated monocytes or monocytes previously treated with HMGBl (Fig. 8a). These data suggest that exposure to HMGBl and Clq drives monocytes into M2 macrophages that do not become antigen presenting cells in the presence of GM-CSF and IL-4. This was tested by using monocytes exposed to HMGBl, Clq or both in an allogeneic mixed lymphocyte reaction. Monocytes exposed to HMGBl induced the strongest mixed lymphocyte reaction; monocytes exposed to both HMGBl and Clq were poor stimulators of T cells (Fig. 8b).

[0092] HMGBl -linker-Clq peptide mimics Clq; cross-links RAGE and LAIR-1 : An in situ proximal ligation assay was performed according to manufacturer's protocol (Duolink, Sigma). Following stimulation with Clq (122 nM) or HMGBl A box-linker Clqa peptide (122 nM) for 15 min, human monocytes were washed three times with ice-cold PBS and were fixed with 4% (wt/vol) PFA for 1 hour at room temperature, seeded (2 xlO ⁵ cells) on slides using cytospin (Shandon), permeablized with chilled MeOH for 4 min and with PBS/0.1% (vol/vol) Triton X-100 and blocked for 1 h at room temperature. Cells were then incubated overnight with a primary antibody pair directed to rabbit anti-RAGE (ab3611, Abeam) and to mouse anti -LAIR-1 (BD Bioscience), respectively. The cells were incubated with corresponding PLA probes conjugated to oligonucleotides (mouse MINUS and rabbit PLUS), then followed by ligation and rolling circle amplification in close proximity. Images were acquired using an Axiolmage Zl (Zeiss) apotome enabled (Zeiss) Fluorescent- intensity analysis of the images was performed using Zen2 (Zeiss). See Fig. 11.

Table 1 : Function of fusion protein components.

Discussion

[0094] It is well established that the healthy mammalian immune system is in a state of dynamic equilibrium, where activating stimuli are constantly balanced by negative feedback loops and inhibitory molecules in order to set a healthy homeostasis. Since autoreactive lymphoid cells have been shown to persist in healthy adults, and myeloid cells respond to molecular patterns that are endogenous (DAMPs) as well as those derived from pathogens (PAMPs), these regulatory mechanisms are of paramount importance in keeping a state of immune quiescence and avoiding unwanted autoimmunity. As an extracellular molecule, HMGB1 represents one of the evolutionarily ancient pro-inflammatory mediators comprising both chemokine and cytokine properties, depending on its redox state. As a cytokine, disulfide HMGB1 activates a program of inflammatory pathways that has been postulated to be essential for the production of anti-DNA antibodies in SLE (37). As a cytosolic molecule, it can regulate the threshold for autophagy and apoptosis, depending in part on its cellular localization (13,38,39). RAGE is a cellular receptor for HMGB1 and its expression determines the strength and duration of an immune response to HMGB1 and its cargo.

[0095] Like HMGB1, Clq is also evolutionarily ancient and has diverse functions.

It can stimulate an antibody response, focusing antigen on follicular dendritic cells and decreasing the threshold for B cell activation (40,41). Clq can either activate or suppress the NLRP3 mflammasome (16). It has also been shown to suppress DC function, blocking monocyte to DC differentiation and DC production of inflammatory cytokines (22). It is a critical component of the process by which natural IgM antibodies mediate attenuation of DAMP- and/or PAMP-induced DC activation (42). It also plays a key role in the activation of intranasal antigen-induced tolerance, presumably because it predisposes DCs to the generation of Tregs (43). Recently, it was demonstrated that apoptotic cells bound by Clq suppress human macrophage and DC-mediated Thl7 and Thl cell activation (44); it appears that there are multiple pathways by which Clq can suppress adaptive immunity.

[0096] This study reveals previously unknown effects of HMGBl and Clq on human monocyte activation and differentiation in inflammatory settings and in SLE. HMGBl and Clq have opposing effects on human monocytes with HMGBl inducing an Ml-like phenotype. More surprisingly, their combined function results in the differentiation to a cell with the characteristics of M2 macrophages more favorable energetics, and which cannot differentiate into DCs. Thus these monocytes are effectively removed from forming the bridge to an adaptive immune response.

[0097] There are many receptors for both HMGBl and Clq (3,15). Herein is disclosed a terra- molecular interaction occurs between RAGE and LAIR-1 and their ligands of HMGBl and Clq (Fig. 9). Clq cross-links LAIR-1 with RAGE and, in the presence of HMGBl, induces co-localization of these receptors to lipid rafts, inhibiting RAGE activity as evidenced by reduced RAGE phosphorylation. As phosphorylated RAGE is a major transporter of HMGBl and its cargo into the cytosol, internalization of HMGBl is significantly inhibited in the presence of Clq. Although Clq alone can bind LAIR-1 on monocytes, and lead to its phosphorylation, presumably by Hck, a more suppressive role of Clq depends on co-stimulation with HMGBl leading to enhanced recruitment of SHP-1. Our model suggests a multi-modal function of Clq. In the absence of inflammatory stimuli, Clq represses the inflammatory properties of the low levels of HMGBl normally present in human serum (Fig. 9b) In the presence of higher levels of HMGBl, however, Clq acts as a molecular switch that drives monocyte differentiation to an immunosuppressive M2-like cell type (Fig. 9c). Finally, when HMGBl levels are too high, Clq fails to dampen monocyte differentiation to Ml like macrophages (Fig. 9a).

[0098] The cooperation of HMGBl with Clq in the inflammatory setting may terminate inflammation through inducing M2 macrophage differentiation with expression of suppressive molecules such as Mer, PDL-1 and IL-10 (45). Moreover, HMGBl- and Clq- exposed monocytes cannot differentiate into dendritic cells and cannot participate in supporting a mixed lymphocyte reaction. This suggests that HMGBl together with Clq would limit monocyte function as APCs in an adaptive immune response. [0099] It is well established that SLE pathology begins after class switching of autoantibodies from IgM to IgG and, further, that IgM autoantibodies can protect against disease onset (46). This model explains how IgM immune complexes are suppressive of innate inflammation while IgG immune complexes provoke an inflammatory response, IgM complexes engage Clq and LAIR-1, while IgG complexes directly engage Fc receptors. Interestingly, it has been reported (37) that immunoglobulin class switching to IgG can be facilitated through engagement of HMGBl to TLR2. Whether Clq can alter this activity of HMGBl is not known.

[00100] These findings emphasize the importance of generating therapeutic approaches to selectively engage RAGE and LAIR-1 to target DAMP-mediated inflammation while preserving other protective immune responses, such as the response to LPS.

[00101] In blood, circulating Clq engages LAIR-1 and maintains quiescence of monocytes. When increased levels of HMGBl are present as a consequence of tissue damage or infection, these cells may differentiate toward macrophages or DCs and migrate to where Clq can be actively secreted by myeloid cells to dampen immune activation. Indeed, we hypothesize that infiltrating monocytes/M2 macrophages engage in resolution of inflammation while tissue-resident myeloid cells may not as they experience LAIR-1 activation through extracellular matrix collagen which fails to crosslink LAIR-1 to RAGE, and so will not lead to M2 like differentiation. It is interesting to consider whether RAGE also binds to other Clq binding partners beyond HMGBl, such as S 100 proteins and amyloid β, which are generated during inflammation

(47,48). Consistent with this hypothesis, dysregulation of Clq has been associated with the development of various inflammatory diseases including rheumatoid arthritis, Alzheimer's disease as well as SLE.

[00102] Taken together, the data demonstrate a mechanism by which Clq regulates the inflammatory properties of HMGBl. Since Clq is produced in sites of inflammation, and considering the overwhelming proportion of Clq deficient patients who manifest with an autoimmune disease, this immune-regulatory mechanism of Clq is evidently of great importance in safeguarding an appropriately regulated immune response. Moreover, the fact that motifs withm Clq and HMGBl can activate an unappreciated natural program of immune quiescence raises the exciting possibility of harnessing this pathway to develop novel mechanism-based lupus therapeutics. [00103] Furthermore, since DWEYS peptide additionally prevents internalization of HMGBl also, and binds RAGE, blocking HMGBl from binding to RAGE, a fusion protein comprising DWEYS and Clq will also be advantageous to administer in certain autoimmune inflammatory conditions, such as SLE or RA. In keeping myeloid cells activated and/or crosslinking and preventing internalization, therapeutic effects are expected.

METHODS

[00104] Reagents: Clq purified from pooled normal human sera was obtained from Complement Technology. Clq tail was purified from whole Clq as previous described (22). Recombinant HMGBl (Calmodulin Binding Protein Epitope, Cbp tagged), reduced or oxidized forms HMGBl and monoclonal anti -HMGBl antibody (2G7) were generated as previously described (49). Human recombinant (hr) LAIR-2 and hrRAGE were purchased from R&D Systems. CpG ODN 2216, FITC-CpG ODN 2216 and ultra pure LPS were purchased from Invivogen. FITC was conjugated to HMGBl using amine-reactive probes (Invitrogen) per the manufacturer's protocol. Biotin labeling of Clq was performed using an EZ-Link Sulfo-NHS-LC Biotinylation kit (Thermo Scientific) per the manufacturer's instructions. SpeedBeads™ streptavidin microparticles were purchased from Thermo Scientific. Fluorochrome-conjugated and unconjugated antibodies were purchased: PE- labeled or unlabeled mouse anti-human LAIR-1 (DX26, BD Bioscience); goat anti-human LAIR-1 (T-15), mouse anti-SHP-1 (D-11), goat anti-calmodulin binding protein tag (Santa Cruz Biotechnology); rabbit anti-RAGE, rabbit anti-flotillin, rabbit anti-NFi B p65 (Abeam); mouse anti-phospho-ΙΚΚ , rabbit anti-phospho-Serine, rabbit anti-RAGE and rabbit anti-phospho-p65 (Cell Signaling); mouse anti-β actin (AC-15, Sigma); mouse anti- Clq (Quidel); FITC-anti-Mer, Pacific Blue-anti-CD l ib, APC-anti-CDl lc, APC-Cy7-anti- CD14, Pacific Blue-CD 16, PE-anti-CD163, PECy7-anti-PDL-l, anti-CD3 (OKT3), FITC- anti-CD4, HRP-anti-goat IgG and isotype-matched control antibodies (eBioscience); Alexa Fluor 594-conjugated anti-rabbit IgG and Cell Trace Violet (Life Technologies); IX RIPA cell lysis buffer (Invitrogen); protease inhibitor cocktail, phosphatase inhibitors (Pierce, Waltham, MA); PBS/4% paraformaldehyde (PFA), Triton X-100, Tween-20 and NP-40 (Sigma). Purified proteins and culture reagents were endotoxin tested (<0.1 EU/ml) either by the manufacturer using a Limulus Amebocyte Lysate (LAL) assay kit performed per the manufacturer's instructions (Endosafe). [00105] Monocyte isolation and stimulation: Human PBMCs were obtained following institutional guidelines of the Feinstein Institute for Medical Research (Feinstein) and isolated from blood of healthy donors by density centrifugation (New York Blood Bank). Monocytes were negatively enriched using a human monocyte enrichment kit (Stem Cell Technology). Purity of monocytes (> 90% CDl lb+CD14+LAIR-l+) was determined by flow cytometry. Purified monocytes (2xl0 ⁶ cells/ml) were cultured in U-bottom 96-well plate and stimulated with HMGB1 (3 or 10 μg ml-1), CpG 2216 (5 uM), LPS (0.1-10 μg/ml), hrLAIR-2 (20 μg/ml) or Clq (25 g ml-1) in X-Vivo 15 serum free medium (Lonza), and harvested at the indicated time points. For Figure If, PBMCs (2x106 cells/ml) were incubated in a flat-bottom 96-well plate with HMGB1 (3 μg/ml) and/or Clq (25 μg/ml) for 6 h, and adherent cells were harvested. In order to differentiate into DCs, monocytes were pre-incubated with Clq and HMGB1 alone or together for 24 h, extensively washed and further cultured for 2 days in the presence of 50 ng ml-1 GM-CSF and 20 ng/ml IL-4 (R&D). Splenic murine monocytes from wild type mice, RAGE deficient or LAIR-1 conditional knockout mice were isolated using EasySep mouse monocyte enrichment kit (Stem Cell Technology). C57BL/6 mice, Lysozyme2-Cre transgenic mice were purchased from Jackson Laboratory. Myeloid specific LAIR-1 conditional knockout mice (Lysozyme2-Cre x LAIR fiVfl) were bred and maintained in our facility (Feinstein) under specific pathogen-free conditions. RAGE deficient mice (Department of Medicine, arolinska Institute Stockholm, Sweden) were maintained at Feinstein. Eight-to ten weeks old male mice were used for experiemnts. All animal procedures were approved by the Feinstein Institutional Animal Care and Use Committee.

[00106] RT-PCR analysis and primers: Total RNA was extracted from cells (l-2xl0 ⁶ cells per sample) with an RNeasy kit (Qiagen, Venlo, Limburg, Netherlands) and cDNA was generated using an iScript cDNA synthesis kit (Bio-Rad laboratories). Real Time-PCR was performed on a Light Cycler 480 II (Roche) using Light Cycler 480 master mix with primers (Applied Biosystems) for IFNal (Hs00256882), IFN 7 (mm02525960), MX1 (Hs00182073, mm01217998), IL-6 (Hs00985639, mm99999064), TNFa (Hs00174128, mm00443258), IL-12a (Hs00168405), Mer (Hs01031973), PDL-1 (HsOl 125301), IL-10 (Hs00961622), CD163 (Hs00174705), HPRTl (Hs99999909, mm01545399) and Polr2a (Hs00172187). The genes of interest were normalized to the expression of house keeping genes and were compared to a control condition with no treatment. The relative induction was calculated by 2-AACt. [00107] Transfection: For RNA interference assays, human monocytes were transfected using an Amaxa Nucleofector kit (Lonza) with a greater than 40% transfection efficiency. siRNAs were obtained from Qiagen. The target sequence of human LAIRl-11 is CAGCATCCAGA AGGTTCGTTA (SEQ ID NO:9). The efficiency of knockdown was determined by flow cytometry and q-PCR.

[00108] Cytokine analysis: Cytokine levels were measured using a Human Proinflammatory 7-plex assay following the manufacturer's protocols (Meso Scale Discovery (MSD). MSD plates were analyzed on the MS2400 imager (MSD). All standards and samples were run in duplicates.

[00109] Immunofluorescence Microscopy: In situ proximal ligation assay was performed according to manufacturer's protocol (Duolink, Sigma). Following stimulation, human monocytes were washed three times with ice-cold PBS and were fixed with 4% (wt/vol) PFA for 1 hour at room temperature, seeded (2 xlO ⁵ cells) on slides using cytospin (Shandon), permeablized with chilled MeOH for 4 min and with PBS/0.1% (vol/vol) Triton X-100 and blocked for 1 h at room temperature. Cells were then incubated overnight with a primary antibody pair directed to rabbit anti-RAGE (ab3611, Abeam) and to mouse anti- LAIR-1 (BD Bioscience), respectively. The cells were incubated with corresponding PLA probes conjugated to oligonucleotides (mouse MINUS and rabbit PLUS), then followed by ligation and rolling circle amplification in close proximity. Images were acquired using AxioVision software and a confocal microscope (Olympus). Quantification was performed using Zen2 (Zeiss). For HMGBl or CpG internalization assays, isolated human monocytes were washed with PBS and stimulated by FITC-labeled HMGBl or CpG ODN 2216 with or without CI q for 15 min at 4°C or 37°C. Cells were washed three times with cold-PBS and fixed with 4% PFA and stained with PECy5-anti -human CD 14 antibody (BD biosciences) or propidium iodide (PI). Before image acquisition, cells were displayed on slides using cytospin (Shandon) and mounted using Dako mounting medium (Agilnet technologies) or 4,6-diamidino-2-phenylinole (DAPI) containing mounting medium (Sigma). PI or DAPI was used for nuclear staining. To analyze nuclear translocation of NFKB p65, cells were stimulated with HMGBl or HMGBl plus Clq for 1 h at 37°C, fixed and permeabilized with PBS/0.5% (vol/vol) Triton-X-100 for 10 min, washed, blocked with 2% (wt/vol) BSA and 2% (vol/vol) goat serum (Life Technologies) for 30 min at room temperature before incubation with anti-rabbit NFKB p65 (Abeam) at 4°C overnight. After washing, cells were stained with Alexa Fluor 594 anti -rabbit IgG (Life Technologies), and mounted DAPI- containing medium. Images were acquired using an Axiolmage Zl (Zeiss) apotome enabled (Zeiss) Fluorescent-intensity analysis of the images was performed using Zen2 (Zeiss).

[00110] Surface Plasmon Resonance analysis (SPR): For real-time binding interaction studies, a BIAcore T200 instrument (GE Healthcare) was used. For RAGE and Clq, HMGBl and Clq binding analyses, Clq (50 μg/ml) or HMGBl (5 u/ml) were immobilized on a CM5 series chip (GE Healthcare). A 1 : 1 mixture of N-hydroxysuccinimide and N- ethyl-N-(dimethyaminopropyl) carbodiimide was used to activate 2 flow-cells of the CM5 chip. One flow-cell was used as a reference and thus immediately blocked upon activation by 1 M ethanolamine. The sample flow-cell was injected with the diluted Clq or HMGBl were injected at a flow rate of 10 ul min-1 The Clq injection was stopped when the surface plasmon resonance reached -2000 response difference (RU); the HMGBl injection was stopped at -60-100 RU. The analytes (RAGE or Clq) were introduced to the immobilized Clq or HMGBl at 5 different concentrations. The analytes were diluted in lx PBS+0.01% (vol/vol) tween-20 buffer. The analytes were sequentially injected at a flow rate of 30 μΐ/min for 60s at 25°C. The KD was dertermined using the BIAcore evaluation software 2.0 (GE Healthcare) supposing a 1 : 1 binding ratio.

[00111] For the RAGE, Clq and HMGBl complex formation assay, RAGE (20 ug/ml) was immobilized on a CM5 chip. The first analyte (Clq fixed at 200 nM) was introduced to the immobilized sRAGE in multiple times until the chip was saturated, and then the second analyte (HMGBl fixed at 500 nM) was injected to the RAGE-Clq complex in multiple times. The dissociation time was set for 1 minute. RAGE, HMGBl and Clq complex formation assay was performed by a similar procedure.

[00112] For the SPR binding assay of different redox states of HMGBl and Clq, high- level immobilization of Clq was immobilized onto a CM5 chip (GE Healthcare). The Clq protein was diluted to a concentration of 20 μg/ml in 10 mM Acetate buffer (pH=4.5). A 1 : 1 mixture of N-hyrdoxysuccinimide and N-ethyl-N-(dimethyaminopropyl) carbodiimide was used to activate 2 flow-cells of the CM5 chip. One flow-cell was used as a reference and thus immediately blocked upon activation by 1 M ethanolamine (pH=8.5). The sample flow-cell was injected with the diluted Clq at a flow rate of 10 ul/min. The Clq injection was stopped when the surface Plasmon resonance reached -2200 RU. The analyte (HMGBl) was diluted in lx HBS-N+0.05% tween-20 buffer (filtered- 0.22um). Three redox states of HMGBl were sequentially injected at a flow rate of 20 ul/min for 60s at 25°C, the dissociation time was set for 3 minutes. The concentration was set at 500 nM. [00113] For, Kinetics assay of different redox states of HMGB1 and Clq, the analytes (Disulfide-HMGB 1 , all thiol-HMGBl, Oxidized-HMGBl) were introduced to the immobilized Clq at 6 different concentrations. The analytes were diluted in lx HBS- N+0.05% tween-20 buffer (filtered - 0.22um). The analytes were sequentially injected at a flow rate of 20 μΐ/min for 60s at 25°C. The dissociation time was set for 3 minutes. The KD for each analyte was determined using the Biacore evaluation software 2.0 supposing a 1 : 1 binding ratio.

[00114] Trimolecular complex assay: Biotinylated-Clq (20 μg/ml) was precoated to SpeedBeads™ (Streptavidin-conjugated microbeads, Thermo Scientific). Beads were saturated with HMGB1, then incubated with RAGE. After extensive washing, RAGE, HMGB1 and Clq were visualized by SDS-PAGE and Western blot.

[00115] Lipid raft fractionation: Lipid rafts were prepared as described (50). Monocytes (5 10 ⁶ ) were lysed in 1 ml TNE buffer (25 mM Tris, 150 rriM NaCl, 5 mM EDTA) containing 1% Triton and incubated for 30 min on ice. Ly sates were homogenized with 10 strokes of a dounce homogenizer, mixed with 2 ml of 80% sucrose in TNE buffer, and transferred to a centrifuge tube. Samples were overlaid with 4 ml of 30% sucrose and 2 ml of 5% sucrose in TNE. After centrifugation for 16 h at 180,000 g in a Beckman Coulter SW41Ti rotor, 0.8 ml fractions were collected from the top of the gradient. Each fraction was subjected to slot blot analysis to identify GM1 -enriched rafts fraction using FITC- conjugated cholera toxin-B subunit (Sigma). Lipid raft fractions were precipitated with TCA and washed with 70% EtOH, then subjected to slot blot analysis or Western blot.

[00116] Immunoprecipitation and Western blot. Total protein extracts were prepared as described (22). Monocytes (2-5x106 cells/ml) were washed in ice-cold PBS and lysed in IX RIPA buffer containing complete protease inhibitor mixture (Roche) and phosphatase inhibitor (Pierce) for 1 h on ice. For immunoprecipitation, anti-LAIR-1 antibody (BD Bioscience) or anti-RAGE antibody (Abeam) was incubated with the lysate overnight at 4°C prior to being incubated with protein G-dynabeads (Life Technologies). Proteins were then separated by SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and immunoblotted with appropriate antibodies. Bands were detected using the ECL reagent (Thermo Scientific) or using the Odyssey Infrared Imaging system (LI-COR) to detect secondary antibodies conjugated with Infrared 680 or 800.

[00117] Phospho-immunoreceptor array: Tyrosine phosphorylated LAIR-1 was determined by human phosphoimmunoreceptor array (Proteome Profiler Array; R&D systems) according to the manufacturer's protocol and as described (22). Phosphorylation levels of individual analytes were determined by average pixel density of duplicate spots; values were obtained after subtraction of background and were normalized to positive control spots.

[00118] Flow cytometry: Cells were suspended in staining buffer containing 2% BSA and incubated with Fc block (Miltenyi Biotec) for 15 min on ice. The cells were then incubated with experimental or isotype matched antibodies and washed. Events were acquired using either an LSRII or Fortessa cell analyzer (BD Biosciences), and data were analyzed using FlowJo (Tree star).

[00119] Cell metabolism analysis using SeaHorse Cellular Flux assay Monocytes (300xl0 ³ cell/ well) from healthy donors were isolated as previously described, seeded in triplicates on SeaHorse XfP plates in X-Vivo 15 culture medium, and treated with HMGBl (3 μg/ml) with or without Clq (25 μg ml-l), or maintained in culture medium alone (untreated control) for 24 h. One hour prior to measurement, culture medium was exchanged for assay medium (unbuffered DMEM (Sigma), supplemented with 10 mM Glucose, 1 mM Pyruvate and 2 mM Glutamine for mitochondrial assays, or with 2 mM Glutamine alone for glycolytic assays. Reagents were injected during the measurement to achieve final concentrations of oligomycin (1 μΜ), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (1 nM), Rotenone/antimycin A (0.5 μΜ). Glucose (10 mM), and 2-Deoxy-D glucose (50 mM). Oxygen consumption rate (OCR) and Extracellular acidification rate (ECAR) values were measured using a SeaHorse XfP instrument (SeaHorse bioscience). Measured values were normalized to the number of live cells present in each well as determined by a trypan blue staining by the end of each run. For OCR/ECAR ratio calculations, an average of the last two basal readings was used for both OCR and ECAR.

[00120] Mixed-lymphocyte reaction: Monocytes (lxlO ⁵ cells per well) were plated on flat-bottom 96 well plates and incubated with or without HMGBl (3 μg/ml) or Clq (25 μg/ml) in X-Vivo 15 medium for 24 h, washed then cultured further 2 more days. Allogeneic CD4 T lymphocytes were isolated from blood using the Naive CD4+ T cell isolation kit II (Miltenyi Biotec.) following the manufacturer's protocol. Cells were analyzed by flow cytometry (>90%). T cells were labeled with 5 μΜ Cell Trace Violet cell proliferation kit (Thermo Fisher Scientific), added to mono/macrophages at a density of 2xl0 ⁵ cells/well (a 1 :2 ratio) in the presence of 1 μg/ml anti-CD3 (OKT3, eBioscience). Control cultures contained medium only or T cells or mono/macrophages alone. After 4 days, cells were stained with FITC-anti-CD4, PerCPCy 5.5-CD14, PE-LAIR-1 and fxable viability dyes (FVD, eBioscience) for 20 min at room temperature and subsequently washed and fixed. Live cells (FVD-negative cells) were then gated on CD4-positive, and cell trace violet was assessed by flow cytometry on a BD LSR II (BD Biosciences). Cell proliferation was analyzed as described (51). The % divided cells was defined as the probability that a cell has divided at least once from the original population. The division index was defined as the average number of cell divisions that a cell in the original population has undergone.

[00121] Statistical analysis: Student's t-test, one-way ANOVA and Kruskal-Wallis were used for statistical analyses with Prism 6.0 (Graphpad, La Jolla, CA) or SPSS 16 (IBM) Adjusted P values (Bonferoni/Dunn) of less than 0.05 were considered significant.

REFERENCES

1. Ronnblom, L. & Elkon, K.B. Cytokines as therapeutic targets in SLE. Nat Rev Rheumatol 6, 339-347 (2010).

2. Mohan, C. & Putterman, C. Genetics and pathogenesis of systemic lupus erythematosus and lupus nephritis. Nature reviews. Nephrology 11, 329-341 (2015).

3. Andersson, U. & Tracey, K.J. HMGB1 is a therapeutic target for sterile inflammation and infection. Annual review of immunology 29, 139-162 (2011).

4. Harris, H.E., Andersson, U. & Pisetsky, D.S. HMGB1: a multifunctional alarmin driving autoimmune and inflammatory disease. Nat Rev Rheumatol 8, 195-202 (2012).

5. Scaffidi, P., Misteli, T. & Bianchi, M E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191-195 (2002).

6. Yanai, H. et al. HMGB proteins function as universal sentinels for nucleic-acid- mediated innate immune responses. Nature 462, 99-103 (2009).

7. Sirois, CM. et al. RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA. J Exp Med 210, 2447-2463 (2013). Tian, J. et al. Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 8, 487-496 (2007). Huebener, P. et al. The HMGBl/RAGE axis triggers neutrophil-mediated injury amplification following necrosis. J Clin Invest 125, 539-550 (2015).

Popovic, P.J. et al. High mobility group Bl protein suppresses the human plasmacytoid dendritic cell response to TLR9 agonists. J Immunol 177, 8701-8707 (2006).

Aikawa, E., Fujita, R., Kikuchi, Y., Kaneda, Y. & Tamai, K. Systemic high-mobility group box 1 administration suppresses skin inflammation by inducing an accumulation of PDGFRalpha(+) mesenchymal cells from bone marrow. Sci Rep 5, 11008 (2015).

Kang, R. et al. The HMGBl/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics. Oncogene 33, 567-577 (2014).

Tang, D. et al. High-mobility group box 1 is essential for mitochondrial quality control. Cell metabolism 13, 701-711 (2011).

Yang, H. et al. MD-2 is required for disulfide HMGB1 -dependent TLR4 signaling. J Exp Med 212, 5-14 (2015).

Kouser, L. et al. Emerging and Novel Functions of Complement Protein Clq. Frontiers in immunology 6, 317 (2015).

Benoit, M.E., Clarke, E.V., Morgado, P., Fraser, D.A. & Tenner, A.J. Complement protein Clq directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells. J Immunol 188, 5682-5693 (2012).

Elkon, K.B. & Santer, D.M. Complement, interferon and lupus. Curr Opin Immunol 24, 665-670 (2012). Galvan, M.D., Greenlee-Wacker, M.C. & Bohlson, S.S. Cl q and phagocytosis: the perfect complement to a good meal. J Leukoc Biol 92, 489-497 (2012).

Botto, M. & Walport, M.J. C lq, autoimmunity and apoptosis. Immunobiology 205, 395-406 (2002).

Kirschfink, M. et al. Complete functional Clq deficiency associated with systemic lupus erythematosus (SLE). Clin Exp Immunol 94, 267-272 (1993).

Botto, M. et al. Homozygous Cl q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 19, 56-59 (1998).

Son, M., Santiago-Schwarz, F., Al-Abed, Y. & Diamond, B. C lq limits dendritic cell differentiation and activation by engaging LAIR-1. Proc Natl Acad Sci U S A 109, E3160-3167 (2012).

Andersson, U. et al. High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 192, 565-570 (2000).

Son, M. & Diamond, B. C lq-mediated repression of human monocytes is regulated by leukocyte-associated Ig-like receptor 1 (LAIR-1). Mol Med 20, 559-568 (2014). Xu, J. et al. Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis. Cell Death Differ 21, 1229-1239 (2014).

Yang, H. et al. A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc Natl Acad Sci U SA 107, 11942-11947 (2010).

Liliensiek, B. et al. Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response. J Clin Invest 113, 1641 -1650 (2004). Chuang, T.H., Lee, J., Kline, L., Mathison, J.C. & Ulevitch, R.J. Toll-like receptor 9 mediates CpG-DNA signaling. J Leukoc Biol l l , 538-544 (2002). Lu, Y.C., Yeh, W.C. & Ohashi, P S. LPS/TLR4 signal transduction pathway. Cytokine 42, 145-151 (2008).

Ma, W. et al. RAGE binds Clq and enhances Clq- mediated phagocytosis. Cell Immunol 274, 72-82 (2012).

Hori, O. et al. The receptor for advanced gly cation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neunte outgrowth and co- expression of rage and amphoterin in the developing nervous system. J Biol Chem 270, 25752-25761 (1995).

Sakaguchi, M. et al. TIRAP, an adaptor protein for TLR2/4, transduces a signal from RAGE phosphorylated upon ligand binding. PloS one 6, e23132 (2011).

Kelly, B. & O'Neill, L.A. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res 25, 771-784 (2015).

Yang, L. et al. PKM2 regulates the Warburg effect and promotes HMGBl release in sepsis. Nat Commun 5, 4436 (2014).

Reales-Calderon, J.A., Aguilera-Montilla, N., Corbi, A.L., Molero, G. & Gil, C. Proteomic characterization of human proinflammatory Ml and anti-inflammatory M2 macrophages and their response to Candida albicans. Proteomics 14, 1503-1518 (2014).

Izquierdo, E. et al. Reshaping of Human Macrophage Polarization through Modulation of Glucose Catabohc Pathways. J Immunol 195, 2442-2451 (2015). Wen, Z. et al. Autoantibody induction by DNA-containing immune complexes requires HMGBl with the TLR2/microRNA- 155 pathway. J Immunol 190, 5411- 5422 (2013).

Zhu, X. et al. Cytosolic HMGBl controls the cellular autophagy/apoptosis checkpoint during inflammation. J Clin Invest 125, 1098-1110 (2015). Liu, L. et al. HMGB1-DNA complex-induced autophagy limits AIM2 inflammasome activation through RAGE. Biochem Biophys Res Commun 450, 851- 856 (2014).

Cutler, A.J. et al. T cell-dependent immune response in Clq-deficient mice: defective interferon gamma production by antigen-specific T cells. J Exp Med 187, 1789-1797 (1998).

Kolev, M., Le Friec, G. & Kemper, C. Complement—tapping into new sites and effector systems. Nat Rev Immunol 14, 811-820 (2014).

Chen, Y., Park, Y.B., Patel, E. & Silverman, G.J. IgM antibodies to apoptosis- associated determinants recruit Clq and enhance dendritic cell phagocytosis of apoptotic cells. J Immunol 182, 6031-6043 (2009).

Fossati-Jimack, L. et al Intranasal peptide-induced tolerance and linked suppression: consequences of complement deficiency. Immunology 144, 149-157 (2015).

Clarke, EN., Weist, B.M., Walsh, CM. & Tenner, AJ. Complement protein Clq bound to apoptotic cells suppresses human macrophage and dendritic cell-mediated Thl7 and Thl T cell subset proliferation. J Leukoc Biol 97, 147-160 (2015).

Manicassamy, S. & Pulendran, B. Dendritic cell control of tolerogenic responses. Immunol Rev 241, 206-227 (2011).

Gronwall, C. et al. IgM autoantibodies to distinct apoptosis-associated antigens correlate with protection from cardiovascular events and renal disease in patients with SLE. Clin Immunol 142, 390-398 (2012).

Deane, R. et al. RAGE mediates amyloid-beta peptide transport across the blood- brain barrier and accumulation in brain. Nat Med 9, 907-913 (2003). Leclerc, E, Fritz, G., Vetter, S.W. & Heizmann, C.W. Binding of SlOO proteins to RAGE: an update. Biochim Biophys Acta 1793, 993-1007 (2009).

Wang, H. et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285, 248-251 (1999).

Hur, E.M. et al. LIME, a novel transmembrane adaptor protein, associates with p561ck and mediates T cell activation. J Exp Med 198, 1463-1473 (2003).

Roederer, M. Interpretation of cellular proliferation data: avoid the panglossian. Cytometry. Part A : the journal of the International Society for Analytical Cytology 79, 95-101 (2011).