TAFA4 POLYPEPTIDE OR POLYNUCLEOTIDE FOR TREATING INFLAMMATORY DISEASE

Abstract

The present invention relates to methods and pharmaceutical composition for treating inflammatory diseases. Inflammation is a defence response to tissue damage or infections that requires tight regulation to prevent impaired healing and to avoid excessive damage and/or autoimmunity. Myeloid cells, including macrophages play a key role in tissue repair and undergo major functional changes during the healing processes, switching from an inflammatory state to a pro-repair phenotype. The inventors have found that TAFA4, a chemokine-like protein, has anti-inflammatory and pro-repair properties. This molecule regulates the phenotype of man monocytes and macrophages by promoting their anti-inflammatory and pro-repair functions. TAFA4 increases macrophage their phagocytic capacities and their production of the anti-inflammatory cytokine IL-10. By contrast, TAFA4 down-regulates the production of the pro-inflammatory cytokines IL-6, IL-113, and TNF-α by human macrophages. Importantly, the inventors have also found that a treatment with TAFA4 has anti-inflammatory effects in vivo and protects mice from LPS-induced endotoxic shock. This protective effect is associated with a reduction in inflammatory cytokine levels and an increase in EL-10 production. Finally, they found that TAFA4 can also exert its anti-inflammatory properties on peripheral blood mononuclear cells from COVID-19 patients, independently of the disease severity. Thus, the present invention relates to a TAFA4 polypeptide or a nucleic acid molecule encoding thereof for use in the treatment of inflammatory diseases.

Claims

1. A method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a TAFA4 polypeptide or a nucleic acid molecule encoding thereof.

2. The method of claim 1 wherein the inflammatory disease is selected from the group consisting of allergy, asthma, preperfusion injury, transplant rejection, sepsis, septic shock, arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), x-linked hyper IgM syndrome, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (A/PGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal gammopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillo sis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis (e.g. chronic pancreatitis), polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Lesihmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, nonalcoholic fatty liver disease and endometriosis.

3. The method of claim 1 wherein the inflammatory disease is sepsis.

4. The method of claim 1, wherein the inflammatory disease is a viral infection which caused lung inflammation.

5. The method of claim 4, wherein the viral infection is severe acute respiratory syndrome.

6. The method of claim 4, wherein the viral infection is Covid-19.

7. The method of claim 1 wherein the TAFA4 polypeptide comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 1.

8. The method of claim 1 wherein the TAFA4 polypeptide is fused to an immunoglobulin constant domain to constitute an immunoadhesin.

9. The method of claim 1 wherein the nucleic acid molecule is included in a suitable vector, such as viral vector (e.g. AAV).

10. The method of claim 1 wherein he TAFA4 polypeptide or the nucleic acid molecule encoding thereof the TAFA4 polypeptide is administered with another active agent used for the treatment of inflammatory disease.

Description

FIGURES

[0049] FIG. 1: In vitro differentiation and polarization of human macrophages. A.

[0050] Experimental protocol used to generate polarized macrophage subsets from human purified monocytes. Monocytes were isolated from human blood and cultured with M-CSF (50 ng/ml) for six days to induce their differentiation in macrophages (My). The cells were then left untreated or treated for two days with specific factors to induce their polarization. Untreated cells remained MO My, IFN-γ (50 ng/ml)+LPS (10 ng/ml)-treated cells became M1 My, IL-4 (10 ng/ml)-treated cells became M2a My, and Dexamethasone (100 nM-1)-treated cells became M2c My. At the end of the culture, the different My types were harvested and used for analysis. B. Expression level (MFI) of different markers specific of My subsets: MO (CD14), M1 (CD80, CD86), M2a (CD86, CD206) and M2c (CD206, CD163). C. Polarized on unpolarized My subsets were cultured for one hour in presence of pHrodo zymosan bioparticles. Bioparticles phagocytosis (mean fluorescence intensity or 1VIFI) was assessed by flow cytometry. D. After the differentiation and polarization steps, My supernatants were harvested and the concentration of IL-6, IL-12p70, TNF-α and IL-10 was assessed by CBA in each individual subset. Data represent mean values ±S.E.M. of the data obtained for six donors. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. One-way ANOVA with Tukey post-hoc test.

[0051] FIG. 2: TAFA4 promotes anti-inflammatory and pro-repair functions of human macrophages. Human macrophages were cultured in conditioning media inducing MO, Ml, M2a or M2c polarization as described in FIG. 1A. From day 6 to day 8, TAFA4 (500 ng/ml) or PBS (Mock) were added to the medium. A. On day 8, My subsets were incubated for one hour in presence of pHrodo zymosan bioparticles to assess their phagocytic functions by flow cytometry. Bioparticle phagocytosis (1W I) was analysed for untreated (in white) and TAFA4-treated (in black) My subsets. B. On day 8 of culture, the concentration of IL-6, IL-12p70, TNF-a, and IL-10 was assessed in the My culture supernatants by CBA. Data for untreated (in white) and TAFA4-treated (in black) conditions were then compared in each My subset. Data represent mean values ±S.E.M. of 6 independent donors. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (Paired t test).

[0052] FIG. 3: TAFA4 treatment protects mice from LPS-induced endotoxic shock. A.

[0053] Experimental protocol used for in vivo experiments. C57BL/6 male mice were i.p. injected with LPS (7.5 μg/g) at day 0 (DO), in the presence or absence of TAFA4 (10 nM.sup.-1). Mouse sera were harvested six hours post-injection and mice were monitored for survival and weight changes during four days. B-C. Survival rate (%) (B) and weight evolution (% of initial weight) (C) of the mice injected with LPS, in absence (full line) or in presence (dotted line) of TAFA4 (10 nM.sup.-1). n=20 mice per group from two independent experiments. D. Production of IFN-γ, IL-1(3 and IL-10 in the serum of mice injected with LPS, in the absence (in white) or in the presence of TAFA4 (in black). Data represent mean values±S.E.M. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Log-rank Mantel Cox test (B); multiple t test (C), Mann-Whitney test (D).

[0054] FIG. 4: TAFA4 inhibits inflammatory cytokine secretion and up-regulates IL-10 production in PBMC from COVID-19 patients. Blood samples were used to isolate peripheral blood mononuclear cells (PBMCs) from healthy donors (CTRL, n=8) and from SARS-CoV-2-infected patients with various forms of the disease: pauci: paucisymptomatic (n=4); Pneumo: mild pneumonia (n=9); and ARDS: severe pneumonia with Acute Respiratory Distress Syndrome (n=9). PBMCs were activated for 24 h with or without LPS (10Ong/ml, LPS or NS respectively), in the presence or absence of TAFA4 (500 ng/ml, T4 or 0 respectively). Concentrations of IL-6 (A), TNF-α (B), and IL-1(3 (C), IL-12p40 (D), and IL-23 (E) were measured in the supernatant of PBMCs. Data represent mean values±S.EM. *P<0.05; **P<0.01; ***P<0.001. Wilcoxon test.

[0055] FIG. 5: TAFA4 displays anti-inflammatory properties in monocytes of healthy donors. Intracellular staining and flow cytometry analysis of cytokine production in PBMC from healthy donors stimulated 6 hours with LPS (100 ng/ml). A. Relative frequency of immune cell subsets in IL-6-, TNF-α- or IL-IP-producing cells upon LPS stimulation. B. Percentages and C. mean fluorescence intensity (MFI) of IL-6, TNF-α and IL-1(3 expression in monocytes from PBMCs stimulated (LPS) or not (NS) and in the presence (T4) or not (0) of TAFA4 (500 ng/ml). Data represent mean values±S.E.M. *P<0.05; **P<0.01; ****P<0.0001. One-way ANOVA with Tukey's multiple comparison test (A); Wilcoxon test (B, C).

Example

[0056] Material & Methods

[0057] Human blood samples

[0058] Over a period of one month (03-27-2020 to 04-24-2020), 22 subjects were recruited from three hospitals (Timone and North University Hospitals, and Laveran Military Hospital, Marseille). Nine of these patients were on mechanical ventilation for COVID-19-related-ARDS (P/F ratio <300) (ARDS group), nine patients required oxygen support at a rate of less than 5 1/min for COVID-19-related pneumonia (pneumonia group). Four patients had a paucisymptomatic form of COVID-19 compatible with outpatient care (paucisymptomatic group). COVID-19 was diagnosed on the basis of positive SARS-CoV-2 RT-PCR on nasopharyngeal samples and/or typical CT-scan findings. We also included eight healthy volunteers (control group), with no fever or symptoms in the days before sampling and negative for SARS-CoV-2 RT-PCR. Peripheral blood mononuclear cells (PBMC) were obtained from blood samples by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare). Freshly isolated PBMC were used for in vitro activations (FIGS. 4 and 5).

[0059] For the experiments on human macrophages (Mq) (FIGS. 1 and 2), blood buffy coat were obtained from the EFS (Etablissement Francais du Sang). PBMC were obtained from blood samples by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare) and used for macrophage differentiation.

[0060] Human Macrophage Differentiation and Polarization

[0061] Monocytes were isolated from PBMC using CD14.sup.+positive selection kit (Stemcell Technologies) according to the manufacturer protocol. Monocytes were then cultured in ImmunoCult-SF macrophage medium (Stemcell Technologies) with M-CSF (50 ng/ml, Stemcell Technologies) for six days to induce their differentiation in macrophages (My). Fresh medium has been added in the culture at day three or four. At day six, the cells were left untreated or treated for two days with specific factors to induce their polarization. Untreated cells remained MO My, IFN-γ (50 ng/ml, Stemcell Technologies)+LPS (10 ng/ml, from Escherichia coli strain 055:B5; Sigma Aldrich)-treated cells polarized into M1 My, IL-4 (long/ml, Stemcell Technologies)-treated cells polarized into M2a My, and Dexamethasone (100 nM.sup.−1, Sigma Aldrich)-treated cells polarized into M2c My. At the end of the culture, macrophage supernatants were collected, centrifuged to remove cellular debris and frozen until quantification of the soluble factors. Then, the different My subsets were harvested using accutase (Stemcell Technologies) according to the manufacturer protocol and used for further experiments.

[0062] PBMC stimulation

[0063] Fresh PBMC (10.sup.6 cells) from control or SARS-CoV-2 infected patients were incubated in 24 well plates in 1 ml of complete medium composed of RPMI (Life Technologies) supplemented with 10% fetal calf serum (FCS, Life Technologies) and 1% antibiotics (penicillin/streptomycin, Life Technologies). Cells were then stimulated with 100 ng/ml lipopolysaccharide (LPS, from Escherichia coli strain 055:B5; Sigma-Aldrich) or mock-stimulated with PBS (Life Technologies) for 6 hours or 24 hours at 37° C. with 5% CO2. After 24h culture, the supernatant were collected, then centrifuged 5 minutes at 1500 rpm to remove floating cells and stored at −80° C. until quantification of the soluble factors. Alternatively, after six hours culture, cells were collected and used for intracellular staining.

[0064] TAFA4 Treatment of PBMC and Macrophages

[0065] Recombinant human TAFA4 (R&D Systems) was reconstituted according to manufacturer procedures and used on different cell types. For PBMC experiments, cells were treated either with TAFA4 (500 ng/ml) or PBS during the 24 hours of stimulation. For My experiments, cells were treated either with TAFA4 (500 ng/ml) or PBS during the two days of polarization. For the phenotypical and functional subsequent analyzes, TAFA4-treated cells were compared to PBS-treated cells from the same donor.

[0066] Macrophage Phenotypic Analysis

[0067] After differentiation and polarization, My were harvested, washed with PBS and stained with Live/Dead Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific) for 30 minutes at room temperature. Nonspecific binding was blocked using PBS supplemented with 10% human serum (Sigma Aldrich). Surface staining was then carried out in PBS supplemented with 1% human serum for 30 minutes at 4° C. The following antibodies were used: CD14-BUV737 (M5E2 clone), and CD8O-Pe-Cy7 (L307.4 clone; BD Technologies), HLA-DR-AF700 (L243 clone), CD86-PE (IT2.2 clone), CD206-Pe-Cy5 (15-2 clone) and CD163-BV510 (GHI/61 clone; BioLegend). Cell phenotype was characterized by multi-parameter flow cytometry analysis on a LSR-FORTESSA X20 cytometer (BD Bioscience). Data were further analyzed using FlowJo v10 software.

[0068] Phagocytosis Assay

[0069] The phagocytic activity of My in various polarizing conditions was measured as the cellular uptake of zymosan bioparticles. Cells were collected and incubated with 0.5 mg/ml pHrodo Zymosan Green BioParticles conjugate (Life Technologies) for 60 min at 37° C. Cells incubated with green pHrodo zymosan bioparticles at 4° C. were used as negative controls to confirm the specificity of the signal observed at 37° C. To stop the phagocytosis and remove the excess of bioparticles, cells were harvested and washed with cold PBS. Finally, the phagocytic activity was evaluated by flow cytometry using a LSR-FORTESSA X20 cytometer (BD Bioscience). Data were further analyzed using FlowJo v10 software.

[0070] Dosage of Human Cytokines and Chemokines

[0071] Cytokine production was assessed in culture supernatants through cytometric bead array (CBA, LEGENDplex, BioLegend), according to the manufacturer protocol. For PBMC stimulation experiments, the following soluble factors were tested: TNF-α, IL-1(3, IL-12p40, and IL-23. IL-6 production was assessed in the supernatants using CBA (BD Technologies) after a hundred-fold dilution. For My experiments, the following soluble factors were tested: TNF-α, IL-6, IL-12p70, and IL-10. Data were then acquired using a FACSCanto II cytometer (BD Bioscience, for PBMC experiments) or a LSR-FORTESSA X20 cytometer (BD Bioscience, for My experiments). Results were then analyzed using LEGENDplex v8 software (BioLegend) or FCAP array v3 software (BD Technologies).

[0072] Intracellular Staining in PBMC

[0073] PBMC were stimulated for 6 hours in the presence or not of LPS (100 ng/ml, from Escherichia coli strain 055:B5; Sigma-Aldrich) and TAFA4 (500 ng/ml; R&D Systems). GolgiPlug (BD Technologies) was added in each well according to the manufacturer procedures. Cells were then collected and stained with Live/Dead Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific) for 30 minutes at room temperature. Surface staining was then carried out in PBS supplemented with mouse serum for 30 minutes at 4° C. The following antibodies were used: CD45-V500 (HI30 clone), CD16-BUV496 (3G8 clone), CD56-BUV395 (B159 clone) and CD3-BUV737 (UCHT1 clone; BD Technologies), HLA-DR-AF700 (L243 clone), CD19-BV711 (HIB19 clone), and CD11b-BV785 (ICRF44 clone; BioLegend). Intracellular staining was then performed using Cytofix/Cytoperm kit (BD Technologies) according to the manufacturer protocol. The following antibodies were used: TNF-APC (MAb 11 clone), IL-10-FITC (AS10 clone) and IL-6-PE (AS12 clone; BD Technologies). Cell phenotype was characterized by multi-parameter flow cytometry analysis on a LSR-FORTESSA X20 cytometer (BD Bioscience). Data were further analyzed using FlowJo v10 software.

[0074] Mouse Experiments

[0075] C57BL/6J mice were purchased from Janvier Lab. All mice were maintained under specific-pathogen—free conditions in the Centre d′Immunologie de Marseille Luminy. Mice were housed under a standard 12 h/12 h light—dark cycle with food and water ad libitum. Nine-week-old male mice were used for all experiments. LPS (from Escherichia coli strain 055:B5; Sigma Aldrich) diluted in PBS was injected intraperitoneally (7.5 μg per gram mouse body weight). Mice were injected either with LPS alone or with LPS+TAFA4 (10 nM.sup.−1, R&D Systems). All LPS injections were performed between 9 a.m. and 10 a.m., and mice were checked every 24 hours for body weight, survival, and signs of distress until day 4. For IL-10 blockade experiments, mice were intraperitoneally injected with 500 μg anti—IL-10 (JES5-2A5), or rat IgG1 (HRPN; all from BioXCell) twenty-four hours before LPS injection.

[0076] Blood samples were obtained using retro-orbital blood collection at six hours post LPS-treatment, and processed to eliminate red blood cells. The same mice were used for blood collection and longitudinal follow-up. Serum samples were then stored at −80° C. until quantification of soluble mediators. The concentrations of various soluble factors was assessed by CBA according to the manufacturer's protocol (BD Biosciences). All samples were diluted by half before quantification.

[0077] Statistical Analyses

[0078] Statistical analyses were performed using Graphpad Prism v8 software. Data represent mean values ±standard error of mean (S.E.M.) and the number of independent donors/mice/experiments is indicated in each figure legend. Paired data were compared with two-tailed paired t tests if the values followed a Gaussian distribution or Wilcoxon test otherwise. Unpaired data were compared with two-tailed Student's t test if the values followed a Gaussian distribution or Mann—Whitney test otherwise. For multiple comparisons, we used one-way Kruskal-Wallis with Dunn's multiple comparison test, analysis of variance (ANOVA) with Tukey post-hoc test or multiple t tests. Differences in survival were evaluated with the Mantel—Cox test. The statistical test used for each panel is stated in figure legend. Data were considered statistically significant if the P value obtained was lower than 0.05. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0079] Results

[0080] TAFA4 promotes human macrophage anti-inflammatory and pro-repair functions.

[0081] We analysed the role of TAFA4 on human macrophages phenotype and functions. We isolated peripheral blood mononuclear cells (PBMCs) from healthy donors and sorted CD14.sup.+monocytes. These monocytes were in vitro differentiated into macrophages (Mq) in the presence of M-CSF during 6 days. Different conditioning media were then used during two additional days to polarize these macrophages in different subtypes as described in FIG. 1A. For each donor, we generated unpolarized My (MO), pro-inflammatory My (M1), pro-repair My (M2a) and regulatory My (M2c). To validate this protocol of macrophage differentiation, we analyzed the phenotype of the different subsets by flow cytometry. We analyzed the expression level of CD14, CD80, CD86, CD206 and CD163 to establish the phenotypic signature of each My subset (FIG. 1B). As expected, compared to the polarized My subsets, MO My expressed high levels of CD14 but low levels of the other markers. M1 My expressed high levels of CD80 and CD86 but low level of CD14. M2a My expressed high levels of CD206 and CD86 but low CD14 levels. Finally, M2c My expressed high levels of CD206 and CD163 with intermediate CD14 levels.

[0082] We then used this in vitro model to assess the effect of TAFA4 on My phagocytic and cytokine production functions. After 6 days of differentiation in M-CSF, My were cultured in conditioning media to induce MO, Ml, M2a or M2c polarization in the presence or in absence (Mock) of TAFA4 (500 ng/ml). We first assessed the phagocytic capacities of the macrophages subtypes by using pHrodo zymosan bioparticles that become fluorescent after internalization and processing in acid compartments (phagosomes). After one hour of culture with the bioparticles, My were harvested and their fluorescence, reflective of their phagocytic ability, was analyzed by flow cytometry. We observed that M1 inflammatory My display reduced phagocytic functions compared to MO, M2a or M2c cells (FIG. 1C). Moreover, TAFA4 induced a significant increase of the phagocytic function in all the macrophage subsets (FIG. 2A). We then analyzed the effect of TAFA4 on the cytokines produced by the My subtypes. We analyzed the production of IL-6, TNF-α, IL-12p70, and IL-10 in the culture supernatants (on day 8) by Cytometric Bead Array (CBA). As expected, we observed that M1 macrophages produced high levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-12p70 (FIG. 1D) whereas M2a and M2c Mq only produced IL-10. TAFA4 modified the profile of cytokine production by macrophages towards a more anti-inflammatory profile. In the supernatant of M1 My, we observed a drastic decrease in the production of pro-inflammatory cytokines, associated with an increased production of the anti-inflammatory cytokine IL-10 (FIG. 2B). Moreover, TAFA4 also up-regulated IL-10 production by M2a and M2c

[0083] In conclusion, these data demonstrate that the neuropeptide TAFA4 promotes the anti-inflammatory and pro-repair functions of human macrophages. TAFA4 can act on all the subtypes of macrophages by increasing their production of IL-10 and reinforcing their phagocytic capacities. Moreover, TAFA4 limits the pro-inflammatory cytokine production of M1 macrophages.

[0084] TAFA4 Treatment Protects Mice from LPS-Induced Endotoxic Shock.

[0085] The human data described above suggest a potential beneficial role of TAFA4 in inflammatory diseases. To test the anti-inflammatory role of TAFA4 in vivo, we used an animal model of systemic inflammation in which a cytokine storm jeopardizing host survival is induced. We injected LPS into mice to induce an endotoxic shock (FIG. 3A). In this model, classically used to mimic sepsis, TAFA4 treatment increased mouse survival (FIG. 3B). Moreover, mice treated with TAFA4 had a reduced weight loss (FIG. 3C). This higher survival rate and lower disease severity were associated lower serum levels of the inflammatory cytokines IFN-γ and IL-1(3 (FIG. 3D). By contrast, IL-10 levels were higher than those in untreated mice (FIG. 3D). Finally, we showed that in this model the main effect of TAFA4 on mouse resistance to endotoxic shock is mediated through its ability to induce IL-10. Indeed, the survival of mice treated with anti-IL-10 neutralizing antibodies, was no longer improved by TAFA4 treatment. However, even in the absence of IL-10, TAFA4 continued to have an effect on reducing serum levels of other inflammatory cytokines/chemokines such as TNF-α, IL-12, CCL3, CCL-4 and CXCL9 (data not shown), showing that TAFA4 can reduce inflammation by controlling multiple pathways.

[0086] These results provide a proof of concept of the potent anti-inflammatory properties of TAFA4 in vivo and demonstrate that TAFA4 treatment can protect mice from an inflammatory disease.

[0087] TAFA4 Acts on Human Monocytes and Reduces Inflammatory Cytokine Production by PBMCs of SARS-CoV-2 Infected Patients.

[0088] SARS-CoV-2 infection can lead to severe forms of COVID-19 characterized by a cytokine storm and the invasion of the lungs by overactivated myeloid cells. In some patients, this disease can lead to severe tissue damage resulting in lung fibrosis and death. In this context and based on our previous results, we hypothesized that treatment with the neuropeptide TAFA4 might be beneficial to treat this disease and other inflammatory pathologies. We therefore studied the anti-inflammatory role of TAFA4 in a cohort of 22 patients infected with SARS-CoV-2. Peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with various forms of the disease (paucisymptomatic, mild pneumonia, severe pneumonia with acute respiratory distress syndrome: ARDS) were activated for 24 hours with LPS in the presence or absence of TAFA4. We showed that TAFA4 reduced the production of the inflammatory cytokines IL-6, TNF-α, IL-1(3, IL-12p40 and IL-23 by PBMC from healthy donors and COVID-19 patients, including patients with ARDS (FIG. 4A-D)

[0089] We also performed intracellular staining and multi-parametric flow cytometry analyses to identify the cellular source of IL-6, TNF-α, and IL-1(3 in the PBMC. We then monitored cytokine expression in B cells (CD19.sup.+), T cells (CD3.sup.+), NK cells (CD56.sup.+CD16.sup.+/-CD3.sup.−), monocytes (CD11b.sup.+HLA-DR.sup.+) and DC-like cells (CD1 lb.sup.-I-ILADR.sup.+). We found that the cytokine-producing cells were mainly monocytes (85%), and DC-like cells to a lower extend (15%) (FIG. 5A). On the contrary, B, T, and NK cells did not produced cytokines after LPS stimulation. Interestingly, we observed that upon TAFA4 treatment, LPS-activated monocytes displayed a significant reduction in their cytokine production both in term frequency (% of producing cells) and quantity (MFI), compared to untreated cells (FIG. 5B-C). These results are consistent with the endogenous anti-inflammatory and pro-repair role of TAFA4 described in our previous study (Hoeffel et al. in revision).

[0090] We then focused our studies on M2c macrophages, which are differentiated in vitro in the presence of glucocorticoids and represent a type of anti-inflammatory cells that promotes tissue repair and could be beneficial in inflammatory diseases. Such M2c-like macrophages, expressing the marker CD163, were already observed in the lung of COVID-19 patients (data from the Vivier's lab: Carvelli et al. In press, Nature). We performed RNA-seq analysis of M2c macrophages differentiated in the presence or absence of TAFA4, and showed that TAFA4 reduces the expression of genes involved in the inflammatory response such as type I and II interferons, and also in the migration of leukocytes to the inflammatory site (data not shown). On the contrary, TAFA4 increases the production of genes involved in the maintenance of homeostasis, and corticosteroid response. The interest of these data is reinforced by results from the RECOVERY trial showing that treatment with corticosteroids improves the clinical course of COVID-19 patients and reduced deaths by one-third in most severe patients (https://www.ox.ac.uk/news/2020-06-16-low-cost-dexamethasone-reduces-death-one-third-hospitalised-pati ents- severe).

[0091] Collectively, these results demonstrate that the neuropeptide TAFA4 can reprogram human monocytes and macrophages toward an anti-inflammatory phenotype. TAFA4 can act on PBMC from COVID-19 patients at any stage of the disease. TAFA4 can also protect from the deleterious effects of over-inflammation in vivo in a mouse preclinical model of acute inflammatory disease. These data strongly support the use of TAFA4 as a therapeutic agent in inflammatory diseases, including sepsis or COVID-19, to reduce the activation and tissue recruitment of immune cells.

REFERENCES

[0092] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0093] 1. D.M. Mosser, J.P. Edwards, Exploring the full spectrum of macrophage activation, Nat. Rev. Immunol. 8 (2008) 958-969. [0094] 2. Y.R Na, S. Je, S.H Seok, Metabolic features of macrophages in inflammatory diseases and cancer. Cancer Letters 413 (2018) 46-58 [0095] 3. I.B. McInnes, G. Schett, The pathogenesis of rheumatoid arthritis, N. Engl. J. Med. 365 (2011) 2205-2219. [0096] 4. K.J. Moore, F.J. Sheedy, E.A. Fisher, Macrophages in atherosclerosis: a dynamic balance, Nat. Rev. Immunol. 13 (2013) 709-721. [0097] 5. Tom Tang, Y. et al. TAFA: a novel secreted family with conserved cysteine residues and restricted expression in the brain. Genomics 83, 727-734, doi:10.1016/j.ygeno.2003.10.006 (2004). [0098] 6. Delfini, M. C. et al. TAFA4, a chemokine-like protein, modulates injury-induced mechanical and chemical pain hypersensitivity in mice. Cell Rep 5, 378-388, doi:10.1016/j.celrep.2013.09.013 (2013). [0099] 7. Wang, W. et al. FAM19A4 is a novel cytokine ligand of formyl peptide receptor 1 (FPR1) and is able to promote the migration and phagocytosis of macrophages. Cellular & molecular immunology 12, 615-624, doi:10.1038/cmi.2014.61 (2015). [0100] 8. Chan, J.F.; Yuan, S.; Kok, K.H.; To, K.K.; Chu, H.; Yang, J.; Xing, F.; Liu, J.; Yip, C.C.; Poon, R. W., et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet 2020, [0101] 9. Huang, C.; Wang, Y.; Li, X.; Ren, L.; Zhao, J.; Hu, Y.; Zhang, L.; Fan, G.; Xu, J.; Gu, X., et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020, 395, 497-506 [0102] 10. Lu, R.; Zhao, X.; Li, J.; Niu, P.; Yang, B.; Wu, H.; Wang, W.; Song, H.; Huang, B.; Zhu, N., et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020, 395, 565-574. [0103] 11. Cao X. COVID-19: immunopathology and its implications for therapy. Nature Reviews. 2020; doi:10.1038/s41577-020-0308-3