New populations of fibroblasts and macrophages as prognosis markers for detection of fibroblasts associated diseases

Abstract

A method is disclosed for detecting if a subject is at risk of a fibroblast associated disease progression or for determining the clinical outcome of subjects suffering of such a disease, wherein the method comprises detecting SFRP1 positive, SFRP4 negative, FAP negative and RAMP1 negative fibroblasts (CXCL-iFibro) and, optionally, CD68 positive, CD206 positive and FOLR2 positive macrophages in a biological sample from said subject. The presence of CXCL-iFibro is indicative of disease progression and of a poor prognosis.

Description

[0001] NEW POPULATIONS OF FIBROBLASTS AND MACROPHAGES AS PROGNOSIS MARKERS FOR

[0002] DETECTION OF FIBROBLASTS ASSOCIATED DISEASES

[0003] Field of the Invention

[0004] The present invention relates to the field of medicine. It provides new markers for fibroblasts associated diseases detection and prognosis.

[0005] Background of the Invention

[0006] Mesenchymal-derived connective tissues including kidney heart, lung, gastrointestinal tract and muscle contain fibroblasts that fulfill specialized functions. Over the years, fibroblasts have been demonstrated as playing an important role in the development and progression of fibrotic diseases such as pulmonary fibrosis or chronic kidney disease. In particular, chronic kidney disease (CKD) is a significant public health burden, affecting 9.1% of the global population, and accounting for 2.6 million deaths. As CKD is becoming one of the leading causes of death, there is an urgent medical need to develop efficient therapeutic strategies and thus to better understand the molecular mechanisms underlying CKD progression.

[0007] CKD results from the development of interstitial fibrosis, which is the consequence of elevated production of extracellular matrix (ECM) proteins by myofibroblasts. Important efforts have been made to decipher myofibroblast origin(s) in kidney fibrosis. Several pieces of evidence previously demonstrated that myofibroblasts mainly originate from resident kidney interstitial cells, with several possible lineages, such as pericytes. Nevertheless, the intermediate step of differentiation toward myofibroblasts, as well as the cellular interactions leading to their differentiation are not yet fully understood.

[0008] Recently, single cell transcriptomic data from human and mouse kidney showed a significant heterogeneity in fibroblast populations during kidney fibrosis development. However, the role of these different fibroblasts in the differentiation processof myofibroblasts, in their interactions with surrounding cellular populations and thus in the development of the disease remains poorly known. One of the peculiarities of CKD is its ability to progress toward end-stage renal disease, even if the initial disease is controlled. Some predictors of CKD progression are well known to date, but the molecular mechanisms underlying this progression are still largely unknown. It is likely that fibroblasts and myofibroblasts are involved in this mechanism. Thus, better characterization of these different fibroblast populations in kidney disease is necessary to better understand this pathology and provide innovative treatments to patients.

[0009] Pulmonary Fibrosis is a chronic irreversible and ultimately fatal disease of unknown etiology. It is characterized by an unpredictable progressive decline in lung function. The disease progression is unpredictable at the time of diagnosis: some subjects may experience episodes of acute respiratory worsening despite being previously stable. It is critically important to distinguish subjects who are expected to progress from those who are expected to remain stable. The identification helps clinicians to make a decision about continuing or switching a treatment, or to refer for lung transplantation at an early stage.

[0010] There is thus a strong need to identify new markers allowing to detect patients at risk of fibroblasts associated diseases progression, to select effective treatments for patients who will really benefit from them.

[0011] There is also a persistent need to develop new strategies to limit or prevent the progression of fibroblasts associated diseases such as CKD in patients. The present invention seeks to meet these and other needs.

[0012] Summary of the invention

[0013] The inventors identified a new pro- inflammatory fibroblast population (CXCL- iFibro), which corresponds to an early state of myofibroblast differentiation. More precisely, the CXCL-iFibro represents an intermediate state in the differentiation process into ECM protein-secreting myofibroblasts. By combining single cell analysis, spatial transcriptomics and in vitro functional assays using fibroblasts isolated from patients, the inventors provide evidence that these pro-inflammatory CXCL-iFibro fibroblasts can attract and activate FOLR2+ macrophages.

[0014] The inventors particularly demonstrate that CXCL-iFibro co-localize with macrophages in kidney and are instrumental for their attraction, accumulation and switch into FOLR2+ macrophages at early stages of fibrosis patients. In turn, macrophages promote the switch of CXCL-iFibro into ECM-secreting myofibroblasts through the WNT / pcatenin- dependent pathway, thereby uncovering a reciprocal crosstalk between these populations of fibroblasts and macrophages during fibroblasts associated disease progression.

[0015] Finally, the inventors demonstrated that the detection of CXCL-iFibro and / or FOLR2+ macrophages at early stages of a fibroblasts associated disease such as CKD is predictive of poor patient prognosis. In addition to CKD, the inventors demonstrated the involvement of CXCL iFibro in liver, heart and lung fibrotic diseases, for a total of four different fibrotic diseases.

[0016] Altogether, by identifying a new population of inflammatory fibroblasts (CXCLiFibro) and its interaction with FOLR2+ macrophages, the inventors show a new mechanism driving fibroblasts associated disease progression, and have identify novel therapeutic targets to limit its progression.

[0017] In a first aspect, the invention concerns an in vitro method for detecting if a subject is at risk of a fibroblasts associated disease progression wherein the method comprises detecting SFRP1+SFRP4TAP-RAMP1’ fibroblasts (CXCL-iFibro), and optionally CD68+CD206+FOLR2+macrophages, in a biological sample from said subject; the presence of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages being indicative of disease progression and optionally, the absence of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages being indicative of the absence of disease progression.

[0018] In a second aspect, the invention concerns in vitro method for selecting a subject at risk of a fibroblasts associated disease, optionally as suitable for a treatment against said fibroblasts associated disease, wherein the method comprises:

[0019] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject, wherein the risk of said subject to have fibroblasts associated disease is proportional to the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample;

[0020] (b) optionally, selecting subjects with high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages as suitable for a treatment against said fibroblasts associated disease.

[0021] In a third aspect, the invention concerns in vitro method for predicting the clinical outcome of a subject affected with a fibroblasts associated disease, wherein the method comprises:

[0022] (a) determining level(s) of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from said subject, wherein high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages are predictive of a poor prognosis and optionally, wherein low level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages are predictive of a good prognosis.

[0023] In particular, a poor prognosis is a poor survival prognosis or an early or fast disease progression and a good prognosis is a good survival prognosis, a cessation of disease progression, a slow disease progression or the absence of early disease progression, preferably a slow disease progression. Preferably, the method further comprises:

[0024] (b) comparing the level(s) of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages higher than their reference level(s) are predictive of a poor prognosis and optionally, and / or level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages lower than or equal to their reference level(s) are predictive of a good prognosis.

[0025] In particular, the CXCL-iFibro have a gene signature comprising, consisting essentially of or consisting of TMEM176A, CTGF, VCAM1, FM03, C2orf40, CTSS, DPEP1, SRGN, NPY1R, HGF, G0S2, ALDH7A1, BIRC3, SDC4, CYB5A, TYMP, FOSB, SLC43A2, SLC2A3, ZFP36, FILIP1L, VMP1, RFTN1, FOS, CNN2, EDNRB, SERPINB9, GGT5, CCL21, NEGRI, MYL12A, THBS1, CCL19, CRISPLD2, DUSP6, JUN, HM0X2, FBLN5, ODF3B, SFRP1, ABCA8, MY ADM, TMEM176B, CYR61, CCBE1, CXCL12, MYO10, EMIDI, CCL2, GFRA1, TSPAN4, C7, GRAMD2B, CEBPD, RND3, IL34, STON1, ANGPTL1, PCSK7, CFHR1, NIDI, PTGER1, SELENOP, COLECI 1 and DNAJB4.

[0026] The level of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages is preferably a percentage of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages in the biological sample, respectively.

[0027] In a fourth aspect, the invention concerns the use of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) optionally with CD68+CD206+FOLR2+macrophages as biomarker(s) of a fibroblasts associated disease progression or for predicting the clinical outcome of a subject suffering from a fibroblasts associated disease.

[0028] In a fourth aspect, the invention concerns an agent targeting SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) for use in the treatment of a fibroblasts associated disease, wherein said agent is a cell depleting agent targeting CXCL-iFibro or is an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction.

[0029] In a fifth aspect, the invention concerns an inhibitor of P-catenin or of P-catenin / TCF interaction for use in the treatment of a fibroblasts associated disease in a subject, wherein the subject has SFRPUSFRP4 FAP RAMP1' fibroblasts (CXCL-iFibro), and optionally CD68+CD206+FOLR2+macrophages, in a biological sample, said inhibitor being preferably selected from the group consisting of iCRT3, Dickkopfl (DKK1), Porcupine inhibitor (PCN), secreted frizzled protein 1 (Sfirpl), Wnt inhibitor 1 (Wif-1), Wnt-C59, Triptonide, Tegatrabetan, Esculetin and Klotho. In a sixth aspect, the invention concerns the use of an agent targeting SFRP1+SFRP4 FAP' RAMPF fibroblasts (CXCL-iFibro) or of a pharmaceutical composition comprising said agent in the manufacture of a medicament for the treatment of a fibroblasts associated disease, wherein said agent is a cell depleting agent targeting CXCL-iFibro or is an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction.

[0030] The invention also concerns the use of an inhibitor of P-catenin or of P-catenin / TCF interaction ) or of a pharmaceutical composition comprising said inhibitor for the manufacture of a medicament for the treatment of a fibroblasts associated disease in a subject, wherein the subject has SFRPl+SFRP4'FAP'RAMPr fibroblasts (CXCL-iFibro), and optionally CD68+CD206+FOLR2+macrophages, in a biological sample, said inhibitor being preferably selected from the group consisting of iCRT3, Dickkopfl (DKK1), Porcupine inhibitor (PCN), secreted frizzled protein 1 (Sfirpl), Wnt inhibitor 1 (Wif-1), Wnt-C59, Triptonide, Tegatrabetan, Esculetin and Klotho.

[0031] The invention also concerns a method for treating a fibroblasts associated disease in a subject in need thereof, wherein the method comprises:

[0032] (a) determining the level(s) of SFRP1+SFRP4TAP'RAMPT fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject,

[0033] (b) selecting the subject as suitable for a treatment against said fibroblasts associated disease if the biological sample comprises:

[0034] - high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages; or

[0035] - level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages higher than a reference level; and c) administering a therapeutic effective amount of the treatment against said fibroblasts associated disease to the selected subject.

[0036] Preferably, in the methods or uses of the invention, the fibroblasts associated disease is selected from the group consisting of Chronic Kidney Disease (CKD), systemic sclerosis, kidney graft chronic rejection, myocardial fibrosis, myocardial infarction, rheumatoid arthritis, liver fibrosis, inflammatory bowel diseases (IBDs) and pulmonary fibrosis, preferably the from the group consisting of renal fibrosis, liver fibrosis, pulmonary fibrosis and cardiac fibrosis, even more preferably is Chronic Kidney Disease (CKD).

[0037] In particular, in the methods or uses of the invention, the biological sample is a kidney sample, a skin sample, a lung sample, a heart sample, a synovial liquid sample, a gut sample, a liver sample, a blood sample or a urine sample.

[0038] Typically, in the methods or uses of the invention: a) the fibroblasts associated disease is CKD and the treatment against CKD is i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation b) the fibroblasts associated disease is pulmonary fibrosis and the treatment against pulmonary fibrosis is corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; c) the fibroblasts associated disease is systemic sclerosis and the treatment against systemic sclerosis is methotrexate, mycophenolate mofetil and / or intravenous cyclophosphamide; d) the fibroblasts associated disease is rheumatoid arthritis and the treatment against rheumatoid arthritis disease is corticosteroids, methotrexate and / or an anti-TNFa treatment; e) the fibroblasts associated disease is kidney graft chronic rejection and the treatment against kidney graft chronic rejection is i) a treatment for limiting kidney graft chronic rejection, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of kidney graft chronic rejection, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) dialysis; or f) the fibroblasts associated disease is myocardial infarction and the treatment against myocardial infarction disease is beta-blockers, renin-angiotensin blockers and / or statins; g) the fibroblasts associated disease is inflammatory bowel diseases (IBDs) and the treatment against IBD is selected from anti-inflammatory drugs such as aminosalicylates; immunosuppressant drugs such as azathioprine, mercaptopurine and methotrexate and / or antibiotics, h) if the disease is cirrhosis the treatment against cirrhosis is diuretics, anti-aldosterone drugs, beta blockers and / or liver transplant.

[0039] The invention finally concerns the use of a kit comprising means for measuring the level of SFRP1+SFRP4 FAP RAMPF fibroblasts (CXCL-iFibro) and optionally CD68+CD206+FOLR2+macrophages in a biological sample for i) predicting the clinical outcome of a subject suffering from a fibroblast associated disease; ii) determining the likelihood of a subject to have a fibroblast associated disease progression; and / or iii) selecting or not a patient having a risk of a fibroblast associated disease progression for a treatment suitable for said fibroblast associated disease. Detailed description of the Invention

[0040] Definition

[0041] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains.

[0042] In order that the present invention may be more readily understood, certain terms are defined hereafter. Additional definitions are set forth throughout the detailed description.

[0043] As used herein, the term “marker” or “biomarker” refers to a measurable biological parameter that helps to detect the presence of cell population such as fibroblasts or macrophages, or to predict the occurrence and / or progression of a disease or condition, in particular chronic kidney disease progression.

[0044] As used herein “+” refers to a cell, especially a fibroblast or a macrophage, expressing a marker. For instance, F0LR2+refers to a cell, in particular a macrophage, expressing F0LR2, in particular that expresses F0LR2 higher than or above a reference level. Alternatively, refers to a cell that does not express the marker. For instance, F0LR2' refers to a cell, in particular a macrophage, that does not express F0LR2 or that expresses F0LR2 less or under a reference level.

[0045] As used herein, the term “diagnosis” refers to the determination as to whether a subject is likely to be affected by a fibroblasts associated disease. The skilled artisan often makes a diagnosis on the basis of one or more diagnosis markers, the presence, absence, or amount of which is indicative of the presence or absence of a fibroblasts associated disease. By “diagnosis”, it is also intended to refer to the provision of information useful for diagnosis.

[0046] As used herein, the term “treatment”, “treat” or “treating” refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis, retardation or cessation of progression of a disease, such as chronic kidney disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease, in particular a fibroblasts associated disease such as CKD.

[0047] The terms “percentage”, “quantity,” “number”, “amount,” and “level” are used interchangeably herein and may refer to an absolute quantification of a molecule or a cell in a sample, or to a relative quantification of a molecule or a cell in a sample, i.e., relative to another value such as relative to a reference value as taught herein.

[0048] The terms “kit”, “product” or "combined preparation", as used herein, defines especially a "kit of parts" in the sense that the combination partners (a) and (b), as defined in the present application can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i.e., simultaneously or at different time points. The parts of the kit of parts can then be administered simultaneously or chronologically staggered, that is at different time points for any part of the kit of parts. The ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied. The combination partners (a) and (b) can be administered by the same route or by different routes.

[0049] The term “and / or” as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.

[0050] The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.

[0051] The term “at least one” means “one or more” or “one or several”. For instance, it refers to one, two, three or more.

[0052] The term “about” as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means any value having an acceptable range of deviation of up to + / - 10% (e.g., + / - 0.5%, + / -1 %, + / -1 .5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / -9.5%). The use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%).

[0053] The methods of the invention as disclosed below may be in vivo, ex vivo or in vitro methods, preferably in vitro or ex vivo methods.

[0054] The term “comprise” or “comprising” can be replaced by “consist” or “consisting” or by “essentially consist” or “essentially consisting” in any aspects or embodiments of the present application. CXCL-iFibro and FOLR2+macrophages

[0055] In a first aspect, the invention concerns the use of SFRPUSFRP4 FAP RAMP1' fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages (FOLR2+macrophages), preferably CXCL-iFibro and optionally FOLR2+macrophages, as biomarker(s) of i) a fibroblasts associated disease, in particular of a fibroblasts associated disease progression; ii) for predicting the risk or likelihood of a subject to have a fibroblasts associated disease progression, in particular the risk of fast fibroblasts associated disease progression; or iii) for predicting the clinical outcome of a subject suffering from a fibroblasts associated disease.

[0056] The detection of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, can be performed by the detection of markers that are expressed by CXCL-iFibro and / or FOLR2+macrophages, or by the detection of a gene signature that characterizes said populations, respectively.

[0057] The detection of CXCL-iFibro is particularly performed by assessing if a fibroblast expresses or not SFRP1, SFRP4, FAP and RAMPL CXCL-iFibro are particularly characterized by the markers SFRPl+SFRP4 FAP RAMPr.

[0058] As used herein the term “fibroblasts” refers to a type of cell that synthesizes the extracellular matrix and collagen, produces structural framework (stroma) for tissues, and plays a critical role in wound healing. The main function of fibroblasts is to maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix. The term “SFRPUSFRP4 FAP RAMP1' fibroblasts” and “CXCL-iFibro” are used interchangeably herein.

[0059] As used herein, the terms “SFRP1”, “Secreted Frizzled Related Protein 1” and “Secreted apoptosis-related protein 2 (SARP-2)” are used interchangeably and refer to the product of the SFRP1 human gene, for example such as described under GenelD: 6422 and UniProt Q8N474 references. Soluble frizzled-related proteins function as modulators of Wnt signaling through direct interaction with Wnts. They have a role in regulating cell growth and differentiation in specific cell types.

[0060] As used herein, the terms “SFRP4” and “Secreted Frizzled Related Protein 4” are used interchangeably and refer to the product of the SFRP4 human gene, for example such as described under GenelD: 6424 and UniProt Q6FHJ7references.

[0061] As used herein, the terms “FAP”, “Fibroblast Activation Protein”, “Prolyl endopeptidase FAP”, “Dipeptidyl peptidase FAP”, “Surface-expressed protease”, “ Sepras”, “Serine integral membrane protease”, “SIMP”, “Integral membrane serine protease”, “Post-proline cleaving enzyme” are used interchangeably and refer to the product of the FAP human gene, for example such as described under GenelD: 2191 and UniProt Q12884 references. FAP is a cell surface glycoprotein serine protease that participates in extracellular matrix degradation and involved in many cellular processes including tissue remodeling, fibrosis, wound healing, inflammation and tumor growth.

[0062] As used herein, the terms “RAMP1“, “Receptor activity -modifying protein 1” and “Calcitonin-receptor-like receptor activity -modifying protein 1” are used interchangeably and refer to the product of the RAMP1 human gene, for example such as described under GenelD: 10267 and UniProt 060894 references.

[0063] In addition to the above markers, other / additional markers specific of CXCL-iFibro can also be used to detect them.

[0064] In some aspects, CXCL12 is an additional marker used to characterize / detect the CXCL- iFibro population.

[0065] As used herein, the term “CXCL12”, “C-X-C chemokine receptor type 12”, “SDF-1” and “Stromal cell-derived factor 1” are used interchangeably and refer to the product of the CXCL12 human gene, for example such as described under GenelD: 6387 and UniProt P48061 references.

[0066] Alternatively or additionally, aSMA is an additional marker used to characterize / detect the CXCL-iFibro population.

[0067] As used herein, the terms “aSMA” or “alpha-Smooth Muscle Actin” are used interchangeably and refer to the product of the ACTA2 human gene, for example such as described under GenelD: 59 and UniProt P62736 references. aSMA is a well-known and characterized protein used for assessment of activated fibroblasts in several tissues and organs.

[0068] In particular, CXCL-iFibro have a gene signature comprising, consisting essentially or consisting of TMEM176A, CTGF, VCAM1, FM03, C2orf40, CTSS, DPEP1, SRGN, NPY1R, HGF, G0S2, ALDH7A1, BIRC3, SDC4, CYB5A, TYMP, FOSB, SLC43A2, SLC2A3, ZFP36, FILIP1L, VMP1, RFTN1, FOS, CNN2, EDNRB, SERPINB9, GGT5, CCL21, NEGRI, MYL12A, THBS1, CCL19, CRISPLD2, DUSP6, JUN, HM0X2, FBLN5, ODF3B, SFRP1, ABCA8, MYADM, TMEM176B, CYR61, CCBE1, CXCL12, MYO10, EMIDI, CCL2, GFRA1, TSPAN4, C7, GRAMD2B, CEBPD, RND3, IL34, STON1, ANGPTL1, PCSK7, CFHR1, NIDI, PTGER1, SELENOP, COLECI 1 and DNAJB4.

[0069] A “gene signature” or “gene expression signature”, as used herein, is a group of genes in a sample with a uniquely characteristic pattern of gene expression. In particular, the gene signature corresponds to the expression of specific genes. When defining a gene signature, “comprising” means that the gene signature may comprise additional genes, in particular less than 30, 25, 20, 15, 10 or 5 additional genes. When defining a gene signature, “essentially consist in” means that the gene signature may comprise 1, 2 or 3 or 4 additional genes maximum. When defining a gene signature, “consist in” means that the gene signature does not include any additional gene.

[0070] The terms “CXCL-iFibro and optionally FOLR2+macrophages” as used herein preferably mean that CXCL-iFibro can be used alone or in combination with FOLR2+macrophages in any embodiments or aspects of the invention. In some preferred aspects and embodiments, the terms “CXCL-iFibro and / or FOLR2+macrophages” can be changed into “CXCL-iFibro and optionally FOLR2+macrophages”.

[0071] The detection of FOLR2+macrophages is particularly performed by assessing if a macrophage expresses or not CD68, CD206 and FOLR2. FOLR2+macrophages are characterized by the markers CD68+CD206+FOLR2+.

[0072] As used herein the term “macrophages” refers to a type of white blood cell of the innate immune system that engulfs and digests pathogens and foreign substances through phagocytosis. Besides phagocytosis, they play a critical role in innate immunity and also help initiate specific defense mechanisms by recruiting other immune cells and acts as antigen presenting cells. The terms “CD68+CD206+FOLR2+macrophages” and “FOLR2+macrophages” are used interchangeably herein.

[0073] As used herein, the terms “CD68”, “Macrosialin” and “Gpl lO” are used interchangeably and refer to the product of the CD68 human gene, for example such as described under GenelD: 968 and UniProt P34810 references.

[0074] As used herein, the terms “CD206”, “Macrophage mannose receptor 1”, “MMR”, “C-type lectin domain family 13 member D” and “Human mannose receptor (hMR)” are used interchangeably and refer to the product of the CD206 human gene, for example such as described under GenelD: 4360 and UniProt P22897 references.

[0075] As used herein, the terms “FOLR2”, “Folate receptor beta” and “FR-beta” are used interchangeably and refer to the product of the FOLR2 human gene, for example such as described under GenelD: 2350 and UniProt P14207 references.

[0076] The above markers of CXCL-iFibro and / or FOLR2+macrophages are useful in methods that includes the detection of CXCL-iFibro and / or FOLR2+macrophages or the determination of CXCL-iFibro and / or FOLR2+macrophages level(s). Then, the invention concerns methods for detecting, determining or diagnosing the risk of a subject to have a fibroblasts associated disease, preferably CKD, progression, based on the presence of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from said subject.

[0077] The invention particularly concerns an in vitro method for detecting if a subject is at risk of a fibroblasts associated disease progression wherein the method comprises detecting SFRP1+SFRP4 FAP RAMP1- fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from said subject; the presence of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages being indicative of disease progression and optionally, the absence of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages being indicative of the absence of disease progression.

[0078] The invention also concerns an in vitro method for detecting if a subject is at risk of chronic kidney disease (CKD) progression, in particular at risk of fast CKD progression, wherein the method comprises detecting SFRP1+SFRP4 FAP RAMPL fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from said subject; the presence of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages being indicative of CKD progression.

[0079] Preferably, the absence of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, is indicative of the absence of a fibroblasts associated disease progression or of a fibroblasts associated disease slow progression.

[0080] In particular, the absence of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, is indicative of the absence of disease progression.

[0081] The invention also concerns an in vitro method for selecting a subject at risk of a fibroblasts associated disease, in particular of subject at risk of a fibroblasts associated disease progression wherein the method comprises:

[0082] (a) determining the level(s) of SFRP1+SFRP4TAP'RAMPT fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from the subject, wherein the risk of said subject to have fibroblasts associated disease is proportional to the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample; (b) optionally, selecting subjects with high level(s) CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, as suitable for a treatment against said fibroblasts associated disease.

[0083] Then, such method may be used for selecting patient at risk of a fibroblasts associated disease for a suitable treatment against said fibroblasts associated disease.

[0084] The invention particularly concerns an in vitro method for selecting a subject at risk of chronic kidney disease (CKD) progression, in particular at risk of fast CKD progression, wherein the method comprises:

[0085] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from the subject, wherein a high level of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample is indicative of a high risk of CKD progression;

[0086] (b) optionally, selecting subjects with high level(s) CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, as suitable for a treatment against CKD.

[0087] Preferably, the method comprises:

[0088] (a) detecting CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from said subject;

[0089] (b) determining the level(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, in said biological sample, wherein a high level of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample is indicative of a high risk of fibroblasts associated disease progression;

[0090] (c) optionally, selecting subjects with high level(s) CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, as suitable for a treatment against fibroblasts associated disease.

[0091] The different treatments against fibroblasts associated diseases are more particularly described hereafter under the section “Treatments”.

[0092] The detection of CXCL-iFibro and optionally FOLR2+macrophages or their level in a biological sample can be performed by any techniques known in the art, in particular based on the above defined biomarkers of said populations, respectively. In some aspects, the detection of the markers of CXCL-iFibro and optionally FOLR2+macrophages is carried out at the protein level, in particular with antibodies that are directed against said markers. Preferably, the detection of CXCL-iFibro and FOLR2+macrophages or their level in a biological sample is determined by measuring the expression of their respective marker proteins.

[0093] The quantity of a protein may be measured by any method known by the skilled person. Usually, these methods comprise contacting the sample with a binding partner capable of selectively interacting with the protein present in the sample. The binding partner is generally a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The antibody can be labelled and / or fused to a detection entity. In particular, the antibody can be labelled with a label selected from the group consisting in a radiolabel, an enzyme label, a fluorescent label, a biotinavidin label, a chemiluminescent label, and the like. The antibody can be labeled by standard labeling techniques well known by the man skilled in the art and labelled antibodies can be visualized using known methods. In particular, labels generally provide signals detectable by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like.

[0094] The quantity of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, may be measured by semi-quantitative Western blots, enzyme- labeled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassay, immunohistochemistry, immunoelectrophoresis or immunoprecipitation, protein or antibody arrays, or flow cytometry, such as Fluorescence-activated cell sorting (FACS). The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. Preferably, the protein expression level of CXCL-iFibro and / or FOLR2+macrophages markers is assessed by FACS or by immunohistochemistry.

[0095] Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.

[0096] Immunohistochemistry (IHC) refers to the process of selectively imaging antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Visualizing the antibody-antigen interaction can be accomplished in a number of ways, well known by the man skilled in the art. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction or is tagged by a fluorophore, such as fluorescein or rhodamine. Immunohistochemistry can be divided into two phases: sample preparation and sample labeling.

[0097] Preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning. A solution of paraformaldehyde is often used to fix tissue, but other methods may be used. The tissue may then be sliced or used whole, dependent upon the purpose of the experiment or the tissue itself. Before sectioning, the tissue sample may be embedded in a medium, like paraffin wax or cryomedia. Sections can be sliced on a variety of instruments, most commonly a microtome, cryostat, or Compresstome tissue slicer. Specimens are typically sliced at a range of 3 pm-50 pm. The slices are then mounted on slides, dehydrated using alcohol washes of increasing concentrations (e.g., 50%, 75%, 90%, 95%, 100%), and cleared using a detergent like xylene before being imaged under a microscope. Depending on the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigen-retrieval is often necessary, and involves pre-treating the sections with heat or protease. These steps may make the difference between the target antigens staining or not staining. Depending on the tissue type and the method of antigen detection, endogenous biotin or enzymes may need to be blocked or quenched, respectively, prior to antibody staining. Although antibodies show preferential avidity for specific epitopes, they may partially or weakly bind to sites on nonspecific proteins (also called reactive sites) that are similar to the cognate binding sites on the target antigen. To reduce background staining in IHC, samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include normal serum, non-fat dry milk, BSA, or gelatin. Methods to eliminate background staining include dilution of the primary or secondary antibodies, changing the time or temperature of incubation, and using a different detection system or different primary antibody. Quality control should as a minimum include a tissue known to express the antigen as a positive control, and negative controls of tissue known not to express the antigen, as well as the test tissue probed in the same way with omission of the primary antibody (or better, absorption of the primary antibody).

[0098] For immunohistochemical detection strategies, antibodies are classified, when necessary, as primary or secondary reagents. Primary antibodies are raised against an antigen of interest and are typically unconjugated (i.e. unlabeled), while secondary antibodies are raised against immunoglobulins of the primary antibody species. The secondary antibody is usually labelled and / or fused to a detection entity as described above.

[0099] The direct method is a one-step staining method and involves a labeled antibody reacting directly with the antigen in tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, in contrast to indirect approaches.

[0100] The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody. The secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter. Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin- or NeutrAvidin proteinbound enzyme.

[0101] In some instance, the CXCL-iFibro and optionally FOLR2+macrophages are detected using primary antibodies against their respective markers (i.e., SFRP1, SFRP4, FAP and RAMP1 and optionally CXCL12 and / or aSMA for CXCL-iFibro; and CD68, CD206 and FOLR2 for FOLR2+macrophages, respectively).

[0102] Antibodies that can be used to measure SFRP1 by FACS or immunohistochemistry are for example: rabbit monoclonal anti-SFRPl Abeam ab267466 or abl26613.

[0103] Antibodies that can be used to measure SFRP4 by FACS or immunohistochemistry are for example: rabbit monoclonal anti-SFRP4 Abeam abl54167.

[0104] Antibodies that can be used to measure FAP by FACS or immunohistochemistry are for example: rabbit monoclonal anti-FAP Abeam ab207178.

[0105] Antibodies that can be used to measure RAMP1 by FACS or immunohistochemistry are for example: mouse monoclonal anti RAMP1 antibody EMD Millipore MABS1904

[0106] Antibodies that can be used to measure aSMA by FACS or immunohistochemistry are for example: DAKO clone 1A4 ref M0851.

[0107] Antibodies that can be used to measure CD68 by FACS or immunohistochemistry are for example: Abeam ab955. Antibodies that can be used to measure CD206 by FACS or immunohistochemistry are for example: BioLegend 321136

[0108] Antibodies that can be used to measure F0LR2 by FACS or immunohistochemistry are for example: Invitrogen MA5-26933 and BioLegend 391704. For example, anti-FOLR2 antibodies are also disclosed in US 2008 / 0260812; US 2014 / 0010756; WO 2012 / 033987.

[0109] In a preferred embodiment, the detection of CXCL-iFibro is performed by FACS. In particular, the detection of CXCL-iFibro may comprise a step of exclusion of non-pertinent cells.

[0110] In particular, the detection of CXCL-iFibro may comprise: - exclusion of the non-fibroblast cells, for example by excluding the CD45+cells; and / or selection of the fibroblast cells, for example by selecting the FSP1+cells (Fibroblast-specific protein 1, also called S100A4); optionally, exclusion of the dead cells, for example by using an intracellular dye such as the violet LIVE / DEAD dye or DAPI and excluding the stained cells; and - detection of SFRPl+SFRP4 FAP RAMPr cells, preferably of SFRPl+SFRP4 FAP RAMPr aSMA+cells and optionally, quantification of SFRP1+SFRP4 FAP RAMP1' cells, preferably of SFRP1+SFRP4‘ FAP RAMPl aSMA+cells.

[0111] In another preferred embodiment, the detection of CXCL-iFibro is performed by immunohistochemistry. Particularly, the detection of CXCL-iFibro in a biological sample may comprise the following steps: identification of fibroblasts in the biological sample, for example on the basis of morphological criteria; and

[0112] - detection of the SFRP1+SFRP4 FAP RAMP1- cells, preferably of SFRP1+SFRP4 FAP RAMP1- aSMA+cells with antibodies against each marker individually for immunostaining.

[0113] In another embodiment, the expression level of CXCL12 in a fibroblast is determined by measuring the activity of the protein, in particular, the enzymatic activity of CXCL12.

[0114] The enzymatic activity of a protein may be measured by any method known by the skilled person. Usually, enzyme assays measure either the consumption of substrate or production of product over time. For example, production of chemokine CXCL12 may be detected by in situ hybridization. To assay CXCL12 enzymatic activity, a non-fluorescent substrate from which a fluorescent product will be released can be used. Alternatively, ELISA kit may be used to assess the production of CXCL12, for example such as CXCL12 ELISA Kit (BT LAB).

[0115] In a preferred embodiment, the detection of FOLR2+macrophages is performed by FACS. In particular, the detection is performed after a positive selection of CD45+ / CD3- cells followed by positive selection of CDl lb+ / CD68+cells.

[0116] Alternatively, the detection of FOLR2+macrophages may comprise a step of exclusion of non-pertinent cells.

[0117] In some aspects, the detection of FOLR2+macrophages by FACS comprises: exclusion of the non-macrophages cells, for example by excluding the endothelial (CD31+) and epithelial cells, and / or selection of the macrophages cells, for example by selecting the CD45+ CD68+ cells; optionally, exclusion of the dead cells, for example by using an intracellular dye such as the violet LIVE / DEAD dye or DAPI and excluding the stained cells; and optionally, positive selection of CD45+ / CD3- cells followed by positive selection of CDl lb+ / CD68+cells; detection of CD68+CD206+FOLR2+cells; and optionally, quantification of CD68+CD206+FOLR2+cells.

[0118] In another preferred embodiment, the detection of FOLR2+macrophages is performed by immunohistochemistry. Preferably, the detection of FOLR2+macrophages in a biological sample may comprise the following steps: identification of macrophages in the biological sample, for example on the basis of morphological criteria; and detection of the CD68+CD206+FOLR2+cells with antibodies against each marker individually for immunostaining.

[0119] Alternatively, the detection of CXCL-iFibro and optionally FOLR2+macrophages or their level in a biological sample can be assessed by measuring the expression of their mRNA. Methods for determining the quantity of mRNA in a cell are well known by the man skilled in the art. mRNA can be detected by hybridization (e. g., Northern blot analysis) in particular by the Nanostring method and / or by amplification (e.g., RT-PCR), in particular by quantitative or semi- quantitative RT-PCR. Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

[0120] Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Taqman probes specific of the protein of interest transcript may be used. In a preferred embodiment, the expression level of the markers of CXCL-iFibro and optionally FOLR2+macrophages and any other protein of interest is determined by measuring the quantity of their mRNA, preferably by quantitative or semi-quantitative RT-PCR or by real-time quantitative or semi-quantitative RT- PCR.

[0121] As used herein, the terms “quantitative RT-PCR”, “qRT-PCR”, “Real time RT-PCR” and “quantitative Real time RT-PCR” are equivalent and can be used interchangeably. Any of a variety of published quantitative RT-PCR protocols can be used (and modified as needed) for use in the present method. Suitable quantitative RT-PCR procedures include but are not limited to those presented in U.S. Pat. No. 5,618,703 and in U.S. Patent Application No. 2005 / 0048542, which are hereby incorporated by reference.

[0122] Quantitative RT-PCR Primers targeting one or more marker(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, can be used to detect these population of cells (i.e., primer or primer pairs targeting SFRP1, SFRP4, FAP and RAMP1 and optionally CXCL12 and / or aSMA for CXCL-iFibro; and primer or primer pairs targeting CD68, CD206 and FOLR2 for FOLR2+macrophages, respectively).

[0123] In another embodiment, the detection of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, or their level in a biological sample can be assessed by measuring the expression of their mRNA by a single cell RNA-Seq.

[0124] In one aspect, the CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, are detected by the expression of specific gene signatures such as described above. The expression of such gene signature can for example be assessed by RNA sequencing or by the Nanostring method. The Nanostring method is a hybridization method that allows to quantify RNA without requiring linear nor exponential amplification. It is a very sensitive method, since only 10 ng of mRNA are needed to perform it, allowing analysis of quantity limited biological samples. The Nanostring method necessitates the use of a pair of probes specifically designed for each mRNA. Nanostring probes targeting one or more marker(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages; can be used to detect these population of cells (i.e., primer or primer pairs targeting SFRP1, SFRP4, FAP and RAMP1 and optionally CXCL12 and / or aSMA for CXCL-iFibro; and primer or primer pairs targeting CD68, CD206 and FOLR2 for FOLR2+macrophages, respectively).

[0125] In the methods of the invention, the detection of CXCL-iFibro and / or FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, by the methods described above indicates their presence and / or their amount in the biological sample.

[0126] In some embodiment, the invention concerns a method for detecting if a subject is at risk of fibroblasts associated disease progression that comprises determining that the subject has an increased likelihood of fibroblasts associated disease progression by detecting an elevated or high number or percentage of CXCL-iFibro fibroblasts and / or FOLR2+ macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, in a sample from said subject.

[0127] In one aspect, the presence of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample is a ratio or percentage of the number of cells of the CXCL-iFibro and / or FOLR2+macrophages, in particular on the total number of cells of the biological sample.

[0128] Preferably, the presence of CXCL-iFibro is a ratio or percentage of the number of cells of CXCL-iFibro on the total number of fibroblasts in the sample, a ratio or percentage of the number of cells of CXCL-iFibro on the total number of fibroblasts in the sample.

[0129] Preferably, the presence of FOLR2+macrophages is a ratio or percentage of the number of cells of FOLR2+macrophages on the total number of macrophages in the sample, a ratio or percentage of the number of cells of FOLR2+macrophages on the total number of macrophages in the sample.

[0130] Preferably, the term “percentage” of CXCL-iFibro or FOLR2+macrophages may refer to any ratio having the number of CXCL-iFibro or FOLR2+macrophages as nominator, respectively, and a number of reference cells as a denominator. Preferably, the term “percentage” may refer to any ratio having the number of CXCL-iFibro and / or FOLR2+macrophages cells as nominator, and a number of reference cells as a denominator.

[0131] A “low percentage” of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL- iFibro and optionally FOLR2+macrophages, corresponds to less than 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1%. It particularly corresponds to less than 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% of CXCL-iFibro in the total number of cells or the total number of fibroblasts or to less than 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% of FOLR2+macrophages in the total number of cells or the total number of macrophages.

[0132] A “high percentage” of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL- iFibro and optionally FOLR2+macrophages, corresponds to more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. It particularly corresponds more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of CXCL-iFibro in the total number of cells or the total number of fibroblasts or more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of FOLR2+macrophages in the total number of cells or the total number of macrophages.

[0133] In some aspects, the presence of CXCL-iFibro and / or FOLR2+macrophages in a biological sample represents the number of CXCL-iFibro and / or FOLR2+macrophages on the total number of cells in a biological sample. Preferably, the presence of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, corresponds to a percentage of at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20% or 30% among the total number of cells in a biological sample.

[0134] Particularly, the presence of CXCL-iFibro in a biological sample of a patient represents the number of CXCL-iFibro on the total number of fibroblasts in a biological sample. Preferably, the presence of CXCL-iFibro corresponds to a percentage of at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40% or 50% among the total number of fibroblasts in a biological sample.

[0135] Alternatively, the presence of FOLR2+macrophages in a biological sample of a patient represents the number of FOLR2+macrophages on the total number of macrophages in a biological sample. Preferably, the presence of FOLR2+macrophages corresponds to a percentage of at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40% or 50% among the total number of macrophages in a biological sample.

[0136] In an aspect, the presence of CXCL-iFibro and / or FOLR2+macrophages, preferably ofCXCL-iFibro and optionally FOLR2+macrophages, in the biological sample, for instance the percentage of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages in the biological sample, is proportional of the risk or likelihood of fibroblasts associated disease progression of said patient.

[0137] By “proportional” it is meant that the more CXCL-iFibro and / or FOLR2+macrophages are in the biological sample, the more the subject suffering from a fibroblasts associated disease is at risk of fibroblasts associated disease progression. These terms do not necessarily imply a direct and measurable correlation factor. Then, the invention also concerns an in vitro method for selecting a subject at risk of fibroblasts associated disease progression, wherein the method comprises:

[0138] (a) determining the level(s) of SFRP1+SFRP4 FAP RAMPL fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from the subject, wherein the risk of said subject to have a fibroblasts associated disease progression is proportional to the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in the biological sample;

[0139] (b) optionally, selecting subjects with high level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages as suitable for a treatment against said fibroblasts associated disease.

[0140] Preferably, the method comprises:

[0141] (a) detecting CXCL-iFibro and / or FOLR2+macrophages in a biological sample from said subject;

[0142] (b) determining the level(s) of CXCL-iFibro and / or FOLR2+macrophages in said biological sample, wherein the risk of said subject to have a fibroblasts associated disease progression is proportional to the level(s) of CXCL-iFibro and / or FOLR2+macrophages in the biological sample;

[0143] (c) optionally, selecting subjects with high level(s) CXCL-iFibro and / or FOLR2+macrophages as suitable for a treatment against said fibroblasts associated disease.

[0144] Preferably, the method comprises:

[0145] (a) detecting CXCL-iFibro and optionally FOLR2+macrophages in a biological sample from said subject;

[0146] (b) determining the level(s) of CXCL-iFibro and optionally FOLR2+macrophages in said biological sample, wherein the risk of said subject to have a fibroblasts associated disease progression is proportional to the level(s) of CXCL-iFibro and optionally FOLR2+macrophages in the biological sample;

[0147] (c) optionally, selecting subjects with high level(s) CXCL-iFibro and optionally FOLR2+macrophages as suitable for a treatment against said fibroblasts associated disease.

[0148] The different treatments against fibroblasts associated diseases are more particularly described hereafter under the section “Treatments”. The detection of CXCL-iFibro and / or FOLR2+macrophages and / or the determination of CXCL- iFibro and / or FOLR2+macrophages level(s) can be performed by any of the techniques disclosed herein.

[0149] In addition to the determination of the level of CXCL-iFibro and / or FOLR2+macrophages per se, the level of CXCL-iFibro and / or FOLR2+macrophages, CXCL-iFibro and optionally FOLR2+macrophages, can be compared to a reference level.

[0150] In some aspects, the level of CXCL-iFibro and / or the level of FOLR2+macrophages in a biological sample is compared to a reference level, respectively. Then, any methods of the invention may further comprise a step of comparing the level(s) of CXCL-iFibro to a reference level and / or FOLR2+macrophages to a refence level. Preferably, any methods of the invention may further comprise a step of comparing the level(s) of CXCL-iFibro to a reference level and optionally of FOLR2+macrophages to a refence level.

[0151] The invention may particularly concern an in vitro method for determining if a subject is at risk of a fibroblasts associated disease progression wherein the method comprises:

[0152] (a) determining level(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL- iFibro and optionally FOLR2+macrophages, in a biological sample from the subject;

[0153] (b) comparing the level(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL- iFibro and optionally FOLR2+macrophages, in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, higher than their reference level(s) are indicative of a high risk of fibroblasts associated disease progression.

[0154] The invention also concerns an in vitro method for selecting a subject at risk of a fibroblasts associated disease progression, wherein the method comprises:

[0155] (a) determining the level(s) of SFRP1+SFRP4TAP'RAMPT fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject,

[0156] (b) comparing the level(s) of CXCL-iFibro and / or FOLR2+macrophages in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and / or FOLR2+macrophages higher than their reference level(s) are indicative of a high risk of a fibroblasts associated disease progression; (c) optionally, selecting subjects with level(s) of CXCL-iFibro and / or FOLR2+macrophages higher than their reference level(s) as suitable for a treatment against said fibroblasts associated disease.

[0157] In some embodiments where level(s) of CXCL-iFibro and / or FOLR2+macrophages are percentages, the reference level(s) is / are reference percentage(s).

[0158] Methods for the determination of reference levels, in particular reference percentages, are well known from the man skilled in the art.

[0159] Preferably, the reference level(s) are determined by measuring the percentage of CXCL- iFibro and / or FOLR2+macrophages in biological sample from cohorts of patients from who a fibroblasts associated disease stage and / or progression is known or who are healthy individual or group of healthy individuals (i.e., that do not suffer from a fibroblasts associated disease).

[0160] Preferably, when the fibroblasts associated disease is CKD, the reference level(s) are determined by measuring the percentage of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in biological sample from cohorts of patients from who CKD stage and / or progression is known or who are healthy individual or group of healthy individuals (i.e., that do not suffer from CKD).

[0161] In the context of the present invention, the level of CXCL-iFibro and / or FOLR2+macrophages is considered to be lower than its reference level if it is at least, when applicable, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25 or 30 % under the reference level.

[0162] In the context of the present invention, the level of CXCL-iFibro and / or FOLR2+macrophages is considered to be higher than its reference level if it is at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25 or 30 % over the reference level.

[0163] The invention may thus concern an in vitro method for selecting a subject at risk of a fibroblasts associated disease progression, wherein the method comprises:

[0164] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from the subject,

[0165] (b) comparing the level(s) of CXCL-iFibro and / or FOLR2+macrophages preferably of CXCL-iFibro and optionally FOLR2+macrophages, in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and / or FOLR2+macrophages higher than their reference level(s) are indicative of a high risk of a fibroblasts associated disease progression; (c) optionally, selecting subjects having level(s) of CXCL-iFibro and / or FOLR2+macrophages preferably of CXCL-iFibro and optionally FOLR2+macrophages, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25 or 30% higher than their reference level(s) as suitable for a treatment against said fibroblasts associated disease.

[0166] The invention particularly concerns an in vitro method for selecting a subject at risk of chronic kidney disease (CKD) progression, wherein the method comprises:

[0167] (a) determining the level(s) of SFRPl+SFRP4'FAP'RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from the subject,

[0168] (b) comparing the level(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and / or FOLR2+macrophages higher than their reference level(s) are indicative of a high risk of CKD progression;

[0169] (c) optionally, selecting subjects having level(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25 or 30% higher than their reference level(s) as suitable for a treatment against CKD.

[0170] In some embodiments, the diagnosis methods disclosed herein further comprises an initial step of providing a biological sample from said patient.

[0171] In some embodiments, the diagnosis methods disclosed herein comprises a further step of administering a therapeutic effective amount of a treatment against a fibroblasts associated disease to the patient having a poor prognosis, in particular a treatment as described below under the section “Treatments”.

[0172] Prognosis methods

[0173] The invention also concerns methods for predicting or monitoring the clinical outcome of a subject affected with a fibroblast associated disease based on the presence or level(s) of CXCL- iFibro and / or FOLR2+macrophages.

[0174] As used herein, the terms “clinical outcome” and “prognosis” are interchangeable and refer to the determination as to whether a subject is likely to be affected by a fibroblast associated disease progression and / or or death. These terms also relate to the survival, in particular the overall survival. “Overall survival” (OS) as used herein refers to the time span from diagnosis until death of the patient from the disease. In embodiments where the fibroblast associated disease is CKD, it particular relates to the length of time after CKD diagnosis that a patient lives until end-stage renal disease (ESRD); total kidney failure or death.

[0175] In embodiments where the fibroblast associated disease is pulmonary fibrosis, it particular relates to the length of time after pulmonary fibrosis diagnosis that a patient lives until lung failure or death.

[0176] In embodiments where the fibroblast associated disease is a cardiac fibrosis, it particular relates to the length of time after cardiac fibrosis diagnosis that a patient lives until hearth failure or death.

[0177] In embodiments where the fibroblast associated disease is liver fibrosis, it particular relates to the length of time after liver fibrosis diagnosis that a patient lives until cirrhosis; total liver failure or death.

[0178] In some aspects, the invention concerns a prognosis method that comprises determining that the subject has an increased likelihood of a fibroblast associated disease progression by detecting an elevated or high number or percentage of CXCL-iFibro fibroblasts and / or FOLR2+ macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a sample from said subject.

[0179] In some embodiment, the invention concerns an in vitro method for predicting the clinical outcome of a subject suffering from a fibroblast associated disease comprising determining the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in said biological sample, wherein high level(s) of CXCL- iFibro and / or CD68+CD206+FOLR2+macrophages are predictive of a poor prognosis.

[0180] Particularly, the invention concerns an in vitro method for predicting the clinical outcome of a subject suffering from a fibroblast associated disease comprising:

[0181] (a) detecting SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from said subject;

[0182] (b) determining the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in said biological sample, wherein high level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages are predictive of a poor prognosis.

[0183] Preferably, low level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages are predictive of a good prognosis; Particularly, the method for predicting the clinical outcome of a subject suffering from a fibroblast associated disease comprises:

[0184] (a) detecting CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from said subject;

[0185] (b) determining the level(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in said biological sample, wherein high level(s) of CXCL-iFibro and / or FOLR2+macrophages are predictive of a poor prognosis; and

[0186] (c) comparing the level(s) of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and / or FOLR2+macrophages higher than their reference level(s) are predictive of a poor prognosis.

[0187] Preferably, level(s) of CXCL-iFibro and / or FOLR2+macrophages egal or lower than their reference level(s) are predictive of a good prognosis.

[0188] In an aspect, the presence of CXCL-iFibro and / or FOLR2+macrophages, preferably of CXCL-iFibro and optionally FOLR2+macrophages, in the biological sample, is inversely proportional of clinical outcome of the patient. By “inversely proportional” it is meant that the more CXCL-iFibro and / or FOLR2+macrophages are in the biological sample, the less the subject suffering from a fibroblast associated disease will have a good prognosis. These terms do not necessarily imply a direct and measurable correlation factor.

[0189] As used herein, the term “poor prognosis” refers to a decreased patient survival and / or an early fibroblast associated disease progression and / or a fast / rapid progression.

[0190] Alternatively, a “good prognosis” is preferably a good survival prognosis, a cessation of fibroblast associated disease progression, a slow progression or the absence of early progression, preferably a slow progression.

[0191] In some embodiments, the prognosis methods disclosed herein further comprises an initial step of providing a biological sample from said patient.

[0192] In some embodiments, the prognosis methods disclosed herein further comprises a further step of selecting patient having a poor prognosis as suitable for a treatment of a fibroblast associated disease, in particular such as described below under the section “Treatments”. Additionally, the prognosis methods may comprise a step of administering a therapeutic effective amount of a treatment of a fibroblast associated disease to the patient having a poor prognosis.

[0193] In the prognosis methods of the invention, the detection of CXCL-iFibro and / or FOLR2+macrophages and / or the level(s) of CXCL-iFibro and / or FOLR2+macrophages and their respective reference levels are determined as described above. Fibroblasts associated diseases

[0194] Preferably, as used herein, the terms “fibroblasts associated disease” or “fibrotic diseases” refer to diseases comprising accumulation of fibrous connective tissue (e.g., components of the extracellular matrix (ECM) such as collagen and fibronectin) in and around inflamed or damaged tissue, which can lead to permanent scarring, organ malfunction and, ultimately, death. Chronic inflammatory diseases are typically fibroblasts associated diseases.

[0195] When it comes to “fibroblasts associated disease progression”, this term refers to the subjects whose status had worsened (for example a subject of stage I can progress in stage II or later stages).

[0196] The “risk of fibroblasts associated disease progression” refers to the likelihood or probability that the disease is worsening or progresses to further stages.

[0197] In particular, the fibroblasts associated disease is selected from the group consisting of Chronic Kidney Disease (CKD), systemic sclerosis, kidney graft chronic rejection, myocardial fibrosis, myocardial infarction, rheumatoid arthritis, liver fibrosis, inflammatory bowel diseases (IBDs) and pulmonary fibrosis. Such conditions and diseases have been described in the art as linked to inflammatory fibroblasts or fibroblasts malfunction.

[0198] In some aspects, the fibroblasts associated disease is selected from the group consisting of liver fibrosis (e.g., cirrhosis) heart fibrosis (e.g., post-myocardial infarction fibrosis) lung fibrosis (e.g., idiopathic pulmonary fibrosis) and renal fibrosis (e.g., CKD).

[0199] In some aspects, the fibroblasts associated disease is selected from the group consisting of cirrhosis, post-myocardial infarction fibrosis, idiopathic pulmonary fibrosis and CKD.

[0200] In some aspects, the fibrotic disease is not asthma. In some aspects, the fibrotic disease is not breast cancer or pancreatic cancer, preferably is not a cancer.

[0201] Preferably, the fibroblast associated disease is Chronic Kidney Disease (CKD).

[0202] As used herein, the terms “chronic kidney disease” or “CKD” are used interchangeably and refer to a long-term condition where the kidneys are impaired. It is a gradual and progressive loss of the ability of the kidneys to excrete wastes, concentrate urine, and conserve electrolytes. CKD can be caused by a variety of diseases and disorders such as genetic diseases (including polycystic kidney disease), glomerulonephritis, diabetes, interstitial nephritis, vascular diseases and / or hypertension.

[0203] There are generally five clinical stages of chronic kidney disease, numbered 1 to 5, wherein stage 1 is the least severe and stage 5 the most severe. In the early stages, e.g., stages 1 and 2, dialysis is not required. As chronic kidney disease progresses to stage 5, a patient may require dialysis treatment. Terminal stage of CKD is end-stage renal disease (ESRD) and / or death which generally occurs at or after stage 5.

[0204] Then, when it comes to “CKD progression”, this term refers to the subjects whose CKD status had worsened (for example a subject of CKD stage I can progress in stage II or later stages). Particularly, the CKD progression is a progression of CKD in end-stage renal disease (ESRD).

[0205] The “risk of CKD progression” refers to the likelihood or probability that the chronic kidney disease is worsening or progresses to further CKD stages, in particular to end-stage renal disease (ESRD).

[0206] In some aspects, the fibroblasts associated disease is scleroderma. “Systemic sclerosis” or “scleroderma” is a rare autoimmune disease that is a type of systemic connective tissue disease (systemic sclerosis). Scleroderma is characterized by progressive skin and connective tissue tightening and hardening. It may also affect subcutaneous tissue, muscles, and internal organs.

[0207] There are five stages of scleroderma symptom development and progression. Stage 1 is related to an immune system malfunction that leads to the development of scleroderma-related pathogenic molecules. Stage II is the raise of circulating pathogenic factors such as autoantibodies, Stage III is linked to microvascular endothelial damages, Stage IV is related to fibrosis and Stage V is the most severe and include organ damages that may lead to death.

[0208] “Scleroderma progression” refers to the subjects whose scleroderma status or stage had worsened (for example a subject of scleroderma stage I can progress in stage II or later stages).

[0209] In some aspects, the fibroblasts associated disease is rheumatoid arthritis. “Rheumatoid arthritis” or “RA” is a long-term autoimmune disorder that primarily affects joints. The disease may also affect other parts of the body, including skin, eyes, lungs, heart, kidney, nerves and blood. The first stage is early RA or synovitis. Stage II is related to the raise of pannus synovial, Stage III is fibrous ankylosis and stage IV is bone ankylosis.

[0210] “Rheumatoid arthritis progression” refers to the subjects whose RA status or stage had worsened (for example a subject of RA stage I can progress in stage II or later stages).

[0211] In some aspects, the fibroblasts associated disease is IBD. “Inflammatory bowel disease” or “IBD” includes two conditions, Crohn's disease and ulcerative colitis, that are characterized by chronic inflammation of the gastrointestinal (GI) tract and intestinal fibrosis is common in IBD patients, resulting in several complications that require surgery, such as a stricture or penetration.

[0212] In some aspects, the fibroblasts associated disease is pulmonary fibrosis, preferably idiopathic pulmonary fibrosis. “Pulmonary fibrosis” or “lung fibrosis” is a condition in which the lungs become scarred over time. Symptoms include shortness of breath, a dry cough, feeling tired, weight loss, and nail clubbing. Complications may include pulmonary hypertension, respiratory failure, pneumothorax, and lung cancer. Pulmonary fibrosis particularly includes idiopathic pulmonary fibrosis.

[0213] In some aspects, the fibroblasts associated disease is hepatic fibrosis, preferably cirrhosis. “Hepatic fibrosis" or “liver fibrosis” is an excessive scarring resulting from the accumulation of connective tissue in the liver. The extracellular matrix is produced in excess and / or insufficiently degraded. Hepatic fibrosis can progress to portal hypertension and / or cirrhosis.

[0214] In some aspects, the fibroblasts associated disease is myocardial fibrosis, preferably myocardial infraction . “Myocardial fibrosis”, “heart fibrosis” or “cardiac fibrosis” is a fibroblast associated disease comprising an increased quantity of collagenous scar tissue in the heart. Three types of myocardial fibrosis have been identified: i) reactive interstitial fibrosis, ii) infiltrative interstitial fibrosis and iii) replacement fibrosis. Myocardial fibrosis may be responsible of endstage heart failure.

[0215] In some aspects, the fibroblasts associated disease is CKTR. “Chronic kidney transplant rejection” or “CKTR” refers to graft failure and rejection beyond 1-year post-transplant, in the absence of acute rejection, drug toxicity and other causes of nephropathy. Progressive stages of kidney transplant rejection can manifest as signs of kidney failure and uremia, including oliguria, nausea, vomiting, a metallic taste, pericardial friction rub, and asterixis.

[0216] Agents targeting CXCL-iFibro and / or FOLR2+ macrophages and uses thereof The invention also concerns an agent targeting CXCL-iFibro and / or FOLR2+macrophages or a pharmaceutical composition comprising said agent.

[0217] As used herein, a “pharmaceutical composition” refers to a preparation of one or more of the active agents, with optional other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism. Compositions of the present invention can be in a form suitable for any conventional route of administration or use. The pharmaceutical composition can be sterilized. It will be understood by one skilled in the art that the formulations of the invention may be isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-freezing type osmometer. In one embodiment, a “pharmaceutical composition” typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally- occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. An “acceptable vehicle” or “acceptable carrier” as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0218] The pharmaceutical composition envisioned herein particularly comprises a therapeutic effective amount of an agent targeting CXCL-iFibro and / or FOLR2+macrophages, and optionally, a pharmaceutical acceptable carrier.

[0219] “An effective amount” or a “therapeutic effective amount” as used herein refers to the amount of active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g., the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The “effective amount” will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

[0220] Preferably, the agent targeting CXCL-iFibro and / or FOLR2+macrophages is a cell depleting agent targeting CXCL-iFibro and / or FOLR2+macrophages or is an inhibitor of CXCL- iFibro and FOLR2+macrophages interaction.

[0221] As used herein, a “cell depleting agent” can be any molecule able to kill or suppress CXCL- iFibro and / or FOLR2+macrophages, such as a peptide, an antibody (e.g., an antibody-drug conjugate or an antibody inducing ADCC), a small molecule, an antisense, a siRNA or shRNA, an aptamer specific to CXCL-iFibro and / or FOLR2+macrophages and having an antagonist activity.

[0222] As used herein, an “inhibitor of CXCL-iFibro and FOLR2+macrophages interaction” refers to any molecule that is able to inhibit, block, disrupt or prevent the interaction or contact between CXCL- iFibro and FOLR2+macrophages, such as a peptide, an antibody or a small molecule. In particular, the inhibitor of CXCL-iFibro and FOLR2+macrophages interaction is able to inhibit the ligand-receptor interaction CXCL12-CXCR4, ANGPTL1-LILRB3 and / or THBS-CD36 that are involved in CXCL-iFibro and FOLR2+macrophages interaction.

[0223] For example, the cell depleting agent targeting FOLR2+macrophages can be EC2319 or aminopterin and / or methotrexate.

[0224] For example, the inhibitor of CXCL-iFibro and FOLR2+macrophages interaction can be Plerixafor.

[0225] In some embodiments, the agent targeting CXCL-iFibro and / or FOLR2+macrophages can be used in combination with another treatment suitable against a fibroblast associated disease, such as described below.

[0226] Treatments

[0227] The invention also concerns an agent targeting CXCL-iFibro and / or FOLR2+macrophages or a pharmaceutical composition comprising said agent, preferably for use as a medicament, even more preferably for use in the treatment of a fibrotic disease disclosed herein. Preferably, said agent is a cell depleting agent targeting CXCL-iFibro or is an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction.

[0228] The invention also relates to an agent targeting CXCL-iFibro and / or FOLR2+macrophages or a pharmaceutical composition comprising said agent for use in the treatment of a fibroblast associated disease.

[0229] The invention further relates to an agent targeting CXCL-iFibro and / or FOLR2+macrophages or a pharmaceutical composition comprising said agent in the treatment of a subject suffering from a fibroblast associated disease such as CKD, in particular a subject having CXCL- iFibro and / or FOLR2+macrophages in a biological sample, preferably a kidney, blood or urine sample.

[0230] The invention also concerns the use of an agent targeting CXCL-iFibro and / or FOLR2+macrophages or a pharmaceutical composition comprising said agent in the manufacture of a medicament for the treatment of a fibroblast associated disease. It also relates to the use of an agent targeting CXCL-iFibro and / or FOLR2+macrophages or of a pharmaceutical composition comprising said agent for treating a fibroblast associated disease in a subject in need thereof. Finally, it relates to a method for treating a subject suffering from a fibroblast associated disease, preferably said subject having CXCL-iFibro and / or FOLR2+macrophages in a biological sample, comprising administering a therapeutically effective amount of an agent targeting CXCL-iFibro and / or FOLR2+macrophages or of a pharmaceutical composition comprising said agent.

[0231] The invention also refers to a method for selecting a subject suffering from a fibroblast associated disease for a treatment with an agent targeting CXCL-iFibro and / or FOLR2+macrophages or a pharmaceutical composition comprising such, comprising:

[0232] (a) determining the level(s) of CXCL-iFibro and / or FOLR2+macrophages in a biological sample from the subject;

[0233] (b) selecting the subject :

[0234] - with high level(s) of CXCL-iFibro and / or FOLR2+macrophages; or

[0235] - with a level of CXCL-iFibro and / or FOLR2+macrophages higher than a reference level; as suitable for a treatment with an agent targeting CXCL-iFibro and / or FOLR2+macrophages.

[0236] Preferably, the method comprises:

[0237] (a) determining the level(s) of CXCL-iFibro and optionally FOLR2+macrophages, in a biological sample from the subject;

[0238] (b) selecting the subject :

[0239] - with high level(s) of CXCL-iFibro and optionally FOLR2+macrophages; or

[0240] - with a level of CXCL-iFibro and optionally FOLR2+macrophages higher than a reference level; as suitable for a treatment with an agent targeting CXCL-iFibro and optionnally FOLR2+macrophages, preferably a cell depleting agent targeting CXCL-iFibro or an inhibitor of CXCL- iFibro and CD68+CD206+FOLR2+macrophages interaction.

[0241] In particular, the determination of the presence or of the level(s) of CXCL-iFibro and FOLR2+macrophages can be determined by any methods disclosed herein.

[0242] Such method may comprise an initial step of providing a biological sample from the subject. Additionally or alternatively, the method may comprise a step of detecting CXCL-iFibro and / or FOLR2+macrophages.

[0243] The invention also concerns an inhibitor of P-catenin or of P-catenin / TCF interaction or a pharmaceutical composition comprising said inhibitor, for use in the treatment of a fibroblast associated disease such as CKD in a subject.

[0244] The invention also concerns an inhibitor of P-catenin or of P-catenin / TCF interaction or a pharmaceutical composition comprising said inhibitor, for use in the treatment of a fibroblast associated disease such as CKD in a subject, wherein the subject has SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample, preferably a kidney, blood or urine sample.

[0245] The invention also concerns the use of an inhibitor of P-catenin or of P-catenin / TCF interaction or of a pharmaceutical composition comprising said inhibitor in the manufacture of a medicament for the treatment of a subject suffering from a fibroblast associated disease such as CKD and having CXCL-iFibro and / or FOLR2+macrophages in a biological sample, preferably a kidney, blood or urine sample. It also relates to the use of an inhibitor of P-catenin or of P- catenin / TCF interaction or of a pharmaceutical composition comprising said inhibitor for treating a fibroblast associated disease such as CKD in a subject in need thereof. Finally, it relates to a method for treating a fibroblast associated disease such as CKD in a subject having CXCL-iFibro and / or FOLR2+macrophages in a biological sample, comprising administering a therapeutically effective amount of an inhibitor of P-catenin or of P-catenin / TCF interaction or of a pharmaceutical composition comprising such.

[0246] Examples of P-catenin inhibitor or of P-catenin / TCF interaction inhibitor are well described in the art and includes but are not limited to iCRT3 (CAS No. 901751-47-1), Dickkopfl (DKK1), Porcupine inhibitor (PCN), Wnt-C59 (CAS No. 1243243-89-1), Triptonide (CAS# 38647-11-9), Tegatrabetan (CAS# 1227637-23-1), Esculetin (CAS# 305-01-1), CWP232228 (CAS# 1144044- 02-9), Agelastatin A (CAS# 152406-28-5) secreted frizzled protein 1 (Sfrpl), Wnt inhibitor 1 (Wif-1) and Klotho.

[0247] In some aspects, the inhibitor of P-catenin or of P-catenin / TCF interaction can be used in combination with a cell depleting agent targeting CXCL-iFibro or an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction such as described herein.

[0248] In some aspects, the inhibitor of P-catenin or of P-catenin / TCF interaction can be used in combination with another treatment against fibroblast associated diseases such as CKD, such as described below.

[0249] In some aspects the invention concerns an in vitro method for selecting a subject as suitable for a treatment against a fibroblasts associated disease, wherein the method comprises:

[0250] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0251] (b) selecting the subject as suitable for a treatment against said fibroblasts associated disease if the biological sample comprises high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages in the biological sample, preferably level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages higher than their reference level(s). Preferably, the treatment against a fibroblasts associated disease is selected from the group consisting of a cell depleting agent targeting CXCL-iFibro, an inhibitor of CXCL-iFibro and FOLR2+macrophages interaction, an inhibitor of P-catenin and an inhibitor of P-catenin / TCF interaction.

[0252] The invention also refers to a method for selecting a subject suffering from a fibroblast associated disease such as CKD for a treatment with an inhibitor of P-catenin or of P-catenin / TCF interaction, comprising:

[0253] (a) determining the level(s) of SFRP1+SFRP4'FAP'RAMPF fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0254] (b) selecting subjects

[0255] - with high level(s) CXCL-iFibro and / or FOLR2+macrophages; or

[0256] - with a level of CXCL-iFibro and / or FOLR2+macrophages higher than a reference level; as suitable for a treatment with an inhibitor of P-catenin or of P-catenin / TCF interaction.

[0257] In particular, the determination of the presence or of the level(s) of CXCL-iFibro and FOLR2+macrophages can be determined by any methods disclosed herein.

[0258] Such method may comprise an initial step of providing a biological sample from the subject. Additionally or alternatively, the method may comprise a step of detecting CXCL-iFibro and / or FOLR2+macrophages.

[0259] Alternatively or additionally to i) the inhibitor of P-catenin or of P-catenin / TCF interaction and / or ii) the cell depleting agent targeting CXCL-iFibro or inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction, treatment described in the art as suitable for fibrotic disease may be used.

[0260] The man skilled in the art is perfectly able to select a suitable treatment depending of the fibrotic disease or organ to treat.

[0261] The treatment against fibroblasts associated diseases can particularly be selected from the group consisting of: a) if the fibroblasts associated disease is pulmonary fibrosis, the treatment against pulmonary fibrosis is preferably corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; b) if the fibroblasts associated disease is systemic sclerosis, the treatment against systemic sclerosis is preferably methotrexate, mycophenolate mofetil and / or intravenous cyclophosphamide; c) if the fibroblasts associated disease is myocardial infarction, the treatment against myocardial infarction disease is preferably beta-blockers, renin-angiotensin blockers and / or statins; d) if the fibroblasts associated disease is rheumatoid arthritis, the treatment against rheumatoid arthritis disease is preferably corticosteroids, methotrexate and / or an anti-TNFa treatment; e) if the fibroblasts associated disease is kidney graft chronic rejection, the treatment against kidney graft chronic rejection is preferably i) a treatment for limiting kidney graft chronic rejection, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or antihypertensive drugs, ii) a treatment for compensating the effect of kidney graft chronic rejection, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) dialysis; f) if the fibroblasts associated disease is CKD, the treatment against CKD is preferably i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation; or g) if the fibroblasts associated disease is inflammatory bowel diseases (IBDs), the treatment against IBD is preferably anti-inflammatory drugs such as aminosalicylates, immunosuppressant drugs such as azathioprine, mercaptopurine and methotrexate and / or antibiotics.

[0262] In some embodiments, if the disease is cirrhosis the treatment against cirrhosis is preferably diuretics, anti-aldosterone drugs, beta blockers and / or liver transplant. In some specific cases, resmetirom, Aramchol, Lanifibranor and / or Belapectin, can be used, alone or in combination with any other treatment against cirrhosis mentioned herein.

[0263] The treatment against fibroblasts associated diseases can particularly be selected from the group consisting of: a) if the fibroblasts associated disease is pulmonary fibrosis, the treatment against pulmonary fibrosis is preferably corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; b) if the fibroblasts associated disease is myocardial infarction, the treatment against myocardial infarction disease is preferably beta-blockers, renin-angiotensin blockers and / or statins; c) if the fibroblasts associated disease is CKD, the treatment against CKD is preferably i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation; d) if the disease is cirrhosis, the treatment against cirrhosis is preferably diuretics, antialdosterone drugs, beta blockers and / or liver transplant. In some specific cases, resmetirom, Aramchol, Lanifibranor and / or Belapectin, can be used, alone or in combination with any other treatment against cirrhosis mentioned herein.

[0264] The invention also refers to a method of treatment of a patient suffering from a fibroblast associated disease, comprising:

[0265] (a) determining the level(s) of SFRP1+SFRP4TAP'RAMPT fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0266] (b) selecting subjects

[0267] - with high level(s) CXCL-iFibro and / or F0LR2+macrophages; or

[0268] - with a level of CXCL-iFibro and / or F0LR2+macrophages higher than a reference level;

[0269] (c) administering a therapeutic effective amount of a fibroblast associated disease treatment to the selected subjects.

[0270] In some aspects, the invention concerns a method for treating a fibroblasts associated disease in a subject in need thereof, wherein the method comprises:

[0271] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject,

[0272] (b) selecting the subject, preferably as suitable for a treatment against said fibroblasts associated disease, if the biological sample comprises:

[0273] - high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages; or

[0274] - level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages higher than a reference level; and c) administering a therapeutic effective amount of the treatment against said fibroblasts associated disease to the selected subject.

[0275] It also refers to a method of treatment of a patient suffering from CKD, comprising:

[0276] (a) determining the level(s) of SFR.P I SFR.P4 FAP R.AMP F fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0277] (b) selecting subjects

[0278] - with high level(s) CXCL-iFibro and / or F0LR2+macrophages; or

[0279] - with a level of CXCL-iFibro and / or F0LR2+macrophages higher than a reference level; (c) administering a therapeutic effective amount of a CKD treatment to the selected subjects.

[0280] It also refers to a method of treating a subject suffering from CKD, comprising:

[0281] (a) determining the level(s) of SFRPl+SFRP4TAP'RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0282] (b) selecting the subject if the biological sample comprises:

[0283] - high level(s) CXCL-iFibro and optionally FOLR2+macrophages; or

[0284] - a level of CXCL-iFibro and optionally FOLR2+macrophages higher than a reference level;

[0285] (c) administering a therapeutic effective amount of a CKD treatment to the selected subject.

[0286] Such method may comprise an initial step of providing a biological sample from the subject. Additionally or alternatively, the method may comprise a step of detecting CXCL-iFibro and / or FOLR2+macrophages.

[0287] In particular, the determination of the presence or of the level(s) of CXCL-iFibro and FOLR2+macrophages can be determined by any methods disclosed herein.

[0288] The invention refers to a method of treatment of a patient suffering from a fibroblast associated disease, comprising:

[0289] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0290] (b) selecting subjects

[0291] - with high level(s) CXCL-iFibro and / or FOLR2+macrophages; or

[0292] - with a level of CXCL-iFibro and / or FOLR2+macrophages higher than a reference level;

[0293] (c) administering a therapeutic effective amount of an agent targeting CXCL-iFibro and / or FOLR2+macrophages or a pharmaceutical composition comprising such to the selected patient, in particular in combination with a treatment suitable for treating said fibroblast associated disease.

[0294] In some aspects the invention concerns an in vitro method for selecting a subject as suitable for a treatment against a fibroblasts associated disease, wherein the method comprises:

[0295] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0296] (b) selecting the subject as suitable for a treatment against said fibroblasts associated disease if the biological sample comprises high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages in the biological sample, preferably level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages higher than their reference level(s).

[0297] Preferably, in such method: a) the fibroblasts associated disease is CKD and the treatment against CKD is i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation b) the fibroblasts associated disease is pulmonary fibrosis and the treatment against pulmonary fibrosis is corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; c) the fibroblasts associated disease is systemic sclerosis and the treatment against systemic sclerosis is methotrexate, mycophenolate mofetil and / or intravenous cyclophosphamide; d) the fibroblasts associated disease is rheumatoid arthritis and the treatment against rheumatoid arthritis disease is corticosteroids, methotrexate and / or an anti-TNFa treatment; e) the fibroblasts associated disease is kidney graft chronic rejection and the treatment against kidney graft chronic rejection is i) a treatment for limiting kidney graft chronic rejection, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of kidney graft chronic rejection, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) dialysis; or f) the fibroblasts associated disease is myocardial infarction and the treatment against myocardial infarction disease is beta-blockers, renin-angiotensin blockers and / or statins; g) the fibroblasts associated disease is inflammatory bowel diseases (IBDs) and the treatment against IBD is selected from anti-inflammatory drugs such as aminosalicylates; immunosuppressant drugs such as azathioprine, mercaptopurine and methotrexate and / or antibiotics.

[0298] The invention also concerns a method for treating a fibroblast associated disease in a subject in need thereof, comprising the determination of the prognosis of a subject, in particular by a prognosis method as described above, and a step of administering a therapeutically effective amount of a fibroblast associated disease treatment in subjects that have a poor prognosis, in particular treatments such as described here above.

[0299] In some preferred aspects, wherein the disease is CKD, the treatment against CKD can particularly be selected from the group consisting of i) a treatment for limiting CKD progression, preferably renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD preferably erythropoietin, vitamin D3, calcium and / or phosphate binders; iii) renal replacement therapy, preferably dialysis or kidney transplantation. The treatment against CKD can particularly be an agent targeting CXCL-iFibro and / or FOLR2+macrophages such as a cell depleting agent targeting CXCL-iFibro and / or FOLR2+macrophages or is an inhibitor of CXCL-iFibro and FOLR2+macrophages interaction, such as described above.

[0300] The invention thus also refers to a method of treatment of a patient suffering from CKD, comprising:

[0301] (a) determining the level(s) of SFRPl+SFRP4'FAP'RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0302] (b) selecting subjects

[0303] - with high level(s) CXCL-iFibro and / or FOLR2+macrophages; or

[0304] - with a level of CXCL-iFibro and / or FOLR2+macrophages higher than a reference level;

[0305] (c) administering a therapeutic effective amount of an agent targeting CXCL-iFibro and / or FOLR2+macrophages, a cell depleting agent targeting CXCL-iFibro or an inhibitor of CXCL- iFibro and FOLR2+macrophages interaction or a pharmaceutical composition comprising such to the selected patient.

[0306] The treatment against CKD can also be an inhibitor of P-catenin or of P-catenin / TCF interaction such as described above. Additionally or alternatively, the treatment against CKD can also be i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation.

[0307] The invention thus also refers to a method of treatment of a patient suffering from CKD, comprising:

[0308] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject;

[0309] (b) selecting subjects

[0310] - with high level(s) CXCL-iFibro and / or FOLR2+macrophages; or

[0311] - with a level of CXCL-iFibro and / or FOLR2+macrophages higher than a reference level;

[0312] (c) administering a therapeutic effective amount of an inhibitor of P-catenin or of P- catenin / TCF interaction or a pharmaceutical composition comprising such to the selected patient.

[0313] The invention thus also refers to a method for treating a fibroblasts associated disease in a subject in need thereof, wherein the method comprises:

[0314] (a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject, wherein the risk of said subject to have fibroblasts associated disease is proportional to the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample;

[0315] (b) selecting subjects with high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages as suitable for a treatment against said fibroblasts associated disease; and c) administering a therapeutic effective amount of the treatment against said fibroblasts associated disease.

[0316] Such method may comprise an initial step of providing a biological sample from the subject.

[0317] Preferably, in such method: a) the fibroblasts associated disease is CKD and the treatment against CKD is i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation b) the fibroblasts associated disease is pulmonary fibrosis and the treatment against pulmonary fibrosis is corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; c) the fibroblasts associated disease is systemic sclerosis and the treatment against systemic sclerosis is methotrexate, mycophenolate mofetil and / or intravenous cyclophosphamide; d) the fibroblasts associated disease is rheumatoid arthritis and the treatment against rheumatoid arthritis disease is corticosteroids, methotrexate and / or an anti-TNFa treatment; e) the fibroblasts associated disease is kidney graft chronic rejection and the treatment against kidney graft chronic rejection is i) a treatment for limiting kidney graft chronic rejection, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of kidney graft chronic rejection, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) dialysis; or f) the fibroblasts associated disease is myocardial infarction and the treatment against myocardial infarction disease is beta-blockers, renin-angiotensin blockers and / or statins; g) the fibroblasts associated disease is inflammatory bowel diseases (IBDs) and the treatment against IBD is selected from anti-inflammatory drugs such as aminosalicylates; immunosuppressant drugs such as azathioprine, mercaptopurine and methotrexate and / or antibiotics; h) if the disease is cirrhosis the treatment against cirrhosis is diuretics, anti-aldosterone drugs, beta blockers and / or liver transplant. The invention also concerns a method for treating CKD in a subject in need thereof, comprising the determination of the prognosis of a subject, in particular by a prognosis method as described above, and a step of administering a therapeutically effective amount of a CKD treatment in subjects that have a poor prognosis. The treatment against CKD can particularly be selected from the group consisting of i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of CKD preferably erythropoietin, vitamin D3, calcium and / or phosphate binders, iii) renal replacement therapy, preferably dialysis or kidney transplantation; iv) an agent targeting CXCL-iFibro and / or FOLR2+macrophages such as a cell depleting agent targeting CXCL-iFibro and / or FOLR2+macrophages ; v) an inhibitor of CXCL- iFibro and FOLR2+macrophages interaction, or vi) an inhibitor of P-catenin or of P-catenin / TCF interaction; such as described herein.

[0318] In any of the methods described herein, the amount of treatment according to the invention or of the pharmaceutical composition according to the invention administered or to be administered can be determined by standard procedure well known by those of ordinary skills in the art. Physiological data of the patient (e.g. age, size, and weight) and the routes of administration have to be taken into account to determine the appropriate dosage, so as a therapeutically effective amount will be administered to the patient.

[0319] The treatment may be administered as a single dose or in multiple doses.

[0320] In some embodiments, the agent targeting CXCL-iFibro and / or FOLR2+macrophages or the inhibitor of P-catenin or of P-catenin / TCF interaction can be administered simultaneously, sequentially or separately with another treatment described herein.

[0321] The suitable treatment for fibrotic diseases envisioned herein can be for example:

[0322] - a reference treatment of a specific fibrotic disease (e.g., as disclosed herein) and agent targeting CXCL-iFibro and / or FOLR2+ macrophages (e.g., a cell depleting agent targeting CXCL-iFibro or an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction);

[0323] - a reference treatment of a specific fibrotic disease (e.g., as disclosed herein) and an inhibitor of P- catenin or of P-catenin / TCF interaction;

[0324] - a reference treatment of a specific fibrotic disease (e.g., as disclosed herein), agent targeting CXCL-iFibro and / or FOLR2+ macrophages (e.g., a cell depleting agent targeting CXCL-iFibro or an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction) and an inhibitor of P-catenin or of P-catenin / TCF interaction; or

[0325] - an agent targeting CXCL-iFibro and / or FOLR2+ macrophages (e.g., a cell depleting agent targeting CXCL-iFibro or an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+ macrophages interaction) and an inhibitor of P-catenin or of P-catenin / TCF interaction.

[0326] As used herein, the term “simultaneous” refers to a pharmaceutical composition, a kit, a product or a combined preparation according to the invention in which the active ingredients are used or administered simultaneously, i.e. at the same time.

[0327] As used herein, the term “sequential” refers to a pharmaceutical composition, a kit, a product or a combined preparation according to the invention in which the active ingredients are used or administered sequentially, i.e. one after the other. Preferably, when the administration is sequential, all the active ingredients are administered in less than about an hour, preferably less than about 10 minutes, even more preferably in less than about a minute.

[0328] As used herein, the term “separate” refers to a pharmaceutical composition, a kit, a product or a combined preparation according to the invention in which the active ingredients are used or administered at distinct time of the day. Preferably, when the administration is separate, the active ingredients are administered with an interval of about 1 hour to about 24 hours, preferably with an interval of about 1 hour and 15 hours, more preferably with an interval of about 1 hour and 8 hours, even more preferably with an interval of about 1 hour and 4 hours.

[0329] Preferably, the treatment is administered regularly, preferably between every day and every month, more preferably between every day and every two weeks, even more preferably between every day and every week.

[0330] The duration of treatment is preferably comprised between 1 day and 24 weeks, more preferably between 1 day and 10 weeks, even more preferably between 1 day and 4 weeks. In a particular embodiment, the treatment last as long as the fibroblast associated disease persists.

[0331] Patient, regimen and administration

[0332] The invention particularly envisions the selection, prognosis, diagnosis and / or treatment of a subject suffering from a fibroblast associated disease such as CKD.

[0333] As used herein, the terms “subject”, “individual” or “patient” are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human. However, the term "subject" can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others.

[0334] The patient is an animal, preferably a mammal, even more preferably a human. However, the patient can also be a non-human animal, in particular mammals such as dogs, cats, horses, cows, pigs, sheep, donkeys, rabbits, ferrets, gerbils, hamsters, chinchillas, rats, mice, guinea pigs and non-human primates, among others, that are in need of treatment. The human patient according to the invention may be a human at the prenatal stage, a newborn, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old or at least 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old, even more preferably an adult of at least 70 years old.

[0335] In some embodiments, the subject has diabetes, overweight, hypertension, glomerulonephritis, polycystic kidney disease, interstitial nephritis a cardiovascular disease and / or is a smoker.

[0336] In some embodiments, the subject has a family history of kidney, lung, liver, gut and / or heart disease.

[0337] In some embodiments, the subject has a family history of kidney disease.

[0338] In some embodiments, the cause of the fibroblast associated disease, in particular CKD, in the subject is not known or has not been determined.

[0339] In some embodiments, the subject has already been diagnosed for fibroblast associated disease by routine examination. CKD can be for example diagnosed using blood or urine tests such as eGFR (estimated glomerular filtration rate).

[0340] In some embodiment, the subject already received a treatment against the fibroblast associated disease, in particular a treatment as disclosed herein for each different fibroblast associated disease . Preferably, the disease is CKD and the treatment against CKD is selected from the group consisting i) a treatment for limiting CKD progression preferably Renin- angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of CKD such as administration of erythropoietin, vitamin D3, calcium and / or phosphate binders and iii) renal replacement therapy (in particular, dialysis or kidney transplantation).

[0341] The amount of treatment, in particular the inhibitor of P-catenin or the inhibitor of P- catenin / TCF interaction or the treatment comprising an agent targeting CXCL-iFibro and / or FOLR2+macrophages, to be administered to the subject is determined by standard procedure well known by those of ordinary skills in the art. Physiological data of the patient (e.g. age, size, weight, and physical general condition) and the routes of administration are taken into account to determine the appropriate dosage, so as a therapeutically effective amount will be administered to the patient.

[0342] In the context of a combination of active ingredients, the active ingredients can be administered to the subject by the same or different routes of administration. Administration routes usually depend on the pharmaceutical compositions used. The methods and uses according to the invention may comprise a step of characterizing a biological sample from a patient. Therefore, the methods according to the invention may comprise an initial step of providing samples from the patient. The sample can, for example, be obtained from a subject by, but not limited to, venipuncture, excretion, biopsy, needle aspirate, lavage sample, scraping, surgical incision, colonoscopy, fibroscopy, endoscopy, surgery or, any combination thereof, and the like.

[0343] The term "biological sample" refers to a sample obtained from a subject or from components (e.g., cells) of a subject. The sample may be of any biological tissue or fluid. The sample may be a "clinical sample" which is a sample derived from a patient. Biological samples may also include sections of tissues such as frozen sections or Formalin-fixed paraffin embedded section (FFPE) section taken for histological purposes. A “biological sample” may also be referred herein to as a "patient’s sample."

[0344] Preferably, the biological sample is a kidney sample, a skin sample, a lung sample, a heart sample, a synovial liquid sample, a gut sample, a liver sample, a blood sample or a urine sample. It will be easily understood by the man skilled in the art that the type of biological sample varies upon the type of fibroblasts associated disease. For example, when the fibroblasts associated disease is CKD, the biological sample is preferably a kidney sample, a urine sample or a blood sample.

[0345] In some embodiments, the biological sample is compared to a normal sample, in particular for determining the expression level of CXCL-iFibro and optionally FOLR2+macrophages. The normal sample can be a sample from the subject before he developed a fibroblast associated disease or from another subject or group of subjects that is healthy (i.e., does not have a fibroblast associated disease). Preferably, the biological sample and the normal sample both provide from the same type of tissue (e.g., both are urine samples or both are kidney samples).

[0346] Kit

[0347] Finally, the present invention provides a kit useful for carrying out the methods disclosed herein, the kit comprising one or several mean(s) capable of detecting CXCL-iFibro fibroblasts and / or one or several mean(s) capable of detecting FOLR2+macrophages.

[0348] Preferably, the present invention provides a kit useful for carrying out the methods disclosed herein, the kit comprising one or several mean(s) capable of detecting CXCL-iFibro fibroblasts and optionally one or several mean(s) capable of detecting FOLR2+ macrophages. As described above, the presence of CXCL-iFibro fibroblasts and / or FOLR2+macrophages can be detected at the nucleic acid level, in particular mRNA, or at the protein level. For example, such mean(s) may be probe(s), primer(s), antibody(ies) and / or aptamer(s), in particular such as described hereabove. Preferably, the means are antibodies, typically antibodies suitable for FACS or immunohistochemistry experiments. The kit preferably comprises at least one antibody against at least one of their respective markers (i.e., SFRP1, SFRP4, FAP and / or RAMP1 and optionally CXCL12 and / or aSMA for CXCL-iFibro; and CD68, CD206 and / or FOLR2 for FOLR2+macrophages, respectively), preferably as described herein.

[0349] Additionally, or alternatively, the kit comprises means, in particular nucleic acid(s), probe(s) or primer(s) targeting at least one gene of the gene signature of CXCL-iFibro. More particular, it comprises means for detecting all the genes of the CXCL-iFibro gene signature.

[0350] The content of the kits may vary based on the method utilized. The man skilled in the art easily knows the means necessary for designing and assessing such methods. For example, when PCR is the method for determining the expression level of the biomarkers of the gene signature, the kit may include primers which facilitate amplification of a marker gene.

[0351] The kit may be a diagnostic kit. The components of the kit may be packaged either in aqueous media or in lyophilized form. The kit may include container means such as at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.

[0352] In some embodiments, means of taking a sample from a subject and / or of assaying the sample may be provided. For example, the kit may comprise a container for collecting urine.

[0353] The kit may also comprise means for containing a sterile, pharmaceutically acceptable buffer(s) and / or other diluent(s). Optionally, a leaflet is provided for guidelines to use such a kit.

[0354] The invention also concerns the use of a kit in the diagnosis of a fibroblast associated disease, wherein said kit comprises at least one antibody targeting SFRPl+SFRP4'FAP'RAMPr fibroblasts (CXCL-iFibro) and optionally at least one antibody targeting CD68+CD206+FOLR2+macrophages.

[0355] The invention finally concerns the use of a kit as disclosed above for: - detecting CXCL-iFibro and / or FOLR2+macrophages in particular in a biological sample from a subject suffering from a fibroblast associated disease such as CKD;

[0356] - determining the level(s) of CXCL-iFibro and / or FOLR2+macrophages in particular in a biological sample from a subject suffering from a fibroblast associated disease such as CKD;

[0357] - predicting the clinical outcome of a subject suffering from a fibroblast associated disease such as CKD;

[0358] - determining the risk / likelihood of a patient to have a fibroblast associated disease progression; and / or

[0359] - selecting or not a patient having a risk of a fibroblast associated disease progression, in particular for a treatment suitable for said fibroblast associated disease, in particular with an agent targeting CXCL-iFibro and / or FOLR2+macrophages or with an inhibitor of P-catenin or of P- catenin / TCF interaction such as described herein

[0360] In particular, the disclosure relates to an in vitro method for detecting if a subject is at risk of a fibroblasts associated disease progression wherein the method comprises detecting SFRP1+SFRP4 FAP RAMP1- fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from said subject; the presence of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages being indicative of disease progression and optionally, the absence of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages being indicative of the absence of disease progression.

[0361] The disclosure concerns an in vitro method for selecting a subject at risk of a fibroblasts associated disease, wherein the method comprises:

[0362] (a) determining the level(s) of SFRP1+SFRP4 FAP RAMPL fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample from the subject, wherein the risk of said subject to have fibroblasts associated disease is proportional to the level(s) of CXCL- iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample;

[0363] (b) optionally, selecting subjects with high level(s) CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages as suitable for a treatment against said fibroblasts associated disease.

[0364] The disclosure concerns an in vitro method for predicting the clinical outcome of a subject affected with a fibroblasts associated disease, wherein the method comprises:

[0365] (a) determining level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in a biological sample from said subject, wherein high level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages are predictive of a poor prognosis and optionally, wherein low level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages are predictive of a good prognosis.

[0366] In particular, a poor prognosis is a poor survival prognosis or an early or fast disease progression and a good prognosis is a good survival prognosis, a cessation of disease progression, a slow disease progression or the absence of early disease progression, preferably a slow disease progression.

[0367] In particular, the method further comprises:

[0368] (b) comparing the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages higher than their reference level(s) are predictive of a poor prognosis and optionally, level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages lower than or equal to their reference level(s) are predictive of a good prognosis.

[0369] In particular, the CXCL-iFibro have a gene signature comprising, consisting essentially of or consisting of TMEM176A, CTGF, VCAM1, FM03, C2orf40, CTSS, DPEP1, SRGN, NPY1R, HGF, G0S2, ALDH7A1, BIRC3, SDC4, CYB5A, TYMP, FOSB, SLC43A2, SLC2A3, ZFP36, FILIP1L, VMP1, RFTN1, FOS, CNN2, EDNRB, SERPINB9, GGT5, CCL21, NEGRI, MYL12A, THBS1, CCL19, CRISPLD2, DUSP6, JUN, HM0X2, FBLN5, ODF3B, SFRP1, ABCA8, MY ADM, TMEM176B, CYR61, CCBE1, CXCL12, MYO10, EMIDI, CCL2, GFRA1, TSPAN4, C7, GRAMD2B, CEBPD, RND3, IL34, STON1, ANGPTL1, PCSK7, CFHR1, NIDI, PTGER1, SELENOP, COLECI 1 and DNAJB4.

[0370] In the methods of the disclosure, the level of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages is preferably a percentage of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample, respectively.

[0371] The fibroblasts associated disease envisioned herein preferably is selected from the group consisting of Chronic Kidney Disease (CKD), systemic sclerosis, kidney graft chronic rejection, myocardial fibrosis, myocardial infarction, rheumatoid arthritis, liver fibrosis, inflammatory bowel diseases (IBDs) and pulmonary fibrosis, preferably is CKD.

[0372] In a particular: a) the fibroblasts associated disease is CKD and the treatment against CKD is i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation b) the fibroblasts associated disease is pulmonary fibrosis and the treatment against pulmonary fibrosis is corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; c) the fibroblasts associated disease is systemic sclerosis and the treatment against systemic sclerosis is methotrexate, mycophenolate mofetil and / or intravenous cyclophosphamide; d) the fibroblasts associated disease is rheumatoid arthritis and the treatment against rheumatoid arthritis disease is corticosteroids, methotrexate and / or an anti-TNFa treatment; e) the fibroblasts associated disease is kidney graft chronic rejection and the treatment against kidney graft chronic rejection is i) a treatment for limiting kidney graft chronic rejection, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of kidney graft chronic rejection, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) dialysis; or f) the fibroblasts associated disease is myocardial infarction and the treatment against myocardial infarction disease is beta-blockers, renin-angiotensin blockers and / or statins; g) the fibroblasts associated disease is inflammatory bowel diseases (IBDs) and the treatment against IBD is selected from anti-inflammatory drugs such as aminosalicylates; immunosuppressant drugs such as azathioprine, mercaptopurine and methotrexate and / or antibiotics.

[0373] In the methods of the disclosure, the biological sample is preferably a kidney sample, a skin sample, a lung sample, a heart sample, a synovial liquid sample, a gut sample, a liver sample, a blood sample or a urine sample.

[0374] The disclosure further concerns the use of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages as biomarker(s) of a fibroblasts associated disease progression or for predicting the clinical outcome of a subject suffering from a fibroblasts associated disease.

[0375] The disclosure further concerns an agent targeting SFRP1+SFRP4'FAP'RAMPF fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages for use in the treatment of a fibroblasts associated disease, wherein said agent is a cell depleting agent targeting CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages or is an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction.

[0376] The disclosure further concerns an inhibitor of P-catenin or of P-catenin / TCF interaction for use in the treatment of a fibroblasts associated disease in a subject, wherein the subject has SFRP1+SFRP4 FAP RAMP1- fibroblasts (CXCL-iFibro) and / or CD68+CD206+FOLR2+macrophages in a biological sample, said inhibitor being preferably selected from the group consisting of iCRT3, Dickkopfl (DKK1), Porcupine inhibitor (PCN), secreted frizzled protein 1 (Sfrpl), Wnt inhibitor 1 (Wif-1), Wnt-C59, Triptonide, Tegatrabetan, Esculetin and Klotho.

[0377] All the references cited in this application, including scientific articles and summaries, published patent applications, granted patents or any other reference, are entirely incorporated herein by reference, which includes all the results, tables, figures and texts of theses references.

[0378] Brief Description of the Drawings

[0379] Figure 1: Identification of distinct mesenchymal clusters in CKD. (a) UMAP of scRNAseq data from 2495 mesenchymal cells (Kuppe et al., 2021) across 12 patients suffering or not from CKD, allowing the visualization of 6 clusters (0 to 5). Colors show the different clusters defined by graph-based clustering method (b) Bar plot showing the percentages of thedifferent clusters according to disease status, i.e. Control or CKD (n=6 and 6 respectively). P-value from Fisher Exact test, (c) UMAP showing new annotations (defined by differential gene expression pathways) of the 6 clusters (0 to 5) identified in Control and CKD patients.

[0380] Figure 2: Accumulation of CXCL-iFibro and ECM-myFibro clusters at distinct stages of chronickidney disease, (a) UMAP showing trajectory inference using Monocle 3. (b) Same UMAP as in (a) showing computed pseudotime by Monocle 3. (c) IHC staining quantification (H-scores in interstitial cells) of SFRP1, FAP, SFRP4 and RAMP1 in PKD patients and the average percentage of fibrosis per quantified fields. N = 12 PKDpatients. (d) IHC staining quantification (H-scores in interstitial cells) of SFRP1, FAP, SFRP4 and RAMP 1 in kidney biopsies from patients with vascular nephropathies, and the average percentage of fibrosis per quantified fields. N = 13 patients with fibrotic kidney. Of note, 3 patients presented with a FAP Score of 0 and a percentage of fibrosis of 0.

[0381] Figure 3: Inflammatory fibroblasts are in close vicinity of FOLR2+ macrophages, (a) Quantifications of IF co-staining of CD68 (pan-macrophage marker) and SFRP1 (inflammatory fibroblast marker) in PKD patients (N = 6). Quantifications show correlations between the number of SFRP1+ cells and the number of CD68+ cells. Left panel represents the average of the numberof cells per patient. Right panel represents the correlation between the number of SFRP1 + cells and the number of CD68+ cells per surface unit, each dot representing a field of 18 600pm2. Each patient is represented by a different color. N = 6 PKD patients, (b) Same as in (a) in fibrotickidney biopsies. N = 10 patients with fibrotic kidney, (c) Quantifications of IF co-staining between CD68 (pan-macrophage marker) and FAP (ECM- secreting myofibroblast marker) in PKD patients. Quantifications show correlations between the number of F AP+ cells and the number of CD68+ cells. Left panel represents the average number of cellsper patient. Right panel represents the correlation between the number of FAP+ cells and the number of CD68+ cells per surface unit, each dot representing a field of 18 600pm2. Each patient is represented by a different color. Scale bar = 20pm. N = 6 PKD patients, (d) UMAP of scRNAseq datafrom 3960 myeloid cells from (Kuppe et al., 2021) across 12 patients suffering or not from CKD, allowing the visualization of 6 myeloid cell clusters (0 to 5). Colors show the different clusters defined by graph- based clustering, (e) Bar plot showing the percentages of each cluster of myeloid cells according to disease status, i.e. Control or CKD. P- value from Fisher Exact test, (f) Quantifications of IF co-staining ofFOLR2, TREM2 (markers of different macrophage subsets) and SFRP1 (inflammatory fibroblast marker) in PKD patients (N = 6). Left panel shows the correlation between the average number of SFRP1+ cells and FOLR2+ cells per patient. Right panel shows the correlation between the average number of FOLR2+ cells and the percentage of fibrosis in each patient. (N = 6 PKD patients), (g) Same as in (f) in fibrotic kidneybiopsies (N = 10 patients with fibrotic kidney), (h) Same as in (f) for FOLR2+ cells and FAP+ cells (ECM-secreting myofibroblasts) in PKD patients (N = 6). (h) Results of the deconvolution using Cell2Location on patient 1 described in Table 1. (i) Non-negative matrix factorization of the deconvolution output with 6 factors highlighting different microenvironment, (j) same as in (i) but with 12 factors (0 to 11)

[0382] Figure 4: CXCL-iFibro attract CD14+ monocytes and induces a switch into FOLR2+ macrophages, (a) Representative images (left panel) and quantifications (right panel) showing the expression of E- Cadherin / CDHl, SFRP1, SFRP4 and aSMA in primary fibroblasts cultured on collagen- (top) or plastic-dishes (bottom). Quantification represents the average MFI of at least 30 cells per condition perindependent experiment. Data are expressed as fold change to the paired collagen condition. N=3 independent experiments. Scale bar = 20pm. (b) Representative western blots (left) and correspondingquantifications (right) showing the expression of SFRP1, SFRP4, FAP and aSMA in primary fibroblasts cultured on collagen- or plastic-dishes. P-values from Mann-Whitney test (n=3 independent experiments), (c) Quantification of the percentage of CD 14+ monocytes transmigrating through atranswell, in presence or not of collagen- or plastic-cultured fibroblasts. P-values from Kruskall-Wallistests (n=3 independent experiments with 3 different cells lines and 2 PBMC healthy donors), (d) Quantification of flow cytometry analysis aiming at characterizing macrophage phenotype after 24h of co-culture of CD 14+ monocytes with either collagen- or plastic-cultured fibroblasts. Quantifications show the percentage of FOLR2+ CD206+ macrophages among alive CD 14+ monocytes. P-values from Kruskall-Wallis test (n=4 independent experiments with 3 different cell lines and 4 healthy PBMC donors).

[0383] Figure 5: Macrophages induce a switch from CXCL-iFibro to ECM-secreting myoFibro through a WNT / pcatenin dependent pathway, (a) Representative images (up) and corresponding quantifications (bottom) of IF showing DAPI (blue), aSMA (green) and SFRP4 (red) staining in fibroblasts plated on collagen- (top) or plastic- (bottom) dishes, cocultured or not for 24h with CD14+ monocytes. Scale bar = 20pm. Quantification shows the average MFI (mean fluorescent intensity) of aSMA (left panel) or SFRP4 protein for atleast 30 cells per condition per independent experiment. Data are expressed as fold change of control condition. P-values from Kruskall-Wallis test (n = 3 independent experiments), (b) Representative images (up) and corresponding quantifications (bottom) of IF showing DAPI (blue), aSMA(green) and SFRP4 (red) staining in fibroblasts cultured in collagen-coated plates, treated or not with 2 different specific agonists of the WNT / pcatenin pathway (WNT agonist 1 : CAS 853220- 52-7; WNT agonist 2: SKL2001). Scale bar = 20pm. Quantification shows the average MFI of aSMA (left) or SFRP4 (right) protein for at least 30 cells per condition per independent experiment. Dataare expressed as fold change of control condition. P-values from Kruskall-Wallis test. N = 3 independent experiments, (c) Representative images (left) and corresponding quantifications (right) of IF showing DAPI (blue) and Pcatenin protein (green) in fibroblasts in collagen- (top) or plastic- (bottom) dishes. Scale bar = 20pm. Right panel shows the quantification of nuclear MFI of Pcatenin staining for at least 100 cells per condition per independent experiment. Data are expressed as fold change to the control condition. P-value from Mann-Whitney test. N=4 independent experiments, (d) Same asin (c) for fibroblasts cultured in collagen-coated dishes either alone or in co-culture with CD14+ monocytes, treated or not with an inhibitor of Pcatenin / TCF interaction (iCRT3). Scale bar = 20pm. Right panel shows the quantification of nuclear MFI of Pcatenin protein for at least 30 cells per condition per independent experiment. Data are expressed as fold change of control condition. P-valuefrom Kruskall-Wallis test. N=4 independent experiments (e) Same as in (c) showing DAPI (blue), aSMA(green) and SFRP4 (red) staining in fibroblasts cultured in collagen- coated dishes either alone or in co-culture with CD14+ monocytes, treated or not with an inhibitor of Pcatenin / TCF interaction (iCRT3). Scale bar = 20pm. Quantification shows the average MFI of aSMA (left panel) or SFRP4 protein for at least 30 cells per condition per independent experiment. Data are expressed as fold changeof control condition. P-values from Kruskall-Wallis test. N=4 independent experiments. Figure 6: Expression of CXCL-iFibro gene signature predicts poor outcome of early CKD patients, (a) Kaplan-Meier curve showing the probability of the events composing the compositeoutcome (ESRD or loss of more than 40% of eGFR) according to the expression of CXCL-iFibro signature. N=134 patients; with 31 events including 4 events in the low- expression group (n=51) vs 27in the high expression group (n=83). High and low expression group were defined by selecting the most significant difference between the 2 groups according to the value of CXCL-iFibro expression score. P-value from Log-rank test, (b) Forest plot showing the results of Cox multivariate analysis according to the following variables: eGFR at biopsy, age of the patient, urinary protein to creatinine ratio (UPCR), presence of hypertension and the expression of the CXCL-iFibro score, (c) Mean expression of CXCL-iFibro gene signature according to the progressor status of CKD patients (Slow progressor N = 99; Fast progressor N = 29). (d) Barplot showing the proportion of CKD patients with low (below median) or high (above median) CXCL-iFibro expression score according to patient progression status. 9% of patients were rapid progressors in the low-score subgroup, compared to 36% patients in the high score subgroup. P-value from Fisher Exact test, (e) Correlation slope between the CXCL-iFibro expression score and the eGFR slope. P-value from Spearman correlation test.

[0384] Figure 7: Identification of CXCL iFibro in liver, heart and lung fibrotic diseases. A- C: reanalysis of the liver single cell RNAseq data from Ramachandran et al. (Nature 2019) on 5 healthy patients or 5 cirrhotic patients (A) UMAP showing the identification of 10 clusters of mesenchymal cells after reanalysis of the cells identified as mesenchymal cells by the authors. (B) violin plot showing the z-score of CXCL iFibro signature expression in the same dataset than A, showing that CXCL iFibro corresponds to a specific cluster (cluster 1). (C) Violin plot showing the z-score of CXCL iFibro signature expression in the same dataset than A, showing that CXCL iFibro are more represented in cirrhotic patients. D-E: reanalysis of the heart single cell RNAseq data from Kuppe et al. (Nature 2022) on 4 control patients or 19 patients with myocardial infarction. (D) UMAP showing the identification of 9 cluster in the cells annotated “fibroblast of cardiac tissue” by Kuppe et al (Nature 2022). (E) violin plot showing the z-score of the signature of CXCL iFibro expression in the same dataset than D, showing its overexpression in cluster 2. F-G: reanalysis of the lung single cell RNAseq data from Adams et al. (science Advances 2020) on mesenchymal cells from 46 healthy patients or 32 patients with idiopathic pulmonary fibrosis. F) UMAP showing the different cell types identified by the authors. G) same as F showing the z- score of the signature of CXCL iFibro expression. H) Quantification of histological score (H- score) of SFRP1 staining by immunohistochemistry on healthy liver (n=l), fibrotic liver (n=5), healthy lung (n=l), fibrotic lung (n=5), healthy heart (n=2) and fibrotic heart (n=3).

[0385] Examples

[0386] Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting.

[0387] MATERIAL AND METHODS

[0388] Cohorts of patients

[0389] The study developed here is based on samples taken from surgical residues or kidney biopsies available after histopathologic analyses and not required for diagnosis. There is no interference with clinical practice. Analysis of kidney samples was performed in accordance with the relevant national law andwith recognized ethical guidelines (Declaration of Helsinki) on the protection of people taking part in biomedical research. All patients hospitalized at Necker or Bichat hospital received a welcome bookletexplaining that their samples may be used for research purposes. All patients included in the study were thus informed by their referring nephrologist that biological samples collected through standard clinical practice could be used for research purposes and they gave their verbal and written informed consent. In the case of patient refusal, which could be either orally expressed or written, residual samples werenot included in the study. The pathology lab of Necker and Bichat hospital are authorized to store andmanage human biological samples according to French legislation (declaration number DC- 2009-955).

[0390] Patients with Polycystic Kidney Diseases (PKD) : PKD is an autosomal dominant kidney disease leadingto the development of cysts and ultimately to end-stage renal disease around the age of 50. Because of the size of these organs, it is sometimes needed to undergo a nephrectomy to make room for a kidney transplant. After macroscopic examination of the explanted kidney, and harvest of the zone of interestfor diagnostic, samples for research were collected, fixed in formol then embedded in paraffin (FFPEsamples).

[0391] Kidney biopsies with interstitial fibrosis'. Kidney biopsies samples were from the routine diagnostic samples stored in the pathology department of Necker hospital. Patients with diagnosis of vascular nephropathy with interstitial fibrosis from 0 to 80% were selected. Briefly kidney biopsy was performed with a 16-gauge needle, and the sample was immediately immerged in acetic acid formaldehyde (AF A) fixative. After fixation, tissue was embedded in paraffin and stored as FFPE samples. Interstitial fibrosis was evaluated independently of the study by a pathologist. Nephrectomy samples with fibrosis for spatial transcriptomics: Nephrectomy samples for spatial transcriptomics were from the routine diagnostic samples stored in the pathology department of Bichathospital. Samples of patients who underwent a total nephrectomy for kidney cancer, and who had non-tumoral tissue available were systematically reviewed by a pathologist. Briefly, if the patient presentedkidney dysfunction at the time of the nephrectomy, a sample of non-tumoral kidney was harvested andexamined by a pathologist, after fixation in formol and paraffin embeddings. Patients with interstitial fibrosis were selected.

[0392] NEPTUNE cohort: NEPTUNE is a multicenter observational, prospective cohort study of children andadults with proteinuric glomerular disease, for which comprehensive clinical and molecular phenotyping data was collected at 21 sites at the time of first clinically indicated renal biopsy (Gadegbeku et al., 2013). Biospecimens were collected after informed consent and with approval of the local ethics committee (Ju et al., 2015). Pathologic diagnosis is confirmed by review of digital whole-slide imagesby study pathologists (Barisoni et al., 2013; Gadegbeku et al., 2013). Patients with secondary glomerulardisease (such as diabetic kidney disease, lupus nephritis, and amyloidosis) were excluded. For this analysis, adult patients with mild to moderate kidney dysfunction, defined as patients with an eGFR calculated by a Modification of Diet in Renal Disease (MDRD) > 45mL / min / 1.73m2 for which transcriptomic data as well as follow up clinical data were available were included in the analysis. Progressor status was defined as a decrease of more than 5mL / min / 1.73m2per year.

[0393] Single cell RNAseq data analysis

[0394] Publicly available single cell RNAseq data for 12 patients with kidney disease from Kuppe et al. (Kuppe et al., 2021) including matrix count and annotations were downloaded from Zenodo data archive (https: / / zenodo.org / record / 4059315, DOI: 10.5281 / zenodo.4059315).

[0395] Stromal cells

[0396] Selection of stromal cells. Quality Control and doublet elimination'. First, stromal cells from the datasetwere selected, based on their annotation by the authors. As a quality-control step, the inventors first filtered outlow-quality cells, empty droplets, and multiplet captures based on the distribution of the unique genesdetected (nonzero count) in each cell for each patient. Cells with less than 200 genes were excluded. Doublets were identified using the DoubletFinder method (https: / / github.com / chris-mcginnis- ucsf / DoubletFinder), homotypic doublet proportion estimation was done using the function modelHomotypic and doublet identification using the doubletFinder_v3 function, high confidence doublet identified were removed for downstream analysis. Normalization and scaling: Library-size normalization of each cell using NormalizeData function with default parameters from Seurat (Hao et al., 2021) was performed. Scaling using ScaleData function from Seurat was performed by regressing on the number of count and patient identity.

[0397] Clustering and Data Visualization: Principal component analysis (PCA) dimensionality reduction was run using default parameters. Number of the included components (PCs) was assessed using the JackStraw procedure implemented in JackStraw and ScoreJackStraw functions. Thirty PCs were conserved. Graph-based clustering approach was used to cluster the cells from the first dataset using FindNeighbours (k = 20) and FindClusters functions (res = 0.6). Thirteen stromal cells clusters were obtained at this resolution. For visualization of the data, the nonlinear dimensional reduction techniqueUMAP was applied using the RunUMAP function from Seurat with default parameters.

[0398] Analysis of Differential Gene Expression and Signaling Pathways: Genes specifically upregulated in each of the 13 clusters of the first dataset were identified using the Seurat function FindAllMarkers with default parameters. For each cluster, functional enrichment was done using the Metascape tool (http: / / metascape.org) using all genes significantly upregulated in each of the 13 initial clusters (one cluster vs. all other clusters; function FindAllMarkers with following parameters: logfc.threshold = 0.25, test = wilcox for Wilcoxon rank sum test). Because cluster 0 to 5 represented more than 85% of the cells, the inventors focused on these clusters.

[0399] Gene Signatures analysis of CAF-S1, CAF-S4 and CAF-S1 Clusters: Specific gene signatures of CAF-S1, CAF-S4 and CAF-S1 clusters were previously published (Kieffer et al., 2020). Z-score average was calculated for each signature, and then plotted on the generated UMAP using FeaturePlot function from Seurat.

[0400] Gene signatures of CXCL-iFibro: Specific gene signature from CXCL-iFibro was defined by performing a differential analysis between clusters (Wilcoxon rank-sum test) with the Seurat func xonk'indAllMarkers. Differentially expressed genes between clusters (one cluster vs. all other clusters) witha Padj < 0.05 were selected. This signature was used for detecting CXCL-iFibro in bulk RNA-seq data from kidney biopsies of the NEPTUNE cohort (see section Bulk RNAseq analysis from the Neptune cohort). Genes defining this signature are detailed in Table 2. Expression score of this signature wascalculated using AddModuleScore function from Seurat, and then plotted on the generated UMAP using FeaturePlot function from Seurat. Violin plot was generated using VlnPlot function from Seurat. Trajectory inference analysis'. Trajectory inference models were performed using Monocle 3 package (Cao et al., 2019). Briefly, after normalization and scaling, a trajectory was defined using the learn graph function from Monocle 3 and plotted with plot cells with default parameters. Root of the traj ectory was defined using 2 methods. First, data from literature defined pericytes as a major sourceof myofibroblast during kidney disease. Then, the function get earliest principal node function already described helped us to define the right root (Cao et al., 2019). A pseudotime was then calculated, allowing to classify cells in order of this pseudotime. Genes of interest have been ordered in pseudotime using the plot genes in pseudotime function.

[0401] Transcription factor inference'. Transcription factor inference was performed using the Dorothea package (Garcia-Alonso et al., 2019). Briefly, Dorothea regulons with high confidence score (level A,B and C) were conserved. Then a transcription activity score was computed using Viper for each cell. After computing the mean scaled activity score per cluster , the 20 most variable transcription factors between clusters of interest were selected for representation (the inventors selected cells at the nodeof differentiation between CXCL-iFibro and ECM-secreting myofibro on the Monocle3 UMAP using choose cells function from Monocle3). CXCL-iFibro corresponds to cluster 0 and ECM-secreting myofibro clusters 3+5).

[0402] Myeloid cells

[0403] Selection of myeloid cells. Quality Control and doublet elimination'. Myeloid cells from the dataset were selected, based on their annotation by the authors. Quality control, normalization and scaling as well as clustering and data visualization were performed similarly than for stromal cells (except a resolution of 0.3 for the FindClusters function).

[0404] Cell-Cell interaction prediction '. To predict cell-cell interaction, the inventors used the Cellchat package (Vu et al., 2022) on fibroblasts and myeloid cells. Briefly, ligand-receptor interactions between cells were estimated through a manually curated database containing 1939 validated molecular interactions. Inference of cell communications were performed through identification of over-expressed ligands andover-expressed receptors in different cell groups. For the purpose of the analysis, CD16+ monocytes and CD16+ RUNX3- monocytes were pooled in one group of cells called CD 16+ monocytes.

[0405] Generation of the cellular atlas

[0406] Publicly available single cell RNAseq data for 12 patients with kidney disease from Kuppe et al. wasused for the generation of the atlas. Cell type annotation was similar to that of the original article, except for the stromal and myeloid populations, for which the inventors used the annotation the inventors defined in the study.

[0407] Bulk RNAseq analysis from the Neptune cohort

[0408] The renal biopsy sample was manually dissected and isolated into tubulointerstitial and glomerular compartments per established protocol (Cohen et al., 2002). The tubulointerstital dataset was used for further analysis. For RNA-sequencing (RNA-seq) profiles, mRNA samples were prepared using the Illumina TruSeq mRNA Sam- pie Prep v2 kit. Multiplex amplification was used to prepare cDNA witha paired-end read length of 100 bases using an Illumina HiSeq2000. RNAseq was performed by the University of Michigan Advanced Genomics Core (https: / / brcf.medicine.umich.edu / cores / advanced- genomics / ). Quality of the sequencing data were assessed using the FastQC tool (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Read counts were extracted from the fastq files using HTSeq (version 0.11). RNA-seq profiles from different batches were voom-transformed and batch corrected using ComBat (Zee et al., 2022). The CXCL-iFibro expression score was defined as the mean expression of genes that compose the CXCL-iFibro signature.

[0409] Isolation and culture of kidney primary fibroblast

[0410] Fresh samples from PKD explant received after surgery were cut into fragment of approximatively 1mm3. Fragments were harvested in either non-coated or type I Collagen coated (9pg / mL, Institut DeBiotechnologie Jacques Boy, #207050357) Petri dishes. Cells were cultured in DMEM (Gibco, #41966- 029) supplemented with 10% heat inactivated FBS (Biosera, #FB-1003-500) and 1% streptomycin andpenicillin (Sigma, #p4333) for 2-3 weeks at 37°C, in an incubator delivering 5% CO2 and 1.5% of 02. Media was renewed every 3 days for 2-3 weeks, until cells reached 50% confluency to perform first passage. All experiments with fibroblasts were performed with fibroblasts from passage 5 to 10, to avoid cellular senescence.

[0411] Isolation of CD14+ PBMC

[0412] PBMC were obtained from healthy donor peripheral blood obtained from “Etablisssement Francais dusang”. PBMC were isolated using Lymphoprep (STEMCELL #07861), and CD14+ PBMC were isolated using a specific isolation kit (Miltenyi #130-050- 201).

[0413] Characterization of fibroblasts phenotype by immunofluorescence

[0414] 2 x 104fibroblast were cultured on glass coverslip coated or not with type I collagen, as described above. On the next day, cells were approximatively at 50% confluency. After PBS wash, cells were fixed in paraformaldehyde 4% for 15min. After several washes with PBS, cells were stained according to the immunofluorescence protocol described below.

[0415] Transwell migration assay

[0416] 3xl04fibroblasts were plated on the lower chamber of transwell 24 wells plate (5 pm pore size, Corning HTS Transwell 24 wells #CLS3421) coated or not with type I Collagen in the lower part of the transwell, in 500pL of DMEM supplemented with 10% heat-inactivated FBS and 1% Penicillin streptomycin at 1.5% 02. After 24h, medium was changed for DMEM supplemented with 1% heat inactivated FBS and 1% Penicillin streptomycin. Then 1.5x105 CD14+ PBMC in 150pL of DMEM supplemented with l%heat inactivated FBS and 1% Penicillin streptomycin were plated in the upper chamber and incubated at 37°C for 6h. After incubation, CD 14+ PBMC were harvested in the upper and lower chamber separately and incubated with 0.5 pl of 10 pm carboxylated beads (Polyscience #18133) and DAPI (3 pM). CD14+ PBMC counting was performed by Flow Cytometry on a LSRII (BD biosciences) using precision beads for normalization and expressed as percentage of migration, being the ratio of the T cell number in thelower chamber by the total number of cells.

[0417] Coculture of fibroblasts and CD14+ PBMC

[0418] For macrophage analysis, 3x104fibroblasts were plated in 24 wells plate coated or not with type I collagen for 24h. Then 1.5xl05CD14+ PBMC in DMEM supplemented with 1% heat inactivated FBSand 1% Penicillin streptomycin were added. After 24 h adherent and nonadherent CD 14+ monocytes were harvested, washed and stained first with LIVE / DEAD dye (1:1000, Thermo Fischer, #L34955) for 10 min at room temperature (RT) in PBS to exclude dead cells. Cells suspensions were then incubatedfor 20 min at RT with antibody mix containing anti-CD14-BV510 (1:50, BD biosciences 563079), anti- CD16-BV650 (1:50, BD biosciences 563692), anti-CD206-BV711 (1:50, BioLegend 321136), anti- FOLR2-PE (1:50, BioLegend 391704), anti-TREM2-biotinilated (1 :50, R&D BAF1828) followed by incubation with streptavidin-PECy5 (1: 100, BioLegend 405205) for 15 min. Isotype controls were BV510 mouse IgGlk (1:50, BD biosciences 56294, BV650 mouse IgGlk (1:50, BD biosciences 563231), BV711 mouse IgGlk (1 :50, BD biosciences 56344), mouse IgGlk (1:50, BioLegend 400112), Goat IgG control (1:50, R&D AB-108-C). Cells were analyzed by LSRFortessaTM analyzer (BD biosciences). Data were examined using FlowJo 10.5.2. For fibroblast phenotype analysis, 2xl04fibroblasts were plated in 24 wells plate with 13mm glass coverslips coated or not with type I collagen for 24h. Then 2xl05CD 14+ PBMC in DMEM supplemented with 1% heat inactivated FBS and 1% Penicillin streptomycin were added. After 24h ofcoculture, cells were washed with PBS, then fixed with 4% PFA for 15 min. After 3 washes with PBS, cells were stained following immunofluorescence protocol described below.

[0419] WNT / p-catenin pathway stimulation

[0420] 2xl04fibroblasts were plated in 24 wells plate with 13mm glass coverslips coated with type I collagenfor 24h. Cells were then stimulated with either control (DMSO), Wnt agonist 1 at the dose of 700nM (Sigma-Aldrich 681665, CAS 853220-52-7), or Wnt agonist 2 at the dose of lOpM (Sigma-Aldrich 681667, SKL2001) for 24h. After that, cells were washed with PBS, then fixed with 4% PFA for 15 min. After 3 washes with PBS, cells were stained following immunofluorescence protocol described below.

[0421] P-catenin / TCF interaction inhibition during fibroblasts-CD14+ PBMC coculture

[0422] 2xl04fibroblasts were plated in 24 wells plate with 13mm glass coverslips coated or not with type I collagen for 24h. Then 2xl05CD14+ PBMC in DMEM supplemented with 1% heat inactivated FBS and 1% Penicillin streptomycin were added. At that time, cells were stimulated with either DMSO or

[0423] Pcatenin / TCF Inhibitor III, iCRT3 (Sigma Aldrich, 219332) at the dose of 20pM. For fibroblast analysis, after 24h of coculture, cells were washed with PBS, then fixed with 4% PFA for 15 min. After 3 washes with PBS, cells were stained following immunofluorescence protocol described below. For macrophages analysis, after 24 h of coculture and stimulation adherent and non-adherent CD 14+ monocytes were harvested, washed and stained first with LIVE / DEAD dye (1 :1000, Thermo Fischer, #L34955) for 10 min at room temperature (RT) in PBS to exclude dead cells. Cells suspensions werethen incubated for 20 min at RT with antibody mix containing anti-CD14-BV510 (1:50, BD biosciences 563079), anti-CD16-BV650 (1:50, BD biosciences 563692), anti-CD206-BV711 (1:50, BioLegend 321136), anti- FOLR2-PE (1:50, BioLegend 391704), anti-TREM2-biotinilated (1:50, R&D BAF1828) followed by incubation with streptavidin-PECy5 (1: 100, BioLegend 405205) for 15 min. Isotype controls were BV510 mouse IgGlk (1:50, BD biosciences 56294, BV650 mouse IgGlk (1 :50, BD biosciences 563231), BV711 mouse IgGlk (1 :50, BD biosciences 56344), mouse IgGlk (1:50, BioLegend 400112), Goat IgG control (1:50, R&D AB-108-C). Cells were analyzed by LSRFortessaTM analyzer (BD biosciences). Data were examined using FlowJo 10.5.2.

[0424] Immunohistochemistry and immunofluorescence studies

[0425] Fibroblasts were fixed in 4% paraformaldehyde for 15 min, permeabilized in PBS containing Triton X- 100 0.1%, BSA 3%, then incubated overnight at 4°C with primary antibodies: mouse monoclonal anti-aSMA (1:200, DAKO clone 1A4 ref M0851) rabbit monoclonal anti-SFRPl (1:50, Abeam ab267466), rabbit monoclonal anti-SFRP4 (1 :400, Abeam abl54167), rabbit monoclonal anti-E-Cadherin (1 :200, Cell Signaling Technology, 4065) mouse monoclonal anti-Pcatenin(l:1000, Bio SB, Ref : BSD 5088). After 3 washes with PBS, cells were incubated in secondary antibody goat anti mouse alexa fluor 488 (1:400, Invitrogen Al 1001) or goat anti rabbit alexa fluor 555 (1:400, Invitrogen A21428). Nuclei were counterstained with Hoescht before mounting with anti-fade diamond mounting medium (Invitrogen P36970).

[0426] Images were acquired by an upright widefield Apotome (Zeiss) or LSM 700 Zeiss confocal. At least 10 fields at 40x were acquired. For quantification of mean fluorescence intensity, Region of Interest (ROI)was defined as the contour of an individual cell. At least 30 cells were quantified per condition, and themean of MFI per condition was then compared.

[0427] For human sections, 3-pm sections of paraffin-embedded kidneys underwent antigen retrieval by microwave heating in a solution of EnVision FLEX Target Retrieval Solution high- pH (Dako, K800421) for 20 minutes. Staining was performed using the Lab Vision Autostainer (Thermo Fisher Scientific). Sections were incubated with the following primary antibodies: FAP (1:100, abeam ab207178), SFRPl (l :200, Abeam abl26613), SFRP4 (1:200 abeam abl54167), RAMP1 (1:400, EMD Millipore MABS1904) CD68 (1 :200, Abeam ab955), aSMA (1:400, Dako clone 1A4 M0851), FOLR2 (1:200, Invitrogen MA5-26933), TREM2 (1: 100, R&D MAB17291) or collagen I (1:400, Invitrogen MAI-26771). For immunohistochemistry, antigen detection was performed using HRP-DAB revelation system, using Vectastain elite ABC kit, Rabbit IgG (for FAP, SFRP1, SFRP4, RAMP1, Vector PK- 6101). For immunofluorescence, antigen detection was performed with alexa-fluor coupled antibodies: alexa fluor488 goat anti mouse (for aSMA, Collagen I, CD68, FOLR2, Invitrogen Al 1001), alexa fluor-555 goatanti rabbit (for FAP, SFRP1, SFRP4 Invitrogen A21428), alexa fluor-647 goat anti rabbit (for FAP, SFRP4 Invitrogen A21245) or alexa fluor-555 goat anti rat (for TREM2 Invitrogen A21434). Slides were counterstained with Mayer hematoxylin freshly prepared (Dako, #S3309) and submitted to serial gradients of xylen and mounted with coverslip in an automatic device (Sakura, Tissue-Tek DRS) for IHC. Nuclei were counterstained with Hoescht and then mounted with anti-fade Diamond mounting medium (Invitrogen P36970). Images were acquired by a Philips scanner for IHC and with an upright widefield Apotome (Zeiss) for immunofluorescence. Costaining experiments for SFRP1 / CD68, FAP / CD68, SFRP1 / TREM2 / FOLR2, FAP / FOLR2 / TREM2, SFRPl / aSMA, SFRP1 / COL1A1 and FAP / COL1A1 were performed as previously described except that the primary and secondary antibodieswere pooled for incubation. Quantification of IHC and immunofluorescence staining

[0428] IHC quantification

[0429] For each slide, staining of fibroblasts markers was evaluated as a histological score (flscore) defined by staining intensity (from 0 to 4) multiplied by the percentage of stained interstitial cells (from 0% to 100%). The whole section was considered, and quantification was performed at 20x magnification. Fibrosis was evaluated as the percentage of fibrotic tissue per field. Fibrosis percentage was evaluatedblindly by 2 independent researchers with very good concordance.

[0430] IF quantification

[0431] For human tissue section. For quantification of SFRP1, FAP CD68, FOLR2 per mm2, each 40xmagnification acquired image was segmented in images of 18.600pm2. Positive cells per field were thenmanually counted. Then, the positive number of cells per mm2could be calculated. For SFRP1 or FAP colocalization with Collagen I, images were segmented in 80x80pm images. Percentage of stained area was quantified after binarization of the staining using thresholding. Correlation between the percentageof the stained area of intserstitial SFRP1 or FAP in these small images is then a reflect of colocalization. fa vitro. For quantification of MFI of markers of interests, at least 10 fields at 40x were acquired. Region of Interest (ROI) was manually defined as the contour of an individual cell. At least 30 cells werequantified per condition per independent experiment, and the average of MFI per condition was then compared. For Pcateninnuclear MFI, nucleus was defined as a ROI using the Hoescht staining by performing binarization and tresholding. Then, MFI of Pcateninstaining was quantified in this ROI.

[0432] Protein extraction and western blot

[0433] Protein extraction: Cells cultured in a 6 well plate coated or not with Collagen I were washed with cold sterile PBS (Gibco #14190) and collected after the addition of 150pl of Laemmli buffer (BioRad, #1610737) supplemented with DTT (Thermosci entific, #11896744) and scratching. The solution wasnext boiled at 95°C for 5 min. Samples were next sonicated for 15 min (cycles of 30 s ON / 1 min 30 sOFF) and centrifuged during 10 min at 13.000 x g at 4°C. The protein extract was then short-term storedat -80°C.

[0434] Western blot. A volume of 15 pl of proteins was loaded onto a NuPAGE Novex 4-12% bis tris midi protein gel 10 wells (Invitrogen, #WG1403BOX). The gel was transfer onto a 0,45pm nitrocellulose membrane (GE Healthcare #10600002) in IX TGS buffer (Biorad #161-0772) at 100V for 2 hr at 4°C. Membrane was next blocked during 30 min in TBS-Tween 0.1% complemented with 5% BSA (Euromedex #04-100-812-C) before blotting with primary antibodies diluted in TBS-Tween 0.1% complemented with 5% BSA at 4°C overnight. Primary antibodies used were FAP (1 : 1000, abeam ab207178), SFRP1 (1 :500, Abeam abl26613), SFRP4 (1 : 1000 abeam abl54167), aSMA(l:1000, Dako clone 1A4 M0851), actin (1 : 10.000; Sigma #A5441). The next day, incubation with secondary antibody anti-mouse (Jackson ImmunoResearch Laboratories, INC., #115-035-003) or anti-rabbit (Jackson ImmunoResearch Laboratories, INC., #115-035-003) in TBS-Tween 0. l%+5% BSA for 1 hr at RT was done. The membrane was incubated for 1 min at RT with ECL (ratio 1 :1) (Western Lightning Plus-ECL, PerkinElmer, #NEL105001EA). The detection of the signal was done in a Chemidoc device for detecting chemiluminescence. The protein bands were analyzed by ImageJ software for protein quantification.

[0435] Statistical analysis

[0436] The graphical representation of the data and statistical analyses were done using R environment (https: / / cran.r-project.org). Bar plots or scatter plots are represented with mean ± standard error of themean (SEM). Statistical tests used agree with data distribution. To assess the normality of the distribution of variables, the inventors first applied the Shapiro- Wilk test. According to normality Shapiro-Wilktest, parametric or non-parametric two-tailed tests were applied. The correlation coefficient and its significance between two independent variables were evaluated by Spearman’s correlation test. Survival curves were established using the Kaplan-Meier method and compared with the Log-rank test using survival R package. Stratification of patients for survival analyses was performed using an iterative method. Patients were separated in 2 groups according to their expression of CXCL-iFibro transcriptomic signature or FOLR2. Each value of expression was tested iteratively. The threshold with the lowest p-value was selected as the threshold differentiating low and high expression levels of CXCL- iFibro transcriptomic signature or FOLR2. Univariate analysis was performed using the Wald test. A multivariate Cox model was applied, using as variable all parameter with a significance of p<0.1 in univariate analysis. All applied statistical tests are indicated in the legends. Differences were statistically significant when p-values were < 0.05.

[0437] RESULTS

[0438] Single cell RNA sequencing identifies different clusters of mesenchymal cells in kidney disease.

[0439] To assess fibroblastic heterogeneity during kidney fibrosis development, the inventors took advantage of anexisting human single cell RNA sequencing (scRNAseq) dataset in patients with or without chronickidney disease (CKD) (Kuppe et al., 2021). After quality control and doublet elimination, 2908 mesenchymal cells were conserved for further analyses. Unsupervised graph-based clustering identified 13 clusters, visualized with the Uniform Manifold Approximation and Projection (UMAP) algorithm (data not shown). The inventors focused on the first 6 clusters, as they represented more than 85% of the total mesenchymal population (Fig. la). All clusters were found in at least 2 patients, albeit in different proportions from one patient to the other. The inventors confirmed that these different clusters were differently distributed between control and CKD patients (Fig. lb). Indeed, the content in clusters 0, 3, 4, and 5 was increased in CKD patients, while clusters 1 and 2 accumulatedin controls (Fig. 1c). The inventors observed that high expression of RGS5 (Regulator of G protein signaling 5), a specific pericyte marker, was mainly detected in clusters 1 and 2, while the pan-fibroblast marker geneDCN (Decorin) was expressed in clusters 0, 3, 4, and 5 (data not shown). The inventors thus determined that clusters 1 and 2 might be pericyteslike and clusters 0, 3, 4, 5 fibroblasts-like. Differential gene expressionanalyses confirmed that each cluster was characterized by a specifictranscriptional profile. Even if all fibroblastic clusters were associated with extracellularmatrix (ECM) remodeling pathways, the inventors could distinguish that cluster 0 was also characterized bycytokine signaling pathway and inflammation, cluster 3 by response to wounding, cluster 4 by complement and coagulation cascade, and cluster 5 by translation elongation and wound-healing. In addition, the pericyte-like cluster 1 was associated with IFNaP-dependent signaling and bloodvessel development, and cluster 2 with muscle system process and oxidative phosphorylation. In agreement with these observations, CXCL12 was highly expressed in cluster 0, and theexpression of COL1A1 and COL3A1 (main ECM components in fibrosis) was mainly detected in clusters3 and 5.

[0440] Interestingly, the inventors found that the aforementioned pathways detected in fibroblasts isolated from kidney diseases were highly reminiscent of those previously identified in specific CAF populations incancer (Bartoschek et al., 2018; Costa et al., 2018; Cremasco et al., 2018; Givel et al., 2018; Biffi et al., 2019; Elyada et al., 2019; Davidson et al., 2020; Dominguez et al., 2020; Kieffer et al., 2020; Sebastian et al., 2020; Wu et al., 2020; Biffi and Tuveson, 2021; Hutton et al., 2021; Krishnamurty et al., 2022; Obradovic et al., 2022). Indeed, the CAF-S1 -specific gene signature (Costa et al., 2018; Givel et al., 2018; Bonneau et al., 2020; Kieffer et al., 2020; Pelon et al., 2020) was highly detected in the fibroblastic clusters (clusters 0, 3,4, 5) in the kidney disease dataset, while the CAF-S4 signature highlighted the pericyte-like cells (clusters 1, 2). On the one hand, the signatures from detox -iCAF, IL-iCAF and IFNy-iCAF subsets previously identified by scRNAseq in the CAF-S1 (FAP+ CAF) population from breast cancer(Kieffer et al., 2020) highlighted mainly the clusters 0 and 4, thereby confirming that these clusters were inflammatory. On the other hand, the ECM-myCAF, TGFP-myCAF and Wound-myCAF signatures from CAF-S1 highlighted the clusters 3 and 5. Interestingly, all gene signatures from CAF-S1 fibroblasts, but not CAF-S4, showed an increased expression in CKD compared to controls, in total agreement with accumulation of clusters 0, 3, 4 and 5 (fibroblast-like) butnot of clusters 1 and 2 (pericyte-like) in CKD patients compared to controls (as shown in Fig. lb). The inventors next sought to focus their further analyses on these clusters 0, 3, 4 and 5, which accumulate in CKDpatients. To define specific markers of these clusters, the inventors performed a pairwise analysis of the genes differentially expressed between the different clusters detected in CKD patients compared to controlsand identified SFRP1 (Secreted frizzled Related Protein 1) for cluster 0, FAP (Fibroblast Activation Protein) for both clusters 3 and 5, SFRP4 (Secreted frizzled Related Protein 4) for clusters 3 and 4 andRAMPl (Receptor Activity Modifying Protein 1) for cluster 5. By this way, cluster 0 could be identified as SFRP1+ SFRP4- FAP- RAMP1-; cluster 4 as SFRP1- SFRP4+ FAP- RAMP1-; cluster 3 as SFRP1- SFRP4+ FAP+ RAMP1- and cluster 5 as SFRP1- SFRP4- FAP+ RAMP1+. Taken as a whole, by combining transcriptomic profiles of these different clusters and expression of specific markers, the inventors defined the CKD clusters 0 and 4 as inflammatory (and thus referred to as iFibro), and the CKD clusters 3 and 5 as ECM-secreting myofibroblasts (then calledmyoFibro). Moreover, based on their expression profiles (data not shown), the inventors annotated CKD clusters moreprecisely as followed: cluster 0, CXCL-iFibro; cluster 4, Detox -iFibro; cluster 3, Wound- myoFibro andcluster 5, TGFP-myoFibro. The pericyte-like clusters 1 and 2, which were down- regulated in CKD, werereferred to as IFNaP-Peri-like and Contractile-Peri-like, respectively (Fig. 1c). Taken as a whole, these data show an important heterogeneity of fibroblasts in CKD.

[0441] CXCL-iFibro and ECM-myFibro clusters accumulate at early and late CKD stages, respectively.

[0442] Inflammatory fibroblasts (iFibro) have been poorly described in kidney fibrosis. To go a step further inthe analysis, the inventors performed trajectory inference and pseudotime analysis using Monocle 3 (Fig. 2a-b). The inventors defined the root of the pseudotime in pericytes-like cells, as several reports identified pericytes as amajor source of myofibroblasts in kidney fibrosis development (Humphreys et al., 2010; Kramann et al., 2013; Kramann and Humphreys, 2014; Kramann et al., 2015; Chang-Panesso et al., 2018; Kuppe et al., 2021). These analyses indicated that CXCL-iFibro (cluster 0) might be an intermediate stage in the differentiation from pericyte-like cells (clusters 1 and 2) toward ECM-secreting myoFibro (clusters 3 and 5) (Fig. 2a, b). Expression of marker genes according to pseudotime confirmed this finding (data not shown), these observations in human CKD being consistent with previous lineage tracing experiments performed inmouse CKD models (Humphreys et al., 2010; Kramann and Humphreys, 2014; Kramann et al., 2015; Chang-Panesso et al., 2018). The inventors next sought to validate the presence of these different clusters of fibroblasts in humantissue sections at different stages of the disease (Fig. 2c-d). In order to capture the fibroblast heterogeneity highlighted in scRNAseq data, the inventors first focused on explants from patients with polycystickidney disease (PKD), an autosomal dominant genetic disease leading to the development of cysts andfinally to end-stage renal disease (Fig. 2c). PKD kidneys exhibited very large fields of fibrosis, which characterized the terminal stage of kidney fibrosis development. Immunohistochemistry (IHC) on PKD explants targeting CXCL-iFibro (SFRP1), ECM-secreting myoFibro (FAP), Wound-myoFibro (SFRP4) and TGFP-myoFibro (RAMP1) validated the existence of these different clusters in tissue sections. Strikingly, CXCL- iFibro (cluster 0) was more abundant in zones with persistent epithelial structures, while ECM- secreting myoFibro (Wound-myoFibro, cluster 3 and TGFP-myoFibro, clusters) were mainly observed in fibrosis-enriched zones. Quantification of the histological scores (H-scores) of the different fibroblast markers, confirmed that the content in CXCL-iFibro (SFRP1+, cluster 0) was anti-correlated with the percentage of fibrosis (evaluated by a nephrologist and a pathologist at diagnosis) within PKD sections (Fig. 2c). In contrast, the inventors found a linear positive correlation between the percentage of fibrosis and markers of ECM-secreting myoFibro, such as FAP, as well as SFRP4 and RAMP1, specific markers of Wound-myoFibro (cluster 3) and TGFP-myoFibro(cluster 5), respectively, in PKD patients (Fig 2c). Because PKD explants correspond to end-stage disease, the inventors next tested the content of these different clusters at earlier stages in kidneys showing lowerpercentage of fibrosis. In that aim, the inventors performed IHC on kidney biopsies from patients suffering from vascular nephropathy with interstitial fibrosis ranging from 0 to 80% of the parenchyma. The CXCL- iFibro and the two ECM-secreting myoFibro clusters, including the Wound-myoFibro and TGFP-myoFibro clusters, were detected in kidney biopsies at these early stages of chronic kidney disease. Interestingly, H-score quantification integrating the proportion of positive cells within the interstitium revealed that CXCL-iFibro expanded in the interstitium at initial stages of fibrosis, before decreasing when the fibrosis percentage increased (Fig. 2d). In contrast, histological scoring of the different ECM-secreting myoFibro markers were linearly correlated with the percentageof fibrosis (Fig. 2d), indicating a constant increase in their content when fibrosis gradually developed in patients. The decrease in the content of the CXCL-iFibro cluster concomitantly to the linear increase inECM-secreting myoFibro clusters when fibrosis was above 50% was consistent with the in-silico analysis using pseudotime and trajectory inference (shown in Fig. 2a-b). Altogether, these data underline the existence of CXCL-iFibro in kidney fibrosis at early stages and suggest that these inflammatory fibroblasts are an early state of differentiation towards ECM-secreting myofibroblasts.

[0443] The content in CXCL-iFibro correlates with FOLR2+ macrophage infiltration in kidney disease

[0444] Based on the accumulation of the CXCL-iFibro cluster at early phase of kidney fibrosis, the inventors next aimedto investigate its functional role in CKD. CXCL-iFibro expressed high levels of several cytokines and chemokines, suggesting potential interactions with immune cells. The inventors first confirmed by immunofluorescence (IF) that SFRP1 + interstitial cells showed low but positive staining for aSMA (smooth-muscle a-actin) (data not shown), similarly as inflammatory CAF-S1 fibroblasts in cancer (Costa et al., 2018; Givel et al., 2018; Bonneau et al., 2020; Kieffer et al., 2020; Pelon et al., 2020; Peltier et al., 2022), confirming that they are activated fibroblasts. Moreover, the CXCL-iFibro accumulated in COL 1 Al -negative zones (data not shown), consistent with the lack of COL1A1 expression in scRNAseq data from CXCL-iFibro. In contrast, the majority of ECM-secreting myoFibro (SFRP1- FAP+) were mainly detected in COL 1 Al -positive zones (data not shown) as expected based on the high expressionof the COL1A1 gene in these clusters.

[0445] As macrophages are well-known to be key players in the development of fibrosis (Guiteras et al., 2016; Tang et al., 2019; Wang et al., 2021; Bell and Conway, 2022; Vlasschaert et al., 2022), the inventors assessed the spatial distribution of CXCL-iFibro and macrophages in PKD explants and fibrotic kidney biopsies byperforming co-staining of both CXCL-iFibro (SFRP1+ FAP-) and macrophages (CD68). Thus, by performing IF staining using specific markers, the inventors confirmed the identity of these cellular clusters in PKD explants and fibrotic kidney biopsies. Interestingly, the inventors observed a proximity and a strong positive correlation between the number of CXCL-iFibro (SFRP1+ FAP-) and the numberof macrophages (CD68+) per mm2in both PKD explants (Fig. 3a) and fibrotic kidney biopsies (Fig. 3b). Interestingly, within each patient tissue section, each field that showed macrophage infiltration also exhibited CXCL-iFibro accumulation in both PKD and fibrotic kidney biopsies (Fig. 3a, b, right panels of quantification). Conversely, each field that did not show CXCL- iFibro infiltration exhibited poor macrophage infiltration, confirming that the colocalization between SFRP1+ and CD68+ cells was not patient-dependent (Fig. 3a, b, right panels of quantification). Importantly, this correlation was not observed between the content in ECM-secreting myoFibro (FAP+) and macrophage infiltration in PKD (Fig 3c). Indeed, while ECM-secreting myoFibro accumulated in the highly fibrotic zones in PKD explants, very few CD68+ cells were observed in these zones and mainly in FAP -negative zones (Fig 3c). The colocalization between pro- inflammatory CXCL-iFibro and CD68+ macrophages suggests that CXCL-iFibro are instrumental in attracting and promoting CD68+ macrophage accumulation at early stages of fibrosis development.

[0446] The inventors next sought to identify more precisely the CD68+ myeloid cells in kidney fibrosis. Recent efforts have been made, especially in cancer, to better characterize the heterogeneity of myeloid cellsand particularly monocytes and macrophages. Several types of tumor-associated macrophages (TAM), such as FOLR2+ (Folate receptor beta) and TREM2+ (Triggering receptor expressed on myeloid cells2) TAM, have been identified (Casanova- Acebes et al., 2021; Nalio Ramos et al., 2022; Timperi et al., 2022). Interestingly, these subtypes of macrophages drive either pro-inflammatory response (FOLR2+) or immunosuppression (TREM2+) (Casanova-Acebes et al., 2021; Nalio Ramos et al., 2022; Timperi et al., 2022). As these macrophage subtypes have not yet been identified in kidney diseases, the inventors first analyzed the different myeloid cell clusters detected in publicly available scRNAseq data from CKD and controls (Kuppe et al., 2021). After quality control and doublet elimination, 3960 myeloid cells were conserved for further analyses. Unsupervised graph-based clustering identified 6 clusters, visualized with the UMAP algorithm (Fig. 3d). These myeloid clusters were differently distributed in control and CKD patients, with a significant decrease in the content of clusters 0, 2 and 5 and accumulation of clusters 1, 3 and 4 in CKD patients compared to controls (Fig. 3e). Among these myeloid clusters, 3 clusters (0, 2 and 3) were identified as monocytes based on VCAN (Versican) expression, and 3 others (clusters 1, 4 and 5) corresponded to macrophages according to C1QA (Complement Clq A chain) andA 7?C7 (Mannose receptor C type 1) expression (data not shown). As previously described (Casanova-Acebes et al., 2021), the inventors identified two types of monocytes, one expressing CD 14 that the inventors annotated CD 14+ monocytes (cluster 0), and the other expressing CD 16, referred to as CD 16+ monocytes (clusters 2 and 3). More precisely, Cluster 2 expressed RUNX3. whereas cluster 3 did not. Interestingly, in the macrophage populations, the inventors could identify specific clusters expressing some of the recently published TAM markers, i.e. TREM2 (left part of cluster 1) and FOLR2 (clusters 4 and 5). It has been described that kidney resident macrophages exhibit a double positivity for CD74 andCD81 (Zimmerman et al., 2019). Interestingly, this population corresponded mainly to cluster 5, which was also positive for FOLR2. The inventors thus defined FOLR2+ CD81+ macrophages (cluster 5), which decreased in CKD, as FOLR2+ resident macrophages and FOLR2+ CD81- macrophages (cluster 4), which accumulated in CKD, as FOLR2+ CKD macrophages (Fig. 3d-e). Finally, cluster 1 was composed of TREM2+ macrophages, also positive for MRC1, CD IE and CD1C expression. The inventors thus annotated this cluster 1 as TREM2+ macrophages (Fig. 3d-e). As these different subtypes of FOLR2+ and TREM2+ macrophages have been poorly described during kidney fibrosis development, the inventors tested if they could detect them in PKD explants and in fibrotic kidney biopsies, and evaluated if they were localized in close vicinity to inflammatory fibroblasts. Co-staining of SFRP1, FOLR2 and TREM2 markers by IF in these tissue samples showed a clear increase in the proportion of FOLR2+ macrophages in CKD upon fibrosis development. Strikingly, the inventors observed a strong correlation between the number of SFRP1+ CXCL-iFibro and F0LR2+ macrophages per mm2(Fig. 3f-g), correlation not detected with FAP+ ECM-secreting myoFibro (Fig. 3h)

[0447] To further validate the spatial distribution of the different fibroblast and myeloid populations the inventors identified in CKD, the inventors performed spatial transcriptomics on 2 patients suffering from kidney fibrosis (Table 1 for clinical characteristics of these patients).

[0448] Table 1: clinical and biological data of patients analyzed by spatial transcriptomics. Abbreviations: CKD, chronic kidney disease; Nx, nephrectomy; eGFR, estimated glomerular filtrationrate; HTN, arterial hypertension.

[0449] To analyze these data, the inventors first built a comprehensive cellular atlas based on scRNAseq data sets from CKD and normal kidney tissues (Kuppe et al., 2021). This cellular atlas was composed of 49 226 cells corresponding to 35 different cell types and states (data not shown). The inventors then mapped the localization and the abundanceof each cell population by performing deconvolution using the Cell2Location algorithm (Kleshchevnikov et al., 2022) with the single cell-based cellular atlas as input. The inventors performed non-negative matrix factorization analysis to identify the different cell types and states that were detected within the same spots. At low-resolution, the inventors confirmed colocalization, such as between glomerular capillaries and podocytes (glomerular compartment), arteriolar endothelium and pericytes, or proximal tubular cells and vasa recta (Fig. 3i). The inventors next observed colocalization between injured tubules, fibroblasts including CXCL-iFibro and immune cells (Fig. 3i). This is of interest, as it has recently been suggested that injured tubules exhibit a pro- inflammatory transcriptomic profile and could trigger immune cell infiltration during CKD progression in mice (Doke et al., 2022; Wu et al., 2022). To go further in the analysis of colocalization between CXCL-iFibro and immune cells, the inventors increased the number of factors in the colocalization analysis, allowing a more resolutive discrimination between the different cell types (Fig 3j). Strikingly, the inventors observeda colocalization between CXCL- iFibro, FOLR2+-CKD macrophages and plasma cells (Fig. 3j). On the other hand, other fibroblast subsets (IFNaP-perilike, Detox-iFibro, wound- myofibro and TGFP-myofibro colocalize together with monocytes, FOLR2 resident macrophages, B cells, T cells, dendritic cells and TREM2+ macrophages (Fig. 3j). Altogether, thesedata show that CXCL-iFibro and FOLR2+ macrophages are co-localized during CKD, suggesting that a reciprocal crosstalk exist between these two populations.

[0450] CXCL-iFibro attract CD14+ monocytes and induce their differentiation into FOLR2+ macrophages

[0451] To decipher the molecular crosstalk between CXCL-iFibro and macrophages, the inventors performed functional assays. To do so, the inventors first generated in vitro cellular models recapitulating the main characteristics of both CXCL-iFibro and ECM-secreting myoFibro clusters. Based on their expertise on iCAF and myCAF isolation from CAF-S1 in cancer (Kieffer et al., 2020), the inventors found that kidney-derived primary fibroblasts acquired distinct phenotypes according to the coating conditions used to expand them in culture (Fig. 4a, b). Indeed, when fibroblasts were expanded on collagen-coated plates, they exhibited higher SFRP1 and lower SFRP4, FAP and aSMA protein levels than cells cultured on plastic-dishes (Fig. 4a, b), showing that collagen-cultured primary fibroblasts were reminiscent of CXCL-iFibro and plastic-cultured fibroblasts of ECM-secreting myoFibro, as previously observed for CAF-S1 (Kieffer et al., 2020). In addition, the inventors observed that the increase in aSMA protein level was a markerdifferentiating CXCL-iFibro from ECM- secreting myoFibro in vitro (Fig. 4a, b). Of note, neither collagen- nor plastic-cultured cells expressed E-Cadherin / CDH1, a marker of epithelial cells (Fig. 4a), thereby confirming that the primary cells established from human kidney are fibroblasts. To investigate interactions between CXCL-iFibro and monocytes / macrophages, the inventors performed an in-silico ligand-receptor interaction study from scRNAseq data using the CellChat algorithm, which quantitatively infers intercellular communications (Vu et al., 2022). By focusing the analysis on CXCL- iFibro and myeloid cells, the inventors observed that the highest number of interactions and the highest interaction strength of CXCL-iFibro was with the CD 14+ monocytes. Interestingly, this in silico analysis suggested that 3 couples of ligandreceptor might drive this interaction, respectively CXCL12-CXCR4, ANGPTL1-LILRB3 and THBS-CD36. These findings led the inventors to design co-culture experiments between collagen-cultured primary fibroblasts (CXCL-iFibro) and CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMC) from healthy donors. The inventors first checked whether CXCL-iFibro could attract CD14+ monocytes by performing transwell migration assays. The inventors observed that CXCL-iFibro stimulated the migration of CD14+ monocytes more efficiently than ECM-secreting myoFibro (Fig. 4c). Moreover, co-culture experiments of CXCL-iFibro or ECM-myoFibro with CD14+ monocytes showed that CXCL- iFibro significantly increased the proportion of FOLR2+CD206+ (MRC1) macrophages from CD14+ monocytes (Fig. 4d). ECM-secreting myoFibro also induced a switch towards FOLR2+ macrophages, but to a much lesser extent than CXCL-iFibro (Fig.4d). Thus, taken together, these data highlight that CXCL-iFibro attract monocytes and promote their differentiation into FOLR2+ macrophages.

[0452] Macrophages induce the transition from CXCL-iFibro to ECM-secreting myoFibro through theWNT / p-catenin pathway

[0453] Based on the crosstalk between CXCL-iFibro and FOLR2+ macrophages, the inventors next tested if, in turn, macrophages could modify the phenotype of kidney-derived primary fibroblasts. The inventors observed that, upon co-culture with CD14+ cells, CXCL- iFibro experienced an increase of both aSMA and SFRP4 proteins to a level close to the one of ECM-secreting myoFibro (Fig. 5a), suggesting that myeloid cellscould induce the switch of CXCL-iFibro into ECM-secreting myoFibro. To decipher the molecular pathways involved in the transition from CXCL-iFibro towards ECM-secreting myoFibro, the inventors performed an in-silico analysis, using transcription factor inference models on the trajectory inferenceanalysis shown in Fig. 2a, in particular on the node between CXCL-iFibro and ECM- secreting myoFibro.

[0454] Using the Dorothea algorithm (Garcia-Alonso et al., 2019), the inventors observed that TCF4 and TCF12, two key mediators of the WNT / p-catenin pathway, were in the toplO of the transcription factors involved in the transition from CXCL-iFibro to ECM-secreting myoFibro. The inventors also validated this finding applying another in-silico approach by using Monocle 3. Monocle 3 classifies differentially expressed genes into modules of genes, which are co-regulated along the trajectory (Cao et al., 2019). The inventors identified 7 gene modules, which were expressed at different points of the trajectory and specifically characterized these distinct states. Interestingly, the inventors identified that module 4 highlighted the transition from CXCL-iFibro to ECM-secreting myoFibro. Indeed, the inventors observed that module 4 signature highlighted both CXCL-iFibro and Wound- myoFibro onthe UMAP, whereas module 3 was exclusively highlighted in CXCL-iFibro. Transcriptomicprofiles of these gene-module 3 and 4 through the Metascape platform revealed that module 3 exhibitedan inflammatory signature, while module 4 showed an ECM-related signature (Table 2), in agreement with the transition from CXCL-iFibro to ECM-secreting myoFibro.

[0455] By doing functional enrichment using the TRRUST database (Han et al., 2015) on module 4 genes, the inventors identified TCF4 as one of the transcription factors involved in the switch from CXCL-iFibro to ECM-secreting myoFibro. Strikingly, the TCF4 transcription factor was thus identified by the 2 approaches, thereby highlighting its relevance. Interestingly, macrophages have been shown to participate in kidney regeneration by activating the WNT / p~ catenin pathway in epithelial cells (Lin et al., 2010). The inventors then aimed to validate the role of the WNT / p-catenin pathway in macrophage-induced switch of CXCL- iFibro into ECM-secreting myoFibro by functional assays. First, the inventors treated CXCL-iFibro with 2 different specific agonists of the WNT / p-catenin pathway (CAS 853220-52-7; SKL2001) and observed that activation of this pathway was concomitant to the increase of aSMA and SFRP4 protein levels (Fig. 5b), suggesting that its activation could drive the phenotype from CXL- iFibro to ECM-secreting myoFibro. Second, the inventors observed that cultured ECM-secreting myoFibro showed more nuclear P-catenin staining than CXCL-iFibro (Fig. 5c), consistent with enhanced activation of the WNT -pathway in ECM- secreting myoFibro compared to CXCL- iFibro. The inventors next tested if the macrophage-induced switch from CXCL-iFibro to ECM-secreting myoFibro could be driven by the WNT / p-catenin pathway. To do so, the inventors co-cultured CD14+ myeloid cells with CXCL-iFibro treated or not with an inhibitor of the P- catenin / TCF interaction (iCRT3). The inventors observed that P-catenin / TCF interaction inhibitor did not affect the proportion of FOLR2+ macrophages upon co-culture (data no shown), indicating that the treatment had noimpact on the differentiation of CD14+ monocytes into FOLR2+ macrophages. Without treatment, the inventors confirmed that coculture of CXCL-iFibro with CD14+ monocytes promoted the nuclear translocation of P- catenin in fibroblasts (Fig. 5d), concomitantly to the up-regulation of ECM-secreting myoFibromarkers (Fig. 5e). Interestingly, the inventors observed that inhibition of P-catenin / TCF interaction prevented the macrophage-induced switch from CXCL-iFibro into ECM-secreting myoFibro (Fig. 5e). Indeed, following inhibition, CXCL-iFibro did not experienced any increase in either aSMA or SFRP4 staining when cocultured with CD 14+ monocytes (Fig. 5e), indicating that the WNT / p-catenin pathway is required for the differentiation of CXCL-iFibro into ECM-secreting myoFibro. Altogether, these data show that macrophages stimulate the differentiation of the CXCL-iFibro into ECM-secreting myoFibro through activation of the WNT / p-catenin pathway. Accumulation of CXCL-iFibro at early stage of chronic kidney disease predicts progression andpoor prognosis of CKD patients.

[0456] As the role of CXCL-iFibro and FOLR2+ macrophages is poorly defined in CKD progression, the inventors sought to assess if their presence could predict CKD progression. As CXCL-iFibro represent an intermediate state in the differentiation process into ECM-secreting myoFibro, the inventors hypothesized that accumulation of CXCL-iFibro might be indicative of CKD progression at early phase of the disease. To address this question, the inventors defined a CXCL-iFibro transcriptomic signature based on the differential expressed genes in the different clusters identified from scRNAseq data (Table 2).

[0457] Table 2: list of genes characterizing the CXCL-iFibro signature.

[0458] The inventors confirmed that thissignature highlighted CXCL-iFibro in the scRNAseq- based cellular atlas the inventors built, thereby validating this gene signature can be used to specifically detect the CXCL-iFibro population in bulk RNAseq data. To evaluate the association of the CXCL-iFibro signature with patient longitudinal outcome, the inventors took advantage of a prospective observational cohort from the Nephrotic Syndrome Study Network (NEPTUNE) with available transcriptomic profiles and longitudinal clinical data (Gadegbeku et al., 2013; Gillies et al., 2018). The inventors selected adult patients with mild to moderate reduction of kidney function (CKD stage 1 to 3a defined by an eGFR>45mL / min / 1.73m2) at the time of the clinically indicated renal biopsy (N = 134 patients) to determine if the CXCL- iFibro signature could predict patient outcome at an early stage of kidney disease. Characteristics of patients were summarized in Table 3.

[0459] Table 3: clinical and biological data of the 134 patients with mild to moderate CKD. Abbreviations: MN, membranous nephropathy; MCD, minimal change disease; IgAN, IgA nephropathy; HTN, hypertension; eGFR, estimated glomerular filtration rate; UPCR, urinary protein-to-creatinine ratio; ESRD, end-stage renal disease. Interestingly, the inventors observed that the expression rate of CXCL-iFibro-specific genes was associated with a composite outcome of end-stage renal disease (ESRD), or reduction of baseline estimated glomerularfiltration rate (eGFR) of more than 40%. Optimal stratification of patients for survival analyses was performed using an iterative method. Patients were ranked by their expression of CXCL-iFibro transcriptomic signature and thresholds was defined for each level of expression. For each threshold, patients were separated in low and high CXCL-iFibro expression scores and Log-rank test was applied. The inventors selected the threshold, which displayed the most significant p-value and separated 50 patients with low-score from 84 patients with high-score. Indeed, patients with high expression score of CXCL-iFibro showed significantly poorer longitudinal outcomes compared to patients with low CXCL-iFibro score (Fig. 6a). Moreover, univariate analysis showed thatthe eGFR at the time of biopsy, presence of arterial hypertension (HTN) at the time of the biopsy, theurinary protein to creatinine ratio (UPCR), and the CXCL-iFibro signature expression were each significantly associated with poor outcomes (Table 4).

[0460] Table 4: results of the univariate analysis of the composite outcome (ESRD or decrease of 40% of eGFR). Abbreviations: eGFR, estimated glomerular filtration rate; HTN, hypertension, UPCR, urinary protein-to-creatinine ratio.

[0461] Strikingly, CXCL-iFibro expression score was one of the most predictive variables, with a hazard ratio at 2.9. The inventors then performed a multivariate analysis using a Cox regression model integrating eGFR, UPCR, HTN, age and CXCL-iFibro expression score, as covariables. The inventors observed that UPCR at the time of biopsy, presence of HTN and high CXCL- iFibro score were independently associated with ESRD or decrease of 40% of eGFR in this cohort of mild-to-moderate CKD patients (Fig. 6b). Finally, the eGFR slope, defined by the change of eGFR per year, was available for 128 patients. The inventors classified patients as fast or slow progressors, based on the level of the eGFR slope (more or less than - 5mL / min / 1.73m2, respectively). The inventors observed that fast progressor patients exhibited a higher CXCL-iFibro expression score than slow progressor patients at the time of kidney biopsy (Fig. 6c). The inventors also defined patients with low- or high-CXCL-iFibro expression score, defined by an expression score below or above the median of the expression score, respectively. The inventors observed that the number of fast-progressor patients was higher in the subgroup with high CXCL-iFibro score than in low-CXCL-iFibro score subgroup (23 vs 6 respectively, p=0.0006, Fig. 6d). Finally, theCXL-iFibro expression score was anticorrelated with the eGFR slope (Fig. 6e). Based on data presented above, the inventors suggest that CXCL-iFibro and FOLR2+ macrophages are interdependent variables during CKD progression. The inventors thus analyzed the FOLR2 expression level in this same cohort(Fig. 6f-j). The inventors found that FOL F2 expression was associated with poor patient outcome, as observed with the CXCL-iFibro score. Indeed, patients with high FOLR2 expression rate experienced poorer outcome than those with low FOLR2 expression (Fig. 6f). Multivariate analysis using Cox regression model showed that UPCR at biopsy, presence of HTN and FOLR2 expression were associated with poor outcome, in an independent manner (Fig. 6g). As for the CXCL-iFibro expression score, the inventors observed that fast progressor patients exhibited a higher FOLR2 expression than slow progressor patients (Fig. 6h), and that a higher proportion of fast progressor patients exhibited a high FOLR 2 expression score (defined by an expression above the median, Fig 6i). Interestingly, Coxregression model integrating eGFR, UPCR, age, presence of HTN, CXCL-iFibro expression score and / 'O / J 2 expression, as covariables, showed that only UPCR at biopsy and the presence of HTN were associated with outcome, indicating that FOLR2 and CXCL-iFibro expression scores were not independent. Consistent with this observation, the expression of FOLR2 and of CXCL-iFibro- specific genes was tightly correlated (Fig. 6j). This finding was in agreement with data showing costaining of SFRP1 (marker of CXCL-iFibro) and FOLR2 protein in patient tissues (as show above Fig.3f-g). Altogether, these data show that CXCL-iFibro and FOLR2+ macrophages are key interconnected players in CKD progression and demonstrate that the findings present herein are relevant in clinical practice.

[0462] CXCL iFibro are involved in liver, heart and lung fibrotic diseases

[0463] To assess the existence of CXCL iFibro in other organs concerned by fibrosis development, the inventors analyzed publicly available data from patients with liver fibrosis (cirrhosis, Ramachandran et al, Nature 2019), post-myocardial infarction fibrosis (Kuppe et al. Nature 2022) and idiopathic pulmonary fibrosis (Adams et al, Science Advance 2020). First, the inventors reanalyzed the single cell RNAseq dataset concerning mesenchymal cells from the liver from patients with or without cirrhosis (Fig.7 A-C). 10 clusters of mesenchymal cells (Fig. 7A) were identified. The z-score of the expression of the CXCL-iFibro signature was calculated, and this signature was dramatically increased in cluster 1 (Fig. 7B). Interestingly, the z-score of CXCL- iFibro signature was increased in patients with cirrhosis compared with healthy controls (Fig. 7C).

[0464] Then, the presence of CXCL-iFibro was assessed in the heart of patient who had suffered from post myocardial infarction. To this end, the inventors reanalysed the single cell RNAseq data generated from the heart from patient with myocardial infarction or not (organ donors, Kuppe et al. Nature 2022). By selecting the cluster annotated “fibroblasts of cardiac tissue” by Kuppe et al., the inventors identified 9 cluster of fibroblasts and myofibroblasts in this dataset (fig. 7D). The z- score of CXCL-iFibro signature was calculated according to these new clusters and identified that the signature was higher in cluster 2 (Fig. 7E).

[0465] Finally, the inventors reanalyzed the single cell RNAseq from the lung of patients with or without idiopathic pulmonary fibrosis (Adams et al Science advance 2020). By focusing on the fibroblasts and myofibroblasts as defined by the Adams et al., (Fig. 7F), the inventors identified that the CXCL-iFibro signature highlighted a specific subset of cells (Fig. 7G).

[0466] Altogether, these data allow the identification of cells that presents similarities with CXCL- iFibro at the transcriptomic level in the liver, the heart and the lung of patients with fibrosis of these organs.

[0467] To validate these findings, the inventors selected tissue sections of patients with healthy or fibrotic liver (n=l and 5 respectively), healthy or fibrotic lung (n=l and 5 respectively), healthy heart or fibrotic heart (n=2 and 3 respectively) and performed an immunostaining of SFRP1 showing the presence of CXCL-iFibro. Interestingly, the inventors identified that normal tissues did not show any SFPR1 positive cells, suggesting the absence of inflammatory fibroblasts (i.e., CXCL-iFibro). On the other hand, all fibrotic tissues showed an infiltration in the fibrotic zones by SFRP1 positive cells (Fig. 7H). Altogether, these results validate the presence of CXCL-iFibro in other organs than kidney, more precisely in fibrotic liver, lung and heart.

[0468] CONCLUSION

[0469] The inventors identified two distinct clusters of an already-known population of ECM- secreting myofibroblasts, and revealed the existence and the function of a new fibroblast population characterized by an inflammatory phenotype, that the inventors named CXCL- iFibro. Interestingly, inflammatory fibroblasts are known in cancer, but have poorly been described in fibrotic diseases such as CKD and liver, lung and heart fibrotic diseases. The inventors validated the existence of the different aforementioned fibroblast populations in human tissue sections by analyzing patients at different stages of CKD, but also in fibrotic diseases such as cirrhosis, myocardial infraction and idiopathic pulmonary fibrosis. By this way, the inventors show that the CXCL-iFibro population expands early during the disease course, before shrinking at late stages while ECM-secreting myofibroblasts grow. Consistent with the accumulation of the CXCL-iFibro population at early stages of kidney disease, the inventors demonstrated its key function in the progression of the pathology by highlighting itsreciprocal crosstalk with FOLR2+ macrophages. Indeed, the inventors show that the inflammatory signature ofCXCL-iFibro is functionally relevant, as CXCL-iFibro are able to attract CD14+ monocytes and to polarize them into FOLR2+ macrophages. In turn, in CKD, FOLR2+ macrophages promote the differentiation of CXCL-iFibro into ECM-secreting myofibroblasts through aWNT / p-catenin dependent mechanism. Finally, the inventors show that detecting CXCL-iFibro or FOLR2 macrophages at early stage of kidney disease in a large cohort of CKD patients is predictive of poor patient outcome. This confirms that the CXCL-iFibro population is a key early player in CKD progression and demonstrates their clinical relevance.

[0470] In addition, the inventors identify several populations of macrophages in CKD, including FOLR2+ and TREM2+ macrophages, two TAM subsets recently discovered in cancer (Cassetta et al., 2019; Casanova-Acebes et al., 2021; Nalio Ramos et al., 2022; Timperi et al., 2022). If the role of macrophages is established in renal fibrosis, the presence of FOLR2+ macrophages has not yet been described, and their role is therefore not known. Interestingly, the inventors identified 2 populations of FOLR2+ macrophages in kidneys, one which corresponds to resident macrophages, and the other one which is more represented in CKD patients. Their different localization within kidney tissue, as revealed by spatial transcriptomics, shows that these 2 populations correspond to 2 different types of macrophages. Here, the inventors reveal thatFOLR2+ macrophages interact with CXCL-iFibro, while the few TREM2+ macrophages detected in CKD are distant from CXCL-iFibro. Interestingly, the inventors decipher that the WNT / p-catenin pathway is a key player in the reciprocal crosstalk between CXCL-iFibro and FOLR2+ macrophages. Sustained activation of WNT / p-catenin pathway is key in cell-to-cell communications and has previously been associated with the development of renal fibrotic lesions. Indeed, while being relatively silent in normal adult kidney, WNT / p-catenin signaling is re- activated in a large number of renal injury mouse models and in human kidney fibrosis (Kawakami et al., 2013; Zhou et al., 2018; Zuo and Liu, 2018; Malik et al., 2020; Li et al., 2022). Moreover, blockade of Wntsecretion by genetic depletion of Wntless, a cargo receptor, in renal tubular epithelial cells in mice markedly reduces myofibroblast activation and kidney fibrosis (Zhou et al., 2017). These data underline the role of WNT / p-catenin pathway in kidney disease through epithelial-to-mesenchymal communication (Surendran et al., 2005; Kawakami et al., 2013; Zuo and Liu, 2018; Malik et al., 2020). In addition, activation of WNT / p-catenin pathway also stimulates macrophage polarization and contributes to kidney fibrosis (Feng et al., 2018). Moreover, macrophage-secreted Wnt ligands, such as Wnt7b, stimulate epithelial responses in injured renal tissue and participate in kidney epithelial repair (Lin et al., 2010). Here, the inventors move a step forward by revealing a new function of the WNT pathway on CXCL-iFibro fibroblasts. Indeed, the inventors show that FOLR2+ macrophages induce the activation of CXCL-iFibro into ECM-secreting myofibroblasts through activation of WNT / pcatenin pathway. Interestingly, inhibition of P-catenin / TCF interaction prevents the activation of myofibroblasts, suggesting that this mechanism could be druggable to preclude kidney fibrosis and CKD progression. The inventors focused on human samples and primary- derived cells. The inventors also had the opportunity to study a large cohort of CKD patients and demonstrate that the detection of CXCL-iFibro at early stages of CKD is clinically relevant. Indeed, very interestingly, the presence of CXCL-iFibro in patients with mild- to moderate-CKD is predictive of poor patient outcome. This shows the early role of CXCL-iFibro in fibrosis development.

[0471] In conclusion, by combining transcriptomic analysis, with imaging and functional assays, the study presented herein unravels new mechanisms driving fibrotic diseases progression, such as CKD. The inventors herein identify CXCL-iFibro as an intermediate fibroblast population, which exhibit pro-inflammatory properties, attracting and activating FOLR2+ macrophages. This new population correlates with poor prognosis in patients with mild-to- moderate CKD. The involvement of CXCL-iFibro in fibrotic diseases such as cirrhosis, myocardial infraction and idiopathic pulmonary fibrosis confirms the interest of the specific population in fibrotic diseases. Therefore, CXCL-iFibro is both a prognostic marker as well as a therapeutic target to prevent fibrotic diseases progression..

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Claims

Claims1. An in vitro method for detecting if a subject is at risk of a fibroblasts associated disease progression wherein the method comprises detecting SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro), and optionally CD68+CD206+FOLR2+macrophages, in a biological sample from said subject; the presence of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages being indicative of disease progression and optionally, the absence of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages being indicative of the absence of disease progression.

2. An in vitro method for selecting a subject at risk of a fibroblasts associated disease, optionally as suitable for a treatment against said fibroblasts associated disease, wherein the method comprises:(a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject, wherein the risk of said subject to have fibroblasts associated disease is proportional to the level(s) of CXCL-iFibro and / or CD68+CD206+FOLR2+macrophages in the biological sample;(b) optionally, selecting subjects with high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages as suitable for a treatment against said fibroblasts associated disease.

3. An in vitro method for predicting the clinical outcome of a subject affected with a fibroblasts associated disease, wherein the method comprises:(a) determining level(s) of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from said subject, wherein high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages are predictive of a poor prognosis and optionally, wherein low level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages are predictive of a good prognosis.

4. The method of claim 3, wherein a poor prognosis is a poor survival prognosis or an early or fast disease progression and a good prognosis is a good survival prognosis, a cessation of disease progression, a slow disease progression or the absence of early disease progression, preferably a slow disease progression.

5. The method of claim 3 or 4, wherein the method further comprises:(b) comparing the level(s) of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages in said biological sample to reference level(s), wherein level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages higher than their reference level(s) are predictive of a poor prognosis and optionally, and / or level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages lower than or equal to their reference level(s) are predictive of a good prognosis.

6. The method of anyone of the preceding claims, wherein the CXCL-iFibro have a gene signature comprising, consisting essentially of or consisting of TMEM176A, CTGF, VCAM1, FM03, C2orf40, CTSS, DPEP1, SRGN, NPY1R, HGF, G0S2, ALDH7A1, BIRC3, SDC4, CYB5A, TYMP, FOSB, SLC43A2, SLC2A3, ZFP36, FILIP1L, VMP1, RFTN1, FOS, CNN2, EDNRB, SERPINB9, GGT5, CCL21, NEGRI, MYL12A, THBS1, CCL19, CRISPLD2, DUSP6, JUN, HM0X2, FBLN5, ODF3B, SFRP1, ABCA8, MY ADM, TMEM176B, CYR61, CCBE1, CXCL12, MYO10, EMIDI, CCL2, GFRA1, TSPAN4, C7, GRAMD2B, CEBPD, RND3, IL34, STON1, ANGPTL1, PCSK7, CFHR1, NIDI, PTGER1, SELENOP, COLECI 1 and DNAJB4.

7. The method of anyone of claims 2-6, wherein the level of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages is a percentage of CXCL-iFibro and optionally of CD68+CD206+FOLR2+macrophages in the biological sample, respectively.

8. The method of anyone of the preceding claims, wherein the fibroblasts associated disease is selected from the group consisting of Chronic Kidney Disease (CKD), systemic sclerosis, kidney graft chronic rejection, myocardial fibrosis, myocardial infarction, rheumatoid arthritis, liver fibrosis, inflammatory bowel diseases (IBDs) and pulmonary fibrosis.

9. The method of anyone of claims 1-7, wherein the fibroblasts associated disease is selected from the group consisting of renal fibrosis, liver fibrosis, pulmonary fibrosis and cardiac fibrosis, preferably is CKD.

10. The method of anyone of claims 2 or 6-9, wherein: a) the fibroblasts associated disease is CKD and the treatment against CKD is i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD,preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation b) the fibroblasts associated disease is pulmonary fibrosis and the treatment against pulmonary fibrosis is corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; c) the fibroblasts associated disease is systemic sclerosis and the treatment against systemic sclerosis is methotrexate, mycophenolate mofetil and / or intravenous cyclophosphamide; d) the fibroblasts associated disease is rheumatoid arthritis and the treatment against rheumatoid arthritis disease is corticosteroids, methotrexate and / or an anti-TNFa treatment; e) the fibroblasts associated disease is kidney graft chronic rejection and the treatment against kidney graft chronic rejection is i) a treatment for limiting kidney graft chronic rejection, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of kidney graft chronic rejection, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) dialysis; f) the fibroblasts associated disease is myocardial infarction and the treatment against myocardial infarction disease is beta-blockers, renin-angiotensin blockers and / or statins; g) the fibroblasts associated disease is inflammatory bowel diseases (IBDs) and the treatment against IBD is selected from anti-inflammatory drugs such as aminosalicylates; immunosuppressant drugs such as azathioprine, mercaptopurine and methotrexate and / or antibiotics; or h) the disease is cirrhosis and the treatment against cirrhosis is diuretics, anti -aldosterone drugs, beta blockers and / or liver transplant.

11. The method of anyone of the preceding claims, wherein the biological sample is a kidney sample, a skin sample, a lung sample, a heart sample, a synovial liquid sample, a gut sample, a liver sample, a blood sample or a urine sample.

12. Use of SFRPl+SFRP4'FAP'RAMPr fibroblasts (CXCL-iFibro) optionally with CD68+CD206+FOLR2+macrophages as biomarker(s) of a fibroblasts associated disease progression or for predicting the clinical outcome of a subject suffering from a fibroblasts associated disease.

13. An agent targeting SFRPUSFRP4 FAP RAMP1' fibroblasts (CXCL-iFibro) for use in the treatment of a fibroblasts associated disease, wherein said agent is a cell depleting agenttargeting CXCL-iFibro or is an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2 macrophages interaction.

14. An inhibitor of P-catenin or of P-catenin / TCF interaction for use in the treatment of a fibroblasts associated disease in a subject, wherein the subject has SFRP1+SFRP4 FAP RAMP1' fibroblasts (CXCL-iFibro), and optionally CD68+CD206+FOLR2+macrophages, in a biological sample, said inhibitor being preferably selected from the group consisting of iCRT3, Dickkopfl (DKK1), Porcupine inhibitor (PCN), secreted frizzled protein 1 (Sfirpl), Wnt inhibitor 1 (Wif-1), Wnt-C59, Triptonide, Tegatrabetan, Esculetin and Klotho.

15. Use of an agent targeting SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) or of a pharmaceutical composition comprising said agent in the manufacture of a medicament for the treatment of a fibroblasts associated disease, wherein said agent is a cell depleting agent targeting CXCL-iFibro or is an inhibitor of CXCL-iFibro and CD68+CD206+FOLR2+macrophages interaction.

16. Use of an inhibitor of P-catenin or of P-catenin / TCF interaction ) or of a pharmaceutical composition comprising said inhibitor for the manufacture of a medicament for the treatment of a fibroblasts associated disease in a subject, wherein the subject has SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro), and optionally CD68+CD206+FOLR2+macrophages, in a biological sample, said inhibitor being preferably selected from the group consisting of iCRT3, Dickkopfl (DKK1), Porcupine inhibitor (PCN), secreted frizzled protein 1 (Sfirpl), Wnt inhibitor 1 (Wif-1), Wnt-C59, Triptonide, Tegatrabetan, Esculetin and Klotho.

17. The use according to claim 12, 15-16, the agent for use according to claim 13 or the inhibitor for use according to claim 14, wherein the fibroblasts associated disease is selected from the group consisting of Chronic Kidney Disease (CKD), systemic sclerosis, kidney graft chronic rejection, myocardial fibrosis, myocardial infarction, rheumatoid arthritis, liver fibrosis, inflammatory bowel diseases (IBDs) and pulmonary fibrosis, preferably the from the group consisting of renal fibrosis, liver fibrosis, pulmonary fibrosis and cardiac fibrosis, even more preferably is Chronic Kidney Disease (CKD).

18. A method for treating a fibroblasts associated disease in a subject in need thereof, wherein the method comprises:(a) determining the level(s) of SFRPl+SFRP4 FAP RAMPr fibroblasts (CXCL-iFibro) and optionally of CD68+CD206+FOLR2+macrophages in a biological sample from the subject,(b) selecting the subject as suitable for a treatment against said fibroblasts associated disease if the biological sample comprises:- high level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages; or- level(s) of CXCL-iFibro and optionally CD68+CD206+FOLR2+macrophages higher than a reference level; and c) administering a therapeutic effective amount of the treatment against said fibroblasts associated disease to the selected subject.

19. The method of claim 18, wherein: a) the fibroblasts associated disease is CKD and the treatment against CKD is i) a treatment for limiting CKD progression, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti-hypertensive drugs, ii) a treatment for compensating the effect of CKD, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) renal replacement therapy, preferably dialysis or kidney transplantation b) the fibroblasts associated disease is pulmonary fibrosis and the treatment against pulmonary fibrosis is corticosteroids and or azathioprine and / or pirfenidone and / or nintedanib; c) the fibroblasts associated disease is systemic sclerosis and the treatment against systemic sclerosis is methotrexate, mycophenolate mofetil and / or intravenous cyclophosphamide; d) the fibroblasts associated disease is rheumatoid arthritis and the treatment against rheumatoid arthritis disease is corticosteroids, methotrexate and / or an anti-TNFa treatment; e) the fibroblasts associated disease is kidney graft chronic rejection and the treatment against kidney graft chronic rejection is i) a treatment for limiting kidney graft chronic rejection, preferably Renin-angiotensin-aldosterone blockers, SGLT2 inhibitors and / or anti -hypertensive drugs, ii) a treatment for compensating the effect of kidney graft chronic rejection, preferably erythropoietin, vitamin D3, calcium and / or phosphate binders and / or iii) dialysis; or f) the fibroblasts associated disease is myocardial infarction and the treatment against myocardial infarction disease is beta-blockers, renin-angiotensin blockers and / or statins; g) the fibroblasts associated disease is inflammatory bowel diseases (IBDs) and the treatment against IBD is selected from anti-inflammatory drugs such as aminosalicylates; immunosuppressant drugs such as azathioprine, mercaptopurine and methotrexate and / or antibiotics,h) if the disease is cirrhosis the treatment against cirrhosis is diuretics, anti-aldosterone drugs, beta blockers and / or liver transplant.

20. Use of a kit comprising means for measuring the level of SFRP1+SFRP4 FAP RAMPF fibroblasts (CXCL-iFibro) and optionally CD68+CD206+FOLR2+macrophages in a biological sample for i) predicting the clinical outcome of a subject suffering from a fibroblast associated disease; ii) determining the likelihood of a subject to have a fibroblast associated disease progression; and / or iii) selecting or not a patient having a risk of a fibroblast associated disease progression for a treatment suitable for said fibroblast associated disease,.

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