Objectives Cytokines released by infiltrating T cells may promote mechanisms leading to fibrosis in scleroderma. The aim of this study was to investigate the role of the Th2 cytokine IL-31, and its receptor IL-31RA, in scleroderma skin and lung fibrosis. Methods IL-31 was measured by ELISA of plasma, and by immunochemistry of fibrotic skin and lung tissue of scleroderma patients. The receptor, IL-31RA, was assayed by qPCR of tissue resident cells. Next-generation sequencing was used to profile the responses of normal skin fibroblasts to IL-31. In wild-type Balb/c mice, IL-31 was administered by subcutaneous mini pump, with or without additional TGFβ, and the fibrotic reaction measured by histology and ELISA of plasma. Results IL-31 was present at high levels in plasma and fibrotic skin and lung lesions in a subset of scleroderma patients, and the receptor overexpressed by downstream cells relevant to the disease process, including skin and lung fibroblasts, through loss of epigenetic regulation by miR326. In skin fibroblasts, IL-31 induced next generation sequencing profiles associated with cellular growth and proliferation, anaerobic metabolism and mineralization, and negatively associated with angiogenesis and vascular repair, as well as promoting phenotype changes including migration and collagen protein release via pSTAT3, resembling the activation state in the disease. In mice, IL-31 induced skin and lung fibrosis. No synergy was seen with TGFβ, which supressed IL-31RA. Conclusion IL-31/IL-31RA is confirmed as a candidate pro-fibrotic pathway, which may contribute to skin and lung fibrosis in a subset of scleroderma patients.
Objective In systemic sclerosis (SSc) a persistent tissue repair process leads to progressive fibrosis of the skin and internal organs. The role of mesenchymal stem cells (MSCs), which characteristically initiate and regulate tissue repair, has not been fully evaluated. We sought to investigate whether dividing metakaryotic MSCs are present in SSc skin, and test whether exposure to the disease microenvironment activates MSCs leading to transdifferentation. MethodsSkin biopsy material from recent onset diffuse SSc patients was examined by collagenase spread of 1mm thick surface-parallel sections, in order to identify metakarytoic dividing stem cells in each tissue plane. Adipose-derived MSCs from healthy controls were treated with dermal blister fluid from diffuse SSc patients, and profiled by next generation sequencing, or evaluated for phenotypic changes relevant to SSc. Differential responses of dermal fibroblasts were studied in parallel. Results MSC-like cells undergoing active metakaryotic division were identified in SSc but not control sections, most prominent in the deep dermis and adjacent to damaged microvessels, in both involved and clinically uninvolved skin. Furthermore, exposure to SSc blister fluid caused selective MSC activation, inducing a myofibroblast signature, whilst reducing signatures of vascular repair and adipogenesis and enhancing migration and contractility. Microenvironment factors implicated in inducing transdifferentiation include the pro-fibrotic growth factor TGFβ, presence of lactate and mechanosensing, whereas the microenvironment Th2 cytokine IL-31, enhanced osteogenic commitment (calcinosis). Conclusion Dividing MSC-like cells are present in the SSc disease microenvironment where multiple factors, likely acting in concert, promote transdifferentiation, leading to a complex and resistant disease state.
BackgroundSystemic sclerosis (SSc) is an autoimmune rheumatic disease associated with fibroblast activation in the skin and visceral organs. In SSc, refractory pruritus is a common symptom in a subgroup of patients. IL-31 is a Th2 cell derived cytokine implicated in severe itch in other conditions including atopic dermatitis and T cell lymphoma. T-lymphocyte-derived factors provide a possible link between autoimmune inflammation and fibrosis reported in SSc. In this study we measured IL-31 levels in SSc tissue fluid and plasma, and sought correlation with itch severity and clinical parameters. Recombinant IL-31 was found to influence fibroblast and fibroblast precursor activity.MethodsIL-31 was measured by ELISA of dermal tissue fluid samples obtained by a suction blister analysis method from the involved skin of SSc patients or matched site of healthy controls (SSc n=28, controls n=15), and matched plasma samples. IL-31 receptor mRNA expression was assayed by qPCR of SSc and control fibroblast lysates, as well as epithelial tissue biopsy samples. Normal skin fibroblasts as well as fat derived mesenchymal stem cells (MSCs) were treated with IL-31 (50 ng/ml) and migratory and fibrotic responses were assayed.ResultsIL-31 levels were increased in dermal interstitial fluid in SSc patients compared to controls (mean of 99.4 pg/ml vs 2.3 pg/ml in controls, P<0.0003) and in plasma samples (mean plasma IL-31 1370 vs 196 pg/ml, P<0.01). Dermal fluid IL-31 levels correlated strongly with itch severity in these patients R=0.72, P<0.0038). Raised blister fluid IL-31 was characteristic of a subgroup of SSc patients with severe pruritus, which included individuals from both limited and diffuse SSc clinical subsets. Both normal and disease fibroblast extracts contained IL-31 receptor mRNA (qPCR relative copy number 6.4 SSc vs 1.4 controls, p<0.01) which was also seen in epidermal tissue extracts (qPCR 18.4 SSc vs 8.2 in control, p NS). Treatment of normal dermal fibroblasts with IL-31 led to induction of type I collagen but not CTGF, and promoted fibroblast and MSC migration dependent on ERK and PI3kinase pathways.ConclusionsThis is the first analysis of IL-31 in systemic sclerosis and we have shown increased expression in the skin and plasma of a subgroup of SSc patients, correlating with severe itch. IL-31 protein induced fibroblasts and MSCs and may link T-cell autoimmune responses to fibroblast and precursor cell activation in SSc. Blocking IL-31 therapeutically may provide effective treatment in a subgroup of SSc patients identified clinically by severe pruritus.Disclosure of InterestNone declared
BackgroundMesenchymal stem cells (MSCs) are pleuripotent bone marrow and tissue resident cells implicated in homeostasis and tissue repair. Systemic sclerosis (scleroderma, SSc) is a severe connective tissue disease characterised by progressive fibrotic thickening of the dermis, accompanied by loss of subcutaneous fat and microvasculature. Aberrant activation of MSCs within the disease microenvironment may underly the persistent fibrotic repair process, or account for the failure of adipogenesis and dysregulated vascular repair.ObjectivesWe sought to: 1) determine whether activated MSCs are present within the SSc involved skin lesions, 2) test whether SSc suction blister fluid (BF) derived from involved forearm skin can induce phenotype changes in MSCs, 3) fully profile the altered gene expression in MSCs exposed to SSc BF, 4) investigate the role of key factors present at increased level in SSc BF (IL-31, lactate).MethodsNovel post-fixation collagenase tissue dissociation techniques applied to 1 mm tissue sections, combined with Feulgen staining of DNA, were used to identify MSCs undergoing metakaryotic division within the involved skin of SSc patients. Fat derived MSCs from healthy controls were treated in tissue culture with blister fluid derived from the fibrotic skin lesions or from matched sites in healthy individuals, or exposed to key constituent factors, including cytokines (IL-31, 50 ng/ml), metabolites (lactate, 25 mM), and enhanced stiffness matrix (50 kPa gels). The responses of MSCs were studied by analysis of next generation sequencing (NGS) and phenotype changes.ResultsMSCs undergoing metakaryotic division were identified in SSc skin biopsy material but not in healthy control (HC) tissue (SSc vs HC, superficial dermis 0 vs 0, mid dermis 1.1 vs 0 p<0.0001, deep dermis 1.4 vs 0 p<0.0001 metakaryotic cells per x20 field). SSc BF (diluted 1:125 in media) induced disease-relevant phenotype changes in MSCs, such as αSMA expression (p<0.05), collagen gel contraction (p<0.002) and scratch wound repair (p<0.016), as well as loss of adipogenic potential, more than control BF or media alone, due in part to elevated IL-31 and lactate. NGS indicated that SSc blister fluid induced treatment-specific gene expression in MSCs (figure 1), more differentially than in normal dermal fibroblasts, consistent with activation of fibrosis, wound repair, migration, osteogenesis, connective tissue formation and loss of angiogenesis/vascular repair. Induction of αSMA in MSCs was dependent on the matrix stiffness in model systems.ConclusionsFactors present at elevated levels in the disease microenvironment, including cytokines and metabolites, as well as the stiffened ECM, are capable of promoting the migration and differentation of fat derived MSCs, towards tissue reparative cells implicated in the fibrotic process. Conversely, the adipogenic and vascular regenerative potential of these cells may be reduced by exposure to the SSc microenvironment.Disclosure of InterestNone declared
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