IntroductionClinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients.MethodsDermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors.ResultsLuminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator.ConclusionsOur data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.

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.