Identification of novel fibroblast subsets in diffuse cutaneous systemic sclerosis.

Abstract

Systemic sclerosis (SSc) is an autoimmune-driven fibrotic disease, characterised by excessive extracellular matrix (ECM) deposition and fibroblast activation. Our study aimed to identify novel fibroblast subpopulations in SSc and determine their functional role. We performed single-cell RNA sequencing on cultured dermal fibroblasts from healthy donors and patients with diffuse cutaneous SSc, verified selected, specific genes at the protein level and used siRNA knockdown experiments to provide evidence for a functional role of these proteins. SSc fibroblasts revealed high heterogeneity and in specific activated subsets strong upregulation of CD9 and four and a half LIM domain 1 (FHL1), previously described as a muscle-related protein. Overexpression of FHL1 and CD9 was also detected in fibroblast subsets in patient skin. CD9 was associated with the regulation of FHL1 expression. Downmodulation of FHL1 in primary skin fibroblasts correlated with downregulation of the vestigial-like family member 3 (VGLL3) gene, which is known to be expressed in myofibroblasts in a stiff and fibrotic environment and to upregulate collagen synthesis. Further, VGLL3 was confirmed to be upregulated in SSc donor skin. Our study identified novel fibroblast subsets in SSc, characterised by CD9 and/or FHL1 upregulation. The data indicate a functional role of FHL1 in fibroblasts and its involvement in the regulation of ECM production and provide a new mechanistic link to VGLL3 regulation. Our findings suggest new avenues for therapeutic exploration targeting the perpetuating fibroblast activation by the fibrotic environment.