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Introduction Fibrosis in the skin, the hallmark of systemic sclerosis (SSc) and other autoimmune diseases, remains poorly understood. Collagen VIII is a short chain non-fibrillar collagen implicated in regulation of extracellular matrix (ECM) organization that is known to be upregulated in several types of cancers but has not been studied in fibrotic diseases of the skin. Our large-scale single-cell RNA sequencing (scRNA-seq) study of SSc skin identified COL8A1 as the primary mediator of pro-fibrotic and pro-inflammatory disease processes (Nat Commun. 2024 Jan 3;15(1):210). Among all fibroblast sub-populations, COL8A1+ fibroblasts expressed the highest levels of fibrotic markers. Furthermore, COL8A1 expression marked the final state of differentiation towards myofibroblasts and the strongest interaction with inflammatory endothelial cells in SSc skin.Material and Methods To assess the prevalence and transcriptomic signatures of COL8A1+ fibroblasts in other skin conditions characterized by fibrosis, we examined skin scRNA-seq and spatial seq data from patients with hypertrophic scar (n=8), keloid scar (n=8), lichen sclerosus (n=2), morphea (n=5), and post-radiation morphea (n=4), or from healthy donors (n=4). Lungs affected by idiopathic pulmonary fibrosis (IPF, n=12) or those from healthy donors (n=2) were also evaluated. Supplementation of endothelial cells and fibroblasts with recombinant full-length collagen VIII was carried out to assess its sufficiency for initiating a pro-fibrotic shift (n=3 each). COL8A1 was silenced in fibroblasts isolated from SSc skin and subjected to scRNA-seq to evaluate efficacy of targeting COL8A1 for anti-fibrotic treatment (n=3).Results COL8A1+ fibroblasts were nearly absent in skin from healthy donors, but prominent in fibrotic diseases, with greatest proportion in keloid scar and hypertrophic scar. ECM module scores were the highest in COL8A1+ fibroblasts, followed by SFRP2+ fibroblasts. The highest ECM signature in COL8A1+ fibroblasts was observed in SSc, followed by lichen sclerosus. Spatial seq revealed a dispersed localization of COL8A1+ fibroblasts in SSc and their containment to the deep dermis in keloid and hypertrophic scars. COL8A1+ fibroblasts were expanded in IPF where they expressed the maximum levels of type 1 collagens and contractile molecules. In vitro supplementation with recombinant COL8A1 induced COL1A1 and smooth muscle actin (SMA) expression as confirmed by qRT-PCR and immunofluorescence. COL8A1 silencing in SSc fibroblasts downregulated multiple fibrotic genes, with top targeted pathways including ECM organization and supramolecular fiber organization.Conclusions We identify COL8A1 as a novel hallmark of multiple fibrotic diseases. Its expression, corelating with ECM remodeling and myofibroblast differentiation, is sufficient to trigger pro-fibrotic reprograming and can be successfully targeted to induce an anti-fibrotic shift.