ACKR1+ Endothelial Cells Mediate Leukocyte Infiltration and Synergize with SFRP2/ASPN+ Fibroblasts to Promote Skin Fibrosis in Systemic Sclerosis

Abstract Background Skin fibrosis is the typical pathological manifestation of systemic sclerosis (SSc) and localized scleroderma (LS); it has an unclear aetiology and few effective treatments. Although excessive collagen secretion by fibroblasts is the primary cause of skin fibrosis, evidence has suggested that vascular damage is the initiating event and that various cell types, including fibroblasts, work together to contribute to the pathogenesis of skin fibrosis. Objectives To explore the relationship between vascular endothelial cell lesions and immune cell infiltration, along with the interactions between various cell types within the fibrotic skin ecosystem. Methods Single-cell RNA sequencing was performed on skin biopsies from three healthy donors and seven patients with SSc. Additional data from three patients with localized scleroderma available in the Gene Expression Omnibus (GSE160536) were integrated by Harmony. CellChat (version 1.5.0) was used to analyse the cell communication network. A Transwell® assay and a bleomycin (BLM) mouse model were used to explore the role of atypical chemokine receptor 1 (ACKR1; ‘Duffy antigen’) in immune cell infiltration. Milo single-cell Western blot was used to show fibroblast subcluster activation. Results A total of 62 295 cells were obtained and subpopulations of stromal and immune cells identified. Interaction network analysis found that multiple chemokines secreted by macrophages, pericytes and proinflammatory fibroblasts could bind with ACKR1, which was highly expressed by endothelial cells in lesional skin. The Transwell® assay revealed that overexpression of ACKR1 in human umbilical vein endothelial cells facilitated leucocyte infiltration following treatment with interleukin-8. BLM mice showed enhanced ACKR1 expression, massive immune cell infiltration and skin fibrosis that was attenuated by ACKR1 inhibition. Furthermore, infiltrated macrophages expressing high levels of transforming growth factor (TGF)-β1 or platelet-derived growth factor B (PDGFB) could activate secreted frizzled-related protein 2 (SFRP2)/asporin (ASPN)+ fibroblasts to contribute to the excessive accumulation of extracellular matrix. It was also found that the SOX4–ASPN axis plays an important role in the TGF-β signalling cascade and the aetiology of skin fibrosis. Conclusions Our results reveal that high expression of ACKR1 by endothelial cells in fibrotic skin tissue promotes immune cell infiltration and that SFRP2/ASPN+ fibroblasts synergize to exacerbate skin fibrosis.