Intercellular communication landscape and its working mechanism in systemic sclerosis-associated interstitial lung disease.

SSc-associated interstitial lung disease (SSc-ILD) is the leading cause of death amongst SSc patients. However, profibrotic factors in SSc-ILD have not been systematically assessed and their working mechanisms remain unclear. We performed an integrated single-cell RNA sequencing (scRNA-seq) analysis of 17 normal and 17 SSc-ILD lung tissues using CellChat and Monocle3. Single-cell ATAC sequencing (scATAC-seq) profiles were analysed by Signac. We performed ELISA to detect specific ligand serum levels using specimens of 55 SSc patients with (n = 32) or without (n = 23) ILD. Bleomycin-induced SSc-associated animal models of pulmonary fibrosis (n = 5) were constructed and used for tissue-level validation using immunofluorescence and immunohistochemistry. Integrative scRNA-seq profiles from 17 healthy control and 17 SSc-ILD lung tissues showed a higher intercellular communication flow in SSc-ILD. SPP1/CCL18-positive macrophages and COL1A2 mesenchymal cells positive for migration inhibitory factor (MIF) or MDK constitute the majority of intercellular communication in SSc-ILD. ELISA showed that serum levels of SPP1, MIF and MDK were increased in SSc-ILD. SPP1 from macrophages generated self-autocrine feedback for CD44-positive macrophages and paracrine activity for ITGB1-positive COL1A2 mesenchymal cells. MIF from COL1A2 mesenchymal cells and SPP1 from macrophages provided co-stimulatory signalling for CD74/CD44-positive macrophages. The MDK-SDC2 pair mediates an autocrine signal that maintains COL1A2-positive mesenchymal cells. Moreover, scATAC-seq analysis showed that SSc-ILD had higher chromatin accessibility for SPP1 (promoter region, intron 4 and intron 7) and SDC2 (promoter region). Ligand-receptor pairs, including SPP1-CD44/ITGB1, MIF-CD74 and MDK-SDC2, constitute a major component of intercellular communication in SSc-ILD. This study provides a panoramic view of the pathogenesis of SSc-ILD in a new dimension.