Macrophage–Fibroblast Signaling Network Identified by Single-Cell RNA Sequencing in Juvenile Systemic Sclerosis

This study aims to address that gap by mapping macrophage-fibroblast signaling using interaction analyses. The current abstract describes the single-cell RNA sequencing (scRNA-seq) profile of 9 SSc patients’ skin compared to 9 healthy subjects. ScRNA-seq was performed on the skin of 9 SSc patients and 9 healthy age-matched subjects. Samples were dissociated into single-cell suspensions and processed using the 10x Genomics® Chromium platform for single-cell RNA sequencing. Data preprocessing and clustering were performed in R using Seurat and Harmony for normalization and batch correction, followed by visualization with UMAP. Cell-to-cell communication was investigated using NicheNet and CellChat to predict ligand–receptor interactions between macrophage and fibroblast populations, including associated signaling pathways and target gene networks. Analysis of scRNA-sequenced data revealed specific fibroblast and macrophage populations driving fibrosis in systemic scleroderma (SSc). Clustering led to 11 unique groups of fibroblasts. Clusters COL11A1+/POSTN+ and STC1+/FZD9+ were markedly expanded in SSc skin and showed activation of TGFβ, WNT, and FGF pathways associated with extracellular matrix (ECM) production. The STC1+/FZD9 cluster was a novel finding, with pathway involvement in pulmonary and hepatic fibrosis signaling and TGFβ pathway activation. Macrophage subsets MMP19+/CTSL+ and MERTK+ emerged as important profibrotic sender cells, expressing ligands such as TGFB1, AREG, and COL6A2 that signal to fibroblast receptors TGFBR1, TGFBR2, FGFR1, FGFR2, and FZD9. NicheNet and CellChat analyses identified a coordinated macrophage–fibroblast signaling network integrating TGFβ, FGF, WNT, IGF, NOTCH, and TNF pathways. Notably, TGFβ signaling persisted through accessory activators (ITGAV, THBS1, and SDC2), suggesting a self-sustaining loop of fibroblast activation independent of accessory inflammatory input. Analysis of scRNA-sequenced data revealed specific fibroblast and macrophage populations driving fibrosis in systemic scleroderma (SSc). Clustering led to 11 unique groups of fibroblasts. Clusters COL11A1+/POSTN+ and STC1+/FZD9+ were markedly expanded in SSc skin and showed activation of TGFβ, WNT, and FGF pathways associated with extracellular matrix (ECM) production. The STC1+/FZD9 cluster was a novel finding, with pathway involvement in pulmonary and hepatic fibrosis signaling and TGFβ pathway activation. Macrophage subsets MMP19+/CTSL+ and MERTK+ emerged as important profibrotic sender cells, expressing ligands such as TGFB1, AREG, and COL6A2 that signal to fibroblast receptors TGFBR1, TGFBR2, FGFR1, FGFR2, and FZD9. NicheNet and CellChat analyses identified a coordinated macrophage–fibroblast signaling network integrating TGFβ, FGF, WNT, IGF, NOTCH, and TNF pathways. Notably, TGFβ signaling persisted through accessory activators (ITGAV, THBS1, and SDC2), suggesting a self-sustaining loop of fibroblast activation independent of accessory inflammatory input. These findings define a macrophage-driven signaling circuit that reinforces chronic fibrosis and identify novel molecular targets that may disrupt this feedback loop. Consistent with adult SSc, fibroblast–macrophage interactions likely sustain fibrosis in SSc, yet the specific fibroblast populations involved are different. TGFβ, FGF, and WNT pathways form an interconnected network that drives fibroblast persistence and ECM accumulation, while NOTCH and TNF signaling amplify immune–fibrotic crosstalk. Targeting key ligand–receptor interactions or accessory activators such as ITGAV, THBS1, and SDC2 may provide novel therapeutic strategies to disrupt chronic fibrotic signaling in SSc. By characterizing these ligand–receptor interactions, our study provides a detailed picture of macrophage–fibroblast communication in juvenile systemic scleroderma.