Multimodal analyses of early, untreated systemic sclerosis skin identify a proinflammatory vascular niche of macrophage-fibroblast signaling
Helen C. Jarnagin, Rezvan Parvizi, Zhiyun Gong, Rosemary Gedert, Xianying Xing, Lam (Alex) C. Tsoi, Rachael Bogle, Madeline J. Morrisson, Laurent Perreard, Patricia A. Pioli, Fred Kolling IV, Johann E. Gudjonsson, Dinesh Khanna, Michael L. Whitfield
Helen C. Jarnagin, Rezvan Parvizi, Zhiyun Gong, Rosemary Gedert, Xianying Xing, Lam (Alex) C. Tsoi, Rachael Bogle, Madeline J. Morrisson, Laurent Perreard, Patricia A. Pioli, Fred Kolling IV, Johann E. Gudjonsson, Dinesh Khanna, Michael L. Whitfield
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Research Article Dermatology

Multimodal analyses of early, untreated systemic sclerosis skin identify a proinflammatory vascular niche of macrophage-fibroblast signaling

Abstract

Uncovering the early interactions and spatial distribution of dermal fibroblasts and immune cells in treatment-naive patients with diffuse cutaneous systemic sclerosis (SSc) is critical to understanding the earliest events of skin fibrosis. We generated an integrated multiomic dataset of early-stage, treatment-naive diffuse cutaneous SSc skin. Skin biopsies were analyzed by single-nuclei multiome sequencing (snRNA-Seq and snATAC-Seq) and two spatial transcriptomic methods to comprehensively determine molecular changes. We identified an immunomodulatory niche within the papillary, hypodermis, and vascular regions enriched for activated myeloid cells and fibroblasts characterized by expression of genes such as CXCL12, APOE, and C7. Pathway analyses showed significant enrichment of PI3K/AKT/mTOR signaling pathway expression in these cellular niches, driven by profibrotic growth factor signaling networks. Macrophage subclustering showed SSc-specific macrophage activation of IL-6/JAK/STAT signaling and enrichment of oxidative phosphorylation pathways. Ligand-receptor analysis revealed that SSc macrophages secrete PDGF and TGF-β to activate SSc-dominant fibroblast subclusters. Spatial transcriptomic analyses showed monocyte-derived MRC1+ macrophages express PDGF near PDGFRhiTHY1hi fibroblasts. Multiomic data integration and spatial transcriptomic neighborhood analysis revealed the colocalization of fibroblasts, macrophages, and T cells around the vasculature. These data suggest that interactions between activated immune cells and immunomodulatory fibroblasts around vascular niches are an early event in scleroderma pathogenesis.

Authors

Helen C. Jarnagin, Rezvan Parvizi, Zhiyun Gong, Rosemary Gedert, Xianying Xing, Lam (Alex) C. Tsoi, Rachael Bogle, Madeline J. Morrisson, Laurent Perreard, Patricia A. Pioli, Fred Kolling IV, Johann E. Gudjonsson, Dinesh Khanna, Michael L. Whitfield

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Figure 7

Single-cell spatial analysis demonstrates distinct, cytokine-rich neighborhoods near vasculature in SSc skin.

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Single-cell spatial analysis demonstrates distinct, cytokine-rich neighb...
(A) Representative Xenium images of fibroblasts (orange), myeloid cells (green), and endothelial cells (yellow). Fibroblasts express PDGF receptors; myeloid cells were more likely to have PDGF ligand expression. PDGF receptors (PDGFR; orange dots), all PDGF ligands (PDGFL; green dots); scale bars: 1,000 μm, 200 μm, and 200 μm (left to right); arrows: infiltrating myeloid cells near fibroblasts. (B) Neighborhood enrichment analysis quantifies likelihood of spatial proximity between cell types;10 most common cell neighbors, disregarding self, shown in table; z scores indicate strength of overrepresented neighbor (z score and P values from z test from randomized cell spatial distribution). (C) Representative images with manually annotated vasculature regions overlaid on histology sections and cellular density with region identification. (D) Odds ratio calculations across all sections show in most sections, myeloid cells more likely to be found in annotated vasculature regions. (E) Differential expression analysis shows top 25 genes (sorted by LogFC, Wilcoxon’s rank-sum test, adjusted P values < 0.05) overexpressed in vasculature regions compared with other sections. Expected genes (e.g., EPAS1 and PECAM1) demonstrate specificity of endothelial cells in these regions. Other genes of interest (e.g., CXCL12, TGFBR2, and CCL14) indicate distinct proinflammatory environment. (F) GO term analysis indicates pathway-level enrichment of key terms: cytokine-mediated signaling, cellular response to cytokine stimulus, and regulation of immune response (sorted by adjusted P value, GSEA, permutation test). (G) Immunofluorescence showing neighboring of CD163-positive cells and α-SMA–positive cells in SSc skin compared with HC tissues. Images represent n = 2 for HC and n = 3 for SSc. Scale bar: 20 μm. (H) Xenium overlay of selected sections. PECAM and CDH5 pseudo-colored yellow to identify endothelial cells; CD163, MRC1, and C5AR1 are combined and pseudo-colored red to identify macrophages; and THY1 pseudo-colored green to identify fibroblasts. H&E overview and inlay displayed alongside DAPI (blue) counterstain overlay and inlay of same regions. Scale bars: 2,000 μm (overview) and 200 μm (inlay).