Background/Purpose: Systemic Sclerosis (SSc) is a rare autoimmune disease characterized by fibrosis of the skin and internal organs. While the tyrosine kinase inhibitor Nintedanib is now FDA-approved to treat lung fibrosis in SSc patients, the mechanism by which this drug alters fibroblast and immune cell activation in SSc is unknown.

Methods: We constructed SSc and control 3D skin-like tissues using fibroblasts, monocytes, keratinocytes, and plasma from SSc patients or healthy donors in the presence or absence of Nintedanib. Multiple samples were pooled from each 3D skin-like tissue and processed for histology, flow cytometry, and single cell RNA-sequencing (scRNA-seq). Data were analyzed in R using Seurat, Signac, monocle3, and nichenetr packages.

Results: Analysis of the 14 SSc and 14 healthy control tissues by scRNA-seq in the absence of drug treatment identified four distinct fibroblast populations and five macrophage populations. To assess the effect of Nintedanib on fibroblast and macrophage representation in SSc skin, we exposed 6 SSc and 6 healthy control 3D skin-like tissues to Nintedanib, which reduced tissue thickness and contractility. SSc-derived saSE tissues displayed measures of increased fibrosis.SSc tissues were significantly thicker (p=0.0007) than healthy control tissues. Notably, the organization of the control tissues was clearly defined with obvious dermal and epidermal layers, while the SSc tissues had a more disorganized appearance with less well-defined layers. There was a statistically significant decrease in both tissue contractility (p=0.0002) and thickness (p=0.0008) after Nintedanib-treatment of SSc tissues; healthy control tissues showed a significant decrease in contractility but did not show a significant difference in tissue thickness. Proportional shifts were noted in two of the four fibroblast populations after Nintedanib treatment. Consistent with prior reports of elevated macrophage numbers in SSc-affected tissues, macrophages were significantly increased in SSc saSE tissues compared to healthy control saSE tissues. Macrophage numbers in SSc saSE tissues were significantly reduced by Nintedanib-treatment. Gene expression analysis of Nintedanib-treated skin demonstrated significant alterations in the expression of matrix and fibrosis-related genes, suggesting these changes underlie drug-mediated anti-fibrotic activity. This included significant reductions in the expression of ELN (p=1.42*1^-61), COL1A1 (p=0.016), TNFAIP3 (p=0.007), TNFAIP2 (p=1.75*10^-8), MMP1 (p=1.26*10^-5), and TNFAIP6 (p=8.27*10^-5).

Conclusion: Our results suggest SSc fibroblast and macrophage populations are plastic and require cell-cell, cell-matrix, and circulating factors to maintain their phenotype in culture. Nintedanib resulted in changes to fibroblasts and macrophage populations, including decreased expression of matrix and secreted factors.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/nintedanib-alters-fibroblast-and-macrophage-diversity-in-a-3d-skin-model-of-systemic-sclerosis-ssc/