Department of Rheumatology, University Hospital Dusseldorf, Medical Faculty of Heinrich Heine University, Dusseldorf, Germany D�sseldorf, Germany
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Background/Purpose: Previous studies showed that monocyte-derived macrophages become pro-fibrotic in systemic sclerosis (SSc) and contribute to fibroblast activation. Macrophages are, however, a heterogeneous population. Macrophages of different ontogenies (monocyte-derived or tissue resident macrophages (TR-Macs)) can be functionally distinct in physiology and disease. However, the role of TR-Macs in the pathogenesis of SSc has not been studied thus far.
Here we aimed to evaluate the role of TR-Macs in SSc skin using induced pluripotent stem cells (iPSC)-derived macrophages and an in vitro human skin equivalent model. Methods: TR-Macs were generated from SSc patients (SSc-TR-Macs) and healthy controls (healthy-TR-Macs). Functional differences were assessed using flow cytometry, phagocytosis assays, and RNA sequencing (RNA-seq). A three-dimensional human skin equivalent model was developed by co-culturing TR-Macs with fibroblasts and keratinocytes. Fluorescence staining was performed to analyze TR-Macs polarization, and single-cell RNA sequencing (scRNA-seq) was used to evaluate the subpopulations of fibroblasts and their communication with macrophages in an SSc skin equivalent in comparison to SSc skin. Results: SSc-TR-Macs expressed higher levels of markers of M2 polarization and showed an increased phagocytic capacity compared to healthy-TR-Macs. Furthermore, SSc-TR-Macs exposed to SSc serum upregulated pro-inflammatory and pro-fibrotic pathways.
Incorporation of SSc TR-Macs in healthy human skin equivalents (containing healthy fibroblasts and keratinocytes) was sufficient to induce fibroblast-to-myofibroblast transition and excessive collagen I deposition without further stimuli. Furthermore, addition of SSc-TR-Macs in a skin equivalent formed by SSc fibroblasts greatly enhanced basal and TGFβ-induced fibroblast activation and collagen I deposition.
ScRNA-seq analysis demonstrated that SSc human skin equivalents (formed by SSc TR-Macs and SSc fibroblasts) recapitulate the composition, shifts in frequencies of fibroblast subpopulations, as well as fibroblasts-macrophages cell-cell communication networks observed in SSc skin.
Treatment with the antifibrotic drug nintedanib, as well as with the CD206-targeting peptide RP-832c (that targets M2 macrophages) effectively reduced collagen deposition in an SSc skin equivalent (containing SSc fibroblasts and SSc TR-Macs). Conclusion: Our study demonstrates a pro-fibrotic role of SSc TR-Macs in SSc skin. These Mac subsets are M2-like polarized and promote fibroblast to myofibroblast transition and fibrotic remodelling in an SSc skin equivalent. The SSc skin equivalent model containing TR-Macs recapitulates key fibroblast and Macs subpopulations and their cell-cell communication in SSc skin. Furthermore, not only drugs that target fibroblasts, but also drugs that specifically target macrophages demonstrate antifibrotic effects in the SSc skin equivalent containing TR-Macs. This model could be thus used for testing of antifibrotic drugs that interfere with the fibroblast-macrophage communication network in SSc.
