Background: Previous studies showed that monocyte-derived macrophages become pro-fibrotic in systemic sclerosis (SSc) and contribute to fibroblast activation [1, 2]. Macrophages are, however, a heterogeneous population, and have distinct roles across tissues, or within specific tissue niches [3]. Furthermore, macrophages of different ontogenies (monocyte-derived (Mo-Macs) or tissue resident macrophages (TR-Macs)) can be functionally distinct in physiology and disease [4]. However, the role of TR-Macs in the pathogenesis of SSc has not been studied thus far.

Objectives: 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 iPSC derived from fibroblasts of SSc patients and healthy controls. 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 capacity of healthy and SSc skin equivalent models containing SSc fibroblasts and TR-Macs to recapitulate SSc-specific fibroblast and macrophage subsets and their cell-cell communication networks observed in human SSc skin.

Results: TR-Macs derived from SSc patients expressed higher levels of the M2 marker CD206 and demonstrated a greater phagocytic capacity compared to those from healthy controls. RNA-seq analysis showed that SSc TR-Macs exposed to SSc serum upregulated pro-inflammatory and pro-fibrotic pathways relative to healthy macrophages. We integrated SSc and healthy TR-Macs in human skin equivalents, and observed that the SSc TR-Macs expressed higher levels of the M2 markers CD206 and CD163 than healthy TR-Macs. Incorporation of SSc TR-Macs in healthy human skin equivalents (containing healthy fibroblasts and keratinocytes) was sufficient to induce fibroblast to myofibroblast transition and 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 indicated that SSc human skin equivalents (formed by SSc TR-Macs and SSc fibroblasts) recapitulate the composition, shifts in frequencies of fibroblast and Macs 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.

REFERENCES: [ 1] Toledo, D.M. & Pioli, P.A. Macrophages in Systemic Sclerosis: Novel Insights and Therapeutic Implications. Curr Rheumatol Rep 21 , 31 (2019).

[2] Bhandari, R , et al. Profibrotic Activation of Human Macrophages in Systemic Sclerosis. Arthritis Rheumatol 72 , 1160-1169 (2020).

[3] Wynn, T.A. & Vannella, K.M. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity 44 , 450-462 (2016).

[4] Ginhoux, F. & Guilliams, M. Tissue-Resident Macrophage Ontogeny and Homeostasis. Immunity 44 , 439-449 (2016).

Acknowledgements: NIL.

Disclosure of Interests: Xuezhi Hong: None declared , Yanhua Xiao: None declared , Shihao Zhu: None declared , Yi-Nan Li: None declared , Linlin Huang: None declared , Martin Regensburger: None declared , Franz Marxreiter: None declared , Tim Filla: None declared , Andrea-Hermina Györfi received lecture fees from Boehringer Ingelheim, James Adjaye: None declared , Juergen Winkler: None declared , Florian Groeber-Becker: None declared , Jörg Distler JHWD is the CEO of 4D Science and scientific lead of FibroCure, JHWD is the CEO of 4D Science and scientific lead of FibroCure, JHWD has consultancy relationships with Active Biotech, Anamar, ARXX, AstraZeneca, Bayer Pharma, Boehringer Ingelheim, Callidatas, Celgene, Galapagos, GSK, Inventiva, Janssen, Kyverna, Novartis, Pfizer, Quell Therapeutics and UCB, JHWD has received research funding from Anamar, ARXX, BMS, Bayer Pharma, Boehringer Ingelheim, Cantargia, Celgene, CSL Behring, Exo Therapeutics, Galapagos, GSK, Incyte, Inventiva, Kiniksa, Kyverna, Lassen Therapeutics, Mestag, Sanofi-Aventis, RedX, UCB and ZenasBio, Alexandru-Emil Matei: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.