Background/Purpose: In the complex pathogenesis of systemic sclerosis (SSc), macrophages are mainly involved in mechanism of progressive tissue fibrosis of skin and internal organs, particularly the lung1,2. Alternatively activated macrophages (M2) seem to drive the process through the synthesis of profibrotic molecules3. The presence on M2 macrophages of pro-fibrotic markers has been previously observed in ILD-SSc lung samples (i.e. presence of mixed M1/M2 cells showing TLR4)3. To characterize phenotype and functional M2 markers in circulating monocytes and cultured monocyte-derived macrophages (MDMs) obtained from SSc patients (pts) with progressive ILD (prog-ILD), versus non-progressive ILD (no-prog-ILD), and without ILD (no-ILD).

Methods: A total of 56 SSc pts, diagnosed according to the 2013 ACR/EULAR criteria, and 20 age-matched healthy controls (HC) were enrolled after providing informed consent. A subgroup of 37 SSc pts (10 with prog-ILD, 14 with no-prog-ILD, and 13 no-ILD; mean age 65±13 years) and 20 HC were assessed for circulating monocytes expressing M2 markers (CD204, CD163, CD206) and the M1 marker toll-like receptor-4 (TLR4) using flow cytometry. Monocyte-derived macrophages (MDMs) obtained from 29 SSc pts (13 with prog-ILD, 10 with no-prog-ILD and 6 no-ILD; mean age 64±14 years) and 5 HC were analyzed for gene expression (real-time PCR) and protein levels (Western blot) of these markers, along with Mer tyrosine kinase (MerTK) and transforming growth factor-β1 (TGF-β1) (ELISA). Statistical analyses were carried out using non-parametric tests.

Results: Prog-ILD SSc pts showed a higher percentage of hybrid (M1/M2) circulating TLR4+CD204+CD206+CD163+monocytes compared to no-prog-ILD and significantly higher compared to no-ILD SSc pts (p < 0.05). Cultured MDMs from prog-ILD SSc pts showed a gene expression and protein synthesis significantly higher for TGF- β1 (p < 0.05) and higher (not significant) for MerTK and CD206 compared to MDMs from no-prog-ILD SSc pts. The gene expression of TGF- β1 and TLR4, as well as the synthesis of TGF- β1, CD206, CD163, and MerTK were significantly increased in cultured MDMs from prog-ILD SSc compared to MDMs from no-ILD SSc pts (TGF- β1, TLR4 and M2 markers: p < 0.05; MerTK: p < 0.01).

Conclusion: The study shows that ILD-SSc pts are characterized by a significant increased percentage of circulating hybrid TLR4+M2 monocytes, and the related cultured MDMs express high levels of MerTK and TGF-β1 selectively in pts with prog-ILD. The detection of increased circulating TLR4+M2 monocytes and MerTK on MDMs from ILD-SSc pts is currently under investigation since might represent a potential solublebiomarker of progressive lung fibrosis.References:1. Volkmann ER, et al Systemic sclerosis. Lancet. 2023 Jan 28;401(10373):304-318. 2. Cutolo M, et al. Pathophysiology of systemic sclerosis: current understanding and new insights. Expert Rev Clin Immunol. 2019 Jul;15(7):753-764. 3. Gotelli E, et al. Prevalence of hybrid TLR4+M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis pts with interstitial lung disease. Front Immunol. 2024 Nov 20;15:1488867.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-expression-of-m2-pro-fibrotic-markers-in-circulating-monocytes-and-cultured-monocyte-derived-macrophages-from-systemic-sclerosis-patients-with-progressive-interstitial-lung-disease-ild/