0963: 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)

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 lung^1,2. 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.