PO:31:167 | Increased M2 pro-fibrotic marker expression in circulating monocytes and cultured monocyte-derived macrophages in systemic sclerosis patients with progressive interstitial lung disease

Vanessa Smith^1|2, Stefano Soldano³, Rosanna Campitiello^3|5, Emanuele Gotelli^3|4, Paola Montagna³, Elvis Hysa^3|5, Tamara Vojinovic^3|4, Carmen Pizzorni^3|4, Sabrina Paolino^3|4, Alberto Sulli^3|4, Maurizio Cutolo^3|4. | ¹Department of Internal Medicine and Department of Rheumatology, Ghent University Hospital, Ghent, Belgium; ²Unit for Molecular Immunology and Inflammation, VIB Inflammation Research Centre, Ghent, Belgium; ³Laboratory of Experimental Rheumatology and Academic Division of Clinical Rheumatology, Department of Internal Medicine, Genova, Italy; ⁴IRCCS Ospedale Policlinico San Martino, Genova, Italy; ⁵Department of Experimental Medicine DIMES, University of Genova, Genova, Italy.

Background. Systemic sclerosis (SSc) is a complex disease in which macrophages play a key role in driving progressive fibrosis of the skin and internal organs, especially the lungs^1,2. Alternatively activated macrophages (M2) seem to promote fibrosis by releasing pro-fibrotic mediators³. Previous studies have identified M2 macrophages expressing pro-fibrotic markers, including hybrid M1/M2 cells expressing toll-like receptor 4 (TLR4), in lung tissues from SSc patients with interstitial lung disease (ILD)³. The aim of the study is to evaluate the 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).

Materials and Methods. A total of 56 SSc pts, diagnosed per 2013 ACR/EULAR criteria, and 20 age-matched healthy controls (HC) were recruited with informed consent. Circulating monocytes from a subset of 37 SSc patients (10 prog-ILD, 14 no-prog-ILD, 13 no-ILD; mean age 65±13) and 20 HC were analyzed by flow cytometry for expression of M2 markers (CD204, CD163, CD206) and the M1 marker (TLR4). MDMs were generated from 29 SSc patients (13 prog-ILD, 10 no-prog-ILD, 6 no-ILD; mean age 64±14) and 5 HC, and evaluated for gene and protein expression of the same markers using RT-PCR, Western blotting, and ELISA for TGF-beta1 and Mer tyrosine kinase (MerTK). Statistical analyses employed 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 pts had significantly increased gene and protein expression of TGF-beta1 (p<0.05), and a trend toward elevated MerTK and CD206 levels compared to no-prog-ILD pts. When compared to no-ILD patients, prog-ILD MDMs showed significantly elevated expression of TGF-beta1, TLR4, and all evaluated M2 markers (p<0.05), with MerTK particularly elevated (p<0.01).

Conclusions. Results highlight that SSc pts with ILD, particularly prog-ILD, exhibit an increased proportion of circulating monocytes with a hybrid M1/M2 phenotype and elevated expression of pro-fibrotic markers. Their derived macrophages also show heightened TGF-beta1 and MerTK levels. These findings suggest that TLR4+M2 monocytes and TGF-beta1-expressing macrophages may serve as potential biomarkers of progressive ILD in SSc.