The role of distinct synovial tissue macrophage populations in shaping synovial tissue microenvironment

We tested the role of distinct STM populations in the modulation of synovial tissue environment in ex vivo STM-FLS micro co-cultures. We first FACS-sorted total synovial tissue fibroblasts as we described previously from biopsies of RA patients. FLS were seeded at 3000/well alone or in contact with either 3000/well FACS-sorted MerTK/CD206neg or MerTK/CD206pos STMs from patients with active or remission RA respectively for 48h. The modulatory effect on the FLS was evaluated by comparing their expression of 446 immune/stromal genes (scRNAseq BD Rhapsody) 32,141 FLS were evaluated. FLS cells cultured alone exhibited 4 distinct activation states: FLS cluster 1 (FLS1) expressed extracellular matrix proteins (e.g. COL1A1, COL1A2) and TGFb (TGFBI, TGFB3); FLS2 expressed cell adhesion molecules (e.g. ITGB2, SELPLG); FLS3 expressed receptors for TGFb and resolvin (e.g.CMKLR1 and TGFBR1); and FLS4 expressed high levels of glycolytic enzymes and proliferation markers (e.g. LDHA, PGK1, ENO1 and PCNA). Interestingly, upon co-culture with MerTK/CD206neg an additional fifth cluster (FLS5) emerged with inflammatory properties In contrast to inflammatory effect of the MerTK/CD206neg STMs on FLS, the MerTK/CD206pos population, especially that isolated from biopsies of RA patients in sustained disease remission, induced repair response of FLS as manifested by increased expression of collagens (e.g. COL1A) and TGFb response genes (e.g. TGFBI) Importantly, MerTK/CD206neg and MerTK/CD206pos STMs isolated from the biopsies of the same patient (either with active RA or RA in remission) elicited these divergent FLS responses. These suggest that these two distinct STM populations play opposing role in synovium (pro-inflammatory versus protective).