Increased macrophage autophagy with unique polarization in rheumatoid synovium

Rheumatoid arthritis (RA) is characterized by synovitis and joint destruction, with macrophages playing a crucial role in pathogenesis. Macroautophagy/autophagy is essential for cellular homeostasis and has been implicated in RA, but its role in macrophage polarization remains unclear. This study aimed to investigate the expression of autophagy-related molecules and macrophage phenotypes in RA synovium. Synovial tissues from patients with RA were analyzed and compared with samples of osteoarthritis (OA) and less-inflammatory synovium (LIS) obtained at immediate surgery for hip fracture. Synovitis severity was histologically assessed, and cellular ultrastructure was examined via electron microscopy. Immunohistochemistry, western blot, and flow cytometry were used to analyze autophagy-related molecules and macrophage phenotypes. RA synovium exhibited significant inflammation, with macrophage-like synovial cells and intimal macrophages showing abundant autophagy-related structures in comparison to those of OA and LIS. Autophagy markers, BECN1 (beclin 1), WIPI2, ATG5, ATG16L1, LC3B, ATG3, SQSTM1, and LAMP1, were highly expressed in CD68⁺ macrophages but less in CD248/TEM-1⁺ fibroblasts. Western blot confirmed higher levels of autophagy-related proteins in RA synovial tissue compared with OA and LIS. Macrophage polarization analysis identified M1-like (NOS2⁺/iNOS⁺, CD86⁺), M2-like (CD163⁺, MRC1/CD206⁺, MERTK⁺), and M1/M2-like (CD86⁺ MRC1⁺ NOS2⁺ CD163⁺) populations. M1/M2-like macrophages showed the highest autophagy-related molecule expression. Autophagy is strongly associated with macrophage polarization in RA synovium. M1/M2-like macrophages, highly enriched in autophagy markers, may play an anti-inflammatory role in modulating inflammation and tissue repair. These findings suggest a potential autophagy-mediated regulatory mechanism in RA macrophage function.<b>Abbreviation</b>s: ATG: autophagy related; BECN1: beclin 1; IL: interleukin; LAMP1: lysosomal associated membrane protein 1; LIS: less-inflammatory synovium; MAP1LC3/LC3: microtubule sssociated protein 1 light chain 3; MFI: mean fluorescence intensity; OA: osteoarthritis; PG: phagophore; RA: rheumatoid arthritis. SQSTM1: sequestosome 1; TNF: tumor necrosis factor; WIPI2: WD repeat domain, phosphoinositide interacting 2.