The Regulation of Macrophage Polarization by Hypoxia-PADI4 Coordination in Rheumatoid Arthritis

Abstract Background : Hypoxia, a common feature of rheumatoid arthritis (RA), induces the overexpression of peptidyl arginine deiminase 4 (PADI4) in fibroblast-like synoviocytes (FLSs) and macrophages. However, the roles of PADI4 and its inducer hypoxia in the regulation of macrophage polarization remain unclear. This study aimed to investigate the role of hypoxia-PADI4 for macrophage polarization in RA patients. Methods : synovial tissue(ST) and synovial fluid(SF) were collected from 3 OA patients and 6 RA patients. The distribution of M1 and M2 in ST and cytokines in SF were examined by immunohistochemical analysis and BioPlex immunoassays. THP-1 macrophages and BMDM polarization were determined under normoxic (21% oxygen) or hypoxic (3% oxygen) conditions. The effects of PADI4 on macrophages were determined by transfection of adenovirus vector-coated PADI4 (AdPADI4) and the use of PADI4 inhibitor. To mimic the environment of RA joints, THP-1 macrophages polarization was examined after coculturing with RA-FLSs. Finally, the roles of PADI4 in joint synovial lesions on macrophage polarization were investigated in collagen-induced arthritis(CIA) rats. Results : We found increased macrophage polarization of M1 and M2 in the RA ST, compared with OA ST. The ratio of M1/M2 for RA and OA was 1.633 ± 0.1443 and 2.544 ± 0.4429, respectively. The concentration of M1- and M2-type cytokines was higher in RA than that in OA patients. Hypoxia contributed to the increase of the gene and protein expression of M1 and M2 markers. M1- but not M2-type gene expression showed a positive relationship with PADI4 expression while the level of expression of M2-type genes showed no significant difference. RA-FLSs could promote the copolarization of M1 and M2, and PADI4 inhibitor reversed M1 activation. The degree of joint swelling and destruction was effectively alleviated, and the number of macrophages especially M1 decreased in CIA rats after downregulating PADI4 expression. Conclusion : Hypoxia is responsible for the copolarization of M1 and M2. Hypoxia-associated PADI4 is responsible for M1 macrophage activation, implying that inflammatory environment can be eased by decreasing PADI4 expression and improving the hypoxic environment.