CTLA4-Ig treatment induces M1–M2 shift in cultured monocyte-derived macrophages from healthy subjects and rheumatoid arthritis patients

Abstract

Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients. Methods Cultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test. Results In LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p<0.01 for all molecules; 12 h p<0.05 for TLR4 and CD86) and significantly upregulated that of M2 markers, primarily after 12 h of treatment (CD163: p < 0.01 and p < 0.05; CD206: p < 0.05 and p < 0.01; CD204: p < 0.05 by 100 mg/mL). Moreover, in these cells, CTLA4-Ig 500 μM increased the protein synthesis of surface M2 markers (p < 0.05). Similarly, in RA-MDMs, the CTLA4-Ig treatment significantly downregulated the gene expression of M1 markers at both concentrations primarily after 12 h (p < 0.05). Furthermore, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD206 (after 3 h of treatment; p < 0.05), CD163, and MerTK (after 12 h of treatment, p < 0.05), whereas CD204 gene expression was significantly upregulated by the high concentration of CTLA4-Ig (p < 0.05). The protein synthesis of all surface markers was increased primarily by CTLA4-Ig 500 μM, significantly for CD204 and CD206 after 24 h of treatment (p < 0.05). Conclusions CTLA4-Ig treatment seems to induce the in vitro shift from M1 to M2 macrophages, of both HS-M1-MDMs and RA-MDMs, as observed by the significant downregulation exerted on selected M1 markers and the upregulation of selected M2 markers suggesting an additional mechanism for its modulation of the RA inflammatory process.

The Role of M1/M2 Macrophage Polarization in Rheumatoid Arthritis Synovitis

“…As is well-demonstrated, in vitro stimulation of circulating human monocytes with LPS induces their differentiation and polarization into a pro-inflamatory M1 phenotype, characterized by the expression of specific surface markers CD80, CD86, HL-DR, TLR2, and 4, and the release of IL-1β, TNFα, and IL-6 ( Figure 2 ) ( 42 , 89 ). In RA synovitis, the best represented and upregulated genes and the secreted proteins are those correlated to M1 macrophages ( 79 ).…”

“…When self-tolerance is lost, inflammation persists evolving to a chronic maladaptive immune response. CD80/CD86 are costimulatory molecules present on these macrophages (among other cells) in response to activating signals finalized to pathogen suppression; these surface proteins bind to CD28 on naive T cells increasing sensitivity to T-cell receptor (TCR) stimulation and T-cell survival ( 42 ).…”

“…The capability of a selected bDMARDs to promote the polarization of pro-inflammatory M1 macrophages to an anti-inflammatory M2 phenotype was recently tested in vitro for the CTLA4-Ig fusion protein in cultured monocyte-derived macrophages obtained from RA patients ( Table 1 ) ( 42 ). These RA monocyte-derived macrophages, which were characterized by a pro-inflammatory M1 phenotype, as demonstrated by their upregulation of CD80, CD86, and TLR4 gene expression, acquired an anti-inflammatory M2 phenotype after treatment with CTLA4-Ig.…”

“…These RA monocyte-derived macrophages, which were characterized by a pro-inflammatory M1 phenotype, as demonstrated by their upregulation of CD80, CD86, and TLR4 gene expression, acquired an anti-inflammatory M2 phenotype after treatment with CTLA4-Ig. This polarization is determined by the downregulation of the gene expression of M1 phenotype markers and the upregulation of the gene and protein expression of M2 cell surface markers CD204, CD163, and CD206 and MerTK, suggesting also an increased induction of their phagocytic activity ( Table 1 ) ( 42 ).…”

Monocytes and Macrophages in Spondyloarthritis: Functional Roles and Effects of Current Therapies

“…Abatacept inhibits CD4 + T cell activation, but also it has been observed that Abatacept reduces the migratory capacity of monocytes from RA patients [115,160]. Moreover, Abatacept downregulates the macrophages TNF production induced by activated T cells [161] and a recent paper has shown that Abatacept is able to directly skew RA macrophages towards a M2 phenotype [162].…”