Background: In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarization into “classically” (M1) or “alternatively activated” (M2) phenotypes and the release of pro-inflammatory cytokines (1). In the active inflammatory phase of RA, circulating intermediate monocytes and synovial tissue macrophages show a M1 phenotype, whereas MerTK ⁺ M2 macrophages seem to characterize the synovial tissue of RA patients under remission (2-4). In RA, CTLA4-Ig fusion protein (abatacept) reduces the pro-inflammatory activity of macrophages by interacting with the costimulatory molecule CD86 on surface cell membrane of activated cells, including macrophages (2).

Objectives: The in vitro study investigated the efficacy of CTLA4-Ig treatment to induce the shift from the M1 phenotype into an M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from active RA patients.

Methods: Cultured MDMs obtained from peripheral blood mononuclear cells of 5 active RA patients (mean age 54±13 years) and 5 age-matched healthy subjects (HSs) after overnight stimulation with phorbol myristate acetate (5ng/ml), were treated with CTLA4-Ig at the concentrations of 100mg/mL or 500mg/mL for 3, 12, 24 and 48 hours. A part of cultured RA-MDMs as wells as cultured HS-MDMs were maintained in growth medium (RPMI at 10% of fetal bovine serum) without any treatment and used as unstimulated cells. Gene expression of CD80, CD86 and toll-like receptor-4 (TLR4), as M1 markers, as well as macrophage scavenger receptors (CD163, CD204), mannose receptor-1 (CD206), as surface M2 markers, and MerTK (functional M2 marker) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by Wilcoxon t-test.

Results: Cultured RA-MDMs showed a high basal gene expression of TLR4, CD80 and CD86 compared to HS-MDMs, confirming to be activated M1 macrophages. In these macrophages, CTLA4-Ig treatment downregulated the gene expression of M1 markers at both concentrations and all timings, but significantly limited to TLR4 and CD80 markers (500mg/mL,12 hours: p<0.05). Conversely, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD163, MerTK and CD206 (p<0.05), whereas only the high concentration of CTLA4-Ig significantly upregulated CD204 gene expression (p<0.05). The protein synthesis of all M2 surface markers was increased after 24 hours of treatment primarily by the high concentration of CTLA4-Ig, and significantly for CD204 and CD206 (p<0.05).

Conclusion: CTLA4-Ig treatment seems to exert an important anti-inflammatory effect by promoting the shift from a M1 to an M2 phenotype in cultured RA macrophages The results suggest a further mechanism for CTLA4-Ig in the modulation of the RA synovitis (5).

REFERENCES:

[1]Yang X et al. Cell Prolif. 2020;53:e12854.doi:10.111/cpr.12854.

[2]Kumar RA et al. Int. Immunol.2018;65:348-59.

[3]Boutet MA et al. Autoimmun Rev.2021;20:102758. doi: 10.1016/j.autrev.2021.102758.

[4]Alivernini S et al. Nat Med. 2020;26:1295-306. 5. Cutolo M et al. Arthritis Res Ther. 2009;11:R176; doi: 10.1186/ar2865.

Disclosure of Interests: Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Celgene, Pfizer, Boehringer Ingelheim, Samuele Tardito: None declared, Emanuele Gotelli: None declared, Paola Montagna: None declared, Rosanna Campitiello: None declared, Sabrina Paolino: None declared, Carmen Pizzorni: None declared, Alberto Sulli Grant/research support from: Laboratories Baldacci, Vanessa Smith Grant/research support from: Boehringer Ingelheim, Janssen-Cilag, Stefano Soldano: None declared