Induction of a non-inflammatory macrophage phenotype M2 (CD206⁺MerTK⁺) by prednisolone and CTLA4-Ig (abatacept) combined treatment in cultured monocyte-derived macrophages from early diagnosed rheumatoid arthritis patients

Macrophages play crucial and bivalent roles in active and remission phases of synovitis in rheumatoid arthritis (RA). Glucocorticoids, usually prednisolone (PD), represent part of the first line of treatment in early RA. Therefore, conventional and then biological disease modifying antirheumatic drugs (DMARDs) are added. The aim of the study was to investigate in cultured monocyte-derived macrophages (MDMs) obtained from early diagnosed RA patients, the efficacy of low doses of CTLA4-Ig (abatacept) added to PD-treated cells, to induce an anti-inflammatory macrophage phenotype polarization (M2), as observed in synovial tissue biopsies of RA in disease remission. Circulating monocytes isolated from nine RA patients naïve to treatment (mean age 52.2±17.8 years, 7 females/2 males) who fulfilled 2010 ACR/EULAR Classification Criteria, and 6 age-matched voluntary healthy subjects (HSs) were differentiated in vitro into MDMs (RA-MDMs and HS-MDMs, respectively). Cultured RA-MDMs were treated with PD (1ng/mL) alone and combined with CTLA4-Ig (0.1 and 1µg/mL) for 3, 16, 24 and 48 h in growth medium. HS-MDMs were stimulated with lipopolysaccharide (LPS 1µg/mL) to induce a pro-inflammatory phenotype (LPS-stimulated HS-MDMs) and then treated as previously described. RA-MDMs and HS-MDMs without any treatment were used as control cells. Gene expression and protein synthesis of CD80, CD86, TLR4 (M1 pro-inflammatory biomarkers), and CD206, CD163, and MerTK (M2 anti-inflammatory biomarkers) were investigated by quantitative real-time PCR and Western blotting. The levels of IL-6, TGFβ1 and IL-10 were evaluated by ELISA in cultured RA-MDMs. CTLA4-Ig added to PD downregulated the gene expression of CD80 and significantly reduced the RA-MDMs production of pro-inflammatory IL-6 after 24 h (p<0.05). On the other hands, the combined treatment significantly reduced the CD80 protein synthesis after 48 hours when compared to the only PD-treated RA-MDMs (p<0.05). Conversely, the addition of CTLA4-Ig enhanced the PD-induced upregulation of CD206 and CD163 gene expression after 24 and 48 hours, and that of MerTK after 48 hours of treatment (p<0.05). In cultured RA-MDMs, a significant increase of TGFβ1 production and CD206 protein synthesis (after 24 and 48 hours of treatment, respectively, p<0.05) was observed when compared to the only PD-treated cells. Of note, CTLA4-Ig (1µg/mL combined with PD) increased the CD163 and MerTK protein synthesis. The CTLA4-Ig capability to reduce M1 pro-inflammatory phenotype biomarkers promoting the increase in gene expression/protein synthesis of M2 anti-inflammatory biomarkers was confirmed also in cultured LPS-stimulated HS-MDMs. Low doses of CTLA4-Ig added to PD-treated RA-MDMs seem to significantly enhance their in vitro polarization toward and anti-inflammatory phenotype (M2), significantly upregulating CD206/MerTK expression, reducing IL-6 production and enhancing TGFβ1 production.