Background/Purpose: Rheumatoid Arthritis (RA) is a chronic autoimmune disorder affecting 1% of the population worldwide [1]. Monocytes/macrophages are known to play a crucial role in modulating the immune inflammatory response through their possible polarization into “pro-inflammatory” (M1) or “anti-inflammatory” (M2) phenotypes [2]. In RA, CTLA4-Ig fusion protein (abatacept) has been found to reduce the proinflammatory activity of macrophages, by interacting with the costimulatory molecule CD86 [3,4].

This study aimed to investigate the in vitro capability of CTLA4-Ig to induce the M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from RA patients and in M1-polarized MDMs obtained from healthy subjects (HSs).

Methods: Five RA patients (mean age 54±13 years), who fulfilled the 2010 ACR/EULAR Classification Criteria for RA, and ten age-matched HSs were enrolled for the study after signed informed consent and local EC approval. Cultured MDMs were obtained from circulating blood mononuclear cells and stimulated with phorbol myristate acetate (PMA, 5ng/ml) for 24 hours (hrs). HS MDMs were then stimulated with lipopolysaccharide (LPS, 1mg/mL) for 4 hrs to induce a pro-inflammatory M1-MDM phenotype. Therefore, both RA-MDMs and M1-MDMs were treated with CTLA4-Ig (100µg/mL or 500μg/mL) for 3, 12, 24 and 48 hrs. Gene expression of CD80, CD86 and TLR4 (M1 markers), CD163, CD204 and CD206 (M2 markers) were evaluated by qRT-PCR. Moreover, protein synthesis of M2 markers was investigated by Western blotting. The statistical analysis was performed by non-parametric Wilcoxon t-test.

Results: In LPS induced M1-MDMs (HS), CTLA4-Ig (100µg/mL and 500μg/mL) significantly downregulated the gene expression of all M1 phenotype markers (CD80, CD86, TLR4: 3 hrs p< 0.01; 12 hrs p< 0.05 for all) and significantly upregulated all M2 markers after 12 hrs of treatment (CD163: p< 0.01 and p< 0.05; CD206: p< 0.05 and p< 0.01; CD204: p< 0.05 by CTLA4-Ig 100μg/mL). Accordingly, CTLA4-Ig (500μg/mL) increased the protein synthesis of M2 markers, significantly after 48 hrs of treatment (p< 0.05).

On the other hands, in RA-MDMs, CTLA4-Ig generally downregulated the gene expression of M1 markers at both concentrations and all timing, but in a significant manner only that of TLR4 and CD80 and only by the highest dose (500μg/mL, 12 hrs, p< 0.05). Of note, CTLA4-Ig (100μg/mL) upregulated significantly the gene expression of CD163 (M2 phenotype marker; p< 0.05). However, gene expression and protein synthesis of all the investigated M2 markers at both concentrations increased in a non-significant manner compared to untreated cells.

Conclusion: CTLA4-Ig seems to induce the in vitro polarization, from M1 to M2, of cultured MDMs obtained from RA patients and of M1-MDMs from HSs, therefore proving a new aspect for the anti-inflammatory activity exerted by abatacept in RA pts.

References:
1. McInnes IB, et al. N Engl J Med 2011;365:2205–19
2. Fujii M, et al. Biochem Biophys Res Commun. 2013;438(1):103-9.
3. Cutolo M, et al. Clin Exp Rheumatol. 2015 Mar-Apr;33(2):250-4.
4. Cutolo M, et al. Clin Exp Rheumatol. 2013 Nov-Dec;31(6):943-6.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/m1-m2-polarization-by-ctla4-ig-abatacept-on-cultured-circulating-monocytes-from-rheumatoid-arthritis-patients-and-healthy-subjects/