AB0057 IN VITRO EFFECT OF CTLA4-IGG ON M1-M2 SHIFT OF MACROPHAGES FROM RHEUMATOID ARTHRITIS PATIENTS
Authors: S. Tardito, S. Soldano, E. Gotelli, P. Montagna, S. Paolino, V. Smith, M. Cutolo
Journal: Annals of the Rheumatic Diseases (2021)

Background: Among the cells involved in the inflammatory process of rheumatoid arthritis (RA) [1], macrophages play a key role through their capacity to polarize into “classically” or “alternatively” activated phenotypes (M1 or M2) and making macrophages important players for the inflammatory cascade or for the anti-inflammatory reaction, respectively [2]. CTLA4-Ig fusion protein (abatacept) has been shown to contribute to macrophage shift from M1 to M2 [3]. Objectives: We aimed to investigate the effects of abatacept to induce the polarization from the pro-inflammatory M1 phenotype into the anti-inflammatory M2 phenotype in cultured human macrophages obtained from RA patients’ and healthy subjects’(HS) circulating monocytes. Methods: Cultured monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of three early RA patients and ten HS, after signing informed consent and Ethics Committee approval. Cells were treated with phorbol myristate acetate (PMA) [5ng/ml] for 24 hours (hrs) to induce their differentiation into monocyte-derived macrophages (MDMs). Therefore, cultured HS MDMs were stimulated with lipopolysaccharides [LPS, 1mg/mL] for 4hrs [4] in order to induce their polarization into a pro-inflammatory M1 phenotype and then treated or not with abatacept at the concentrations of 100mg/mL and 500mg/mL for 3, 12, 24 and 48hrs. Cultured RA MDMs, were directly treated with abatacept as previous described. Cultured HS and RA MDMs without any pro-inflammatory stimuli and abatacept treatment were used as respective control. The transition of MDMs from M1 to M2 phenotype was evaluated through gene expression and protein synthesis of M2 macrophage markers, namely scavenger receptors (CD163 and CD204), and mannose receptor-1 (CD206) by quantitative real-time polymerase chain reaction (PCR) and by Western blotting. The statistical analysis evaluation was carried out by GraphPad Prism 8 analysis software using the Wilcoxon non-parametric t-test. Any p-value lower than 0.05 was considered as statistically significant. Results were indicated as median±standard deviation (SD). Results: In cultured RA MDMs (three cases), abatacept upregulated the gene expression of all investigated M2 markers, specifically after 12hrs of treatment with the concentration of 100mg/mL. In these cells, abatacept upregulated only the CD204 protein synthesis with more evidence at 24hrs of treatment and with the 500mg/mL concentration. In cultured HS MDMs (ten cases), abatacept upregulated the gene expression of M2 markers, significantly for that of CD206 [at 3hrs with 100mg/mL concentration, p= 0.0312] and CD163 [at 12hrs with 500mg/mL concentration, p= 0.0312]. Moreover, in these cells, abatacept significantly upregulated the protein synthesis of CD206 [at 48hrs with 500mg/mL concentration, p= 0.0195] and CD204 [at 24hrs with 100mg/mL concentration, p= 0.0156; both at 24 and 48hrs with 500mg/mL concentration, p= 0.0234]. Conclusion: Preliminary data seem to indicate that abatacept can promote the in vitro shift from the M1 into the M2 macrophage phenotype, by upregulating specific markers (CD163, CD204, CD206) in cultured M1-MDMs from RA patients and in M1 macrophages induced from HS. 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. Arthritis Res Ther. 2009;11:R176. [4]Pelegrin P., Surprenant, A. EMBO J. 2009 Jul 22; 28(14): 2114–2127. Disclosure of Interests: Samuele Tardito: None declared, Stefano Soldano: None declared, Emanuele Gotelli: None declared, Paola Montagna: None declared, Sabrina Paolino: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene.