P008\u2005Influence of M-CSF polarisation of monocytes and activation of the M-CSF macrophages by pro-inflammatory or other polarising cytokines, on expression of peptidylarginine deiminases 2 and 4

Abstract

Career situation of first and presenting author Post-doctoral fellow. Introduction Autoantibodies to citrullinated proteins (ACPA) are specifically associated to rheumatoid arthritis (RA) and likely involved in its pathophysiology. ACPA are produced in the inflamed synovial tissue (ST) where peptidylarginine deiminase (PAD) 2 and 4, responsible for fibrin citrullination, generate the ACPA-targeted epitopes. PAD2 and 4 are expressed in the intima and the subintimal inflammatory infiltrates by CD68+ mononuclear cells.1 That made macrophages (Mφ) of the ST suspect to synthesize PADs. Moreover, we demonstrated that among various polarised subsets, the M-CSF-Mφ present the highest pro-inflammatory response to ACPA-containing immune complexes.2 Recently, we confirmed that PAD2 and 4 are expressed in monocytes and showed that PAD2 is expressed at various degrees in monocyte-derived polarised Mφs generated in the presence of IFN-γ, IL-4, IL-10 or M-CSF, while PAD4 is only detected in the IFN-γ Mφ. Objectives To evaluate expression of PAD2 and 4 in Mφ polarised by M-CSF, activated by various polarising or pro-inflammatory cytokines, expressed in the ST. Methods CD14+ monocytes from healthy donors were differentiated in Mφ in the presence of M-CSF and were subsequently exposed for 18\u2009hour either to polarising cytokines or to pro-inflammatory cytokines such as TNF-α, IL-6, IL-8 or IL-17. PADI2 and 4 gene expression was measured by RT-qPCR and expression of the related proteins evaluated by immunoblotting on total cell extracts. Results PADI2 mRNAs are less detected in M-CSF-Mφ than in the monocytes while PADI4 gene expression is totally suppressed. All the polarised M-CSF-Mφ subsets retain PADI2 gene expression, and interestingly, after IFN-γ stimulation PADI2 mRNAs are detected at a higher relative rate. However IFN-γ, IL-4 or IL-10 does not induce PADI4 gene re-expression. Whereas PAD4 protein remains undetectable, PAD2 is detected in all the polarised M-CSF-Mφ subsets but not more after IFN-γ stimulation. M-CSF-Mφ activation by TNF-α, IL-8 or IL-17 does not modulate expression of the PADI2 and 4 genes. IL-6 induces a significant decrease of PADI2 gene expression and has no effects on the PADI4 gene. Finally, the protein expression of PAD2 is not modulated in the various activated M-CSF-Mφs, and PAD4 is never induced. Conclusions This study shows that in M-CSF-Mφ PADI2 gene expression can be modulated by pro-inflammatory cytokines while PADI4 gene expression cannot be re-induced. Our results reinforce the hypothesis of monocytes and Mφ involvement in generation of the ACPA epitopes in the ST of RA patients. References Foulquier, et al. Arthritis Rheum 2007. Clavel, et al. Ann Rheum Dis 2016. Acknowledgements The technical assistance of C. Thomann, G. Offer and I. Belhaouane is gratefully acknowledged. Disclosure of Interest None declared.