SAT0034 Nicotine Is A Potent Driver of Netosis and Accelerates Collagen-Induced Arthritis

BackgroundCigarette smoking is a major risk factor for rheumatoid arthritis (RA). The contribution of nicotine, the most ubiquitous and addictive component in the traditional as well as electronic cigarettes (e-cigarettes), to the development of RA remains unclear. Recent evidences implicate aberrant Neutrophil Extracellular Trap (NET) formation in the pathogenesis of RA.ObjectivesThis study was undertaken to investigate the effect of nicotine on NETosis and its ability to potentiate arthritis in a murine model of RA.MethodsHuman neutrophils were incubated with nicotine in the presence of DNA-binding dye (Sytox) to quantify NETosis. In parallel, levels of NET-associated myeloperoxidase (MPO)/DNA complexes were measured using a novel NET-specific ELISA. Immunofluorescent staining was used to perform microscopic kinetic analysis of NETosis and to assess the presence of nicotinic acetylcholine receptors (nAChRs) in these neutrophils. NETosis were measured after stimulating neutrophils with GTS-21 (α7-specific nAChR agonist) or mecamylamine (an AChR antagonist). Finally, the effect of systemic nicotine exposure on arthritis development and progression was investigated using subcutaneous osmotic nicotine pumps in the collagen-induced arthritis (CIA) mouse model.ResultsNeutrophils derived from current smokers displayed significantly higher level of spontaneous- and PMA-induced NETosis as measured by Sytox assay and by NET-specific ELISA. In line with this, nicotine enhanced NETosis in a dose-dependent manner in vitro. Furthermore, incubation with APCA-immune complexs or TNF-α facilitated priming of neutrophils to undergo nicotine-induced NETosis. Immunofluorescent staining revealed that nicotine induced robust formation of NET-like structure in these neutrophils. Cell surface staining with an α7-specific agonist (α-bungarotoxin) and anti-α7-specfic antibody demonstrated co-localization on the plasma membrane. Nicotine-induced NETosis was reproduced when stimulated with GTS-21 and was abrogated when neutrophils were pretreated with mecamylamine. A significantly increased arthritis score as measured by paw swelling and thickness was observed in CIA mice treated with nicotine compared with vehicle treated controls.ConclusionsOur data indicate that nicotine is a potent inducer of NETosis and may play an important role in the initiation and propagation of RA. Furthermore, our study provides molecular evidence for public awareness and clinical guidance against the use