Background:The autoimmune regulator gene (AIRE) has very important role in self-tolerance. Estrogen induced hypermethylation of AIRE promoter was put forward as one of the reasons for susceptibility to autoimmunity in females. [1] Sulphasalazine is a commonly used drug in rheumatology and is known to be responsible for various autoimmune side effects.Objectives:We aimed to investigate the effects of commonly used treatments on the methylation status of AIRE and levels of interleukin-16 (IL16), interleukin-1β (IL1β), and interferon-γ (IFNγ), in lipopolysaccharides (LPS)-induced RAW264.7 macrophage cells which mimic inflammatory state in rheumatoid arthritis in vitro.[2]Methods:RAW264.7 cells were stimulated by LPS (1μ/mL). Cell viability test was performed to determine drug concentrations. Following drug treatments, cell media was isolated for the determination of IL16, IL1β, and IFNγ levels by ELISA. Also, the cells detached by trypsin-EDTA were used for DNA isolation and bisulfite modification. Then, the promoter methylation status of AIRE was analyzed with methylation-specific PCR and agarose jel electrophoresis.Results:Our results demonstrated that the AIRE promoter is highly methylated in absence of any inflammatory stimulus.LPS treatment changed methylation status to unmethylated form. Leflunomide(LEF), sulfasalazine(SLZ) and methotrexate(MTX) significantly altered methylation status as the methylated (p=0.041,p<0.001,p<0.001, respectively). Surprisingly, SLZ treatment caused significantly more methylation in AIRE promoter than other drugs (p<0.001,Fig 1A). Analysis of IL16, IL1β and IFNγ levels demonstrated that LPS caused potent increase in all three cytokines (p<0.001, Fig 1B-D). LEF and SLZ significantly prevented LPS-induced increase in these cytokines (p=0.025, p<0.001 for IL-16, p<0.001 for IL-1β and IFNγ, Fig 1B-D). Although MTX exacerbated LPS-induced increase in IL16 levels, it inhibited LPS induced increase in IL1β and IFNγ levels (p<0.001, Fig 1B-D).Figure 1.PCR gel image. All data expressed as mean±SD. ***p<0.001 versus control, ###p<0.001, #p<0.05 versus LPS, +++p<0.001 versus sulfasalazine.Conclusion:Our results suggest that although LEF,SLZ and MTX potently suppresses LPS-induced inflammatory cytokines, they affect AIRE methylation differently.Changes in the methylation status of AIRE are important for autoimmunity. Relative increase in methylation of AIRE promoter by SLZ usage can be responsible for its autoimmune side effects, like drug induced lupus or hypersensitivity reactions, which are not common in MTX and LEF usage.IL16 is a key regulator of biological properties of CD4+T cells, it also regulates migration of CD4+CD25+Treg cells.[3] MTX treatment was shown to increase Treg cells in early rheumatoid arthritis patients. LEF and SLZ’s effect on Treg cells was shown to be different from that of MTX.[4] Our results demonstrated that MTX exacerbated LPS-induced increase in IL16 levels in contrary to LEF and SLZ. This difference may also be responsible for the different effect of these medications on Treg functions.The different effect of commonly used disease modifying drugs on IL16 levels and methylation of AIRE promoter is interesting and deserves attention.References:[1]Berrih-Aknin, Le Panse R, Dragin N. AIRE:a missing link to explain female susceptibility to autoimmune diseases. Ann.N.Y.Acad.Sci, 2018;1412(1):21-32.[2]Lin Y-Y, Jean Y-H, Lee H-P et al. Excavatolide B attenuates rheumatoid arthritis through the inhibition of osteoclastogenesis. Marine Drugs, 2017; 15(1):9.[3]Skundric DS, Cruikshank WW, Montgomery PC et al. Emerging role of IL-16 in cytokine-mediated regulation of multiple sclerosis. Cytokine, 2015;75(2):234-248.[4]Oh JS, Kim Y-G, Lee SG et al. The effect of various disease modifying anti-rheumatic drugs on suppressive function of CD4+CD25+ regulatory Tcells. Rhematol Int. 2013;33:381-388.Disclosure of Interests:None declared
Screening system for drug-induced arrhythmogenic risk combining a patch clamp and heart simulator
