The Mechanisms Of Cholinergic Anti-inflammatory Pathway In Protecting Rheumatoid Arthritis:from The Perspective Of Helper T Cell Differentiation

Part1The effect of vagus nerve and acetylcholine receptor agonist nicotine on the distribution of helper T cells in collagen induced arthritisObjectives. The cholinergic anti-inflammatory pathway can inhibit the inflammation of collagen induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). However, the immunologic mechanisms that provide a therapeutic effect against the auto-inflammatory disease are not yet elucidated. The present study explores the effect of cholinergic anti-inflammatory pathway on helper T cell (Th cell) distribution in CIA.Methods. Forty DBA/1mice were divided into4groups:a control group, a CIA group, a vagotomy group, and a nicotine group. The degree of arthritis was measured by arthritis score and hematoxylin&erosin. ELISA was used to detect the serum concentration of IFN-γ, IL-4and IL-17A. Flow cytometry was used to detect the cytokines and transcription factors (TFs)(the TFs of Thl, Th2, and Th17cells are T-bet, RORγτ and GATA3respectively) in the spleen. Immunohistochemistry was used to analyze RORyi expression in the joint synoviumResults. Arthritis in the nicotine group was significantly lightened compared with that in the CIA group and in the vagotomy group. Nicotine attenuated Th17lineage by reducing IL-17A production and RORγτ expression. The expressions of IL-4and GATA3were increased in the same setting. However, the expressions of IFN-y and T-bet had no difference between the nicotine and the CIA group. Nicotine induce a shift to the Th2lineage and improve the Thl/Th2imbalance.Conclusions. Nicotine ameliorates the clinical arthritis of CIA. Nicotine reduces Th17cell distribution in the peripheral blood, spleen and synovium of CIA, increases the Th2cell distribution in the peripheral blood and spleen, but has no effect on the Th1cell distribution, nicotine improves the Th1/Th2imbalance in CIA.Part2Effect of acetylcholine receptor agonists on differentiation of Th17cells from CD4+T cells of patients with RAObjectives.Previous researches found that nicotine can inhibit the inflammation of rheumatoid arthritis (RA). However, the immunologic mechanisms that provide a therapeutic effect against the auto-inflammatory disease are not yet completely elucidated. The effect of acetylcholine receptor agonists on Th17cells differentiation, which play an important role in the inflammation associated with RA has not been extensively studied in humans.Methods. In this study, peripheral blood mononuclear cells (PBMCs) and CD4+T cells were separated from treatment-naive patients with RA. We investigated the effect of nicotine and GTS-21on PBMCs stimulated with anti-CD3/anti-CD28and CD4+T cells in the context of Th17cell differentiation. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to analyze IL-17A+T cell percentage and IL-17A level, and western-blot was employed to detect the level of extracellular signal regulated kinase1/2(ERK1/2) and Th17specifical transcription factor retinoic orphan receptor c (RORc).Results. The results showed that nicotine and GTS-21reduce IL-17A levels in stimulated peripheral blood mononuclear cells (PBMCs) from treatment-naive patients with RA. Meanwhile, under conditions of Th17cell differentiation, nicotine and GTS-21reduced the percentage of IL-17A+CD3+CD8-Tcells and decreased IL-17A production in culture supernatant. Furthermore, nicotine and GTS-21inhibited the expression of the Th17cell-specific transcription factor RORc. The effects of nicotine and GTS-21were blocked by the a7-nicotinic acetylcholine receptor (a7nAchR) antagonist α-bungarotoxin (α-Bgt), which increases the expression of IL-17A and RORc. Nicotine induced ERK1/2phosphorylation. MEK1/2blocker reversed the activating effect of nicotine on ERK1/2.Conclusions. Nicotine and GTS-21suppress RA Th17cell differentiation through activation of a7nAchR, and the effect of nicotine is related to the MEK1/2-ERK1/2pathway.Part3Effect of acetylcholine receptor agonists on differentiation of Thl cells and Th2cells from CD4+T cells of patients with RAObjectives. To probe the effect of acetylcholine receptor agonists nicotine and GTS-21on differentiation of Thl cells and Th2cells from CD4+T cells of patients with RA.Methods. Freshly separated PBMCs from RA patients was added different concentration of nicotine and GTS-21.The level of IFN-γ and IL-4from culture supernatant was detected by ELISA. CD4+T cells were separated from RA patients by magnetic beads, added nicotine or GTS-21in Thl and Th2polarizing mediums30minutes after addition of a-bungarotoxin (a-Bgt). The level of IFN-γ and IL-4in the culture supernatant and the ratio of Thl cells and Th2cells were respectively tested by ELISA and flow cytometry. T-bet and GATA3was measured by Western blot.Results. GTS-21, but not nicotine reduced the level of IFN-y in culture supernatant of PBMCs. GTS-21reduced the percentage of IFN-y+T cells, IFN-y level and T-bet expression in RA CD4+T cells during Thl differentiation, while a-Bgt reversed the inhibitory effect of GTS-21on Thl differentiation. Nicotine and GTS-21increased the level of IL-4in culture supernatant of PBMCs and upregulated IL-4+T cell percentage, IL-4level and GAT A3expression in RA CD4+T cells during Th2differentiation, while a-Bgt reversed the effect of nicotine and GTS-21.Conclusions. GTS-21but not nicotine inhibits the differentiation of Th1through activation of a7nAchR; nicotine and GTS-21promote the differentiation of Th2through activation of a7nAchR. Both improve the balance of Thl/Th2.