Investigation Of The Mechanism Of Cholinergic Anti-inflammatory System In Central Nervous System Autoimmune Disease

Objective:The exogenous and endogenous cholinergic system is crucial for the maintenance of the immune homeostasis and keeps balance against the over-activated inflammatory responses.The central nervous system autoimmune disease comes along with the infiltration of peripheral immune cells,which lead to the loss of myeline basic protein?MBP?and causes multiple sclerosis?MS?.As the exogenous agonist of the cholinergic receptor,nicotine could efficiently ameliorate infiltrated immune cells and alleviated inflammatory damage.This project investigated on the effect of the exogenous cholinergic system on the immune cells development,differentiation and migration;and also further explored the endogenous cholinergic system of NK cells,which are finely modulated along with the disease progression and act as an endogenous factor to maintain immune homeostasis.This research provides evidences for better understanding the cholinergic system and leads to the exploration of new therapeutic strategy.Methods:?1?The pumps with nicotine were put subcutaneously in mice with EAE to investigate the exogenous cholinergic effect on the clinical manifestation and immune cells phenotypic distribution.?2?Sorted NK cells from MACS or FACS were collected and indentified of the cholinergic system existence by PCR and Western blot.?3?EAE was induced in Ch AT-e GFP mice for Ch AT expression compared between normal and EAE mice.?4?Ch AT+ and Ch AT-NK cells were collected for the whole genome analysis and enrichment analysis.?5?Ch AT+ and Ch AT-NK cells were implanted into the cerebral ventricles of CX3CR1-mice for the comparison of their functions in CNS microenvironment.?6?The dynamic distribution of immune cells and the subsets in CNS were detected by FACS analysis.?7?The immunofluorescent staining and transwell experiments were conducted to shed light on the co-localization and chemoattraction of Ch AT+ NK cells and CCR2+ Ly6 Chi monocytes.?8?The immunofluorescent staining was performed for the identification of the ?7n ACh R and Perforin distribution with the synapses.?9?The intracellular concentration of acetylcholine in peripheral NK cells of MS patients was detected by HPLC-MS/MS.Results:?1?Nicotine could efficiently reduce the infiltration of neutrophils and CCR2⁺Ly6C^hi monocytes as the exogenous cholinergic agonist.?2?NK cells possessed the entire cholinergic system for acetylcholine synthesis,secretion and metabolism.?3?Ch AT expression in NK cells increased in EAE,and the expression was higher in periphery than in the bone marrow.?4?Ch AT expression was closed related to the expression of CX3CR1 and NKG2 A.?5?The whole genome analysis of Ch AT+ NK cells and Ch AT-NK cells revealed that they were distinguished in cytokine and chemokine production.?6?Ch AT+ NK cells implantation into the cerebral ventricles of CX3CR1-mice further confirmed the immune regulatory effect of those cells through inhibition of the CCR2⁺Ly6C^hi cells.?7?Ch AT+ NK cells and CCR2⁺Ly6C^hi formed immune synapse with the polarization of ?7n ACh R and Perforin.?8?The loss of immune tolerance towards Ch AT+ NK cells was due to the down-regulation of Qa-1 on CCR2⁺Ly6C^hi cells,which was mediated by the cholinergic effect from Ch AT+ NK cells.?9?The up-regulation of Ch AT expression in NK cells were observed in the peripheral blood of MS patients,with the most obvious increase in the CD56 bright subset.Conclusion:?1?The exogenous cholinergic system alleviated CNS inflammation through inhibiting immune cells infiltration.?2?The immune regulatory NK cells of the CNS were capable for acetylcholine synthesis and secretion.?3?Ch AT+ NK cells exert distinct immune regulatory effect through inhibiting CCR2⁺Ly6C^hi cells.?4?The cholinergic system of NK cells under went up-regulation in MS patients,which showed positive correlation with the severity of disease.