肺泡巨噬细胞CD39/ATP轴调控非嗜酸性粒细胞性哮喘Th17/Treg极化失衡的机制研究

The imbalance of Th17/Treg polarization is a key pathogenesis to non-eosinophilic asthma (NEA), but its regulatory mechanism is still unclear.Our previous study showed that ATP accumulated in the airway of NEA model mouse，activated NLRP3 inflammasome in alveolar macrophages (AM), and produced IL-1β,which promotes the differentiation and polarization of Th17 cell; while the expression of CD39, which can hydrolyze extracellular ATP, was significantly reduced on AM. Further, blocking CD39 expression on AM led to the ATP accumulation in airway and Th17 polarization. Accordingly, we assume that the down-regulation of CD39 expression on AM leads to ATP accumulation in airway, and activates NLRP3 inflammasome in AM, and results in the diseqilibrium of Th17 / Treg polarization. Therefore, this study intends to validate that CD39/ATP axis has a regulatory role in NLRP3 inflammasome activation and Th17/Treg polarization in NEA model mouse; The co-culture experiments of AM and Th0 cells will explore the effects of CD39/ATP axis on regulating Th17 differentiation in vitro; By using the adoptive AM and the interventions in CD39/ATP axis, we ultimately clarify the mechanism of NLRP3 inflammasome-mediated CD39/ATP axis in regulating Th17/Treg polarization of NAE, and provide a new thought in preventing and combating NEA.

Th17/Treg失衡是介导非嗜酸性粒细胞性哮喘（NEA）的关键，但内在调控机制尚不清楚。我们前期研究发现，NEA小鼠气道内ATP聚集，并激活肺泡巨噬细胞(AM) NLRP3炎性小体，产生正向调控Th17分化的IL-1β，促进Th17极化；而ATP水解酶CD39在AM上表达明显下调，进一步阻断小鼠体内CD39，则加重气道内ATP聚集以及Th17极化。据此假设，AM通过下调CD39，导致气道内ATP聚集，激活NLRP3炎性小体，促进Th17/Treg失衡。故本课题拟明确NEA小鼠肺泡巨噬细胞CD39/ATP轴与NLRP3炎性小体表达、Th17/Treg失衡的关联；体外AM及Th0共培养探讨CD39/ATP轴调控Th17分化的作用机制；体内AM过继并干预CD39/ATP轴，最终阐明NLRP3炎性小体介导肺泡巨噬细胞CD39/ATP轴调控Th17/Treg极化失衡的机制，为防治NEA提供新思路。

背景：Th17/Treg失衡是介导非嗜酸性粒细胞性哮喘（NEA）的关键机制，但内在调控机制尚不清楚。.主要研究内容：本研究着重探讨CD39/ATP轴以及阻断HMGB1信号调控非嗜酸性粒细胞性哮喘Th17/Treg极化失衡的机制。.重要结果：本研究通过动物实验发现和证实外源性ATP是导致非嗜酸性粒细胞哮喘气道炎症和Th17极化的重要介质，阻断ATP可以减少中性粒细胞气道炎症、气道高反应性及Th17细胞反应，ATP有可能成为非嗜酸性粒细胞性哮喘的标志物；本研究通过动物实验发现和证实运用sRAGE阻断HMGB1可以改善非嗜酸性粒细胞哮喘气道炎症和Th17极化，HMGB1有可能成为非嗜酸性粒细胞性哮喘的标志物或者靶标。.关键数据：（1）非嗜酸性粒细胞性哮喘小鼠肺泡灌洗液中ATP水平明显升高。体外实验中ATP可以促进CD4+T细胞向Th17细胞分化。在非嗜酸性粒细胞性哮喘小鼠抗原激发前给予ATP受体阻断剂suramin阻断ATP信号，可以减轻小鼠气道的中性粒细胞性炎症、气道高反应性及肺组织中Th17的极化失衡。ATP酶中和气道内ATP后同样可以减轻中性粒细胞气道炎症。因此，非嗜酸性粒细胞哮喘小鼠模型中ATP明显增加，ATP可以促进Th17的极化，ATP抑制剂减轻非嗜酸粒细胞哮喘小鼠的气道炎症。（2）前期研究已发现能非嗜酸性粒细胞性哮喘小鼠肺组织HMGB1信号表达显著增强。后期进一步研究发现非嗜酸性粒细胞性哮喘小鼠肺泡灌洗液中sRAGE水平明显降低。非嗜酸性粒细胞性哮喘小鼠抗原激发前给予sRAGE阻断HMGB1信号后，可以减轻小鼠气道的中性粒细胞性炎症以及肺组织中Th17的极化失衡。sRAGE可以通过抑制气道CD11c+抗原提呈细胞RAGE、IL-23的表达来产生抗哮喘作用。在体外，sRAGE抑制rHMGB1激活的树突状细胞诱导的Th17细胞极化。在小鼠哮喘模型中回输rHMGB1和sRAGE活化的CD11c+DC细胞明显减轻气道中性粒细胞炎症。因此，非嗜酸性粒细胞哮喘小鼠模型中sRAGE缺乏，从而HMGB1信号亢进，促进Th17的极化和气道中性粒细胞性炎症，是非嗜酸粒细胞哮喘小鼠重要发病机制。.科学意义：本课题揭示了ATP和HMGB1信号在非嗜酸性粒细胞哮喘发病中的作用和机制，为临床治疗NEA提供潜在靶点和治疗新策略。