滤泡辅助性T细胞在DEHP与PM2.5联合暴露诱导哮喘中的作用及机制研究

Environmental pollutants particulate matter (PM) 2.5 and di-(2-ethylhexyl) phthalate (DEHP) are believed to be closely related to an increased prevalence of allergic asthma especially in children. A new subset of T helper cells termed T follicular helper cell (Tfh) has been identified as a key player that potently assists B cells to mediate humoral immune response. Our previous research found that PM2.5 could directly cause hypersensitivity through its contained allergen components. And DEHP could adjuvantly induce humoral immunity hyperfunction in ovalbumin-sensitized mice ascribing to Tfh cells. Therefore we hypothesize that DEHP acts as an adjuvant to promote PM2.5 to induce the imbalanced humoral immune response by disrupting Tfh cells differentiation and function, leading to the occurrence and exacerbation of asthma. In this project, using weanling mice model co-exposed to DEHP and PM2.5, we plan to explore the effects of DEHP on PM2.5 induced-asthma. Meanwhile we intend to analyze the influence of DEHP and PM2.5 co-exposure on Tfh cells differentiation, localization, function and transcriptional regulation by detecting the expression of Tfh cells specific surface molecules, characteristic cytokines and nuclear transcription factors. Furthermore, in order to determine the target cell of immunotoxicity caused by DEHP and PM2.5, we will perform DEHP and PM2.5 co-exposure to CD4 -/- weanling mice reconstituted with normal wild type adoptive Tfh cells, non-Tfh cells or Th2 cells and examine the effects of co-exposure on those transplanted mice. The present study can provide theoretical foundation to clarify the underlying mechanism of asthma induced by DEHP and PM2.5 co-exposure and search for the target of prevention and therapeutic treatment.

环境污染物PM2.5和DEHP都被证实与哮喘有关。Tfh细胞是新确认的辅助B细胞介导体液免疫应答的关键Th亚群。本课题组前期研究发现：PM2.5能通过其含有的致敏原成分直接诱发超敏反应；DEHP可靶向作用于Tfh细胞，以佐剂样作用造成OVA致敏小鼠体液免疫亢进。我们提出假说：DEHP作为佐剂促进PM2.5致敏原诱导Tfh细胞分化和功能紊乱，导致体液免疫失调并引起哮喘发生和加重。本项目建立DEHP与PM2.5联合暴露小鼠模型，探讨DEHP在PM2.5诱导哮喘中的作用；检测Tfh细胞相关分子表达，分析DEHP与PM2.5联合暴露对Tfh细胞分化、定位、功能及转录调控的影响；分别将正常Tfh、非Tfh或Th2细胞过继输入CD4基因敲除小鼠体内后实施联合暴露，确定DEHP与PM2.5联合产生体液免疫毒性作用的靶细胞。本研究为阐明DEHP与PM2.5联合暴露致哮喘作用机制及寻找防治靶点提供理论基础。

环境污染物PM2.5和DEHP都被证实与过敏性哮喘有关。Tfh细胞是新近确认的辅助B细胞介导体液免疫应答的关键Th亚群。本研究旨在从Tfh细胞这个新的切入点探讨DEHP作为免疫佐剂促进PM2.5致敏原诱发和加重哮喘的作用及其机制。首先建立生长期小鼠DEHP与PM2.5联合暴露模型，结果显示DEHP能够发挥类似佐剂样毒性作用促进PM2.5致敏原引起气道反应性升高、支气管肺泡灌洗液中嗜酸性粒细胞和中性粒细胞浸润加剧、肺组织病理损伤加重。鉴于体液免疫应答是过敏性哮喘炎性病变的基础，本研究利用上述动物模型继续观察到DEHP能够以类似佐剂样作用促进PM2.5致敏原引起血清中IgE和IgG1类抗体产生增多、外周淋巴器官CD19+CD138+GL-7+浆细胞和CD4+CXCR5+ICOS+/CD4+CXCR5+PD-1+ Tfh细胞分化增加；进一步分析还发现DEHP能够以佐剂样作用促进PM2.5致敏原引起Tfh胞内细胞因子IL-21和IL-4合成增多、核转录因子Bcl-6和c-Maf表达增加。为了明确DEHP与PM2.5联合产生免疫毒性作用的靶细胞，本研究分别将正常Tfh细胞或非Tfh细胞过继性输入裸鼠体内后实施DEHP与PM2.5联合暴露，结果表明二者联合暴露对气道高反应性、支气管-肺泡炎症反应、体液免疫应答亢进以及浆细胞分化过度的诱导作用可能直接起因于Tfh细胞。由此得出结论：DEHP可作为免疫佐剂促进PM2.5致敏原诱导Tfh细胞分化紊乱和功能异常，其辅助B细胞介导的体液免疫应答失调，产生过多的速发型超敏反应相关抗体，继而引起过敏性哮喘发生和症状加重。本研究为今后评估环境中DEHP与PM2.5联合暴露对人类健康的影响提供了科学依据，也为深入阐明DEHP与PM2.5联合暴露致哮喘作用机制及寻找防治靶点提供了理论基础。