肽段免疫抑制嗜碱性粒细胞对哮喘气道炎症防治作用研究

Asthma is characterized by chronic airway inflammation, which is mainly mediated by Th2 immune response. Peptide immunotherapy (PIT) could induce immune tolerance without adverse reactions which were observed in specific immunotherapy, however, the understanding of the mechanism of peptide immunotherapy (PIT) is limited. Recently, the basophils were found that it not only was an effector cell but also could initiate Th2 immune response, enhance immune memory, and result in the inflammation in asthma. In our preliminary experiment, when basophils were deleted, the airway inflammation induced by OVA alleviated markedly, Th2 cell differentiation was inhibited, as well as OVA-specific IgE level decreased, which indicated that this cell could promote cell immune and humoral immunity in asthma. While after subcutaneous injection with OVA323-339MAP, the airway inflammation alleviated, along with the number of basophils decreased, the activity and antigen present function attenuated. We postulate that PIT may play its protected role by influencing the basophills function, and then inhibiting Th2 cell differentiation and B cell function. This project will validate this hypothesis, and these findings will provide us new directions to investigate the mechanism of PIT.

支气管哮喘是以Th2反应为主、由细胞与体液免疫共同参与的气道炎症。肽段免疫治疗（PIT）既能诱导对特异性抗原的耐受，又可避免类似特异性免疫治疗（SIT）引发的不良反应，但其诱导耐受的机制尚未完全明了。新近发现嗜碱性粒细胞不仅作为效应细胞参与哮喘发病，还可启动Th2反应，强化免疫记忆应答，导致炎症迁延不愈。我们前期实验发现：嗜碱性粒细胞剔除小鼠，OVA诱发的气道炎症明显减轻，Th2细胞分化受抑，OVA特异性IgE下降，提示该细胞在哮喘中可促进细胞及体液免疫应答； 采用皮下注射OVA323-339MAP治疗后气道炎症减轻，嗜碱性粒细胞数量、活性及抗原递呈功能均受抑。据此我们推测PIT可能通过影响嗜碱性粒细胞抗原递呈功能，抑制Th2分化并干扰对B细胞的促进作用而发挥保护效应。本项目拟通过动物模型及体外细胞培养对上述假设进一步验证，以期为PIT的作用机制提供新视角，为临床治疗提供新思路。

本项目在前期工作基础上，进一步对多聚抗原肽（MAP）- OVA323-339作为肽段免疫治疗（PIT）的手段，干预小鼠过敏性气道炎症中的作用进行了探讨。首先通过体内实验，摸索了MAP-OVA323-339 干预效应以及保护小鼠气道炎症反应的最佳剂量，分析了量效关系。进一步发现MAP-OVA323-339 干预还可抑制Th2免疫应答，有助于调节小鼠体内Th1/Th2免疫平衡。此外，MAP-OVA323-339不仅能降低血清中OVA特异性IgE水平，还可上调OVA特异性IgG1的表达，提示MAP-OVA323-339可通过调节免疫球蛋白类别转换而诱导免疫耐受。随后，我们利用小鼠过敏性气道炎症模型发现，BASO在始动Th2免疫应答，摄取并递呈抗原方面发挥重要作用，而通过MAP-OVA323-339 干预后，小鼠外周血、纵隔淋巴结及肺组织中BASO的数量减少，活性下降，细胞表面MHC-II类分子和共刺激分子CD40、CD86的表达显著下调，抗原摄取能力减弱；且将经MAP-OVA323-339处理后的BASO回输至正常小鼠体内的促炎性也明显低于未经干预组。继而通过体外实验显示，MAP-OVA323-339 干预后，BASO促Th2细胞分化的作用明显受抑。在此基础上，我们体外模拟了Th2免疫应答时局部微环境，发现IL-4和IgE的大量存在可增强BASO的抗原摄取能力，验证了BASO在Th2免疫应答中可发挥抗原递呈的作用。为更深入探讨MAP-OVA323-339对BASO的干预效应机制，我们计划外观察到经MAP-OVA323-339干预后，小鼠肺组织中BASO成熟所需的关键细胞因子IL-3的表达下调，结合体外实验中IL-3可增强BASO抗原摄取能力的结果，推测MAP-OVA323-339可能通过下调IL-3水平抑制嗜碱性粒细胞的成熟及功能；此外我们进一步发现，通过MAP-OVA323-339 干预，可上调小鼠肺组织IL-10水平以及嗜碱性粒细胞表面IL-10受体水平，而体外实验也证实IL-10的存在可明显抑制BASO的抗原摄取能力，故推测IL-10/IL-10R途径可能是MAP-OVA323-339对BASO发挥抑制作用的重要机制，有待进一步探讨。总之，本项目对PIT的作用机制研究提供了新的视角，并为临床应用奠定了实验基础。