AHR通路协同下的NLRC4炎症小体在低过敏原性免疫毒素PEA残存的超敏反应中的作用机制

The clinical application of Pseudomonas aeruginosa exotoxin A (PEA) has been studied for several decades, with the clinical efficacy debated by its high allergenicity. Our previous work showed that the mutants (mPEA), obtained through amino acid substitutions which reduces the HLA binding affinity, induced a significantly lower level of IL-5 and IL-13 compared to wtPEA, while that of IL-4 and IL-6 also declined but still significantly exceeded the blank control. We therefore hypothesize that the residual hypersensitivity may have resulted from the densely populated aromatic amino acid residues and/or leucine residues in certain segments of mPEA endocytosed by epithelial and/or dendritic cells, through activation of the aryl hydrocarbon receptor (AHR) pathway and the NLRC4 inflammasome. In this study, mPEA will be used as the basic template and control. Two types of short peptides composed of different aromatic amino acid and/or leucine residues, and ns(mPEA), nanoscale particles assembled by immunostimulatory DNAs and mPEA, will be used along with their counterpart controls to stimulate the cell lines 16HBE and THP-1 and conditional knock-out mice. The cellular responses will be monitored in real-time by LightSheet Fluorescence Microscopy, accompanied by the investigation of the indicators including CRYP1A1, IL-1β, and caspase-1, etc., in order to confirm the involvement of these two inflammatory events, relating to both AHR and NLRC4 inflammasome, and the causality between the specific peptide(s) and the events. Humanized mice with specific HLA genotypes will be created by adoptively transferring urine stem cells from specific allergic patients to investigate any crosstalk between these two pathways. In conclusion, this study aims to uncover the mechanism by which mPEA induces the residual hypersensitivity, and thus will provide new therapeutic targets and agents for the treatment of allergy.

绿脓杆菌外毒素PEA抗原性高副作用强而临床效用差。前期分析改造获低过敏原性突变体mPEA，IL-5、IL-13产率极显著降低并与空白对照持平，IL-4、IL-6产率虽显著降低却显著高于对照。故认为：内吞入上皮细胞后，mPEA中密集的芳香族残基/亮氨酸残基启动多环芳香烃受体AHR通路、激活NLRC4炎症小体而导致残存的超敏反应。本研究以mPEA为基本模板及对照，合成两类残基不同配置的短肽、免疫刺激性DNA装载无超敏反应的纳米材料ns(mPEA)为试材，刺激细胞系16HBE和THP-1及条件敲除小鼠，LightSheet记录细胞命运，检测CYP1A1、IL-1β、Caspase-1等指标，确证AHR、NLRC4两类炎症事件发生及与特定肽的关联；构建特异HLA基因型的人源化小鼠，基于多个成对材料发掘其间的级联通路，揭示mPEA诱导残存超敏反应的机制，为变态反应哮喘的治疗提供新靶点及新材料。

基于绿脓杆菌外毒素A（PEA）的重组免疫毒素治疗会使患者立即产生超敏反应。我们在前期发现过敏原性弱化后的PEA仍存在残存的超敏反应，提示PEA可能通过非经典途径引起过敏。因此，本项目旨在研究PEA参与非经典途径下超敏反应的机制。我们的结果如下：（1）利用内毒素功能缺陷的ClearColi® BL21 (DE3)原核表达系统表达野生型重组PEA（rPEA）和过敏原性弱化的突变型PEA（rPEA-I241D），并纯化获得目的蛋白。（2）首次利用rPEA成功构建小鼠哮喘模型。rPEA激发的小鼠出现气道高反应性、严重气道炎症和气道重塑特征的重度哮喘。嗜中性粒细胞、II型固有免疫细胞（ILC2）和IL-17A+细胞在rPEA激发的小鼠中显著升高，产生non-Th2细胞来源的非典型炎症混合细胞因子（IL-4、IL-5、IL-13和IL-17A）。（3）与野生型的rPEA相比，在rPEA-I241D激发的小鼠中BALF的中性粒细胞和肺实质中的炎性细胞数量以及肺组织匀浆中IL-23、IL-4、IL-5和IL-13的水平显著减少。在体外培养的TDDC细胞中检测到rPEA-I241D的刺激导致部分细胞因子（IL-23、IL-6和IL-1β）水平降低。这些结果验证了过敏原性弱化后的rPEA可以减轻炎症反应，但仍无法完全消除炎症。（4）在多环芳烃受体（AHR）抑制剂CH223191处理小鼠后，rPEA激发的小鼠的BALF炎性细胞数量和肺组织炎性细胞浸润没有变化。然而CH223191可以抑制小鼠的Th17细胞的产生，这提示了CH223191可能影响Th细胞的极化。（5）rPEA激发的小鼠中炎症小体极少被激活，表明炎症小体对rPEA引起的炎症贡献甚微。（6）rPEA激发的小鼠中AHR阳性的巨噬细胞和树突状细胞水平上升。（7）rPEA刺激TDDC细胞后，我们检测到AHR通路被激活、色氨酸代谢酶、KYN、IL-1β和HLA-DR的水平上调，并且rPEA通过溶酶体途径被TDDC内吞。这些结果表明，rPEA通过溶酶体途径被DC细胞内吞，经过降解后进入色氨酸代谢，其代谢产物激活AHR通路，并促进IL-1β和HLA-DR的表达，从而参与哮喘的发生。因此，过敏原PEA可以通过激活AHR通路参与残存的超敏反应。