日本血吸虫SjP40蛋白Th1型表位肽鉴定及对过敏性哮喘的抑制作用及机制研究

Asthma is a common disease that affects 300 million people worldwide. It is increasingly thought that asthma is a heterogenicity disease and consists of multiple phenotypes, early-onset allergic asthma, late-onset eosinophilic asthma, exercise-induced asthma, obesity asthma and neutrophilic asthma. It is recognized that Th2 cells and their cytokines (IL-4, IL-5, and IL-13) are responsible for initiating and maintaining allergic asthma. . TSLP-DCs-OX40L axis has been recently implicated as potential molecular target against allergic disorders. Thymic stromal lymphopoietin (TSLP) is produced predominantly by damaged epithelial cells. TSLP-activated DCs can promote the differentiation of naive CD4+ T cells into a Th2 phenotype producing IL-4, IL-5, IL-13, and TNF, but not IL-10 in a unique manner dependent on expression of OX40L and lack of production of IL-12.. Currently, it was shown that using Th1 response-inducing antigen could alter the ability of TSLP to induce the inflammatory Th2 type responses, which depended on both repression of OX40 ligand and induction of IL-12 production from DCs. Bacillus Calmette  GueÁrin (BCG), which is the most widely used Th1-inducing vaccine, could inhibit asthmatic reactions by balancing the Th1/Th2 response. Furthermore, it was found that BCG could function as potent inhibitors of OX40L expression on DCs, while they induce IL-12 production by DCs, even in the presence of TSLP. . Several studies showed that SmP40 and its peptide 234-246 from Schistosoma mansoni could elicit a strikingly immunodominant Th1 polarized response in sensitized or acute-infected mice. SmP40 is the most abundant egg component of S. mansoni, with homology to alpha-crystallins and Drosophila small heat shock proteins. SjP40 is the similar egg antigen with SmP40 from Schistosoma japonicum, which is the only specifies of Schistosoma epidemic in China. The amino acid sequence showed about 74.5% identity between SjP40 and SmP40.. In the previous result, 20/10 mer overlapping peptides spanning the whole SjP40 were synthesized. Splenocytes from BALB/c mice infected with S. japonicum cercaria were stimulated with those peptides and IFN-gamma in supernatant was measured by ELISA. Four regions containing Th1 epitopes were identified. Interestingly, we found that three Th1 epitope peptides immunizaiton could induce IL-12 production from DCs and inhibit the allergic asthma response in mice. Based on that, we will further map the epitopes by SjP40 15/12 mer overlapping peptides, and the core residues will be identified by alanine substitute peptides. Then, we will test the effect of Th1 epitope peptides immunization and their adoptive transfer DCs on OVA-induced allergic asthma in mice, and check whether Th1 epitope peptides immunization could inhibit up-regulation of OX40L on DCs in the presence of TSLP and inhibit the generation of Th2 cells. These research will provide a new way for allergic asthma prevention and treatment.

过敏性哮喘是由变应原介导的Th2型免疫反应。当呼吸道粘膜受变应原刺激时可产生大量TSLP，由TSLP激活DCs表达OX40L，是启动哮喘Th2型免疫反应的重要因素。据报道，利用Th1型免疫刺激剂如BCG能诱导DCs表达IL-12，从而对TSLP-DCs-OX40L通路进行靶向抑制。日本血吸虫SjP40蛋白能诱导以Th1型为主的免疫应答。前期实验中我们通过20/10重叠肽确定了SjP40蛋白Th1型表位分布区间；发现Th1型表位肽免疫能够显著减轻OVA诱导的小鼠过敏性哮喘。本研究将利用15/12重叠肽及丙氨酸替代法进一步鉴定SjP40蛋白中Th1型表位肽及其核心氨基酸；并确实其对小鼠哮喘的抑制作用及对Th1/Th2平衡的影响；同时，检测Th1型表位肽免疫对TSLP-DCs-OX40L信号通路的干预作用以及过继转移DCs对小鼠过敏性哮喘的抑制作用，阐明作用机制。这将为过敏性哮喘防治提供新依据。

过敏性哮喘是由变应原介导的Th2型免疫反应。当呼吸道粘膜受变应原刺激时可产生大量TSLP，由TSLP激活DCs表达OX40L，是启动哮喘Th2型免疫反应的重要因素。据报道，利用Th1型免疫刺激剂如BCG能诱导DCs表达IL-12，从而对TSLP-DCs-OX40L通路进行靶向抑制。日本血吸虫SjP40蛋白能诱导以Th1型为主的免疫应答。前期实验中我们通过20/10重叠肽确定了SjP40蛋白Th1型表位分布区间；发现Th1型表位肽免疫能够显著减轻OVA诱导的小鼠过敏性哮喘。本研究利用15/12重叠肽及丙氨酸替代法进一步鉴定SjP40蛋白中Th1型表位肽及其核心氨基酸；并确实其对小鼠哮喘的抑制作用及对Th1/Th2平衡的影响；同时，检测Th1型表位肽免疫对TSLP-DCs-OX40L信号通路的干预作用以及过继转移DCs对小鼠过敏性哮喘的抑制作用，阐明作用机制。本研究顺利鉴定出了日本血吸虫SjP40蛋白的Th1型表位肽P5，P6，P17，P25，P26，P30，并且分析出了其核心氨基酸，并验证了其多小鼠过敏性哮喘的抑制作用。本研究首次报道了日本血吸虫SjP40蛋白中Th1型表位及其核心氨基酸，这将为后续研究提供重要的线索。目前，利用血吸虫感染和血吸虫分泌性抗原对过敏性哮喘进行抑制已有大量文献报道，但是血吸虫感染具有危险性，而其分泌性抗原成分复杂，均不能用于临床研究。本研究从日本血吸虫Th1型表位肽入手，研究其免疫后对小鼠过敏性哮喘的抑制作用，表位肽具有易合成、纯度高、特异性强等诸多优势，具有很强的应用前景，将为过敏性哮喘防治提供新依据。