结核分枝杆菌分泌蛋白HtdY诱导树突状细胞成熟的信号通路及Th1/Th2极化研究

The pathogenesis of tuberculosis is the product of the interaction between bacterial virulence and host resistance. Currently, it is significant to screen and identify key proteins associated with the interaction between Mycobacterium tuberculosis (Mtb) and host, and to investigate how Mtb impact host via these pathogen proteins. Dendritic cells (DCs) are vitally linked to the regulation of innate immune responses transferring to adaptive immunity during Mtb infection. Previous studies demonstrated that mature DCs express high levels of the NF-κB family of transcription factors. In addition, it was reported that mitogen activated protein kinase (MAPK) signaling pathways may play an important role in DCs maturation. Thus, it is assumed that NF-κB and MAPK signaling are rate-limiting step in DCs maturation, and in turn regulate the immune response to pathogen. Previous studies have shown that many of the cell wall-associated or secreted antigens of pathogenic mycobacteria could activate DCs maturation; nevertheless, the information related to the effects of these antigens on the maturation and functions of human DCs, and the underlying signaling events remains scanty. In addition, the diversifications of Thl/Th2 polarization drived by DCs maturation were also detected. Accordingly, it is still unknown how these antigens trigger the protective immune response. Based on our preliminary studies of immunoproteomics, a novel immunostimulatory antigen of Mtb, named HtdY, was found. It is a secretory protein, involving in the biosynthesis of mycolic acids in Mtb. Based on the bioinformatics, the possible antigen epitopes of HtdY were predicted. Further experimental study revealed the immunodominant nature of HtdY antigen, and HtdY protein could not only activate humoral immune response but also cell-mediated immune response. Especially, based on its reactivity with blood obtained from patients with active TB and healthy controls, IFN-γ release assay results showed that HtdY antigen induced a strong immune response compared with ESAT-6 and CFP-10 antigens, which are particularly robust inducers of recall IFN-γ responses. However, there have been no reports on the molecular mechanism of the immunogenicity of HtdY. This project is to investigate the signaling pathways such NF-κB and MAPK in DCs maturation, and polarization of Th1/Th2 cells via flow cytometry, immunofluorescence, and immunoprecipitation assay. Taken together, our findings would strengthen the need to delineate the interaction of bacteria with host to effectuate deeper insights into disease pathogenesis as well as to identify and develop credible vaccine candidates.

系统地筛选和鉴定结核分枝杆菌（Mtb）中与宿主相互作用过程中的关键蛋白，并揭示其诱导树突状细胞（DCs）成熟所涉及的信号通路及其免疫机制是当前国际结核病领域的研究热点。基于结核免疫蛋白组学的研究，我们发现了一种新的免疫优势抗原HtdY。HtdY是一种与Mtb细胞壁毒力因子分枝菌酸生物合成相关的分泌蛋白；通过生物信息学分析预测其具有多个免疫优势表位；进一步实验证明HtdY能体外刺激T细胞产生IFN-γ。据此我们推测HtdY是诱导DCs成熟并参与结核感染免疫过程的重要蛋白质。本研究在此基础上，拟通过细胞形态、细胞表面标记分子、细胞因子释放、受体结合情况和药物处理信号通路关键蛋白变化等分析，深入探索HtdY诱导DCs成熟中NF-κB和MAPK等信号通路及Th1/Th2极化的机制。本研究成果将为揭示结核病发生过程中宿主-病原体相互作用机制提供新的视角，并为结核病新的免疫策略提供理论依据。

结核病是一种由结核分枝杆菌（Mtb）引起的在世界范围内死亡人数最多的感染性疾病。随着耐多药和广泛耐药结核病的急剧增加，卡介苗疫苗的保护效应不稳定，以及结核病与艾滋病共感染患者的增多，探究结核免疫机制及在此基础上寻找新的诊断、预防和治疗措施正日益引起世界各国的重视。本课题组依据项目研究目标任务，验证HtdY是参与结核感染免疫过程的重要蛋白质这一假设。按项目计划，首先进行HtdY重组蛋白制备和鉴定。同时进一步进行HtdY在结核病发展过程的表达确认。此外，进一步对HtdY在结核病人治疗前后病人体内抗HtdY抗原的IgG水平以及HtdY引起免疫C57BL/6小鼠和TB病人体内的抗原特异性T细胞免疫反应进行了研究。对C57BL/6小鼠免疫。初步揭示HtdY 参与的病原体与宿主免疫反应的调控机制，结果显示HtdY 抗原和巨噬细胞相互作用，活化p38MAPK、ERK1/2和JNK1/2 MAPK信号以及NF-κB 信号，促进巨噬细胞上调表达COX-2，参与负向免疫调控。并对Rv3117等RD区特异性蛋白和结核特异性胞壁脂聚糖LAM的免疫原性进行了评估。首次发现LAM能诱导树突状细胞成熟以及Th1细胞的分化，其中国内流行的北京基因型菌株LAM有诱导Th17细胞分化的潜力。在已发现的HtdY蛋白基础上，首次引入仿生亲和解析蛋白质组学BiAC技术筛选结核分枝杆菌CFPs关键蛋白，迄今已有300多个CFPs蛋白通过生物信息学或其他方法确定为分泌型蛋白。并首次揭示了Rv3457c和Rv3248c两个免疫优势蛋白抗原。这些研究为深入揭示结核分枝杆菌特异性抗原对宿主的毒力和致病机制，探索宿主细胞—病原体相互作用的分子机制提供了新的视角，并为结核病新的免疫策略提供理论依据。