IL23R致病变异通过阻断信号转导抑制Th17细胞免疫应答在麻风发病中的分子机制研究

IL23R has been identified as one of the susceptibility genes of leprosy. Extensive high-throughput re-sequencing has determined the rare variant rs76418789 (G149R), which was located in the coding region of IL23R, might be the causal variant regarding to the IL23R region. Analysis of IL23R G149R protein structural suggests that this coding variant has a strong damaging effect on the protein structure, indicating that the rare variant may be involved in pathogenesis of leprosy via influence the function of IL23R molecule, but the exact mechanism remains unclear. To answer that question, rs76418789 (G149R) will be genotyped in 3000 leprosy cases and 3000 health controls to validate the genetic signal. Furthermore, other molecular experiments, such as RT-PCR, immunochemistry staining, flow cytometry and immunofluorescence will be conducted to clarify how the candidate causal variant affects the function of IL23R. The mechanism of candidate causal variant regarding to the phenotype analysis of the formation granuloma and relative genes expression will be explored by using gene knock-down Zebrafish. This study will greatly advance the biological understanding of leprosy as well as other mycobacterial infectious diseases.

课题组前期发现IL23R为麻风的易感基因，该基因的深度测序显示其编码区域的变异位点rs76418789与疾病的相关性最强，蛋白结构分析结果也发现该变异影响IL23R蛋白的结构稳定性，提示rs76418789可能通过阻断IL23R信号转导进而导致疾病的发生，但具体机制有待实验证实。后续课题组拟在3000例麻风和3000例正常对照中对测序所得变异位点进一步验证以明确致病变异，而后利用RT-PCR、免疫组化、流式细胞术和免疫荧光等技术阐述候选致病变异对IL23R基因功能的影响；同时利用吗啉代反义寡核苷酸Knock-down斑马鱼在体内条件下探讨候选致病变异对麻风肉芽肿形成及相关基因表达的影响，以最终阐释麻风的发病机理，为疾病风险预测、疾病干预等累积基础。

麻风是由麻风分枝杆菌感染易感个体、特异性破坏皮肤与外周神经系统，晚期可致残的一种慢性传染病，以往研究证实其具有很强的遗传性，遗传度达到57%。课题组前期进行了系列麻风遗传学研究，发现IL23R为麻风的易感基因。深度测序显示，IL23R基因编码区域的变异位点rs76418789（G149R）与疾病的相关性最强，然后我们在3000例麻风及3000例对照中进一步证实了rs76418789即为麻风的致病变异。课题组通过蛋白结构分析发现该变异影响IL23R蛋白的结构稳定性，可能通过破坏/阻断IL-23/IL23R信号转导进而导致疾病的发生。接下来课题组利用qPCR和免疫印迹技术对麻风和正常对照的外周血及皮肤组织中IL23R的表达模式进行分析，发现麻风皮损部位IL23R表达明显上调，可是由于麻风分枝杆菌感染所致，也证实了该基因参与到麻风发病过程中；为了进一步证实以上现象，我们利用海鱼分枝杆菌诱导外周血PBMC细胞，发现在感染6-12小时后，IL-17A、IL-17F、IL-22及IL23R细胞因子表达明显升高，充分证实了Th17细胞参与到机体对麻风分枝杆菌的免疫反应中；然后为探索rs76418789（G149R）对IL23R基因功能的影响，我们通过流式细胞术对IL-23诱导的Th17细胞的增值与分化进行了检测，发现rs76418789（G149R）可明显减低IL23R的反应性，导致Th17细胞分化降低。以上结果为最终阐释麻风的发病机理，为疾病风险预测、疾病干预等累积了基础。