LILRA3对炎症性肠病肠黏膜内巨噬细胞激活的作用及机制研究

Inflammatory bowel disease (IBD) is relapsed and its long duration makes it hard to cure. Its pathogenesis remains unclear. Recently it is thought that genetic susceptibility and abnormalities of immunity play important roles in IBD by genome wide association study. The novel immunoregulatory molecular LILRA3 is associated with many autoimmune diseases. Our preliminary study has shown that LILRA3 gene polymorphisms were associated with IBD and its expression was much higher in intestinal macrophages of IBD patients as well as colons of rat with colitis model. We also found that LILRA3 could decrease TNF-α, IFN-γ secretion in monocytes. Accordingly, we speculate that LILRA3 may be involved with IBD pathogenesis, whereas there are no studies reporting the relationship between LILRA3 and IBD. The present study aims to demonstrate the relationship between LILRA3 and IBD in a larger cohort study, to detect LILRA3 expression in different types of immunocytes by Flow cytometry. Moreover, both LILRA3 knock-down and over-expression cells will be established to investigate the specific role of this gene in inflammation. Levels of inflammatory cytokines and key enzymes in related inflammatory signaling pathway will be detected. We will construct a LILRA3 transgene rat model with adeno-assioated virus (AAV) to further identify the role of LILRA3 in inflammation by inducing colitis with TNBS. Co- immunoprecipitation and mass spectrometry will be applied to explore the possible receptors for this gene. We then knock out the candidate receptor using CRISPR/Cas9, and the related inflammatory cytokines and key enzymes in inflammatory signaling pathway will be tested again to clarify the molecular mechanism of LILRA3 in IBD, and thus providing certain theoretical basis for new drug discovery for IBD.

炎症性肠病(IBD)发病机制不明，遗传易感性和免疫异常发挥重要作用。新型免疫调节分子LILRA3与IBD的关系尚无文献报道，我们前期研究发现该基因基因多态性与IBD相关，且主要表达于肠道巨噬细胞，在病人和肠炎大鼠体内高表达，能抑制单核细胞分泌TNF-α、IFN-γ，据此我们推测该基因可能参与IBD发病。本项目将通过扩大样本明确LILRA3同IBD的关系，构建LILRA3敲低和过表达巨噬细胞，检测炎症因子、炎症相关信号通路蛋白变化情况，探索该基因在巨噬细胞激活及肠道炎症反应中的作用，AAV腺相关病毒构建LILRA3转基因大鼠，制备结肠炎模型，动物水平进一步验证该基因在炎症反应中的作用，蛋白质谱法研究LILRA3的可能受体，CRISPR/Cas9敲除候选受体，观察敲除前后相关炎症因子、信号通路蛋白变化情况，阐明LILRA3调控巨噬细胞激活和肠道炎症反应的分子机制，为IBD新药研发提供重要思路。

LILRA3被报道同多种自身免疫性疾病相关，很可能是一种新的免疫调节因子。它与炎症性肠病（IBD）的关系尚无研究报道。我们课题组利用飞行质朴方法分析克罗恩病（CD）、溃疡性结肠炎（UC）患者以及健康对照组外周血基因组进行测序发现，LILRA3基因SNP位点 rs410852与中国IBD患者显著相关。收集IBD患者及健康对照者外周血及肠粘膜标本，RT-PCR提示LILRA3基因水平在CD、UC均显著增高。进一步免疫组化及westernblot实验发现同对照组相比，其蛋白水平也显著增高，免疫组化提示LILRA3主要表达在肠粘膜固有层炎症细胞，为了进一步明确LILRA3的细胞定位，我们采用特异性荧光抗体分别标记不同免疫细胞及LILRA3，荧光双标提示LILRA3主要表达在固有层巨噬细胞，这与之前的研究报道一致。接下来我们研究了LILRA3是如何调控巨噬细胞从而参与IBD发病机制的，我们首先利用慢病毒转染方法构建LILRA3过表达U937单核细胞株，发现LILRA3过表达后，细胞分泌TNF-α、IFN-γ、IL-6减少，相反IL-10分泌增多，提示该基因具有一定的抗炎作用。进一步功能研究发现，LILRA3过表达后可以增加单核细胞表面CD36分子数量促进其吞噬功能；抑制单核细胞分泌CCL2、CCL3及IL-8等驱化因子抑制其迁移能力；除此之外，它还可以通过同时活化PI3K-AKT、PI3K-MEK信号通路促进单核细胞增殖。通过予SD大鼠腹腔注射重组LILRA3蛋白发现，LILRA3可以减轻DSS诱导的大鼠肠道炎症，具体表现为炎症细胞浸润减少，肠粘膜组织完整性较好，且结肠长度显著长于对照组。.综上所述，我们的研究提示LILRA3做为一种抗炎因子在IBD疾病发生发展过程中发挥重要作用，该发现为IBD药物治疗提供了新的思路和方向。