LIGHT/HVEM信号调控T细胞活化在特应性皮炎发病中的作用及机制研究

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. It’s widely accepted that T cells plays a crucial role in the onset and progression of AD, however the key point and mechanism in T cells regulation is not clear. Our previous work indicated that LIGHT, as the 14th member of TNFSF, was significantly increased in serum of AD patients than that of healthy controls. The level of LIGHT was positively correlated with SCORAD index and IgE level. The receptor of LIGHT – HVEM, showed higher expression on the T cells of AD patients than that of healthy controls. In addition, LIGHT can promote T cells proliferation and induce production of inflammatory cytokine. In this study, We will (1) investigate the expression and distribution of LIGHT as well as its receptor in AD lesions; (2) confirm the AD acceleration though LIGHT/HVEM pathway and its effect on CD4+ T cells, by injecting recombinant LIGHT or receptor antagonist in Nc/Nga mice model; (3) identify the signaling pathway and mechanism of LIGHT/HVEM cositimulating T cell activity using the methods of the RNAi technology and specific inhibitor in CD4+ T cell line; (4) determine the possibility of LIGHT as a potential therapeutic target for clinical management of AD, by knocking-out LIGHT gene using CRISPR/cas9 technology in Nc/Nga mice. This study will discover a central factor in the mechanism AD and provide a promising way to AD therapy.

特应性皮炎(AD)是一种常见慢性炎性皮肤病，T细胞应答在其炎症启动及维持中发挥重要作用，但T细胞活化调控的关键因子及作用机制尚不清楚。我们前期研究发现肿瘤坏死因子超家族分子LIGHT及其受体HVEM在AD患者中高表达且与病情严重度正相关，并可体外刺激T细胞增殖及分泌细胞因子。本课题拟进一步研究LIGHT/HVEM信号通过调控T细胞活性在AD发病中的作用及机制：1. 明确LIGHT及HVEM在AD皮损组织中的表达分布；2. 通过在Nc/Nga鼠AD模型中过表达LIGHT或阻断其受体，证实LIGHT/HVEM促进AD皮损发生及调控T细胞的作用；3. 采用CD4+T细胞体外培养体系，探索AD发病中该信号通路双向调控T细胞活性的作用及机制；4. 通过CRISPR/cas9技术，敲除鼠AD模型LIGHT基因，验证其作为AD治疗靶点的可行性。本研究将揭示AD发病的新机制并为其治疗提供新方向。

在特应性皮炎（AD, atopic dermatitis）发病机制中，T细胞的应答在其炎症的启动及维持中都起到的关键性作用，其具体机制尚不明确。TNF超家族成员LIGHT（TNFSF14）是共刺激分子，与树突状细胞成熟、T细胞活化和免疫调节密切相关。因此探讨LIGHT调控T细胞的活化在AD发病和进展中的作用研究有重要作用。.本项目分别从动物模型、细胞水平和治疗靶点可行性研究三方面开展。我们首先在人体组织水平证实了LIGHT及其受体在AD患者皮损组织中的高表达分布。以此为探究基础，构建了AD小鼠模型，比较了AD鼠与对照鼠耳组织LIGHT/HVEM及LTβR蛋白量的差异，证实了AD鼠内源性LIGHT/HVEM表达异常，且外源性LIGHT蛋白可进一步影响LIGHT/HVEM的表达。同时，AD鼠给予外源性LIGHT蛋白后，淋巴结Th2细胞比例升高，Th1细胞比例降低。耳组织TNF-α、IL-2、IL-6、IL-4、IL-10、IL—9细胞因子分泌增加、下游炎症信号通路NF-κB、MAPK被激活。给予HVEM阻断剂后，可下调Th2细胞比例，且NF-κB、MAPK中分子的磷酸化激活减少。因此，LIGHT/HVEM信号通路可能在AD的炎症反应中起重要作用。随后，我们从小鼠脾脏分离出CD4+T细胞，与可溶性LIGHT蛋白共培养，分别加入抗HVEM单抗或抗BTLA单抗干预，对比了不同干预情况下，CD4+T细胞的增殖能力和细胞因子分泌能力的变化。结果证实，LIGHT可促进CD4+T细胞增殖，上调TH1、2、17、22相关的细胞因子表达量。而LIGHT/aHVEM或LIGHT/aBTLA共培养可显著抑制CD4+T细胞IL-4的分泌。说明LIGHT通过HVEM促进T细胞的活化，诱导其分化，提高其分泌IL-4的能力。通过本研究，证实了LIGHT调控T细胞的活化在AD发病和进展中发挥了重要作用，有望成为一个新的药物治疗靶点。发表项目标注的SCI论著2篇，英文学术专著1篇，已接收待发表中文核心期刊论文1篇。