甲羟戊酸通路对皮肤炎症中γδT细胞调控作用的研究

We previously found that the genomic variations on mevalonate pathway can cause porokeratosis. It is well-known that multiple phosphorylated mevalonate metabolites are potent agonists of γδT cells. Thus, we supposed that mevalonate pathway might be involved in the pathogenesis of skin inflammation. In this project, porokeratosis and psoriasis vulgaris are served as paradigms to comparatively study the regulation of γδT cells by mevalonate pathway in skin inflammatory diseases. First, targeted proteomics and flow cytometry are used to detect the abnormal mevalonate metabolites and with accompanying changes of inflammatory cytokines, respectively; Secondly, Seahorse extracelluar flux analyzer and RNA in situ hybridization are applied to identify the intracellular and extracellular dual regulation of γδT cells by mevalonate pathway; Thirdly, CRISPR/Cas9 techniques are used to construct cell models of site-directed mutagenesis in genes on mevalonate pathway, and different groups are designed to intervene in mevalonate pathway at the cellular level, whose direct and indirect effects on the regulation of γδT cells are analyzed. This study would provide new ideas for the treatment of skin inflammation.

我们发现甲羟戊酸通路上的遗传变异可导致汗孔角化症，已知该通路上的多种磷酸化代谢产物能够有效激活γδT细胞，由此，我们推测该通路可能参与皮肤炎症的发生机制。本项目以汗孔角化症和寻常型银屑病为范例，对皮肤炎症性疾病中甲羟戊酸通路调控γδT细胞的作用机制进行比较研究。首先，分别采用靶向蛋白质学技术和流式细胞仪来检测甲羟戊酸通路的异常情况和皮肤炎症反应程度；其次，采用海马代谢分析和RNA原位杂交来明确甲羟戊酸通路调控γδT细胞的内外双重机制；第三, 采用CRISPR/Cas9技术构建甲羟戊酸通路上基因定点突变的细胞模型，在细胞水平上进行分组干预甲羟戊酸通路，分析其对γδT细胞调控的直接和间接影响，为皮肤炎症的治疗提供新思路。

已知甲羟戊酸通路上的多种磷酸化代谢产物能够有效激活γδT细胞，参与皮肤炎症反应。嗜乳脂蛋白3A（butyrophilin3A，BTN3A/CD277）可呈递甲羟戊酸通路多种磷酸化代谢产物，有效激活Vγ9Vδ2 T 细胞。本项目以寻常型银屑病和汗孔角化症为范例，研究CD277对皮肤炎症性疾病中γδT细胞的调控机制。首先，采用流式细胞术检测寻常型银屑病和汗孔角化症患者外周血Vγ9Vδ2 T 细胞数量及致炎功能。结果显示与对照组比较，患者的外周血Vγ9Vδ2 T 细胞数量下降、增殖和炎症因子分泌能力增加；其次，检测外周血CD14+细胞在提呈抗原活化Vγ9Vδ2 T 细胞的能力及其CD277表达水平。结果显示Vγ9Vδ2 T 细胞活化主要依赖于CD14+细胞呈递抗原，患者外周血CD14+细胞数量增加并出现CD277表达水平上调，促炎因子(IFN-γ和TNF-α)可使CD14+细胞CD277表达上调，阻断CD277后患者外周血Vγ9Vδ2 T 细胞过度活化状态被抑制；第三，通过免疫荧光法来验证患者皮损处是否与其外周血的变化相符合。结果显示患者皮损处Vγ9Vδ2 T 细胞浸润增加，CD14+细胞表面CD277表达上调且与Vγ9Vδ2 T 细胞发生密切接触。简而言之，炎症性皮肤疾病中Vγ9Vδ2 T 细胞异常活化与CD14+细胞表面CD277表达上调有关，通过干预CD277表达有望参与调控皮肤炎症，为治疗皮肤炎症性疾病提供新思路。