Gaq对Th17细胞分化的调控及其调控的分子机制研究

Galphaq, the alpha subunit of Gq protein, is ubiquitously expressed in mammalians. Gq transduces many external signals into the cell, and controls many biological functions. Its important roles in centrol nerve system, cardiovascular system, endocrine and metabolism were well addressed. However, the roles of Gaq in the immune system were not well addressed until 2007. With Gnaq knockout mice, we first studied the role of Gaq in immune regulation. We found that, Gaq controls DC and neutrophils migration. Further, we found that chimeric mice with only Gaq deficiency in their immune system could spontaneously develop autoimmune disease. We have proven that Gaq regulated B cell development and survival. Most recently, we reported that Gaq expression in peripheral blood lymphocytes (PBLs) from patients with rheumatoid arthritis was significantly reduced, and the reduction was correlated with disease activity. This result further confirmed the role of Gaq in the pathogenesis of human autoimmune diseases. Meanwhile, we found Gaq also regulate T cell proliferation and survival. Since, Th17 is a subset of CD4 cells, and Th17 played a critical role in the development of autoimmunity. Therefore, Gaq may regulate Th17 development and involve in the development of autoimmunity. With this idea, we analyzed the expression of Gaq and IL-17 by RT-PCR in PBMC of RA patients, and found a strong correlationship between them. Further, we detected the Th17 cells in Gaq knockout mice, and found the Th17 cells are significantly higher in knockout mice than wildtype controls. Based on those findings, we hypothesize that Gaq regulate Th17 cell differentiation and is involved in the development of autoimmunity. To prove our hypothesis, we'll use Gaq knockout mice to clarify the role of Gaq in Th17 differentiation and address the underlying molecular mechanism. Then, explore the role of Gaq in Th17 cell differentiation in T cells with low Gaq expression RA patients in comparison with T cells from high Gaq expression from RA patients. T cells from healthy individuals will be used as control. The success of this study will deepen our knowledge in the role of Gaq in immune regulation and autoimmunity.

Ｇaq是由Gnaq编码的Gq蛋白alpha亚单位，广泛表达于各器官组织。申请人既往研究发现：Gaq在免疫系统缺失的小鼠可以自发产生自身免疫病。进一步研究发现Gaq调控B细胞的的分化和功能。临床研究证实Gaq在RA患者外周血中的表达降低，且与疾病活动度相关。以上研究确立了Gaq在自身免疫病发生中的重要作用。我们未发表的研究结果显示，Gaq对T细胞的增殖和死亡有调控作用。由于Th17细胞属于CD4阳性T细胞，且与自身免疫病的发病密切相关。因此推测Gaq可能调控Th17细胞的分化与功能。并进行了预实验，结果发现：Gaq基因缺失的小鼠体内Th17细胞的数量明显增多。在RA患者PBMC中Gaq 与IL-17的表达呈明显负相关。本研究将应用Gaq基因敲除小鼠，结合RA患者外周血，详细研究Gaq在Th17细胞的分化与功能中的作用，并阐明其作用的分子机制，进一步探讨Gaq在自身免疫病发病中的作用。

Gαq是Gq蛋白的alpha亚单位，我们既往研究发现：Gαq在免疫系统缺失的小鼠自发产生自身免疫病。由于Th17细胞与自身免疫病的发病密切相关。本研究应用Gnaq-/-小鼠，及RA患者外周血，研究了Gαq在Th17细胞的分化与功能中的作用，阐明了其作用的分子机制，进一步探讨了Gαq在自身免疫病发病中的作用。本研究取得的重要成果包括：1、我们收集了RA、AS患者外周血单个核细胞检测Gαq的mRNA表达，同时检测血清中IL-17A的水平。分析Gαq与IL-17A表达水平的相关性，证实炎性关节炎患者中Gαq负调控Th17的分化。2、完成了Gαq对Th17细胞体外分化的调控的影响及分子机制的研究。研究结果示 Gnaq-/- CD4+ T细胞IL-17A阳性的细胞比例、分泌的IL-17A及相关基因（IL-17A、IL-17F、IL-22、GM-CSF、IL-23R）表达水平明显高于WT的CD4+ T细胞，说明Gnaq-/- CD4+ T细胞更趋于向Th17分化，Gαq负向调控Th17的分化。其次，我们研究了Gαq对STAT3, RORγt 及RORα三个转录因子的表达影响，结果示Gnaq-/- CD4+ T细胞STAT3, p-STAT3、RORγt 及RORα的表达水平均显著高于WT CD4+T细胞。最后，我们通过使用ERK抑制剂PD98059发现，ERK1/2的抑制使WT CD4+ T细胞更趋于向Th17分化，并且STAT3及RORα的表达水平均相应增高，而ERK1/2的抑制对Gnaq-/- CD4+ T细胞的Th17的分化无显著影响，其STAT3及RORα的表达水平无明显变化。提示Gαq可能通过影响ERK1/2活性调控STAT3及RORα的表达进而影响Th17的分化。 3、完成了Gαq对iTreg细胞体外分化及功能的调控。我们发现，Gnaq-/- CD4+T细胞体外诱导产生的iTreg比例明显减少，且分泌的IL-10及TGF-β水平明显减少。说明Gαq正向调控iTreg的分化。我们还做了iTreg细胞抑制CD4+CD25-细胞增殖的实验。结果显示， Gnaq-/- iTreg细胞抑制CD4+CD25-细胞增殖的功能明显受损。以上数据证实了Gαq作为自身免疫性疾病治疗靶点的可行性。