S1PR1通过STAT1/BCL6通路抑制SLE滤泡辅助性T细胞的分化及机制研究

Sphingosine-1-phosphate receptor 1 (S1pr1) has been shown to be substantially dysregulated in various types of autoimmune diseases, and small-molecular drugs targeted S1pr1 are used in clinic one after another in order to relieve a variety of inflammatory diseases, including multiple sclerosis and rheumatoid arthritis. However, the role of S1pr1 has not been well determined during the pathogenesis of systemic lupus erythematosus (SLE). In this study, we first generated a line of transgenic mice harboring a mice S1pr1 cDNA as well as Flag peptide cDNA under the control of the hCD2 promoter by Cas9/RNA system gene targeting method. Expression pattern of S1pr1 in the immune-related tissues of transgenic mice line was examined by Real-time RT-PCR as well as flow cytosorting. Then we systematically investigated the lupus phenotypes in WT and S1pr1-transgenic mice with pristane-induced SLE. And we found that overexpression of S1pr1 was positively correlated with remissive lupus phenotype as well as decreased T follicular helper (Tfh) cells in vivo. As we all know, Tfh cells are a distinct subset of CD4+ helper T (Th) cells that regulate the development of antigen-specific B cell immunity, and dysregulation of Tfh cells is believed to be involved in angioimmunoblastic T cell lymphoma and underlie several autoimmune diseases, including SLE and Sjogren's syndrome. Tfh cells are identified by elevated expression levels of multiple surface proteins and Bcl-6, as well as enhanced IL-21 secretion. The high expression levels of these proteins correlate with Tfh cells' enhanced capacity to facilitate antibody production. Our preliminary results showed that S1pr1 was negatively correlated with Bcl6 and IL-21 in spontaneous lupus mice’s spleen. This result impels us to suggest this hypothesis: S1pr1 might influence the pathogenesis of systemic lupus erythematosus by altering the function of Tfh cells. Further study demonstrated that S1pr1 could suppress the activity of Stat1/Bcl6 pathway in vitro and in vivo, however, the detailed molecular mechanism remains unclear. So the purpose of this study is to explore the detailed mechanism as well as the role of Stat1/Bcl6 pathway regulated by S1pr1 in the process of Tfh cells differentiation under pathological conditions by using adoptive transplantation experiment in vivo or stimulating differentiation experiment (Polarization of naive T cells toward Tfh cells) in vitro. The results of this proposed study might not only elucidate the biological function of S1pr1, but also make for identification of potential targets towards SLE.

1-磷酸鞘氨醇受体1（S1PR1）在多种自身免疫病中发挥抑炎作用，但该基因在系统性红斑狼疮（SLE）发病中的功能尚未明确。为证实S1PR1参与SLE发病，我们构建了S1pr1 T细胞特异性转基因（S1pr1-TG）小鼠，发现过表达S1pr1可缓解药物诱导的狼疮小鼠表型并伴随免疫组织内滤泡辅助性T（Tfh）细胞比例下调。前期实验也发现S1pr1与Tfh细胞分化的关键因子Bcl6显著负相关，并初步证实S1pr1负向调控Stat1/Bcl6信号途径，据此我们提出假说：S1pr1可通过调控Stat1/Bcl6信号途径抑制Tfh细胞分化进而影响SLE发病。为验证该假说，我们将在系统分析S1pr1-TG狼疮小鼠表型的基础上，通过体外诱导分化实验、体内过继移植实验和细胞水平实验等研究手段探究S1pr1介导Stat1/Bcl6信号在Tfh细胞分化和SLE发病中的作用，以期为研究SLE发病机制提供理论依据。

SLE是一种慢性异质性自身性免疫性疾病。其病因复杂，发病机制目前尚不完全明确。课题组研究初期发现S1PR1在SLE患者PBMC和狼疮小鼠脾脏细胞中显著低表达，提示该基因可能参与SLE的发生发展过程。本项目通过构建S1pr1转基因小鼠和Pristane诱导的狼疮小鼠模型系统探究了S1pr1在SLE发生中的作用。通过分析S1pr1-TG狼疮小鼠和对照组小鼠的表型，发现S1pr1-TG狼疮小鼠的脾脏重量明显减少，肾脏损伤程度减轻，免疫复合物沉积降低，这表明高表达S1pr1可显著缓解狼疮小鼠的病情。Tfh细胞是一类与SLE发生密切相关的CD4+ T细胞亚群，我们发现S1pr1-TG狼疮小鼠脾脏和淋巴结中Tfh细胞比例减少，提示S1pr1可抑制Tfh细胞的分化过程.Bcl6是Tfh细胞分化及功能的重要调控因子，我们检测了小鼠脾脏CD4+ T细胞内S1pr1与Bcl6的表达相关性，结果显示S1pr1与Bcl6显著负相关。对比分析Pristane诱导狼疮模型下S1pr1-TG及WT小鼠脾脏CD4+ T细胞中Bcl6的含量，发现S1pr1显著下调Bcl6的mRNA，提示S1pr1可通过调控Bcl6表达影响Tfh细胞的分化过程。通过慢病毒感染的方式建立了S1pr1低表达及过表达的L615细胞系，并利用Western blot检测Ifnar1及Bcl6的表达情况，发现干扰S1pr1表达可上调Ifnar1及Bcl6表达，过表达S1pr1显著下调Ifnar1及Bcl6表达。课题组在S1pr1-TG及对照狼疮小鼠脾脏CD4+ T细胞中对上述结论进行了验证，结果显示S1pr1-TG小鼠脾脏CD4+ T细胞中Ifnar1、p-Stat1及Bcl6的表达均显著低于WT小鼠。这就进一步证实了S1pr1调控Tfh细胞关键转录因子Bcl6表达。.鉴于狼疮患者和狼疮小鼠模型体内S1PR1 mRNA和蛋白水平异常低表达，猜测该基因可能受转录水平的调控，因此课题组对基因上游表达调控机制进行系统分析。通过构建系列截短载体和双荧光素酶报告基因系统分析了S1PR1基本启动子，证实S1PR1转录起始点-29 — -12 bp区域存在转录因子的结合。我们利用生物信息学分析，结合ChIP、EMSA和细胞实验，证实STAT1可通过结合-29 — -12 bp区域正向调控S1PR1的表达。