Fractalkine/CX3CL1通过活化辅助性T细胞17调控系统性红斑狼疮的机制研究

T cell immunity in systemic lupus erythematosus (SLE) has become increasingly appreciated. T helper 17 (Th17) cells are a unique CD4+ T subset characterized as preferential producers of interleukin-17 (IL-17). However, the function of Th17 cells in SLE is indefinite. Fractalkine/CX3CL1 (FKN), the unique member of the CX3C chemokine family, mediates both chemotaxis and adhesion of inflammatory cells via its highly selective receptor CX3CR1 and attracts T cells for efficient antigen presentation. Our previous studies showed that FKN was up-regulated in the serum and renal glomerulus of patients with lupus nephritis. Moreover, CX3CR1 deficiency can protect inflammatory reaction through decreasing specific responses of Th17 cells. However, it is unclear that how FKN regulates the pathogenesis of SLE through activating Th17. In our project, we determined the effects of FKN on the differentiation, phenotype and function of Th17 cells in CD4+ T cells, derived from lupus mice, with overexpressing or depleting FKN using the CRISPR/Cas9 system technology. In the MRL/lpr, (NZB/W) F1 and Pristane-induced lupus mice, the regulatory effects and molecular mechanisms of FKN on the activation of Th17 cells in the pathogenesis of SLE were investigated by molecular biology, immunology and pathology methods. Furthermore, the effects of FKN depletion in lupus mice models on the cytokines and signaling pathways were explored as the potential treatments. Overall, our study could provide a rationale for targeting FKN in SLE as a novel and promising therapy.

系统性红斑狼疮（systemic lupus erythematosus, SLE）的T细胞免疫已成为研究热点，然而辅助性T细胞17（T helper type 17, Th17）在SLE中的功能尚未明确；趋化因子Fractalkine/ CX3CL1（FKN）可吸引T细胞的高效抗原呈递，FKN受体CX3CR1缺陷可通过减少Th17细胞反应实现对炎症的保护作用，但在FKN通过Th17细胞调控SLE方面的研究有待于进一步深入；本项目拟以CRISPR/Cas9基因组编辑技术，研究狼疮小鼠CD4+ T细胞过表达和缺陷表达FKN对Th17细胞活化的影响，以狼疮小鼠、基因敲除小鼠作为研究模型，探讨FKN通过活化Th17细胞对SLE的调控机制，阐明FKN拮抗可否通过调控Th17细胞功能来治疗SLE，并发现参与此过程的细胞因子和信号通路，研究将有助于理解SLE发病机理及开发靶向治疗药物。

目的：课题组前期研究证实fractalkine（FKN）的表达水平与狼疮性肾炎（LN）的发病呈正相关，尤其与病人的疾病活动度和肾脏损害密切相关。此外，FKN通过其受体CX3CR1发挥作用，也被报道可调节缺血再灌注损伤后小鼠肾间质纤维化。因此，大量的数据表明FKN在狼疮肾间质纤维化的发展中起着重要的作用。自身免疫性疾病的发病机制通常是由Treg和Th17细胞相互排斥的方式产生的。然而，尚未有关于狼疮性肾炎（LN）中FKN通过影响Treg/Th17细胞失衡。我们提出FKN通过促进Th17细胞和抑制Treg细胞的产生，促进促炎T细胞反应进而激活LN免疫系统的假设。.方法：用抗FKN抗体（aFKN）、重组FKN（rFKN）或同型抗体（IgG）分别处理MRL/lpr和C57BL/6小鼠。分离小鼠脾脏提取淋巴细胞，使用TGF-β 和 IL-2刺激使其增殖并分化为Treg细胞，用IL-6、TGF-β、IL-23、抗IFN和抗IL-4刺激使其增殖分化为Th17细胞，然后分别用抗FKN抗体、重组FKN或者IgG抗体刺激。测定肾脏、Treg细胞和Th17细胞中FKN、Foxp3、IL-10、IL-7、IL-6的表达水平，以及肾脏中Treg/Th17的比例。.结果：在体内，用抗FKN抗体治疗后的狼疮小鼠的肾损伤有改善，肾小管间质损害减少，肾脏中的Foxp3、IL-10表达水平上调，而IL-7、IL-6表达水平下调。脾脏中的Th17细胞的数量减少，Treg细胞的数量增多。然而，用重组FKN处理狼疮小鼠，其作用结果相反。在体外，用抗FKN抗体分别处理Treg细胞和Th17细胞，检测结果发现Foxp3和IL-10在Treg细胞中的表达上调，IL-7和IL-6在Th17细胞中的表达下调。然而，用重组FKN处理后结果相反。.结论：FKN促进狼疮小鼠淋巴细胞中炎症因子水平增加和肾小管间质病变，其作用可能是通过改变Treg/Th17细胞的平衡而导致。