关节滑膜肥大细胞的区域免疫特性及其介导的Th17活化和相互作用在类风湿关节炎发病中的研究

Although Th17 is the main pathogenic cell of rheumatoid arthritis (RA), especially in chronic inflammation and joint injury, it is not clear how Th17 is activated in the regional immune abnormalities in RA. Mast cell (MC) is an important cell in regional immunity, which can release inflammatory mediators, specific proteases and newly synthesized lipid mediators, and participate in the development of autoimmune diseases and inflammation. Whether MC is involved in the activation of Th17 and the maintenance of immune abnormalities in the focal area, and the relationship between the regional immunity and system immunity has not been known. .In this application, first, we want to observe the regional characteristics of MC in RA inflammed joints, relationship among synovial mast cells, Th17 and RA disease severity in RA patients. Then we intend to study the effect of lesion microenvironment on mast cells, and observe the effects of mast cells to adaptive immune response and inflammation under lesion microenvironment in vitro. At last, we analysis the relationship among synovial mast cells, Th17 and disease severity in collagen-induced arthritis in mice, observe the change of disease severity and immunological changes after blocking relating molecules between MC and Th17, appropriate timing of intervention with different anti-MC and/or anti-Th17 combination treatment in RA..So as to clarify the regional characteristics of MC, the pathway of MC, which act as innate immune cells and effector cells, to promote the Th17 activation, the mechnaism to cause disease exacerbation whether is through positive feedback to expand the inflammatory effect in RA.These series study could provide new perspectives on the mechanism of RA from the regional immune system to the systemic immune system, rich the theory for understanding the pathogenesis of RA and provide new theoretical and experimental basis for therapeutic intervention in RA.

Th17虽是类风湿关节炎（RA）主要致病细胞，尤其在慢性炎症和关节损伤中起重要作用，但在RA区域免疫异常中，其如何被活化尚不明确。肥大细胞（MC）是区域免疫的重要细胞，可释放炎症介质、预存特异性蛋白酶和新合成脂质介质参与自身免疫病的发生与炎症维持。MC是否参与Th17活化与病灶部位区域免疫异常的维持，以及从区域免疫影响到系统免疫对RA的发生发展关系尚不清楚。我们拟通过①临床：关节滑膜病灶MC的区域特点、与Th17、RA病情关系；②体外：病灶微环境对MC区域特性影响，以及MC促进活化致炎的机制；③动物模型：CIA小鼠MC促进Th17、疾病严重关系、阻断相互作用分子对疾病影响、适当时机组合干预MC、Th17的治疗策略。籍此阐明MC免疫区域特性、MC作为固有免疫细胞致适应性应答炎症通路，从全新角度阐释RA中从区域免疫到系统性免疫的作用机理，深入理解RA发病机制，为靶向治疗提供新的理论和实验依据。

Th17虽是类风湿关节炎（RA）主要致病细胞，但其如何被活化尚不明确。肥大细胞（MC）可释放炎症介质、预存特异性蛋白酶和新合成脂质介质参与自身免疫病的发生与炎症维持。MC是否参与Th17活化与病灶部位区域免疫异常的维持和对RA的发生发展关系尚不清楚。我们通过临床分析RA关节滑膜病灶MC与Th17、RA病情关系；探讨病灶微环境低氧对MC影响，以及MC促进活化致炎和Th17的具体作用机制；探究CIA小鼠MC对Th17和疾病严重的影响。籍此从全新角度阐释RA中MC的作用机理，深入理解RA发病机制，为靶向治疗提供新的理论和实验依据。.研究发现RA患者外周血和关节滑膜Th1和Th17及相关细胞因子升高；滑膜T和MC浸润且依靠紧密，提示存在T和MC相互作用。与OA相比，RA关节滑膜MC蛋白组学检测显示存在265个差异蛋白；KEGG和GO分析分析显示富集通路和差异蛋白多与线粒体代谢、三羧酸循环和糖代谢等相关；GSEA分析发现与氧化磷酸化、细胞呼吸和脂肪酸代谢相关，提示RA微环境对MC进行了代谢重塑。.MC促进CD4+T增殖及向Th17方向的分化，主要依赖于细胞间的直接接触，但同时可溶性免疫分子也起到一定作用；并发现人MC具有抗原提呈能力；MC脱颗粒和分泌介质主要涉及PLCγ-Ca2+以及AKT和ERK信号通路。RA滑膜微环境缺氧条件会显著抑制Th17和Treg的分化，MC的加入显著促进Th17分化，抑制Treg的分化。RNAseq分析了缺氧处理MC细胞后的基因变化，GO富集和KEGG通路富集亦显示糖酵解信号通路表达的上调，提示缺氧使MC细胞发生糖酵解代谢重编程，进而可导致功能发生变化。实验证实缺氧会显著上调Il6、Vegfa、Tpsab1的表达，提示可能由此促进炎症发展；缺氧使作为转录因子的二聚体PKM2明显上调，其可通过作用MC的相关基因进而促进对CD4+T的作用。.CIA小鼠过继转移MC的实验表明，过继转移组Th1与Th17细胞比例升高、抗CII抗体升高、IFNγ和IL-17明显上升、滑膜中肥大细胞数目增多，伴随T细胞浸润的明显增多，且T细胞的定位从滑膜下层延申到了滑膜衬里层，进一步提示肥大细胞对于T细胞的增殖和促进迁移作用。