关节免疫炎症时特异SLC38A调节巨噬细胞活化的分子机制

Macrophages possess the integral effects on the occurrence and development of rheumatoid arthritis (RA). And the activated macrophages during immune inflammation of joints may be regulate by metabolism reprogramming. Our recent preliminary data showed that special SLC38A is feasibly involved in the macrophage activation. So we propose the hypothesis that special amino acid transporter SLC38A may participate in the occurrence and development of immune inflammation of joints by regulating the function of macrophages. We intend to prove our hypothesis by the following experiments. Firstly, the macrophages are collected in arthritis rats and activated macrophages in vitro. And the expressions of SLC38A family members which are associated with macrophage function are screened. Secondly, the special SLC38A molecule is studied for its mechanism in regulating macrophage activation from 2 aspects. The RNA and protein expression profiles are performed and analyzed in special SLC38A knock-out macrophages by RNA-sequencing and iTRAQ to obtain the signal molecules and the downstream effect molecules which are associated with special SLC38A. The special amino acid transported by the special SLC38A is screened out by culturing and activating special SLC38A deficient or overexpressed macrophages in culture media lacking single amino acid. And the metabolite of this special molecule could be measured by HPLC. Thirdly, the regulatory molecules of the special SLC38A in activated macrophage are pinpointed in transcriptional and post-transcriptional levels. Finally, the function of special SLC38A in arthritis development is being further confirmed by establishing the CIA animal model using macrophage conditional knock-out mice and human RA samples. Association study between SLC38A gene polymorphism and arthritis is performed in over 200 pairs of RA patients and healthy control to verify whether the special SLC38A alleles could affect the susceptibility to RA. We insist that the results should offer strong evidence to support the hypothesis that the special amino acid transporter SLC38A may participate in the occurrence and development of immune inflammation of joints by regulating the function of macrophages. Also, the findings will provide new insights into understanding the pathogenesis of RA and supply novel molecular targets for the prevention, diagnosis, therapy and new drug development of arthritis.

关节免疫炎症时巨噬细胞（Mφ）活化可受代谢再编程的影响，预实验也提示有特异氨基酸转运子参与了Mφ活化。故认为特异SLC38A影响Mφ活化和功能，进而参与关节免疫炎症的发生发展。本项目首先在关节炎实验动物和RA样本中，验证已筛选出的特异SLC38A与Mφ活化的关系；继而在特异SLC38A敲除／过表达的Mφ活化模型上，通过检测基因表达谱等获得其介导的下游信号通路及效应分子；同时检测其转运的特异氨基酸及下游小分子，验证相互作用分子及转运的氨基酸对Mφ活化和功能的机制；再利用生物信息学及分子生物学方法，从转录和转录后水平解析调控特异SLC38A表达的机制；最后在SLC38A敲除小鼠上诱导关节炎、RA患者样本中检测相关分子及特异SLC38A的SNP与RA的关联分析，确定SLC38A与Mφ活化及关节炎发生发展中的作用。结果不但丰富人们对关节炎发病机制的认识，也为关节炎的诊断与治疗提供理论和实验依据。

类风湿性关节炎时，巨噬细胞的活化受到代谢重编程的影响，但代谢重编程是如何影响巨噬细胞活化，又如何调控关节炎的进展知之甚少。我们的研究发现了一个全新的氨基酸转运子SLC38A，在实验性关节炎的巨噬细胞中表达增强，且与关节炎的进程相关。在敲除了SLC38A之后，小鼠关节炎模型无论从临床评分还是从患病率上都有显著减轻，且活化的CD4阳性T细胞在其引流淋巴结的各种淋巴细胞比例也显著下降，证实了关键SLC38A确实参与了关节炎的进展。与野生型巨噬细胞相比，关键SLC38A的巨噬细胞无法有效应答由LPS和IFNγ引起的巨噬细胞活化过程，在RNAi或CRISP-Cas9构建的关键SLC38A的敲低或敲除细胞系中也出现了同样的结果，说明关键SLC38A调控巨噬细胞的活化过程。利用质谱等方法检测了关键SLC38A敲除巨噬细胞及野生型巨噬细胞中各种氨基酸的含量，结果显示，谷氨酰胺、丝氨酸、苏氨酸、天冬酰胺和酪氨酸的含量远低于野生对照细胞内的氨基酸含量。在巨噬细胞培养体系中缺乏谷氨酰胺后，再诱导巨噬细胞活化，巨噬细胞也无法有效的产生IL1β和甘露糖受体。通过荧光共定位和亚细胞器分离，发现关键SLC38A主要定位于巨噬细胞的线粒体膜上，转运谷氨酰胺入线粒体进行代谢过程。敲低关键SLC38A会抑制谷氨酰胺的分解代谢酶的表达，促进谷氨酰胺合成酶的表达；反之，高表达关键SLC38A则会促进谷氨酰胺分解酶的表达。这些结果也说明关键SLC38A通过调控线粒体内谷氨酰胺的代谢参与巨噬细胞活化过程进而影响关节炎的进展。这些结果的获得，不但丰富了代谢重编程通过调控巨噬细胞内氨基酸代谢过程参与调控巨噬细胞的活化过程，也为关节炎的治疗提供了更多的靶点，为新药的研制提供了研究思路、理论和实验依据，为更好的为广大人民的健康生活提供了科学依据。