VSIG4通过衔接蛋白MS4a6D抑制NLRP3炎性小体活化的机制研究

The hyperactivation of NLRP3 inflammasome contributes to the pathogenesis of multiple diseases whereas the mechanisms negatively regulating this process are poorly understood. We here illustrated that V-set immunoglobulin domain-containing 4 (VSIG4, also called complement receptor of the immunoglobulin superfamily, CRIg), which expresses specifically in resting macrophages, attenuates NLRP3 inflammasome activation and IL-1β secretion. Macrophages lacking Vsig4 have increased basal and LPS-induced NLRP3 and proIL-1β expression in vitro. In parallel, Vsig4-/- mice are more susceptible to develop experimental autoimmune encephalomyelitis (EAE) as well as suffer from high fat diet (HFD)-caused obesity and insulin tolerance due to overburden of NLRP3 inflammasome- mediated IL-1β in vivo. Moreover, enhancing IL-1β also protected Vsig4-/- mice from dextran sulfate sodium (DSS)-induced colitis. Conversely, deletion of NLRP3, Caspase-1 or IL-1 receptor-1 (IL-1R1) in Vsig4-/- mice, respectively, dramatically reverses these effects. Mechanically, VSIG4 promotes the expression of A20, which inhibits the activation of NF-kB and thus lead to attenuate the gene transcription encoding for NLRP3-inflammasome components, through interacting with MS4A6D, a four-pass membrane protein of the MS4A family. In this project, we would detect and confirm that MS4a6D controls the expression of NLRP3 components by upregulating Akt/PI3K-ERK-STATs cascades- mediated A20 expression. Furthermore, MS4A6D-/- animals would be used to analyze whether they phenocopy Vsig4-/- mice and deteriorate the development of several kinds of inflammatory diseases including EAE, DSS-caused colitis et al. Finally, WT mice would be injected with VSIG4 agonist mAbs to confirm that this administration would be suitable for disease therapy. These results reveal that VSIG4 attenuates NLRP3 inflammasome activation and manipulation of VSIG4/MS4A6D signaling would be usefully for inflammatory disease therapy.

NLRP3炎性小体异常活化可加剧多种炎症疾病的进程，因此，抑制NLRP3炎性小体活化可改善疾病的进展。VSIG4是特异表达与巨噬细胞表面的B7家族分子，前期研究我们发现VSIG4通过下游的衔接蛋白MS4a6D向巨噬细胞传递信号并促进A20表达，进而负调NLRP3及proIL-1β基因转录从而改善结肠炎、EAE及胰岛素抵抗等多种疾病的进展，但VSIG4/MS4a6D复合体上调A20的机制未知。本研究拟使用RNAi、Co-IP及Western-blot等技术探讨其通过活化Akt/PI3K-ERK-STATs通路促进A20表达的分子机制；利用MS4a6D KO小鼠研究其对多种炎症性疾病进程的影响；最后使用VSIG4激动型抗体于体内探讨增强VSIG4信号对EAE等疾病的干预效果。本研究结果将明确VSIG4抑制NLRP3炎性小体活化的分子机制，为NLRP3介导的多种炎症疾病的临床治疗提供新策略。

VSIG4是特异表达与巨噬细胞表面的B7家族分子，前期研究我们发现VSIG4通过下游的衔接蛋白MS4a6D向巨噬细胞传递信号并促进A20表达，进而负调NLRP3及proIL-1β基因转录从而改善结肠炎、EAE及胰岛素抵抗等多种疾病的进展，但VSIG4/MS4a6D复合体上调A20的机制未知。本研究拟使用RNAi、Co-IP及Western-blot等技术探讨其通过活化Akt/PI3K-ERK-STATs通路促进A20表达的分子机制；利用MS4a6D KO小鼠研究其对多种炎症性疾病进程的影响；最后使用VSIG4激动型抗体于体内探讨增强VSIG4信号对EAE等疾病的干预效果。本研究结果将明确VSIG4抑制NLRP3炎性小体活化的分子机制，为NLRP3介导的多种炎症疾病的临床治疗提供新策略。经过4年期的NSFC资助，获得了以下成果：1、首次证明VSIG4可作为NLRP3异常活化导致的炎症性疾病包括EAE、肥胖及胰岛素抵抗等疾病的靶标，而使用激动型抗体增强VSIG4信号可延缓这些疾病的进展；2、本研究阐明VSIG4是负调Nlrp3和proIL-1β mRNA转录及蛋白表达的宿主内源分子；同时仔细研究VSIG4的信号传导依赖于下游的A20及其介导的NF-κB炎性途径；3、开发了针对VSIG4的机动性抗体VG11，临床前研究发现其可调节EAE等多种AID的进展。此外，4、发现VSIG4及MS4A6D形成二聚体，构建了该MS4A6D基因敲除小鼠，其可拮抗DSS-诱导的肠炎、LPS-诱导的内毒素休克及卡拉胶诱导的足垫肿胀等模型；5、该MS4A6D分子可形成可通过半胱氨酸Ser237介导形成同源二聚体，在急性炎症条件下与巨噬细胞表面的MHC-II分子结合，进而活化下游的SYK-CREB等信号通路及NF-KB信号通路，导致巨噬细胞线粒体代谢和促炎细胞因子产生，从而促进巨噬细胞炎症分泌；6、构建了Ser137及Tyr237位点特异性基因敲除小鼠，证明同源二聚体及Tyr的活性对巨噬细胞的活化是关键的位点，而这两个位点突变的小鼠也可延缓LPS诱导内毒素休克和卡拉胶诱导的足垫肿胀进展，成果已经投稿Science Immunology (2022)。7、新型免疫干预药物PLA-O-66A（一种全新的冬凌草甲素的衍生物）通过VSIG4-MS4A6D信号通路下调炎症可临床转化。

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