髓系细胞来源的MCPIP1对肠道稳态的免疫调节机制研究

Both polymorphonuclear neutrophils (PMN) and macrophages play an important role in maintaining intestinal mucosal homeostasis, and MCP-1 induced protein 1 (MCPIP1) functions as a RNase and deubiquitinase that regulates mRNA degradation by binding to 3’-untranslated region (3’-UTR) and negatively regulates NF-kB signaling pathway by deubiquitinating TRAFs, respectively. Our previous studies have demonstrated that myeloid cell-specific MCPIP1 knockout (MCPIP1△Mye) mice developed spontaneous multiple organ disorders at 8 wk of age. Intestinal epithelia including Paneth cells and goblet cells were more proliferated and the proportion of Proteobacteria in the feces was increased in these mice compared with those in controls. Interestingly, MCPIP1△Mye mice also developed more severe colitis induced by dextran sulfate sodium (DSS) in the drinking water compared with wild-type controls, characterized by increased expression of IL-1b, IL-6, CXCLs and CCLs, impaired development of monocyte-macrophage in inflamed colon and increased proportion of Clostridia in the feces. In vivo analysis showed that the release of ROS and NET formation were significantly downregulated in PMN from myeloid cell-specific MCPIP1 transgenic mice and that the differentiation of bone marrow-derived macrophages (BMDM) into alternatively activated macrophages was compromised in MCPIP1△Mye mice compared with controls. RNA sequencing analysis of MCPIP1△Mye BMDM revealed that MCPIP1 could modulate macrophages via NF-kB and Rho GTPase signaling pathway. In addition, MCPIP1 was significantly increased in inflamed mucosa of patients with inflammatory bowel disease compared with healthy controls. Therefore, we hypothesized that myeloid cell-derived MCPIP1 may play an important role in regulating intestinal stem cell (ISC) differentiation, intestinal microbiota and metabolic homeostasis, and mucosal inflammation. In this study, we aim to detect the expression of MCPIP1 in peripheral blood cells (e.g., monocytes, neutrophils) and inflamed mucosa from patients with inflammatory bowel disease, and investigate the underlying mechanisms of MCPIP1 in regulating PMN and macrophage function, ISC differentiation, microbiota composition, metabolic substrate production, and intestinal mucosal inflammation induced by DSS in the drinking water or Citrobacter rodentium infection in myeloid cell-specific MCPIP1 knockout mice and myeloid cell-specific MCPIP1 transgenic mice. Our study will provide new theoretical basis for target immune therapy in inflammation-mediated intestinal diseases, importantly in inflammatory bowel disease.

中性粒细胞和巨噬细胞在维持肠道稳态中起着重要作用。MCPIP1通过RNase和去泛素化调控免疫应答反应。我们前期研究发现MCPIP1△Mye小鼠发生自发性多器官炎症伴粪便变形菌门增多；DSS诱导该小鼠结肠炎症加重，促炎细胞因子和趋化因子增高，单核巨噬细胞活化受阻，粪便梭菌纲减少。体外研究发现MCPIP1抑制中性粒细胞迁移和NET形成，调控巨噬细胞NF-kB和Rho GTPase信号通路，而缺失MCPIP1抑制巨噬细胞向M2极化。据此我们提出髓系细胞来源的MCPIP1可能参与调控肠上皮干细胞分化、肠道菌群和代谢产物平衡以及肠道炎症发生发展。本课题将利用炎症性肠病肠黏膜标本、髓系细胞特异性MCPIP1基因敲除和转基因小鼠，构建结肠炎和肠道感染模型，分析其在维持肠道稳态与调控肠道炎症发生过程中的效应作用，旨在阐明MCPIP1参与维持肠道稳态的免疫调节机制，为临床治疗肠道炎症疾病提供新的理论依据。

中性粒细胞和巨噬细胞在维持肠道稳态中发挥着重要作用。MCPIP1通过RNase和去泛素化调控免疫应答反应。本课题首先构建了髓系细胞MCPIP1基因特异性敲除（即Mcpip1∆Mye）小鼠，给予2% DSS饮用水诱导急性结肠炎发生，发现该小鼠发生肠道炎症显著加重，肠黏膜固有层组织内髓系细胞（特别是单核-巨噬细胞、中性粒细胞）浸润明显增加，尤其是促炎型P1群细胞占比显著增加，而抑炎型P3/P4群细胞占比显著降低；单细胞测序分析发现Mcpip1∆Mye小鼠肠黏膜固有层内单核-巨噬细胞促炎型P1细胞群比例较对照组（Mcpip1fl/fl）小鼠显著增多，而成熟的抑炎型P3/P4群占比显著降低。对比单核巨噬细胞RNA测序与单细胞测序数据，发现Mcpip1∆Mye巨噬细胞表达Atf3、Ap1s2增加，Atf3蛋白可结合Ap1s2分子的启动子区域，促进其表达，而此信号通路受到MCPIP1的抑制调控。进一步体内抑制Ap1s2表达，可有效地阻止Mcpip1∆Mye小鼠实验性肠炎发生，提示抑制Atf3-Ap1s2信号通路具有抑制炎症进展，维持肠道免疫稳态。.另外，我们利用髓系细胞MCPIP1基因特异性过表达（即Mcpip1 Mye-tg）和Mcpip1∆Mye小鼠，发现Mcpip1 Mye-tg小鼠骨髓来源的中性粒细胞（PMN）产生ROS/MPO、释放中性粒细胞胞外诱捕网（NETs）及迁移能力明显下降；而Mcpip1∆Mye小鼠骨髓的PMN产生ROS/MPO、释放NETs及迁移能力增强。构建沙门氏伤寒杆菌急性腹腔感染模型，发现Mcpip1 Mye-tg小鼠外周血、腹腔和肝脏PMN迁移减少，腹腔感染及肝脏炎症加重；而Mcpip1 ∆Mye小鼠外周血、腹腔和肝脏PMN迁移增多，腹腔感染及肝脏损伤明显缓解。RNA-Seq测序及RIP分析发现MCPIP-1可结合并降解CIRBP mRNA。使用腺病毒转染干扰HL-60细胞系CIRBP表达，可抑制ROS/MPO产生、释放NETs及迁移能力，证实了MCPIP1通过降解CIRBP调控PMN的免疫功能。.本课题研究阐明了MCPIP1参与维持肠道稳态的免疫调节机制，为临床治疗肠道炎症疾病提供新的理论依据。