RESUMEN
BACKGROUND: Many studies have demonstrated that the expression of methyltransferase- like 3 (METTL3) is altered in various inflammatory diseases. Its specific mechanistic role in the intestinal inflammatory response during sepsis remains limited and requires further investigation. OBJECTIVES: Explore the potential mechanism of METTL3 in the intestinal inflammatory response during sepsis. MATERIALS AND METHODS: Immunohistochemical analysis was utilized to detect the expression of METTL3 in the necrotic intestine of patients with intestinal necrosis and the small intestine of cecal ligation and puncture (CLP) mice. Mice were subjected to the CLP and Sham surgeries, intestine tissue was harvested and performed HE staining, and ELISA to examine intestinal inflammatory responses, while TUNEL staining was applied to detect intestinal cell apoptosis. Additionally, ELISA was used to detect diamine oxidase (DAO) and intestinal fatty acid binding protein (I-FABP) levels in intestinal tissue. Immunohistochemistry and RT-qPCR were also employed to examine the mRNA and protein expression levels of Zona Occludens 1 (ZO-1) and Claudin-1. Finally, transcriptomic sequencing was performed on the small intestine tissues of METTL3 Knock-out (KO) and Wild-type (WT) mice in response to sepsis. RESULTS: METTL3 exhibited lower expression level in the necrotic intestine of patients and the small intestine of CLP mice. Loss of METTL3 in CLP mice triggered significantly higher expression of TNF-α and IL-18, down-regulated expression of ZO-1 and claudin-1, and decreased expression of DAO and I-FABP in the intestinal tissue. KEGG enrichment analysis showed that the differential genes were significantly enriched in immune-related pathways. CONCLUSION: This study reveals a novel mechanism responsible for exacerbated intestinal inflammation orchestrated by METTL3. Particularly, METTL3 null mice displayed decreased ZO- 1 and Claudin-1 expression, which largely hampered intestinal epithelial barrier function, resulting in bacterial and toxin translocation and intestinal immune activation and inflammation against sepsis.
RESUMEN
The enzymatic core component of m6A writer complex, Mettl3, plays a crucial role in facilitating the development and progress in gastric and colorectal cancer (CRC). However, its underlying mechanism in regulating intestinal inflammation remains unclear and poorly investigated. Firstly, the characteristics of Mettl3 expression in IBD patients were examined. Afterwards we generated the mice line with IECs-specific deletion of Mettl3 verified by various experiments. We continuously recorded and compared the physiological status including survival rate etc. between the two groups. Subsequently, we took advantage of staining assays to analyze mucosal damage and immune infiltration of Mettl3WT and Mettl3KO primary IECs. Bulk RNA sequencing was used to pursuit the differential expression of genes (DEGs) and associated signaling pathways after losing Mettl3. Pyroptosis-related proteins were to determine whether cell death was caused by pyroptosis. Eventually, CyTOF was performed to probe the difference of CD45+ cells, especially CD3e+ T cells clusters after losing Mettl3. In IBD patients, Mettl3 was highly expressed in the inner-nucleus of IECs while significantly decreased upon acute intestinal inflammation. IECs-specific deletion of Mettl3 KO mice triggered a wasting phenotype and developed spontaneous colitis. The survival rate, body weight and intestinal length observed from 2 to 8-week of Mettl3KO mice was significantly lower than Mettl3WT mice. The degree of mucosal damage and immune infiltration in Mettl3KO were even more serious than their WT littermate. Bulk RNA sequencing demonstrated that DEGs were dramatically enriched in NOD-signaling pathways due to the loss of Mettl3. The colonic epithelium were more prone to pyroptosis after losing Mettl3. Subsequently, CyTOF revealed that T cells have altered significantly in Mettl3KO. Furthermore, there were abnormal proliferation of CD4+ T and markedly exhaustion of CD8+ T in Mettl3KO mice. In severe IBD patients, Mettl3 located in the inner-nucleus of IECs and declined when intestinal inflammation occurred. Subsequently, Mettl3 prevented mice from developing colitis.
