Functions And Mechanisms Of OTUD4 In Regulating Intestinal Inflammation And Bacterial Infection

Inflammatory bowel disease（IBD）is a chronic intestinal inflammatory disorder consisting of Crohn’s disease（CD）and ulcerative colitis（UC）.Its clinical manifestations usually include abdominal pain,diarrhea,bloody stools and weight loss.In addition to causing damage to the intestines and triggering inflammation in other parts of the body,IBD is also an important inducement of intestinal tumors.At present,the pathogenesis of IBD is not well understood,but studies have shown that genetic sensitivity,immune abnormalities,gut microbiota and other environmental factors can trigger IBD.Since the beginning of the 21st century,the incidence of IBD has continued to rise and become a global public health problem.IBD has complex etiology and unpredictable recurrence,and it is difficult to completely cure it with current medical means.Therefore,it is of great significance to further explore the pathogenesis of IBD and find new targets and strategies for the treatment of IBD.Antimicrobial peptides（AMPs）are endogenous antibiotics with antibacterial activity,which play an important role in maintaining the ecological balance of gut microbiota,anti-microbial infection and and regulating intestinal inflammation.The disturbance of AMPs is closely related to IBD and bacterial infection,and the related molecular mechanisms are still not fully understood.This study found that Ovarian tumor family deubiquitinase 4（OTUD4）promoted intestinal inflammation and bacterial infection by inhibiting the expression of AMPs.By analyzing transcriptomic data from the colonic mucosa of patients with UC,this study found that OTUD4 was specifically highly expressed in the inflammatory mucosa of patients.Subsequently,this study constructed OTUD4-specific knockout mice in different cell types and found that the mice with OTUD4-specific knockout in intestinal epithelial cells(Vil-Cre;Otud4^fl/fl)were more resistant to colitis induced by sodium dextran sulfate（DSS）than the control(Otud4^fl/fl)mice.This study further isolated the IECs from the Vil-Cre;Otud4^fl/fl and the Otud4^fl/fl mice after DSS treatment and performed transcriptomic sequencing and quantitative real-time PCR assays.The results suggested that OTUD4 knockout in IECs up-regulated the expression of AMPs.In addition,this study collected feces from the Vil-Cre;Otud4^fl/fl and the Otud4^fl/fl mice after DSS treatment and analyzed the microbial composition in the feces by 16S r RNA sequencing assays.The results showed that DSS treatment significantly decreased the richness and diversity of gut microbiota and OTUD4 knockout in IECs inhibited this process.In addition,The Vil-Cre;Otud4^fl/fl mice shaped more microbes of the phylum Firmicutes and fewer microbes of the phylum Proteobacteria in comparison to the Otud4^fl/fl mice,suggesting that a healthier composition of gut microbiota in the Vil-Cre;Otud4^fl/fl mice.Salmonella typhimurium（S.t.）is a kind of pathogenic bacteria that can trigger intestinal inflammation.This study found that the Vil-Cre;Otud4^fl/fl mice were more resistant to S.t.infection compared to the Otud4^fl/fl mice,and the expression of AMPs in IECs of the Vil-Cre;Otud4^fl/fl mice was higher than the Otud4^fl/fl mice.AMPs in the gut are produced mainly by Paneth cells（a specialized type of IECs）.This study further constructed the mice with OTUD4-specific knockout in Paneth cells(Def-Cre;Otud4^fl/fl)and found that the Def-Cre;Otud4^fl/fl mice were more resistant to DSS-induced colitis and S.t.infection and the AMP expression in IECs was higher compared to the Otud4^fl/fl mice.These results indicate that OTUD4 affects the composition of gut microbiota and promotes intestinal inflammation and bacterial infection by inhibiting the expression of AMPs.In order to further explore the mechanism by which OTUD4 regulates the expression of AMPs,this study first confirmed the interaction between OTUD4 and MyD88 in IECs of the wild-type mice through immunoprecipitation experiment.Subsequently,tandem ubiquitin binding entity（TUBE）enrichment ubiquitin and western blotting were performed and the results suggested that the K63polyubiquitination modification of MyD88 in IECs of the Vil-Cre;Otud4^fl/fl mice was significantly enhanced and the phosphorylation levels of NF-κB and MAPKs which are related to AMP expression were significantly up-regulated compared to the Otud4^fl/flmice after DSS treatment or S.t.infection.After stimulation with lipopolysaccharide（LPS）or peptidoglycan（PGN）or infection with S.t.,the expression level of AMPs in the intestinal organoids of the Vil-Cre;Otud4^fl/fl mice was significantly increased,while ST2825（MyD88 inhibitor）significantly decreased the high expression level of AMPs in the intestinal organoids of the Vil-Cre;Otud4^fl/fl mice.These results suggest that OTUD4 negatively regulates the K63 polyubiquitination modification of MyD88 and the activation of NF-κB and MAPK to restrict the expression of AMPs.In summary,this study revealed that OTUD4 negatively regulates the K63-linked ubiquitination of MyD88 and the activation of NF-κB and MAPKs,thereby restricting the expression of AMPs and promoting intestinal inflammation and bacterial infection,providing a new target and intervention strategy for the treatment of intestinal inflammation and bacterial infection.