Study On The Mechanism Of TRIM21 Regulating NF-κB Pathway Mediated Inflammation Caused By Corynebacterium Pseudotuberculosis Infection

Corynebacterium pseudotuberculosis（Cp）,the pathogen of Category III animal epidemics,can infect many animals and people,mainly causing inflammatory suppurative reactions.Tripartite motif（TRIM）is a family of E3 ligases involved in important life processes such as tumor killing,virus replication inhibition and apoptosis.TRIM21,a member of Trim family,is a protein that regulates immune response such as antiviral infection,cytokine and chemokine secretion.Many studies have emphasized the importance of TRIM21 in antiviral immunity,but little is known about its role in resisting bacteria or other pathogens.Previous studies in our laboratory confirmed that TRIM21 negatively regulates Cp-induced inflammation,but its regulatory mechanism is still unclear.Therefore,in this experiment,firstly,the signal pathways that may be involved in TRIM21 regulating inflammation process of Cp-infected macrophages were screened by transcriptome sequencing.Secondly,the role and mechanism of key signal pathways in TRIM21 regulating inflammatory response of Cp-infected macrophages were screened by Trim21 gene knockout or overexpression.At the same time,the deletion strain of Cp PLD coding gene（pld）was used to infect mouse macrophages to study the relationship between PLD and TRIM21 regulating the inflammatory effect in Cp-infected macrophages.The main results are as follows:1.Based on transcriptome sequencing,the signal pathway of TRIM21 regulating inflammation caused by Cp-infected macrophages was analyzed.Wild-type（WT）and Trim21 knockout(Trim21^-/-)mice were infected with Cp ATCC19410 with the multiplicity of infection（MOI）of 10,and the transcriptome was sequenced.At the same time,the transcriptome sequencing results were verified by real-time quantitative PCR（q PCR）.A total of 1757 differentially expressed genes（DEGs）were screened out in this sequencing.The results of q PCR showed that the m RNA expression of the selected genes was consistent with the expression trend of the transcriptome sequencing results,indicating that the transcriptome sequencing data was highly accurate.KEGG enrichment analysis of DEGs showed that DEGs could be enriched in NF-κB,MAPK,Ras and Fox O after Cp infection compared with uninfected WT and Trim21^-/-mouse macrophages.Compared with Cp-infected WT mouse macrophages,DEGs that in infected Trim21^-/-mouse macrophages can be enriched in Jak-STAT,Fox O and NF-κB signaling pathways,suggesting that these pathways may be related to TRIM21 regulating inflammation in Cp-infected macrophages.2.Trim21 regulates the phosphorylation level of key molecules of NF-κB and p38MAPK signaling pathway in Cp-infected mouse macrophages.Based on the transcriptome analysis of the inflammatory pathway of Cp infection and the previous research in our laboratory,the NF-κB and p38 MAPK signaling pathways were further studied.The peritoneal macrophages of WT mice were infected with Cp with MOI of 10,and the phosphorylation levels of key molecules of NF-κB and p38 MAPK were detected by Western blot at different time points.The results showed that the phosphorylation levels of IκBαand p65,which the key molecules of NF-κB signaling pathway,increased after Cp infection for 2 hours compared with the uninfected group.In addition,the expression level of p-p38increased after Cp infection.These results suggest that NF-κB and p38 MAPK signaling pathways are involved in the response of macrophages to this pathogen infection.In order to further clarify the role of TRIM21 in this process,Western blot was used to detect the expression levels of p65,IκBα,p38 and their phosphorylated proteins in macrophages of WT and Trim21^-/-mice infected with Cp.The results showed that compared with infected WT macrophages,the expression levels of p-p65,p-IκBαand p-p38 increased significantly after Cp infected Trim21^-/-macrophages.At the same time,the Trim21 was overexpressed,and found that the expression levels of p-p65 and p-IκBαwere significantly decreased.The results showed that TRIM21could negatively regulate the inflammatory effect of Cp-infected mice macrophages by reducing the phosphorylation levels of IκBα,p65 and p38 molecules.3.Trim21 regulates the ubiquitination of key molecules of NF-κB signaling pathway in Cp-infected mouse macrophages.Besides phosphorylation,ubiquitination modification pathway is also related to the activation of NF-κB signaling pathway.In order to study the effect of TRIM21 on the ubiquitination of key molecules in the activation of NF-κB signaling pathway in Cp-infected macrophages,WT mouse macrophages were first infected with Cp with MOI of 10,and whether TRIM21 could interact with p65 and IκBαwas detected by Co-IP.The results showed that TRIM21 could interact with p65 and IκBαbefore and after Cp infection.In order to determine whether Cp infection could cause ubiquitination of key molecules in NF-κB pathway,the ubiquitination level of total protein and the ubiquitination of p65 and IκBαat K48 and K63 sites after Cp infection in WT macrophages were detected by Western blot and IP respectively.The results showed that the ubiquitination level of total protein and IκBαin K48 site of macrophages infected with Cp were increased,but the ubiquitination level of IκBαin K63 site did not change significantly.It is suggested that Cp infection can enhance the ubiquitination level of total cell proteins,and IκBαis mainly ubiquitinated at K48 site after the pathogen infected macrophages.In order to further study the effect of TRIM21 on ubiquitination of key molecules of NF-κB signaling pathway in mouse macrophages infected with Cp,Trim21 gene overexpression and knocking-out macrophages were infected with Cp,respectively,and the ubiquitination levels of total protein and IκBαin infected macrophages were detected.The results showed that compared with infected WT macrophages,the ubiquitination level of total protein was decreased after Cp infected Trim21^-/-macrophages,and the ubiquitination level of IκBαat K48 site was also weakened,while the ubiquitination level of total protein and IκBαat K48 site in infected macrophages were increased after Trim21 gene overexpression.It is suggested that TRIM21 played a positive regulatory role in the ubiquitination of IκBαat K48 site in Cp infected mouse macrophages.4.The role of phospholipase D in the activation of NF-κB signaling pathway in mouse macrophages infected with Cp.WT mouse macrophages were infected with ATCC19410 and its pld deletion strain（ATCC19410Δpld）with MOI of 10.The phosphorylation level of key proteins in the pathway was detected by Western blot,and the ubiquitination level of IκBαat K48 site and total protein was detected by IP or Western blot.The results showed that the absence of pld had no significant effect on the protein expression levels of p65,p-p65,IκBαand p-IκBαin Cp-infected macrophages,suggesting that PLD was not involved in the phosphorylation of IκBαand p65 in the activation of NF-κB signaling pathway.Compared with uninfected macrophages,the ubiquitination level of total protein and IκBαat K48 were increased after ATCC19410 and ATCC19410Δpld infected macrophages,but compared with macrophages infected with wild strains,the ubiquitination level of total protein and IκBαat K48 were decreased after pld deletion strain infection,suggesting that PLD is related to the ubiquitination of IκBαpromoted by Cp infected mouse macrophages.To sum up,this study showed that TRIM21 negatively regulated the phosphorylation of key molecules IκBαand p65 of NF-κB pathway and the phosphorylation level of key molecule p38 of p38MAPK pathway after Cp infected macrophages,but positively regulated the ubiquitination of key molecules IκBαof NF-κB pathway in infected macrophages.Deletion of pld did not affect the phosphorylation of IκBαand p65 mediated by Cp infected macrophages,but it was related to the ubiquitination of IκBαpromoted by this pathogen infected macrophages.