SETD4 Regulates The Expression Of Inflammatory Cytokines In Endotoxmia

Sepsis is a systemic inflammatory response syndrome which could further induce sepsis shock and multiple organ dysfunction syndrome(MODS),even death.Currently,however,there is no effective strategy to prevent and treat sepsis because of the complexity of its pathogenesis.Thus,it remains an important and key issue to explore the exact molecular mechanism of sepsis in the related fields.It has been demonstrated that LPS,as a prototypical molecule of pathogen associated molecular patterns(PAMPs),could induce and activate substantial inflammatory cytokines in sepsis,which is critical to induce sepsis and sepsis shock.It has suggested that activation of Toll like receptor(TLR)by PAMPs and MAPK and NF-?B signaling pathways mediate the production of inflammatory cytokines.Recently,studies have shown that histone modifications such as histone methylation,and chromatin remodeling are also involved in the cytokines production.Histone methylation is mainly catalyzed by the SET family proteins,all of which contains a highly conservative SET domain.SET proteins play a critical role in inflammation and its related diseases through methylation of histone.SETD4 was found a differential protein in mice with endotoxic shock through using proteomics technology in our laboratory.Currently,however,studies for SETD4 remain limited.To fully understand the molecular mechanisms of SETD4 regulating cytokines expression in endotoxin shock,we constructed Setd4 knockout mice to investigate whether SETD4 can regulate cytokine production and affect the survival rate of mice with endotoxic shock.And we also further explored the methylation substrates,sites and methylation level associated with SETD4,which might partially demonstrate the molecular mechanism of cytokine production.The important findings in this study:1.Compared with Setd4+/+ mice,the expression of cytokines in serum from Setd4-/-mice such as IL-1??TNF??IL-6?MCP-1 and IFN-? was obviously decreased,indicating that SETD4 positively regulates the production of cytokines.2.Knockout Sed4 can reduce the mortality of mice with endotoxin shock,suggesting that the expression of cytokines regulated by SETD4 plays a key role in endotoxic shock.3.In comparison with BMDMs from Setd4+/+mice,mRNA level of IL-6?TNF-? and IL-1? was significantly suppressed after BMDMs from Setd4-/-mice were stimulated with LPS,which was consistent with serum cytokine protein change in Setd4-/-mice,implying that SETD4 may regulate the production of cytokines in the transcriptional level.4.We found that the phosphorylation of related molecules in MAKP and NF-?B signaling pathways was increased in both of BMDMs from Setd4+/+ and Setd4-/-mice after LPS stimulation,but activation levels had no significant difference.These results disclose that transcription of cytokines regulated by SETD4 may be localized at the downstream of MAPK and NF-?B signaling pathways.5.Both mass spectrum and product detection of in vitro methylation reaction with specific antibodies revealed that SETD4 can enhance of H3K4me2.The methylation of H3K4mel was enhanced by SETD4 as well when detected by specific antibodies.6.Based on the results of BMDMs from Setd4-/-and Setd4+/+ mice,we found that the methylation level of H3K4meland H3K4me2 was obviously reduced when Setd4 was deleted.7.When compared with BMDMs from Setd4+/+,the recruitment of H3K4mel and H3K4me2 showed a significant reduction in the promoter of Tnf and Il-6 after BMDMs from Setd4-/-mice were treated with LPS.Based on the above results,the following preliminary conclusions can be get,SETD4 is able to enhance the methylation of H3K4mel and H3K4me2,which,in turn,promotes the transcription of Tnf and 1l-6.As a result,inflammatory cytokines are substantially induced in endotoxemia,which further amplifies the inflammation response and results in animal death.Collectively,our study demonstrates that SETD4 plays a critical role in the development and progression of endotoxemia.