A Preliminary Study Of The Regulatory Mechanisms For IRAK-M Expression

Background and objectivesSepsis is defined as the host inflammatory response to severe,life-threatening infection with the presence of organ dysfunction.Sepsis initiates a complex interplay of host pro-inflammatory and anti-inflammatory processes,and it is the proper balance between the often competing pro-and anti-inflammatory pathways that determines the fate of the individual.In recent years,it has been recognized that immunosuppression is the main cause of death in patients with sepsis and the underlying mechanism is the major focus of the related studies,however,the treatment for sepsis-induced immunoparalysis is lacking.Toll-like receptor is one of the most important pattern recognition receptors(PPRs)in the host.Pattern recognition receptors recognition of pathogen-associated molecular patterns(PAMPs)is a primary modulator of sepsis,which partcipate in the regulation of the innate and adaptive immunity by activating various signaling pathways.It has known that endotoxin tolerance can lead to immunesuppression,which is the main cause of death in patients with sepsis.Studies show that IRAK-M is related to the pathogenesis of endotoxin tolerance.Interleukin-1 receptor associated kinase M(IRAK-M),a serine / threonine kinase,is an important negative regulation factor of TLRs pathway.IRAK-M mainly expressed in monocytes/macrophages and some epithelial cells.There have been reported that the expression of IRAK-M is closely related to the occurrence and development of sepsis,asthma,pneumonia,autoimmune disease and tumors.Although IRAK-M expression is typically induced through TLR signaling,IRAK-M can also be expressed in response to various endogenous and exogenous soluble factors as well as cell surface and intracellular signaling molecules.However,the regulation mechanism of IRAK-M is still less reported.In the present study,we detected the expression of IRAK-M in peripheral blood mononuclear cells from patients with sepsis again,as well as the expression of IRAK-M introduced by LPS in THP1 cells.Furthermore,we constructed reporter plasmids which contain human IRAK-M gene promoter,tested their activity and analyzed its key transcriptional regulatory elements.Meanwhile,we investigated the effect of major cytokines during sepsis,such as TNF-?,IL-10,TGF-? on the expression of IRAK-M.We also analyzed of the expression of mi R-320 a in monocytes of patients with sepsis,and its effect on IRAK-M expression and its mechanisms.Besides,we investigated the role of resveratrol on the expression of IRAK-M and its possible mechanisms.In this study,we preliminary explored the regulatory mechanisms of IRAK-M expression by in-vitro experiments,in order to lay the foundation for the further understanding of the mechanisms of the expression of IRAK-M in sepsis-induced immunoparalysis.Methods1.The m RNA levels of IRAK-M in peripheral blood mononuclear cells separated from sepsis patients and normal person were detected by RT-PCR.THP1 were stimulated with different concentrations of LPS,0,100ng/ml,1?g/ml,then collected cells at 12 h to detect the m RNA levels of IRAK-M by RT-PCR,and collected cells at 24 h to detect the protein levels of IRAK-M by Western Blot.THP1 were stimulated with a final concentration of 100ng/ml LPS,then collected cells at 0h,3h,6h,9h,12 h,24h,48 h respectively.The m RNA levels were detected by RT-PCR.2.We established the six reporter plasmids containing of different lengths of IRAK-M gene promoter,P1442,P970,P500,P300,P200,P100.Dual luciferase reporter assay was performed to detect the transcriptional activity of these promoter reporter plasmids in 293 T,A549,BEAS-2B cells.The region with stronger transcriptional activity was analyzed by bioinformatics analysis.By construction of two mutant variants P100-delete and P100-mutant,we explored the key transcriptional element of the promoter sequences.Silence of the key transcription factor by si RNA,dual luciferase reporter assay was performed to detect the transcriptional activity of the promoter,and RT-PCR was carried out to detect its effect on the expression of IRAK-M.3.THP1 cells were treated with different concentrations of IL-10 0,5ng/ml,10ng/ml,20ng/ml,then collected cells at 12 h to detect the m RNA levels of IRAK-M by RT-PCR,and collected cells at 24 h to detect the IRAK-M protein levels by Western Blot.THP1 were stimulated with a final concentration of 100ng/ml LPS,then collected cells at 0h,6h,12 h,24h respectively.The m RNA levels were detected by RT-PCR.THP1 cells were treated with different concentrations of TGF-? 0,5ng/ml,10ng/ml,20ng/ml,12 hours later,RT-PCR was performed to detect the m RNA levels of IRAK-M.Different concentrations of TNF-? 0,10ng/ml,20ng/ml,50ng/ml were treated THP1 cells,RT-PCR was performed to detect the m RNA levels of IRAK-M after 6 hours,and Western Blot was carried out to detect IRAK-M protein expression after 24 h.4.The m RNA levels of mi R-320 a in peripheral blood mononuclear cells isolated from sepsis patients and normal person were detected by RT-PCR.THP1 cells were transfected with different concentrations of NC and 10 n M,50 n M,100 n M of mi R-320 a mimics.RT-PCR and Western Blot were performed to detect the impact of mi R-320 a in the expression of IRAK-M.Bioinformatics method was used to predict the binding sites of mi R-320 a in IRAK-M 3'UTR.Then,we built a reporter plasmid named p GLO-IRAKM-WT,which contained two binding sites between mi R-320 a and IRAK-M 3'UTR.mi R-320 a and p GLO-IRAKM-WT co-transfected into A549 cells,dual luciferase reporter assay was carried out to analysis the activity of IRAK-M 3'UTR.Next,three mutant reporter plasmids of IRAK-M 3'UTR were constructed,636 point mutant p GLO-IRAKM-mut1,745 site mutant p GLO-IRAKM-mut2,and 633,745 sites double mutant p GLO-IRAKM-mut3.These reporter plasmids and mi R-320 a were co-transfected into A549 cells,luciferase activity was used to test the 3'UTR activity.5.We first constructed a human IRAK-M expression plasmid.20?M MG132 and 5m M E64 were respectively used to treat THP1 cells and 293 T cells which transfected with flag-IRAK-M plasmid.Western Blot was used to detect changes of IRAK-M protein expression.Flag-IRAK-M and HA-Nedd4 L plasmids were co-transfected in Hela cells,then we performed Western Blot,co-immunoprecipitation,and immunofluorescence to detect the interaction between exogenous IRAK-M and Nedd4 L.Different concentrations of resveratrol were added in Hela cells which transfected with exogenous flag-IRAK-M plasmids for 20 hours,the expression of Nedd4 L and IRAK-M protein were tested by Western Blot.IRAK-M protein expression was also detected in resveratrol treated THP1 cells.Results1.The expression of IRAK-M m RNA in human peripheral blood mononuclear cells from sepsis patients was significantly higher than normal person.And in LPS treated THP1 cells,the m RNA and protein expression of IRAK-M were both increased significantly.2.Six luciferase reporter gene vectors containing IRAK-M promoter region were successfully constructed,P1442,P970,P500,P300,P200,P100.By testing their transcriptional activity,we found that the regions from-100 to +161 bp had the highest transcriptional activity.Then,mutated the transcription factor AP-1 binding sequence,the promoter activity was decreased.By knocking down of the subunits of AP-1,the promoter activity was also suppressed and the expression of IRAK-M was reduced.3.Pro-inflammatory cytokine TNF-? can up-regulate the expression of IRAK-M,but TNF-? had no obvious effect on IRAK-M promoter activity.However,various concentrations of anti-inflammatory cytokines IL-10 and TGF-? can't impact the expression of IRAK-M.4.The expression of mi R-320 a in human peripheral blood mononuclear cells from sepsis patients was decreased than that of normal person.In-vitro experiments showed that mi R-320 a can significantly inhibit m RNA and protein expression of IRAK-M.Meanwhile,mi R-320 a can inhibit the activity of IRAK-M 3'UTR,and the activity of IRAK-M 3'UTR recovered when the mi R-320 a binding sites in 3'UTR.5.The ubiquitin-proteasome pathway inhibitor MG132 can inhibit the degradation of IRAK-M protein.E3 ubiquitin ligase Nedd4 L inhibited the expression of IRAK-M protein.There was an interaction between exogenous Nedd4 L and IRAK-M.Resveratrol can increase the expression of Nedd4 L and significantly down-regulated the expression of IRAK-M protein.ConclusionIRAK-M expression in peripheral blood mononuclear cells in patients with sepsis was significantly up-regulated,while the expression of mi R-320 a was decreased.Transcription factor AP-1 played an important role in the regulation of basal transcription of IRAK-M.mi R-320 a directly targeted with IRAK-M 3'UTR and inhibited the expression of IRAK-M at the post transcriptional level.Resveratrol inhibited the expression of IRAK-M probably through up-regulating of Nedd4 L,and the specific regulatory mechanisms remained to further study.