The Regulatory Role And Molecular Mechanism Of Aryl Hydrocarbon Receptor On The Expression Of IL-10 In Inflammatory Macrophages

Backgrounds:Sepsis is a life-threatening condition that arises when the body 's response to infection causes injury to its own tissues and organs.It is a serious complication in patients with acute and critical diseases such as infection,burn,trauma,shock,with a dangerous condition and a high morbidity and mortality.At present,there is no specific method of clinical prevention and treatment of sepsis.The mechanism is generally believed that systemic inflammatory response and immune suppression cause imbalance of immune and disorder of inflammatory cytokines prodution.Therefore,accurate regulation of inflammatory cytokine production can improve the development and progression of sepsis.In innate immune and adaptive immune responses,macrophages play a major role in the inflammatory cytokine secretion.Its dysfunction plays an important role in the pathogenesis of sepsis.Investigate the pathological mechanism of macrophages related signaling pathway in the pathogenesis of sepsis,and explore the new adjustment strategy,can provide innovative means for sepsis prevention.Aryl hydrocarbon receptor(AhR)is a ligand activation transcription factor.Previous evidences have identified that AhR plays a role in the metabolism of environment toxins and chemical substance,regulating biological rhythm,reproduction,oxidative stress.In recent years,it has been found to have important effects on innate and adaptive immune responses,autoimmune diseases,infections and inflammation.In the immune response,AhR not only plays an important role in T cells,B cells,monocytes,neutrophils,and dendritic cells,but also is involved in the inflammatory gene expression and the activity of macrophages.AhR is indispensable in LPS-induced inflammatory response and the sepsis tolerance,and the mice with sepsis characterized by the activation of AhR can effectively resist the fatal attack of gram-positive and gram-negative bacteria.The pro-inflammatory/anti-inflammatory balance of AhR activated macrophages is involved in the host state of sepsis susceptibility/tolerance.Activation of AhR is involved in the inflammatory gene expression and regulation of macrophages.AhR can downregulate pro-inflammatory cytokines expression including IL-6 and IL-1?,but for anti-inflammatory cytokines,such as IL-10,the mechanism should be elucidated.AhR knockout macrophages can secrete more inflammation cytokines under LPS stimulation,such as TNF-?,IL-6,IL-1? and IL-18,excluding IL-10.IL-10 is a multicellular source cytokine,mainly comes from monocytes and macrophages.IL-10 has the biological functions of anti-inflammatory,immune stimulation/suppression and negative feedback regulation in the control of inflammatory responses caused by pathogens and microorganisms.In natural killer cells,T cells,bone marrow dendritic cells and other immune cells,AhR can regulate the production of IL-10 in different ways,but the mechanism of AhR regulating IL-10 in innate immune macrophages needs to be clarified.We conducted experiments in mouse peritoneal macrophages,BMDM and spleen macrophages.In the AhR knockout mice macrophages and AhR overexpression RAW264.7 cells,we built the LPS-induced sepsis model in vivo and in vitro.To elucidate the mechanism of AhR expression in inflammatory macrophages,to explore the influence of AhR on IL-10 expression,to clarify the molecular mechanisms of AhR regulating IL-10 expression,regulating the expression of AhR and IL-10 to improve peritonitis process of septic mice.Methods:Part ?:Effect of AhR on IL-10 expression in LPS stimulated macrophages.1.Mice peritoneal and spleen macrophages were extracted and bone marrow derived macrophages were prepared.After purification and identification,they were cultured in vitro.We established LPS-stimulated model in vitro,and the AhR protein expression was measured by WB.2.In mouse peritoneal macrophages after LPS stimulation at different times,we used methods of qRT-PCR,Western blot and cell immunofluorescence to detect the expression levels of AhR mRNA and protein.3.Mice peritoneal macrophages was pretreated with NF-?B signaling pathway inhibitor PDTC,and then was stimulated with LPS.AhR protein expression was measured by WB.4.Adult male and female AhR+/-mice were mated in cages to produce AhR-/-mice.The mouse DNA was extracted and genotyping was performed.AhR overexpression RAW264.7 cell line was constructed and identified.5.Peritoneal macrophages and BMDMs from WT and AhR KO mice were used to establish LPS-stimulated model in vitro.The AhR protein expression was measured by WB,and the expression of IL-10 protein and mRNA were detected by ELISA and qRT-PCR.6.RAW/NC cells and RAW/AhR cells were used to establish LPS-stimulated model in vitro.We detected the expression of AhR protein and mRNA levels using WB and qRT-PCR,and detected the expression of IL-10 protein and mRNA levels using ELISA and qRT-PCR.7.LPS-stimulated model in vitro was established in RAW/NC and RAW/AhR cells,and M1/M2 phenotype markers of macrophages were detected by qRT-PCR.Part ?:The molecular mechanism of AhR regulating IL-10 expression in LPS-stimulated macrophages.1.Mice peritoneal macrophages were pretreated with different AhR ligands,and then stimulated by LPS.The expression of CYP1A1 mRNA and IL-10 protein in cell supernatant were detected by qRT-PCR and ELISA.2.The AhR binding sites on IL-10 gene promoter were predicted and DNA probes were constructed.In LPS-stimulated RAW/NC and RAW/AhR cells,the binding activity of nucleoprotein and DNA was detected by EMSA.3.The pGL3 plasmid and the plasmid containing IL-10 promoter region were respectively transfected into 293T/NC and 293T/AhR cells,and the reporter gene activity of the IL-10 promoter was detected by the dual-luciferase reporter gene system.4.In LPS-stimulated RAW/NC and RAW/AhR cells,the changes of STAT3,Src,mTOR and MAPK signaling pathways were detected by Western blot.5.RAW/NC and RAW/AhR cells were pretreated with IL-10 blocking antibodies,STAT3 and Src siRNAs and inhibitors,and then stimulated by LPS.The expression of STAT3 and Src protein and phosphorylation level were detected by Western blot,at the same time,IL-10 mRNA and protein expression level in cell supernatant were detected by qRT-PCR and ELISA.Part ?:Effect of regulating AhR and IL-10 expression in macrophages on peritonitis in mice.1.Peritoneal macrophages of WT and AhR KO mice were infected with AhR expression adenovirus and control adenovirus in vitro,and then stimulated by LPS.The green fluorescence intensity of the infected cells was detected by flow cytometry,and the AhR expression,the phosphorylation of Src and STAT3 were measured by WB.2.Peritoneal macrophages of WT donor mice were infected with AhR expression adenovirus and control adenovirus in vitro,and then were injected into WT recipient mice.The peritonitis model stimulated by LPS was established.The protein expressions of IL-10,IL-6 and IL-1? in peritoneal lavage fluid were detected by ELISA.3.Peritoneal macrophages of WT donor mice were infected with AhR expression adenovirus and control adenovirus in vitro,and then were injected into WT recipient mice.A lethal model of peritonitis stimulated by LPS was established.We observed the situation of mice and counted the survival of mice.Results:Part ?:Effect of AhR on IL-10 expression in LPS stimulated macrophages.1.The identification results of macrophages suggested that the proportion of peritoneal macrophages and BMDMs were about 95%,and the proportion of spleen macrophages was about 30%.LPS could increase the expression of AhR protein in peritoneal macrophages,spleen macrophages and BMDMs.2.In LPS-stimulated peritoneal macrophages,the expression of AhR in mRNA and protein levels could be enhanced in a time-dependent manner.After LPS stimulation,the mRNA levels of AhR were increased at 1 h,reached a peak at 2 h,then gradually decreased,and returned to the level at rest at 12 h.AhR protein expression levels were increased at 2 h after LPS stimulation,increased rapidly up to 12 h,and peaked at 8 h.LPS stimulation had no significant effect on the expression of AhR chaperone ARNT and HSP90.The results of cell immunofluorescence also showed that the protein expression of AhR increased after 6 h and 8 h of LPS stimulation.3.PDTC could inhibit the nuclear translocation of P65 and P50 proteins and the activation of NF-?B signaling pathway,and thereby reducing the expression level of AhR protein in inflammatory macrophages.4.Compared with WT mice,AhR was not expressed in peritoneal macrophages and BMDMs of AhR KO mice.In LPS-stimulated peritoneal macrophages and BMDMs,AhR knockout reduced the expression of IL-10 at mRNA and protein levels.5.RAW/AhR cells showed significantly higher mRNA and protein levels than RAW/NC and normal RAW264.7 cells.In LPS-stimulated RAW264.7 cells,AhR overexpression increased the expression of IL-10 at mRNA and protein levels.6.In LPS-induced AhR overexpression macrophages,the level of most M1-type surface markers decreased,while the level of M2-type surface markers increased.Part II:The molecular mechanism of AhR regulating IL-10 expression in LPS-stimulated macrophages.1.The agonist and antagonist of AhR did not affect the expression of IL-10 in inflammatory macrophages.2.AhR binding sites on IL-10 promoter were predicted to be-631 to-607 bp AhR1 and-319 to-343 bp AhR2,respectively.In LPS-stimulated RAW264.7 cells,AhR did not affect the DNA binding activity of IL-10 promoter in inflammatory macrophages.3.AhR did not affect the DNA transcription activity of IL-10 promoter.4.In inflammatory macrophages,AhR overexpression increased the phosphorylation level at Tyr705 of STAT3 without affecting the quantity of STAT3 protein and the phosphorylation level at Ser727 of STAT3.The activation of STAT3 was independent of the IL-10 autocrine loop.5.In inflammatory macrophages,AhR knockout reduced the phosphorylation levels of Src Tyr416 and STAT3 Tyr705,but did not affect the quantity of Src and STAT3 proteins.6.AhR promoted the expression of IL-10 in inflammatory macrophages independent of mTOR pathway and MAPK pathway.7.In AhR overexpression RAW264.7 cells,knockdown of Src and inhibition of Src Tyr416 phosphorylation not only reduced the phosphorylation level of STAT3 Tyr705,but also prevented the expression of IL-10 after LPS stimulation.Knockdown of STAT3 and inhibition of STAT3 Tyr705 phosphorylation did not affect Src and Src phosphorylation levels,but still prevented the expression of L-10 after LPS stimulation.Part ?:Effect of regulating AhR and IL-10 expression in macrophages on peritonitis in mice.1.Green fluorescent protein was expressed in primary macrophages of adenovirus-infected mice,and the average FITC fluorescence intensity of the cells increased.2.In AhR KO and WT peritoneal macrophages,the up-regulation of AhR expression by adenovirus infection could not only increase the phosphorylation level of Src Tyr416 and STAT3 Tyr705 after LPS stimulation,but also increase the expression of IL-10.3.In LPS-induced peritonitis mice,adoptive transfer of AhR overexpression peritoneal macrophages could improve the level of inflammatory cytokines in the peritoneal lavage fluid of mice,increase the level of anti-inflammatory factor IL-10,reduce the level of pro-inflammatory factors IL-6 and IL-1?,and improve the survival rate of peritonitis mice.Conclusions:1.LPS can promote AhR expression by activating the NF-?B signaling pathway in macrophages.2.The knockout of AhR can reduce the expression of IL-10 in inflammatory macrophages,while the overexpression of AhR can increase the expression of IL-10,indicating that AhR positively regulates the production of IL-10 in LPS-stimulated macrophages.3.The up-regulation of AhR expression can increase the phosphorylation level of Src Tyr416 and STAT3 Tyr705 in inflammatory macrophages,and further promote the production of IL-10.4.AhR-overexpression macrophages adoptive transfer to peritonitis mice can reduce the production of inflammatory cytokines in vivo,increase the level of IL-10,and reduce the mortality.