The Regulation Of Interferon-γ On Intestinal Epithelial Cell Necroptosis,Proliferation,and Differentiation

BackgroundDuring the past decades,inflammatory bowel diseases(IBD)have emerged as a public health challenge in China,but traditional drugs are not effective for the treatment of IBD.The incidence of IBD is closely related to the impaired barrier function of the intestinal mucosa that depends on the epithelial tight junction permeability and the integrity of the intestinal epithelium.Previous studies showed that IFN-γ increases the permeability of the epithelial tight junction by activating myosin light chain kinase(MLCK).However,it is still unclear whether IFN-γ affects the occurrence and progression of IBD by regulating intestinal epithelial cell survival,proliferation,and differentiation.AimsOur study aimed to investigate the mechanisms of how IFN-γ regulates the mucosal barrier function by affecting intestinal epithelial cell survival,proliferation,and differentiation.Besides,our study aimed to reveal the relationship between IFN-γand the occurrence and development of IBD and to explore whether IFN-γ can be used as a new target to develop new therapeutic drugs for IBD.Methods1.To explore the mechanism that IFN-γ regulated intestinal epithelial cell necroptosis,the following methods were used:(1)Two types of experimental colitis models in mice were established:1)DSS(dextran sulfate sodium)-induced colitis model;and 2)T-cell adoptive transfer colitis model.Colon tissues were collected at different time points during the development of colitis.The correlation between the expression of IFN-γ and marker genes of cell death at different stages of colitis were analyzed;(2)Intestinal organoids from wild-type mice were cultured in ENR(EGF,Noggin,and R-spondin)or WRN(Wnt,R-spondin,Noggin)medium in vitro.The effect of IFN-γ on the survival of intestinal organoids was evaluated through bright-field observation under the microscopy;(3)The survival of intestinal cells was determined by relevant cell death staining,and the expression of marker genes of cell death was determined by quantitative Real-time(qPCR)and western blotting;(4)The specific inhibitors of apoptosis and necroptosis were used to rescue IFN-y-treated intestinal organoids to explore which type of cell death is dominant in IFN-y-induced cell death.Intestinal organoids were cultured from Ripk3-1-or Mlkl-1-mice,in which the necroptosis pathway was blocked,to further investigate the type of cell death induced by IFN-y;(5)A pGL3-luciferase vector that contained the mouse Mlkl gene promoter was constructed,transfected into Hela cells,and Mlkl gene promoter activity was evaluated by the dual-luciferase reporter assay;(6)Kinase inhibitors of JAK were used to rescue the IFN-y induced intestinal organoids death by blocking the JAK/STAT1 signaling pathway;(7)Wild-type mice were injected with a high dose of IFN-y.Intestinal mucosal injury and cell death marker gene expression were detected by pathological analysis,qPCR,and western blot.The effect of excessive IFN-y on intestinal epithelial cell injury was studied in vivo.2.To explore the mechanism that IFN-y regulated intestinal epithelial cells proliferation and differentiation,the following methods were used:(1)Intestinal organoids from wild-type mice were cultured in ENR medium and the growth status was recorded and analyzed after IFN-y treatment.Intestinal organoids were stained with EdU and the number of proliferating cells was analyzed by flow cytometry.Quantitative Real-time PCR was used to determine the expression of marker genes of proliferative and transition-amplified(TA)cells;(2)Intestinal organoids from Lgr5-EGFP knock-in mice were cultured in ENR medium and the number of intestinal stem cells was analyzed by fluorescence staining and flow cytometry in organoids with IFN-γ treatment.Quantitative Real-time PCR and western blotting were used to determine the expression of marker genes of activate and quiescent stem cells in organoids;(3)Quantitative Real-time PCR and western blotting were used to determine the expression of marker genes of absorptive enterocytes in organoids;(4)Immunofluorescence staining was used to analyze the number of various secretory epithelial cells in IFN-y treated organoids.Quantitative Real-time PCR was used to determine the expression of marker genes of secretory epithelial cells in organoids;(5)Quantitative Real-time PCR and western blotting were used to determine the expression of genes in Wnt and Notch in organoids with IFN-γ treatment.Results1.IFN-γ regulates intestinal epithelial cell necroptosis(1)The expression of Ifn-y in experimental colitis was positively correlated with the expression of the necroptosis marker genes Ripk3 and Mlkl.All of these genes gradually increased with the occurrence of colitis and decreased when after the recovery of colitis;(2)IFN-γ treatment inhibited the normal growth of intestinal organoids(cultured in ENR or WRN)in vitro,and the number of survived intestinal organoids gradually decreased with increased concentration of IFN-γ.(3)PI and cleaved caspase3/7 staining and the following flow cytometry analysis indicated that a large number of dead cells were detected in cultured intestinal organoids with IFN-y treatment.Quantitative Real-time PCR and western blotting showed the expression of apoptosis marker genes Caspase3 and Caspase8,and necroptosis marker genes Ripk3 and Mlkl,were significantly increased with IFN-y treatment.(4)Compared with the pan-caspase inhibitor zVAD,Necrostatin-1(Nec-1),a specific necroptotic inhibitor of Receptor-interacting serine/threonine-protein kinase 1(RIPK1),more effectively rescued organoids death induced IFN-y.Intestinal organoids cultured from Ripk3-/-or Mlkl-/-mice,in which the necroptosis pathway was inhibited,grow normally with IFN-γ addition in the culture medium.(5)Dual-luciferase reporter assay showed that in Hela cells,IFN-γ increased the promoter activity of Mlkl gene through pSTAT1,thus may increase the expression of MLKL protein.(6)Inhibition of the JAK/STAT signaling pathway by the addition of JAK inhibitor I in the cultured medium could rescue intestinal organoids damage caused by IFN-γtreatment and therefore,increased the number of intestinal organoids in vitro.(7)Excessive administration of IFN-γ in mice induced the damage of intestinal mucosa and caused a large number of cell death in the epithelium.Excessive administration of IFN-y activated the JAK/STAT1 pathway and increased the expression of necroptotic markers RIPK3 and MLKL in epithelial cells.2.IFN-y regulates intestinal epithelial cell proliferation and differentiation(1)IFN-y increased the volume of cultured intestinal organoids and accelerated the proliferation of epithelial cells in organoids.The expression of CyclinD1 mRNA significantly increased with IFN-y treatment.In contrast,the expression of TA cells marker genes significantly decreased with IFN-y treatment.(2)IFN-y treatment reduced the number of Lgr5+intestinal stem cells in intestinal organoids.Consistently,the expression of active stem cell marker genes such as Lgr5 and Olfm4 barely expressed with IFN-y treatment.The expression of quiescent stem cell marker genes such as Bmil and Lrig was significantly reduced.In addition,IFN-y significantly increased the expression of fetal stem cell marker gene Scal.(3)IFN-y treatment reduced the mRNA and protein expression of absorptive enterocytes,indicating the disruption of maturation and differentiation of absorptive cells in cultured intestinal organoids.(4)IFN-y treatment significantly reduced the expression of marker genes and the number of various secretory cells in cultured intestinal organoids.(5)IFN-y treatment significantly reduced the expression of Wnt3 and its downstream genes including β-catenin and Axin2.Besides,the expression of the Notch signaling pathway such as notch ligands Dll1 and Jagged1,as well as notch receptors Notch1 and Notch2 were significantly reduced in IFN-y treated organoids.These results suggested that IFN-γ inhibited Wnt and Notch signaling pathways in intestinal organoids.ConclusionsBased on the above results,we conclude that:(1)Using in vitro organoids and in vivo mice models,we found that excess administration of IFN-y activated JAK/STAT1 signaling pathway and increased the phosphorylation level of STAT1 in intestinal epithelial cells,and thereby upregulated the expression of the necroptotic gene Mlkl,which could lead to intestinal epithelial cell necroptosis and mucosal damage.Blocking of the necroptosis or JAK/STAT1 pathway could reduce the damage of intestinal cells induced by IFN-y;(2)Using the intestinal organoid culture model in vitro,we found that IFN-y accelerated the proliferation while inhibiting Wnt and Notch signaling pathways in cultured intestinal organoids.Interestingly,IFN-y affected intestinal stem cell maintenance and cell differentiation simultaneously.IFN-y treatment significantly reduced the number of adult stem cells,TA cells,and mature intestinal epithelial cells(both absorptive and secretory cells).By contrast,the number of fetal stem cells was significantly increased after IFN-y treatment.Based on these results,we revealed the molecular mechanism of IFN-y on regulating intestinal epithelial cell necroptosis,proliferation,and differentiation.We demonstrated that IFN-y aggravated colitis and hindered the repair of the damaged mucosa by impairing the mucosal barrier and the integrity of epithelium.Our study will be helpful for further understanding of the role of inflammatory cytokines such as IFN-y in regulating intestinal epithelium homeostasis and provides specific targets for the development of new drugs for IBD therapy.