PBLD Attenuates Intestinal Epithelial Inflammatory Response And Improves Intestinal Barrier Function By Inhibiting NF-?B Signaling

Background and ObjectionIntestinal epithelial cells(IECs)are important regulators of the intestinal barrier function and immune homeostasis.Intestinal barrier dysfunction and dysregulated immune response are the two key factors in the pathogenesis of ulcerative colitis(UC).The destruction of the intestinal barrier function causes microbial exposure to the mucosal immune system,leading to uncontrolled inflammation in the colon.The normal performance of the intestinal barrier function depends on the cell junctions between IECs,especially tight junctions(TJ).In addition to the barrier function,IECs also have immunoregulatory function.In response to pro-inflammatory cytokine stimuli or microbial invasion,IECs can secrete cytokines and chemokines to orchestrate interaction between the gut microbiome and mucosal immune system.This process is mainly regulated by NF-?B signaling pathway.Therefore,IECs play an essential role in intestinal homeostasis,and finding the molecules that regulate IECs functions may provide a new perspective for the pathogenesis of UC.Our previous study found that phenazine biosynthesis-like domain-containing protein(PBLD)was significantly decreased in the colonic mucosa of patients with UC,and it was negatively correlated with the severity of the patient's clinical condition,suggesting that PBLD may play a role in the progression of UC.PBLD is a tumor suppressor gene which can inhibit the occurrence and development of hepatocellular carcinoma,breast cancer and gastric cancer.However,the role of PBLD in the pathogenesis of UC remains unclear.Studies have reported that overexpression of PBLD in gastric cancer cells could increase the expression of E-cadehrin which is an intercellular junction molecule.Whether PBLD can regulate the expression of other intercellular junction molecules(such as TJ)and thus affect the intestinal barrier function is still unclear.In addition,studies have shown that PBLD inhibits the occurrence and development of hepatocellular carcinoma by inhibiting NF-?B signaling pathway.NF-?B signaling pathway is significantly activated in patients with UC,and inhibiting NF-?B signaling pathway can alleviate mouse colitis,however,whether PBLD can inhibit the pathogenesis of UC by inhibiting NF-?B signaling pathway is still unclear.Therefore,this study focused on elucidating the role of PBLD in the pathogenesis of UC and its molecular mechanism.Methods and results1.PBLD is significantly decreased in patients with UCWe evaluated the expression of PBLD,inflammation-related molecules and tight junction molecules in paired colon tissue of patients with UC.PBLD was significantly decreased in the lesions of patients with UC.The expression of PBLD was positively correlated with the expression of tight junction molecules,and negatively correlated with the expression of pro-inflammatory cytokines.Mice were treated with 2.5%DSS for 5 days,followed with regular water for 3 days to induced colitis,and the expression of PBLD in the intestinal tissue was detected by western blot and IHC.PBLD was decreased in DSS-induced colitis,and PBLD was mainly expressed in intestinal epithelial glands.2.Epithelial PBLD deficiency exacerbates DSS-induced and TNBS-induced colitisWe constructed intestinal epithelial PBLD knockout mice(PBLDIEC-/-mice)and wild-type mice(WT mice)by using CRISPR/Cas9 system.PBLDIEC-/-mice were generally similar to WT mice,and no abnormal phenotypes were observed under normal conditions.Intestinal epithelial PBLD knockout does not affect the structure and function of the intestine in mice.PBLDIEC-/-mice and WT mice were treated with 2.5%DSS for 5 days,followed with regular water for 3 days to induce acute colitis.Compared with WT mice,PBLDIEC-/-mice lost more body weight and had higher disease activity scores,shorter colon length,and higher histopathological scores.PBLDIEC-/-mice and WT mice were treated with DSS for three cycles,with each cycle containig with DSS for 7 days,followed with regular water 14 days to induce chronic colitis.Compared with WT mice,PBLDIEC-/-mice lost more body weight and had a higher mortality rate,shorter colon length,and higher histopathological scores.PBLDIEC-/-mice and WT mice were administrated with TNBS by enema to make TNBS-induced colitis.Compared with WT mice,PBLDIEC-/-mice lost more body weight,had shorter colon length and higher histopathological scores.3.Epithelial PBLD deficiency aggravates intestinal barrier dysfunction in DSS-induced colitisThe intestinal permeability assay was used to evaluate the intestinal barrier function of mice with DSS-induced colitis.The intestinal permeability of PBLDIEC-/-mice with colitis was significantly increased compared to WT mice with colitis.Western blot,RT-qPCR and immunofluorescence were used to detect the expression of tight junction molecules in the colon tissue of mice with DSS-induced colitis.Compared to WT mice with colitis,the expressions of ZO1 and Occludin in the colon tissue of PBLDIEC-/-mice with colitis were sigificantly decreased.In addition,the numbers and distribution of goblet cells in the colon tissue of colitis mice were evaluated by IHC.Compared to WT mice with colitis,PAS-positive and MUC2-positive cells in the colon tissue of PBLDIEC-/-mice with colitis were significantly decreased.4.PBLD overexpression enhances Caco2 monolayer barrier function by improving TJ expressionCaco2 cells were transfected with lentivirus to construct cell line with vector(Caco2-Vector)or overexpressing PBLD(Caco2-PBLD),and then these cells were inoculated into transwell chambers and cultured for 21 days to confluence into monolayer.The barrier function of monolayer was evaluated by transepithelial resistance(TEER)and FITC-dextran flux assay.In the absence of TNF?/IFN?,TEER values in Caco2-PBLD monolayer was similar to that in Caco2-Vector monolayer.In the presence of TNF?/IFN?,TEER values in Caco2-PBLD monolayer decreased slower than that in Caco2-Vector monolayer.Regardless of the presence or absence of LPS stimulation,less FITC-dextran passed through Caco2-PBLD monolayer compared with Caco2-Vector monolayer.Western blot and RT-qPCR analysis showed that the expression of ZO1 and Occludin in the Caco2-PBLD monolayer was significantly increased compared to those in Caco2-vector monolayer.Caco2-vector and Caco2-PBLD monolayers were treated with TNF?/IFNy for 24 hours,then the changes of tight junction were detected by immunofluorescence and western blot.Compared with Caco2-vector monolayer,enforced expression of PBLD in Caco2 monolayer not only inhibited reduction of TJ proteins,but also improved the disrupted location of TJ proteins induced by TNF?/IFN-y.Moreover,enforced expression of PBLD in Caco2 monolayer also inhibited NF-?B activation induced by TNF?/IFN-y.5.Ablation of PBLD in IECs increases inflammatory cells infiltration in murine colitis and enhances NF-?B activation in IECs of murine colitisInflammatory cells infiltration in the colon tissue of WT and PBLDIEC-/-mice with DSS-induced colitis was evaluated by IHC.The results showed that F4/80 and MPO-positive cells were significantly higher in PBLDIEC-/-mice than those in WT mice with colitis.Inflammatory cells infiltration in the colonic mucosa propria and mesenteric lymph nodes of mice with colitis was assessed by flow cytometry.The results showed that compared to WT mice with colitis,PBLDIEC-/-mice with colitis had significantly higher levels of macrophages,neutrophils and monocytes in the colonic mucosa propria and mesenteric lymph nodes.The expression levels of pro-inflammatory cytokines in the colon tissues of mice with colitis was evaluated by ELISA.The expression levels of TNFa and IL-6 in the colon tissue of PBLDIEC-/-mice with colitis were significantly higher than those in WT mice with colitis.The intestinal epithelial cells of WT and PBLDIEC-/-mice with DSS-induced colitis were extracted,and western blot was used to evaluate the activation of NF-?B pathway in intestinal epithelial cells of mice with colitis.Compared to WT mice with colitis,NF-?B was significantly activated in the intestinal epithelial extract of PBLDIEC-/-mice with colitis.The production of pro-inflammatory cytokines and chemokines in the intestinal epithelial cells of mice with colitis was assessed by RT-qPCR.Compared to WT mice with colitis,the mRNA expression of TNF?,IL-6,IFN?,IL-1?,CCL20 and IL-17c were significantly increased in intestinal epithelial extract of PBLDIEC-/-mice with colitis.Intestinal epithelial cells of WT and PBLDIEC-/-mice were extracted and stimulated with TNFa for 1 hour.Western blot analysis showed that the activation of NF-?B in the intestinal epithelial cells of PBLDIEC-/-mice was significantly increased compared with that in WT mice.6.PBLD inhibits IECs inflammatory response by supressing NF-?B signalling pathwayFHC or HT29 cells were transinfected with lentivirus to construct empty(FHC-vector or HT29-vector)and over-expressing PBLD cell lines(FHC-PBLD or HT29-PBLD),then TNFa was used to stimulate these cells for 1 hour.The effect of PBLD in intestinal epithelial cells was assessed by RT-qPCR.Compared with FHC-vector or HT29-vector,the mRNA expression levels of TNF?,IL-1?,IL-6 and IL-8 were significantly decreased in FHC-PBLD or HT29-PBLD cells.The effect of PBLD on NF-?B activation in intestinal epithelial cells was evaluated by western blot and immunofluorescence.The expressions of pI?B? and pp65 were significantly dcreased,and the translocation of p65 from the cytoplasm to the nucleus were decreased in FHC-PBLD cells compared to FHC-vector cells induced by TNF?.In addition,the dual luciferase reporter gene experiment found that overexpression of PBLD significantly reduced the activity of NF-?B in FHC cells.FHC cells were transfected with small interference RNA(siRNA)to construct empty(siNC)and PBLD knockdown(siPBLD)FHC cells,then pretreated with NF-?B inhibitor(IKK-16)for 2 hours,and followed by TNF? treatment for 1 hour.The effect of PBLD knockdown on the inflammatory response of intestinal epithelial cells was detectd by RT-qPCR.Compared with siNC,siPBLD significantly increased the mRNA expression of IL-1?,IL-6 and IL-8 under TNFa stimulation,while IKK 16 could reverse the changes caused by siPBLD.The effect of PBLD knockdown on the activation of NF-?B in intestinal epithelial cells was assessed by western blot.Compared with siNC,siPBLD enhanced NF-?B activation under TNFa stimulation,while IKK16 could reverse the changes caused by siPBLD.Co-immunoprecipitation(Co-IP)assay demonstrated that PBLD could interact with IKKa and IKK? in FHC-PBLD cells.Conclusions1.PBLD is decreased in patients with ulcerative colitis and in mice with DSS-induced colitis.2.Epithelial PBLD deficiency exacerbates DSS-induced colitis characterized by more impaired intestinal barrier function and more immune cells infiltration in colon tissue.3.PBLD enhances intestinal epithelial cells barrier function by improving tight junction expression.4.In vitro,PBLD inhibits the activity of IKK complex by interacting with IKKa and IKK?,thereby inhibiting NF-?B activation and reducing the inflammatory response of intestinal epithelial cells.