Study Of Lactobacillus Plantarum Activating Type Ⅰ Interferon Pathway Of IPEC-J2 Via STING Against Pathogenic Infection

Intrinsic immunity is the first line to protect host against pathogenic infection,which is regulated by a variety of signaling pathways.c GAS-STING-mediated type I interferon production pathway is one of the classical ones.As cytosolic DNA sensor,c GAS can be activated by viral or bacterial DNA,triggering a downstream cascade reaction to produce type I interferon（IFN-I）and related cytokines through the activation of STING to display anti-infection.IFN-I mainly includes IFN-αand IFN-β,which play important roles in both antiviral and intracellular bacterial infections.As an important member of the intestinal flora,lactic acid bacteria can regulate the intrinsic immunity through a variety of mechanisms.In recent years,certain strains of Lactobacillus have been found to activate the STING or MAVS-mediated IFN-I production pathway and thus exert anti-infective effects.However,the exact mechanism of LP on regulating anti-vital or anti-intracellular bacterial immunity is unclear.Herein,we studied the STING in IPEC-J2 cells regulated by Lactobacillus plantarum（LP）,a probiotic strain obtained from piglet intestine.Base on the porcine rotavirus and Salmonella typhimurium（intracellular pathogens）,which are important pathogens for the intestinal health of piglets,as subjects,we revealed the potential mechanisms by which LP regulates the STING to mediate the anti-infective effects of IPEC-J2 cells.The aim is to lay the foundation for research into the regulation of host intrinsic immunity against intracellular pathogenic infections by lactic acid bacteria.1.Effect of Lactobacillus plantarum on the c GAS-STING pathway in IPEC-J2 cellsTo determine the condition（ratio and time）for co-culture of LP and IPEC-J2 cells,we compared the effects of LP on cell proliferation and apoptosis on IPEC-J2 cells at different ratio of bacteria:cell（MOI=10,100）and incubating time（1 h,4 h and 6 h）by CCK-8 and flow cytometry.The adhesion effect of LP on IPEC-J2 cells was analyzed using fluorescence microscopy and plate counting.The results showed that LP did not adversely affect the viability and apoptosis of IPEC-J2 cells,and the adhesion effect of LP to IPEC-J2 cells was better under MOI=100 or incubating 6 h.The results showed that LP had no adverse effect on the viability and apoptosis of IPEC-J2 cells.To further clarify the effect of LP-mediated STING on the IFN-Ⅰpathway in porcineintestinal epithelial cells.In this study,the expression of IFN-β,TNF-α,IL-6 and IL-1βin supernatants was detected by ELISA.The transcriptional of intracellular IFN-βwere detected by fluorescence quantitative PCR（RT-q PCR）.The phosphorylation of STING and IRF3 in the c GAS-STING pathway detected by Western blot.The results showed that the secretion of IFN-β（p<0.01）and transcription of intracellular IFN-βwere significantly higher of LP,to compare with that of control group（p<0.05）.The secretion TNF-αand IL-6 were also significantly up-regulated for 6 h-treatment of LP（p<0.01）,which showed a positive correlation with the incubating time.The secretion of IL-1βwas also elevated,with no significant difference（p>0.05）.The phosphorylation of STING and IRF3 were both significantly increased in IPEC-J2 cells under LP treatment（p<0.01）,indicating that LP mediates STING to promote IFN-βproduction in IPEC-J2 cells.2.Effect of c GAS/STING pathway of IPEC-J2 cells on inhibition of Po RV replication regulated by Lactobacillus plantarumLP and IPEC-J2 cells were co-cultured at MOI=100 for 6 h and then inoculated with Po RV at 0.5 TCID₅₀/cell for 12 h（LP+Po RV group）,LP treatment without Po RV infection treatment（LP group）,Po RV infection only without LP pretreatment（Po RV group）,and blank control（CON）.TCID₅₀ and RT-q PCR were used to determine the virus titer and viral NSP4 gene copy number in each group.The results showed that the viral titer of Po RV in the LP+Po RV group was significantly lower than that in the Po RV group（p<0.05）and the viral copy number in the LP+Po RV group was significantly lower than that in the Po RV group（p<0.001）.It was suggested that LP was able to inhibit the replication of Po RV in IPEC-J2 cells.Notably,the transcription and expression levels of IFN-βwere significantly higher in the LP+Po RV group than in the Po RV group（p<0.05）.It was probably that LP might achieve the effect of suppressing Po RV by promoting IFN-βproduction in IPEC-J2 cells.To clarify IFN-βproduction mediated by the STING pathway,we detected the expression of STING and IRF3phosphorylation,key proteins in this pathway,by Western blot.LP+Po RV group significantly enhanced STING expression to compare with CON（p<0.05）.In addition,the phosphorylation of IRF3 protein in the LP+Po RV group was significantly more than that in the LP and CON groups（p<0.05）and slightly higher than that in the Po RV group with no significance（p>0.05）.These indicated that LP treatment following Po RV challenge activated STING.Given that IFN-βfurther promotes the expression of interferon-stimulated genes（ISGs）,the transcript levels of IFIT2,CXCL10 and ISG15,representative genes of ISGs,were examined by RT-q PCR.The results showed that the transcript of IFIT2,CXCL10 and ISG15 genes were significantly higher in the LP+Po RV group than that in LP and Po RV group（p<0.05）.It is thus hypothesized that LP may induce IFN-βproduction in IPEC-J2 cells through activation of the STING pathway,thereby inhibiting rotavirus replication.To clarify whether STING plays a key role or not,this assay utilized the STING covalentinhibitor C-170 for blocking experiments.The results showed that the expression and transcription of IFN-βsignificantly decreased in all groups with C-170 blockade（p<0.05）.The phosphorylation of IRF3 significantly decreased（p<0.05）.In addition,the transcription of interferon-stimulated genes IFIT2,CXCL10 and ISG15 was significantly inhibited（p<0.05）.C-170 supplement did not affected the viral titer and viral copy number,between LP+Po RV group and Po RV group（p>0.05）,indicating that STING played a key role in regulation of LP on Po RV replication in IPEC-J2 cells.That is to say,LP regulation the anti-Po RV infection in IPEC-J2 cells dependened on STING-IRF3-IFN-βpathway.3.Effect of c GAS/STING pathway on Lactobacillus plantarum regulation of IPEC-J2 cells against Salmonella typhimurium infectionLP was co-cultured with IPEC-J2 cells for 1 h,and then infected with Salmonella typhimurium（S.Tyhimurium,ST）for 1 h（LP+ST group）,no ST infection（LP group）,ST infection alone（ST group）and blank control（CON）.The effects of LP on ST adhesion and invasion into IPEC-J2 cells were evaluated by exclusion assay（LP stimulated IPEC-J2 cells before ST）,competition assay（LP stimulated IPEC-J2 cells simultaneously with ST IPEC-J2cells）and replacement assay（LP stimulated IPEC-J2 cells later than ST）,the effects of LP on ST adhesion and invasion of IPEC-J2 cells were evaluated by rejection（LP stimulated IPEC-J2cells before ST）,competition（LP stimulated IPEC-J2 cells simultaneously with ST）and replacement assays.The results of the exclusion assay showed that the LP+ST group was significantly lower than the ST group（p<0.05）,and there was no significant difference between LP+ST group and the ST group in the competition and replacement assay,indicating that LP pre-stimulation with IPEC-J2 cells before ST displayed more effectively defense against ST adhesion and invasion.The expression of TNF-α,IL-6,IL-1βand IFN-βwere significantly increased in the LP+ST group compared to the ST group（p<0.05）when the cytokines secreted by IPEC-J2 cells were measured by ELISA.Western blot results showed that the phosphorylation of STING was significantly higher in the LP+ST group than in the CON group（p<0.05）,but there was no significant change in the phosphorylation level of IRF3 protein between the groups（p>0.05）.This suggests that STING does not promote IFN-I production through activating IRF3.Presumably,phosphorylation of IκBαwas activated for triggering downstream signals to produce IFN-β.The results showed that IκBαprotein phosphorylation expression levels were significantly higher in the LP+ST group than in the CON group（p<0.05）,with an up-regulation trend compared to the LP group and ST group with no difference（p>0.05）.This suggests that LP may mediate STING-IκBα-IFN-βpathway of IPEC-J2 cells to defense against S.Tyhimurium infection.In conclusion,Lactobacillus plantarum mediates STING activation of the IFN-Ⅰsignaling pathway in IPEC-J2 to produce IFN-βand downstream interferon-stimulated genes,which in turn inhibited Po RV replication and interfered with S.Tyhimurium adhesion and invasion,preliminarily revealing the role of the STING in the regulation of IPEC-J2 cells against infection by Lactobacillus plantarum.This study will lay the foundation for the study on the regulation of intrinsic immunity against intracellular pathogen infection by Lactobacillus plantarum.