| Objective:Salmonella is a facultative intracellular pathogen that is a leading cause of food-and water-borne diseases in mammals.The pathogenicity of the Salmonella and the host inflammatory response determine the course and outcome of the infection.NLRP6,a member of the NLRs,participates in mediation of the communication networks involving the intestinal immune system and the gut microbiota.However,the mechanism of NLRP6 regulates inflammation and host defense against microorganisms remains elusive.Here,we focus on the influence of NLRP6 on the intestinal mucosal mechanical barrier.By establishing the mice and cells infection models,and combining with the pathophysiology of Salmonella infection,we investigated the role and mechanism of NLRP6 in the process of Salmonella infection.Methods:?.Role of NLRP6 in Salmonella infection1.The effect of Salmonella infection on the expression of NLRP6.The WT mice were administered orally with SL1344 at a dose of 5×107 CFU to establish the mice infection models.Total colon protein of mice was extracted,the expression levels of NLRP6 were evaluated by Western blot to analyze the effect of Salmonella infection on the expression of NLRP6 in mouse colon.Caco-2 cells were co-cultured with SL1344 at a MO I of 100 to establish infection model.Total protein was extracted,and NLRP6 expression was detected by Western blot.Immunofluorescence labeling NLRP6 was used to analyze the effect of Salmonella infection on the expression and distribution of NLRP6 in intestinal epithelial cells.2.The effect of NLRP6 on the process of Salmonella infection.The WT or Nlrp6-/-mice were administered orally with SL1344 at a dose of 5×107 CFU to establish the mice infection models and the following experiments were performed:After infection,the body weight of mice was recorded and the weight change curve was drawn;The death and survival of mice were recorded,and a survival curve was drawn according to the survival rate of the mice;The liver,spleen,mesenteric lymph nodes and colon of mice were collected to enumerate bacterial counts respectively by plate colony count;The colon tissues of mice were collected to observe the pathological changes by HE staining.The effects of NLRP6 on the process of Salmonella infection were analyzed by comparing weight changes,survival rates,bacterial counts and pathological changes between different infection groups.?.The influence of NLRP6 on the intestinal mucosal mechanical barrier1.The effect of NLRP6 on the proliferation of intestinal epithelial cells in the process of Salmonella infection.The WT or Nlrp6-/-mice were administered orally with SL1344 at a dose of 5×107 CFU to establish the mice infection models.The colon tissues were separated 3 d post infection,and the proliferation of colonic epithelial cells was evaluated by immunohistochemical labeling Ki67.2.The influence of NLRP6 on the intestinal mucosal mechanical barrier in vivo.The mice infection models were established as mentioned above.The mice were orally gavaged with non-metabolizable 4 kDa FITC-dextran 4 h prior to sacrifice.Blood was collected and FITC-dextran levels in serum were measured;The mucus,epithelial cell intercellular,lamina propria and epithelial cell extracellular fractions from the colon tissues were separated to enumerate bacterial counts respectively by plate colony count.The influence of NLRP6 on the intestinal mucosal mechanical barrier in vivo was analyzed by comparing the differences in bacterial counts of 4 parts from colon tissues and FITC-dextran permeability between different infection groups.3.The influence of NLRP6 on AJC of intestinal epithelial cells.The mice infection models were established as mentioned above.The colonic epithelial cells were collected to detect the expression of AJC-related transmembrane proteins ZO-1,Claudin-1,Occludin and E-cadherin.The colon tissues of mice were collected and AJC-related above proteins were labeled with immunofluorescence to observe their distribution at cell membranes and in the cytoplasm by confocal laser scanning microscope(CLSM).The influence of NLRP6 on AJC of intestinal epithelial cells was analyzed by comparing the distribution and expression of AJC-related proteins between different infection groups.?.Mechanism research on the influence of NLRP6 on the intestinal mucosal mechanical barrier1.The relationship between NLRP6 affecting the intestinal mucosal mechanical barrier and commensal bacteria.WT and Nlrp6-/-mice were co-housed together in 1:1 ratio for 4 weeks.After cohousing,WT and the Nlrp6-/-mice were infected with SL1344 at a dose of 5×107 CFU.The liver,spleen,mesenteric lymph nodes and colon of mice were collected to enumerate bacterial counts respectively by plate colony count;The mucus,epithelial cell intercellular,lamina propria and epithelial cell extracellular fractions from the colon tissues were separated to enumerate bacterial counts respectively by plate colony count.The relationship between NLRP6 affecting the intestinal mucosal mechanical barrier and commensal bacteria was analyzed by comparing bacterial counts between different infection groups.2.The cell types of NLRP6 affecting the integrity of intestinal mucosal mechanical barrier.Bone marrow chimeras were generated then infected with SL1344 at a dose of 5×107 CFU and the following experiments were performed:The mice were orally gavaged with non-metabolizable 4 kDa FITC-dextran 4 h prior to sacrifice.Blood was collected and FITC-dextran levels in serum were measured;The mucus,epithelial cell intercellular,lamina propria and epithelial cell extracellular fractions from the colon tissues were separated to enumerate bacterial counts respectively by plate colony count;The colon tissues of mice were collected to observe the pathological changes by HE staining.The cell types of NLRP6 affecting the integrity of intestinal mucosal mechanical barrier was analyzed by comparing the differences in bacterial counts of 4 parts from colon tissues,FITC-dextran permeability and pathological changes between different infection groups.3.Construct the NLRP6 knockout Caco-2 cell line.The NLRP6 knockout Caco-2 cell line was constructed through CRISPR/Cas9 system.Two group of sgRNA were designed.After PCR amplification using pUC57 plasmid as template,the recombinant vector pGL3-NLRP6 was obtained through single digestion,ligation and transformation of Esp3I.Then the recombinant plasmid was confirmed by Xhol or sequencing.Co-transfection the pGL3-NLRP6 recombinant plasmid and the Cas9 plasmid into Caco-2 cells.Screening the monoclonal cell strains via finiting dilution procedure.Finally,NLRP6 levels of monoclonal cell strains were evaluated by Western blot to confirm whether the NLRP6 knockout Caco-2 cell line was successfully constructed.4.Forms of intestinal epithelial cells NLRP6 regulating the integrity of the intestinal mucosal mechanical barrier.Caco-2 or NLRP6-/-Caco-2 cells were co-cultured with SL1344 at a MOI of 100 to establish infection model and the following experiments were performed:The expression of the ASC,Pro-Caspase-1 and Cleaved-Caspase-1(p20)were detected respectively by Western blot;The expression levels of IL-1? and IL-18 mRNA were evaluated by qPCR;NLRP6,ASC and Caspase-1 were labeled with immunofluorescence to observe their co-localization.Forms of intestinal epithelial cells NLRP6 regulating the integrity of the intestinal mucosal mechanical barrier was analyzed by comparing the differences in the expression of ASC,Pro-Caspase-1 and Cleaved-Caspase-1(p20),the expression levels of IL-1? and IL-18 mRNA and NLRP6,ASC and Caspase-1 co-localization between different infection groups.5.Establishment of the Caco-2 monolayer model.Caco-2 cells originate from human colorectal adenocarcinoma cell line.After the proliferation,fusion and differentiation of the cells on the Transwell chambers,the Caco-2 monolayer model acting like intestinal epithelial cells can be used to simulate the human intestinal mucosa mechanical barrier.In this experiment,Caco-2 cells were seeded into Transwell chambers and TEER values were determined by Millicell ERS-2 Voltohmmeter.On the 3rd,13th and 21st days after cell seeded,the culture medium in the apical and basolateral sides of chambers was collected respectively to detect the activity of alkaline phosphatase(ALP).According to TEER and ALP values we confirm the integrity and permeability of the monolayer.6.The effect of NLRP6 on the structural and functional integrity of AJC.Caco-2 or NLRP6-/-Caco-2 monolayers were co-cultured with SL1344 at a MOI of 100 to establish infection model and the following experiments were performed:The TEER values were monitored every 0.5 h;4 kDa FITC-dextran was added to the apical sides of chambers with the bacteria suspension,the culture medium in the basolateral sides of chambers was collected to detect FITC-dextran levels at 3 h post infection;The culture medium in the basolateral sides of chambers was collected to enumerate bacterial translocation by plate colony count of CFU at 3 h post infection;AJC-related above proteins were measured by Western blot and immunofluorescence.The effect of NLRP6 on the structural and functional integrity of AJC was analyzed by comparing TEER values,FITC-dextran permeability,bacterial translocation,distribution and expression of AJC-related proteins between different infection groups.7.Mechanism research on the effect of NLRP6 on integrity of AJC.The assembly,disassembly and phosphorylation of AJC related proteins can be regulated by many signaling pathways,including PI3K/Akt,MLCK,MAPK and PKC.In this study,Western blot was used to detect the key regulatory targets of the above-mentioned signaling pathways,and to screen the related signaling pathways of NLRP6 impact on AJC.Treatment of key proteins of the target pathway with corresponding interventions,mechanism research on the effect of NLRP6 on integrity of AJC was analyzed by comparing TEER values,FITC-dextran permeability,bacterial translocation,distribution and expression of AJC-related proteins between different infection groups.Results:?.Role of NLRP6 in Salmonella infection1.The effect of Salmonella infection on the expression of NLRP6.The results of Western blot in colon of mice showed that Salmonella infection can promote colonic NLRP6 expression.The results of Western blot and immunofluorescence showed that Salmonella infection can promote the expression of NLRP6 in intestinal epithelial cells,indicating that NLRP6 is related to the anti-Salmonella infection.2.The effect of NLRP6 on the process of Salmonella infection.The weight curve results showed that the body weight of mice infected with Salmonella decreased gradually with the infection process,but the weight loss of Nlrp6-/-infected group was lower than that of WT infected group,and the body weight of Nlrp6-/-infected mice had a trend of recovery after 6 days post.The survival curve results showed that all WT mice died 7 days post infection,and 20%of Nlrp6-/-mice survived more than 10 days post infection.There was a significant difference in mortality between the two infected groups.The results of bacterial counts showed that bacterial counts in liver,spleen and mesenteric lymph nodes in WT infected mice were significantly higher than that in Nlrp6-/-infected mice.The pathological changes of colon were more serious in WT infected mice.The above results indicate that NLRP6 can increase the susceptibility to Salmonella infection.?.The influence of NLRP6 on the intestinal mucosal mechanical barrier1.The effect of NLRP6 on the proliferation of intestinal epithelial cells in the process of Salmonella infection.The results of immunohistochemistry showed that there were more Ki67-positive cells in the intestinal epithelial crypts of Nlrp6-/-mice than in WT mice,indicating that Nlrp6-/-mice's intestinal epithelial cells had strong proliferation ability and intestinal mucosal barrier function during Salmonella infection.2.The influence of NLRP6 on the intestinal mucosal mechanical barrier in vivo.The results of the FITC-dextran permeability and bacterial counts in different parts from mice colon showed that:The FITC-dextran concentration in the serum of Nlrp6-/-infected mice was lower than WT infected mice;Bacterial counts of the epithelial cells,epithelial extracellular and lamina propria fractions in Nlrp6-/-infected mice were significantly less than that in WT infected mice.There were no differences in bacterial counts of the mucus between different infection groups.These results suggest that NLRP6 promotes Salmonella to impair the function of the intestinal mucosal mechanical barrier.3.The influence of NLRP6 on AJC of intestinal epithelial cells.The results of immunofluorescence and Western blot showed that:The expression of the AJC-related proteins(Claudin-1,Occludin and ZO-1)in colonic epithelial cells of Nlrp6-/-infected mice was higher than WT infected mice.Compared with WT infected mice,the above AJC-related proteins Claudin-1,Occludin and ZO-1 in Nlrp6-/-infected mice were found more localization at cell junctions.These results suggest that NLRP6 promotes Salmonella to impair the function of the AJC of intestinal epithelial cells.?.Mechanism research on the influence of NLRP6 on the intestinal mucosal mechanical barrier1.The relationship between NLRP6 affecting the intestinal mucosal mechanical barrier and commensal bacteria.The results of the bacterial counts showed that bacterial counts in liver,spleen and mesenteric lymph nodes in Co-WT infected mice were significantly higher than that in Co-Nlrp6-/-infected mice;The epithelial cells,epithelial extracellular and lamina propria fractions in Co-Nlrp6-/-infected mice were significantly less than that in Co-WT infected mice.These results suggest that NLRP6 affecting the intestinal mucosal mechanical barrier was not related to commensal bacteria.2.The cell types of NLRP6 affecting the integrity of intestinal mucosal mechanical barrier.The results of the bacterial counts showed that bacterial counts in epithelial cells,epithelial extracellular and lamina propria fractions in Nlrp6-/--Nlrp6-/-infected mice were significantly less than that in WT-WT infected mice.There were no differences in bacterial counts of 4 parts from colon tissues between WT-Nlrp6-/-infected mice and Nlrp6-/-Nlrp6-/-infected mice.There were no differences in bacterial counts of 4 parts from colon tissues between Nlrp6-/--WT infected mice and WT-WT infected mice either;The results of FITC-dextran permeability were consistent with the bacterial counts;Compared with the Nlrp6-/--Nlrp6-/-infected mice,the pathological changes of colon were more serious in WT-WT infected mice.There were no differences in colonic pathological changes between WT-Nlrp6-/-infected mice and Nlrp6-/--Nlrp6-/-infected mice.There were no differences in colonic pathological changes betweel Nlrp6-/--WT infected mice and WT-WT infected mice either.These results suggest that NLRP6 derived from non-hematopoietic compartments is detrimental for Salmonella to impair the function of the intestinal mucosal mechanical barrier.3.Construct the NLRP6 knockout Caoo-2 cell line.The NLRP6 knockout Caco-2 cell line was constructed through CRISPR/Cas9 system.The results of enzyme digestion and DNA sequencing show that the recombinant plasmid of pGL3-NLRP6 was successfully constructed.Western blot results confirm that the NLRP6 knockout Caco-2 cell line was successfully constructed.4.Forms of intestinal epithelial cells NLRP6 regulating the integrity of the intestinal mucosal mechanical barrier.The results of Western blot showed that the expression levels of ASC,Pro-Caspase-1 and Cleaved-Caspase-1(P20)were significantly increased in the two infection groups,but there was no significant difference between the two groups;The results of qPCR showed that the transcription levels of IL-1? and IL-18 in the two infection groups were significantly increased,and no significant difference was seen;No co-localization of NLRP6 with ASC and Caspase-1 during Salmonella infection.These results suggest that intestinal epithelial cells NLRP6 itself can regulate the integrity of the intestinal mucosal mechanical barrier.5.Establishment of the Caco-2 monolayer model.The TEER values were stable at the 200 ?·cm2 levels about 14 days after cell seeded.The ALP activity of the culture medium in apical sides of wells was significantly higher than that in the basolateral sides at 13 and 21 days after cell seeded.These results suggested that the Caco-2 monolayer was successfully established from 13 to 21 days after cell seeded and could be used in subsequent experiments.6.The effect of NLRP6 on the structural and functional integrity of AJC.The results of TEER values,FITC-dextran permeability,bacterial translocation,distribution and expression of AJC-related proteins showed that:The decrease of TEER values in NLRP6-/-infection group was significantly less than that in WT infection groups;The FITC-dextran concentration in the basolateral sides of chambers in NLRP6-/-infection group was significantly lower than WT infection groups at 3 h p.i.;Bacterial translocation to the basolateral sides of chambers in NLRP6-/-infection group was significantly less than WT infection groups at 3 h p.i.;The expression of the AJC-related proteins(Claudin-1,Occludin and ZO-1)in Nlrp6-/-infection group was higher than WT infection group;Compared with WT infection group,the above AJC-related proteins in Nlrp6-/-infection group were found more localization at cell junctions.These results suggest that NLRP6 promotes Salmonella to impair the integrity of AJC.7.Mechanism research on the effect of NLRP6 on integrity of AJC.Western blot was used to detect the key regulatory targets of the PI3K/Akt,MLCK,MAPK and PKC signaling pathways,and showed that NLRP6 did not affect the phosphorylation of MLC and PCK,and NLRP6 inhibited the expression of p-Akt and pp38.It is indicated that NLRP6 contributes to disruption of epithelial tight junctions via Akt-dependent pathway.After pretreatment with Akt inhibitor MK2206,the results of TEER values,FITC-dextran permeability,bacterial translocation,expression of AJC-related proteins showed that:Compared with the control group,the TEER value of each infection group decreased in a time-dependent manner,but there was no significant difference between WT+MK2206 and Nlrp6-/-+MK2206 infection groups;The FITC-dextran concentration in the Nlrp6-/-+DMSO infection group was significantly lower than that in the WT+DMSO infection group,but the FITC-dextran concentration in the Nlrp6-/-+MK2206 infection group was not statistically different from the WT+MK2206 infection group;The results of bacterial translocation were consistent with the FITC-dextran permeability;The expression levels of Occludin,Claudin-1 and ZO-1 in WT infection group were significantly lower than that in Nlrp6-/-infection group before and after infection,and the difference between the two groups disappeared after MK2206 treatment.The above results indicate that NLRP6 contributes to disruption of epithelial tight junctions via Akt-dependent pathway and promotes paracellular bacterial invasion.Conclusions:1.Nlrp6-/-mice are resistant to Salmonella infection.2.NLRP6's contribution to disruption of AJC promotes Salmonella to impair the function of the intestinal mucosal mechanical barrier.3.NLRP6 affecting the intestinal mucosal mechanical barrier was not related to commensal bacteria.4.Intestinal epithelial cells NLRP6 itself contributes to disruption of epithelial tight junctions via Akt-dependent pathway and promotes paracellular bacterial invasion. |
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