| Object:Intestinal tract is not only the organ that digest and absorpt food to supply nutrient for human body,it is also the body’s largest immune organ especially.It not only contacts with a lot of food antigen and is also the main place for many kinds of intestinal commensal bacteria to settle.In order to maintain the intestinal balance and protect the intestinal from pathogens infection,intestinal mucosal immunity need to keep the symbiotic bacteria resistance and initiate a rapid immune response to against pathogens at the same time.Intestinal immune cells(like:intestinal intraepithelial lymphocytes)and pattern-recognition receptors(like:toll-like receptor)in intestine play an important role in this process,but the mechanism of intestinal mucosal immunity tolerance to intestinal commensal bacteria and distinguish pathogenic bacteria from intestinal commensal bacteria has not been fully elucidated.Intest:ine epithelium is the first line of defense against invading pathogens.And it is constituted by single layer epithelial cells and intestinal intraepithelial lymphocytes between the intestinal epithelial cells.After stimulated by antigen,intestinal intraepithelial lymphocytes can quickly arouse the immume response,product inflammatory cytokines to eliminate invading pathogens,and intestinal intraepithelial lymphocytes can also exert cytotoxicity to infected epithelial cells to eliminate pathogen.Epithelial cells express rich toll-like receptors,this receptor can recognize pathogen-associated molecular pattern of pathogens and cause inflammation to secret cytokines,which enhance activation and function of intestinal intraepithelial lymphocytes.Toll-like receptors play an important role in interreaction between intestinal epithelial cells and intestinal intraepithelial lymphocytes.But at present,the mechanism of toll-like receptors in the maintenance of intestine homeostasis and intestinal defense against pathogen infection has not been fully elucidated.S.typhimurium(ST),a kind of facultative intracellular Gram-negative bacteria and an important food-borne pathogen,can cause a broad range of clinical manifestations in human and animal hosts.It causes a self-limited gastroenteritis,characterized by fever,diarrhea,acute intestinal inflammation,and the presence of neutrophils in stool samples.There are four TLR ligands have been found in S.typhimurium,bacterial lipoproteins can be recognized by TLR2,lipopolysaccharide(LPS)can be recognized by TLR4,flagellin can be recognized by TLR5 and CpG DNA can be recognized by TLR9.In this paper,we used the model of S.typhimurium infection and TLR deficiency mice to analyse the role of TLR2,TLR4 and TLR9 in murine S.typhimurium oral infection.Through detection of life cycle,intestine inflammation and bacteria load in small intestinal tissue mesenteric lymph nodes,we demonstrated that TLR9 knockout mice were significantly more susceptible to oral S.typhimurium infection compare with WT mice.And then,we explore the role of TLR9 in protection of intestine integrity and intestinal defense against S.typhimurium infection using WT and TLR9-/-mice which were orally infected with S.typhimurium.Methods:1.Mice were orally infected with S.typhimurium,to explore the roles of TLR2,TLR4 and TLR9 in the defense against S.typhimurium infection through detecting life cycle,the weight of body,small intestine,liver,spleen,histopathology of small intestine,bacteria load in small intestinal tissue and mesenteric lymph nodes,etc.2.The numbers,percentages and activation(CD69)of different iIELs subpopulations in WT and TLR9-/-mice after S.typhimurium infection by FACS.3.The cytolytic activity of iIELs against IECs and MODE-K cells assessed by the LDH assay.4.The expression of NKG2D and CD 107a in different iIELs subpopulations of WT and TLR9-/-mice after S.typhimurium infection was detected by FACS.5.Real-time PCR and FACS were used to investigate the expression of all known NKG2D ligands(MULT1,Rael and H60)on IECs of WT and TLR9-/-mice after S.typhimurium infection.6.IL-1β,IL-18,IL-8 levels in IECs and culture supernatant of intestinal tissue in WT and TLR9-/-mice after S.typhimurium infection were detected by real-time PCR and ELISA.7.Western blot and real-time PCR detected the expression of NF-κB、p-NF-κB、NLRP3、pro-caspase-1、caspase-1、pro-IL-1β、IL-1β in IECs of WT and TLR9-/-mice after S.typhimurium infection.8.IL-1RI expression in iIELs of WT and TLR9-/-mice after S.typhimurium infection and CD69,NKG2D,CD 107a expression in different iIELs subpopulations after stimulated by IL-1β were detected by FACS.9.The cytolytic activity of iIELs against MODE-K cells after stimulated by IL-1(3 or blocked by anti-IL-1β antibody assessed by the LDH assay.10.Liver,spleen and MLN bacterial loads were detected after anti-γδTCR Ab treatment.11.Histopathology observation by hematoxylin-eosin staining after S.typhimurium infection in iIELs transfer mice and bone marrow chimeric mice.12.Liver,spleen and MLN bacterial loads were detected after S.typhimurium infection in iIELs transfer mice and bone marrow chimeric mice.13.Bacterial loads were detected after S.typhimurium infection in peritoneal macrophage of WT and TLR9-/-mice.14.ROS and the expression of CD86 were detected by FACS after S.typhimurium infection in peritoneal macrophage of WT and TLR9-/-mice.15.NO was detected by Nitric Oxide Assay Kit after S.typhimurium infection in peritoneal macrophage of WT and TLR9-/-mice.16.MLN,liver and spleen bacterial loads were detected after S.typhimurium infection in macrophage transfer mice.Result:1.TLR9 knockout mice were significantly more susceptible to oral S.typhimurium infection compare with WT,TLR2-/-and TLR4-/-mice.WT,TLR2-/-,TLR4-/-and TLR9-/-mice(6-8 weeks)were orally infected with S.typhimurium at a dose of 5×04 CFU.TLR9-/-mice had significantly shorter survival times than WT mice,weight changes of small intestine,hepatosplenomegaly,the histopathology of small intestine and S.typhimurium burden were more obvious than WT mice.However,there were no significant changes in TLR2-/-and TLR4-/-mice compare with WT mice,suggesting TLR9 knockout mice were the most susceptible to oral S.typhimurium infection.2.The activation and cytotoxicity of iIELs are augmented in TLR9-/-mice after S.typhimurium infection.After S.typhimurium infection,we found that the total numbers of iIELs increased significantly at day 7 in WT mice,while in TLR9-/-mice,the numbers increased significantly from day 5 and sustained high levels at day 7 after infection;the percentage and number of CD8α+TCRγδ+iIELs increased more obviously than WT mice;and activation of CD8α+TCRγδ+ iIELs in TLR9-/-mice increased more obviously than WT mice at day 5 and 7.Further analyse the function of iIELs,TLR9-/-iIELs exhibited much stronger cytotoxicity against both infected IECs and MODE-K cells at each E:T ratio than WT iIELs in vitro;FACS results show that the expressions of NKG2D and CD 107a were much higher in each iIEL subpopulation from TLR9-/-mice than from WT mice;the levels of NKG2D ligands(MULT-1,RAE-1 and H60)on IECs after S.typhimurium infection in WT and TLR9-/-mice have similar phenomenon;in vitro,blocking of NKG2D significantly attenuated the cytotoxicity of iIELs against S.typhimurium-infected IECs,and the cytolytic activity of iIELs from TLR9-/-mice reduced to nearly the same level to that of iIELs from WT mice,indicated that iIELs exerts NKG2D-dependent cytotoxicity against IECs and the cytolytic activity increased significantly.3.The activation of inflammatory pathway and the expression of NKG2D ligands are augmented in TLR9-/-miceReal time-PCR and ELISA results show that the levels of IL-1β and IL-8 were upregulated both in WT and TLR9-/-mice after S.typhimurium infection,and more obvious upregulation were observed in TLR9-/-mice;the activation of NF-κB was further detected by western blot,a higher level of NF-κB activation was found in IECs of TLR9-/-mice compared with WT mice;the blockage of NF-κB signaling using PDTC could completely eliminate the increase of NKG2D ligands in IECs,show that the expression of NKG2D ligands is related to NF-κB signaling pathway.Further observed that a higher level of NLRP3 inflammatory activation and IL-1β expression was found in IECs of TLR9-/-mice compared with WT mice by Western blot;Infect MODE-K cells in vitro and inhibit caspas-1 at the same time,the secretion of IL-1βwas significantly decreased,the result further confirm that the produce of IL-1β is associated with NLRP3 inflammatory pathways;NKG2D ligands(MULT-1,RAE-1 and H60)expressed on MODE-K cells were upregulated after stimulated by IL-1β in vitro,suggestting that the lack of TLR9 enhance the activation of NF-κB signaling pathway and NLRP3 inflammasome in IECs druing S.typhimurium infection,IL-1βmature after cleaved by caspase-1 and up-regulated the expression of NKG2D ligands.4.IL-1β stimulated iIELs activation and upregulated cytotoxicity of iIELs.The expression of IL-1RI on iIELs from WT and TLR9-/-mice during S.typhimurium infection was detected by FACS,and more obvious upregulation were observed in TLR9-/-mice;the expression of activation and cytotoxicity related molecules on iIELs was upregulated after stimulated by IL-1β in vitro;the cytotoxic potential of iIELs was measured after the stimulation or blockage of IL-1β,to futher confirm the influence of IL-1β to cytotoxicity of iIELs.5.The lack of TLR9 leads to excessive damage of intestinal during S.typhimurium infectioniIELs isolated from WT or TLR9-/-mice were adoptively transferred into WT mice separately,no significant differences in bacteria burden of liver,spleen and MLN were found between the two groups,H&E staining and the levels of inflammatory cytokines showed no differences in inflammation and intestine injury,these results suggested that TLR9 deficiency in iIELs did not influence the susceptibility and intestine injury of mice in response to S.typhimurium infection.Bone marrow chimeras were used to futher confirm the role of TLR9 in IECs,more bacteria burden in liver,spleen and MLN,and more inflammatory and intestine injury were found in TLR9-/-recipient mice,these data demonstrated that it is the expression of TLR9 in IECs could protect intestine from excessive injury.6.TLR9 promotes the activation and bactericidal activity of macrophagesThe number of bacteria in macrophages from TLR9-/-mice was obviously higher than that in macrophages from WT mice,the production of ROS(MFI)and NO by macrophages was reduced from TLR9-/-mice,confirmed that TLR9 promotes the activation and bactericidal activity of macrophages.Transfer experiment futher demonstrated that the activation and bactericidal activity of macrophages was significantly weakened when TLR9 is absence,leading to the dissemination of S.typhimurium.TLR9 plays an important role in host defense against intracellular bacterial infection.Conclusion:1.We found that TLR9-/-mice were significantly more susceptible to oral S.typhimurium infection compare with WT mice;2.iIELs,particularly CD8α+TCRγδ+iIELs,are critical for the detection of pathogenic bacteria during S.typhimurium infection,the percentage and activation of CD8α+TCRγδ+ iIELs in TLR9-/-mice increased more obviously than WT mice,and cytotoxicity of iIELs is higher than that of WT mice;3.During S.typhimurium infection,the expression of NKG2D on iIELs and NKG2D ligands on IECs were more obviously than WT mice,the recognition of NKG2D to its ligands mediated the susceptible of IECs to the cytotoxicity of iIELs;4.TLR9 inhibits excessive secretion of IL-1β in IECs through regulate NF-κB and NLRP3 inflammasome signaling pathways;5.IL-1β increased the activation and the expression of NKG2D ligands on IECs,upregulated cytotoxicity of iIELs to IECs,lead to serious intestine damage;6.TLR9 promotes the activation and bactericidal activity of macrophages,plays an important role in host defense against intracellular bacterial infection. |
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