AIM To investigate the effects of active vitamin D3 on autophagy and interleukin(IL)-1β expression in Salmonella-infected intestinal epithelial cells(IECs).METHODS Caco-2 cells, NOD2 siR NA-, Atg16L1 siR NA- or vitam...AIM To investigate the effects of active vitamin D3 on autophagy and interleukin(IL)-1β expression in Salmonella-infected intestinal epithelial cells(IECs).METHODS Caco-2 cells, NOD2 siR NA-, Atg16L1 siR NA- or vitamin D receptor(VDR) siR NA-transfected Caco-2 cells were pretreated with 1,25-dihydroxyvitamin D3(1,25D3), and then infected by wild-type S. typhimurium strain SL1344. The conversion of LC3-I to LC3-II was detected by Western blot analysis and LC3+ autophagosome was analyzed by immunofluorescence. Caco-2 cells or VDR si RNA-transfected cells were pretreated with 1,25D3, and then infected by SL1344. Membrane protein and total RNA were analyzed by Western blot and RT-PCR for VDR and Atg16L1 protein and m RNA expression, respectively. Atg16L1 si RNA-transfected Caco-2 cells were pretreated by 1,25D3 and then infected with SL1344. Total RNA was analyzed by RT-PCR for IL-1β mR NA expression.RESULTS The active form of vitamin D, 1,25D3, showed enhanced VDR-mediated Atg16L1 mR NA expression, membranous Atg16L1 protein expression leading to autophagic LC3 II proteins expression and LC3 punctae in Salmonella-infected Caco-2 cells which was counteracted by Atg16L1 and VDR si RNA, but Atg16L1 mediated suppression of IL-1β expression. Thus, active vitamin D may enhance autophagy but suppress inflammatory IL-1β expression in Salmonella-infected IECs.CONCLUSION Active vitamin D might enhance autophagic clearance of Salmonella infection, while modulation of inflammatory responses prevents the host from detrimental effects of overwhelming inflammation.展开更多
AIM To test whether Nox1 plays a role in typhlitis induced by Salmonella enterica serovar Typhimurium(S. Tm) in a mouse model.METHODS Eight-week-old male wild-type(WT) and Nox1 knockout(KO) C57BL6/J(B6) mice were admi...AIM To test whether Nox1 plays a role in typhlitis induced by Salmonella enterica serovar Typhimurium(S. Tm) in a mouse model.METHODS Eight-week-old male wild-type(WT) and Nox1 knockout(KO) C57BL6/J(B6) mice were administered metronidazole water for 4 d to make them susceptible to S. Tm infection by the oral route. The mice were given plain water and administered with 4 different doses of S. Tm by oral gavage. The mice were followed for another 4 d. From the time of the metronidazole application, the mice were observed twice daily and weighed daily. The ileum, cecum and colon were removed for sampling at the fourth day post-inoculation. Portions of all three tissues were fixed for histology and placed in RNAlater for m RNA/c DNA preparation and quantitative real-time PCR. The contents of the cecum were recovered for estimation of S. Tm CFU.RESULTS We found Nox1-knockout(Nox1-KO) mice were not more sensitive to S. Tm colonization and infection than WT B6 mice. This conclusion is based on the following observations:(1) S. Tm-infection induced similar weight loss in Nox1-KO mice compared to WT mice;(2) the same S. Tm CFU was recovered from the cecal content of Nox1-KO and WT mice regardless of the inoculation dose, except the lowest inoculation dose(2 × 106 CFU) for which the Nox1-KO had one-log lower CFU than WT mice;(3) there is no difference in cecal pathology between WT and Nox1-KO groups; and(4) there are no S. Tm infection-induced changes in gene expression levels(IL-1b, TNF-α, and Duox2) between WT and Nox1-KO groups. The Alpi gene expression was more suppressed by S. Tm treatment in WT than the Nox1-KO cecum. CONCLUSION Nox1 does not protect mice from S. Tm colonization. Nox1-KO provides a very minor protective effect against S. Tm infection. Using NOX1-specific inhibitors for colitis therapy should not increase risks in bacterial infection.展开更多
基金Supported by the Ministry of Science and Technology[MOST 103-2314-B-182-032(in part)]the Chang Gung Memorial Hospital,No.CMRPG8B1431,No.CMRPG8B1481 and No.CMRPG880443the Stem Cell Research Core Laboratory (grant CLRPG8B0052) for technical support
文摘AIM To investigate the effects of active vitamin D3 on autophagy and interleukin(IL)-1β expression in Salmonella-infected intestinal epithelial cells(IECs).METHODS Caco-2 cells, NOD2 siR NA-, Atg16L1 siR NA- or vitamin D receptor(VDR) siR NA-transfected Caco-2 cells were pretreated with 1,25-dihydroxyvitamin D3(1,25D3), and then infected by wild-type S. typhimurium strain SL1344. The conversion of LC3-I to LC3-II was detected by Western blot analysis and LC3+ autophagosome was analyzed by immunofluorescence. Caco-2 cells or VDR si RNA-transfected cells were pretreated with 1,25D3, and then infected by SL1344. Membrane protein and total RNA were analyzed by Western blot and RT-PCR for VDR and Atg16L1 protein and m RNA expression, respectively. Atg16L1 si RNA-transfected Caco-2 cells were pretreated by 1,25D3 and then infected with SL1344. Total RNA was analyzed by RT-PCR for IL-1β mR NA expression.RESULTS The active form of vitamin D, 1,25D3, showed enhanced VDR-mediated Atg16L1 mR NA expression, membranous Atg16L1 protein expression leading to autophagic LC3 II proteins expression and LC3 punctae in Salmonella-infected Caco-2 cells which was counteracted by Atg16L1 and VDR si RNA, but Atg16L1 mediated suppression of IL-1β expression. Thus, active vitamin D may enhance autophagy but suppress inflammatory IL-1β expression in Salmonella-infected IECs.CONCLUSION Active vitamin D might enhance autophagic clearance of Salmonella infection, while modulation of inflammatory responses prevents the host from detrimental effects of overwhelming inflammation.
基金Supported by Federal funds from the National Cancer Institute(NCI)under Contract,No.HHSN261200800001E(to Chu FF)Research reported in this publication included work performed in the Animal Resources Center Core supported by the National Cancer Institute of the National Institutes of Health under award No.P30CA033572
文摘AIM To test whether Nox1 plays a role in typhlitis induced by Salmonella enterica serovar Typhimurium(S. Tm) in a mouse model.METHODS Eight-week-old male wild-type(WT) and Nox1 knockout(KO) C57BL6/J(B6) mice were administered metronidazole water for 4 d to make them susceptible to S. Tm infection by the oral route. The mice were given plain water and administered with 4 different doses of S. Tm by oral gavage. The mice were followed for another 4 d. From the time of the metronidazole application, the mice were observed twice daily and weighed daily. The ileum, cecum and colon were removed for sampling at the fourth day post-inoculation. Portions of all three tissues were fixed for histology and placed in RNAlater for m RNA/c DNA preparation and quantitative real-time PCR. The contents of the cecum were recovered for estimation of S. Tm CFU.RESULTS We found Nox1-knockout(Nox1-KO) mice were not more sensitive to S. Tm colonization and infection than WT B6 mice. This conclusion is based on the following observations:(1) S. Tm-infection induced similar weight loss in Nox1-KO mice compared to WT mice;(2) the same S. Tm CFU was recovered from the cecal content of Nox1-KO and WT mice regardless of the inoculation dose, except the lowest inoculation dose(2 × 106 CFU) for which the Nox1-KO had one-log lower CFU than WT mice;(3) there is no difference in cecal pathology between WT and Nox1-KO groups; and(4) there are no S. Tm infection-induced changes in gene expression levels(IL-1b, TNF-α, and Duox2) between WT and Nox1-KO groups. The Alpi gene expression was more suppressed by S. Tm treatment in WT than the Nox1-KO cecum. CONCLUSION Nox1 does not protect mice from S. Tm colonization. Nox1-KO provides a very minor protective effect against S. Tm infection. Using NOX1-specific inhibitors for colitis therapy should not increase risks in bacterial infection.
