| OBJECTIVESalmonella is a group of bacteria threatening human and animal health,and regarded as model bacteria for infection and immunity research.It is of great significance to further explore Salmonella pathogenicity in order to control infection and develop novel strategies to prevent and treat infectious diseases.Iron,acting as an essential nutrient for Salmonella survival,growth and pathogenicity,is also one of the most abundant microelements which are closely associated with human life.Nutritional immunity is designated as a host defense mechanism by iron withdrawal during infection,but the underlying mechanisms remains poorly understood.Salmonella may also provide corresponding countermeasures to meet their own nutrient requirements.However,which virulence strategy utilized by Salmonella to contribute to interference with host iron homeostasis remained to be illuminated.Here,the project aims to shed light on the underlying mechanisms of dysregulation of host iron metabolism caused by Salmonella infection from the double perspective of host nutritional immunity and Salmonella effector,revealed the key molecules and signaling pathways involved in this process and provide new insights into prevention and treatment of infectious diseases.METHODS1.The effects and underlying mechanisms of the pattern recognition receptor NLRP6 on Salmonella-associated dysregulation of host iron metabolism1.1 Effects of NLRP6 on host iron metabolism during Salmonella infectionTo investigate the role of NLRP6 in Salmonella-associated dysregulation of host iron metabolism,streptomycin-pretreated WT and Nlrp6-/-mice were orally infected with 1 × 107 CFUs of S.Typhimurium SL1344.Survival and weight loss was monitored.Bacterial load in duodenum,colon,MLN,liver and spleen were determined by plating.HE staining of duodenum,liver and spleen,and Perls’Prussian Blue staining of spleen were measured.Serum iron and non-heme iron concentrations were measured with a colorimetric assay.Heme levels were measured with a fluorescence assay.1.2 The effects and underlying mechanisms of NLRP6 on macrophage iron metabolism during Salmonella infectionTo investigate the effects of NLRP6 on modulating macrophage iron metabolism,WT and Nlrp6-/-BMDMs were infected with SL1344.After administering the iron chelator DFP,intracellular bacterial load was determined by plating.Cellular iron contents and iron export were measured with a colorimetric assay.Tfrc,Dmtl,Ftl,Fth and Fpn levels were determined by quantitative PCR.FTL,FTH and FPN protein levels were analyzed by western blot.After administering Fpn siRNA,intracellular bacterial load was determined by plating and iron contents were measured.1.3 The underlying mechanisms of NLRP6 on FPN transcriptionTo determine whether NLRP6 decreases FPN at the transcriptional level and the underlying mechanism,WT and Nlrp6-/-BMDMs were infected with SL1344.BMDMs were treated with the RNA polymerase Ⅱ inhibitor actinomycin D and Fpn levels were determined by quantitative PCR.SL1344-infected BMDMs were transfected with Arnt siRNA,Mtfl siRNA or Nrf2 siRNA,Fpn levels were determined by quantitative PCR and cellular iron contents were measured with a colorimetric assay.Moreover,NRP2,Nqol and Hmoxl levels were determined by quantitative PCR and western blot.1.4 Effects of NLRP6 on macrophage autophagyTo investigate the effects of NLRP6 on macrophage autophagy,WT and Nlrp6-/BMDMs were infected with SL1344.LC3 protein levels were analyzed by western blot.Cells were treated with the autophagy inhibitor BafAl,LC3 protein levels were analyzed by western blot.HeLa cells expressing RFP-GFP-LC3 were co-transfected with the vector control or pCMV-NLRP6,the number of LC3-positive puncta was counted.1.5 The effects and underlying mechanisms of NLRP6 on NRF2 expression during Salmonella infectionTo elucidate the mechanism underlying NLRP6-mediated downregulation of NRF2,WT and Nlrp6-/-BMDMs were infected with SL1344.The protein levels of autophagy receptors NBR1,TAX1BP1,OPTN,p62 and NDP52 were analyzed by western blot.BMDMs were treated with the mTOR inhibitor Rapamycin or the AKT inhibitor MK2206,the protein levels of p-p62(S351),4EBP1,p-4EBP1(Thr37/46),AKT and p-AKT(S473)were analyzed by western blot.293T cells were co-transfected with pcDNA3.1-Flag-AKT and pCMV-His-NLRP6 plasmid,cell lysates were subjected to immunoprecipitation with Flag-tag antibody,followed by western blot analysis.1.6 The effects and underlying mechanisms of NLRP6 on KEAP1 expression during Salmonella infectionTo further examine whether KEAP1 involves in NLRP6-mediated NRF2 reduction,WT and Nlrp6-/-BMDMs were infected with SL1344.KEAP1 expression levels were determined by quantitative PCR and western blot.BMDMs were treated with the AKT inhibitor MK2206,p-FOXO3A(S253)and FOXO3A protein levels were analyzed by western blot,and Keapl levels were determined by quantitative PCR.1.7 The effects and underlying mechanisms of NLRP6 on intestinal epithelial cells iron metabolism in Salmonella infectionTo investigate the effects of NLRP6 on iron metabolism in intestinal epithelial cells during Salmonella infection,WT and NLRP6-/-Caco-2 cells were infected with SL1344,Cellular iron contents were measured with a colorimetric assay.FPN expression levels were determined by quantitative PCR and western blot.The protein levels of NRF2,KEAP1,p-AKT(S473),AKT,LC3 and autophagy receptors NBR1,TAX1BP1,OPTN,p62 and NDP52 were analyzed by western blot.Caco-2 cells were treated with the AKT inhibitor MK2206,and cellular iron contents were measured with a colorimetric assay.1.8 The effects of the NRF2/FPN pathway on NLRP6-associated dysregulation of host iron metabolism during Salmonella infection in vivoWT and Nlrp6-/-mice were orally infected with SL1344 as previously described.The protein levels of NRF2 and FPN in liver,spleen and duodenum were analyzed by western blot.SL1344-infected mice were administered intravenously with Nrf2 shRNA.Bacterial load in liver and spleen was determined by plating.Non-heme iron contents of liver,spleen and liver macrophages were measured with a colorimetric assay.The protein levels of NRF2 and FPN in liver macrophages were analyzed by western blot.1.9 The effects and underlying mechanisms of AKT on NLRP6-associated dysregulation of host iron metabolism during Salmonella infection in vivoSL1344-infected mice were administered orally with the AKT inhibitor MK2206.Bacterial load in MLN,liver and spleen was determined by plating.Serum iron levels and non-heme iron contents of liver,spleen and liver macrophages were measured with a colorimetric assay.The protein levels of FPN,NRF2 and KEAP1,as well as FOXO3A,AKT and 4EBP1 phosphorylation in liver macrophages were analyzed by western blot.2.The effects and underlying mechanisms of Salmonella effector SpvB on dysregulation of host iron metabolism2.1 Effects of Salmonella effector SpvB on host systemic iron metabolismTo investigate the role of SpvB in Salmonella dysregulation of host systemic iron metabolism,streptomycin-pretreated mice were orally infected with 1 ×107 CFUs of either the WT or the ⊿spvB mutant S.Typhimurium strain,Hepatic bacterial load a was determined by plating.Hepatic iron concentrations and serum iron contents were measured with a colorimetric assay.Perls’ Prussian Blue staining of the liver were measured.Salmonella-infected mice were administered with DFX,and hepatic bacterial load was determined by plating.2.2 Effects of Salmonella effector SpvB on the Hamp/FPN regulatory axisTo investigate the effects of NLRP6 on host Hamp/FPN axis,Salmonella-infected mouse model was used and hepatic Hamp levels were determined by quantitative PCR.Hepatic FPN protein levels were analyzed by western blot.Streptomycin-pretreated WT,Hamp+/-and Hamp-/-mice were orally infected with 1 ×107 CFUs of the WT or the⊿spvB mutant S.Typhimurium strain.Hepatic bacterial load was determined by plating.2.3 The effects and underlying mechanisms of SpvB on hepcidin expressionTo elucidate the mechanisms underlying SpvB-increased hepcidin expression,Salmonella-infected mouse model was used.The phosphorylation level of SMAD1/5/9 and STAT3 in the liver were analyzed by western blot.Salmonella-infected mice were further administered intraperitoneally with the STAT3 inhibitor Stattic.Hepatic bacterial burden was determined by plating.Serum iron levels were measured with a colorimetric assay.Hepatic iron content was determined on the basis of a multiscan spectrum.Hamp levels were determined by quantitative PCR.The protein levels of FPN were analyzed by western blot.2.4 The relationship between SpvB and liver inflammationTo investigate the influence of SpvB on liver inflammation,Salmonella-infected mouse model was used.Histopathological analysis of the liver was measured.Flow cytometry analysis of hepatic non-parenchymal cells and percentage of liver-infiltrated myeloid cells,liver macrophages,Kupffer cells and inflammatory monocyte-derived macrophages were counted.2.5 The effects of SpvB on hepatic cytokine and chemokine expressionTo further investigate the influence of SpvB on hepatic inflammatory mediator,Salmonella-infected mouse model was used.Hepatic Ccl2,Ccl3,Cxcl10,Il1β,Tnfα and 116 levels were determined by quantitative PCR.IL6 protein levels in the liver were analyzed by western blot.2.6 Effects of SpvB on the master hepatic inflammatory regulator TREM-1To investigate the effects of SpvB on hepatic inflammatory regulator TREM-1,Salmonella-infected mouse model was used,and the expression of TREM-1 was determined by quantitative PCR and western blot.Salmonella-infected mice were administered intraperitoneally with the TREM-1 inhibitor LP17.Hepatic bacterial load was determined by plating.Hepatic cytokines and chemokines were determined by quantitative PCR.2.7 Effects of TREM-1 on SpvB-mediated iron metabolic disorderTo investigate the influence of TREM-1 on SpvB-mediated dysregulation of host iron metabolism,Salmonella-infected mice were administered intraperitoneally with the TREM-1 inhibitor LP17.Hepatic iron content and serum iron levels were measured with a colorimetric assay.Hepatic Hamp levels were determined by quantitative PCR.The protein levels of FPN and pSTAT3 were analyzed by western blot.2.8 Effects of macrophage/hepatocyte interaction on SpvB-associated iron metabolic disorderTo explore the effects of macrophage/hepatocyte interaction on SpvB interfering with hepcidin-FPN axis,HepG2 cells were infected with the WT,AspvB or c-spvB S.typhimurium strain.HAMP levels were determined by quantitative PCR,THP-1/HepG2 co-culture model was further established and infected with different S.typhimurium strains mentioned above.When appreciated,HepG2 cell were transfected with HAMP siRNA,THP-1 cells were transfected with TREM-1 siRNA or pEGFP-spvB plasmid.HAMP levels in co-cultured HepG2 cells were determined by quantitative PCR.FPN levels in co-cultured THP-1 cells were analyzed by western blot.Cellular iron contents in co-cultured THP-1 cells were measured with a colorimetric,assay.RESULTS1.The effects and underlying mechanisms of the pattern recognition receptor NLRP6 on Salmonella-associated dysregulation of host iron metabolism1.1 Effects of NLRP6 on host iron metabolism during Salmonella infectionSalmonella-infected Nlrp6-/-mice displayed a higher ratio of survival and a lower proportion of weight loss as well as less pathological injury.Salmonella-infected Nlrp6-/-mice contained lower bacterial burdens when compared to Salmonella-infected WT mice.Meanwhile,Salmonella-infected Nlrp6-/-mice showed higher serum iron levels and lower non-heme iron content of liver,hepatic non-parenchymal cells and liver macrophages than that detected in Salmonella-infected WT mice.There were no apparent differences in heme levels between two groups of Salmonella-infected mice.1.2 NLRP6 altered iron export and further interfered with iron homeostasis through inhibiting FPN expressionSalmonella-infected Nlrp6-/-BMDMs contained a lower bacterial burden when compared to Salmonella-infected WT cells.The iron chelator DFP treatment eliminated the significant difference in bacterial load between two groups of Salmonella-infected BMDMs.Data showed a higher level of FPN and iron export ratio in Salmonella-infected Nlrp6-/-BMDMs.After administrating Fpn siRNA,no significant difference in cellular iron content were found between two groups of Salmonella-infected BMDMs.1.3 NLRP6 decreases FPN transcription via a NRF2-dependent mannerAfter actinomycin D treatment,no apparent differences in Fpn levels were found between Salmonella-infected WT BMDMs and Nlrp6-/-BMDMs.After administrating Arnt or Mtfl siRNA,Fpn levels remained significantly higher in Salmonella-infected Nlrp6-/-BMDMs than in Salmonella-infected WT cells.However,Nrf2 siRNA treatment eliminated differences in the transcription levels of Fpn and cellular iron content between these Salmonella-infected cells.Moreover,Salmonella-infected Nlrp6-/BMDMs showed higher lever of NRF2,Nqol and Hmoxl than Salmonella-infected WT cells.1.4 Effects of NLRP6 on macrophage autophagySalmonella-infected Nlrp6-/-BMDMs displayed a lower ratio of LC3-Ⅱ/LC3-Ⅰthan Salmonella-infected WT cells.After administrating the autophagy inhibitor BafA1,LC3-Ⅱ/LC3-Ⅰ levels in Nlrp6-/-BMDMs remained lower than those in WT cells.HeLa cells overexpressing NLRP6 contained a higher number of RFP-LC3 dots and GFP-LC3 dots during Salmonella infection.1.5 NLRP6 decreases NRF2 expression via inhibiting AKT/mTOR-mediated p62 phosphorylationSalmonella-infected Nlrp-/-BMDMs showed higher level of p62 and NDP52 than Salmonella-infected WT cells,while there was no apparent difference in NBR1,OPTN and TAXI BP 1 expression between these Salmonella-infected cells.Salmonella-infected Nlrp6-/-BMDMs showed higher levels of p62(S351),4EBP1(Thr37/46)and AKT(S473)phosphorylation when compared to Salmonella-infected WT cells.The mTOR inhibitor Rapamycin or the AKT inhibitor MK2206 treatment abrogated the significant difference in p62(S351)phosphorylation between two groups of Salmonella-infected BMDMs.Immunoprecipitation data showed NLRP6 interacted with AKT.1.6 NLRP6 increases the transcription level of KEAP1 through inhibiting AKT/FOX03 A pathwaySalmonella-infected Nlrp6-/-BMDMs showed a lower level of KEAP1 expression and a higher level of FOXO3A phosphorylation than Salmonella-infected WT cells.After administrating the AKT inhibitor MK2206,there were no apparent differences in Keapl transcription and FOXO3A phosphorylation between Salmonella-infected Nlrp6-/-and WT BMDMs.1.7 NLRP6 interferes intestinal epithelial cells iron metabolism via decreasing NRF2-mediated FPN expressionSalmonella-infected NLRP6-/-Caco-2 cells showed a lower cellular iron content than Salmonella-infected WT cells,Meanwhile,there was a higher protein level of FPN,NRF2,p-AKT,p62 and NDP52,and a lower expression level of LC3-Ⅱ/LC3-Ⅰ and KEAP1 in Salmonella-infected NLRP6-/-Caco-2 cells.After the AKT inhibitor MK2206 treatment,there were no apparent differences in cellular iron content between these Salmonella-infected cells.1.8 NLRP6 contributes to Salmonella dysregulation of host iron metabolism via a NRF2/FPN dependent manner in vivoThere were higher protein levels of FPN and NRF2 in liver,spleen and duodenum of Salmonella-infected Nlrp6-/-mice when compared to those of Salmonella-infected WT mice.After administering intravenously with Nrf2 shRNA,there were no significant differences in hepatic and splenic bacterial burden,non-heme iron content of liver,spleen,hepatic non-parenchymal cell and liver macrophages,as well as FPN protein levels between Salmonella-infected Nlrp6-/-mice and WT mice.1.9 NLRP6 interfering with AKT activation contributes to the dysregulation of iron homeostasis in vivoAfter administering orally with the AKT inhibitor MK2206,there were no apparent differences between Salmonella-infected Nlrp6-/-mice and WT mice,including bacterial load of MLNs,liver and spleen,serum iron content,non-heme iron content of liver,spleen and liver macrophages,protein levels of FPN,NRP2 and KEAP1,as well as AKT,4EBP1 and FOXO3A phosphorylation.2.The effects and underlying mechanisms of Salmonella effector SpvB on dysregulation of host systemic iron metabolism2.1 Effects of Salmonella effector SpvB on host systemic iron metabolismAlthough there were no apparent differences at 1 day post-infection,mice infected with the WT strain contained a significantly higher hepatic bacterial burden than mice infected with the ⊿spvB strain at 3 days post-infection.DFX eliminated the significant difference in hepatic bacterial burden between two groups of Salmonella-infected mice.Mice infected with the WT strain showed significant lower serum iron levels and higher hepatic iron content than that detected in mice infected with the ⊿spvB strain.2.2 Effects of Salmonella effector SpvB on the Hamp/FPN regulatory axisAlthough there were no apparent differences at 1 day post-infection,mice infected with the WT strain showed significantly higher hepcidin expression and lower FPN levels than mice infected with the ⊿spvB strain at 3 days post-infection.There was no apparent difference in the number of Salmonella in the liver of Hamp-/-mice infected with the WT strain as compared to the ⊿spvB strain.2.3 SpvB increases hepcidin expression via the STAT3-dependent pathwayThere were no apparent differences in pSMAD 1/5/9 expression,however,mice infected with the WT strain showed a significantly higher level of STAT3 phosphorylation as compared to mice infected with the ⊿spvB strain.After Static treatment,there were no apparent differences in hepatic bacterial load,serum iron levels,hepatic iron content,Hamp and FPN expression level between two groups of Salmonella-infected mice.2.4 SpvB results in hepatic inflammatory cell infiltrationHistopathological appearance of the liver showed severe inflammatory responses,such as more inflammatory cells around the hepatocytes,in mice infected with the WT strain as compared to mice infected with the ⊿spvB strain.Meanwhile,mice infected with the WT strain showed a higher number of hepatic non-parenchymal cells,macrophages and inflammatory monocyte-derived macrophages.2.5 SpvB promotes hepatic cytokine and chemokine expressionThe mRNA levels of CCL2 in the liver of WT-infected mice were significantly higher than those in the liver of ⊿spvB-infected mice at 1 day post-infection,while there were no apparent differences in IL1β,TNFα,IL6,CCL3 and CXCL10 expression.At 3 days post-infection,the mRNA levels of chemokines CCL2 and CXCL10,as well as cytokines IL1β and TNFa in the liver of WT-infected mice were significantly higher than those in the liver of ⊿spvB-infected mice.Hepatic mRNA level and protein level of IL6 of WT-infected mice were significantly higher than those in ⊿spvB-infected mice.2.6 Effects of SpvB on the master hepatic inflammatory regulator TREM-1WT-infected mice showed a significantly higher level of TREM-1 in the liver than those of ⊿spvB-infected mice.The TREM-1 inhibitor LP17 treatment eliminated the significant differences in hepatic bacterial load,cytokines and chemokines expression levels between WT-infected mice and ⊿spvB-infected mice.2.7 SpvB-mediated iron metabolic disorder is ameliorated by the TREM-1 inhibitor LP17After administrating the TREM-1 inhibitor LP17,no apparent differences in serum iron levels and hepatic iron contents were found between WT-infected mice and⊿spvB-infected mice.Meanwhile,no apparent differences in hepatic Fpn mRNA levels,FPN protein levels and STAT3 phosphorylation were found between these infected mice.2.8 SpvB interferes host systemic iron metabolism when macrophages interacted with hepatocytesThere were no apparent differences in HAMP expression among HepG2 cells infected with the WT,⊿spvB,or c-spvB strain.HepG2 cells/THP-1 macrophages co-culture model was established,and cells infected with the WT or the c-spvB strain displayed a higher mRNA level of hepcidin and a lower protein level of FPN.HAMP knockdown in co-cultured HepG2 cells or TREM-1 knockdown in co-cultured THP-1 macrophages could reverse the effects of SpvB on systemic iron metabolism.CONCLUSIONS1.Observations from this study revealed that intracellular pattern recognition receptor NLRP6 is involved in Salmonella dysregulation of host iron metabolism.NLRP6 interacted with AKT and decreased its phosphorylation,and then on the one hand decreased NRF2 expression through downregulating AKT/mTOR pathway associated p62 phosphorylation;on the other hand,increased KEAP1 transcription via downregulating AKT/FOXO3A pathway mediated FOXO3A phosphorylation,thereby inhibiting NRF2-mediated FPN transcription,which ultimately result in dysregulation of host iron metabolism and benefit for Salmonella survival and replication.2.Findings from this study revealed that SpvB is an effective strategy utilized by Salmonella to scavenge iron from the host in order to achieve their own nutrient requirements.By activating TREM-1 signaling,SpvB contributes to extensive and severe hepatic inflammation which subsequently perturb the hepcidin-FPN axis in an IL6/JAK/STAT3-dependent manner,and eventually interfering with the host nutritional immune strategy of iron withhold and facilitating the pathogenesis of Salmonella. |
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