Expression,Regulation And Function Of Cathepsin C In Helicobacter Pylori Chronic Infection

BackgroundsHelicobacter pylori（H.pylori）is a gram-negative helical bacterium that colonizes in the stomach,infecting nearly half the world’s population.Persistent H.pylori infection can result in many gastric diseases,including chronic gastritis,peptic ulceration,and most seriously,increase the risk of gastric adenocarcinoma.The mechanism of severe outcome in H.pylori infection is still not fully understood,and it seems to be multifactorial.Several studies have reported the immunosuppressive microenvironment by bacterial virulence factors in the sites of H.pylori infection plays important roles in the failure of clearing bacteria.And clinical chronic gastritis is on the basis of the persistence colonization of H.pylori in the gastric mucosa.Cathepsin C（CtsC）,also known as dipeptidyl peptidase I（DPPI）,is one of the cathepsins with a lysosomal exo-cysteine protease activity,which functions as a central coordinator for activation of many serine proteases in immune cells.The serine proteases activated by CtsC are secreted to the microenvironment,and are involved in proinflammatory effects.CtsC plays the inflammatory regulation roles in many inflammation diseases.The depletion of CtsC is a useful protective mechanism of inflammation,but it also loses the bactericidal capacity in different diseases.However,CtsC expression in gastric mocusal and its role in H.pylori infection remain unclear.Therefore,focusing on the regulation of CtsC expression and its pathophysiological role in H.pylori infection is helpful to demonstrate the mechanism of persistent infection and inflammation.Objectives1.To discover the expression of CtsC in gastric mucosa during H.pylori infection and the source cell type of CtsC;2.To investigate the regulatory mechanisms of CtsC expression in H.pylori infection;3.To study the founction of CtsC in the microenvironment of H.pylori infection;4.To clarify the mechanisms of CtsC on activating neutrophils and its host defense role in H.pylori infection.Methods1.The expression of CtsC in gastric mucosa and gastric epithelial cells during H.pylori infectionThe human gastric biopsy specimens and blood samples were collected from H.pylori-infected and uninfected patients.H.pylori infection was determined by ¹⁴C urea breath test and rapid urease test of biopsy specimens taken from the antrum,and subsequently conformed by real-time PCR（RT-PCR）for 16S rDNA and serology test for specific anti-H.pylori antibodies（Abs）.CagA was also detected by RT-PCR,and the result is as a basis for grouping into cagA+and cagA-.SPF C57 mice were oralgastrically inoculated by H.pylori NCTC 11637（WT H.pylori）and cagA-knockout mutant H.pylori NCTC 11637（ΔcagA）.The gastric mucosa from human and mice were isolated and detected by RT-PCR,immunohistochemistry and Western blot to evaluate CtsC expression.Human primary gastric mucosas were also infected by WT H.pylori andΔcagA,and the expression and secretion of CtsC were detected by RT-PCR,ELISA and protein chip.The gastric mucosa from human and mice were immunofluorescence stained by CtsC and EpCam（CD326）Abs.Human gastric epithelial cell lines（AGS,GES-1,HGC-27）and primary gastric epithelial cells were stimulated with WT H.pylori,ΔcagA,or H.pylori26695 at different multiplicity of infection（MOI）and at different time piont.Cts family expressions were detected by RT-PCR,and CtsC protein was detected by Western blot and ELISA.2.To investigate the regulatory mechanisms of CtsC expression in H.pylori infectionAGS cells were pre-treated with a variety of common signaling pathway inhibitors,and then were stimulated by WT H.pylori orΔcagA.The effect of inhibitors in the expression of CtsC and the phosphorylation of signal molecules were evaluated by RT-PCR,ELISA and Western blot.Dual-luciferase reporter assay was used to test the CtsC promoter activity of full-length and the different fragment sequences in WT H.pylori infection manner.Also,the promoter activity was assayed with WT H.pylori orΔcagA infection with or without signaling inhibitors.The TGF-β1 expression in gastric mocusa from H.pylori-infected human or mice,or in H.pylori-stimulated AGS cells were tested by RT-PCR and ELISA.Moreover,the correlation between CtsC and TGF-β1 expression in H.pylori-infected human gastric mocusa was evaluated.Furthermore,AGS cells were stimulated with WT H.pylori and/or TGF-β1,or with the culture supernatants of WT H.pylori-stimulated AGS cells in the presence or absence of a neutralizing antibody against TGF-β1,TGF-βreceptor antagonist LY364947,ERK inhibitor U0126 or STAT3 inhibitor AG490.The expression of CtsC was detected by RT-PCR and ELISA,while the phosphorylation of ERK and STAT3 were assayed by Western blot.3.The founction of CtsC in the microenvironment of H.pylori infectionThe correlation between CtsC expression and H.pylori colonization in H.pylori infected human gastric mocusas was evaluated.One day after infection,mice were injected intraperitoneally（i.p.）with recombinant mouse active CtsC,and the injection was repeated every week until the mice were sacrificed.The H.pylori colonization in the stomach of mice was detected by 16S rDNA RT-PCR and calculated as the numbers of bacterial genomes per nanogram of host genomic DNA.Flow cytometry was used to detect the percentages of each major immunity cells and the activity of neutrophils（pHrodo,CD11b,CD62L）.4.The mechanisms of CtsC activating neutrophils and playing host defense role in H.pylori infectionWe depleted neutrophils through anti-Ly6G Abs injection i.p.and activated neutrophils through fMLP injection i.p..The H.pylori colonization in the stomach of mice was also detected by 16S rDNA RT-PCR.Flow cytometry was used to assay the effect of neutrophils depletion and the activity of neutrophils（pHrodo,CD11b,CD62L,ROS）in active CtsC or proCtsC stimulation experiment in vitro.Moreover,neutrophils were pre-treated with ginkgolic acid（GA,a SUMO inhibitor）,BAY11-7082（a NF-κB inhibitor）or NEMO siRNA,and then atimulated by active CtsC.The SUMOylation and phosphorylation of signal molecules in NF-κB pathway were detected by Western blot,while activity of neutrophils was assayed using flow cytometry.Furthermore,human neutrophils were stimulated with indicated concentrations of recombinant active CtsC,and assayed for neutrophil opsonophagocytic killing effect in vitro.Results1.The expression of CtsC in gastric mucosa and gastric epithelial cells during H.pylori infectionCtsC mRNA and protein were decreased in gastric mucosa of H.pylori-infected patients,and this decrease was more significantly in cagA+H.pylori compared to cagA-H.pylori infection.Similar observations were found in mice experiment at 56 d after infection.CtsC was mainly expressed in CD326+gastric epithelial cells in gastric mucosa of uninfected donors or mice.AGS cells,GES-1 cells,HGC-27 cells and human primary gastric epithelial cells showed the decrease of CtsC expression with H.pylori stimulation,and the lower expression of CtsC was shown with WT H.pylori compared toΔcagA stimulation.The expression of CtsC was MOI and infection time point dependent in AGS cells,and the downregulation of mature subunits of active CtsC was also observed in AGS supernatant.Moreover,11637 H.pylori and 26695 H.pylori had the similar effect in downregulation of CtsC.2.To investigate the regulatory mechanisms of CtsC expression in H.pylori infectionWT H.pylori significantly increase ERK and STAT3 phosphorylation.When inhibited these signaling pathways by specific inhibitors,the effect of CtsC downregulation by WT H.pylori was partly abolished.Moreovere,inhibiting cagA phosphorylation by blocking Src could decrease the ERK phosphorylation,and increase the CtsC expression,but had no effect on STAT3 phosphorylation.Furthermore,WT H.pylori decreased the promoter activity of CtsC,blocking Src,ERK,STAT3 or knockout cagA all reversed the decrease.The TGF-β1 expression was increased in human and mice gastric mocusas and AGS cells in H.pylori infection,and CtsC expression was negatively correlated with TGF-β1expression in gastric mucosa of H.pylori-infected patients.Moreover,TGF-β1 exerted synergistic effect to downregulate CtsC with H.pylori expression.And inhibiting the ERK and STAT3 signaling pathway could abolish the synergistic effect of TGF-β1.Furthtermore,neutralizing TGF-β1 or blocking TGF-βreceptor in AGS cells stimulated with the culture supernatants from H.pylori-stimulated AGS cells could effectively inhibit the downregulation of CtsC.3.The founction of CtsC in the microenvironment of H.pylori infectionCtsC expression was negatively associated with H.pylori colonization in human gastric mucosa.Administration of active CtsC also significantly decreased gastric bacterial burden and increase the activity（increase CD11b and decrease CD62L）and phagocytosis（increase pHrodo）of neutrophils,while had no effect on the percentages of immune cells in stomach and blood.4.The mechanisms of CtsC on activating neutrophils and its host defense role in H.pylori infectionThe results of administration of anti-Ly6G Abs and fMLP experiments showed that H.pylori colonization increased in neutrophils deleption manner and decreased in neutrophils activation manner,and anti-Ly6G Abs also abolished the decreased H.pylori colonization by active CtsC.In vitro active CtsC stimulation experimrnt indicated active CtsC could activate fresh human neutrophils（increase CD11b,pHrodo,ROS and decrease CD62L）,and promote NEMO SUMOylation and NF-κB p65 phosphorylation,but this activation could be inhibited by SUMO inhibitor,NF-κB inhibitor and NEMO siRNA.In vitro bactericidal experiment demonstrated bactericidal capacity of active CtsC depended on the activity of neutrophils.Conclusion1.CtsC decreases in H.pylori-infected gastric mucosa and gastric epithelial cells;2.H.pylori and TGF-β1 exert synergistic effect to downregulate CtsC of gastric epithelial cells via Src/ERK and JAK/STAT3 pathway;3.Active CtsC induces neutrophil activation and inhibits bacteria colonization in gastric mucosa during H.pylori infection;4.Active CtsC contributes to host defense against H.pylori by activating neutrophils via NF-kB pathwaySignificanceOur finding identified a mechanism that H.pylori abrogate CtsC to impair neutrophil activation and to ensure persistence in gastric mucosa,and provided new insights into how H.pylori escape from neutrophil clearance in H.pylori-associated chronic gastritis.Efforts to enable and boost this neutrophils activation pathway by active CtsC may therefore become valuable strategies in treating H.pylori infection.