Abstract BHLHE40, a member of the basic helix‐loop‐helix transcription factor family, has been reported to play an important role in inflammatory diseases. However, the regulation and function of BHLHE40 in Helicobacter pylori ( H pylori )‐associated gastritis is unknown. We observed that gastric BHLHE40 was significantly elevated in patients and mice with H pylori infection. Then, we demonstrate that H pylori‐ infected GECs express BHLHE40 via cagA ‐ERK pathway. BHLHE40 translocates to cell nucleus, and then binds to cagA protein ‐ activated p‐STAT3 (Tyr705). The complex increases chemotactic factor CXCL12 expression (production). Release of CXCL12 from GECs fosters CD4 + T cell infiltration in the gastric mucosa. Our results identify the cagA ‐BHLHE40‐CXCL12 axis that contributes to inflammatory response in gastric mucosa during H pylori infection.
Background & AimsRev-erbα represents a powerful transcriptional repressor involved in immunity. However, the regulation, function, and clinical relevance of Rev-erbα in Helicobacter pylori infection are presently unknown.MethodsRev-erbα was examined in gastric samples from H pylori-infected patients and mice. Gastric epithelial cells (GECs) were isolated and infected with H pylori for Rev-erbα regulation assays. Gastric tissues from Rev-erbα–/– and wild-type (littermate control) mice or these mice adoptively transferred with CD4+ T cells from IFN-γ–/– and wild-type mice, bone marrow chimera mice and mice with in vivo pharmacological activation or inhibition of Rev-erbα were examined for bacteria colonization. GECs, CD45+CD11c–Ly6G–CD11b+CD68– myeloid cells and CD4+ T cells were isolated, stimulated and/or cultured for Rev-erbα function assays.ResultsRev-erbα was increased in gastric mucosa of H pylori-infected patients and mice. H pylori induced GECs to express Rev-erbα via the phosphorylated cagA that activated ERK signaling pathway to mediate NF-κB directly binding to Rev-erbα promoter, which resulted in increased bacteria colonization within gastric mucosa. Mechanistically, Rev-erbα in GECs not only directly suppressed Reg3b and β-defensin-1 expression, which resulted in impaired bactericidal effects against H pylori of these antibacterial proteins in vitro and in vivo; but also directly inhibited chemokine CCL21 expression, which led to decreased gastric influx of CD45+CD11c–Ly6G–CD11b+CD68– myeloid cells by CCL21-CCR7-dependent migration and, as a direct consequence, reduced bacterial clearing capacity of H pylori-specific Th1 cell response.ConclusionsOverall, this study identifies a model involving Rev-erbα, which collectively ensures gastric bacterial persistence by suppressing host gene expression required for local innate and adaptive defense against H pylori. Rev-erbα represents a powerful transcriptional repressor involved in immunity. However, the regulation, function, and clinical relevance of Rev-erbα in Helicobacter pylori infection are presently unknown. Rev-erbα was examined in gastric samples from H pylori-infected patients and mice. Gastric epithelial cells (GECs) were isolated and infected with H pylori for Rev-erbα regulation assays. Gastric tissues from Rev-erbα–/– and wild-type (littermate control) mice or these mice adoptively transferred with CD4+ T cells from IFN-γ–/– and wild-type mice, bone marrow chimera mice and mice with in vivo pharmacological activation or inhibition of Rev-erbα were examined for bacteria colonization. GECs, CD45+CD11c–Ly6G–CD11b+CD68– myeloid cells and CD4+ T cells were isolated, stimulated and/or cultured for Rev-erbα function assays. Rev-erbα was increased in gastric mucosa of H pylori-infected patients and mice. H pylori induced GECs to express Rev-erbα via the phosphorylated cagA that activated ERK signaling pathway to mediate NF-κB directly binding to Rev-erbα promoter, which resulted in increased bacteria colonization within gastric mucosa. Mechanistically, Rev-erbα in GECs not only directly suppressed Reg3b and β-defensin-1 expression, which resulted in impaired bactericidal effects against H pylori of these antibacterial proteins in vitro and in vivo; but also directly inhibited chemokine CCL21 expression, which led to decreased gastric influx of CD45+CD11c–Ly6G–CD11b+CD68– myeloid cells by CCL21-CCR7-dependent migration and, as a direct consequence, reduced bacterial clearing capacity of H pylori-specific Th1 cell response. Overall, this study identifies a model involving Rev-erbα, which collectively ensures gastric bacterial persistence by suppressing host gene expression required for local innate and adaptive defense against H pylori.
Background: Adrenomedullin (ADM) is a multifunctional peptide that is expressed by many surface epithelial cells, but its relevance to Helicobacter pylori (H. pylori)-induced gastritis is unknown.Methods: The expression of ADM was detected and localized in H. pylori infected patients and mice gastric. The signaling pathway of ADM expression was detected. The differentiation and proliferation of T cells was detected after stimulated with ADM. IL-12 was detected in macrophages after stimulated with ADM.Findings: Gastric ADM expression was elevated in gastric mucosa of H. pylori-infected patients and mice. In H. pylori-infected human gastric mucosa, ADM expression was positively correlated with the degree of gastritis, accordingly, blockade of ADM resulted in decreased inflammation within the gastric mucosa of H. pylori-infected mice. During H. pylori infection, ADM production was promoted via PI3K-AKT signaling pathway activation by gastric epithelial cells in a cagA-dependent manner, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterized by the increased IFN-γ-producing T cells, whose differentiation was induced via the phosphorylation of AKT and STAT3 by ADM derived from gastric epithelial cells. ADM also induced macrophages to produce IL-12, which promoted the IFN-γ-producing T-cell responses, thereby contributing to the development of H. pylori associated gastritis. Accordingly, blockade of IFN-γ or knockout of IFN-γ decreased inflammation within the gastric mucosa of H. pylori-infected mice.Interpretation: This study identifies a novel regulatory network involving H. pylori, gastric epithelial cells, ADM, macrophages, T cells, and IFN-γ, which collectively exert a pro-inflammatory effect within the gastric microenvironment.Funding Statement: This work was funded by National Key Research and Development Program of China (grant number: 2016YFC1302200) and National Natural Science Foundation of China (grant number: 81870394).Declaration of Interests: The authors declare that they have no conflict of interest.Ethics Approval Statement: The study was approved by the Ethics Committee of XinQiao Hospital and Southwest Hospital of Third Military Medical University. All human subjects were adult, and the written informed consent was obtained from each subject. All animal experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China. All breeding and experiments were undertaken with review and approval from the Animal Ethical and Experimental Committee of Third Military Medical University.
To investigate the effects of tumor necrosis factor-alpha-inducing protein-alpha (Tipalpha) released from Helicobacter pylori (Hp) on human gastric epithelial cells.Human gastric epithelial cells of the line GES-1 were cultured. Tipalpha gene located in Hp 0596 was extracted from the genome sequence of the Hp strain 26695 and its open reading frame were cloned into the eukaryotic expressing vector pcDNA3.1. The recombinant plasmid pcDNA3.1-Tipalpha thus obtained and the blank plasmid pcDNA3. 1 were transfected into the GES-1 cells, and the G418-resistent clones were screened. The GES-1 cells transfected with blank vector pcDNA3.1 and normal GES-1 cells were used as controls. RT-PCR and Western blotting were used to detect the expression of Tipalpha. The influences of Tipalpha protein on the cells proliferation, apoptosis and cell cycles, concentration of related cytokines such as TNF-alpha, IL-1beta, and IL-8, and the expression of Bcl-2 and P53 genes were respectively observed by MTT assay, flow cytometry, ELISA, and Western blotting.1. A eukaryotic expression vector of Hp Tipalpha was successfully constructed. Steadily transfected strains were screened by G418. 2. MTT method showed that the growth curve of the GES-1 cells transfected with GpcDNA3.1-Tipalpha was higher, showing a faster growth. 3. Flow cytometry showed an increase in the proportion of the S-phase and a decrease in the G1-phase in the GpcDNA3.1-Tipalpha-transfected cells [(45.33 +/- 1.03)% vs (38.24 +/- 1.5)%, (33.94 +/- 1.67)%, (41.39 +/- 0.08)% vs (49.74 +/- 0.12)%, (49.27 +/- 0.15)%], and the apoptotic rate of the GpcDNA3.1-Tipalpha-transfected cells was 0.76 +/- 0.04, significantly lower than those of the GpcDNA3.1-transfected cells and non-transfected cells (16.84 +/- 2.16 and 8.36 +/- 1.07 respectively, both P < 0.05). 4. ELISA showed that there was no significant difference in intracellular TNF-alpha concentration among the 3 GES-1 cell groups (all P > 0.05), however, the extra-cellular TNF-alpha level of the GpcDNA3.1-Tipalpha-transfected cells was significantly higher than those of the 2 control groups (both P < 0.05), and that the intra- and extra-cellular IL-1beta and IL-8 concentrations of the GpcDNA3.1-Tipalpha-transfected cells were both significantly higher than those of the 2 control groups (all P < 0.05). 5. The expression level of Bcl-2 gene of the GpcDNA3.1-Tipalphac-transfected cells was obviously higher than those of the 2 control groups, and the expression of P53 gene of the GpcDNA3.1-Tipalphac-transfected cells was lower than those of the 2 control cells.The GpcDNA3.1-Tipalpha-transfected cells steadily and highly express Tipalpha protein, that induces the high expression of TNF-alpha, IL-1beta, and IL-8, proinflammatory cytokines, enhances cell proliferation, and upregulates Bcl-2 gene and down-regulates P53 gene. Tipalpha may play an important carcinogenic role in gastric cancer progression.
Abstract Background Staphylococcus aureus is the major cause of hospital-acquired and community-acquired pneumonia. Host defense to S.aureus infection is largely mediated by the innate immune system. γδ T cells play an important role in innate immunity to many infectious diseases. However, less is known about the role of these cells during S.aureus -induced pneumonia. In this study, we examined the response and the role of γδ T cells to pulmonary S.aureus infection. Results Mice infected with S. aureus intranasally showed rapid γδ T cells accumulation in the lung. Deficiency of γδ T cells led to attenuated bacterial clearance and less tissue damage in lung compared with WT mice. Moreover, TCR-δ −/− mice exhibited impaired neutrophil recruitment and reduced cytokine production at the site of infection. The γδ T cells in response to pulmonary S. aureus infection mainly secreted IL-17 and γδ T cells deficiency reduced IL-17 production, which might regulate the production of neutrophil-inducing cytokine/chemokine in the S. aureus -infected lungs. Conclusions Accumulation of γδ T cells in the lungs to S. aureus infection is beneficial for bacteria clearance and also contributes to the tissue damage. These cells were the primary source of IL-17, which might influence the recruitment of neutrophils at the early stage of infection.
Regulated in development and DNA damage responses-1 (REDD1) is a conserved and ubiquitous protein, which is induced in response to multiple stimuli. However, the regulation, function and clinical relevance of REDD1 in Helicobacter pylori-associated gastritis are presently unknown.
Helicobacter pylori infection is characterized as progressive processes of bacterial persistence and chronic gastritis with features of infiltration of mononuclear cells more than granulocytes in gastric mucosa. Angiopoietin-like 4 (ANGPTL4) is considered a double-edged sword in inflammation-associated diseases, but its function and clinical relevance in H. pylori -associated pathology are unknown. Here, we demonstrate both pro-colonization and pro-inflammation roles of ANGPTL4 in H. pylori infection. Increased ANGPTL4 in the infected gastric mucosa was produced from gastric epithelial cells (GECs) synergistically induced by H. pylori and IL-17A in a cagA -dependent manner. Human gastric ANGPTL4 correlated with H. pylori colonization and the severity of gastritis, and mouse ANGPTL4 from non-bone marrow-derived cells promoted bacteria colonization and inflammation. Importantly, H. pylori colonization and inflammation were attenuated in Il17a −/− , Angptl4 −/− , and Il17a −/− Angptl4 −/− mice. Mechanistically, ANGPTL4 bound to integrin αV (ITGAV) on GECs to suppress CXCL1 production by inhibiting ERK, leading to decreased gastric influx of neutrophils, thereby promoting H. pylori colonization; ANGPTL4 also bound to ITGAV on monocytes to promote CCL5 production by activating PI3K–AKT–NF-κB, resulting in increased gastric influx of regulatory CD4 + T cells (T regs ) via CCL5–CCR4-dependent migration. In turn, ANGPTL4 induced T reg proliferation by binding to ITGAV to activate PI3K–AKT–NF-κB, promoting H. pylori -associated gastritis. Overall, we propose a model in which ANGPTL4 collectively ensures H. pylori persistence and promotes gastritis. Efforts to inhibit ANGPTL4-associated pathway may prove valuable strategies in treating H. pylori infection.
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