Helicobacter pylori-controlled c-Abl localization promotes cell migration and limits apoptosis.

BACKGROUND: Deregulated c-Abl activity has been intensively studied in a variety of solid tumors and leukemia. The class-I carcinogen Helicobacter pylori (Hp) activates the non-receptor tyrosine kinase c-Abl to phosphorylate the oncoprotein cytotoxin-associated gene A (CagA). The role of c-Abl in CagA-dependent pathways is well established; however, the knowledge of CagA-independent c-Abl processes is scarce. METHODS: c-Abl phosphorylation and localization were analyzed by immunostaining and immunofluorescence. Interaction partners were identified by tandem-affinity purification. Cell elongation and migration were analyzed in transwell-filter experiments. Apoptosis and cell survival were examined by FACS analyses and MTT assays. In mice experiments and human biopsies, the involvement of c-Abl in Hp pathogenesis was investigated. RESULTS: Here, we investigated the activity and subcellular localization of c-Abl in vitro and in vivo and unraveled the contribution of c-Abl in CagA-dependent and -independent pathways to gastric Hp pathogenesis. We report a novel mechanism and identified strong c-Abl threonine 735 phosphorylation (pAblT735) mediated by the type-IV secretion system (T4SS) effector D-glycero-ß-D-manno-heptose-1,7-bisphosphate (ßHBP) and protein kinase C (PKC) as a new c-Abl kinase. pAblT735 interacted with 14-3-3 proteins, which caused cytoplasmic retention of c-Abl, where it potentiated Hp-mediated cell elongation and migration. Further, the nuclear exclusion of pAblT735 attenuated caspase-8 and caspase-9-dependent apoptosis. Importantly, in human patients suffering from Hp-mediated gastritis c-Abl expression and pAblT735 phosphorylation were drastically enhanced as compared to type C gastritis patients or healthy individuals. Pharmacological inhibition using the selective c-Abl kinase inhibitor Gleevec confirmed that c-Abl plays an important role in Hp pathogenesis in a murine in vivo model. CONCLUSIONS: In this study, we identified a novel regulatory mechanism in Hp-infected gastric epithelial cells by which Hp determines the subcellular localization of activated c-Abl to control Hp-mediated EMT-like processes while decreasing cell death.

Lymphotoxin ß receptor signalling executes Helicobacter pylori-driven gastric inflammation in a T4SS-dependent manner.

OBJECTIVE: Lymphotoxin ß receptor (LTßR) signalling has been implicated in inflammation-associated tumour development in different tissues. We have analysed the role of LTßR and alternative NF-κB signalling in Helicobacter pylori-mediated gastric inflammation and pathology. DESIGN: We analysed several ligands and receptors of the alternative NF-κB pathway, RelB, p52 nuclear translocation and target genes in tissue samples of H. pylori-infected patients with different degrees of gastritis or early gastric tumours by in situ hybridisation, immunohistochemistry, Western blot and real-time PCR analyses. Molecular mechanisms involved in LTßR activation by H. pylori were assessed in vitro using human gastric cancer cell lines and distinct H. pylori isolates. The effects of blocking or agonistically activating LTßR on gastric pathology during challenge with a human pathogenic H. pylori strain were studied in a mouse model. RESULTS: Among the tested candidates, LT was significantly increased and activated alternative NF-κB signalling was observed in the gastric mucosa of H. pylori-infected patients. H. pyloriinduced LTßR-ligand expression in a type IV secretion system-dependent but CagA-independent manner, resulting in activation of the alternative NF-κB pathway, which was further enhanced by blocking canonical NF-κB during infection. Blocking LTßR signalling in vivo suppressed H. pylori-driven gastritis, whereas LTßR activation in gastric epithelial cells of infected mice induced a broadened pro-inflammatory chemokine milieu, resulting in exacerbated pathology. CONCLUSIONS: LTßR-triggered activation of alternative NF-κB signalling in gastric epithelial cells executes H. pylori-induced chronic gastritis, representing a novel target to restrict gastric inflammation and pathology elicited by H. pylori, while exclusively targeting canonical NF-κB may aggravate pathology by enhancing the alternative pathway.

Effective treatment of allergic airway inflammation with Helicobacter pylori immunomodulators requires BATF3-dependent dendritic cells and IL-10.

The prevalence of allergic asthma and other atopic diseases has reached epidemic proportions in large parts of the developed world. The gradual loss of the human indigenous microbiota has been held responsible for this trend. The bacterial pathogen Helicobacter pylori is a constituent of the normal gastric microbiota whose presence has been inversely linked to allergy and asthma in humans and experimental models. Here we show that oral or i.p. tolerization with H. pylori extract prevents the airway hyperresponsiveness, bronchoalveolar eosinophilia, pulmonary inflammation, and Th2 cytokine production that are hallmarks of allergen-induced asthma in mice. Asthma protection is not conferred by extracts from other enteropathogens and requires a heat-sensitive H. pylori component and the DC-intrinsic production of IL-10. The basic leucine zipper ATF-like 3 (BATF3)-dependent CD103(+)CD11b(-) dendritic cell lineage is enriched in the lungs of protected mice and strictly required for protection. Two H. pylori persistence determinants, the γ-glutamyl-transpeptidase GGT and the vacuolating cytotoxin VacA, are required and sufficient for asthma protection and can be administered in purified form to prevent asthma. In conclusion, we provide preclinical evidence for the concept that the immunomodulatory properties of H. pylori can be exploited for tolerization strategies aiming to prevent allergen-induced asthma.

Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance.

Infection with the gastric bacterial pathogen Helicobacter pylori is typically contracted in early childhood and often persists for decades. The immunomodulatory properties of H. pylori that allow it to colonize humans persistently are believed to also account for H. pylori's protective effects against allergic and chronic inflammatory diseases. H. pylori infection efficiently reprograms dendritic cells (DCs) toward a tolerogenic phenotype and induces regulatory T cells (Tregs) with highly suppressive activity in models of allergen-induced asthma. We show here that two H. pylori virulence determinants, the γ-glutamyl transpeptidase GGT and the vacuolating cytotoxin VacA, contribute critically and nonredundantly to H. pylori's tolerizing effects on murine DCs in vitro and in vivo. The tolerance-promoting effects of both factors are independent of their described suppressive activity on T cells. Isogenic H. pylori mutants lacking either GGT or VacA are incapable of preventing LPS-induced DC maturation and fail to drive DC tolerization as assessed by induction of Treg properties in cocultured naive T cells. The Δggt and ΔvacA mutants colonize mice at significantly reduced levels, induce stronger T-helper 1 (Th1) and T-helper 17 (Th17) responses, and/or trigger more severe gastric pathology. Both factors promote the efficient induction of Tregs in vivo, and VacA is required to prevent allergen-induced asthma. The defects of the Δggt mutant in vitro and in vivo are phenocopied by pharmacological inhibition of the transpeptidase activity of GGT in all readouts. In conclusion, our results reveal the molecular players and mechanistic basis for H. pylori-induced immunomodulation, promoting persistent infection and conferring protection against allergic asthma.

Caspase-1 has both proinflammatory and regulatory properties in Helicobacter infections, which are differentially mediated by its substrates IL-1ß and IL-18.

The proinflammatory cysteine protease caspase-1 is autocatalytically activated upon cytosolic sensing of a variety of pathogen-associated molecular patterns by Nod-like receptors. Active caspase-1 processes pro-IL-1ß and pro-IL-18 to generate the bioactive cytokines and to initiate pathogen-specific immune responses. Little information is available on caspase-1 and inflammasome activation during infection with the gastric bacterial pathogen Helicobacter pylori. In this study, we addressed a possible role for caspase-1 and its cytokine substrates in the spontaneous and vaccine-induced control of Helicobacter infection, as well as the development of gastritis and gastric cancer precursor lesions, using a variety of experimental infection, vaccine-induced protection, and gastric disease models. We show that caspase-1 is activated and IL-1ß and IL-18 are processed in vitro and in vivo as a consequence of Helicobacter infection. Caspase-1 activation and IL-1 signaling are absolutely required for the efficient control of Helicobacter infection in vaccinated mice. IL-1R(-/-) mice fail to develop protective immunity but are protected against Helicobacter-associated gastritis and gastric preneoplasia as a result of their inability to generate Helicobacter-specific Th1 and Th17 responses. In contrast, IL-18 is dispensable for vaccine-induced protective immunity but essential for preventing excessive T cell-driven immunopathology. IL-18(-/-) animals develop strongly accelerated pathology that is accompanied by unrestricted Th17 responses. In conclusion, we show in this study that the processing and release of a regulatory caspase-1 substrate, IL-18, counteracts the proinflammatory activities of another caspase-1 substrate, IL-1ß, thereby balancing control of the infection with the prevention of excessive gastric immunopathology.

MicroRNA-155 is essential for the T cell-mediated control of Helicobacter pylori infection and for the induction of chronic Gastritis and Colitis.

MicroRNAs govern immune responses to infectious agents, allergens, and autoantigens and function by posttranscriptional repression of their target genes. In this paper, we have addressed the role of microRNA-155 (miR-155) in the control of Helicobacter pylori infection of the gastrointestinal tract and the development of H. pylori-induced chronic gastritis and associated gastric preneoplastic pathology. We show that miR-155 is upregulated in the gastric mucosa of experimentally infected mice and that miR-155(-/-) mice fail to control H. pylori infection as a result of impaired pathogen-specific Th1 and Th17 responses. miR-155(-/-) mice are also less well protected against challenge infection after H. pylori-specific vaccination than their wild-type (wt) counterparts. As a consequence of their impaired T cell responses to H. pylori, miR-155(-/-) mice develop less severe infection-induced immunopathology manifesting as chronic atrophic gastritis, epithelial hyperplasia, and intestinal metaplasia. T cells from miR-155(-/-) mice that are activated by CD3/CD28 cross-linking expand less and produce less IFN-γ and IL-17 than wt T cells. Finally, we show in this paper using adoptive transfers that the phenotypes of miR-155(-/-) mice are likely due to T cell-intrinsic defects. In contrast to wt T cells, miR-155(-/-) T cells from infected donors do not control H. pylori infections in T cell-deficient recipients, do not differentiate into Th1 or Th17 cells, and do not cause immunopathology. In addition, naive miR-155(-/-) T cells fail to induce chronic Th17-driven colitis in an adoptive transfer model. In conclusion, miR-155 expression is required for the Th17/Th1 differentiation that underlies immunity to H. pylori infection on the one hand and infection-associated immunopathology on the other.

The C-terminally encoded, MHC class II-restricted T cell antigenicity of the Helicobacter pylori virulence factor CagA promotes gastric preneoplasia.

Chronic infection with the human bacterial pathogen Helicobacter pylori causes gastritis and predisposes carriers to an increased gastric cancer risk. Consequently, H. pylori-specific vaccination is widely viewed as a promising strategy of gastric cancer prevention. H. pylori strains harboring the Cag pathogenicity island (PAI) are associated with particularly unfavorable disease outcomes in humans and experimental rodent models. We show in this study using a C57BL/6 mouse model of Cag-PAI(+) H. pylori infection that the only known protein substrate of the Cag-PAI-encoded type IV secretion system, the cytotoxin-associated gene A (CagA) protein, harbors MHC class II-restricted T cell epitopes. Several distinct nonoverlapping epitopes in CagA's central and C-terminal regions were predicted in silico and could be confirmed experimentally. CagA(+) infection elicits CD4(+) T cell responses in mice, which are strongly enhanced by prior mucosal or parenteral vaccination with recombinant CagA. The adoptive transfer of CagA-specific T cells to T cell-deficient, H. pylori-infected recipients is sufficient to induce the full range of preneoplastic immunopathology. Similarly, immunization with a cholera toxin-adjuvanted, CagA(+) whole-cell sonicate vaccine sensitizes mice to, rather than protects them from, H. pylori-associated gastric cancer precursor lesions. In contrast, H. pylori-specific tolerization by neonatal administration of H. pylori sonicate in conjunction with a CD40L-neutralizing Ab prevents H. pylori-specific, pathogenic T cell responses and gastric immunopathology. We conclude that active tolerization may be superior to vaccination strategies in gastric cancer prevention.

Helicobacter pylori-specific protection against inflammatory bowel disease requires the NLRP3 inflammasome and IL-18.

BACKGROUND: The Gram-negative bacterium Helicobacter pylori is a constituent of the human gastric microbiota. Chronic infection with H. pylori causes gastritis and predisposes to gastric carcinoma but has also been inversely linked to various allergic and chronic inflammatory conditions. In particular, large meta-analyses have documented an inverse association between H. pylori infection and the risk of developing ulcerative colitis and Crohn's disease. METHODS: We investigated possible protective effects of experimental H. pylori infection and of regular treatment with H. pylori extract in 2 mouse models of colitis and in mouse models of type I diabetes and multiple sclerosis. The mechanism of protection was examined in mouse strains lacking specific innate immune recognition pathways and cytokines. RESULTS: We show here that experimental infection with H. pylori and administration of regular doses of H. pylori extract both alleviate the clinical and histopathological features of dextran sodium sulfate-induced chronic colitis and of T-cell transfer-induced colitis. High resolution endoscopy of the protected animals revealed the accumulation of large amounts of colonic mucus upon H. pylori exposure, which could be attributed to transcriptional activation of the mucin 2 gene. The protection against dextran sodium sulfate-induced colitis was dependent on the NLRP3 inflammasome and interleukin-18 signaling. Other autoimmune diseases, i.e., experimental autoimmune encephalomyelitis and type I diabetes, were not controlled by H. pylori. CONCLUSIONS: In summary, we propose here that the immunomodulatory activity of an ancient constituent of the gut microbiota, H. pylori, may be exploited for the prevention and/or treatment of inflammatory bowel diseases.

The role of Th cell subsets in the control of Helicobacter infections and in T cell-driven gastric immunopathology.

Chronic infection with the gastric bacterial pathogen Helicobacter pylori causes gastric adenocarcinoma in a particularly susceptible fraction of the infected population. The intestinal type of gastric cancer is preceded by a series of preneoplastic lesions that are of immunopathological origin, and that can be recapitulated by experimental infection of C57BL/6 mice with Helicobacter species. Several lines of evidence suggest that specific T cell subsets and/or their signature cytokines contribute to the control of Helicobacter infections on the one hand, and to the associated gastric preneoplastic pathology on the other. Here, we have used virulent H. pylori and H. felis isolates to infect mice that lack α/ß T cells due to a targeted deletion of the T cell receptor ß-chain, or are deficient for the unique p35 and p19 subunits of the Th1- and Th17-polarizing cytokines interleukin (IL)-12 and IL-23, respectively. We found that α/ß T cells are absolutely required for Helicobacter control and for the induction of gastric preneoplastic pathology. In contrast, neither IL-12-dependent Th1 nor IL-23-dependent Th17 cells were essential for controlling the infection; IL-12p35(-/-) and IL-23p19(-/-) mice did not differ significantly from wild type animals with respect to Helicobacter colonization densities. Gastritis and gastric preneoplastic pathology developed to a similar extent in all three strains upon H. felis infection; in the H. pylori infection model, IL-23p19(-/-) mice exhibited significantly less gastritis and precancerous pathology. In summary, the results indicate that neither Th1 nor Th17 cells are by themselves essential for Helicobacter control; the associated gastric pathology is reduced only in the absence of Th17-polarizing IL-23, and only in the H. pylori, but not the H. felis infection model. The results thus suggest the involvement of other, as yet unknown T cell subsets in both processes.

DC-derived IL-18 drives Treg differentiation, murine Helicobacter pylori-specific immune tolerance, and asthma protection.

Persistent colonization with the gastric bacterial pathogen Helicobacter pylori causes gastritis and predisposes infected individuals to gastric cancer. Conversely, it is also linked to protection from allergic, chronic inflammatory, and autoimmune diseases. We demonstrate here that H. pylori inhibits LPS-induced maturation of DCs and reprograms DCs toward a tolerance-promoting phenotype. Our results showed that DCs exposed to H. pylori in vitro or in vivo failed to induce T cell effector functions. Instead, they efficiently induced expression of the forkhead transcription factor FoxP3, the master regulator of Tregs, in naive T cells. Depletion of DCs in mice infected with H. pylori during the neonatal period was sufficient to break H. pylori-specific tolerance. DC depletion resulted in improved control of the infection but also aggravated T cell-driven immunopathology. Consistent with the mouse data, DCs infiltrating the gastric mucosa of human H. pylori carriers exhibited a semimature DC-SIGN(+)HLA-DR(hi)CD80(lo)CD86(lo) phenotype. Mechanistically, the tolerogenic activity of H. pylori-experienced DCs was shown to require IL-18 in vitro and in vivo; DC-derived IL-18 acted directly on T cells to drive their conversion to Tregs. CD4(+)CD25(+) Tregs from infected wild-type mice but not Il18(-/-) or Il18r1(-/-) mice prevented airway inflammation and hyperresponsiveness in an experimental model of asthma. Taken together, our results indicate that tolerogenic reprogramming of DCs ensures the persistence of H. pylori and protects against allergic asthma in a process that requires IL-18.