Interleukin-6 induction by Helicobacter pylori in human macrophages is dependent on phagocytosis

BACKGROUND: The colonization of the gastric mucosa with Helicobacter pylori is accompanied by elevated levels of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and IL-8. The aim of our study was to determine the mechanisms of IL-6 stimulation in phagocytes upon H. pylori infection. MATERIALS AND METHODS: We investigated the secretion of IL-6 by different professional phagocytes from murine and human origin, including granulocyte- and monocyte-like cells and macrophages derived from human peripheral blood monocytes (PBMCs). The influence of viability, phagocytosis, and the impact of different subcellular fractions of H. pylori bacteria were evaluated. RESULTS: IL-6 levels induced by H. pylori were low in cell lines derived from murine and human monocytes and in human granulocyte-like cells. By contrast, macrophages derived from human PBMCs were highly responsive to both H. pylori and Escherichia coli. IL-6 induction was blocked by inhibition of actin-dependent processes prior to infection with H. pylori, but not with E. coli or E. coli lipopolysaccharide (LPS). Using cell fractionation, the most activity was found in the H. pylori membrane. H. pylori LPS exhibited a 10(3)- to 10(4)-fold lower biologic activity than E. coli LPS, suggesting a minor role for toll-like receptor 4 (TLR4)-mediated signalling from the exterior. CONCLUSIONS: From these data, we conclude that macrophages may be a major source of IL-6 in the gastric mucosa upon H. pylori infection. The IL-6 induction by H. pylori in these cells is a multifactorial process, which requires the uptake and presumably degradation of H. pylori bacteria.     

Host response to Helicobacter pylori infection before initiation of the adaptive immune response

Helicobacter pylori persistently colonizes the human stomach. In this study, immune responses to H. pylori that occur in the early stages of infection were investigated. Within the first 2 days after orogastric infection of mice with H. pylori, there was a transient infiltration of macrophages and neutrophils into the glandular stomach. By day 10 postinfection, the numbers of macrophages and neutrophils decreased to baseline levels. By 3 weeks postinfection, an adaptive immune response was detected, marked by gastric infiltration of T lymphocytes, macrophages, and neutrophils, as well as increased numbers of H. pylori-specific T cells, macrophages, and dendritic cells in paragastric lymph nodes. Neutrophil-attracting and macrophage-attracting chemokines were expressed at higher levels in the stomachs of H. pylori-infected mice than in the stomachs of uninfected mice. Increased expression of TNFalpha and IFNgamma (Th1-type inflammatory cytokines) and IL-17 (a Th17-type cytokine) was detected in the stomachs of H. pylori-infected mice, but increased expression of IL-4 (a Th2-type cytokine) was not detected. These data indicate that a transient gastric inflammatory response to H. pylori occurs within the first few days after infection, before the priming of T cells and initiation of an adaptive immune response. It is speculated that inappropriate waning of the innate immune response during early stages of infection may be a factor that contributes to H. pylori persistence.     

Increased production of matrix metalloproteinases in Helicobacter pylori-associated human gastritis

BACKGROUND AND AIMS: Helicobacter pylori infection results in an active, chronic inflammation of the gastric mucosa. Previous studies have highlighted the importance of matrix metalloproteinases (MMPs) in diseases involving mucosal inflammation, prompting us to investigate MMP activity in H. pylori-induced gastritis. METHODS: Gastric biopsies were obtained from H. pylori-infected and uninfected volunteers, and MMP activity was assessed using substrate gel electrophoresis. MMP production was also evaluated by immunohistochemistry and real time-polymerase chain reaction. In parallel, tissue inhibitors of MMPs (TIMP) levels and TIMP-MMP complexes were examined in corresponding tissues using enzyme-linked immunosorbent assays and Western blotting. Finally, MMP production by gastric macrophages was determined after stimulation with H. pylori. RESULTS: Antral mucosa of H. pylori-infected subjects demonstrated a 19-fold higher MMP-9 activity than that of uninfected individuals. MMP-2 was present at lower levels, but was also increased in H. pylori-infected individuals, while there was no difference in the total levels of TIMP-1 and TIMP-2 between the groups of volunteers. Significant numbers of MMP-9-containing cells were only found in the H. pylori-infected antral mucosa. Tissue-resident macrophages were significantly increased in H. pylori-infected individuals, and double-staining showed MMP-9 colocalized to macrophages. Furthermore, gastric macrophages secreted MMP-9 in response to H. pylori bacteria. A corresponding 10-fold increase of gene expression of MMP-9 was seen in patients infected with H. pylori compared to uninfected individuals. CONCLUSIONS: Helicobacter pylori infection results in a substantial increase in MMP-9 and MMP-2 activity in the gastric mucosa, probably contributed to in large part by tissue-resident macrophages, while no changes were seen in the TIMP levels. The net increase in gastric MMP activity is likely to contribute to tissue damage during H. pylori-associated gastritis.     

Helicobacter pylori heat shock protein 60 mediates interleukin-6 production by macrophages via a toll-like receptor (TLR)-2-, TLR-4-, and myeloid differentiation factor 88-independent mechanism

Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which H. pylori induces IL-6 production in macrophages, however, has not been investigated. To identify the H. pylori factor responsible for this activity, we fractionated soluble proteins from H. pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as H. pylori heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of H. pylori HSP60, soluble protein extracts of H. pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with H. pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of H. pylori HSP60 responses from these signaling molecules. H. pylori HSP60-induced IL-6 mRNA expression, and NF-kappaB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by H. pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.     

Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction

Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.     

Does Helicobacter pylori infection per se cause gastric cancer or duodenal ulcer? Inadequate evidence in Mongolian gerbils and inbred mice

A role for Helicobacter pylori infection in the development of gastric cancer in humans is well established; however, evidence for its carcinogenicity in animals remains inadequate. Mongolian gerbils and mice are commonly used to investigate the carcinogenicity of H. pylori, yet it is unclear whether H. pylori infection per se causes gastric cancer or duodenal ulcers in these animal models. Gastric adenocarcinoma in the gerbils was reported over 10 years ago, but this species has proved an unreliable model for studying H. pylori infection-associated gastric cancer. Helicobacter pylori infection alone appears insufficient to induce gastric cancer in these animals; additional carcinogenic insult is required. The development of invasive adenocarcinoma in inbred mice is rare regardless of the mouse or bacterial strain, and many long-term studies have failed to induce gastric cancer in these animals. Helicobacter pylori infection is also an established causative factor for duodenal ulcer in humans. However, few studies have attempted to develop animal models of H. pylori infection-induced duodenal ulcer. We therefore conclude that both Mongolian gerbils and inbred mice may be inadequate models for studying H. pylori infection-associated gastric cancer and that there is no animal model of H. pylori infection-induced duodenal ulcer.     

Accumulation of 8-nitroguanine in human gastric epithelium induced by Helicobacter pylori infection

Helicobacter pylori infection causes chronic inflammation, which can lead to gastric carcinoma. A double immunofluorescence labeling study demonstrated that the level of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) apparent in gastric gland epithelium was significantly higher in gastritis patients with H. pylori infection than in those without infection. A significant accumulation of proliferating cell nuclear antigen, a prognostic factor for gastric cancer, was observed in gastric gland epithelial cells in patients with H. pylori infection as compared to those without infection, and its accumulation was closely correlated with the formation of 8-nitroguanine and 8-oxodG. These results suggest that nitrosative and oxidative DNA damage in gastric epithelial cells and their proliferation by H. pylori infection may lead to gastric carcinoma. 8-Nitroguanine could be not only a promising biomarker for inflammation but also a useful indicator of the risk of gastric cancer development in response to chronic H. pylori infection.     

Genome of Helicobacter pylori strain 908

Helicobacter pylori is a genetically diverse and coevolved pathogen inhabiting human gastric niches and leading to a spectrum of gastric diseases in susceptible populations. We describe the genome sequence of H. pylori 908, which was originally isolated from an African patient living in France who suffered with recrudescent duodenal ulcer disease. The strain was found to be phylogenetically related to H. pylori J99, and its comparative analysis revealed several specific genome features and novel insertion-deletion and substitution events. The genome sequence revealed several strain-specific deletions and/or gain of genes exclusively present in HP908 compared with different sequenced genomes already available in the public domain. Comparative and functional genomics of HP908 and its subclones will be important in understanding genomic plasticity and the capacity to colonize and persist in a changing host environment.     

Helicobacter pylori infection influences expression of genes related to angiogenesis and invasion in human gastric carcinoma cells

Infection with Helicobacter pylori (H. pylori) is considered a risk factor for gastric carcinoma. The purpose of this study was to clarify whether H. pylori infection plays a role in progression of gastric carcinoma. We examined the expression of genes encoding angiogenic factors and proteases by human gastric carcinoma cell lines (MKN-1 and TMK-1) co-cultured with or without H. pylori by cDNA microarray analysis. Co-culture with H. pylori increased expression of mRNAs encoding interleukin (IL)-8, vascular endothelial growth factor (VEGF), angiogenin, urokinase-type plasminogen activator (uPA), and metalloproteinase (MMP)-9 by gastric carcinoma cells. Up-regulation of these genes at the mRNA and protein levels was confirmed by Northern blot analysis, semi-quantitative RT-PCR analysis, and ELISA. In vitro angiogenic and collagenase activities of conditioned medium from the gastric carcinoma cells were also stimulated by co-culture with H. pylori. These results indicate that H. pylori infection may regulate angiogenesis and invasion of human gastric carcinoma.     

Cell-cycle inhibition by Helicobacter pylori L-asparaginase

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.     

Monocyte and Macrophage Killing of Helicobacter pylori: Relationship to Bacterial Virulence Factors

Background:  Helicobacter pylori infection is an important health problem, as it involves approximately 50% of the world's population, causes chronic inflammatory disease and increases the risk of gastric cancer development. H. pylori infection elicits a vigorous immune response, but this does not usually result in bacterial clearance. We have investigated whether the persistence of H. pylori in the host could be partly due to an inability of macrophages to kill this bacterium.
Materials and Methods:  Monocytes and macrophages isolated from the peripheral blood of normal human controls were infected in vitro with five H. pylori isolates. The isolates were characterized for known H. pylori virulence factors; vacuolating cytotoxin (VacA), the cag pathogenicity island ( cag PAI), urease, and catalase by Western blot and polymerase chain reaction analysis. The ability of primary human monocytes and macrophages to kill each of these H. pylori strains was then defined at various time points after cellular infection.
Results:  The five H. pylori strains showed contrasting patterns of the virulence factors. There were different rates of killing for the bacterial strains. Macrophages had less capacity than monocytes to kill three H. pylori strains. There appeared to be no correlation between the virulence factors studied and differential killing in monocytes.
Conclusions:  Primary human monocytes had a higher capacity to kill certain strains of H. pylori when compared to macrophages. The VacA, cag PAI, urease, and catalase virulence factors were not predictive of the capacity to avoid monocyte and macrophage killing, suggesting that other factors may be important in H. pylori intracellular pathogenicity.