Peter J. M. Ceponis

Helicobacter pylori Activates Toll-Like Receptor 4 Expression in Gastrointestinal Epithelial Cells

ABSTRACT Helicobacter pylori activates the transcription factor NF-κB, leading to proinflammatory cytokine production by gastric epithelial cells. However, the receptors for the initial bacterial interaction with host cells which activate downstream signaling events have not been completely defined. Recently, it has been shown that microbial components activate Toll-like receptors (TLRs), thereby leading to AP-1- and NF-κB-dependent transcription and resulting in the production of proinflammatory cytokines. The aim of this study was to determine whether H. pylori activates TLR4. Reverse transcription-PCR showed that both type I and type II H. pylori clinical isolates induced TLR4 mRNA expression in AGS cells compared with that by uninfected controls. H. pylori upregulated TLR4 protein expression in two gastric epithelial cell lines (AGS and MKN45) and one intestinal epithelial cell line (T84). Monoclonal TLR4 antibody inhibited lipopolysaccharide-induced interleukin-8 secretion from THP-1 macrophages but not from gastric epithelial cells infected with H. pylori. H. pylori demonstrated increased adherence to CHO TLR4-transfected cells compared with that to both CHO TLR2-transfected and nontransfected CHO cells (P < 0.01). These results indicate that H. pylori activates TLR4 expression in epithelial cells and that TLR4 can serve as a receptor for H. pylori binding.

Escherichia coli shiga-like toxins induce apoptosis and cleavage of poly(ADP-ribose) polymerase via in vitro activation of caspases

ABSTRACT Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% ± 1.8% and 81.7% ± 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% ± 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.

Helicobacter pylori Induces Apoptosis of Macrophages in Association with Alterations in the Mitochondrial Pathway

ABSTRACT Helicobacter pylori is a gastric bacterial pathogen that evades host immune responses in vivo and is associated with the development of gastritis, peptic ulcer disease, and gastric cancers. Induction of macrophage apoptosis is a method employed by multiple pathogens to escape host immune responses. Therefore, we hypothesized that H. pylori induces apoptosis of infected macrophages. RAW 264.7 cells were infected with H. pylori strain 60190, and apoptosis was assessed. Transmission electron microscopy and fluorescence microscopy showed that infected macrophages displayed morphological features characteristic of apoptosis. Quantification by acridine orange-ethidium bromide fluorescent-dye staining showed that apoptosis was dose and time dependent, and apoptosis was further confirmed by increased binding of annexin V-fluorescein isothiocyanate (FITC) to externalized phosphatidylserine of infected but not of control macrophages. Macrophages infected with isogenic mutants of H. pylori strain 60190 deficient in either cagA or vacA induced significantly less apoptosis than the parental strain, as assessed by increased binding of annexin V-FITC. Western blot analysis of whole-cell protein lysates revealed that infection with strain 60190 induced a time-dependent increase in cleavage of procaspase 8 and disappearance of full-length Bid compared with uninfected cells. Furthermore, pharmacological inhibition of caspase 8 caused a decrease in levels of apoptosis. Finally, infection caused a time-dependent increase in mitochondrial-membrane permeability and release of cytochrome c into the cytosol. These results suggest that H. pylori induces apoptosis of macrophages in association with alterations in the mitochondrial pathway. Elimination of this key immunomodulatory cell may represent a mechanism employed by the bacterium to evade host immune responses.

Enterohemorrhagic Escherichia coli O157:H7 Disrupts Stat1-Mediated Gamma Interferon Signal Transduction in Epithelial Cells

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a clinically important bacterial enteropathogen that manipulates a variety of host cell signal transduction cascades to establish infection. However, the effect of EHEC O157:H7 on Jak/Stat signaling is unknown. To define the effect of EHEC infection on epithelial gamma interferon (IFN-γ)-Stat1 signaling, human T84 and HEp-2 epithelial cells were infected with EHEC O157:H7 and then stimulated with recombinant human IFN-γ. Cells were also infected with different EHEC strains, heat-killed EHEC, enteropathogenic E. coli (EPEC) O127:H6, and the commensal strain E. coli HB101. Nuclear and whole-cell protein extracts were prepared and were assayed by an electrophoretic mobility shift assay (EMSA) and by Western blotting, respectively. Cells were also processed for immunofluorescence to detect the subcellular localization of Stat1. The EMSA revealed inducible, but not constitutive, Stat1 activation upon IFN-γ treatment of both cell lines. The EMSA also showed that 6 h of EHEC O157:H7 infection, but not 30 min of EHEC O157:H7 infection, prevented subsequent Stat1 DNA binding induced by IFN-γ, whereas infection with EPEC did not. Immunoblotting showed that infection with EHEC, but not infection with EPEC, eliminated IFN-γ-induced Stat1 tyrosine phosphorylation in both dose- and time-dependent fashions and disrupted inducible protein expression of the Stat1-dependent gene interferon regulatory factor 1. Immunofluorescence revealed that EHEC infection did not prevent nuclear accumulation of Stat1 after IFN-γ treatment. Also, Stat1 tyrosine phosphorylation was suppressed by different EHEC isolates, including intimin-, type III secretion- and plasmid-deficient strains, but not by HB101 and heat-killed EHEC. These findings indicate the novel disruption of host cell signaling caused by EHEC infection but not by EPEC infection.

Conditioned Medium from Enterohemorrhagic Escherichia coli-Infected T84 Cells Inhibits Signal Transducer and Activator of Transcription 1 Activation by Gamma Interferon

ABSTRACT Gamma interferon (IFN-γ) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-γ-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-γ and protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-γ receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-γ signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.

Helicobacter pylori Disrupts STAT1-Mediated Gamma Interferon-Induced Signal Transduction in Epithelial Cells

ABSTRACT Infection with Helicobacter pylori is chronic despite a vigorous mucosal immune response characterized by gastric T-helper type 1 cell expansion and gamma interferon (IFN-γ) production. IFN-γ signals by activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1); however, the effect of H. pylori infection on IFN-γ-STAT1 signaling is unknown. We infected human gastric (MKN45 and AGS) and laryngeal (HEp-2) epithelial cell lines with type 1 and type 2 H. pylori strains and then stimulated them with IFN-γ. Western blotting of whole-cell protein extracts revealed that infection with live, but not heat-killed, H. pylori time-dependently decreased IFN-γ-induced STAT1 tyrosine phosphorylation. Electrophoretic mobility shift assay of nuclear protein extracts demonstrated that H. pylori infection reduced IFN-γ-induced STAT1 DNA binding. STAT1 was unable to translocate from the cytoplasm to the nucleus in H. pylori-infected HEp-2 cells examined by immunofluorescence, and reverse transcription-PCR showed that IFN-γ-induced interferon regulatory factor 1 expression was inhibited. These effects were independent of the cagA, cagE, and VacA status of the infecting H. pylori strain. Furthermore, neither H. pylori culture supernatants nor conditioned medium from H. pylori-infected MKN45 cells inhibited IFN-γ-induced STAT1 tyrosine phosphorylation, suggesting that inhibition is independent of a soluble epithelial or bacterial factor but is dependent on bacterial contact with epithelial cells. H. pylori disruption of IFN-γ-STAT1 signaling in epithelial cells may represent a mechanism by which the bacterium modifies mucosal immune responses to promote its survival in the human host.

Helicobacter pylori Infection Interferes with Epithelial Stat6-Mediated Interleukin-4 Signal Transduction Independent of cagA, cagE, or VacA

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA+/cagE+/VacA+), LC20 (type 2; cagA−, cagE−, VacA−), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain α or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.

Helicobacter pylori infection induces interleukin-18 production in gastric epithelial (AGS) cells.

Helicobacter pylori colonization of the stomach results in a chronic–active gastritis characterized by mucosal infiltration of both neutrophils and lymphocytes. A T helper lymphocyte (Th1) profile predominates, which promotes the chronic and persistent inflammatory changes in the gastric mucosa in response to this bacterial pathogen. The cytokine interleukin-18 induces production of interferon-γ by activated T lymphocytes and promotes a Th1 profile. An in vitro model system was utilized to determine the role of interleukin-18 in response to infection of gastric epithelial cells by H. pylori. H. pylori isolates, characterized with respect to cagE and cagA and VacA status, were employed to infect AGS gastric epithelial cells. Interleukin-18 production was determined by immunoassay. Infection of AGS cells with H. pylori resulted in a 1.8-fold increase in interleukin-18 compared to uninfected cells (22.7 ± 2.4 vs. 12.7 ± 2.2 pg/ml; P&<0.005). This interleukin-18 response was independent of the cagE status of infecting strains (23.3 ± 1.9 vs. 26.3 ± 3.6 pg/ml; P = NS). Exposure of AGS cells to recombinant interleukin-18 resulted in dose-dependent and time-dependent secretion of interleukin-8 that was maximal following exposure to 100 pg/ml interleukin-18 for 24 hr (292 ± 5 pg/ml, versus 102 ± 14 pg/ml in unstimulated cells; P&<0.001). Interleukin-8 secretion was inhibited following pretreatment of cells with anti-interleukin-18 antibody and by pharmacological inhibition of the nuclear transcription factor, NF-κB. These findings demonstrate that interleukin-18 can enhance host chemokine response to H. pylori infection.

Epithelial cell signaling responses to enterohemorrhagic Escherichia coli infection

Enterohemorrhagic Escherichia coli, including the serotype O157:H7 that is most commonly identified with human disease, cause both sporadic cases and outbreaks of non-bloody diarrhea and hemorrhagic colitis. In about 10% of infected subjects, the hemolytic uremic syndrome (hemolytic anemic, thrombocytopenia, and acute renal failure) develops, likely as a consequence of systemic spread of bacterial-derived toxins variously referred to as Shiga-like toxin, Shiga toxin, and Verotoxin. Increasing evidence points to a complex interplay between bacterial products--for example, adhesins and toxins--and host signal transduction pathways in mediating responses to infection. Identification of critical signaling pathways could result in the development of novel strategies for intervention to both prevent and treat this microbial infection in humans.

Modulation of Host Cell Signal Transduction Pathways by Helicobacter Pylori Infection

Bacterial pathogens modulate host cell signal transduction responses to establish infection and cause disease. The purpose of the present summary, first presented at the Canadian Helicobacter Study Group meeting, is to discuss current knowledge of specific Helicobacter pylori factors, including the vacuolating cytotoxin, cytotoxin-associated gene A and the type four secretion system encoded by the cytotoxin-associated gene pathogenicity island and review the host cell signal transduction cascades that they modulate.