No abstract

Helicobacter pylori activates the transcription factor NF-B, 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-B-dependent transcription and resulting in the production of proinflammatory cytokines. Helicobacter pylori is a gram-negative bacterium that plays an etiologic role in the development of gastritis, peptic ulceration, and gastric adenocarcinoma (2). Several bacterial factors are proposed to play a role in disease pathogenesis. Type I H. pylori strains contain a pathogenicity island, which carries a number of virulence factors, including cagA and cagE (7), and is associated with more severe gastroduodenal disease (2). Studies using isogenic mutants demonstrate that certain genes carried on the cag pathogenicity island, including cagE but not cagA, are responsible for nuclear factor-B (NF-B) activation resulting in the transcription of a number of proinflammatory genes such as interleukin-8 (IL-8), IL-1␤, gamma interferon, and tumor necrosis factor alpha (20,29). However, the eukaryotic receptors involved in H. pylori activation of the innate immune response have not been clearly defined.Toll-like receptors (TLRs) play a crucial role in host innate and adaptive immune responses to microbial pathogens and their products (1). TLRs have leucine-rich motifs in their extracellular domains similar to those of other pattern-recognition proteins that promote ligand binding (1). TLR proteins also contain a cytoplasmic tail that is homologous to the IL-1 and IL-18 receptor and hence can trigger intracellular signaling pathways (23). To date, 10 TLRs have been described (31), with TLR2 and TLR4 the two best characterized. TLR2 responds to peptidoglycan, lipoteichoic acid (24), and bacterial lipoproteins (19). TLR4 is activated by the lipopolysaccharide (LPS) of gram-negative bacteria (3). Recently, it has been demonstrated that TLR2 and TLR4 are expressed on human intestinal epithelial cell lines (4, 5) and that Escherichia coli (O26:B6)-derived LPS induces TLR4 trafficking in epithelial cells (3). Maeda et al. (20) showed that TLR4 mRNA is also expressed on gastric epithelial MKN45 cells. In contrast to macrophages, TLR4 is not involved in H. pylori-induced NF-B activation in gastric epithelia (21). Therefore, the precise function(s) of TLR4 in gastric epithelial cells is still not known. MATERIALS AND METHODSReagents. H. pylori-derived LPS was kindly provided by Mario Monteiro (Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada). Polyclonal anti-TLR4 and anti-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.), and monoclonal anti-TLR4 (HTA125) antibody was purchased from eBioscience (San Diego, Calif.). Polyclonal H. pylori immune serum was purchased from DAKO (...

Interleukins 4 and 13 can affect their target cells by activation of signal transducer and activator of transcription 6 (STAT 6) or phosphatidylinositol 3-kinase (PI3K). We examined the signal transduction events involved in IL-4 and IL-13 regulation of epithelial paracellular permeability using T84 cells, a model human colonic epithelium. T84 cells treated with IL-4 or IL-13 displayed virtually identical dose-and time-dependent STAT 6 activation as assessed by electrophoretic mobility shift assay (EMSA) and decreases in transepithelial resistance (TER). STAT 6 DNA binding activity was maximal in nuclear extracts 30 min after exposure to IL-4 or IL-13, and TER was maximally reduced by 24 h posttreatment. Pretreatment of epithelia with transcription factor decoys (phosphorothioated DNA oligonucleotides containing the STAT 6 binding site) dramatically reduced STAT 6 activation as detected by EMSA, but did not attenuate the TER reduction by IL-4 or IL-13. In contrast, although the PI3K inhibitors wortmannin and LY294002 did not affect IL-4 or IL-13 STAT 6 activation, they significantly inhibited the ability of either cytokine to lower TER. Thus, we provide evidence for PI3K as the major proximal signaling event in IL-4 and IL-13 regulation of TER and speculate that pharmacological targeting of enterocytic PI3K activity may represent a means to manipulate epithelial permeability.

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-␥)-Stat1 signaling, human T84 and HEp-2 epithelial cells were infected with EHEC O157:H7 and then stimulated with recombinant human IFN-␥. 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-␥ 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-␥, whereas infection with EPEC did not. Immunoblotting showed that infection with EHEC, but not infection with EPEC, eliminated IFN-␥-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-␥ 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.Infection of humans with the bacterial enteropathogen enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 causes outbreaks of hemorrhagic colitis and hemolytic uremic syndrome; the latter is related to production of Shiga-like toxins (5) and is an important cause of acute kidney failure (5, 54). The related pathogen enteropathogenic E. coli (EPEC) is an important cause of prolonged watery diarrhea in infants in underdeveloped countries (5,11,51). Once ingested, these bacteria establish infection by manipulating host epithelial cell signal transduction cascades (5, 11, 51). For example, activation of phospholipase C␥, phosphoinositide 3-kinase, and 5-lipoxygenase (20) and mobilization of the second messengers inositol trisphosphate and calcium (6) are important for forming characteristic attaching and effacing lesions and rearrangements of the host cell cytoskeleton. However, these bacteria disrupt intestinal epithelial barrier function by manipulation of differential signal transduction cascades; EHEC does this via protein kinase C and the myosin light-chain kinase (38), and EPEC does it via a myosin light-chain kinase-dependent, protein kinase C-in...

Shiga-like toxin-producing

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 (

Gamma interferon (IFN-␥

. Interleukin-4-and -13-induced hypercontractility of human intestinal muscle cells-implication for motility changes in Crohn's disease.