Regulation of the Helicobacter pylori cellular receptor decay-accelerating factor

Abstract

Chronic gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulceration and distal gastric cancer, and adherence of H. pylori to gastric epithelial cells is critical for induction of inflammation. One H. pylori constituent that increases disease risk is the cag pathogenicity island, which encodes a secretion system that translocates bacterial effector molecules into host cells. Decay-accelerating factor (DAF) is a cellular receptor for H. pylori and a mediator of the inflammatory response to this pathogen. H. pylori induces DAF expression in human gastric epithelial cells; therefore, we sought to define the mechanism by which H. pylori up-regulates DAF and to extend these findings into a murine model of H. pylori-induced injury. Co-culture of MKN28 gastric epithelial cells with the wild-type H. pylori cag(+) strain J166 induced transcriptional expression of DAF, which was attenuated by disruption of a structural component of the cag secretion system (cagE). H. pylori-induced expression of DAF was dependent upon activation of the p38 mitogen-activated protein kinase pathway but not NF-kappaB. Hypergastrinemic INS-GAS mice infected with wild-type H. pylori demonstrated significantly increased DAF expression in gastric epithelium versus uninfected controls or mice infected with an H. pylori cagE(-) isogenic mutant strain. These results indicate that H. pylori cag(+) strains induce up-regulation of a cognate cellular receptor in vitro and in vivo in a cag-dependent manner, representing the first evidence of regulation of an H. pylori host receptor by the cag pathogenicity island.

FIGURE 1.

H. pylori induces the transcriptional up-regulation of DAF in gastric epithelial cells. MKN28 gastric epithelial cells were pretreated with either actinomycin D (ActD) or cycloheximide (CHX) at the indicated concentrations and then co-cultured with the H. pylori cag⁺ strain J166 for 24 h at an m.o.i. = 100. A, Western blot analysis was performed using an anti-DAF antibody as described under “Experimental Procedures.” -, cells incubated with medium alone. A representative blot is shown. Anti-GAPDH blots served as normalization controls. B, densitometry represents data from three independent experiments. Error bars, S.E. ^*, p < 0.001 J166 versus control cells or ActD-treated cells. §, p < 0.01 J166 versus cycloheximide-treated cells.

FIGURE 2.

H. pylori induction of daf requires viable bacteria. MKN28 cells were incubated with medium alone (-) or live, heat-killed, or sonicates of H. pylori strain J166 for 2 h. Levels of daf mRNA were determined by real-time qRT-PCR as described under “Experimental Procedures” and normalized to corresponding levels of gapdh mRNA. Results are expressed as -fold increase in daf mRNA in H. pylori-infected versus uninfected samples. Error bars, S.E. ^*, p < 0.001 versus uninfected cells.

FIGURE 3.

DAF induction is mediated by a functional cag secretion system but not CagA. MKN28 cells were co-cultured with wild-type (WT) H. pylori strain J166 or isogenic cagA^- or cagE^- mutants at an m.o.i. = 100. A, levels of daf mRNA were determined by real-time qRT-PCR after 2 h of co-culture and were normalized to corresponding levels of gapdh mRNA. Results are expressed as -fold increase in daf mRNA in H. pylori-infected versus uninfected samples. Error bars, S.E. ^*, p < 0.05 versus uninfected cells. B, cell extracts were used for Western blot analysis using an anti-DAF antibody. A representative blot of multiple repetitions performed on three occasions is shown. Anti-GAPDH blots served as normalization controls.

FIGURE 4.

NF-κB is not required for H. pylori induction of daf. MKN28 cells were transiently transfected with a NF-κB-responsive luciferase reporter construct and either a dominant negative-IκBα (dnIκBα) or a dominant negative IκB kinase β (dnIKKβ) expression construct. Cells were then co-cultured with H. pylori strain J166 at a m.o.i. = 100. A, NF-κB-driven firefly luciferase activity was assayed on a luminometer after 6 h of co-culture and normalized to Renilla luciferase activity. Error bars, S.E. ^*, p < 0.001 versus uninfected cells. B, levels of daf mRNA were determined by real-time qRT-PCR after 2 h of co-culture with the H. pylori strain J166 and were normalized to corresponding levels of gapdh mRNA. Results are expressed as -fold increase in daf mRNA in H. pylori-infected versus uninfected samples. Error bars, S.E. ^*, p < 0.05 versus uninfected cells.

FIGURE 5.

Activation of p38 is required for daf up-regulation by H. pylori. MKN28 cells were pretreated with pharmacological inhibitors of MEK1/2 (MEKi, PD98095, 50 μm), p38 (SB203580, 10 μm), JNK (JNK inhibitor II (JNKi), 10 μm), or vehicle control (DMSO) (-) for 30 min and then co-cultured with the H. pylori strain J166 at a m.o.i. = 100. Levels of daf mRNA were determined by real-time qRT-PCR after 2 h of co-culture and were normalized to corresponding levels of gapdh mRNA. Results are expressed as -fold increase in daf mRNA in H. pylori-infected versus uninfected samples. Error bars, S.E. ^*, p < 0.05 versus uninfected cells.

FIGURE 6.

Inactivation of a component of the cag secretion system attenuates H. pylori induction of DAF in vivo. A, INS-GAS mice were challenged with Brucella broth (BB) control, wild-type (WT) H. pylori cag⁺ strain 7.13, or an isogenic 7.13 cagE^- mutant for 4, 12, or 24 weeks. A, immunohistochemical staining of DAF was performed and scored on an ordinal scale from 0 to 4 by a single pathologist. Error bars, S.E. ^*, p < 0.05 WT 7.13 versus Brucella broth or cagE^-. B–D, representative DAF IHC-stained sections from mice challenged for 12 weeks with wild-type H. pylori strain 7.13 (B), 7.13 cagE^- isogenic mutant (C), or Brucella broth alone (D). Magnification, 20×.

FIGURE 7.

H. pylori-induced expression of DAF is mediated by the cag secretion system in vivo. A–C, gastric epithelial cells were harvested from frozen tissue obtained 12 weeks post-challenge from INS-GAS mice challenged with Brucella broth (n = 5), wild-type H. pylori strain 7.13 (n = 6), or an isogenic 7.13 cagE^- mutant (n = 4), stained with phycoerythrin (PE)-conjugated anti-DAF antibodies and fluorescein isothiocyanate (FITC)-conjugated anti-cytokeratin antibodies, and then used for flow cytometric analysis. A, representative dot plots of DAF and cytokeratin expression demonstrate increased DAF expression on gastric epithelial cells from mice challenged with wild-type (WT) H. pylori strain 7.13 compared with either Brucella broth (BB) alone or a cagE^- isogenic mutant. B, representative histogram of DAF expression on gastric epithelial cells shows an increase in DAF fluorescence as a shift in fluorescence profile to the right in cells infected with wild type strain 7.13 versus uninfected cells. C, epithelial expression of DAF as determined by flow cytometry was significantly higher in mice infected with wild-type H. pylori (n = 6) when compared with mice challenged with either the cagE^- isogenic mutant (n = 4) or Brucella broth control (n = 5). Error bars, S.E. ^**, p < 0.01 versus uninfected cells. D, total RNA was isolated from frozen gastric tissue, and daf mRNA expression was analyzed by real-time RT-PCR. daf mRNA expression was significantly increased in mice infected with wild-type H. pylori strain 7.13 (n = 5) versus mice challenged with the cagE^- isogenic mutant (n = 4) or Brucella broth alone (n = 5). Error bars, S.E. ^*, p < 0.05 versus uninfected cells.