Ells were co-cultured with heat-killed (HK) H. Docosahexaenoyl ethanolamide supplier pylori or viable wildtype H. pylori added to a transwell (TW) system that separates bacteria from gastric epithelial cells, respectively (Figure 2D). Heat-killed H. pylori, but not viable H. pylori within the transwell, induced KLF5 expression, similar to co-culture with the wild-type H. pylori strain 60190. To assess whether the highly conserved H. pylori cell wall component, lipopolysaccharide (LPS), altered KLF5 expression levels, gastric epithelial cells were treated with physiologic concentrations of purified H. pylori LPS. Treatment of cells with either 10 ng/ml or 100 ng/ml H. pylori LPS did not alter KLF5 expression compared to uninfected controls (Figure 2E).H. pylori induces inflammation and KLF5 expression in a cag-independent manner within gastric epithelium in vivoTo extend our in vitro results into an in vivo model of H. pylori infection, C57BL/6 mice were challenged with Brucella broth as a negative uninfected (UI) control, wild-type mouse-adapted cag+ H. pylori strain PMSS1, or a PMSS1 cagE2 isogenic mutant for 4 or 8 weeks (Figure 3A). Colonization efficiency, defined as the percentage of successfully colonized mice, was 100 for wildtype PMSS1 and the cagE2 isogenic mutant at all time points (data not shown). As expected, infection with H. pylori strain PMSS1 resulted in Clavulanate (potassium) significantly increased inflammation within the gastricFigure 3. H. pylori induces inflammation in an in vivo C57BL/6 murine model. (A) C57BL/6 mice were challenged with Brucella broth, as an uninfected (UI) negative control, with the mouse-adapted wildtype cag+ H. pylori strain PMSS1, or a PMSS1 cagE2 isogenic mutant for 4 or 8 weeks. (B and C) A single pathologist, blinded to treatment groups, assessed and scored inflammation at 4 weeks (B) and 8 weeks (C). Acute and chronic inflammation in both the antrum and corpus was scored on a scale of 0?, leading to a possible maximum score of 12. Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE2infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups. doi:10.1371/journal.pone.0054344.gmucosa compared to uninfected controls while levels of inflammation following infection with the cagE2 isogenic mutant were no different than controls (Figure 3B and 3C). To assess upregulation of KLF5 in this model, immunohistochemistry (IHC) was performed. KLF5 immunostaining was maximal within the gastric epithelial proliferative zone (isthmusKLF5 and H. Pylori-Mediated Gastric Carcinogenesiszone) within the 24195657 antrum of uninfected mice (Figure 4A). The intensity and magnitude of KLF5 immunostaining increased significantly in H. pylori-infected gastric epithelial cells, compared to uninfected gastric tissue, and expression expanded beyond the isthmal zone to include nearly all cells (Figure 4B, 4C, 4D, and 4E). Expression of KLF5 was predominantly nuclear, consistent with its function as a transcription factor; however, significant cytoplasmic staining was also evident in infected epithelial cells. Despite differences in levels of gastric inflammation in miceinfected with PMSS1 versus the cagE2 isogenic mutant (Figure 3B), both strains induced significantly increased levels of KLF5 compared to uninfected controls (Figure 4A ). These findings are consistent with our in vitro data de.Ells were co-cultured with heat-killed (HK) H. pylori or viable wildtype H. pylori added to a transwell (TW) system that separates bacteria from gastric epithelial cells, respectively (Figure 2D). Heat-killed H. pylori, but not viable H. pylori within the transwell, induced KLF5 expression, similar to co-culture with the wild-type H. pylori strain 60190. To assess whether the highly conserved H. pylori cell wall component, lipopolysaccharide (LPS), altered KLF5 expression levels, gastric epithelial cells were treated with physiologic concentrations of purified H. pylori LPS. Treatment of cells with either 10 ng/ml or 100 ng/ml H. pylori LPS did not alter KLF5 expression compared to uninfected controls (Figure 2E).H. pylori induces inflammation and KLF5 expression in a cag-independent manner within gastric epithelium in vivoTo extend our in vitro results into an in vivo model of H. pylori infection, C57BL/6 mice were challenged with Brucella broth as a negative uninfected (UI) control, wild-type mouse-adapted cag+ H. pylori strain PMSS1, or a PMSS1 cagE2 isogenic mutant for 4 or 8 weeks (Figure 3A). Colonization efficiency, defined as the percentage of successfully colonized mice, was 100 for wildtype PMSS1 and the cagE2 isogenic mutant at all time points (data not shown). As expected, infection with H. pylori strain PMSS1 resulted in significantly increased inflammation within the gastricFigure 3. H. pylori induces inflammation in an in vivo C57BL/6 murine model. (A) C57BL/6 mice were challenged with Brucella broth, as an uninfected (UI) negative control, with the mouse-adapted wildtype cag+ H. pylori strain PMSS1, or a PMSS1 cagE2 isogenic mutant for 4 or 8 weeks. (B and C) A single pathologist, blinded to treatment groups, assessed and scored inflammation at 4 weeks (B) and 8 weeks (C). Acute and chronic inflammation in both the antrum and corpus was scored on a scale of 0?, leading to a possible maximum score of 12. Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE2infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups. doi:10.1371/journal.pone.0054344.gmucosa compared to uninfected controls while levels of inflammation following infection with the cagE2 isogenic mutant were no different than controls (Figure 3B and 3C). To assess upregulation of KLF5 in this model, immunohistochemistry (IHC) was performed. KLF5 immunostaining was maximal within the gastric epithelial proliferative zone (isthmusKLF5 and H. Pylori-Mediated Gastric Carcinogenesiszone) within the 24195657 antrum of uninfected mice (Figure 4A). The intensity and magnitude of KLF5 immunostaining increased significantly in H. pylori-infected gastric epithelial cells, compared to uninfected gastric tissue, and expression expanded beyond the isthmal zone to include nearly all cells (Figure 4B, 4C, 4D, and 4E). Expression of KLF5 was predominantly nuclear, consistent with its function as a transcription factor; however, significant cytoplasmic staining was also evident in infected epithelial cells. Despite differences in levels of gastric inflammation in miceinfected with PMSS1 versus the cagE2 isogenic mutant (Figure 3B), both strains induced significantly increased levels of KLF5 compared to uninfected controls (Figure 4A ). These findings are consistent with our in vitro data de.
