Transcriptional profiling of gastric epithelial cells infected with wild type or arginase-deficient Helicobacter pylori

Abstract

Background: Helicobacter pylori causes acute and chronic gastric inflammation induced by proinflammatory cytokines and chemokines secreted by cells of the gastric mucosa, including gastric epithelial cells. Previous studies have demonstrated that the bacterial arginase, RocF, is involved in inhibiting T cell proliferation and CD3ζ expression, suggesting that arginase could be involved in a more general dampening of the immune response, perhaps by down-regulation of certain pro-inflammatory mediators.

Results: Global transcriptome analysis was performed on AGS gastric epithelial cells infected for 16 hours with a wild type Helicobacter pylori strain 26695, an arginase mutant (rocF-) or a rocF+ complemented strain. H. pylori infection triggered altered host gene expression in genes involved in cell movement, death/growth/proliferation, and cellular function and maintenance. While the wild type strain stimulates host inflammatory pathways, the rocF- mutant induced significantly more expression of IL-8. The results of the microarray were verified using real-time PCR, and the differential levels of protein expression were confirmed by ELISA and Bioplex analysis. MIP-1B was also significantly secreted by AGS cells after H. pylori rocF- mutant infection, as determined by Bioplex. Even though not explored in this manuscript, the impact that the results presented here may have on the development of gastritis, warrant further research to understand the underlying mechanisms of the relationship between H. pylori RocF and IL-8 induction.

Conclusions: We conclude that H. pylori arginase modulates multiple host signaling and metabolic pathways of infected gastric epithelial cells. Arginase may play a critical role in anti-inflammatory host responses that could contribute to the ability of H. pylori to establish chronic infections.

Figure 1

Differential gene expression profiles of AGS gastric epithelial cells infected with WT,rocF-and therocF⁺complementedH. pyloristrains. A. Representative portion of the Log10 ratio between the net expression values between the infected and the non-infected cells, as described in Materials and Methods. The analysis was done using four replicates of each treatment. The marked areas above the heat map show genes associated with different cellular functions. B. Venn diagram showing the number of genes affected (up- and down-regulated) by the infection of AGS cells with the WT, rocF-, or rocF + strains of H. pylori. The green number (262) indicates the number of genes that are common to all treatments; the black numbers indicate unique genes in each treatment; the total shaded area represent 583 genes that are neither common nor unique (similar genes).

Figure 2

Network interactions in AGS cells infected withH. pylori. A. Expanded central node of a network (RelA (p65), NFkB, c-IAP2, NFkBIA, and MUC1) generated using the net gene expression values of the different H. pylori infections of the AGS cells. Green arrow = positive regulation; green icons represent receptor ligands (IL-8, VEGFA); red icons represent transcription factors (NFKB1, STAT3); yellow icon represent generic enzyme (p300). Thicker arrows indicate stronger association. B. Heatmap showing the similarity of the different replicates, using the Log10 ratio of the expression values, as explained in Figure 1. Both Figures were generated using data from four replicate independent experiments.

Figure 3

Infection with theH. pylori26695rocF-mutant induces significantly higher levels ofIL-8than its wild type orrocF⁺counterparts. Fold induction of genes depicted in Figure 2, performed as explained in Materials and Methods using GAPDH as housekeeping gene and one internal calibrator. * p < 0.0001, as compared to the induction in response to the infection with H. pylori rocF-. Values represent the average expression ± SEM of three independent replicates.

Figure 4

TheH. pylori rocF-mutant induces more IL-8 and MIP-1βin AGS cells than wild typeH. pylori,as determined by Bioplex. Supernatants from H. pylori infected-AGS cells were collected and used to determine the concentration of IL-8 and MIP-1β (pg/ml) A. Levels of IL-8; one-way ANOVA p < 0.0001; *p = 0.0001 (rocF- vs NS); #p = 0.0249 (rocF- vs WT); **p = 0.044 (rocF- vs rocF⁺); B. Levels of MIP-1B; one-way ANOVA p < 0.0001; *p < 0.0001 (rocF- vs NS); #p < 0.0001 (rocF- vs WT); p = 0.0001 (rocF- vs rocF⁺). Values in both Figures represent the average signal ± SEM of four independent replicates.

Figure 5

TherocFmutant ofH. pyloriinduces more IL-8 in AGS cells compared with wild typeH. pylori,as determined byELISAanalysis. Please see legend on Figure 4 for IL-8. One-way ANOVA p = 0.0002; *p = 0.0003 (rocF- vs NS); #p = 0.045 (rocF- vs WT); **p = 0.0185 (rocF- vs rocF⁺). Values represent the average signal ± SEM of four independent replicates.