Interleukin-8 is the single most up-regulated gene in whole genome profiling of H. pylori exposed gastric epithelial cells

Abstract

Background: The association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA⁺ and OMPLA⁻ H. pylori variants.

Results: Interleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the epithelial cell response to H. pylori infection and in the pathological processes leading to gastric disease. MAPK and NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response. There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation. We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory epithelial cell gene response in this experiment.

Conclusion: In whole genome analysis of the epithelial response to H. pylori exposure, IL-8 demonstrated the most marked up-regulation, and was involved in many of the most important cellular response processes to the infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24 h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA⁺/⁻ did not affect the acute inflammatory response to H. pylori.

Figure 1

Immunofluorescence images of AGS cells exposed to H. pylori. AGS cells were non-exposed, or exposed to OMPLA⁺ and OMPLA^- H. pylori at a MOI of 300:1 and co-cultured for 3 and 6 h. The bacteria were stained with rabbit anti-Helicobacter antibody. Images were captured by fluorescent microscopy.

Figure 2

Venn diagrams of significantly regulated genes. Venn diagrams of differentially expressed genes of H. pylori-infected AGS cells compared to control cells (p < 0.05). The intersecting circles indicate overlapping genes at the indicated time points. AGS = non-infected control AGS cells.

Figure 3

Hiarchical clustering of the most significantly differentially regulated genes. Hiarchical clustering of significantly differentially regulated genes (log₂FC > 1.5, p < 0.05). Arrow points at IL-8.

Figure 4

Time-course of IL-8 mRNA expression in AGS cells co-cultured with H. pylori. Quantitative PCR analysis of IL-8 expression in H. pylori-infected AGS cells at six different sampling points over 24 h. Data points are the values of three cell culture replicates from two independent experiments, A and B. Lines represent the calculated mean within each of the experiments.

Figure 5

Time-course of IL-8 protein expression in AGS cells co-cultured with H. pylori. ELISA analysis of IL-8 protein expression in H. pylori-infected AGS cells at six different sampling points over 24 h. Data points are the values of three cell culture replicates from two independent experiments, A and B. Lines represent the calculated mean within each of the experiments.

Figure 6

Dose-response of IL-8 mRNA expression in AGS cells co-cultured with H. pylori. Quantitative PCR analysis of IL-8 expression in H. pylori-infected AGS cells, co-incubated for 3 h. Data points are the values of three cell culture replicates from two independent experiments, A and B. Lines represent the calculated mean within each of the experiments.