Determinants of Helicobacter pylori density in the human stomach and implications in disease

Abstract

Introduction and aims: Helicobacter pylori establishes a chronic infection in the stomach of almost half the world's population. A small proportion of patients go on to develop peptic ulcer disease and in some cases gastric cancer. Numerous factors influence the outcome of infection, including host, bacterial and environmental factors. The impact of bacterial colonisation density on the outcome of disease is poorly understood. The aim of this study was to investigate the determinants of bacterial density and the implications in disease. Methods: Gastric biopsies were collected from 50 H. pylori infected patients attending the Queen's Medical Centre, Nottingham for a routine upper GI endoscopy. Patient disease status was determined as having gastritis, duodenal ulcer disease (DUD), or gastric ulcer disease (GUD). Histological analysis for inflammation, activity, H. pylori density, atrophy and intestinal metaplasia were assessed according to the Sydney scoring system. Bacterial strains were cultured from gastric biopsies. Isolated bacterial DNA was used for genotyping strain virulence for CagA, VacA and DupA status. In order to determine accurate levels of colonisation in gastric tissue samples, a quantitative real-time PCR (qPCR) assay was developed based on the quantification of H. pylori 16S rRNA copy number, relative to the number of human GAPDH copy numbers. Quantification of IL8, ILIO, TGFß, FOXP3, h8, Dl and LL37 mRNA levels in gastric tissue were assessed by real-time RTPCR. The impact of host factors on bacterial density was assessed in vitro using H. pylori infected or uninfected AGS gastric epithelial cells, treated with recombinant cytokine. Colony forming units (CFUs) were enumerated in culture suspensions. The in vitro model was also used to examine the impact of cagA, cage, and vacA isogenic deletion mutant strains on bacterial density. Also, h, 8D1 expression levels were measured in culture suspensions, and IL-8 secretion levels in were assessed by ELISA. Results and conclusions: The pattern of colonisation in the gastric mucosa was found to be associated with the disease profile that develops, with an antral-predominant colonisation was associated with DUD, whereas corpus-predominant colonisation was associated with GUD. Bacterial factors that may drive the pattern of bacterial density in the stomach were then investigated, and strains expressing the dupA virulence factor were coincident with antral-predominant colonisation in the gastric mucosa. cagA and vacA were not found to be associated with bacterial density in vivo or in vitro. To assess the relationship between colonisation density and the host response, firstly IL8 mRNA expression levels were measured in gastric biopsies. An inverse relationship between bacterial load and expression of this pro-inflammatory cytokine was observed, but only at high IL8 expression levels and with higher H. pylori density. The inflammatory cytokines IL-10, TNFa, IL-17 and IL-22 did not impact CFU counts in vitro. No association was observed between bacterial load and expression levels of the immunosuppressive genes ILIO, TGFBI and FOXP3. However, increased CFU counts were found on treatment of H. pylori infected AGS cells with recombinant IL-10 and TGFß in vitro. Also, reduced expression of these immunosuppressive genes was associated with the presence of atrophy and intestinal metaplasia. In addition, ILIO and FOXP3 expression levels were negatively associated with mononuclear cell (MNC) infiltration. Reduced expression levels of the antimicrobial peptide (AMP) defensin hß, Dl were found with H. pylori infection in vivo and in vitro, and a negative association between hßDl expression and bacterial load was observed in vivo. A small increase in hßDl expression was observed in AGS cells infected with the cagPAI defective mutant strain in comparison to the wild type strain, suggesting a possible role for the CagPAI in modulating hßDl expression. In contrast, expression levels of the antimicrobial peptide LL37 were upregulated in the H. Mori infected gastric mucosa. In conclusion, the localisation of H. pylori in the gastric mucosa is associated with the disease profile that develops in the stomach. The patterns of colonisation observed may be influenced by the dupA virulence determinant. Host immune responses, were not associated with colonisation density. However, increased Treg response may be associated with protecting the host from the deleterious effects of inflammation. The direct modulation of bacterial load may be regulated by AMPs. In the H. pylori infected mucosa, reduced hßDl expression was observed which may assist persistent colonisation