Helicobacter pylori: reduced phagocytic killing and altered phagosome maturation in primary human macrophages.

Abstract

Helicobacter pylori (H. pylori) colonises the human gastric mucosa and is the principal causative agent of gastric and duodenal ulcers. Long term infection with H. pylori represents a major risk for the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. It is estimated that half of the world’s population is infected with H. pylori with rates of infection up to 90% in the developing world. Despite eliciting a vigorous and sustained immune response in the host, H. pylori is able to persist in the gastric mucosa for life. In this study we have developed an in vitro infection model to (1) investigate the ability of primary human monocytes and macrophages to effectively kill H. pylori and (2) examine the process of H. pylori phagosome maturation in infected macrophages. Five H. pylori strains were selected on the basis of their clinical phenotype and characterised for the VacA (vacuolating cytotoxin), cagPAI (cag Pathogenicity Island), urease and catalase virulence factors by Western blot and PCR analysis. Each strain possessed a unique combination of virulence factors and there was only limited correlation between molecular typing results and clinical phenotype. These five H. pylori strains were then used to individually infect in vitro cultures of primary human monocytes and macrophages. At various time points after infection, the infected monocytes and macrophages were lysed and the remaining viable bacteria were counted to determine phagocytic killing efficacy. Primary human monocytes had a higher capacity to kill certain strains of H. pylori when compared to macrophages. Three of the H. pylori strains were killed by monocytes after 48 hours whereas none of the H. pylori strains were killed by macrophages over the same time. There appeared to be no correlation between the virulence factors studied and differential killing in monocytes. The virulence factors studied were not predictive of the capacity for H. pylori to avoid monocyte and macrophage killing. The process of H. pylori phagosome maturation was then investigated using the same in vitro infection model. Macrophages were infected with H. pylori and the amount of early endosome (Rab5 and EEA1), late endosome (Rab7 and CD63) and lysosome (LAMP-1 and LAMP-2) markers that co-localised with phagosomes was determined over a four hour time course. There was a dramatic change in the kinetics of phagosome maturation between H. pylori phagosomes and control E. coli phagosomes and it was proposed that this could contribute to the reduced killing of H. pylori observed in macrophages. H. pylori phagosomes retained the characteristics of early and late endosomes despite gaining lysosome markers. This demonstrated a fundamental change in phagosome maturation whereupon the H. pylori phagosome underwent normal fusion with the elements of the endocytic network, but blocked the subsequent fission part of the interaction. Macrophages are the critical regulatory component of the innate and adaptive immune responses in the stomach. Restoring the normal process of phagosome maturation in H. pylori infection may realise a strategy, enabling the effective killing of H. pylori. Reinstating the efficacy of the immune response generated by H. pylori to ultimately clear an H. pylori infection would have enormous benefits, particularly in the developing world where H. pylori infection has a very high prevalence.