The active form of Helicobacter pylori vacuolating cytotoxin induces decay-accelerating factor CD55 in association with intestinal metaplasia in the human gastric mucosa

Abstract

High-level expression of decay-accelerating factor, CD55, has previously been found in human gastric cancer (GC) and intestinal metaplasia (IM) tissues. Therapeutic effects of CD55 inhibition in cancer have been reported. However, the role of Helicobacter pylori infection and virulence factors in the induction of CD55 and its association with histological changes of the human gastric mucosa remain incompletely understood. We hypothesised that CD55 would be increased during infection with more virulent strains of H. pylori, and with more marked gastric mucosal pathology. RT-qPCR and immunohistochemical analyses of gastric biopsy samples from 42 H. pylori-infected and 42 uninfected patients revealed that CD55 mRNA and protein were significantly higher in the gastric antrum of H. pylori-infected patients, and this was associated with the presence of IM, but not atrophy, or inflammation. Increased gastric CD55 and IM were both linked with colonisation by vacA i1-type strains independently of cagA status, and in vitro studies using isogenic mutants of vacA confirmed the ability of VacA to induce CD55 and sCD55 in gastric epithelial cell lines. siRNA experiments to investigate the function of H. pylori-induced CD55 showed that CD55 knockdown in gastric epithelial cells partially reduced IL-8 secretion in response to H. pylori, but this was not due to modulation of bacterial adhesion or cytotoxicity. Finally, plasma samples taken from the same patients were analysed for the soluble form of CD55 (sCD55) by ELISA. sCD55 levels were not influenced by IM and did not correlate with gastric CD55 mRNA levels. These results suggest a new link between active vacA i1-type H. pylori, IM, and CD55, and identify CD55 as a molecule of potential interest in the management of IM as well as GC treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: CD55; Helicobacter pylori; decay-accelerating factor; gastric cancer; intestinal metaplasia; vacuolating cytotoxin; virulence factor.

Figure 1

Infection with Helicobacter pylori increases CD55 mRNA in human gastric antrum. The relative levels of CD55 mRNA in gastric mucosal tissues from 42 infected and 42 uninfected patients were analysed by RT‐qPCR. Data were normalised to ACTB mRNA and expressed relative to pooled RNA from antral tissue samples from uninfected patients. Levels were compared between (A) uninfected and infected patients in the antrum and corpus; (B) infected patients with and without atrophy or IM in the antrum; (C) the antrum of infected patients with varying IM score; and (D) varying infiltration scores of neutrophils and MNCs in infected antral samples. Each dot represents an individual patient's data. The horizontal line in each box represents the median value, with the boxes representing the interquartile range. Lines extend from the box to the highest and lowest values. P values were calculated using (A) two‐way ANOVA with Tukey's post hoc test and (B–D) Kruskal–Wallis with Dunn's post hoc test. AG, atrophic gastritis; Hp, H. pylori; IM, intestinal metaplasia; MNCs, mononuclear cells; Mod, moderate; Mar, marked.

Figure 2

CD55 in H. pylori‐infected gastric epithelium is associated with intestinal metaplasia but not inflammation. Gastric antrum biopsy tissues from 36 infected and 11 uninfected patients were stained immunohistochemically for CD55. (A) Representative images of stained sections. (B) Each section was scored semi‐quantitatively based on the distribution and intensity of mucosal staining. (C, D) CD55 IHC scores of infected epithelium samples were stratified by IM score and the amount of neutrophil and MNC infiltration. Each dot represents an individual patient's data. The horizontal line in each box represents the median value, with the boxes representing the interquartile range. Lines extend from the box to the highest and lowest values. P values were calculated using (B) two‐way ANOVA with Tukey's post hoc test and (C, D) Kruskal–Wallis with Dunn's post hoc test. AG, atrophic gastritis; Hp, H. pylori; IM, intestinal metaplasia; MNCs, mononuclear cells; Mod, moderate; Mar, marked.

Figure 3

Gastric expression of CD55 and intestinal metaplasia are associated with H. pylori strains of vacA i1 genotype. (A) CD55 mRNA; (B) mucosal CD55 IHC scores; and Sydney System scores (0, none; 1, mild; 2, moderate; and 3, marked) for (C) IM and (D) neutrophil infiltration, in the gastric antrum of 37 patients infected with H. pylori strains of different virulence genotypes (cagA ⁺, n = 27; cagA ⁻ , n = 10; vacA i1, n = 25; vacA i2, n = 12). Each dot represents an individual patient's data. The horizontal line in each box represents the median value, with the boxes representing the interquartile range. Lines extend from the box to the highest and lowest values. P values were calculated using a Mann–Whitney U‐test. IM, intestinal metaplasia.

Figure 4

Increased CD55 expression and secretion by H. pylori are mediated via vacA and cagT4SS. (A, B) AGS or MKN28 cells were co‐cultured with H. pylori strain 60190 or isogenic mutants for 24 h at an MOI of 20. (C, D) AGS or MKN28 cells were incubated for 24 h in the presence of 100 μg/ml total protein of water extracts of H. pylori strain 60190 or isogenic vacA ⁻ mutant. (A, C) sCD55 concentrations in culture supernatants were determined by ELISA. Mean ± SEM from three or four independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one‐way ANOVA with Tukey's post hoc test. (B, D) Western blot of CD55 in cell lysates. wt, wild type.

Figure 5

CD55 knockdown reduces IL‐8 production but has no effect on H. pylori binding to gastric epithelial cells. AGS or MKN28 cells transfected with CD55 siRNA (siCD55) or non‐targeting control (siNT) were co‐cultured with H. pylori strain 60190. (A) Representative images of cell morphology 24 h post‐infection. (B) Cell viability 24 h post‐infection as measured using an MTT assay. (C) IL‐8 concentrations in cell culture supernatants determined by ELISA 24 h post‐infection. (D) Adherence of H. pylori to epithelial cells at 4 and 24 h post‐infection. Mean ± SEM from four independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by two‐way ANOVA with Tukey's post hoc test.

Figure 6

Comparison of plasma sCD55 levels in patients with gastric atrophy and intestinal metaplasia. (A) sCD55 concentrations in plasma samples from 39 infected and 38 uninfected patients were quantified by ELISA. (B) sCD55 concentrations in infected patients were stratified by Operative Link on Gastritis (OLGA) and Operative Link on Gastritis based on IM (OLGIM) stage. Each dot represents an individual patient's data. The horizontal line in each box represents the median value, with the boxes representing the interquartile range. Lines extend from the box to the highest and lowest values. Hp, H. pylori; AG, atrophic gastritis; IM, intestinal metaplasia.