Introduction Helicobacter pylori (Hp) infection may result in peptic ulcers or gastric cancer. Disease risk is associated with more virulent strains, such as those with the cag pathogenicity island (cagPAI), which induce higher levels of gastritis. Disease is also associated with an insufficient anti-inflammatory regulatory T-cell (Treg) response to Hp. Preliminary studies of circulating T-cell adhesion molecule expression highlighted an increased frequency of CCR6+ Tregs in the blood of Hp+ patients. This chemokine receptor is expressed by both T-helper 17 (Th17) cells and Tregs. It has been demonstrated that the Hp-infected human gastric mucosa contains significantly raised concentrations of the CCR6 ligand, CCL20. We hypothesised that CCL20 may play a role in the migration of Tregs to the infected mucosa, and therefore aimed to investigate the mechanisms by which CCL20 expression is induced by Hp, and to quantify and characterise gastric mucosal CCR6+ Tregs.
Methods 24 Hp+ and 34 Hp− patients attending the Queen's Medical Centre, Nottingham, donated gastric biopsies and peripheral blood with informed consent and ethics approval. Isolated CD4 cells were stained for Treg markers (CD25hi, FOXP3+, CD127lo) and CCR6, prior to analysis by flow cytometry. Gastric epithelial cell lines (AGS, MKN28 and MKN45) were cultured with Hp (Wild type, and the following null mutants: ΔcagA, ΔcagE, ΔvacA and Δslt) at a range of multiplicities of infection (MOI), with or without chemical inhibitors, for up to 48 h. CCL20 in culture supernatants was quantified by ELISA.
Results CCL20 levels were threefold higher in biopsies from Hp+ patients than Hp− patients (p=0.015). >80% of Tregs extracted from gastric biopsies were CCR6+, and 3.5-fold higher numbers of Tregs were present in samples from infected compared to uninfected patients (p=0.050). twofold higher proportions of Tregs in the peripheral blood of Hp+ patients were CCR6+ (p=0.021). In cell lines, cagPAI+ Hp strains induced a dose-dependent increase in CCL20 production, with a sixfold increase after 24 h at MOI 1 (p=0.01). The ΔcagE (p<0.01) and Δslt (p<0.01) but not ΔcagA (p=0.18) inactivations reduced CCL20 induction by multiple Hp strains, indicating CagA-independent, cagPAI-dependent signalling.
Conclusion Hp induces CCL20 production by gastric epithelial cells in a cagPAI‑dependent manner. Higher gastric CCL20 levels in Hp+ patients correspond to increased gastric infiltration of CCR6+ Tregs and to the proportions of these cells in the peripheral blood. We speculate that CCL20/CCR6 is the main homing system for Tregs to the stomach in Hp infection and thus central to pathogenesis. Migration assays are being performed in vitro prior to in vivo studies in mouse models.
Competing interests None declared.