PWE-173 Increased CCL20 and CCR6+ regulatory T-cell responses in the<i>Helicobacter pylori</i>infected human gastric mucosa
Authors: K Cook, D Letley, J Atherton, K Ragunath, K Robinson
Journal: Gut (2012)

<h3>Introduction</h3> <i>Helicobacter pylori</i> (<i>Hp</i>) infection may result in peptic ulcers or gastric cancer. Disease risk is associated with more virulent strains, such as those with the <i>cag</i> pathogenicity island (<i>cag</i>PAI), which induce higher levels of gastritis. Disease is also associated with an insufficient anti-inflammatory regulatory T-cell (Treg) response to <i>Hp</i>. Preliminary studies of circulating T-cell adhesion molecule expression highlighted an increased frequency of CCR6<sup>+</sup> Tregs in the blood of <i>Hp</i>+ patients. This chemokine receptor is expressed by both T-helper 17 (Th17) cells and Tregs. It has been demonstrated that the <i>Hp</i>-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 <i>Hp,</i> and to quantify and characterise gastric mucosal CCR6+ Tregs. <h3>Methods</h3> 24 <i>Hp</i>+ and 34 <i>Hp</i>− patients attending the Queen9s Medical Centre, Nottingham, donated gastric biopsies and peripheral blood with informed consent and ethics approval. Isolated CD4 cells were stained for Treg markers (CD25<sup>hi</sup>, FOXP3<sup>+</sup>, CD127<sup>lo</sup>) and CCR6, prior to analysis by flow cytometry. Gastric epithelial cell lines (AGS, MKN28 and MKN45) were cultured with <i>Hp</i> (Wild type, and the following null mutants: Δ<i>cagA</i>, Δ<i>cagE</i>, Δ<i>vacA</i> and Δ<i>slt</i>) 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. <h3>Results</h3> CCL20 levels were threefold higher in biopsies from <i>Hp</i>+ patients than Hp− patients (p=0.015). &gt;80% of Tregs extracted from gastric biopsies were CCR6<sup>+</sup>, 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 <i>Hp</i>+ patients were CCR6<sup>+</sup> (p=0.021). In cell lines, <i>cag</i>PAI+ <i>Hp</i> strains induced a dose-dependent increase in CCL20 production, with a sixfold increase after 24 h at MOI 1 (p=0.01). The Δ<i>cagE</i> (p&lt;0.01) and Δ<i>slt</i> (p&lt;0.01) but not Δ<i>cagA</i> (p=0.18) inactivations reduced CCL20 induction by multiple <i>Hp</i> strains, indicating CagA-independent, <i>cag</i>PAI-dependent signalling. <h3>Conclusion</h3> <i>Hp</i> induces CCL20 production by gastric epithelial cells in a <i>cag</i>PAI‑dependent manner. Higher gastric CCL20 levels in <i>Hp+</i> patients correspond to increased gastric infiltration of CCR6<sup>+</sup> 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 <i>Hp</i> infection and thus central to pathogenesis. Migration assays are being performed in vitro prior to in vivo studies in mouse models. <h3>Competing interests</h3> None declared.