Cytokine Profile of CCR6+ T-Helpers Isolated from the Blood of Patients with Peptic Ulcer Associated with Helicobacter pylori Infection.

Abstract

We previously found that the number of CCR6⁺ T-helpers with the phenotype of effector/effector memory T cells increases in the blood of patients with H. pylori-associated peptic ulcer. The mature phenotype and the expression of the chemokine receptor CCR6, which is involved in migration of lymphocytes to the inflamed mucous membrane of the gastrointestinal tract, suggests that these cells are involved in the immune response observed in this clinical condition. To better understand the pathogenetic role of these cells, it is necessary to study their functional activity, specifically, the production of pro-inflammatory cytokines involved in the pathogenesis of the disease. The aim of the study was to evaluate changes in the blood level of pro-inflammatory types of mature CCR6⁺ T-helpers in H. pylori-associated peptic ulcer disease.

Materials and methods: CCR6⁺ T-helpers were isolated from the blood by using immuno-magnetic separation adapted to this study. The number of T-helpers of types 1 and 17 (Th1 and Th17) and cells with mixed properties of Th1 and Th17 (Th1/Th17) was determined by intracellular cytokine assay.

Results: Initially, we planned to activate unseparated peripheral blood mononuclear cells ex vivo and evaluate the number of cytokine producers among mature CCR6⁺ T-helper cells by gating them during the flow cytometry. However, dramatic changes in the phenotype of T-helpers upon activation did not allow us to reliably identify the cells of interest. Subsequently, we used a two-stage immunomagnetic separation procedure to obtain functionally active mature CCR6⁺ T-helpers with a purity of >90%. The quantitative yield of these cells from the blood of patients with gastric and duodenal peptic ulcer associated with H. pylori was 9 times higher than that from the blood of healthy donors. Activation of CCR6⁺ T-helpers purified from blood of ulcer patients revealed an increased content of Th1, Th17, and Th1/Th17. One ml of the patient's blood yielded 18.1 times more CCR6⁺ Th1, 19.4 times more CCR6⁺ Th17, and 21.1 times more CCR6⁺ Th1/Th17 compared with the blood of healthy subjects.

Conclusion: The content of mature CCR6⁺ T-helper cells with pro-inflammatory activity significantly increases in the blood of patients with peptic ulcer associated with H. pylori infection.