Efeito do número de sítios EPIYA C da proteína CagA de Helicobacter pylori na produção in vitro de citocinas associadas à carcinogênese gástrica e avaliação das vias de sinalização

Abstract

Subjects infected with cagA-positive H. pylori strains are at increased risk for gastric carcinoma. cagA (cytotoxin associated gene A) encodes the CagA protein that is injected into host gastric epithelial cells via a type 4 secretion system (T4SS) where it undergoes tyrosine phosphrorylation by Src family protein kinases and binds SHP-2 (Src Homology 2 Domain-Containing Tyrosine Phosphatase-2) phosphatase at several repeated sites in the C-terminal region containing EPIYA C motifs. The complex CagA/SHP-2 leads to sustained activation of several intracellular events, mainly linked to the signalling pathways, SHP-2/MAPK/ERK1/2 and JAK/STAT3, which alters host cell activities and functions including apoptosis, proliferation, morphogenesis and motility, considered relevant in the carcinogenesis. However, up to now there are no studies evaluating the effect of the number of CagA EPIYA C motifs on the production of cytokines, also considered relevant in carcinogenesis. Therefore, in the present study, we evaluated the effect of the number of CagA EPIYA C motifs on the production of cytokines related to Th17 cell commitment (IL-1β, IL-6, IL-17A, IL-23 and TGF-β), as well as IL-8 and IL-11, in peripheral blood mononuclear cells (PBMC) of healthy H. pylori-negative subjects (qRT-PCR and ELISA) and AGS cells (ELISA) stimulated by isogenic H. pylori strains with different number of EPIYA C motifs. Signalling pathways involved in CagA interaction were evaluated by immunoblot and the results were confirmed by using specific inhibitors of each pathway. Statistical analyses were performed by using SPSS and the differences were considered significant when p values were ≤ 0.05. We demonstrated that CagA protein is injected, broken down and phosphorylated in the cytosol of PBMC after stimulation. CagA, most prominently the strain with two and three EPIYA C motifs, bound to SHP-2 leading to activation of MAPK/ERK1/2 signalling pathway. Otherwise, JAK/STAT3 signalling pathway was mainly activated by cagA-positive strain with one EPIYA C motif. As consequence of the activation of these signalling pathways, an increased secretion of IL-6, IL-11, IL-17A, IL-23 and TGF-β by PBMC and IL-11 by AGS cell was observed. Cytokine secretion was proportional to the number of EPIYA C. By contrast, the IL-1β and IL-8 secretion by PBMC and AGS cells was independent of the number of EPIYA C motifs. In conclusion, we demonstrated, for the first time, that H. pylori CagA strains with different number of EPIYA C motifs were injected into cytosol of the immune response cells, where it was broken down and phosphorylated leading to activation of SHP-2/MAPK/ERK1/2 signalling pathway. JAK/STAT3 signalling pathway was also activated, but inversely proportional to the number of EPIYA C motifs. Altogether the alterations resulted in heightened production of cytokine related to carcinogenesis.