Helicobacter-aktive regülatör B hücrelerinde gen ifadesinin belirlenmesi

The immune system provides the primary defense against the invasion of foreign organisms including; bacteria, viruses, fungi or any antigen recognized as a foreign antigen by immune cells. Every moment inside our bodies, the immune system encounters many antigens around it. For that reason, the immune system should discriminate the non-self pathogens from self-antigens and respond appropriately. Broadly, the immune system is divided into innate and adaptive arms. The innate immune cells are the early line of defense in front of any pathogen in a nonspecific manner and they proceed to an inflammation. In addition, the most important innate immune cells are neutrophils and macrophages, which are responding quickly to local infection. On the other hand, the adaptive immune cells are T- and B- lymphocytes, both have specific cell surface markers, and mainly take time to recognize the specific foreign antigen. The innate and adaptive immune system often work in harmony to remove pathogens or foreign invaders. Helicobacter pylori (H. pylori) is a gram-negative bacterium that selectively colonizes the gastric epithelium and is the most common bacterial infection worldwide. More than 50% of the world's population are H. pylori infected. Approximately, 60% of adults and 30% of children. Clear evidence indicates that once the Helicobacter infection is acquired, it usually persists for lifelong unless it is eradicated by antimicrobial therapy. H. pylori is located in gastric mucosal cells. All of the H. pylori-infected patients mainly develop asymptomatic chronic gastric inflammation. The presence of genes such as Cag pathogenicity island (cag-PAI) and vacuolating cytotoxin A (vacA) that increases the risk for gastric atrophy, gastric adenocarcinoma development, recruitment and activation of immune cells in the underlying mucosal host tissue. The present study reported that the relative risk of gastric cancer is 1.7-5.3 times higher in H. pylori - infected individuals compared to uninfected individuals. The mechanism of interaction between H. pylori and host lymphocytes as well as antigen presenting cells is complicated. Therefore, the regulatory B and T cells have a pivotal role in the balance between immune pathogenicity and protection. Recently, it has been shown that the regulatory T cells could reduce H. pylori-induced gastritis in mice. At the same time, the regulatory T cells allow the bacterium to colonize the mucosa at higher densities. In addition, H. pylori-specific CD4+ CD25+ regulatory T cells suppress memory T-cell responses against H. pylori in infected humans. In addition to that, the role of regulatory B cells in Helicobacter - driven immune response was investigated. It was reported that IL-10- producing B cells were activated upon Helicobacter infection. The IL-10- producing B cells were activated through Toll-Like Receptor 2 (TLR-2)-myeloid differentiation primary-response protein 88 (MyD88) signaling. In fact, the IL-10- producing B cells lead to differentiation of T regulatory-1 (Tr-1) cells from naive T cells. The interaction xxvi between Tr-1 and IL-10-producing B cells may prevent the gastric precancerous lesions and serve as a good immune modulator against Helicobacter. While phenotype of H. felis stimulated (Hfstim) B cells were investigated two H. felis induced Breg subsets were determined; Hfstim-IL-10+ B and Hfstim-IL-10– B cells. Both subsets were identified with various surface markers and cytokine profiles. Hfstim-IL-10+ B cells (CD1d+CD5+ CD21+CD23+) and produce IL-10, and Hfstim-IL-10– B cells (CD21+CD23lo) and produce IgM, IgG2b and TGF-β. Furthermore, we assessed the level of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in Hfstim-IL-10+ B and in Hfstim-IL-10– B cells. Both the m RNA and protein levels of TNF-α and IL-6 cytokines are expressed significantly higher in Hfstim-IL-10– B cells compared to Hfstim-IL-10+ B. Based on ours and others findings, we aimed to identify the functional role of Hfstim-Bregs upon interaction with naive CD4+ T cells. Previous studies showed the transcriptome profile of B10 cells by using RNA-Seq and microarray analysis. In our study, we aimed to investigate the expression profile of Helicobacter - stimulated regulatory B cells (Hfstim-Bregs) by microarray analysis. Our samples include four groups; unstimulated B cells, Hfstim-total B cells, Hfstim-IL-10+ B and Hfstim-IL-10– B cells. Our microarray data indicated that 1615 genes are up-regulated and 794 genes are down–regulated in Hf stim- IL-10+ B cells compared to Hf stim- IL-10– B cells. CD9 and PDCD1 were shown to be highly expressed in IL-10+ B cells by a recent publication. Based on our microarray and real -time PCR data, we found differential expression of CD9, PDCD1, and NRP2 together with two other candidate genes (S100A8 and Trib1) in Hfstim-IL-10+ B cells in comparing to Hfstim-IL-10– B cells. Afterward, the surface expression of PD-1 and CD9 was investigated by flow cytometry analysis in Hfstim-IL-10+ B and Hfstim-IL-10– B cells. The PD-1 and CD9 protein levels are significantly high in Hfstim-IL-10+ B cells compared to Hfstim-IL-10– B cells. Of note Of noteOf note , both q-RT-PCR results and flow analysis confirmed the expression of PD-1 in Hfstim-IL-10+ B cells. Accordingly, we pointed out to identify the functional implication of a PDCD1 in Helicobacter stimulated Bregs. The flow cytometric analysis is applied to assess CD9, PD-1, CD19, CD4, IL-10, PD-L1, and PD-L2 expression on B cell groups. In line with the previous reports, we found high expression of PD-1 in mRNA and protein level in Hf stim-IL-10+ B cells. While, there was a distinct surface expression of PD-L1 in Hf stim-IL-10– B cells compared to Hf stim-IL-10+ B cells. Besides the m RNA level of PD-L1 was high in Hf stim-IL-10+ B cells compared to Hf stim-IL-10– B cells. In this regard, we blocked PD-1 or PD-L1 in Hfstim-IL-10+ B or in Hfstim-IL-10– B cells prior to co-culturing B cells with naive CD4+ T cells. The anti PD-1 or anti PD-L1was used to block the PD-1-PD-L1 axis pathway. Remarkably, blockade of PD-1 or PD-L1 in Hfstim-IL-10+ B cells led to a pronounced down regulation of IL-10+ CD4+ Tr1 cell differentiation. Interestingly, blockade of PD-1 or PD-L1 on CD4+ T cell prior to co-culturing with Hfstim-IL-10+ B / Hfstim-IL-10– B cells also decrease the percentage of Tr1 differentiated cells. Therefore, PD-1/ PD-L1 interaction between B and T cells is important in inducing Tr-1 cell differentiation. Also, the Hfstim-PD-1+ Bregs have a potential role in inducing Tr1 cells. Furthermore, the PD-1+ Bregs could be used, in future, as a therapeutic target to provoke the immune response and prevent the progression of gastric cancer. Currently, it was reported that PD-1 expression was high in CD4+ T cells of H. pylori infected patients. Notably, they found a significant correlation between H. pylori xxvii density with CD4+ PD-1+ T cell frequency or CD4+ CD25hi Tregs. Therefore, we studied PD-1 mRNA expression in Helicobacter-infected gastric tissue. H. pylori infection status of gastric tissue in this study was confirmed previously using ure A and ure B qPCR. Here, we determined the expression levels of PD-1 in gastric tissue of H. pylori negative individuals and in H. pylori infected gastritis and ulcer patients. The PD-1 mRNA is expressed significantly higher in ulcers compared to gastritis. Further studies would be performed to investigate the correlation between the human PD-1 in H. pylori infected gastric tissue with the IL-10- producing B cells or IL-10- producing T cells.