LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

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http://hdl.handle.net/11527/19401

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  • Öge
    The impact of metabolic reprogramming in helicobacter-activated B cell survival, differentiation, and proliferation
    (Graduate School, 2022-12-20) Deniz, Bahar ; Yazgan Sayı, Ayça ; 521191103 ; Molecular Biology-Genetics and Biotechnology
    Helicobacter pylori (H.pylori) is a helical-shaped, gram-negative, and microaerophilic bacterium that was identified by Robbin Warren and Barry Marshall in 1982. The bacterium, selectively colonized in the gastric mucosa, causes approximately 90% of duodenal ulcers and 80% of gastric ulcers. Affecting approximately half of the world's population, H. pylori is the most widespread bacterial infection in humans. Although the majority of infections do not result in symptoms or gastrointestinal disease, bacterially induced inflammation can lead to peptic ulcer disease (PUD), gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. Helicobacter felis (H.felis) is a spiral-shaped, gram-negative, and microaerophilic bacterium isolated from a cat's gastric mucosa. H.felis has been used for investigating the underlying mechanisms of Helicobacter-induced gastric pathology in mouse models. B lymphocytes which originated from hematopoietic stem cells in the bone marrow are responsible for the humoral immune responses. To be able to gain their antigen specificity, B cells go through several developmental stages in the bone marrow. Compared to T lymphocytes, B lymphocytes do not need antigen presenting cells (APCs) to be able to recognize antigens. With the help of their unique antibodies which they expressed on the cell surface, B cells can directly recognize antigens. Activation of naive B cells requires recognition via the Ig receptor and also additional signals. These additional signals can come from a CD4+ T cell, which is called thymus-dependent activation, or from microbial components which is called thymus-independent activation. Secondary lymphoid tissues, such as the spleen and lymph nodes provide a suitable environment for interactions between T cells and B cells and APCs, such as macrophages and DCs. Toll-Like Receptors (TLRs) are important members of receptors that provide the first line of defense against pathogens and microbes. They have an important role in connecting innate and adaptive immunity and they are able to recognize both pattern recognition receptors (PAMPs) and damage-associated molecular patterns (DAMPs). TLR stimulation causes the activation of signaling cascades which leads to the secretion of inflammatory cytokines and immune modulators. B cells are highly activated via TLR ligation, resulting in an increase in B cell survival, surface molecule expression, cytokine and antibody production, and antigen presentation. Proinflammatory responses are important for the efficient clearance of infections and the killing of malignant cells. On the other hand, anti-inflammatory responses are necessary for preventing chronic inflammation. Providing a fine balance between proinflammatory and anti-inflammatory responses is critical for a healthy immune system. Besides contributing to immune response via antibody production, B cells were also found to have a central role in the regulation of immunity. In cancer, autoimmune diseases, allergy, transplantation, and infection, several types of murine and human regulatory B cells have been reported that suppress inflammation via IL 10, IL-35, and TGF-β secretion or cell membrane-bound molecules ( programmed death-ligand 1, CD39, CD73) interaction. In mice and humans, several types of B cells with regulatory functions have been identified which differ in phenotypic characteristics and the suppressive molecules. B1-a cells which are also called innate immune-like B cells have CD5 on their cell surface as a marker and are a major source of IL-10, in mice. Also, another subset of B1-a cells has been discovered in the mouse spleen called the 'killer B cell.' These killer B cells express Fas ligand (FasL), induced CD4+ T-cell death, and suppressed arthritis. B10 cells which are CD5+CD1dhi can secrete high amounts of IL-10 when stimulated with LPS, PMA, and ionomycin. In various disease models such as autoimmune encephalomyelitis (EAE), intestinal inflammation, and allergic inflammation, the immunosuppressive functions of B10 cells are very well characterized. Another IL-10-producing B cell type was found that expresses CD9 and has regulatory functions such as suppressing Th2- and Th17-mediated inflammation, increasing the Treg/effector T-cell ratio, and inducing apoptosis of effector T cells. B cells have been shown to negatively regulate adaptive immune responses to bacterial pathogens such as H.felis, which is a close relative of the human gastrointestinal pathogen H. pylori and TL2 ligand. When activated with Helicobacter, B cells induce IL-10–producing CD4+ CD25+ T regulatory-1 (Tr-1)–like cells. TCR signaling and a direct T-/B-interaction through CD40/CD40L and CD80/CD28 are necessary for his Tr-1 conversion. Various populations of murine B cells have been shown to suppress the proliferation of CD4+ T cells and the production of proinflammatory cytokines in an IL-10-dependent manner in autoimmunity models such as antigen-induced arthritis, spontaneous lupus, type 1 diabetes, colitis, and EA. Metabolism is an energy source for cells, tissues, and organisms and fuels all kinds of biological programs like development, proliferation, differentiation, and the effector functions. Recent studies showed the importance of cellular metabolism in terms of supporting immune cell maintenance and development. Through their life cycle, immune cells experience dynamic and adaptive metabolic changes. İnvestigating the impact of metabolism on immune cell activation, differentiation, and regulation is becoming a new area for understanding infectious or autoimmune disease progression and cancer. Naive B cells require low levels of catabolic metabolism for maintaining energy homeostasis. This state of naive B cells is also called metabolically quiescent. Upon activation, B cells remodel their metabolic program to be able to meet the energetic and biosynthetic requirements of proliferation. Compared to naive B cells, glucose uptake, oxygen consumption, and lactate secretion were found to be increased in activated B cells. Both glycolysis and OXPHOS are thought to play a role in the activation of B cells. Studies have shown that glycolysis inhibition with glycolytic inhibitor 2-DG and OXPHOS inhibition with ATP synthase inhibitor oligomycin have a suppressive effect on B cell proliferation, survival, and function. The main aim of this study is to elucidate the impact of metabolism on Helicobacter-activated B-cell survival, differentiation, and proliferation. For this purpose, B cells were magnetically isolated from spleens of C57BL/6 mice or IL-10 GFP reporter (VertX IL10 egfp) mice and treated with H. felis antigen, PAM3CSK4, and LPS in the presence or absence of increasing doses of oligomycin (ATP synthase inhibitor ) (0.1-0.5-1 nM) and 2-DG (glycolytic inhibitor) (0.1-0.5-1 mM) for 6 h, 24 h, and 72 h. Afterward, cells were collected at respective time points for viability, IL-10 secretion, CD86, and PD-L1 surface markers expression and proliferation analysis. For analyzing the survival of B cells, 7-AAD viability dye was used. IL-10 production levels were determined by IL-10 ELISA and flow cytometry. The expression levels of surface markers CD86 and PD-L1 were also analyzed by flow cytometry. Moreover, the proliferation capacity of the cells was investigated via Carboxyfluorescein succinimidyl ester (CFSE) labeling. Our results showed that 1nM oligomycin decreased IL-10 secretion and viability at 24 h. Nevertheless, none of the doses of oligomycin significantly affected either proliferation or IL-10 secretion of B cells at 72 h. On the other hand, 2-DG decreased IL-10 secretion in a dose-dependent manner both at 24 h and 72 h without causing significant cell death. Additionally, it suppressed the proliferation of Helicobacter-activated B cells in a dose-dependent manner at 72 h. Moreover, 0.5 mM 2-DG significantly decreased PD-L1 and CD86 expressions of Helicobacter-activated B cells at 24 h. According to our results, glycolysis has a pivotal role in B cell proliferation, survival, IL-10 production, and CD86 and PD-L1 surface markers expression compared to OXPHOS inhibition. This study showed that the glycolytic pathway takes a role in both the expansion and differentiation of Helicobacter-activated B cells.