LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

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

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Konu "B-cell" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma
  • Öge
    Investigation of glucose metabolism on helicobacter-activated B cells
    (Graduate School, 2022-12-13) Akdağ, Işılay ; Yazgan Sarı, Ayça ; 521191115 ; Molecular Biology-Genetics and Biotechnology
    Helicobacter pylori is a gram-negative and spiral-shaped bacterium that may lead to gastritis, ulcer, and pathological scenario after its infection in humans. Its role in gastritis and gastric ulcer disease which is discovered by Barry Marshall and Robin Warren was awarded with the Nobel Prize in Physiology or Medicine, in 2005. Mostly, the prevalence of the H. pylori-positive population is 80% in developing countries and they are infected at an early age. A high percentage of the positive population do not show any clinical sign in a short time. H. felis (Helicobacter felis) is a gram-negative, spiral-shaped bacterium similar to H. pylori and one of the species that have zoonotic potential. Due to the lack of an efficient immune response against H.pylori in murine; mice have been infected with Helicobacter felis (H. felis) to create a mouse model of Helicobacter infection. B lymphocytes are one of the responsible cells of adaptive immunity, assisted by antigen-presenting cells, and provide an advanced response. B lymphocytes quite mobile, migrate into different sites of the body to show their functions through the lymphatic and blood circulation system in favor of adhesion molecules and chemokines receptors. Activation of B cells is provided by some molecular events that are binding of antigens to BCR( B cell receptor) , clustering of BCRs, and constitution of signaling cascade by several components including kinases, phosphatases, and scaffolds under the BCR. BCR activation leads to the initiation of B cell activation process which requires some changes in cell metabolism, actin cytoskeleton, and gene expressions. In addition to BCR activation, secondary signals assist full activation of B cells which causes their proliferation and differentiation. Secondary signals can be provided by pathogens and their particles which can enable TLR( Toll-like receptor) activation and signaling process. Toll-like receptors are evolutionary conserved and important receptor family of pattern recognition receptors (PRRs) for recognizing microbes and their particles which are external pathogen-associated molecular patterns (PAMPs) and internal damage-associated molecular patterns (DAMPs). Unique nucleic acids, some types of proteins, and lipids that are synthesized by microbes such as LPS, and unmethylated CpG DNA sequences are several examples of PAMPs. TLRs are located on the membrane of both innate immune cells, as well as on adaptive immune cells like B and T lymphocytes, and also other body cells such as the brain, skeletal muscle, testis, placenta, kidney, etc. Their amount on the surface can be arranged depending on the response to specific molecules derived from invading pathogens or damaged host cells. Studies have shown that TLR-signaling plays a crucial role in eliciting a humoral immune response and affects B cells response and activation such as clonal expansion and differentiation. Studies showed that TLR expression profiles and TLR signaling exhibit variations on different B cell subsets. Regulatory B cells play an important role with their suppressive functions to maintain tolerance, prevent excessive immune response, and support immune homeostasis. Their functional properties are provided by cytokines including IL-10 (Interleukin-10, IL-35 (Interleukin-35), and also TGF- (Transforming growth factor-). Dominantly, the IL-10-producing subset of regulatory B cells plays an essential role during bacterial infections. TLRs and CD-40-mediated activations support the differentiation of regulatory B cells and also there is a piece of recent evidence that pro-inflammatory cytokines, including IL-1β and IL-6, can induce IL-10-producing Breg differentiation. In vitro LPS stimulation induces the differentiation of B10 subsets that are IL-10-producing Breg subtypes and have CD1dhiCD5+ and CD24hiCD27+ surface markers on mice and human, respectively. Additionally, lacking TLRs and their signaling molecules can lead to autoimmune diseases. Studies showed that in mice lacking MyD88, which is a TLR-signaling molecule, TLR2 and TLR4 activation in B cells induce the development of chronic experimental autoimmune encephalomyelitis (EAE). On the other hand, previous studies investigated that the H.felis activation via MyD88-dependent TLR2 signaling of B cells restricts the Th1-driven gastric pathologies by the Breg and Treg collaborations. Bregs acquire the ability to induce Tr-1 cells by the H.felis activation. B cells alter their metabolism in different ways during their activation; glucose is an essential molecule in the activation process to sustain biomolecule synthesis. Additionally, glucose is the key precursor for other macromolecules such as ribose, deoxyribose, glycoproteins, etc. Glucose uptake is driven by glucose transporters (GLUTs) and sodium-glucose cotransporters (SGLTs) via facilitated diffusion. Activation of B cells leads to metabolic reprogramming in many different ways. TLR-4 through an activation with LPS which is the TLR-4 agonist, changes and increases the glucose import, expression of glucose transporter GLUT1, and also OXPHOS of B cells. B cell activation via BCR increases glucose uptake and expression of glucose transporter GLUT1 by the PI3K. With the TLR-9 activation via CpG, which is the TLR-9 agonist, B cells rapidly increase their glycolytic and mitochondrial capacities with the changes in the gene expression of related mediators such as glycolytic enzymes, GLUT1 protein, and GLUT3 protein. Similar to B cells, regulatory B cells also induce their glycolytic activity and IL-10 production after their activation with LPS, IgM, and anti-CD40. In this study, the effect of Helicobacter felis antigens on the glucose metabolism of B cells was investigated at the short-term and long-term stimulation. H.felis stimulation leads to IL-10-producing B cells via TLR-2-mediated signaling in murine B cells. Because of the importance of the IL-10-producing B cells in various bacterial infections and gastrointestinal pathologies, the reprogramming of glucose metabolism was investigated within 48 hours. Results demonstrated that the H.felis antigen, Pam3CSK4, and LPS stimulation increase the glucose uptake level after 24 hours and the Helicobacter-activated B cells undergo metabolic reprogramming via increasing glucose uptake at 24 hours and a further increase was seen at 48 hours. After that, the mRNA and protein expression of glucose transporters, GLUT1, GLUT3, and GLUT4, are investigated whether affected by Helicobacter stimulation at 6,24, and 48 hours in live cells. mRNA expression levels of the Glut1 gene were induced. Also, the Glut3 mRNA expression level of Helicobacter-activated B cells was lower than the expression level of unstimulated B cells. Intracellular protein staining of glucose transporters (GLUT1, 3, and 4) demonstrated that the GLUT4 protein level did not change after H.felis-stimulation, but GLUT1 and GLUT3 proteins increased when compared to the unstimulated control group. Additionally, results showed two different positive populations which are expressing high or low protein levels at 24 and 48 hours. The intracellular GLUT protein staining showed that high-GLUT1 and GLUT3-expressing populations are positive for IL-10 at 24 and 48 hours. A higher increase was detected for the GLUT3 protein compared to the GLUT1 protein in the Helicobacter-stimulated IL10+ or IL-10- B cells. Glycolysis is the vital pathway for every living cell and pyruvate is the key molecule to form precursors of many biomolecules including, cholesterol, lactic acids, plasma proteins, and fatty acids. Previous studies have shown that the glycolytic activity of B cells increases after their stimulation via IgM, LPS, and CpG for 24 hours. Thus, in this study, the glycolysis enzymes including; hexokinase2, pyruvate kinase2, and lactate dehydrogenase expressions were investigated in mRNA levels. Results showed that the Helicobacter activation upregulates the hexokinase2, pyruvate kinase2, and lactate dehydrogenase gene expressions at 6, 24, and 48 hours. Their expression levels were at their highest level at 24 hours. Collectively, Helicobacter-mediated stimulation increased the glucose metabolism of B cells. This study is the first investigation that clarifies the reprogramming of glucose metabolism of Helicobacter-stimulated B cells.
  • Öge
    Investigation of the energy metabolism of helicobacter-activated B cells
    (Graduate School, 2022-07-07) Çalçı, Mehmet ; Yazgan Sayı, Ayça ; 521181128 ; Molecular Biology-Genetics and Biotechnology
    Helicobacter pylori is a gram-negative, spiral-shaped and microaerophilic bacterium. It has been declared as type 1 carcinogen by the World Health Organization since it causes stomach cancer. Although more than 50 % of the world's population is infected with this bacterium, only 10-20 % of them have serious gastrointestinal diseases. For the development of infection-disease murine models, H.felis which is instead of H.pylori can be used. The immune system is the system of defense and protection against any infectious disease. It is categorized into two groups as innate and adaptive. An early line of defense against microbes is formed by innate immunity. Innate immunity includes both cellular and biochemical defense mechanisms that are present even before infection and are ready to respond immediately to infections. On the other hand, adaptive immunity becomes active when the organism encounters an infectious agent. Memory response generation is one of the most basic features of adaptive immunity. B cells play a significant role in the regulation of immune response due to the key functions including antibody production and antigen presentation. The regulatory role of B cells was shown with the use of mice lacking B cells which could not overcome autoimmune encephalitis. Helicobacter felis stimulates B cells via TLR2 and Myd88 and suppress immune responses. This suppression brings about persistency of bacteria in gastric mucosa Therefore, a balance between regulatory and effector response could determine the fate of infection. As demonstrated by our previous laboratory studies, H. felis-mediated activation of B cells induces two different B cell subgroups: IL-10- producing IL-10+ B cells and TGF-β-producing IL- 10- B cells. When a resting immune cell encounters an antigen, it undergoes metabolic reprogramming. The metabolic profile of immune cells for energy production plays a significant role in the regulation of the immune system. The energy (ATP) production mechanism required by immune cells during proliferation and differentiation into an effector cell differs from cell to cell. Glucose which is one of the key biomolecules used as the basis for ATP production is taken into the cell via glucose transporters and metabolized during glycolysis. The pyruvate formed at the end of glycolysis can pass into the mitochondria and enter the TCA cycle, increasing oxidative phosphorylation, or it can remain in the cytoplasm and produce lactate. The oxidative phosphorylation pathway can also utilize fatty acids and amino acids. Glycolysis and oxidative phosphorylation are the two main pathways involved in energy production in the cell. Studies have shown that there is a decrease in the proliferation of LPS-stimulated B cells treated with 2-DG, a glycolysis inhibitor. In addition, it has been reported that treatment of B cells with oligomycin, an ATPsynthase inhibitor, during stimulation with anti-CD-40 and IL-4, decreases the activation marker CD86 and cell viability. However, there is no information in the literature about how B cells undergo metabolic reprogramming when stimulated by Helicobacter species. B cells that do not receive stimulation in cell culture can not maintain their viability for a long time. In a study, a low dose of recombinant B cell-activating factor (rBAFF) was added to B cell culture and increased the viability of the cells. Based on this information, it was aimed to investigate the effect of rBAFF on Helicobacter-activated B cell viability and function since cell viability was very critical in the metabolic analysis studies that would follow. B cells isolated by magnetic separation from C57BL/6 mouse spleen were stimulated with H.felis sonicate, PAM3CSK4, and LPS for 6 and 24 hours. The main purpose of choosing 6h time point is to investigate how cells respond only in the presence of stimuli before they proliferate, and the main reason for choosing the 24h time point is that B cells begin to secrete the anti- inflammatory cytokine IL-10. To increase the viability of B cells in the culture, the effect of rBAFF added at a low dose on the viability is determined by the flow cytometry, its effect on the function with the expression levels of CD86 and CD9 surface markers in the flow cytometry again, and the effect on the IL-10 secretion level by the IL-10 ELISA method. researched with. Although rBAFF added to the B cell culture medium significantly increased the viability of the control group, which did not receive stimulation, it was excluded from the experimental plan because it would affect the results of metabolism studies since it changed the IL-10 levels of the cells. Because rBAFF was intended to be given to the control group, which was not stimulated in metabolism studies, in case it did not show any effect on the function while increasing B cell viability, rBAFF was not given to the experimental groups since it slightly changed the IL-10 production levels of B cells. Another aim of the study is to investigate how the metabolic profile of splenic B cells stimulated with H.felis is characterized. The metabolic flux analysis method was used to determine which pathways are activated for the required energy production while becoming an effector by being exposed to stimuli from the resting state. B cells were cultured with H.felis sonicate, PAM3CSK4, and LPS for 6 and 24 hours, and then the change in energy metabolism was measured in XFe96 device by extracellular flux analysis method. Since the number of dead cells exceeding 20 % would adversely affect the results of metabolism studies, dead cell removal was performed by the magnetic separation method, especially in the control groups. Only cells with high viability were included in the metabolism studies. The proportion of viable cells in the non-stimulated groups increased from 50% to 80% after the magnetic removal of dead cells. Since the viability data of the stimulus groups were suitable for metabolism experiments, no dead cell separation was made in these groups. According to the results obtained from our study, as a result of stimulation of B cells with H.felis sonicate, PAM3CSK4 and LPS for 24 hours, glycolysis and glycolytic capacity tend to increase significantly as a result of the measured ECAR data. Although the groups that received stimulation at 6 hours increased both glycolysis and glycolytic capacity compared to the control groups that did not receive stimulation, this rate is much less when compared to 24-hour stimulation. Basal and maximum OCR values of cells are directly proportional to the amount of oxygen they consume during OXPHOS. As a result of stimulation of B cells with H.felis sonicate, PAM3CSK4 and LPS for 24 hours, both basal and maximum OCR values increased significantly compared to the non-stimulated groups. This increased rate is much lessin the 6-hour warning groups. The OCR data also gives information about the mitochondrial ATP production of the cells. B cells stimulated with H.felis sonicate, PAM3CSK4 and LPS also significantly increased their mitochondrial ATP production after 24 hours. As a result of this study, the effect of rBAFF on cell viability and function was investigated for the first time in mouse splenic B cells stimulated with H.felis for both short (6 hours) and long (24 hours) periods. At the same time, the specificity of the study is high since it was investigated for the first time how the metabolic profile required for energy production is shaped in mouse splenic B cells stimulated with H.felis for both short (6 hours) and long (24 hours) periods.