Investigation of the energy metabolism of helicobacter-activated B cells

Abstract

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.