LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

Bu koleksiyon için kalıcı URI

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

Gözat

Yazar "Akdağ, Işılay" 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.