Investigation of the mitochondrial metabolism of Helicobacter-activated B cells

Abstract

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, and spiral-shaped bacterium and a member of the Helicobacteraceae family. H. pylori was discovered in 1982 by Warren and Marshall. Helicobacter infection can lead to multiple gastro pathologies such as chronic gastritis, gastric cancer, peptic ulcer disease, and mucosa-associated lymphoid tissue lymphoma. Whereas more than 50% of the world population has been infected with H. pylori, 80% of them are asymptomatic. Similar to H. pylori; H. felis is a gram-negative, urease-positive, spiral-shaped, and microaerophilic bacteria. Studies have shown that H. felis can induce gastric atrophy, metaplasia, dysplasia, chronic and persistent inflammatory response, and gastric cancer in mice models. Because H. pylori have less capacity to activate an efficient immune response in mice compared to Helicobacter felis (H. felis); H. felis is used to generate mice models for studying this pathogen. B cells play critical roles in adaptive immunity with antigen presentation to T cells and antibody production. Recently, new B cell subsets have been shown to exert anti-inflammatory and immune suppressive properties were discovered. These B cells are termed regulatory B cells (Bregs) by Bhan and colleagues. In mice; mainly CD19+CD21hiCD23hiCD24hi transitional 2 marginal-zone precursor cells (T2-MZP), IL-10 producing CD1dhiCD5+ B10 cells, CD19+CD21hi CD23- marginal-zone (MZ) B cells, Tim-1+ B cells, CD19+CD5+ B1a cells, CD9+ B cells, CD138+ plasma B cells, and CD138+CD44hi plasma blasts are identified as Breg subsets. For maintaining host immune tolerance and balance effector immune responses, these Breg cells secrete IL-10, IL-35, and TGF-ꞵ cytokines. In addition to cytokines, Breg cells also use cell membrane-bound molecules such as CD39, CD73, programmed death-ligand 1(PD-L1), or aryl hydrocarbon receptors for their functions. Stimulation of B cells with H. felis; signals via TLR2 and MyD88 and results with IL-10 producing Bregs. Bregs can induce differentiation of naive CD4+ T cells to IL-10-producing regulatory Tr1 cells by direct B and T cell interactions for suppressing Helicobacter-associated pathologies. Metabolism is the collection of all anabolic and catabolic reactions which are the generation and breakdown of cellular substances respectively. Oxidative phosphorylation (OXPHOS) is one of the major metabolic pathways inside the cell. It occurs in the mitochondria and consists of the tricarboxylic acid cycle (TCA) and electron transport chain reactions for generating ATP. Shortly, in OXPHOS electrons formed from the tricarboxylic acid cycle (TCA); are combined with molecular oxygen (final acceptor of electron transport chain) and this results in many oxidation/reduction reactions where energy is released for the production of ATP from ADP. Mitochondria are double membrane organelles that have both outer and inner mitochondrial membranes (OMM and IMM) and are found in eukaryotic cells. Membrane transporters and electron transport chain (ETC) complexes of mitochondria localize on the inner mitochondrial membrane. IMM encloses a viscous structure called a mitochondrial matrix. Enzymes, mitochondrial DNA (mtDNA), ribosomes, and nucleotides are placed in the mitochondrial matrix. Mitochondrial DNA encodes 37 mitochondrial genes including 22 transfer RNAs, 2 ribosomal RNAs, and 13 important oxidative phosphorylation polypeptides: ND1, ND2, ND3, ND4L, ND4, ND5, ND6 (parts of Complex I); Cytochrome b (parts of Complex III), COI, COII, COIII (parts of Complex IV) and ATP6, ATP8 (parts of Complex V). To provide expression of a mitochondrial gene, mitochondrial DNA needs to be transcribed by mitochondrial transcription factor A (Tfam), mitochondrial RNA polymerase (POLRMT), and mitochondrial transcription factor B1 and B2 (Tfb1m and Tfb2m). Two of the most critical features of multicellular life are metabolism and immunity. These can be explained as the need to distribute nutrients across cells, tissues & organs and protect from injury and inflammation. In recent years, studies have focused on elucidating the metabolism of immune cells in the context of their survival, activation, differentiation, and functions. For the activation of immune cells, signals which are triggered by metabolic intermediates and ATP molecules are required. In order to maintain proper immune cell activation, differentiation, and function, mitochondrial metabolism which generates energy plays a critical role. Studies have demonstrated that B cell activation with B cell receptor (BCR) or different Toll-like receptor (TLR) ligands changes mitochondrial dynamics. LPS-stimulated B cells enhance mitochondrial mass, and co-stimulation of B cells with BCR ligand IgM and TLR9 ligand CpG increases mitochondrial biogenesis. In addition to that, anti-CD-40 and IL-4 stimulated B cells to undergo OXPHOS. However, there is no information in the literature about the mitochondrial metabolism of Helicobacter-activated B cells. The main aim of this study is to elucidate the mitochondrial metabolism of Helicobacter-infected B cells. For this purpose, B cells were magnetically isolated from spleens of C57BL6 mice and treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Afterwards, cells were collected at respective time points for mitochondrial mass and membrane potential staining by using Mitoview Green and Mitoview 633 or TMRE dyes respectively in the flow cytometry. The supernatant of these cells is used for the IL-10 ELISA experiments for checking their IL-10 secretion. H. felis, PAM3CSK4, and LPS-stimulated B cells increased their IL-10 production most noticeably at 24h and 48h indicating the suppressive capacity of that cells. Also, compared to the unstimulated control group, all of the stimulant groups increased both the mass and membrane potential of mitochondria at 24h and 48h time points. The second aim of our study was to investigate whether B cells with high mitochondrial membrane potential (Mitoview 633+ B cells) produce IL-10 or not. For that, after B cells were isolated from IL-10 GFP reporter (VertX IL10 egfp) mice, they were treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Mitochondrial membrane potential were analyzed by flow cytometry. Afterwards, at 6h, 24h and 48h we evaluated mitochondrial membrane potential of the IL-10 producing B cells with quadrant analysis using flow cytometry. At 6h time there were no significant changes on IL-10+ Mitoview 633+ B cells. But at 24h and 48h time points; H.felis, PAM3CKS4, and LPS-stimulated B cells increased IL-10+ Mitoview 633+ B cells. These data show that in all stimulated B cell groups; high portion of the IL-10 producing B cells also have high mitochondrial potential. Our data shows, H. felis, PAM3CSK4, and LPS-stimulated Mitoview 633 + B cells increased their IL-10 GFP signal both at 24h and 48h time points compared to the unstimulated control group. The third aim of our study was to investigate mitochondrial biogenesis markers: mtDNA:nDNA ratio and mitochondrial transcription factor A (Tfam) gene expression. To perform Q-PCR experiments isolated and treated B cells were collected for DNA and RNA isolation at 6h, 24h, and 48h. For mtDNA:nDNA ratio analysis in Q-PCR; respectively cytochrome c oxidase subunit I (COX1) and 18S ribosomal subunit gene (RPS18) were targeted by using gene-specific primers. While H. felis, PAM3CSK4, and LPS-stimulated B cells decreased their mtDNA:nDNA ratio, they increased Tfam expression level compared to the unstimulated control group. This study showed that H. felis-activated B cells have active/functional mitochondria, and increased oxidative phosphorylation for energy production and their IL-10 production can be related to their mitochondrial metabolism.