Thus, vagus nerve stimulation controls the production of proinflammatory cytokines

ion of glycolysis or B cell-specific deletion of Glut1 suppressed antibody production in vivo. Therefore, B cells rely on Glut1 and targeting B cell metabolic regulation and glycolytic pathways may provide a new tool to prevent B cell proliferation and autoantibody production. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Mice Materials and Methods C57BL/6, RAG1-/-, Hif1fl/fl, MD4 ML5, and CD19-Cre transgenic mice were obtained from Jackson Laboratories. BAFF transgenic mice that express full length BAFF driven by the myeloid cell specific CD68 promoter were generously provided by D. Nemazee . Mycfl/fl mice were backcrossed six generations onto the C57BL/6 background. Both Mycfl/fl and Hif1fl/fl were crossed with ROSA26CreERT2. Glut1fl/fl mice were crossed to CD19-Cre transgenics. The acute deletion of Myc or HIF1 was achieved through in vivo delivery of Tamoxifen three days before B cell isolation. Some animals were treated with dichlroroacetate. For bone marrow reconstitution, RAG1-/- mice were lethally irradiated with two doses of 4.5Gy, and provided wild type bone marrow by tail vein injection. Sex matched 7-12 week old mice were used throughout. Mice were housed and cared for at Duke University or St. Jude Children’s Research Hospital under Institutional Animal Care and Use Committee approved protocols. Human B cells were isolated from healthy donor peripheral blood. J Immunol. Author manuscript; available in PMC 2015 April 15. Caro-Maldonado et al. Page 4 Cell isolation and reagents Splenic nave B or T cells or human peripheral blood B cells were isolated by magnetic bead negative selection and cultured in RPMI 1640 supplemented with 10% FBS, HEPES, and ME. B cells were stimulated with 10 g/ml of LPS, 20 g/ml of F2 anti-IgM, or ODN. T cells were treated in plates coated with 10 g/ml of CD3 and CD28. Unstimulated B cells were maintained in 20ng/ml of BAFF to maintain in vitro viability. Some cultures were treated as indicated with 2-DG, dichloroacetate, or low dose rotenone. Flow cytometric analysis and antibodies Cytometry analysis was performed with a MACSQuant Analyzer and analyzed with FlowJo software. Anti-mouse CD19-APC, CD69-PE, IgM-FITC and IgDVioblue or anti-human CD69-FITC were used to measure purity and B cell activation. Cells were incubated 30 minutes with 200nM of Mitotracker Green, and washed to measure mitochondrial content. Proliferation was analyzed by CFSE staining and flow cytometric measurement of CFSE dilution. Glut1 expression was measured by intracellular flow cytometry of fixed cells using monoclonal anti-Glut1 in the presence of rat serum and Fc Block, followed by anti-rabbit-PE before flow analysis. Quantitative RT-PCR RNA was harvested from purified B cells ex vivo or following stimulation with anti-IgM or LPS and reverse transcribed to perform SYBR Green-based quantitative RT-PCR of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844094 Glut1 and cMyc. Results were normalized to Beta-2-Microglobulin. Western Blot Cells were lysed in a low detergent buffer for one hour with protease and phosphatase inhibitors. Nitrocellulose membranes were hybridized with anti-phospho S232-PDH-E1, total PDH-E1 antibodies, Glut1 rabbit monoclonal, Glut3 rabbit polyclonal, actin, cMyc or Hif1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Metabolic assays Glucose uptake, glycolytic flux, purchase LY341495 hexokinase activity, fatty acid -oxidation, glucose oxidation, glutamine oxidation, and pyruvate oxidation were measured as previously