Nutrients and Metabolism in the regulation of humoral immunity: a role for HIFs

Abstract

Humoral immunity, mediated by B cells, is vital for antibody-mediated protection against microbes. After first encounter with antigen, B cells proliferate, undergo selection for high affinity receptors, and differentiate into B memory cells (Bmem) and long-lived plasma cells (LLPC). Hypoxia-inducible factor (HIF) is a transcription factor related to several metabolic roles in various cell subtypes by reducing O2 consumption within the cells through a metabolic shift toward glycolysis. However, the role of HIF has not been assessed in B cell development, function and metabolism. Here, we hypothesize that HIFs regulate humoral immunity, both in primary antibody responses and in the generation or maintenance of antigen-specific Bmem, and that this function is mediated at least in part through promotion of metabolic fitness. Therefore, the aim of this study is to explore the oxygen sensing pathways mediated by the hypoxia-inducible transcription factor(s) HIF-1 and HIF2 in regulating B cell development, antibody responses and memory cell function/development. We will use C57BL/6J wild type (WT) mice and a model where HIF-1± and HIF-2± will be conditionally deleted specifically from the B cell lineage due to expression of tamoxifen-inducible Cre under the huCD20 promoter expressed exclusively on B cells. First, sorted-B untouched B cells cells from WT mice will be cultured in normoxic or hypoxic conditions along with different activators (LPS, CpG, anti-CD40 and/or anti-IgM F(ab')2 fragments) to verify the impact of HIF activation on the metabolic profile of B cells and plasma cell differentiation/function by using flow cytometry and metabolic assays (including reactive oxygen species in mitochondria and cells, lipid peroxidation, and redox balance, nutrient transporter expression, glutaminolysis, glycolysis, mitochondrial homeostasis - number and morphology). The same experiments will be performed by using B cell-specific HIF knockout cells after activation. We will also validate fluorescent and CreERT2 reporter constructs whose transcription is regulated by HIF-responsive elements (HRE) and use the retrovectors with these constructs to purify hypoxic cells in lymphoid organs. Moreover, the RNA profiling of fluorescent hypoxia-responsive element-reporter (positive vs negative) Germinal Centre B cells and plasma cells that are HIF-1±- or 2± -deficient will be performed. Finally, we will also immunize tamoxifen-treated mice with hapten-carrier protein and screen for NP-specific antibody secreting cells, circulating anti-NP antibody, and NP-specific Bmem of IgM and IgG1 isotypes at various times by using ELISA (NP20-BSA to capture anti-NP antibody), ELISpot and flow cytometry. The proposed study will establish key insights into the molecular and metabolic programming of recall responses and memory in the B cell lineage. Such approach may lead to discovery of new forms of pharmacological targets.