Abstract P785: The B Cell Response to Extracellular Glutamate in the Ischemic Brain

Abstract

Background: Neuronal networks require significant neurotrophic support for functional plasticity after stroke. We showed that B cells exhibit a cell-specific migration pattern in the post-stroke brain. Post-stroke B cell depletion impedes neurogenesis, increases anxiety, and exacerbates memory deficits in mice; deficits generally mediated by brain regions occurring outside the initial infarct. We hypothesize that the post-stroke microenvironment can enhance neurotrophic capacities of B cells to promote plasticity. Methods: Splenic B cells were isolated from 3-5 mo-old male C57Bl/6J mice. B cell N-methyl-D-aspartate receptor (NMDAR) subunits were identified by confocal microscopy. The acute (8 min) Ca 2+ response to 1uM glutamate (glu) +/- NMDAR antagonists (10uM DAPV (competitive NMDAR inhibitor), 30uM ifenprodil (ifen., GluN2B subunit inhibitor), and 10uM TCN201 (GluN2A subunit inhibitor)) was assessed via flow cytometry in B cells (+/- 5ug/mL LPS). B cell viability and neurotrophin (NT)-related genes were assessed by flow cytometry and qPCR, respectively, in B cells (+/- LPS) treated with glu +/- NMDAR antagonists for 24h. Data were analyzed in Graphpad Prism. Results: B cells express functional GluN2A- and GluN2B-containing NMDARs that influx Ca 2+ in response to extracellular glu (*p<0.05). While LPS did not impact NMDAR-dependent Ca 2+ influx in most B cell subsets, Ca 2+ influx was significantly reduced by NMDAR antagonists in LPS-stimulated B cells (Effector B cells (DAPV *p<0.05, ifen **p<0.01), Bregs (DAPV *p<0.05, Ifen *p<0.05), B220 + antibody-secreting cells (ifen *p<0.05, TCN201 *p<0.05)). Furthermore, a 24h glu treatment increased NT (BDNF: 2.28-fold, IL-10: 27.16-fold), NT receptor (TrkB: 1.33-fold) and NMDAR (GluN2A: 2.01-fold, GluN2B: 1.27-fold) expression in LPS-stimulated B cells (vs. untreated controls). Conclusions: Our studies show that B cells respond to glu via NMDARs. Our data suggests that exposure to physiologic levels of glu enhance NMDAR-dependent signaling and upregulate NTs and NT receptors. These results are the first to indicate a glu-induced neurotrophic role for B cells in the ischemic brain. Future studies will determine whether B cell-derived NTs can protect neurons after stroke.