NMDA receptor modulation of glutamate release in activated neutrophils.

Ana Gutierrez Del Arroyo,Seth G.N Grant,Alexander V Gourine,Tom D Nightingale,Jenifer Sanchez,Simon Lambden,Anna Hadjihambi,Vitaly Kasymov,David Cain,Egor Turovsky,Gareth L Ackland

doi:10.1016/j.ebiom.2019.08.004

Abstract

BackgroundNeutrophil depletion improves neurologic outcomes in experimental sepsis/brain injury. We hypothesized that neutrophils may exacerbate neuronal injury through the release of neurotoxic quantities of the neurotransmitter glutamate.MethodsReal-time glutamate release by primary human neutrophils was determined using enzymatic biosensors. Bacterial and direct protein-kinase C (Phorbol 12-myristate 13-acetate; PMA) activation of neutrophils in human whole blood, isolated neutrophils or human cell lines were compared in the presence/absence of N-Methyl-d-aspartic acid receptor (NMDAR) antagonists. Bacterial and direct activation of neutrophils from wild-type and transgenic murine neutrophils deficient in NMDAR-scaffolding proteins were compared using flow cytometry (phagocytosis, reactive oxygen species (ROS) generation) and real-time respirometry (oxygen consumption).FindingsBoth glutamate and the NMDAR co-agonist d-serine are rapidly released by neutrophils in response to bacterial and PMA-induced activation. Pharmacological NMDAR blockade reduced both the autocrine release of glutamate, d-serine and the respiratory burst by activated primary human neutrophils. A highly specific small-molecule inhibitor ZL006 that limits NMDAR-mediated neuronal injury also reduced ROS by activated neutrophils in a murine model of peritonitis, via uncoupling of the NMDAR GluN2B subunit from its' scaffolding protein, postsynaptic density protein-95 (PSD-95). Genetic ablation of PSD-95 reduced ROS production by activated murine neutrophils. Pharmacological blockade of the NMDAR GluN2B subunit reduced primary human neutrophil activation induced by Pseudomonas fluorescens, a glutamate-secreting Gram-negative bacillus closely related to pathogens that cause hospital-acquired infections.InterpretationThese data suggest that release of glutamate by activated neutrophils augments ROS production in an autocrine manner via actions on NMDAR expressed by these cells.FundGLA: Academy Medical Sciences/Health Foundation Clinician Scientist. AVG is a Wellcome Trust Senior Research Fellow.

Highlights

Neurologic dysfunction is strongly linked to adverse outcomes and excess mortality in critical illness yet is often unrelated to the inciting pathology [1]
Phosphorylation of protein kinase C was not affected by pharmacologic GluN2B inhibition in primary human neutrophils, as assessed by immunoblot and flow cytometry (Supplementary Fig. 5). We examined whether this may be explained by a critical feature of N-methyl-d-aspartate receptor (NMDAR) neuronal biology, which requires protein kinase-C (PKC) activation to increase the probability of NMDA channel opening by reducing voltagedependent Mg2+ block of NMDA-receptor channels [20]
The principal finding of our studies is that glutamate may contribute to several aspects of neutrophil function via activation of complexes formed by the NMDAR and associated scaffolding proteins

Summary

Introduction

Neurologic dysfunction is strongly linked to adverse outcomes and excess mortality in critical illness yet is often unrelated to the inciting pathology [1]. The mechanisms underlying neurologic morbidity in critical illness remain incompletely understood, but systemic inflammation [2] and alterations in N-methyl-d-aspartate receptor (NMDAR) signalling are common features [3,4]. The mechanisms underlying neurologic morbidity in critical illness remain incompletely understood, but systemic inflammation and alterations in N-methyl-D-aspartate receptor signalling are common features. Our data suggest that autocrine release of potentially neurotoxic quantities of glutamate in activated neutrophils augments ROS production. Pharmacological NMDAR blockade reduced both the autocrine release of glutamate, D-serine and the respiratory burst by activated primary human neutrophils. Interpretation: These data suggest that release of glutamate by activated neutrophils augments ROS production in an autocrine manner via actions on NMDAR expressed by these cells.

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