The Contribution of Mitochondrial Calcium Exchange in Neutrophil Activation

Abstract

Neutrophils are the primary immune cells, that combat intrinsic and extrinsic stresses by various host defense mechanisms. Studies have reported hyperglycemia as an intrinsic inducer of neutrophil activation, which functions by increasing intracellular calcium and ROS. The current work investigated the role of mitochondrial calcium efflux in neutrophil activation brought on by high glucose in vitro. For this purpose, different concentrations of glucose were used to stimulate neutrophils in both whole blood (WB) and platelet lacking whole blood preparation (WBP). Isolated neutrophils were stimulated with 25mM glucose and neutrophil activation along with nuclear changes were analyzed by Sytox Green signal. Myeloperoxidase (MPO)-based degranulation in glucose-stimulated neutrophils was measured through spectrophotometry. To understand the role of mitochondrial calcium homeostasis neutrophils were pre-treated with CGP-37157 to inhibit NCLX, the calcium export channel in mitochondria. Under a bright field microscope, neutrophils in blood smears of WB and WBP showed activated morphology with more nuclear damage at high glucose concentrations that increased with time from 30 minutes to 5 hours. Fluorescence microscopy of isolated neutrophils showed the release of Sytox Green-positive chromatin filaments from activated neutrophils at high glucose concentrations. We also observed a maximum release of MPO within 15 minutes of in vitro incubation with glucose. Our brightfield microscopy data demonstrated that neutrophil activation triggered with glucose was amplified by CGP-37157. The observations of fluorescence microscopy also indicated that inhibition of NCLX increased the Sytox Green-positive neutrophils in glucose-stimulated samples. We also observed a decrease in the total cell count of neutrophils in CGP-pretreated and glucose-stimulated neutrophils which highlights the decreased viability probably due to NCLX inhibition. However, degranulation of MPO decreased in NCLX-inhibited glucose-stimulated neutrophils which might be due to a decrease in ATP generation after blocking NCLX. Overall, our data showed a significant contribution of mitochondrial calcium efflux in glucose-induced neutrophil activation. Keywords: Neutrophil, Mitochondrial Calcium Homeostasis, MCU, NCLX, Neutrophil Activation, MPO Degranulation