The Contribution of Mitochondrial Calcium Efflux in Escherichia Coli-Induced Neutrophil Activation

Abstract

Neutrophils are the most abundant and fundamental components of innate immune cells that prevent the host from invading pathogens through antimicrobial strategies including phagocytosis, ROS, degranulation, and NETs extrusion. Several studies have reported the involvement of microbes to stimulate neutrophils. The current study was designed to in vestigate the role of mitochondria calcium efflux in Escherichia coli (E. coli)-induced neutrophil activation via pharmacological blockage of Na+/Ca2+ exchanger (NCLX). As the expression of NCLX in neutrophils is not known, we started with in silico analysis for evaluating its expression and 3-dimensional structures using various computational tools. Our bioinformatics analysis revealed the presence of NCLX in the myeloid lineage. Moreover, its predicted 3-dimensional structure showed the presence of several alpha hel ices indicating its role as a transmembrane protein acting as a channel. Further, whole blood (WB) was incubated with E.coli at varying time points to explore the effect of time on E.coli-induced activation of neutrophils. Different concentrations of NCLX inhibitor, CGP-37517, were used to evaluate the role of NCLX in E.coli-induced neutrophils stimu lation. Morphological characterization of E.coli-stimulated neutrophils illustrated various damage hallmarks including toxic vacuolization, DNA delobulation, decondensation, and ruptured plasma and nuclear membranes. An increase in neutrophil damage was observed with an increase in the infection time of E.coli. Interestingly, a slight elevation in E.coli induced neutrophil damage was noted in CGP-37157-pretreated WB. Similarly, there was a tendency toward the accelerated release of myeloperoxidase (MPO) and cell-free DNA in E.coli-treated enriched neutrophils with a further rise upon treatment with CGP-37167. In summary, the experimental data of the present study shows that mitochondrial calcium efflux probably has a role in the E.coli-induced activation of neutrophils. Keywords: E.coli, Neutrophils, Mitochondria, Mitochondrial Calcium Efflux, NCLX.