Histone H2A and Bovine Neutrophil Extracellular Traps Induce Damage of Besnoitia besnoiti-Infected Host Endothelial Cells but Fail to Affect Total Parasite Proliferation.

Grob Grob,Zhou Zhou,Taubert Taubert,Conejeros Conejeros,Hermosilla Hermosilla,Velásquez Velásquez,Salecker Salecker

doi:10.3390/biology8040078

Abstract

Besnoitia besnoiti tachyzoites infect and develop in bovine endothelial cells in vivo and trigger the release of neutrophil extracellular traps (NETs) from bovine polymorphonuclear neutrophils (PMN). The purpose of this study was to analyze if pure B. besnoiti tachyzoite-triggered NETs would damage endothelial host cells and subsequently influence intracellular development and proliferation of B. besnoiti tachyzoites in primary bovine endothelial cells. For comparison purposes, isolated A23187-induced NETs were also used. Thus, we here evaluated endothelial host cell damage triggered by histone 2A (H2A) and B. besnoiti tachyzoite-induced NET preparations and furthermore estimated the effects of PMN floating over B. besnoiti-infected endothelium under physiological flow conditions on endothelial host cell viability. Overall, all treatments (H2A, B. besnoiti-triggered NETs and floating PMN) induced endothelial cell death of B. besnoiti-infected host cells. However, though host cell damage led to significantly altered intracellular parasite development with respect to parasitophorous vacuole diameter and numbers, the total proliferation of the parasite over time was not significantly affected by these treatments thereby denying any direct effect of NETs on intracellular B. besnoiti replication.

Highlights

The obligate intracellular parasite Besnoitia besnoiti (B. besnoiti) replicates in vivo in endothelium and represents the causal agent of besnoitiosis in cattle
Markers of neutrophil extracellular traps (NETs) of granular origin, such as neutrophil elastase, were detected colocalizing with histone and DNA when bovine polymorphonuclear neutrophils (PMN) were confronted with B. besnoiti tachyzoites at 1:4 ratio for 3 h and observed under confocal microscopy after immunostaining (Figure 1A) or after DNA staining with Sytox Orange (Supplementary Figure S4)
Using the NETs quantification method proposed by González et al [40] it was estimated that 15% of bovine PMN that were confronted with B. besnoiti tachyzoites release NETs (Supplementary Figure S2)

Summary

Introduction

The obligate intracellular parasite Besnoitia besnoiti (B. besnoiti) replicates in vivo in endothelium and represents the causal agent of besnoitiosis in cattle. Bovine besnoitiosis has a high impact on animal welfare and cattle production. Polymorphonuclear neutrophils (PMN) and endothelium are key players of host innate immune responses both interacting with B. besnoiti stages during acute infection [4]. In addition to classical effector mechanisms, such as reactive oxygen species (ROS) production, phagocytosis and degranulation, PMN are able to extrude chromatin structures decorated with granular proteins that are able to ensnare and eventually kill pathogens. These structures were first reported by Brinkmann et al [5] and named

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Results