Abstract
Neospora caninum represents a relevant apicomplexan parasite causing severe reproductive disorders in cattle worldwide. Neutrophil extracellular trap (NET) generation was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites. In vitro interactions of bovine PMN with N. caninum were analyzed at different ratios and time spans. Extracellular DNA staining was used to illustrate the typical molecules of NETs [i.e., histones (H3), neutrophil elastase (NE), myeloperoxidase (MPO), pentraxin] via antibody-based immunofluorescence analyses. Functional inhibitor treatments were applied to reveal the role of several enzymes [NADPH oxidase (NOX), NE, MPO, PAD4], ATP-dependent P2Y2 receptor, store-operated Ca++entry (SOCE), CD11b receptor, ERK1/2- and p38 MAPK-mediated signaling pathway in tachyzoite-triggered NETosis. N. caninum tachyzoites triggered NETosis in a time- and dose-dependent manner. Scanning electron microscopy analyses revealed NET structures being released by bovine PMN and entrapping tachyzoites. N. caninum-induced NET formation was found not to be NOX-, NE-, MPO-, PAD4-, ERK1/2-, and p38 MAP kinase-dependent process since inhibition of these enzymes led to a slight decrease of NET formation. CD11b was also identified as a neutrophil receptor being involved in NETosis. Furthermore, N. caninum-triggered NETosis depends on Ca++ influx as well as neutrophil metabolism since both the inhibition of SOCE and of P2Y2-mediated ATP uptake diminished NET formation. Host cell invasion assays indicated that PMN-derived NETosis hampered tachyzoites from active host cell invasion, thereby inhibiting further intracellular replication. NET formation represents an early and effective mechanism of response of the innate immune system, which might reduce initial infection rates during the acute phase of cattle neosporosis.
Highlights
Neospora caninum is an apicomplexan obligate intracellular parasite with comparable characteristics in structure and development to Toxoplasma gondii [1]
NETosis is known as a NADPH oxidase (NOX)-dependent mechanism [10, 12, 13, 17, 19], which leads to the extrusion of nuclear and cytoplasmic granule enzymes leading to the formation of DNA-rich networks adorned with different histones (H1, H2A/H2B, H3, H4) and antimicrobial granular effector molecules, such as neutrophil elastase (NE), myeloperoxidase (MPO), pentraxin, lactoferrin, cathepsins, gelatinase, bacterial permeability-increasing protein, peptidoglycan recognition proteins, calprotectin, and other leukocyte proteins [10, 16, 17, 20, 21]
Scanning electron microscopy analyses revealed tachyzoitetriggered generation of a fine network of grosser and slimmer strands of fibers produced by bovine polymorphonuclear neutrophils (PMN) and being solidly adhered to tachyzoites
Summary
Neospora caninum is an apicomplexan obligate intracellular parasite with comparable characteristics in structure and development to Toxoplasma gondii [1] It has a wide intermediate host range and is responsible for reproductive disorders mainly in cattle but is associated with clinical reproductive and neural infections in dogs, horses, goats, sheep, and deer [2,3,4]. Yipp et al [27] recently demonstrated that PMN, which undergo NETosis without cell lysis, remain viable and retain their ability to phagocytise bacteria In agreement with these findings, PMN seem to be able to release small-sized NETs of mitochondrial origin without suffering cell death [28]. Blood vessel analyses of P. falciparum-infected patients [29] and intestinal tissue samples of Eimeria-infected goats and cattle proved apicomplexan parasite-triggered NETosis to happen in vivo [38]
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