Bovine Polymorphonuclear Neutrophils Research Articles

Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling.

Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC50 of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

Cryptosporidium parvum-induced neutrophil extracellular traps in neonatal calves is a stage-independent process.

Infections with the apicomplexan obligate intracellular parasite Cryptosporidium parvum lead to cryptosporidiosis-a worldwide zoonotic infection. C. parvum is one of the most common diarrheal pathogens in young calves, which are the main reservoir of the pathogen. Cryptosporidiosis leads to severe economic losses in the calf industry and being a major contributor to diarrhea morbidity and mortality in children. Polymorphonuclear neutrophils (PMN) are part of the innate immune system. Their effector mechanisms directed against invasive parasites include phagocytosis, production of antimicrobial molecules as well as the formation of so-called neutrophil extracellular traps (NETs). Like other leukocytes of the innate immune system, PMN are thus able to release chromatin fibers enriched with antimicrobial granular molecules extracellularly thereby immobilizing and partially killing invasive bacteria, viruses, fungi and parasites. In vitro interactions of neonatal bovine PMN and C. parvum-oocysts and sporozoites were illustrated microscopically via scanning electron microscopy- and live cell imaging 3D holotomographic microscopy analyses. C. parvum-triggered NETosis was quantified via extracellular DNA measurements as well as verified via detection of NET-typical molecules [histones, neutrophil elastase (NE)] through immunofluorescence microscopy analysis. To verify the role of ATP in neonatal-derived NETosis, inhibition experiments were performed with NF449 (purinergic receptor antagonist with high specificity to P2X1 receptor). Using immunofluorescence- and SEM-based analyses, we demonstrate here for the first time that neonate bovine PMN are capable of forming NETs against C. parvum-sporozoites and oocysts, thus as a stage-independent cell death process. Our data further showed that C. parvum strongly induces suicidal neonatal NETosis in a P2X1-dependent manner, suggesting anti-cryptosporidial effects not only through firm sporozoite ensnarement and hampered sporozoite excystation, but also via direct exposure to NETs-associated toxic components.

Dynamics of cell cycle proteins involved in Toxoplasma gondii-induced bovine NET formation.

Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by chromatin and proteins with microbicidal and signaling activity. So far, there is one report on Toxoplasma gondii-triggered NETs in cattle, however, exact mechanisms, including signalling pathways and dynamics governing this reaction remain largely unknown. Recently, involvement of cell cycle proteins was demonstrated for phorbol myristate acetate (PMA)-triggered human PMN-derived NETs. Here, we studied the involvement of cell cycle proteins in T. gondii-induced NETs in exposed bovine PMN. Through confocal and transmission electron microscopy we discovered that Ki-67 and lamin B1 signals are upregulated and relocated during T. gondii-induced NETosis. Nuclear membrane disruption was also observed as a hallmark of NET formation in bovine PMN confronted with viable T. gondii tachyzoites, mimicking some steps of mitosis. However, we did not observe centrosome duplication as previously described for human PMN-derived NET formation stimulated with PMA.

Bovine neutrophil chemotaxis to Listeria monocytogenes in neurolisteriosis depends on microglia-released rather than bacterial factors

BackgroundListeria monocytogenes (Lm) is a bacterial pathogen of major concern for humans and ruminants due to its neuroinvasive potential and its ability to cause deadly encephalitis (neurolisteriosis). On one hand, polymorphonuclear neutrophils (PMN) are key players in the defense against Lm, but on the other hand intracerebral infiltration with PMN is associated with significant neural tissue damage. Lm-PMN interactions in neurolisteriosis are poorly investigated, and factors inducing PMN chemotaxis to infectious foci containing Lm in the central nervous system (CNS) remain unidentified.MethodsIn this study, we assessed bovine PMN chemotaxis towards Lm and supernatants of infected endogenous brain cell populations in ex vivo chemotaxis assays, to identify chemotactic stimuli for PMN chemotaxis towards Lm in the brain. In addition, microglial secretion of IL-8 was assessed both ex vivo and in situ.ResultsOur data show that neither Lm cell wall components nor intact bacteria elicit chemotaxis of bovine PMN ex vivo. Moreover, astrocytes and neural cells fail to induce bovine PMN chemotaxis upon infection. In contrast, supernatant from Lm infected microglia readily induced chemotaxis of bovine PMN. Microglial expression and secretion of IL-8 was identified during early Lm infection in vitro and in situ, although IL-8 blocking with a specific antibody could not abrogate PMN chemotaxis towards Lm infected microglial supernatant.ConclusionsThese data provide evidence that host-derived rather than bacterial factors trigger PMN chemotaxis to bacterial foci in the CNS, that microglia have a primary role as initiators of bovine PMN chemotaxis into the brain during neurolisteriosis and that blockade of these factors could be a therapeutic target to limit intrathecal PMN chemotaxis and PMN associated damage in neurolisteriosis.

Glycolysis, monocarboxylate transport, and purinergic signaling are key events in Eimeria bovis-induced NETosis.

The protozoan parasite Eimeria bovis is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis—an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)—is elicited by E. bovis stages. So far, the metabolic requirements of E. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in E. bovis sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied via chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished via PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in E. bovis sporozoite-induced NETosis.

Adverse Effects of Single Neutrophil Extracellular Trap-Derived Components on Bovine Sperm Function.

Simple SummaryNeutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract. However, the individual exposure of bull spermatozoa to NET-derived components has not yet been explored. The aim of this study was to evaluate the individual effects of the main NET-derived proteins on sperm parameters. Sperm were selected and incubated for 4 h with different NET-derived proteins. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. All NET-derived proteins/enzymes showed cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and advancement of spermatozoa in the female reproductive tract.Neutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract (FRT). The deleterious effects of NETs on spermatozoa have been previously described; however, individual exposure to NET-derived components in bull spermatozoa has not been explored. The aim of this study was to evaluate the effects of the main NET-derived proteins, histone 2A (H2A), neutrophil elastase (ELA), myeloperoxidase (MPO), pentraxin 3 (PTX), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), at concentrations of 1, 10, and 30 μg/mL, on sperm parameters. Sperm were selected and incubated with different NET-derived proteins for 4 h. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. Bovine polymorphonuclear neutrophil (PMN)/sperm co-cultures were evaluated by scanning electron microscopy and immunofluorescence. All NET-derived proteins/enzymes resulted in a reduction in membrane integrity, acrosome integrity, and lipoperoxidation at a concentration of 30 μg/mL. Bovine PMN/sperm co-cultures showed marked NET formation in the second hour. In conclusion, all NET-derived proteins/enzymes exerted cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and the advancement of spermatozoa in the FRT.

ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by Cryptosporidium parvum under Physioxia Conditions.

Simple SummaryCryptosporidium parvum, a zoonotic apicomplexan, is one of the leading causes of severe diarrheal disease worldwide and a major contributor to early infant and neonatal mortality. In vivo, sporozoites of C. parvum must invade enterocytes where they develop in a unique intracellular extracytoplasmic niche. Polymorphonuclear neutrophils (PMN) are the most common leukocytes in the immune defense system of the host, the first to be transferred to C. parvum-infected gut mucosa, and are increasingly recognized as important cells in the fight against other intestinal parasites. We investigated the role of ATP purinergic receptor P2X1, glycolysis, plasma membrane monocarboxylate transporters (MCTs) of lactate, extracellular acidification rates (ECAR), and mitochondrial oxygen consumption rates (OCR), as well as Notch signaling in C. parvum-triggered NETs formation in exposed bovine PMN under intestinal physioxic (5% O2) as well as hyperoxic (21% O2) conditions. Both C. parvum stages, i.e., sporozoites and oocysts, strongly induce suicidal NETosis in a P2X1-dependent manner, suggesting anti-cryptosporidial effects not only through firm sporozoite entrapment and hampered sporozoite excystation, but also probably through direct exposure to NETs-derived toxic molecules.Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by Cryptosporidium parvum. Neutrophil extracellular traps (NETs), also known as suicidal NETosis, are a powerful and ancient innate effector mechanism by which polymorphonuclear neutrophils (PMN) battle parasitic organisms like protozoa and helminths. Here, C. parvum oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN under both 5% O2 (physiological conditions within small intestinal tract) and 21% O2 (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O2) and hyperoxia (21% O2). Besides, C. parvum-induced suicidal NETosis was affirmed by total break of PMN, co-localization of extracellular DNA decorated with pan-histones (H1A, H2A/H2B, H3, H4) and neutrophil elastase (NE) by means of confocal- and immunofluorescence microscopy investigations. C. parvum-triggered NETs entrapped sporozoites and impeded sporozoite egress from oocysts covered by released NETs, according to scanning electron microscopy (SEM) examination. Live cell 3D-holotomographic microscopy analysis visualized early parasite-induced PMN morphological changes, such as the formation of membrane protrusions towards C. parvum while undergoing NETosis. Significant reduction of C. parvum-induced suicidal NETosis was measured after PMN treatments with purinergic receptor P2X1 inhibitor NF449, under both oxygen circumstances, this receptor was found to play a critical role in the induction of NETs, indicating its importance. Similarly, inhibition of PMN glycolysis via 2-deoxy glucose treatments resulted in a reduction of C. parvum-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in C. parvum-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce C. parvum-mediated NET extrusion. Concerning Notch signaling, no significant reduction was detected after PMN treatments with two specific Notch inhibitors, i.e., DAPT and compound E. Overall, we here describe for the first time the pivotal role of ATP purinergic receptor P2X1 in C. parvum-mediated suicidal NETosis under physioxia (5% O2) and its anti-cryptosporidial properties.

The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation.

Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites. Results: we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation. Conclusions: This is the first report on E. bovis-induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.

Semen extender and seminal plasma alter the extent of neutrophil extracellular traps (NET) formation in cattle

During artificial insemination in bovine, the deposition of semen into the uterus results in an immune reaction which is based on polymorphonuclear neutrophils activity, including the formation of neutrophil extracellular traps. The formation of neutrophil extracellular traps as a reaction of neutrophils to spermatozoa was recently described. However, it is not completely clear which components of the semen are responsible for this reaction. The objective of this study was to quantify and compare the formation of neutrophil extracellular traps following in vitro incubation of bovine polymorphonuclear neutrophils with semen and extenders of different origins and conditions. We investigated the interactions between bovine polymorphonuclear neutrophils and different semen extenders, various seminal plasma concentrations from young and old bulls as well as sexed and non-sexed semen and their corresponding extenders. Three different semen extenders from two companies in fresh and frozen-thawed conditions were compared. Fresh semen extenders showed higher neutrophil extracellular traps induction than did frozen-thawed ones. The formation of neutrophil extracellular traps were also dependent on the presence of seminal plasma. We could show that seminal plasma alone, without any sperm cells, induced the reaction and that the addition of at least 1% seminal plasma already resulted in the formation of neutrophil extracellular traps. Furthermore, seminal plasma from young bulls led to significant higher neutrophil extracellular traps induction. No difference between non-sex-sorted and sex-sorted sperm and its extenders was observed. Taken together, different semen extenders as well as the amount and origin of seminal plasma influence neutrophil extracellular traps formation, whereas sex-sorted sperm did not affect the reaction.

Trypanosoma brucei brucei Induces Polymorphonuclear Neutrophil Activation and Neutrophil Extracellular Traps Release.

Trypanosoma brucei brucei trypomastigotes are classical blood parasites of cattle, these stages might become potential targets for circulating polymorphonuclear neutrophils (PMN). We here investigated NETs extrusion and related oxygen consumption in bovine PMN exposed to motile T. b. brucei trypomastigotes in vitro. Parasite exposure induced PMN activation as detected by enhanced oxygen consumption rates (OCR), extracellular acidification rates (ECAR), and production of total and extracellular reactive oxygen species (ROS). Scanning electron microscopy (SEM) showed that co-cultivation of bovine PMN with motile trypomastigotes resulted in NETs formation within 120 min of exposure. T. b. brucei-induced NETs were confirmed by confocal microscopy demonstrating co-localization of extruded DNA with neutrophil elastase (NE) and nuclear histones. Immunofluorescence analyses demonstrated that trypomastigotes induced different phenotypes of NETs in bovine PMN, such as aggregated NETs (aggNETs), spread NETs (sprNETs), and diffuse NETs (diffNETs) with aggNETs being the most abundant ones. Furthermore, live cell 3D-holotomographic microscopy unveiled detailed morphological changes during the NETotic process. Quantification of T. b. brucei-induced NETs formation was estimated by DNA and nuclear area analysis (DANA) and confirmed enhanced NETs formation in response to trypomastigote stages. Formation of NETs does not result in a decrease of T. b. brucei viability, but a decrease of 26% in the number of motile parasites. Referring the involved signaling pathways, trypomastigote-induced NETs formation seems to be purinergic-dependent, since inhibition via NF449 treatment resulted in a significant reduction of T. b. brucei-triggered DNA extrusion. Overall, future studies will have to analyze whether the formation of aggNETs indeed plays a role in the outcome of clinical disease and bovine African trypanosomiasis-related immunopathological disorders, such as increased intravascular coagulopathy and vascular permeability, often reported to occur in this disease.

Fasciola hepatica induces weak NETosis and low production of intra- and extracellular ROS in exposed bovine polymorphonuclear neutrophils

Fasciola hepatica is the causative agent of fasciolosis, a worldwide distributed zoonotic disease, leading to hepatitis in humans and livestock. Newly excysted juveniles (NEJ) of F. hepatica are the first invasive stages to encounter leukocytes of host innate immune system in vivo. Among leukocytes, polymorphonuclear neutrophils (PMN) are the most abundant granulocytes of blood system and first ones to migrate into infection sites. PMN are able to cast neutrophil extracellular traps (NETs), also known as NETosis, consisting of nuclear DNA, decorated with histones, enzymes and antimicrobial peptides, which can entrap and eventually kill invasive parasites. Given that only few large parasitic helminths have been identified as potent NETosis inducers, here we studied for first time whether different F. hepatica stages can also trigger NETosis. Therefore, isolated bovine PMN were co-cultured with viable F. hepatica-NEJ, -metacercariae, -eggs and soluble antigen (FhAg). Interestingly, all stages failed to induce considerable levels of NETosis as detected by immunofluorescence- and scanning electron microscopy (SEM) analyses. NEJ remained motile until the end of incubation period. In line, NETosis quantification via nuclear area expansion (NAE) analysis revealed NEJ as weak NETosis inducers. However, bovine PMN frequently displaced towards motile NEJ and were found attached to NEJ surfaces. Functional PMN chemotaxis assays using vital F. hepatica-NEJ revealed a slight increase of PMN migration when compared to non-exposed controls. Additional experiments on intra- and extracellular reactive oxygen species (ROS) production revealed that soluble FhAg failed to induce ROS production of exposed PMN. Finally, mitochondrial oxygen consumption rates (OCR), extracellular acidification rates (ERAC) and proton production rates (PPR) were not significantly increased in FhAg-stimulated PMN. In summary, data suggest that F. hepatica might effectively evade PMN activation and NETosis by secreting parasite-specific molecules to either resolve NETs or to impair NETosis signaling pathways. We call for future molecular analysis not only on F. hepatica-derived NETosis modulation but also on its possible role in fasciolosis-associated pathology in vivo.

Pro-Inflammatory Response of Bovine Polymorphonuclear Cells Induced by Mycoplasma mycoides subsp. mycoides.

Mycoplasma mycoides subsp. mycoides (Mmm) is the etiological agent of contagious bovine pleuropneumonia (CBPP), one of the major diseases affecting cattle in sub-Saharan Africa. Some evidences suggest that the immune system of the host (cattle) plays an important role in the pathogenic mechanism of CBPP, but the factors involved in the process remain largely unknown. The present study aimed to investigate the cell response of bovine polymorphonuclear neutrophils (PMNs) after Mmm in vitro exposure using one step RT-qPCR and Western blotting. Data obtained indicate that gene and protein expression levels of some pro-inflammatory factors already change upon 30 min of PMNs exposure to Mmm. Of note, mRNA expression level in Mmm exposed PMNs increased in a time-dependent manner and for all time points investigated; targets expression was also detected by Western blotting in Mmm exposed PMNs only. These data demonstrate that when bovine PMN cells are triggered by Mmm, they undergo molecular changes, upregulating mRNA and protein expression of specific pro-inflammatory factors. These results provide additional information on host-pathogen interaction during CBPP infection.

Bovine sperm samples induce different NET phenotypes in a NADPH oxidase-, PAD4-, and Ca++-dependent process†.

Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMNs). Furthermore, sperm-mediated neutrophil extracellular trap (NET) formation (NETosis) was recently reported to occur in different mammalian species, including humans. We, here, investigated the interactions of bovine PMN with different semen-derived samples and analyzed in more depth molecular aspects of this effector mechanism. Overall, confrontation of PMN with sperm/cell preparation (SCP) resulted in a rapid and dose-dependent NET formation leading to effective spermatozoa entrapment. Thereby, spermatozoa induced different phenotypes of NETs. Immunostaining analyses revealed the presence of histones (H3), neutrophil elastase (NE), and pentraxin (PTX) in sperm-triggered NET structures. Fresh SCP strongly induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SCs) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm SNs but not purified SCs as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as an NADPH oxidase- and peptidylarginine deiminase 4-dependent process and proved to be dependent on intra- and extracellular Ca++ influxes while myeloperoxidase activity and as ERK1/2- and PI3K-related signaling pathways did not seem to play a pivotal role in this effector mechanism. From these findings, we speculate that sperm-derived NETosis might also occur in vivo during artificial insemination and might therefore play a role related to reduced fertility.

Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosis.

Besnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.

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

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.

Simultaneous and Positively Correlated NET Formation and Autophagy in Besnoitia besnoiti Tachyzoite-Exposed Bovine Polymorphonuclear Neutrophils.

Given that B. besnoiti tachyzoites infect host endothelial cells of vessels in vivo, they become potential targets for professional phagocytes [e.g., polymorphonuclear neutrophils (PMN)] when in search for adequate host cells or in case of host cell lysis. It was recently reported that B. besnoiti-tachyzoites can efficiently be trapped by neutrophil extracellular traps (NETs) released by bovine PMN. So far, the potential role of autophagy in parasite-triggered NET formation is unclear. Thus, we here analyzed autophagosome formation and activation of AMP-activated protein kinase α (AMPKα) in potentially NET-forming innate leukocytes being exposed to B. besnoiti tachyzoites. Blood was collected from healthy adult dairy cows, and bovine PMN were isolated via density gradient centrifugation. Scanning electron microscopy confirmed PMN to undergo NET formation upon contact with B. besnoiti tachyzoites. Nuclear area expansion (NAE) analysis and cell-free and anchored NETs quantification were performed in B. besnoiti-induced NET formation. Interestingly, tachyzoites of B. besnoiti additionally induced LC3B-related autophagosome formation in parallel to NET formation in bovine PMN. Notably, both rapamycin- and wortmannin-treatments failed to influence B. besnoiti-triggered NET formation and autophagosome formation. Also, isolated NETs fail to induce autophagy suggesting independence between both cellular processes. Finally, enhanced phosphorylation of AMP activated kinase α (AMPKα), a key regulator molecule of autophagy, was observed within the first minutes of interaction in tachyzoite-exposed PMN thereby emphasizing that B. besnoiti-triggered NET formation indeed occurs in parallel to autophagy.

Procoagulant activity of bovine neutrophils incubated with conditioned media or extracellular vesicles from Histophilus somni stimulated bovine brain endothelial cells

Histophilus somni is a Gram negative coccobacillus that causes respiratory, reproductive and central nervous system disease in cattle. The hallmark of H. somni infection is diffuse vasculitis and intravascular thrombosis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because neutrophils are major players in the pathophysiology of septic meningitis, we sought to determine their role in H. somni-induced fibrin clot formation in vitro. Bovine brain endothelial cells (TBBE cells) were exposed to H. somni cells at a 1:25 ratio, respectively. Conditioned media (CM) were collected after a 6 h incubation at 37 °C with 5% CO2, and then incubated with bovine peripheral blood polymorphonuclear neutrophils (PMNs). Following incubation, fibrin clot formation and tissue factor activity were assessed by a re-calcified plasma clotting assay. We found greater tissue factor activity in cell lysates and CM from H. somni-stimulated TBBE cells than unstimulated control TBBE cells. In addition, PMNs exposed to CM or extracellular vesicles from H. somni-stimulated TBBE cells expressed von Willenbrand factor, exhibited increased fibrin clot formation, and displayed greater tissue factor activity than PMNs exposed to CM or extracellular vesicles from unstimulated control TBBE cells. These results suggest that bovine PMNs might acquire extracellular vesicles from endothelial cells that leads to thrombus formation in bovine brain microvasculature and contribute to the process that characterizes TME.

14 Composition of Semen Extenders and Varying Seminal Plasma Concentrations Affects Formation of Neutrophil Extracellular Traps in the Bovine System

Neutrophils are recruited into the female reproductive tract following insemination to eliminate excess spermatozoa and bacteria introduced by the breeding process. In natural breeding, bovine semen is deposited in the vagina and sperm migrate into the uterus, leaving the bulk of seminal plasma (SP) behind. Current artificial insemination protocols introduce variable amounts of SP and semen extender into the uterus. Beside phagozytosis and secretion of immune modulators, polymorphonuclear neutrophils (PMN) are able to form neutrophil extracellular traps (NETs) extruding their DNA into the extracellular environment and ensnare pathogens including sperm cells. Recently, a time-dependent increase in bovine NETs formation has been reported. In the absence of SP and extender only a low degree of NETs formation by the sperm cells alone was detected (Fichtner et al. 2017 Reprod. Domest. Anim. 52(S1), 16). The aim of the present study was to investigate the effect of different semen extenders with various supplements or varying SP concentrations on NETs formation. Bovine PMN were isolated via Ficoll gradient centrifugation from peripheral blood. Semen extenders, purchased from 2 companies, were supplemented either with no animal protein or fresh egg yolk or “egg yolk-like substances” (gamma-irradiated sterile egg yolk or phospholipids as liposomes). The SP collected from ejaculates of eight 5-year-old bulls was added to the incubation medium in concentrations of 1, 3, 5, 10, 15, and 20%. For NETs induction, PMN and semen extenders or SP concentrations were co-cultured for 60 min; PMN and incubation medium alone served as negative controls. After incubation, the samples were treated with micrococcal nuclease and stained with PicoGreen. Quantification was performed by spectrofluorometric analyses using an automated plate monochrome reader (Varioscan Flash; Thermo Scientific, Waltham, MA, USA). Relative fluorescence intensities (FI in arbitrary units, AU) were calculated by subtracting the values of the negative controls from the ones obtained from the different samples. For statistical analyses, a one-way ANOVA followed by Tukey’s HSD test was used. Experiments were repeated at least 9 times. No significant differences in the relative FI were detected between the 2 animal protein-free extenders. A significantly higher FI was observed in one of the extenders supplemented with egg yolk compared with the other. The same held true for one extender completed with an egg yolk-like substance. Relative FI significantly increased from 1 to 5% SP, stayed constant up to 10%, followed by a slight decrease up to a concentration of 20%. These data indicate that formation of NETs in vitro depends on the composition of the extender itself and the protein source used by different companies. Furthermore, formation of NETs is also dependent on the dose of SP. From these results, it could be speculated that semen extenders plus additives and SP may affect fertility. The financial support of the Förderverein Bioökonomieforschung e.V. (FBF) is gratefully acknowledged.

Amiloride interferes with platelet- activating factor-induced respiratory burst and MMP-9 release in bovine neutrophils independent of Na+/H+ exchanger 1.

Cytoplasmic pH homeostasis is required for an appropriate response in polymorphonuclear neutrophils (PMNs). In these cells, chemotaxis and reactive oxygen species (ROS) production are reduced by the use of Na+/H+ exchanger (NHE-1) inhibitors, but these results are mainly obtained using amiloride, a non-selective NHE-1 inhibitor. In bovine PMNs, the role of NHE-1 in functional responses has not been confirmed yet. The aim of this study was to determine the role of NHE-1 using amiloride and zoniporide in pH regulation, ROS production, matrix metalloproteinase 9 (MMP-9) release and calcium flux in bovine PMNs induced by the platelet activation factor (PAF), additionally we evaluated the presence of NHE-1 and NHE-2 mRNA Our data show the presence only of NHE-1 but not NHE-2 in bovine PMNs. Amiloride or zoniporide inhibited the intracellular alkalization induced by PAF without affecting calcium flux. Amiloride diminished ROS production and MMP-9 release, while zoniporide enhanced ROS production without change the MMP-9 release induced by PAF. Our work led us to conclude that changes in intracellular pH induced by PAF are regulated by NHE-1 in bovine neutrophils, but the effects of amiloride on ROS production and MMP-9 release induced by PAF are not NHE-1 dependent.

Bovine Polymorphonuclear Neutrophils Cast Neutrophil Extracellular Traps against the Abortive Parasite Neospora caninum.

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.