3D holotomographic monitoring of Ca++ dynamics during ionophore-induced Neospora caninum tachyzoite egress from primary bovine host endothelial cells.

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.

Propionate induces the release of granules from bovine neutrophils.

Short-chain fatty acids (SCFA) are produced by bacterial fermentation in the rumen of cattle and are the primary energy source in ruminants. Propionate is one of the main SCFA and it can exert multiple effects on the inflammatory process and neutrophil function via calcium (Ca(2+)) release, reactive oxygen species, and intracellular pH changes. However, currently no evidence has shown whether propionate can induce granule release from bovine neutrophils. The purpose of this study was to analyze the effect of propionate on granule release and to evaluate the expression of two G-protein coupled receptors-GPR41 and GPR43-that are activated by propionate. Neutrophil degranulation was assessed by quantifying the release of the neutrophil enzymes myeloperoxidase (MPO), lactoferrin, and matrix metalloprotease-9 (MMP-9) as markers of azurophil, specific granules, and gelatinase granules, respectively. Isolated bovine neutrophils were treated with millimolar concentrations of propionate (0.3, 3 and 30mM), and the cell-free supernatants were recovered. The stimulation of neutrophils with 0.3mM propionate induced the release of lactoferrin and MMP-9 as revealed by ELISA and gelatin zymography, respectively. Propionate at 30mM induced the release of MPO as demonstrated using an enzymatic assay. The role of intracellular Ca(2+) influx and the signaling pathways that may regulate the propionate effect on granules release were also determined. Reverse transcription (RT)-PCR and real-time PCR were performed to analyze the expression of GPR41 and GPR43 mRNA in bovine neutrophils. Both mRNA were detected, whereas the expression of GPR43 was higher than that of GPR41, and the synthetic agonists for this receptor, phenylacetamides 1 and 2, caused an increase in intracellular Ca(2+), lactoferrin, and MMP-9 release. These results support that propionate-induced granule release is mediated by intracellular Ca(2+) influx and activation of extracellular signal-regulated kinase ERK 1/2. We also propose a potential role of GPR43 in propionate-induced granule release from bovine neutrophils that may be involved in regulatory effects of propionate in the innate immune response in cattle.

2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 µM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 µM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle.

2-Aminoethoxydiphenyl borate (2-APB) reduces respiratory burst, MMP-9 release and CD11b expression, and increases l-selectin shedding in bovine neutrophils.

This study describes the effect of 2-aminoethoxydiphenyl borate (2-APB), a putative store-operated calcium (Ca(2+)) entry (SOCE) inhibitor, on reactive oxygen species (ROS) production, matrix metalloproteinase 9 (MMP-9) release, CD11b and l-selectin (CD62L) expression, size changes and apoptosis in bovine neutrophils stimulated with platelet-activating factor (PAF). It was observed that doses ⩾1µM 2-APB significantly reduced ROS production, whereas 50 and 100µM 2-APB reduced MMP-9 release induced by PAF. Moreover, concentrations ⩾10µM 2-APB reduced CD11b expression and increased l-selectin shedding. PAF induced size changes in neutrophils, and this effect was inhibited by 2-APB. From this work it is possible to conclude that 2-APB at concentrations that inhibit SOCE responses was able to inhibit ROS and MMP-9 release and CD11b expression, and increase l-selectin shedding, suggesting that the Ca(2+) channel involved in SOCE is a potential target for the development of new anti-inflammatory drugs in cattle.

D-lactic acid interferes with the effects of platelet activating factor on bovine neutrophils.

D-lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of D-lactic acid and measured intracellular pH using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that D-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100 nM platelet-activating factor (PAF). D-lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease L-selectin expression, and induces late apoptosis. We concluded that D-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10mM D-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis.

Calcium influx, a new potential therapeutic target in the control of neutrophil-dependent inflammatory diseases in bovines.

Neutrophils are the first line of defense against pathogens in bovines; however, they are also one of the most aggressive cells during the inflammatory process, causing injury in surrounding tissues. At present, anti-inflammatory drugs are limited in acute diseases, such as pneumonia, mastitis and endometritis, because neutrophils are mostly insensitive. One of the earliest events during neutrophil activation is the increase in intracellular calcium concentration. The calcium movement is attributed to the release from intracellular stores and influx through the calcium channels in the plasma membrane, a process called store operated calcium entry (SOCE). Recently, several calcium influx blockers have been shown to have strong effects on bovine neutrophils, and this suggests that the manipulation of this pathway can be useful in the control of neutrophil functions during acute inflammatory processes. In this paper, we will review the role of calcium influx as a potential anti-inflammatory target and summarize the most recent evidences for this in bovine neutrophils.

Induction of reactive oxygen species in bovine neutrophils is CD11b, but not dectin-1-dependent.

Neutrophils play a key role in initiating an innate immune response, being the first type of immune cell arriving at the site of injury or infection. These cells are able to mount a direct anti-bactericidal response by the production of reactive oxygen or reactive nitrogen species (ROS/RNS). An important component of the host innate immune response is recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are an important family of PRRs and, are a key component in activation of innate immune mechanisms. In the present study we described the presence of mRNA transcripts for TLR1, TLR2, TLR4, TLR6, TLR7 and TLR10 in bovine neutrophils. In contrast, the presence of mRNA transcripts for TLR3 varied between animals, whereas no transcripts were detected for TLR5, TLR8, TLR9 or the C-type lectin receptor dectin-1 in neutrophils isolated from bovine blood. Additionally, zymosan, a dectin-1/TLR2 ligand, induced ROS, but not RNS production in a CD11b-, but not dectin-1-dependent manner. This effect was dependent on Store Operated Calcium Entry (SOCE), and partially inhibited using monoclonal antibodies to CD11b. Taken together, our data describe the presence of specific PRRs transcripts in the mRNA isolated from bovine neutrophil and show a CD11b-/Ca(2+) dependent ROS production by these cells.

New potential targets to modulate neutrophil function in inflammation.

The importance of neutrophils in human disease such as rheumatoid arthritis, asthma, adult respiratory distress syndrome, and COPD has prompted the search for drugs capable to slow down neutrophil-dependent inflammation, without interference with innate immune responses. In this review, we summarize new potential drugs targets against neutrophils mediated inflammatory responses.