Eimeria bovis is an important coccidian parasite that causes high economic losses in the cattle industry. We recently showed that polymorphonuclear neutrophils (PMN) react upon E. bovis sporozoite exposure by neutrophil extracellular trap (NET) formation. We focused here on the molecular mechanisms that are involved in this process. The sporozoite encounter led to an enhanced surface expression of neutrophil CD11b suggesting a potential role of this receptor in E. bovis-mediated NETosis. Antibody-mediated blockage of CD11b confirmed this assumption and led to a significantly decreased sporozoite-triggered NET. In addition, E. bovis-induced NETosis was found to be Ca2+-dependent since the inhibition of store-operated calcium entry (SOCE) significantly diminished NET. Furthermore, NADPH oxidase, neutrophil elastase (NE) and myeloperoxidase (MPO) were confirmed as key molecules in sporozoite-triggered NETosis, as inhibition thereof blocked parasite-triggered NET. PMN degranulation analyses revealed a significant release of matrix metalloprotease-9 containing granules upon sporozoite exposure. We further show a significantly enhanced phosphorylation of ERK1/2 and p38 MAPK in sporozoite-exposed PMN indicating a key role of this signaling pathway in E. bovis-mediated NETosis. Accordingly, ERK 1/2 and p38 MAPK inhibition led to a significant decrease in NET formation. Finally, we demonstrate that sporozoite-induced NETosis is neither a stage-, species-, nor host-specific process.

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Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(−) lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(−) lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(−) lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET) production (NETosis) in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(−) lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(−) lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1). d(−) lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(−) lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(−) lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

Andrographolide is a labdane diterpene and the main active ingredient isolated from the herb Andrographis paniculata. Andrographolide possesses diverse biological effects including anti-inflammatory, antioxidant, and antineoplastic properties. Clinical studies have demonstrated that andrographolide could be useful in therapy for a wide range of diseases such as osteoarthritis, upper respiratory diseases, and multiple sclerosis. Several targets are described for andrographolide, including the interference of transcription factors NF-κB, AP-1, and HIF-1 and signaling pathways such as PI3K/Akt, MAPK, and JAK/STAT. In addition, an increase in the Nrf2 (nuclear factor erythroid 2–related factor 2) signaling pathway also supports its antioxidant and anti-inflammatory properties. However, this scenario could be more complex since recent evidence suggests that andrographolide targets can modulate glucose metabolism. The metabolic effect of andrographolide might be the key to explaining the diverse therapeutic effects described in preclinical and clinical studies. This review discusses some of the most recent evidence about the anti-inflammatory and metabolic effects of andrographolide.

During an inflammatory process, shift in the cellular metabolism associated with an increase in extracellular acidification are well-known features. This pH drop in the inflamed tissue is largely attributed to the presence of lactate by an increase in glycolysis. In recent years, evidence has accumulated describing the role of lactate in inflammatory processes; however, there are differences as to whether lactate can currently be considered a pro- or anti-inflammatory mediator. Herein, we review these recent advances on the pleiotropic effects of lactate on the inflammatory process. Taken together, the evidence suggests that lactate could exert differential effects depending on the metabolic status, cell type in which the effects of lactate are studied, and the pathological process analyzed. Additionally, various targets, including post-translational modifications, G-protein coupled receptor and transcription factor activation such as NF-κB and HIF-1, allow lactate to modulate signaling pathways that control the expression of cytokines, chemokines, adhesion molecules, and several enzymes associated with immune response and metabolism. Altogether, this would explain its varied effects on inflammatory processes beyond its well-known role as a waste product of metabolism.

Background Acute ruminal acidosis (ARA) is a metabolic disease of cattle characterized by an aseptic synovitis. ARA is the result of an increased intake of highly fermentable carbohydrates that frequently occurs in dairy cattle subjected to high production requirements. In human joint diseases such as rheumatoid arthritis and gout, several pro-inflammatory molecules are increased in the synovial fluid, including cytokines, prostaglandin E 2 (PGE 2 ), metalloproteinases, and neutrophil extracellular traps (NETs). The aim of this study was to identify the presence of proinflammatory mediators and neutrophils in the synovial fluid of heifers with ARA, induced by an oligofructose overload. Five heifers were challenged with an oligofructose overload (13 g/kg BW) dissolved in water. As a control, a similar vehicle volume was used in four heifers. Synovial fluid samples were collected from the tarso-crural joint and PGE 2 , IL-6, IL-1β, ATP, lactate dehydrogenase (LDH), albumin, glucose, matrix metalloproteinase-9 (MMP-9), cellular free DNA, NETs, and serpin B1 were analyzed at 0, 9, and 24 h post treatment. Results At 9 h post oligofructose overload, an increase of IL-1β, IL-6, PGE 2 , serpin B1 and LDH was detected in the joints when compared to the control group. At 24 h, the synovial fluid was yellowish, viscous, turbid, and contained abundant neutrophils. An increase of DNA-backbone-like traps, histone 3 (H 3 cit), aggregated neutrophil extracellular traps ( agg NETs), and serpin B1 were observed 24 h post treatment. Furthermore, albumins, LDH, ATP, MMP-9, IL-6, and IL-1β were increased after 24 h. Conclusions The overall results indicate that IL-1β, IL-6 and PGE 2 , were the earliest proinflammatory parameters that increased in the synovial fluid of animals with ARA. Furthermore, the most sever inflammatory response in the joint was observed after 24 h and could be associated with a massive presence of neutrophils and release of agg NETs. Electronic supplementary material The online version of this article (10.1186/s12917-019-1974-x) contains supplementary material, which is available to authorized users.

Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood neutrophils. Blood neutrophils and plasma were obtained by jugular venipuncture, while ruminal samples were collected using rumenocentesis. Lactic acid from plasma and ruminal samples was measured by HPLC. PAF-induced ROS production and L-selectin shedding were measured in vitro in bovine neutrophils by a luminol chemiluminescence assay and flow cytometry, respectively. A significant increase in ruminal and plasma lactic acid was recorded in these animals. Specifically, a decrease in PAF-induced ROS production was observed 8 h after oligofructose overload, and this was sustained until 48 h post oligofructose overload. A reduction in PAF-induced L-selectin shedding was observed at 16 h and 32 h post oligofructose overload. Overall, the results indicated that neutrophil PAF responses were altered in heifers with ruminal acidosis, suggesting a potential dysfunction of the innate immune response.

Non-esterified fatty acids (NEFAs) such as oleic acid (OA) and linoleic acid (LA) are associated with a higher incidence of infectious diseases such as metritis and mastitis during the bovine peripartum. Fatty acids can induce an increase in the release of ATP, and changes in the expression levels of purinergic receptors in bovine polymorphonuclears (PMN) during peripartum have also been reported. PMN respond to inflammatory processes with production of ROS, release of proteolytic and bactericidal proteins, and formation of neutrophil extracellular traps (NETs). NETs formation is known to require ATP production through glycolysis. Studies have shown that the above-mentioned metabolic changes alter innate immune responses, particularly in PMN. We hypothesized that NEFAs induce the formation of NETs through ATP release by Pannexin 1 and activation of purinergic receptors. In this study, we found that OA and LA induce NET formation and extracellular ATP release. Carbenoxolone, a pannexin-1 (PANX1) inhibitor, reduced OA-and LA-induced ATP release. We also found that P2X1, P2X4, P2X5, P2X7, and PANX1 were expressed at the mRNA level in bovine PMN.Additionally, NEFA-induced NET formation was completely abolished with exposure to NF449, a P2X1 antagonist, and partially inhibited by treatment with etomoxir, an inhibitor of fatty acid oxidation (FAO). Our results suggest that OA and LA induce NET formation and ATP release via PANX1 and activation of P2X1. These new data contribute to explaining the effects of NEFA high concentrations during the transition period of dairy cattle and further understanding of pro-inflammatory effects and outcome of postpartum diseases.