Andrographolide Ameliorates Inflammatory Changes Induced by D-Lactate in Bovine Fibroblast-like Synoviocytes.

During acute ruminal acidosis, the manifestation of aseptic polysynovitis and lameness in cattle has been observed. Evidence suggests that joint inflammation can be attributed to the metabolic alterations induced by D-lactate in fibroblast-like synoviocytes (FLSs). We aimed to investigate whether andrographolide could mitigate the inflammation and metabolic alterations induced by D-lactate in bovine fibroblast-like synoviocytes (bFLSs). To assess this, bFLSs were cultured in the presence or absence of andrographolide. We evaluated its potential interference with the expression of proinflammatory cytokines, COX-2, HIF-1α, and LDHA using RT-qPCR. Furthermore, we investigated its potential interference with PI3K/Akt signaling and IκBα degradation through immunoblotting and flow cytometry, respectively. Our observations revealed that andrographolide reduced the elevation of IL-6, IL-8, COX-2, HIF-1α, and LDHA induced by D-lactate. Additionally, andrographolide demonstrated interference with the PI3K/Akt and NF-κB pathways in bFLSs. In conclusion, our findings suggest that andrographolide can potentially reverse the inflammatory effects and metabolic changes induced by D-lactate in bFLSs, showing promise as a therapeutic intervention for managing these conditions associated with lameness.

d-lactate-induced ETosis in cattle polymorphonuclear leucocytes is dependent on the release of mitochondrial reactive oxygen species and the PI3K/Akt/HIF-1 and GSK-3ß pathways.

d-lactate is a metabolite originating from bacterial metabolism that accumulates as a result of dietary disturbances in cattle, leading to ruminal acidosis. d-lactate exerts functions as a metabolic signal inducing metabolic reprogramming and extracellular trap (ET) release in polymorphonuclear leucocytes (PMNs). We previously demonstrated that d-lactate induces metabolic reprogramming via hypoxia-induced factor 1 alpha (HIF-1α) stabilization in bovine fibroblast-like synoviocytes (FLSs). In the present study, the role of HIF-1 in ET formation induced by d-lactate was assessed. HIF-1α stabilization in PMNs was controlled by mitochondrial reactive oxygen species (mtROS) release. Furthermore, inhibition of mitochondrial complex I and scavenging of mtROS decreased d-lactate-triggered ETosis. d-lactate-enhanced HIF-1α accumulation was dependent on the PI3K/Akt pathway but independent of GSK-3ß activity. Pharmacological blockade of the PI3K/Akt/HIF-1 and GSK-3ß axes inhibited d-lactate-triggered ETosis and downregulated PDK1 and LDHA expression. However, only GSK-3ß inhibition decreased the expression of glycogen metabolism enzymes and prevented the decline in glycogen stores induced by d-lactate exposure. The results of this study suggest that mtROS, PI3K/Akt/HIF-1 and GSK-3ß axes regulate carbohydrate metabolism adaptations that support d-lactate-induced ET formation in cattle.

Bovine tumor necrosis factor-alpha Increases IL-6, IL-8, and PGE2 in bovine fibroblast-like synoviocytes by metabolic reprogramming.

Lameness is a common condition in dairy cattle caused by infectious or noninfectious agents. Joint lesions are the second most common cause of lameness and can be diagnosed in association with the presentation of digit injuries. Fibroblast-like synoviocyte (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the expression of proinflammatory mediators. Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine involved in cyclooxygenase 2 (COX-2) and proinflammatory cytokine expression in FLS. Previously, TNF-α was demonstrated to increase hypoxia-inducible Factor 1 (HIF-1), a transcription factor that rewires cellular metabolism and increases the expression of interleukin (IL)-6 in bovine FLS (bFLS). Despite this, the proinflammatory effects of TNF-α in bFLS on metabolic reprogramming have been poorly studied. We hypothesized that TNF-α increases glycolysis and in this way controls the expression of IL-6, IL-8, and COX-2 in bFLS. Results first, gas chromatography/mass spectrometry (GC/MS)-based untargeted metabolomics revealed that bTNF-α altered the metabolism of bFLS, increasing glucose, isoleucine, leucine, methionine, valine, tyrosine, and lysine and decreasing malate, fumarate, α-ketoglutarate, stearate, palmitate, laurate, aspartate, and alanine. In addition, metabolic flux analysis using D-glucose-13C6 demonstrated an increase of pyruvate and a reduction in malate and citrate levels, suggesting a decreased flux toward the tricarboxylic acid cycle after bTNF-α stimulation. However, bTNF-α increased lactate dehydrogenase subunit A (LDHA), IL-6, IL-8, IL-1ß and COX-2 expression, which was dependent on glycolysis and the PI3K/Akt pathway. The use of FX11 and dichloroacetate (DCA), an inhibitor of LDHA and pyruvate dehydrogenase kinase (PDK) respectively, partially reduced the expression of IL-6. Our results suggest that bTNF-α induces metabolic reprogramming that favors glycolysis in bFLS and increases IL-6, IL-8, IL-1ß and COX-2/PGE2.

d-lactate-triggered extracellular trap formation in cattle polymorphonuclear leucocytes is glucose metabolism dependent.

D-lactic acidosis is a metabolic disease of cattle caused by the digestive overgrowth of bacteria that are highly producers of d-lactate, a metabolite that then reaches and accumulates in the bloodstream. d-lactate is a proinflammatory agent in cattle that induces the formation of extracellular traps (ETs) in polymorphonuclear leucocytes (PMN), although information on PMN metabolic requirements for this response mechanism is insufficient. In the present study, metabolic pathways involved in ET formation induced by d-lactate were studied. We show that d-lactate but not l-lactate induced ET formation in cattle PMN. We analyzed the metabolomic changes induced by d-lactate in bovine PMN using gas chromatography-mass spectrometry (GC-MS). Several metabolic pathways were altered, including glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, galactose metabolism, starch and sucrose metabolism, fructose and mannose metabolism, and pentose phosphate pathway. d-lactate increased intracellular levels of glucose and glucose-6-phosphate, and increased uptake of the fluorescent glucose analog 2-NBDG, suggesting improved glycolytic activity. In addition, using an enzymatic assay and transmission electron microscopy (TEM), we observed that d-lactate was able to decrease intracellular glycogen levels and the presence of glycogen granules. Relatedly, d-lactate increased the expression of enzymes of glycolysis, gluconeogenesis and glycogen metabolism. In addition, 2DG (a hexokinase inhibitor), 3PO (a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 inhibitor), MB05032 (inhibitor of fructose-1,6-bisphosphatase) and CP-91149 (inhibitor of glycogen phosphorylase) reduced d-lactate-triggered ETosis. Taken together, these results suggest that d-lactate induces a metabolic rewiring that increases glycolysis, gluconeogenesis and glycogenolysis, all of which are required for d-lactate-induced ET release in cattle PMN.

Metabolic Reprogramming and Inflammatory Response Induced by D-Lactate in Bovine Fibroblast-Like Synoviocytes Depends on HIF-1 Activity.

Acute ruminal acidosis (ARA) occurs after an excessive intake of rapidly fermentable carbohydrates and is characterized by the overproduction of D-lactate in the rumen that reaches the bloodstream. Lameness presentation, one of the primary consequences of ARA in cattle, is associated with the occurrence of laminitis and aseptic polysynovitis. Fibroblast-like synoviocytes (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the chances of the release of pro-inflammatory cytokines. Increased D-lactate levels and disturbances in the metabolism of carbohydrates, pyruvates, and amino acids are observed in the synovial fluid of heifers with ARA-related polysynovitis prior to neutrophil infiltration, suggesting an early involvement of metabolic disturbances in joint inflammation. We hypothesized that D-lactate induces metabolic reprogramming, along with an inflammatory response, in bovine exposed FLS. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics revealed that D-lactate disrupts the metabolism of bovine FLS, mainly enhancing glycolysis and gluconeogenesis, pyruvate metabolism, and galactose metabolism. The reverse-transcription quantitative PCR (RT-qPCR) analysis revealed an increased expression of metabolic-related genes, including hypoxia-inducible factor 1 (HIF-1)α, glucose transporter 1 (Glut-1), L-lactate dehydrogenase subunit A (L-LDHA), and pyruvate dehydrogenase kinase 1 (PDK-1). Along with metabolic disturbances, D-lactate also induced an overexpression and the secretion of IL-6. Furthermore, the inhibition of HIF-1, PI3K/Akt, and NF-κB reduced the expression of IL-6 and metabolic-related genes. The results of this study reveal a potential role for D-lactate in bFLS metabolic reprogramming and support a close relationship between inflammation and metabolism in cattle.

Role of Lactate in Inflammatory Processes: Friend or Foe.

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.

Eimeria bovis infections induce G1 cell cycle arrest and a senescence-like phenotype in endothelial host cells.

Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced ß-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.

Andrographolide, an Anti-Inflammatory Multitarget Drug: All Roads Lead to Cellular Metabolism.

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.

D-Lactate Increases Cytokine Production in Bovine Fibroblast-Like Synoviocytes via MCT1 Uptake and the MAPK, PI3K/Akt, and NFκB Pathways.

Acute ruminal acidosis (ARA) is caused by the excessive intake of highly fermentable carbohydrates, followed by the massive production of D-lactate and the appearance of neutrophilic aseptic polysynovitis. Bovines with ARA develop different lesions, such as ruminitis, polioencephalomalacia (calves), liver abscess and lameness. Lameness in cattle with ARA is closely associated with the presence of laminitis and polysynovitis. However, despite decades of research in bovine lameness as consequence of ruminal acidosis, the aetiology and pathogenesis remain unclear. Fibroblast-like synoviocytes (FLSs) are components of synovial tissue, and under pathological conditions, FLSs increase cytokine production, aggravating inflammatory responses. We hypothesized that D-lactate could induce cytokine production in bovine FLSs. Analysis by qRT-PCR and ELISA revealed that D-lactate, but not L-lactate, increased the expression of IL-6 and IL-8 in a monocarboxylate transporter-1-dependent manner. In addition, we observed that the inhibition of the p38, ERK1/2, PI3K/Akt, and NF-κB pathways reduced the production of IL-8 and IL-6. In conclusion, our results suggest that D-lactate induces an inflammatory response; this study contributes to the literature by revealing a potential key role of D-lactate in the polysynovitis of cattle with ARA.

Metabolomics analysis of bronchoalveolar lavage fluid samples in horses with naturally-occurring asthma and experimentally-induced airway inflammation.

The present work characterized the metabolomic profile of bronchoalveolar lavage fluid (BALF) in healthy horses, experimentally-induced airway inflammation by lipopolysaccharide (LPS) nebulization, and naturally-occurring asthma (n = 3 in each group). All animals underwent clinical and upper airway endoscopic examinations, and bronchoalveolar lavage. BALF supernatant samples were subjected to metabolic analysis based on gas chromatography-mass spectrometry (GC-MS). Overall, 67 peaks were obtained from BALF GC-MS analysis, corresponding to 53 metabolites which were categorized according to chemical class, such as organic acids, fatty acids, nucleosides or their derivatives, amino acids, peptides or their derivatives, carbohydrates, and other compounds. Our results showed that the airway inflammation induction model with LPS produced the same pattern of metabolite changes as in horses with naturally occurring asthma. Metabolic pathway analysis was done by means of Fisher's exact test, for detection of metabolites over-represented in asthma affected-horses and LPS-induced airway inflammation as compared with healthy horses. The most significant altered metabolic pathways were fatty acid biosynthesis, galactose metabolism and citrate cycle. These results suggest that the airway inflammation induction model with LPS is a good study model for asthma-affected horses, due to the similarity of the profile of inflammatory cells (specifically neutrophils) and similar metabolic alterations found in BALF that occur during the inflammatory process of the airways. Further research may increase understanding of metabolomics disturbances and their significance in the pathogenesis of equine asthma.

Hydrocortisone decreases metacognitive efficiency independent of perceived stress.

It is well established that acute stress produces negative effects on high level cognitive functions. However, these effects could be due to the physiological components of the stress response (among which cortisol secretion is prominent), to its psychological concomitants (the thoughts generated by the stressor) or to any combination of those. Our study shows for the first time that the typical cortisol response to stress is sufficient to impair metacognition, that is the ability to monitor one's own performance in a task. In a pharmacological protocol, we administered either 20 mg hydrocortisone or placebo to 46 male participants, and measured their subjective perception of stress, their performance in a perceptual task, and their metacognitive ability. We found that hydrocortisone selectively impaired metacognitive ability, without affecting task performance or creating a subjective state of stress. In other words, the single physiological response of stress produces a net effect on metacognition. These results inform our basic understanding of the physiological bases of metacognition. They are also relevant for applied or clinical research about situations involving stress, anxiety, depression, or simply cortisol use.

Mitochondria-derived ATP participates in the formation of neutrophil extracellular traps induced by platelet-activating factor through purinergic signaling in cows.

Neutrophil extracellular trap (NET) formation eliminates/prevents the spread of infectious agents. Platelet activating factor (PAF) is involved in infectious diseases of cattle because it recruits and activates neutrophils. However, its ability to induce NET release and the role of metabolism in this process is not known. We investigated if inhibition of glycolysis, mitochondrial-derived adenosine triphosphate (ATP) synthesis and purinergic signaling though P2X1 purinoceptors interfered with NET formation induced by PAF. We inhibited bovine neutrophils with 2-deoxy-d-glucose, rotenone, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and NF449 to evaluate PAF-mediated NET extrusion. PAF induced mitochondrial hyperpolarization and triggered extracellular ATP release via pannexin-1. Inhibition of mitochondrial metabolism prevented extracellular ATP release. Inhibition of glycolysis, complex-I activity and oxidative phosphorylation prevented NET formation induced by PAF. Inhibition of P2X1 purinergic receptors inhibited mitochondrial hyperpolarization and NET formation. We concluded that PAF-induced NET release is dependent upon glycolysis, mitochondrial ATP synthesis and purinergic signaling.

Oleic and Linoleic Acids Induce the Release of Neutrophil Extracellular Traps via Pannexin 1-Dependent ATP Release and P2X1 Receptor Activation.

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.

Glycolysis and mitochondrial function regulate the radical oxygen species production induced by platelet-activating factor in bovine polymorphonuclear leukocytes.

Dairy cows undergo metabolic disturbances in the peripartum period, during which infectious inflammatory diseases and detrimental polymorphonuclear leukocytes (PMN) functions, such as radical oxygen species (ROS) production, are observed. Platelet-activating factor (PAF) is a key pro-inflammatory mediator that increases PMN ROS production. To date, the role of glycolysis and mitochondria in PAF-induced ROS production in bovine PMN has not been known. The aim of this study was to assess whether inhibition of glycolysis and disruption of mitochondrial function alter the oxidative response induced by PAF. We isolated PMN from non-pregnant Holstein Friesian heifers and pre-incubated them with 2-deoxy-d-glucose (2-DG; 2 mM, 30 min), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 5 µM, 5 min), oligomycin (10 µM, 30 min) or rotenone (10 µM, 30 min). Respiratory burst was measured by luminol-chemiluminescence assay, while mitochondrial ROS (mtROS) were evaluated by MitoSOX probe and flow cytometry. Also, we detected the presence of mitochondria by MitoTracker Deep Red FM probe and changes in mitochondrial membrane potential (Δψm) were assessed by JC-1 probe and flow cytometry. We observed that all inhibitors separately were able to reduce PAF-induced ROS production. Presence of mitochondria was detected and PAF increased the Δψm, while CCCP reduced it. 2-DG and rotenone reduced the mtROS production induced by PAF. CCCP did not alter the mtROS and oligomycin administered independently increased mtROS production. We concluded that PAF-induced ROS production is glycolysis- and mitochondria-dependent. Bovine PMN have a functional mitochondrion and PAF induced mtROS via glycolysis and mitochondrial complex-I activity. Our results highlight an important modulation of cellular metabolism in the oxidative response induced by proinflammatory agents, which could contribute to PMN disfunction during peripartum in cattle.

Free Fatty Acid Receptor 1 Signaling Contributes to Migration, MMP-9 Activity, and Expression of IL-8 Induced by Linoleic Acid in HaCaT Cells.

Keratinocytes and neutrophils are the main cellular components in wound healing during re-epithelization and inflammation. Free fatty acids such as linoleic acid (LA) present beneficial properties for wound healing by modulating the inflammatory response. LA is a natural ligand of free fatty acids receptor 1 (FFA1), a G protein-coupled receptor (GPCR), able to modulate inflammatory process; however, the role of FFA1 in keratinocytes and wound healing remains poorly understood. In this study, we investigated the role of FFA1 signaling in migration, matrix metalloproteinase-9 (MMP-9) activity, and IL-8 expression induced by LA in keratinocytes. We confirmed that HaCaT cells, a human keratinocyte cell line, expresses the FFA1 receptor and GW1100, a selective antagonist of FFA1, decreased LA-induced migration of HaCaT cells. Also, GW9508, a synthetic agonist of FFA1, increased migration of these cells. Furthermore, ERK1/2 and p38 MAPK inhibitors abolished the LA-induced increase in cell migration. Besides, HaCaT cells stimulated with LA or GW9508 increased the activity of MMP-9 and the expression of IL-8. GW1100 partially inhibited both responses. We further evaluated the effects of HaCaT cells conditioned media stimulated with LA or GW9508 on neutrophil chemotaxis. Conditioned media induced neutrophil chemotaxis. Furthermore, IL-8 secreted by HaCaT cells stimulated with LA or GW9508, contributed to neutrophil chemotaxis. In conclusion, LA increased migration, MMP-9 activity, and expression of IL-8 from HaCaT cells via FFA1. Hence, these results showed that the effects induced by LA in keratinocytes can be mediated through FFA1, thus explaining a possible mechanism by which this fatty acid could accelerate wound healing.

Pro-inflammatory mediators and neutrophils are increased in synovial fluid from heifers with acute ruminal acidosis.

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 E2 (PGE2), 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 PGE2, IL-6, IL-1ß, 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-1ß, IL-6, PGE2, 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 (H3cit), aggregated neutrophil extracellular traps (aggNETs), and serpin B1 were observed 24 h post treatment. Furthermore, albumins, LDH, ATP, MMP-9, IL-6, and IL-1ß were increased after 24 h. CONCLUSIONS: The overall results indicate that IL-1ß, IL-6 and PGE2, 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 aggNETs.

Functional expression of the free fatty acids receptor-1 and -4 (FFA1/GPR40 and FFA4/GPR120) in bovine endometrial cells.

Endometrial epithelial cells play a key defensive role as part of the innate immune response of cow uterus. An association between risk of acquiring infectious diseases and increased levels of free fatty acids postpartum has been suggested, and the use of omega-3 fatty acids such as docosahexaenoic acid (DHA) has been proposed as a beneficial strategy to improve immunity and fertility. The goal of our study was to demonstrate the presence of free fatty acid (FFA)-1 and 4 receptors in endometrial cells and to investigate their role on DHA interference in lipopolysaccharide (LPS)-induced inflammatory endometrial activation. We demonstrated that the bovine endometrial (BEND) cells line and bovine endometrium express both FFA1 and FFA4 receptors. FFA1 and FFA4 receptors were localized in the epithelium lining the endometrial cavity and in endometrial glands whereas in BEND cells a characteristic cell membrane localization of both receptors was observed. DHA, a FFA4 natural agonist, increased intracellular calcium mobilization in BEND cells, but the FFA1 agonists oleic and linoleic acids did not increase this response. DHA-induced intracellular calcium mobilization was inhibited by the FFA4 and FFA1 antagonists AH7614 and GW1100, respectively. DHA significantly reduced LPS-induced prostaglandin E2 (PGE2) production, but none of the antagonists reduced the effect produced by DHA. On the contrary, linoleic acid increased LPS-induced PGE2 production. In conclusion, endometrial cells express FFA4 and FFA1 receptors, and DHA induces intracellular calcium release via FFA4 and FFA1 receptors. DHA reduces PGE2, but this response was not mediated by FFA4 or FFA1 receptors.

Transcriptional analysis of flagellar and putative virulence genes of Arcobacter butzleri as an endocytobiont of Acanthamoeba castellanii.

Arcobacter butzleri is an emerging foodborne zoonotic pathogen that has been isolated from environmental water sources. This pathogen establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba found in environmental matrices such as soil and water. The principal aim of this study was to analyse the transcriptional pattern of flagellar (flaA-flaB-flgH-motA) and other putative virulence genes (ciaB-cadF-mviN-pldA) of A. butzleri during its interaction with A. castellanii by quantitative real-time PCR. The transcriptional analysis showed up-regulation of all genes analysed before A. butzleri became established as an endocytobiont of A. castellanii. In contrast, while A. butzleri remains an endocytobiont, a significant and sustained decrease in the transcription of all analysed genes was observed. Our findings suggest that A. butzleri requires a biphasic transcriptional pattern of flagellar and other putative virulence genes to establish an endosymbiotic relationship with A. castellanii.

Metabolic disturbances in synovial fluid are involved in the onset of synovitis in heifers with acute ruminal acidosis.

Acute ruminal acidosis (ARA) is the result of increased intake of highly fermentable carbohydrates, which frequently occurs in dairy cattle and is associated with aseptic polysynovitis. To characterise the metabolic changes in the joints of animals with ARA, we performed an untargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis of synovial fluid. Seven heifers were challenged with an intraruminal oligofructose overload (13 g/kg of body weight [BW]) dissolved in water. Synovial fluid samples were collected at 0, 9 and 24 h post-overload. Metabolome analysis revealed the presence of 67 metabolites. At 9 h post-overload, glyceric acid, cellobiose, fructose and lactic acid were all increased, whereas at 24 h, sorbitol, lactic acid and fructose levels were all increased >10-fold. At 24 h, citric acid and threonine levels were significantly reduced. We detected increased L- and D-lactate, and the presence of interleukin-6 (IL-6) in synovial fluid. Furthermore, using bovine fibroblast-like synoviocytes, we observed that D-lactate induces IL-6 synthesis. Our results suggest that ARA produces severe metabolomic changes in synovial fluid, including disturbances in starch and sucrose metabolism, and increased lactate levels. These changes were observed prior to the appearance of synovitis, suggesting a potential role in the onset of polysynovitis.

d(-) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression.

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.