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This study aimed to investigate the protective effects and mechanisms of myricetin on acute liver failure in mice induced by lipopolysaccharide (LPS)/D-galactosamine (D-Gal). Our results showed myricetin (25, 50 and 100 mg/kg) pretreatment significantly improved the pathological changes of liver tissues, decreased serum ALT and AST (p < 0.001) induced by LPS/D-GalN. Moreover, MDA and MPO levels were reduced (p < 0.001), CAT and SOD activities were increased (p < 0.001) with myricetin (50 and 100 mg/kg) pretreatment. Likewise, inflammatory cytokines TNF-α and IL-6 mRNA in liver tissues were markedly decreased (p < 0.001) by myricetin. Besides, Nrf2 protein expression was drastically elevated (p < 0.001) by myricetin (25, 50 and 100 mg/kg). All these findings imply that myricetin may protect against acute liver failure by suppressing inflammation and regulating oxidative stress via Nrf2 signaling, and that it may be a possible strategy to avoid liver damage.

It is well-established that lysine-specific demethylase 1 (LSD1) is the first identified histone demethylase. Based on its demethylase enzymatic activity, LSD1 plays a pivotal role in vast range of cellular processes and cancers, but the understanding of its effects on inflammation is relatively limited. Using in vivo models of lipopolysaccharide (LPS)-induced inflammation and in vitro assays in mouse mammary epithelial cells, we identified the novel regulatory roles and underlying mechanisms of LSD1 on LPS-induced mastitis. Mammary gland and cells were collected for the following experiments after treatment. Histological changes were determined by H&E. Western blot analysis was used to detect the protein expression. ELISA and real-time PCR were used to evaluate protein and mRNA expression of inflammatory genes. Our results showed that LPS treatment resulted in a significant increase in LSD1 protein expression. GSK-LSD1 is a selective inhibitor of LSD1 enzyme activity. Treatment of mice with GSK-LSD1 inhibited LSD1 activity, reduced inflammatory cells recruitment to tissues and attenuated LPS-induced damage in mammary gland. Mechanistic investigations suggested that LSD1 inhibition led to the increase of histone H3K4me2 and H3K9me2. Furthermore, GSK-LSD1 inhibition of LSD1 further inhibited nuclear factor κ-B (NF-κB) signaling cascades, and subsequently inhibited the production of cytokines (TNF-α, IL-6 and IL-1β) in mammary gland. Taken together, our data reveal LSD1 as a potential regulator of inflammation and improve our understanding of epigenetic control on inflammation.

Mastitis is an inflammation of mammary gland, which directly affects the milk production performance and causes huge economic losses in the dairy industry. During mastitis, the blood-milk barrier (BMB) loses its integrity and aggravates the severity of mastitis. Exogenous DNase I has been exerted protective effects in different model of tissue injury. Here, we designed a study to investigate the effects of DNase I on inflammation and BMB in a mice model of Staphylococcus aureus-induced mastitis. In the model, we found that DNase I treatment significantly alleviated the inflammatory response through decrease of inflammatory cells in mammary alveoli, MPO activity and cytokines in mammary gland. Furthermore, immunofluorescent staining and western blotting demonstrated that exogenous DNase I obviously reduced BMB permeability and changed the expression of tight junction proteins to support the re-establishment of the barrier integrity. Mechanismly, DNase I treatment inhibited NF-κB and enhanced AKT signaling pathways. Therefore, our results indicate that DNase I may be an effective treatment for attenuating mastitis.

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.

Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease that affects both, animal welfare and cattle productivity. Neutrophil extracellular trap (NET) formation represents an important innate effector mechanism of polymorphonuclear neutrophils (PMN) acting against various pathogens. Recently, NETs release was reported in response to B. besnoiti tachyzoites. However, limited knowledge is available on the role of metabolic pathways during parasite-triggered NET formation, nowadays known as NETosis. By analysing metabolic signatures of tachyzoite-exposed PMN and applying functional inhibition experiments, we here aimed to investigate the importance of distinct PMN-derived metabolic pathways for effective B. besnoiti tachyzoite-induced NETosis. Overall, exposure to heat-inactivated tachyzoites induced an increase in glucose and serine consumption, a drop in glutamine consumption and an enhancement of glutamate and alanine production in bovine PMN. Moreover, tachyzoite-induced formation of cell free NETs was significantly diminished via PMN pretreatments with dichloroacetate (pyruvate dehydrogenase inhibitor), oxythiamine (inhibitor of pyruvate dehydrogenase kinase) and oxamate (inhibitor of lactate dehydrogenase), thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. In line, NET formation was also induced by enhanced pH, however, blockers (AR-C141900, AR-C151858) of lactate transporters failed to influence NETosis. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X1 antagonist) which suggested a pivotal role of ATP availability in this effector mechanism. In contrast, treatments with inhibitors of early steps of glycolysis or glutaminolysis did not affect parasite-triggered NETosis. These current data will provide a better understanding of metabolic pathways involved in B. besnoiti tachyzoite-induced NETosis.Moreover, tachyzoites of B. besnoiti induced LC3B-related autophagosome formation in parallel to NETosis in bovine PMN. Notably, both rapamycin- and wortmannin-treatments failed to influence B. besnoiti-triggered NETosis and autophagosome formation. Also, isolated NETosis failed to induce autophagy suggesting independence between both cellular processes. Interestingly, enhanced phosphorylation of AMPKα, a key regulator molecule of autophagy, was observed within the first minutes of interaction in tachyzoite-exposed bovine PMN thereby emphasizing that B. besnoiti-triggered NETosis indeed occurred in parallel to autophagy.In addition, we investigated early effector mechanisms of bovine PMN being exposed to vital B. besnoiti bradyzoite stages, which were isolated from skin tissue cysts of a naturally infected animal presenting characteristic symptoms of bovine besnoitiosis. Histopathological examinations confirmed the presence of typical roundish, large-sized cysts in subdermal biopsies. After PMN:B. besnoiti bradyzoites co-cultures, scanning electron microscopy (SEM)- and epifluorescence microscopy-analyses demonstrated a fine network of NET-like structures being released by bovine PMN and efficiently ensnaring bradyzoites. Classical NETosis-associated components were confirmed in these extracellular structures via immunofluorescence analyses on extracellular DNA, histone (H1-H4) and neutrophil elastase (NE) colocalization. Besides NETosis, an increased formation of autophagosomes (visualized by specific-LC3B staining) was observed in bradyzoite-stimulated PMN. Statistical analyses revealed a significant positive correlation (p = 0.042) between the occurrence of NETosis and autophagy in these immunocompetent cells. These findings suggest NETosis plays a pivotal role in early innate host responses against bradyzoite stages and furthermore deliver first evidence on autophagy being associated with B. besnoiti bradyzoite-induced NETosis., Besnoitia besnoiti ist der Erreger der Rinderbesnoitiose, einer Erkrankung, die die Tierproduktivität beeinträchtigt und auch tierschutz-relevant ist. Die Bildung sogenannter ´neutrophile extracellular traps (NETs) stellt einen wichtigen angeborenen Effektormechanismus von Neutrophilen dar, der gegen verschiedene Pathogene wirkt. Vor kurzem wurde über die Freisetzung von NETs, die auch als NETosis bezeichnet wird, als Reaktion auf B. besnoiti-Tachyzoiten berichtet. Über die Rolle von Stoffwechselwegen bei durch Parasiten ausgelöster NETosis ist jedoch nur ein begrenztes Wissen verfügbar. In der vorliegenden Arbeit wurden Stoffwechselsignaturen von Tachyzoiten-exponierter Neutrophilen (PMN) analysiert und Experimente zur funktionellen NETosis-Hemmung durchgeführt. Weiterhin wurde die Bedeutung verschiedener PMN-abgeleiteter Stoffwechselwege für eine durch B. besnoiti-Tachyzoiten und -Bradyzoiten induzierte NETosis untersucht. Insgesamt führte die Exposition gegenüber hitzeinaktivierten Tachyzoiten zu einem Anstieg des Glukose- und Serin-Verbrauchs, einem Rückgang des Glutamin-Verbrauchs und einer Steigerung der Glutamat- und Alanin-Produktion in exponierten Rinder-PMN. Darüber hinaus wurde die Tachyzoiten-induzierte Bildung zellfreier NETs durch PMN-Vorbehandlung mit Dichloracetat (Pyruvatdehydrogenase-Inhibitor), Oxythiamin (Inhibitor der Pyruvatdehydrogenase-Kinase) und Oxamat (Inhibitor der Lactatdehydrogenase) signifikant reduziert, was auf eine Schlüsselrolle der Pyruvat- und Laktat-vermittelten Stoffwechselwege für die richtige Tachyzoit-vermittelte NETosis hinweist. In der Folge wurde die NETosis-Bildung auch durch einen erhöhten pH-Wert induziert, jedoch konnten Laktat-Transporter-Blocker (AR-C141900, AR-C151858) die NETosis nicht beeinflussen. Eine signifikante Reduktion der Tachyzoiten-induzierten NET-Bildung wurde auch durch Behandlung mit Oligomycin (Inhibitor der ATP-Synthase) und NF449 (purinergischer Rezeptor P2X1-Antagonist) erreicht, was auf eine zentrale Rolle der Verfügbarkeit von ATP hindeutet. Im Gegensatz dazu beeinflussten Behandlungen mit Inhibitoren der frühen Schritte der Glykolyse oder Glutaminolyse die durch Parasiten ausgelöste NETosis nicht. Diese genauen Daten vermitteln ein besseres Verständnis der Stoffwechselwege, die an der Bildung von durch B. besnoiti-Tachyzoiten induzierten NETosis beteiligt sind.Weiterhin induzierten Tachyzoiten von B. besnoiti die Bildung von LC3B-verwandten Autophagosomen parallel zur NETosis in Rinder-PMN. Bemerkenswerterweise beeinflusste weder eine Rapamycin- noch eine Wortmannin-Behandlung die durch B. besnoiti ausgelöste NET-Bildung und die Bildung von Autophagosomen. Auch isolierte NETs induzierten keine Autophagie, was die Unabhängigkeit zwischen beiden zellulären Prozessen nahelegt. Interessanterweise wurde innerhalb der ersten Minuten der Wechselwirkung in Tachyzoiten-exponierten Rinder-PMN eine verstärkte Phosphorylierung von AMPKα, einem der wichtigsten Regulatormoleküle der Autophagie, beobachtet. Dies unterstreicht, dass die durch B. besnoiti ausgelöste NET-Bildung tatsächlich parallel zur Autophagie auftritt.Außerdem wurden frühe Effektormechanismen von Rinder-PMN, die vitalen, aus Hautgewebszysten eines infizierten Tieres isolierten B. besnoiti-Bradyzoiten ausgesetzt waren untersucht. Histopathologische Untersuchungen bestätigten das Vorhandensein von typischen runden, großformatigen Zysten in Hautbiopsien. Nach PMN:B.besnoiti- Bradyzoiten Co-Kulturen, Rasterelektronen- und Fluoreszenzmikroskopieanalysen zeigten ein feines Netzwerk NETosis-ähnlicher Strukturen, die von PMN von Rindern und verstrickten Bradyzoiten freigesetzt wurden. Klassische NETosis-Komponenten wurden in diesen Strukturen durch Immunfluoreszenzanalysen an extrazellulärer DNA, Histone (H1-H4) und Neutrophilen-Elastase (NE)-Co-Lokalisation, bestätigt. Neben NETosis wurde in Bradyzoiten-stimulierten PMN auch eine erhöhte Bildung von Autophagosomen (sichtbar durch spezifische LC3B-Färbung) beobachtet. Statistische Analysen zeigten eine signifikante positive Korrelation (p = 0,042) zwischen dem NETosis-Auftreten und der Autophagie in diesen angeborenen Immunzellen. Diese Ergebnisse legen nahe, dass NETosis eine zentrale Rolle bei frühen angeborenen Wirtsreaktionen gegen B. besnoiti-Bradyzoiten spielt und liefern darüber hinaus erste Beweise für die Autophagie, die mit der durch B. besnoiti-Bradyzoiten induzierten NET-Bildung assoziiert ist.

Chlorogenic acid (CGA), one of the most abundant polyphenols in the diet, has been reported to have potent anti-inflammatory properties. However, the effect of CGA on lipopolysaccharide (LPS)-induced mice mastitis has not been investigated. The purpose of the present study was to elucidate whether CGA could ameliorate the inflammation response in LPS-induced mice mastitis and to clarify the possible mechanism. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. CGA was administered intraperitoneally with the dose of 12.5, 25, and 50mg/kg respectively 1h before and 12h after induction of LPS. In this study, the effect of CGA on LPS-induced mice mastitis was assessed through histopathological examination, ELISA assay, and western blot analysis. The results showed that CGA significantly reduced TNF-α, IL-1β, and IL-6 production compared with LPS group. Besides, western blot analysis showed that CGA could inhibit the expression of TLR4 and the phosphorylation of NF-κB and IκB induced by LPS. These results suggested that anti-inflammatory effects of CGA against LPS-induced mastitis may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathway. Therefore, CGA may be a potent therapeutic reagent for the prevention of the immunopathology encountered during Escherichia coli elicited mastitis.