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ABSTRACT: Gliotoxin (GT) belongs to the epipolythiodioxopiperazine (ETP) family, which is considered a crucial virulence determinant among the secondary metabolites produced by Aspergillus fumigatus. The metabolites are commonly found in food and feed, contributing to the invasion and immune escape of Aspergillus fumigatus, thereby posing a significant threat to the health of livestock, poultry, and humans. Heterophil extracellular traps (HETs), a novel form of innate immune defense, have been documented in the chicken's innate immune systems for capturing and eliminating invading microbes. However, the effects and mechanisms of GT on the production of duck HETs in vitro remain unknown. In this study, we first confirmed the presence of HETs in duck innate immune systems and further investigated the molecular mechanism underlying GT-induced HETs release. Our results demonstrate that GT can trigger typical release of HETs in duck. The structures of GT-induced HETs structures were characterized by DNA decoration, citrullinated histones 3, and elastase. Furthermore, NADPH oxidase, glycolysis, ERK1/2 and p38 signaling pathway were found to regulate GT-induced HETs. In summary, our findings reveal that gliotoxin activates HETs release in the early innate immune system of duck while providing new insights into the immunotoxicity of GT towards ducks.

ABSTRACT: Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus and parasitic aspergillus, mainly existing in cereals, peanuts, corn, and other crops, which seriously endanger poultry, human health, and environment. Morin, a flavonoid compound extracted from moraceae plants, possess antioxidant, antibacterial, and anti-inflammatory effects. However, whether morin has a protective effect on AFB1-induced liver and kidney damage in chicks has not been specifically reported. In this study, we mainly confirmed the protective effect of morin on AFB1-induced liver and kidney damage in chicks and clarified its mechanism. It was found that morin can significantly reduce the liver biochemical indicators of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and kidney indicators of creatinine (CRE) and urea nitrogen (BUN) levels. Meanwhile, histopathological examination showed that morin effectively relieved AFB1-caused liver damage, including hepatocyte disruption, swelling, and inflammatory cell infiltration, and effectively relieved kidney damage, including renal cell necrosis, exfoliation, and vacuolization. Further investigation of its mechanism demonstrated that morin significantly inhibited AFB1-induced heterophil extracellular traps (HETs) release, and decreased the level of malondialdehyde (MDA) but increased the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in vivo. Moreover, quantitative real-time PCR (qRT-PCR) analysis showed that morin also significantly decreased AFB1-induced mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), caspase-1, caspase-3, and caspase-11. In conclusion, all results confirmed that morin could protect AFB1-caused liver and kidney damage by inhibiting HETs release, regulating oxidative stress, and inhibiting inflammatory response, suggesting that morin can be utilized as a potential drug for prevention and treatment of aflatoxicosis in poultry breeding industry.

Molybdenum (Mo) is not only an important rare metal that is widely used in industrial production but also an essential trace element for plants and animals. Nevertheless, in Mo polluted areas, excess Mo intake will not only cause gout in humans but also cause diarrhea in livestock and growth inhibition of chickens. Heterophils extracellular traps (HETs) are an important way to clear pathogens in the innate immune system of the chicken. However, the effects of Mo on the innate immune responses of HETs formation in chicken, and the mechanism undergoing this phenomenon remain unknown. In the study, we firstly aim to investigate the effects of sodium molybdate (Na2MoO4) on chicken HETs formation in vitro, and further to explore its related metabolic requirements and molecular mechanisms. Chicken heterophils were cultured with Na2MoO4, and Na2MoO4-induced HETs structures were analyzed by confocal microscopy. Moreover, Na2MoO4-induced HETs were quantified by Quant-iT PicoGreen® dsDNA Assay kits and fluorescence microplate. It has been shown that Na2MoO4 truly triggered HETs-like structures that were composed of DNA decorated with citrullinated histone 3 (citH3) and elastase. The inhibitors of NADPH oxidase, ERK1/2 and p38 MAPK signaling pathway significantly reduced Na2MoO4-induced HETs formation. Further experiments on energy metabolism involving Na2MoO4-induced HETs formation showed that Na2MoO4-induced HETs release was relevant to glucose, and the inhibitors of glycolysis including 3PO, AZD23766 and 3-Bromopyuvic acid, the inhibitors of glucose transport including STF31 and Ritonavir and NSC23766 significantly decreased Na2MoO4-induced HETs formation. In summary, these results demonstrate that Mo does induce chicken HETs formation in vitro, and the formation of HETs is a process relying on glucose transport 1 (GLUT1)，glucose transport 4 (GLUT4), glycolysis, and ROS production depended on the activation of NADPH oxidase, ERK1/2 and p38 signaling pathways, which also reflects the early innate immune responses of chicken against excessive molybdenum intake.

Cyanidin-3-O-β-glucoside (C3G) (CAS number 7084-24-4), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. However, the effect of C3G on lipopolysaccharide (LPS)-induced mastitis and the molecular mechanisms have not been investigated. In this study, we detected the protective effects of C3G on a LPS-induced mouse mastitis model and investigated the molecular mechanisms in LPS-stimulated mouse mammary epithelial cells (MMECs). Our results showed that C3G could attenuate mammary histopathologic changes and myeloperoxidase activity, and inhibit TNF-α, interleukin (IL)-1β, and IL-6 production caused by LPS. Meanwhile, C3G dose-dependently inhibited TNF-α and IL-6 in LPS-stimulated MMECs. C3G suppressed LPS-induced nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) activation. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol. Moreover, C3G activated liver X receptor (LXR)-ABCG1-dependent cholesterol efflux. Knockdown of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced mastitis. The promising anti-inflammatory mechanisms of C3G are associated with upregulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol, thereby suppressing toll-like receptor 4-mediated NF-κB and IRF3 signaling pathways induced by LPS.