Pathological characteristics and congenital immunological responses of pigeons-infected with Neospora caninum.

Pigeons are natural intermediate host of Neospora caninum (N. caninum). In comparison to ruminants, N. caninum causes milder clinical symptoms and less financial loss to pigeons. Natural infectious rates and high prevalence of N. caninum in pigeons, and death cases of N. caninum-infected pigeons under experimental conditions have been reported, but the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum remain not well described. In this study, pigeons were infected intraperitoneally with 107 N. caninum tachyzoites. N. caninum in tissues was detected by qPCR. Pathological changes of tissues were examined by hematoxylin-eosin staining. Blood smears were prepared for counting eosinophils changes in blood. Heterophil extracellular traps (HETs) in vivo and in vitro were quantified by Pico Green. N. caninum-induced HETs structures were observed by immunofluorescence staining. The model of pigeons-infected with N. caninum was successfully established. Lung and duodenum were the main target organs of pigeons-infected with N. caninum. N. caninum caused hemorrhage, edema and inflammatory cell infiltration in liver, pulmonary congestion and hemorrhage, organizational destruction in lung, and shorter villi or even disappear in duodenum. N. caninum also increased the number of eosinophils in blood of pigeons. Moreover, N. caninum-induced HETs release in the congenital immunological system of pigeons were first demonstrated, and the HETs structures were consisted of DNA as the skeleton and modified with citH3 and elastase. N. caninum-induced HETs release was related with NADPH oxidase, TLR 2 and 4, ERK1/2 and p38 MAPK signaling pathways, and glycolysis. In summary, it is the first report on the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum, which may provide theoretical basis for the prevention and control of Neosporosis in pigeons.

Toxoplasma gondii triggers heterophil extracellular traps via NADPH oxidase, ERK1/2 and P38 signalling pathways, glycolysis and autophagy in chickens.

Toxoplasma gondii is a zoonotic parasite with a global distribution. Heterophil extracellular traps (HETs) are a novel innate immune mechanism of chickens against pathogens, but whether T. gondii can induce HETs release in chickens has not been reported. The effects of T. gondii on heterophils viability were assessed by using Cell Counting Kit-8. T. gondii-induced HETs were observed and analysed by the immunofluorescence method. T. gondii-induced reactive oxygen species (ROS) was determined by the DCFH-DA method. The mechanisms underlying T. gondii-triggered HETs were investigated by inhibitors and fluorescence microplate reader. T. gondii did not significantly affect heterophils viability at a 1:1 ratio within 1 h. It was demonstrated for the first time that T. gondii could induce HETs release in chicken, and the structure of HETs was comprised of DNA, elastase and citrullinated histone 3 (citH3). T. gondii increased ROS production in a dose-dependent manner. Inhibitors of NADPH oxidase, extracellular signal-regulated kinase 1/2 (ERK1/2 ) and P38 signalling pathways, glycolysis and autophagy significantly decreased the release of T. gondii-induced HETs. Taken together, T. gondii can induce HETs release in chickens, and ROS, NADPH oxidase, ERK1/2 and P38 signalling pathways, glycolysis and autophagy participate in the process of HETs release, which provides new insights into the innate immune mechanism of chickens against T. gondii infection.

Chicken heterophils extracellular traps act as early effectors against cyclopiazonic acid dependent upon NADPH oxidase, ROS and glycolysis.

Cyclopiazonic acid (CPA) is a secondary metabolite produced by Aspergillus and Penicillium, which is present in contaminated crops and food, causing severe toxicity to humans and animals. Heterophil extracellular traps (HETs) are a novel host innate immune mechanism of chicken heterophils against pathogen infection. However, whether CPA can cause immunotoxicity of heterophils on HETs release remains unclear. Here, we attempt to detect the effects of CPA on HETs release, and further investigate the molecular mechanisms underlying these processes. We exposed heterophils to 2.5, 5, 10 µM CPA for 90 min. The results showed that CPA induced the release of HETs in heterophils, consisting of DNA-modified citrullinated histone 3 and elastase. The quantitative analysis of HETs content was positively correlated with CPA concentration. CPA also promoted reactive oxygen species production and phosphorylation of ERK1/2 and p38. In addition, CPA-triggered HETs formation was reduced by NADPH oxidase, ERK1/2, and p38 signaling pathway and glycolysis inhibitors, indicating that CPA-induced HETs were related to the production of ROS dependent on NADPH oxidase, ERK1/2, and p38 signaling pathways, as well as glycolysis. Our study describes the underlying mechanism of CPA-induced HETs release, which may provide a further understanding of the immunotoxicology of CPA poisoning.

The release of FB1-induced heterophil extracellular traps in chicken is dependent on autophagy and glycolysis.

Fumonisin B1 (FB1), a worldwide contaminating mycotoxin produced by Fusarium, poses a great threat to the poultry industry. It was reported that extracellular traps could be induced by FB1 efficiently in chickens. However, the relevance of autophagy and glycolysis in FB1-triggered heterophil extracellular trap (HET) formation is unclear. In this study, immunostaining revealed that FB1-induced HETs structures were composed of DNA coated with histones H3, and elastase, and that heterophils underwent LC3B-related autophagosome formation assembly driven by FB1. Western blotting showed that FB1 downregulated the phosphorylated phosphoinositide 3-kinase3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin complex 1 (mTORC1) axis and raised the AMP-activated kinase α (AMPKα) activation protein. Furthermore, rapamycin- and 3-Methyladenine (3-MA)-treatments modulated FB1-triggered HET formation according to the pharmacological analysis. Further studies on energy metabolism showed that glucose/lactate transport and glycolysis inhibitors abated FB1-induced HETs. These results showed that FB1-induced HET formation might interact with the autophagy process and relied on glucose/monocarboxylic acid transporter 1 (MCT1) and glycolysis, reflecting chicken's early innate immune responses against FB1 intake.

Quercetin alleviates gliotoxin-induced duckling tissue injury by inhibiting oxidative stress, inflammation and increasing heterophil extracellular traps release.

Aspergillus fumigatus causes aspergillosis with high morbidity and mortality in the duck industry. As a vital virulence factor produced by A. fumigatus, gliotoxin (GT) is widely present in food and feed, threatening duck industry and human health. Quercetin is a polyphenol flavonoid compound from natural plants with anti-inflammatory and antioxidant functions. However, the effects of quercetin on ducklings with GT poisoning are unknown. The model of ducklings with GT poisoning was established, and the protective effects and molecular mechanisms of quercetin on ducklings with GT poisoning were investigated. Ducklings were divided into control, GT, and quercetin groups. A model of GT (2.5 mg/kg) poisoning in ducklings was successfully established. Quercetin protected GT-induced liver and kidney functions and alleviated GT-induced alveolar wall thickening in lungs, cell fragmentation, and inflammatory cell infiltration in liver and kidney. Quercetin decreased malondialdehyde (MDA) and increased superoxide dismutase (SOD) and catalase (CAT) after GT treatment. Quercetin significantly reduced GT-induced mRNA expression levels of inflammatory factors. Furthermore, quercetin increased GT-reduced heterophil extracellular traps (HETs) in serum. These results indicated that quercetin protected ducklings against GT poisoning by inhibiting oxidative stress, inflammation and increasing HETs release, which confirms the potential applicability of quercetin in treating GT-induced duckling poisoning.

Aflatoxin B1-activated heterophil extracellular traps result in the immunotoxicity to liver and kidney in chickens.

Aflatoxin B1 (AFB1) is a mycotoxin with strong toxicity and play a large proportion in aspergillosis. Heterophil extracellular traps (HETs) was considered as an innate immune response of chickens to resist pathogens. AFB1 has been reported to trigger macrophages extracellular traps (METs) in THP-1 cells and RAW264.7 cells, but whether AFB1 could also activate HETs release, and the mechanism underlying AFB1-activated HETs in chicken remains unclear. In this study, we confirmed that AFB1could induce HETs release, which was a network of DNA-based structures consist of citrullinated histone 3 (citH3) and elastase. Meanwhile, AFB1-activated HETs rely on the glycolytic process to provide energy, NADPH oxidase and p38 signaling pathway. Moreover, it has been verified that AFB1-activated HETs release could significantly increase the biochemical indexes of liver (ALT and AST) and kidney (CRE and BUN) in serum. In addition, histopathological observation showed that AFB1 caused swelling, necrosis and vacuolation of hepatocytes in liver, and necrosis, exfoliated of nephrocyte in kidney. Further investigation demonstrated that AFB1 significantly decreased the levels of SOD and GSH-PX but increased the level of MDA, and meanwhile induced the mRNA expressions of TNF-α, IL-6 and IL-1ß, iNOS, COX-2, NLRP3, caspase-1, caspase-3 and caspase-11. However, all these AFB1-induced biochemical indexes and histopathological changes were effectively alleviated by DNase I (the standard degradant for HETs). In conclusion, it has preliminary confirmed that AFB1-activated HETs formation contributed to the immunotoxicity in chicken and provide new strategies for the therapy in aspergillosis.

Fumonisin B1 induces chicken heterophil extracellular traps mediated by PAD4 enzyme and P2 × 1 receptor.

Fumonisin B1 (FB1) is a common mycotoxin contamination in agricultural commodities being considered as a significant risk to human and livestock health, while the mechanism of FB1 immunotoxicity are less understood, especially in chicken. Given that extracellular traps as a novel defense mechanism of leukocytes play an important role against foreign matters, in this study we aimed to investigate the effects of FB1 on chicken heterophil extracellular traps (HETs) formation. Our result showed that FB1 induced HETs release in chicken heterophils observed via immunostaining, and it was concentration-dependent during 10 to 40 µM. Moreover, in 40 µM FB1-exposed chicken heterophils, reactive oxygen species (ROS) level was increased, while catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity and glutathione (GSH) content were decreased. Simultaneously, FB1 (40 µM) activated ERK and p38 MAPK signaling pathways via increasing the phosphorylation level of ERK and p38 proteins. However, pretreatment of SB202190, U0126, and diphenyleneiodonium chloride (DPI) did not change FB1-triggered ROS production and HETs formation, suggesting FB1-induced HETs was a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p38, and extracellular regulated protein kinases (ERK) signaling pathways-independent process. Inhibition of peptidyl arginine deiminase 4 (PAD4) enzyme and P2 × 1 receptor showed their vital role in 40 µM FB1-triggered HETs. This study reported for the first time that 40 µM FB1 induced the release of HETs in heterophils, and it was related to ROS production, PAD4, and P2 × 1, but was independent of NADPH oxidase, p38 and ERK signaling pathways, which might provide a whole novel perspective of perceiving and understanding the role of FB1 in immunotoxicity.

Citrinin stimulated heterophil extracellular trap formation in chickens.

Citrinin (CTN), a secondary fungal metabolite produced by several Aspergillus, Penicillium, and Monascus genera species, is a toxin with a wide range of biological activities. Neutrophil extracellular traps represent a novel potential mechanism of the neutrophil response to foreign matters, and chicken heterophils can release similar heterophil extracellular traps (HETs). In this study, we aimed to investigate the effect of CTN on HET formation. Density gradient centrifugation was used to isolate chicken peripheral blood heterophils, and then immunofluorescence was used to observe the effects of CTN on HET formation. The mechanisms of HET formation were analyzed using pharmacological inhibitors and quantification of extracellular DNA, and the production of reactive oxygen species was detected with a fluorescent probe. Our results revealed that CTN (50-400 µM) had no cytotoxic effect on heterophils. CTN exposure induced the release of HETs composed of chromatin decorated with histones and elastase, and CTN-triggered HETs were dose- and time-dependent to some extent. Furthermore, CTN increased ROS production and activated p38 and ERK1/2 signaling pathways in heterophils. However, inhibition of the p38 signaling pathway, ERK1/2 signaling pathway, and NADPH oxidase pathway did not block HET formation induced by CTN. Inhibition of peptidyl arginine deiminase 4 (PAD4) enzyme and P2×1 receptor decreased HET formation after CTN stimulation, suggesting that HET formation exposed to CTN was mediated by PAD4 and P2×1 receptor. In conclusion, these findings may suggest a canonical mechanism relevant to the innate immunity caused by mycotoxins in chickens.

Morin alleviates aflatoxin B1-induced liver and kidney injury by inhibiting heterophil extracellular traps release, oxidative stress and inflammatory responses in chicks.

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-1ß (IL-1ß), 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.

Presence of DNA extracellular traps but not MUC5AC and MUC5B mucin in mucoid plugs/casts of infectious laryngotracheitis virus (ILTV) infected tracheas of chickens.

Although it has been speculated that the tracheal obstructions and asphyxiation during acute infectious laryngotracheitis (ILT) are due to mucoid plugs/casts formed by mucus hypersecretion, there are no reports demonstrating this. Hence, in the present study, we first examined if the main respiratory mucins, MUC5AC and MUC5B, are expressed in the mucosae of larynx, trachea and bronchi of mock-inoculated and ILTV infected chickens. Second, the tracheas with plugs/casts were stained for mucins (MUC5AC and MUC5B) and nuclear material (traps). MUC5AC and MUC5B were produced by the mucosae of larynx, trachea and bronchi of mock-inoculated chickens. Interestingly, MUC5AC and MUC5B were exclusively present in the dorsal tracheal region of the cranial and middle part of trachea of mock-inoculated chickens. In ILTV infected chickens, the tracheal lumen diameter was almost 40% reduced and was associated with a strongly increased tracheal mucosal thickness. MUC5AC and MUC5B were scarcely observed in larynx, trachea and bronchi, and in tracheal plugs/casts of ILTV infected birds. Surprisingly, DNA fibrous structures were observed in connection with nuclei of 10.0±7.3% cells, present in tracheal plugs/casts. Upon inoculation of isolated blood heterophils with ILTV, DNA fibrous structures were observed in 2.0±0.1% nuclei of ILTV inoculated blood heterophils at 24hours post inoculation (hpi). In conclusion, the tracheal obstructions and suffocation of ILTV infected chickens are due to a strong thickening of the mucosa (inflammation) resulting in a reduced tracheal lumen diameter and the presence of mucoid plugs/casts containing stretched long DNA-fibrous structures (traps) but not MUC5AC and MUC5B mucins.