Differential expression of innate immune and metabolic genes in LPS-challenged chickens
ABSTRACT: Salmonella causes inflammation in infected hosts. Inflammation is a well-characterized defensive mechanism of innate immunity. The recognition and engagement of lipopolysaccharide (LPS) endotoxins in the outer membranes of Salmonella to Toll-like receptor 4 of immune cells (macrophages and dendritic cells) trigger inflammatory responses characterized by secretion of pro-inflammatory cytokines, including TNF-beta, IL-1 and IL-6. These cytokines cause fever, anorexia, bodyweight losses, and catabolism of skeletal muscles and adipose tissues. However, molecular events underlying innate immune responses and metabolic activities during the later stage of inflammation are poorly understood. Additionally, the effects of prebiotics and antibiotics on innate immunity and nutrient metabolism are not yet reported. The objective of this study is to investigate the effects of a mannanoligosaccharide (MOS) prebiotic and virginiamycin (VIRG) sub-therapeutic antibiotic on innate immunity and glucose metabolism during late inflammation. We induced Salmonella LPS-systemic inflammation in a chicken model. Differentially regulated gene expressions were measured using 2 colour focussed oligonucleotide chicken-specific microarrays. Microarray analysis was performed on liver, intestinal and skeletal muscle tissues. We found that late inflammation was principally modulated by interleukin 3 (IL 3) and that glucose was mobilized from gluconeogenesis occurring in the intestines only. MOS and VIRG modulated innate immunity and metabolic genes differently. In contrast to VIRG, MOS terminated inflammatory responses earlier. Our results indicate IL 3 gene up-regulation in VIRG-fed chickens. To meet the higher energy requirements of VIRG chickens, genes for intestinal gluconeogenesis and liver glycolysis were respectively induced. Our study reveals the potential mechanisms by which prebiotic and antibiotic modulated innate immunity and glucose metabolism during late inflammation. 14-day old chickens were injected i.p. with saline or LPS. For each tissue and experimental conditions (saline or LPS challenge), a total of 12 microarrays (6 MOS birds + 6 VIRG birds) were used in a 2 x 2 factorial design and complete interwoven loop arrangement. We compared gene expression from prebiotic-fed birds with antibiotic-fed birds without including reference RNA. LPS challenge, antibiotic or prebiotic, innate immunity, glucose metabolism
Project description:BACKGROUND: Salmonella causes acute systemic inflammation by using its virulence factors to invade the intestinal epithelium. But, prolonged inflammation may provoke severe body catabolism and immunological diseases. Salmonella has become more life-threatening due to emergence of multiple-antibiotic resistant strains. Mannose-rich oligosaccharides (MOS) from cells walls of Saccharomyces cerevisiae have shown to bind mannose-specific lectin of Gram-negative bacteria including Salmonella, and prevent their adherence to intestinal epithelial cells. However, whether MOS may potentially mitigate systemic inflammation is not investigated yet. Moreover, molecular events underlying innate immune responses and metabolic activities during late inflammation, in presence or absence of MOS, are unknown. METHODS AND PRINCIPAL FINDINGS: Using a Salmonella LPS-induced systemic inflammation chicken model and microarray analysis, we investigated the effects of MOS and virginiamycin (VIRG, a sub-therapeutic antibiotic) on innate immunity and glucose metabolism during late inflammation. Here, we demonstrate that MOS and VIRG modulated innate immunity and metabolic genes differently. Innate immune responses were principally mediated by intestinal IL-3, but not TNF-α, IL-1 or IL-6, whereas glucose mobilization occurred through intestinal gluconeogenesis only. MOS inherently induced IL-3 expression in control hosts. Consequent to LPS challenge, IL-3 induction in VIRG hosts but not differentially expressed in MOS hosts revealed that MOS counteracted LPS's detrimental inflammatory effects. Metabolic pathways are built to elucidate the mechanisms by which VIRG host's higher energy requirements were met: including gene up-regulations for intestinal gluconeogenesis (PEPCK) and liver glycolysis (ENO2), and intriguingly liver fatty acid synthesis through ATP citrate synthase (CS) down-regulation and ATP citrate lyase (ACLY) and malic enzyme (ME) up-regulations. However, MOS host's lower energy demands were sufficiently met through TCA citrate-derived energy, as indicated by CS up-regulation. CONCLUSIONS: MOS terminated inflammation earlier than VIRG and reduced glucose mobilization, thus representing a novel biological strategy to alleviate Salmonella-induced systemic inflammation in human and animal hosts.
Project description:Worldwide Campylobacter jejuni is a leading cause of foodborne disease. Contamination of chicken meat with digesta from C. jejuni-positive birds during slaughter and processing is a key route of transmission to humans through the food chain. Colonization of chickens with C. jejuni elicits host innate immune responses that may be modulated by dietary additives to provide a reduction in the number of campylobacters colonizing the gastrointestinal tract and thereby reduce the likelihood of human exposure to an infectious dose. Here we report the effects of prebiotic galacto-oligosaccharide (GOS) on broiler chickens colonized with C. jejuni when challenged at either an early stage in development at 6 days of age or 20 days old when campylobacters are frequently detected in commercial flocks. GOS-fed birds had increased growth performance, but the levels of C. jejuni colonizing the cecal pouches were unchanged irrespective of the age of challenge. Dietary GOS modulated the immune response to C. jejuni by increasing cytokine IL-17A expression at colonization. Correspondingly, reduced diversity of the cecal microbiota was associated with Campylobacter colonization in GOS-fed birds. In birds challenged at 6 days-old the reduction in microbial diversity was accompanied by an increase in the relative abundance of Escherichia spp. Whilst immuno-modulation of the Th17 pro-inflammatory response did not prevent C. jejuni colonization of the intestinal tract of broiler chickens, the study highlights the potential for combinations of prebiotics, and specific competitors (synbiotics) to engage with the host innate immunity to reduce pathogen burdens.
Project description:Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confer better immunity to the chickens in the paradigm of mammalian milk that enhances immunity. Therefore, we investigated the effect of hatchery eggshell membranes (HESM) as a short term feed supplement on growth performance and immunity of chickens under bacterial lipopolysaccharide (LPS) challenged condition. Three studies were conducted to find the effect of HESM supplement on post hatch chickens. In the first study, the chickens were fed either a control diet or diets containing 0.5% whey protein or HESM as supplement and evaluated at 5 weeks of age using growth, hematology, clinical chemistry, plasma immunoglobulins, and corticosterone as variables. The second and third studies were done to compare the effects of LPS on control and HESM fed birds at 5 weeks of age following at 4 and 24 h of treatment where the HESM was also sterilized with ethanol to deplete bacterial factors. HESM supplement caused weight gain in 2 experiments and decreased blood corticosterone concentrations. While LPS caused a significant loss in body weight at 24 h following its administration, the HESM supplemented birds showed significantly less body weight loss compared with the control fed birds. The WBC, heterophil/lymphocyte ratio, and the levels of IgG were low in chickens fed diets with HESM supplement compared with control diet group. LPS challenge increased the expression of pro-inflammatory cytokine gene IL-6 but the HESM fed birds showed its effect curtailed, also, which also, favored the up-regulation of anti-inflammatory genes compared with control diet fed chickens. Post hatch supplementation of HESM appears to improve performance, modulate immunity, and increase resistance of chickens to endotoxin.
Project description:BACKGROUND: The emergence and spread of antibiotic resistance in pathogens have led to a restriction on the use of antibiotic growth promoters (AGPs) in animal feed in some countries. The potential negative after-effects of a ban on AGPs could be mitigated by improving animal intestinal health with prebiotic dietary fibers such as xylo-oligosaccharides (XOS). However, the mechanism(s) by which an antibiotic or prebiotic contributes to the health and growth of animals are not well understood. Here, we evaluated XOS and virginiamycin (VIRG)-mediated changes in gut microbiota of broiler chickens using pyrosequencing of the 16S rRNA gene. RESULTS: There was a significant change in the relative abundance of certain bacteria, but the overall microbial diversity was not affected by treatment with either XOS or VIRG. Supplementation of HXOS (2 g XOS/kg diet) increased the proportion of Lactobacillus genus in the cecum, whereas Propionibacterium and Corynebacterium genera were enriched in the ileum of VIRG (16 mg/kg) treated birds. Furthermore, an increase in the cecal concentrations of acetate and propionate was observed in HXOS- and VIRG-fed chickens, respectively. These two groups of birds had better feed conversion efficiencies in comparison with the control group from day 7 to 21. In addition, temporal variations in the gut microbiota were evident in the chickens of different ages. CONCLUSIONS: Treatments with XOS or VIRG modified the relative abundance but not the presence or absence of specific microbial genus. The increase in both Lactobacillus spp. and acetate production in the cecum of HXOS-treated chickens may promote intestinal health.
Project description:Oyster mushroom waste (OMW) is a by-product of the agriculture industry with valuable antimicrobial, antioxidant, antifungal, and prebiotic properties. This by-product might be a useful alternative to antibiotic growth stimulators in poultry nutrition. The purpose of this research was to test the impact of OMW on the immune responses and on the morphology of intestine of broiler chickens. Four dietary therapies with five replicas of 15 birds in each, totalling 300 day- Ross 308 broiler chickens, were utilized in this study. Control chickens were fed a mixed diet that included a maize-soybean meal complemented by 1 and 2% OMW in addition to the basal diet. Furthermore, Enramycin (125 g/kg) was added to the control diet as an antibiotic. Throughout this experiment, performance was studied as well as the immune response to the Newcastle Disease Virus (NDV) and intestinal morphological traits. A substantial surge was noted in body weight gain (BWG) and feed intake (FI) of chickens after the addition of 1% OMW (p ? 0.05). In contrast, feed supplementation with 2% OMW, compared with the control diet, produced no noteworthy increase in BWG or the feed conversion rate (FCR). Antibiotic addition, on the other hand, increased serum cholesterol (p ? 0.05). After 42 days, neither OMW nor antibiotic addition affected organ mass. In contrast, antibiotic addition reduced the small intestine percentage, crypt depth and villus height (p ? 0.05). The Newcastle disease vaccine (NDV) antibody titer improved after feed supplementation with 1% OMW comparing with the control and antibiotic diet group. Furthermore, OMW supplementation decreased the heterophil-to-lymphocyte H/L ratio (p ? 0.05). The use of OMW led to a reduction in the malondialdehyde (MDA) content of the breast and liver and an increase in glutathione peroxidase. It helped to reduce glutathione, glutathione reductase, and glutathione S-transferase. In conclusion, the impact of OMW were dose-dependent, and the use of 1% OMW in broiler diets enhanced their growth and immunity. Nonetheless, supplementation with 2% OMW produced conflicting results.
Project description:Commercially produced chickens have become key food-producing animals in the global food system. The scale of production in industrial settings has changed management systems to a point now very far from traditional methods. During the perinatal period, newly hatched chicks undergo processing, vaccination and transportation, which introduces a gap in access to feed and water. This gap, referred to as the hatching window, dampens the potential for microflora inoculation and as such, prevents proper microbiome, gastrointestinal system and innate immunity development. As a consequence, the industrial production of chickens with a poor microbial profile leads to enteric microbial infestation and infectious disease outbreaks, which became even more prevalent after the withdrawal of antibiotic growth promoters on many world markets (e.g., the EU).This review presents the rationale, methodology and life-long effects of in ovo stimulation of chicken microflora. In ovo stimulation provides efficient embryonic microbiome colonization with commensal microflora during the perinatal period. A carefully selected bioactive formulation (prebiotics, probiotics alone or combined into synbiotics) is delivered into the air cell of the egg on day 12 of egg incubation. The prebiotic penetrates the outer and inner egg membranes and stimulates development on the innate microflora in the embryonic guts. Probiotics are available after the mechanical breakage of the shell membranes by the chick’s beak at the beginning of hatching (day 19). The intestinal microflora after in ovo stimulation is potent enough for competitive exclusion and programs the lifespan condition. We present the effects of different combinations of prebiotic and probiotic delivered in ovo on day 12 of egg incubation on microflora, growth traits, feed efficiency, intestinal morphology, meat microstructure and quality, immune system development, physiological characteristics and the transcriptome of the broiler chickens.We discuss the differences between in ovo stimulation (day 12 of egg incubation) and in ovo feeding (days 17–18 of egg incubation) and speculate about possible future developments in this field. In summary, decades of research on in ovo stimulation and the lifelong effects support this method as efficient programming of lifespan conditions in commercially raised chickens.
Project description:Lipid mediators are known to play important roles in the onset and resolution phases of the inflammatory response in mammals. The phospholipid platelet-activating factor (PAF) is a pro-inflammatory lipid mediator which participates in vascular- and innate immunity-associated processes by increasing vascular permeability, by facilitating leukocyte adhesion to the endothelium, and by contributing to phagocyte activation. PAF exerts its function upon binding to its specific receptor, PAF receptor (PAFR), which is abundantly expressed in leukocytes and endothelial cells (ECs). In chickens, lipid mediators and their functions are still poorly characterized, and the role of PAF as an inflammatory mediator has not yet been investigated. In the present study we demonstrate that primary chicken macrophages express PAFR and lysophosphatidylcholine acyltransferase 2 (LPCAT2), the latter being essential to PAF biosynthesis during inflammation. Also, exogenous PAF treatment induces intracellular calcium increase, reactive oxygen species release, and increased phagocytosis by primary chicken macrophages in a PAFR-dependent manner. We also show that PAF contributes to the Escherichia coli lipopolysaccharide (LPS)-induced pro-inflammatory response and boosts the macrophage response to E. coli LPS via phosphatidylinositol 3-kinase/Akt- and calmodulin kinase II-mediated intracellular signaling pathways. Exogenous PAF treatment also increases avian pathogenic E. coli intracellular killing by chicken macrophages, and PAFR and LPCAT2 are upregulated in chicken lungs and liver during experimental pulmonary colibacillosis. Finally, exogenous PAF treatment increases cell permeability and upregulates the expression of genes coding for proteins involved in leukocyte adhesion to the endothelium in primary chicken endothelial cells (chAEC). In addition to these vascular phenomena, PAF boosts the chAEC inflammatory response to bacteria-associated molecular patterns in a PAFR-dependent manner. In conclusion, we identified PAF as an inflammation amplifier in chicken macrophages and ECs, which suggests that PAF could play important roles in the endothelium-innate immunity interface in birds during major bacterial infectious diseases such as colibacillosis.
Project description:Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked ?-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.
Project description:Microbial colonization of the gut early in life is crucial for the development of the immune and nervous systems, as well as influencing metabolism and weight gain. While early life exposure to antibiotics can cause microbial dysbiosis, prebiotics are non-digestible substrates that selectively promote the growth of beneficial gut microbiota. Our objective was to examine the effects of dietary prebiotic administration on the consequences of maternal antibiotic intake on offspring body weight, behavior, and neuroimmune responses later in life. Sprague-Dawley rat dams were given low-dose penicillin (LDP), prebiotic fiber (10% oligofructose), or both, during the third week of pregnancy and throughout lactation. Anxiety-like behavior, weight gain, body composition, cecal microbiota composition, and microglial responses to lipopolysaccharide (LPS) were assessed in offspring. Male and female prebiotic offspring had lower body weight compared to antibiotic offspring. Maternal antibiotic exposure resulted in lasting effects on select offspring microbiota including a lower relative abundance of Streptococcus, Lactococcus, and Eubacterium at 10 weeks of age. Maternal antibiotic use impaired microglial response to LPS in the hypothalamus compared to control, and this phenotype was reversed with prebiotic. Prebiotic fiber warrants further investigation as an adjunct to antibiotic use during pregnancy.
Project description:Salmonella enteritidis can cause significant morbidity and mortality in humans and economic loss in the animal industry. Improving the innate immunity is an effective method to prevent S. enteritidis infection. Pediococcus pentosaceus is a Gram-positive coccus which had probiotics properties. Numerous previously published studies reported that probiotics were beneficial to gut microbiota by changing the intestinal flora structure and inhibiting the harmful microbial growth to enhance the innate immunity. We investigated the immunological effects of P. pentosaceus on Salmonella-infected chickens by the following experiment. A total of 120 broilers from AA line were fed and divided into 2 groups (treated and control groups) for the experiment from day 1. The control group was fed with the basic diet, while the treated group was fed with the basic diet adding P. pentosaceus microcapsule with the bacterial concentration of 1?g/kg in the feed and bacterial counts 2.5 × 109?CFU/g. All the birds were given with 0.5?ml of S. enteritidis bacterial suspension (109?CFU/ml) through oral cavity at day 9. The number of dead birds was recorded and used in the analysis. The bacterial culture method and quantitative real-time PCR analysis were used to evaluate the effects of P. pentosaceus on chickens infected with S. enteritidis and to ascertain the mechanism of the effect. The results showed that the P. pentosaceus could restrain the pathogenicity of S. enteritidis and reduce the death rate from 44.4% to 23.3%. The flora in the caecum exhibited "rising-declining" trends, and the gene (TLR4, MyD88, TRAF6 NF-?B, IFN-?, TNF-a, IL6, and IL8) expression pattern was different between the experimental and control group. P. pentosaceus as a probiotic may competitively inhibit the growth of S. enteritidis and control the inflammatory response through regulating the gene expression which involved in the toll-like receptor pathway and inflammation pathway.