Innate Immune Training in Chickens for Improved Defense against Pathogens: A Review.

The avian immune system plays a vital role in poultry production to obtain good productibility and products that are safe and of high quality. Historically, adaptive immunity has been the main target of vaccination. However, over the past decade, innate immunity has been reported to be enhanced in different animals through vaccination and feed additives. This enhancement is due to innate immune memory termed "trained immunity," in which epigenetic and metabolic reprogramming play significant roles. Although reports on trained immunity in poultry are limited, several studies have suggested that vaccinations and feed additives affect the innate immunity. This review discusses the possible effects of vaccination and ß-glucan on innate immunity for potential incorporation in advanced strategies to enhance the defense function in poultry while considering the information on trained immunity in mammals.

Effects of Newcastle Disease/Infectious Bronchitis Vaccine and Feeding Yeast Products on the Innate Immune System in the Proventriculus and Ileum of Broiler Chicks.

The aim of this study was to determine whether Newcastle disease/infectious bronchitis (ND/IB) vaccination and yeast product diet supplementation modulate the expression of innate immune molecules in the proventriculus and ileum of broiler chicks. One-day-old male broiler chicks were divided into four groups (V-Y- (control), V-Y+, V+Y-, and V+Y+ groups, where V and Y represent vaccination and yeast product supplementation, respectively). Chicks in the V+Y- and V+Y+ groups were immunized with the live ND/IB vaccine, whereas chicks in the V-Y- and V-Y+ groups were not. Chicks in the V-Y+ and V+Y+ groups received feed containing yeast products from day 4, whereas chicks in the V-Y- and V+Y- groups did not. The proventriculus and ileum were collected on day 7 to analyze the expression of seven Toll-like receptors (TLRs) and Dectin-1. In the proventriculus, compared with those of the V-Y- control group, the TLR7 and TLR21 expression levels were higher in the V+Y- group; however, there were no differences in the expression levels of any TLR or Dectin-1 in the ileum. There were also no differences in the expression of avian ß-defensins and cathelicidin-1 in the proventriculus and ileum between the control and treatment groups. The expression of granzyme in cytotoxic cells and interleukin (IL)-1B was upregulated by ND/IB vaccination in the proventriculus. Supplementation with yeast products upregulated only granzyme expression in the ileum and downregulated IL-6 expression in the proventriculus in chicks immunized with the ND/IB vaccine. Thus, we concluded that ND/IB vaccination is effective at enhancing the innate immune system in the proventriculus of chicks, at least until day 7 post-hatching, whereas the effects of diet supplementation with yeast products may be limited, at least under the present study conditions.

Modulation of the innate immune system by lipopolysaccharide in the proventriculus of chicks inoculated with or without Newcastle disease and infectious bronchitis vaccine.

This study aimed to determine whether the innate immune system in the proventriculus of broiler chicks responds to lipopolysaccharide (LPS) and whether this response is affected by Newcastle disease and infectious bronchitis (ND/IB) vaccination. Chicks were divided into 4 groups: nonvaccinated and injected with PBS or LPS (V-L- and V-L+), and vaccinated and injected with PBS or LPS (V+L- and V+L+). Vaccination was performed on d 1, and LPS was intraperitoneally injected on d 11 of age. The gene expression and protein levels of immune molecules, including toll-like receptors (TLRs), antimicrobial peptides, interleukin-1ß (IL-1B), and immunoglobulin A (IgA) in the proventriculus and serum were analyzed. The results showed that the expression levels of TLR21 were higher in vaccinated (V+L-) group than in nonvaccinated (V-L-) group. Gene expression levels of avian ß-defensin (AvBDs) and cathelicidin1 (Cath1) were not different among the 4 groups. However, the results of LC/MS analysis showed that the levels of AvBD2, 6, and 7 significantly increased after the LPS challenge in nonvaccinated and vaccinated chicks; the levels were higher in V-L+ and V+L+ than in V-L- and V+L-, respectively. Immunohistochemistry analysis revealed the localization of AvBD1 protein in the epithelial cells of the surface glands and AvBD2 and CATH1 in the heterophil-like cells in the lamina propria of surface glands. Although IL-1B gene expression and protein concentration in the proventriculus tissues were not different among the 4 groups, serum IL-1B levels were upregulated by LPS in both the nonvaccinated and vaccinated groups (V-L- vs. V-L+, V+L- vs. V+L+). Moreover, IgA levels in the proventriculus and serum were not affected by vaccination or LPS challenge. Taken together, we conclude that LPS derived from gram-negative bacteria upregulates the innate immune system, including antimicrobial peptide synthesis in the proventriculus. ND/IB vaccination may not significantly affect antimicrobial peptide synthesis in response to LPS; however, TLR21 expression is upregulated by that vaccination. The antimicrobial peptides synthesized in the proventriculus probably prevent pathogenic microbes from entering the intestine.

Effect of temporary cessation of milking on the innate immune components in goat milk.

Temporary cessation of milking is widely used during the dry period of dairy cows. Temporary cessation of milking induces an increase in the somatic cell count (SCC) and level of several inflammatory components of milk, which is believed to be a local adaptation and defense mechanism of the mammary gland. In Japan, temporary cessation of milking combined with antibiotic administration is widely used to treat mastitis. The present study aimed to elucidate the role of the innate immune system during temporary cessation of milking in a goat model by investigating the concentration of several innate immune components in milk during and around the temporary cessation. In experiment 1, 6 goats were subjected to cessation of milking for 3 d in both udder halves, whereas in experiment 2, 6 other goats were subjected to cessation of milking for 3 d only in 1 udder half. In experiment 1, the milk yield was lower on d 5 and 6, whereas the mean SCC was higher on d 5 compared with d 0 before temporary milking cessation. The concentrations of goat DEFB1, S100A7, cathelicidin-2 and 7 (CATHL-2 and 7), IgA, and lactoferrin were increased after temporary cessation of milking. In experiment 2, the milk yield was lower between d 5 and 7, whereas the mean SCC was higher between d 4 and 7 compared with d 0. The concentrations of CATHL-2, IgA, and lactoferrin were increased after temporary cessation of milking only in the udder half subjected to milking cessation. These results suggest that temporary cessation of milking increase the SCC and concentration of several innate immune components in milk without infection, which may contribute to mastitis treatment.

Effects of Probiotics Lactobacillus reuteri and Clostridium butyricum on the Expression of Toll-like Receptors, Pro- and Anti-inflammatory Cytokines, and Antimicrobial Peptides in Broiler Chick Intestine.

The aim of this study was to determine the effects of live probiotics Lactobacillus reuteri (LR) and Clostridium butyricum (CB) on the expression of genes of innate immune system in broiler chick ileum and cecum. Chicks were administered 500 µl water with or without LR or CB, daily from day 1 to 6 after hatching. The ileum and cecum were collected on day 7 for analysis of gene expression of Toll-like receptors (TLRs), pro- and anti-inflammatory cytokines, and antimicrobial peptides (AMPs) using real-time PCR. The expression of TLR2-1 was upregulated by CB in the ileum and that of TLR5 was upregulated by both LR and CB. Expression of IL-1ß and TGFß2 in the ileum and of TGFß3 and TGFß4 in the cecum was upregulated by both LR and CB. The gene expressions of avian ß-defensin (AvBD) 1 and cathelicidin (CATH) 3 were upregulated by CB and that of AvBD4 was upregulated by LR in the cecum. However, the expression of CATH2 in the ileum was downregulated by LR. These results suggest that probiotic LR and CB treatments affect a part of the innate defense system in the ileum and cecum by modulating the expression of innate immune molecules including TLRs, pro- and anti-inflammatory cytokines, and AMPs.

Effects of avian infectious bronchitis with Newcastle disease and Marek's disease vaccinations on the expression of toll-like receptors and avian ß-defensins in the kidneys of broiler chicks.

The aim of this study was to determine the effect of vaccinations for avian infectious bronchitis with Newcastle disease (IB/ND) and Marek's disease (MD) on the expression of toll-like receptors (TLR) that recognize viral RNA and microbial DNA, and AvBD in chick kidneys. Day-old chicks were vaccinated with MD or IB/ND vaccines or received no treatment (control group). The gene expression of TLR and AvBD in the kidneys of 3-day-old chicks and 10-day-old chicks was examined using real-time PCR. The localization of AvBD2 and AvBD4 was examined by immunohistochemistry at day three only. At 3 days of age, the expression of TLR7 and TLR21 was significantly higher in the IB/ND group (but not in the MD group) than in the control group. Conversely, at 10 days of age there was no significant difference in the expression of the three TLR between groups. In the 3-day-old chicks the expression levels of AvBD4, 5, 6, and 7 were higher in the MD group than in the control group. Furthermore, at this age, the expression levels of other AvBD were not significantly different between the control and vaccination (MD and IB/ND) groups. At 10 days of age, no AvBD expression was affected by MD and IB/ND vaccinations. Immunohistochemistry results localized AvBD2 in the leukocytes in the interstitial tissue and AvBD4 in the surface of microvillus epithelial cells of renal tubules, and in the epithelial cells of the collecting ducts and ureter. The localization of AvBD2 and AvBD4 was identified in all chicks. We suggest that the expression of innate immune molecules (including TLR and AvBD) in kidneys could be modulated by MD and IB/ND vaccination when performed at the day-old stage. Although the effects of both vaccinations may subside within 10 days, the enhanced expression of those innate immune molecules may support the innate immunodefense function in the kidneys of young chicks.

Translocation of intrauterine-infused bacterial lipopolysaccharides to the mammary gland in dexamethasone-treated goats.

Our previous study showed that intrauterine-infused lipopolysaccharide (LPS) can be translocated to the mammary gland to induce weak inflammation. This study aimed to determine whether dexamethasone treatment facilitated the translocation of LPS from the uterus to the mammary gland to induce a heavy inflammatory response. Sixteen goats were divided into control and LPS groups, subjected to daily dexamethasone administration before saline or LPS infusion. Milk and blood samples were collected before and after LPS infusion to determine the milk yield and somatic cell count (SCC) and blood leucocyte count (BLC), cytokines, antimicrobial peptides and serum amyloid A (SAA) concentrations. Mammary gland tissues were collected from two goats before and 24 hr after LPS infusion for immunohistochemical analysis of LPS. The mean SCC in the LPS group was significantly higher, whereas the milk yield was significantly lower than that in the control group after LPS infusion. The mean BLC in the LPS group was significantly lower than in the control group after LPS infusion. Furthermore, milk concentrations of IL-1ß, S100A8 and lactoferrin were higher in the LPS group than in the control group after infusion. LPS was detected in the connective tissues and inner alveolar spaces of the mammary glands 24 hr after LPS infusion. We concluded that dexamethasone administration facilitated the translocation of intrauterine-infused LPS to the mammary gland, where it induced an inflammatory response. Therefore, LPS translocated from other organs, such as the uterus, can induce heavy inflammation in the mammary gland under immunosuppressive conditions.

Effects of Toll-like Receptor Ligands on the Expression of Proinflammatory Cytokines and Avian ß-defensins in Cultured Chick Intestine.

Few studies have focused on the regulation of cytokine and avian ß-defensin (AvBDs) expression for promoting immune defense in the avian intestine. The aim of this study was to investigate the effects of different Toll-like receptor (TLR) ligands (bacterial patterns) on the expression of proinflammatory cytokines (IL-1ß and IL-6) and AvBDs (AvBD1, AvBD4, and AvBD7) in the chick intestine. The ileum and cecum of 3-day-old chicks were collected and examined histologically to identity the cells present in the intestinal mucosa. Other tissues were cultured with or without the TLR2, TLR4, and TLR21 ligands-Pam3CSK4, LPS, and CpG-ODN-for 1 or 3 h. The gene expression profiles of proinflammatory cytokines and AvBDs were determined in these tissues using real-time polymerase chain reaction (PCR). The mucosa of the ileum and cecum contained leukocytes, luminal and crypt epithelial cells, and other enterocytes. Pam3CSK4 tended to downregulate the expression of IL-1ß, AvBD1, and AvBD7 in the ileum but upregulated their expression in the cecum. LPS downregulated the expression of IL-1ß and IL-6 in both the ileum and the cecum, whereas it upregulated the expression of AvBD1, AvBD4, and AvBD7 in the cecum. CpG-ODN upregulated the expression of IL-6 and AvBD7 in the ileum and IL-1ß in the cecum, and downregulated the expression of IL-1ß and AvBDs in the ileum. We suggested that the expression levels of proinflammatory cytokines and AvBDs in the chick intestine are affected by TLR2, TLR4, and TLR21 ligands. Thus, these innate immune factors may be modulated by the luminal microbe complex in the intestine.

Seasonal variations in the concentration of antimicrobial components in milk of dairy cows.

The incidence of bovine mastitis and the bulk milk somatic cell count (BMSCC) are influenced by season, which may be associated with innate immune functions, including antimicrobial components in mammary glands. Therefore, the present study was conducted to examine the effect of season on antimicrobial components in milk. Rectal temperature and plasma cortisol, thyroxine, and derivatives of reactive oxygen metabolites (d-ROMs) were measured as stress parameters. Concentrations of lactoferrin (LF), lingual antimicrobial peptide (LAP), psoriasin (S100A7), and Immunoglobulin A (IgA) in milk were measured as indicators of innate immune function. LF and LAP concentrations were significantly lower in summer than in winter and spring, respectively, whereas the concentration of S100A7 was significantly lower in winter than in spring and autumn. The rectal temperature was significantly higher in summer than in other seasons, whereas plasma cortisol, thyroxine, and d-ROMs did not exhibit any seasonal variation. In conclusion, even though stress parameters were not changed, the concentration of antimicrobial components, such as LF and LAP, decreased in summer, which may explain the frequent occurrence of mastitis during this season.

Lactobacillus reuteri Enhances the Mucosal Barrier Function against Heat-killed Salmonella Typhimurium in the Intestine of Broiler Chicks.

Salmonella Typhimurium (ST) infection in chickens inhibits their growth and can lead to food-borne diseases in humans. Probiotics are expected to enhance the function of host intestinal barrier against pathogen infection. The aim of our study was to determine the effect of viable Lactobacillus reuteri (LR) on the response of the mucosal barrier function to antigen stimulation in broiler chicks. Day-old male (n=8) and female (n=4) broiler chicks were orally administered either 1 × 108 LR or a water-only control, every day for 7 days. After 7 days, either 1 × 108 heat-killed ST (k-ST), or a buffer-only control, was administered via intra-cardiac injection. The ileum and cecum were collected 3 h post-injection, and paraffin sections were prepared for either mRNA extraction (males), or gut permeability tests (females). Villus and crypt lengths were determined via histological analysis. Real-time PCR was used to calculate expression levels of Toll-like receptors (TLRs), pro-inflammatory cytokines, anti-inflammatory cytokines, avian ß-defensins, and tight-junction-associated molecules. Gut permeability was assessed using the inverted intestine method. We found that (1) expression of TLR2-1, TLR21, TGF-ß2 and TGF-ß3 were reduced following k-ST stimulation, but were unaffected by LR-treatment; (2) oral administration of LR led to increased Claudin1, Claudin5, ZO2, and JAM2 expression following k-ST stimulation; (3) cecal permeability was reduced by co-treatment with LR and k-ST, but not by treatment with LR or k-ST alone. These results suggest that LR, as used in this study, may enhance the intestinal mucosal physical barrier function, but not the expression of other immune-related factors in newly hatched chicks.

Effects of oral administration of colostrum whey in peripartum goat on antimicrobial peptides in postpartum milk.

The present study was conducted to examine whether colostrum supplementation in peripartum goats increases the antimicrobial peptides in their milk. Goats were orally administered 2 ml of colostrum whey products (colostrum group) or water (control group) daily, from 2 weeks before until 2 weeks after kidding. Body weights of mothers and kids were measured. Blood, milk, and fecal samples were collected from the mothers, and blood samples were collected from the kids. Concentrations of milk antimicrobial peptides (beta-defensin, cathelicidin, lactoferrin, S100A7, lactoperoxidase, and immunoglobulin A [IgA]) were determined. IgA and nutritional parameters (glucose, total cholesterol, triglyceride, ketone bodies, and non-esterified fatty acids) were also determined in the blood of mothers and kids. Milk IgA and lactoferrin concentrations were higher in the colostrum group than in the control group. Conversely, lower milk concentrations of S100A7 were observed in the colostrum group than that in the control group. Plasma IgA concentrations were higher for kids from the colostrum group than for those from the control group. These results suggest that oral administration of colostrum in pregnant goats increases IgA concentration in postpartum milk, which can subsequently improve the health of their kids.

Effects of Oral Administration of Lactobacillus reuteri on Mucosal Barrier Function in the Digestive Tract of Broiler Chicks.

Probiotic bacteria are known for their beneficial effects on the intestinal immune function of the host animal. However, their effects on mucosal barrier function in chicks are not completely understood. The aim of this study was to determine the effects of the probiotic bacterium, Lactobacillus reuteri (LR), on the gastrointestinal mucosal barrier function of broiler chicks. One day-old male broiler chicks were orally injected water (300 µL) with or without 1 × 108 cfu of LR (5 mg FINELACT, Asahi Calpis Wellness Co. Ltd.) every morning for 7 days (day 0 to 6). The crop, duodenum, ileum, and cecum were collected on day 7 and were used for histological analysis and RNA extraction. Then, the thickness of the mucosal structures and the number of goblet cells in the digestive tract were assessed using histological analysis. The expression of Mucin 2, factors related to the formation of tight junctions (Claudin1, 5, and 16, ZO2, and JAM2), cytokines (IL-6, CXCLi2, and IL-10), and avian ß-defensin 10 (AvBDs) (AvBD2, 10, and 12) in the crop, duodenum, ileum, and cecum were analyzed using real-time polymerase chain reaction (PCR). Results showed that oral administration of LR increased ileal villus height and crypt depth, decreased Claudin16 level in the crop and increased JAM2 level in the crop and ileum, and decreased the expression of AvBD10 in the ileum and cecum and that of AvBD12 in the crop. It did not affect goblet cell number and Mucin 2 expression. These results suggested that LR used in this study may enhance mucosal barrier function by regulating tight junctions in the upper gastrointestinal tract.

Slight Disruption in Intestinal Environment by Dextran Sodium Sulfate Reduces Egg Yolk Size Through Disfunction of Ovarian Follicle Growth.

Intestinal environments such as microbiota, mucosal barrier function, and cytokine production affect egg production in laying hens. Dextran sodium sulfate (DSS) is an agent that disrupts the intestinal environment. Previously, we reported that the oral administration of dextran sodium sulfate (DSS: 0.9 g/kg BW) for 5 days caused severe intestinal inflammation in laying hens. However, the DSS concentration in the previous study was much higher to induce a milder disruption of the intestinal environment without heavy symptoms. Thus, the goal of this study was to determine the effects of a lower dose of DSS on the intestinal environment and egg production in laying hens. White Leghorn laying hens (330-day old) were oral administered with or without 0.225 g DSS/kg BW for 28 days (DSS and control group: n = 7 and 8, respectively). Weekly we collected all laid eggs and blood plasma samples. Intestinal tissues, liver, ovarian follicles, and the anterior pituitary gland were collected 1 day after the final treatment. Lower concentrations of orally administered DSS caused (1) a decrease in the ratio of villus height/crypt depth, occludin gene expressions in large intestine and cecal microbiota diversity, (2) a decrease in egg yolk weight, (3) an increase in VLDLy in blood plasma, (4), and enhanced the egg yolk precursor accumulation in the gene expression pattern in the follicular granulosa layer, (5) an increase in FSH and IL-1ß gene expression in the pituitary gland, and (6) an increase in concentration of plasma lipopolysaccharide binding protein. These results suggested that the administration of the lower concentration of DSS caused a slight disruption in the intestinal environment. This disruption included poor intestinal morphology and decreased cecal microbiome diversity. The change in the intestinal environment decreases egg yolk size without decreasing the VLDLy supply from the liver. The decrease in egg yolk size is likely to be caused by the dysfunction of egg-yolk precursor uptake in ovarian follicles. In conclusion, the oral administration of a lower dose of DSS is an useful method to cause slight disruptions of intestinal environment, and the intestinal condition decreases egg yolk size through disfunction of ovarian follicle.

Effects of intrauterine infusion of bacterial lipopolysaccharides on the mammary gland inflammatory response in goats.

This study aimed to determine if intrauterine-infused lipopolysaccharides (LPS) can be translocated to the mammary glands and induce an inflammatory response. Thirty-seven goats were divided into two experiments. Nineteen goats (control group, n = 9; LPS group, n = 10) were subjected to intravenous injection of LPS, and eighteen goats (control group, n = 8; LPS group, n = 10) were subjected to intrauterine infusion of LPS. Milk and blood samples were collected before and after the LPS challenge, to measure the blood leukocyte count (BLC), plasma LPS-binding protein (LBP), milk yield, milk somatic cell count (SCC), lactoferrin (LF), milk lactoperoxidase (LPO) activity, and pro- and anti-inflammatory cytokines in plasma and milk. Mammary gland tissues were collected from the parenchyma before and after the LPS challenge, for immunohistochemistry of LPS. In the intravenous injection experiment, the BLC (P < 0.001) and milk yield (P = 0.009) were lower, whereas the LF concentration (P < 0.001) and milk LPO activity (P < 0.001) were higher in the LPS group compared to that in the control group. LPS was detected in the mammary gland 3 and 24 h after intravenous injection of LPS. In the intrauterine infusion experiment, the mean concentrations of IL-1ß and IL-6 in milk were higher in the LPS group compared to that in the control group (P = 0.004 and P = 0.017, respectively), whereas there were no changes in milk yield or SCC. LPS was detected in the connective tissues and interepithelial spaces of the alveoli of the mammary glands 24 h after intrauterine infusion of LPS. We conclude that intrauterine-infused LPS can be translocated to the mammary glands from the uterus, however, the amount of translocated LPS might not be enough to induce symptoms of clinical or subclinical mastitis.

Effects of Salmonella enteritidis Vaccination on the Expression of Innate Immune Molecules and Histone Modifications in the Follicular Theca of Laying Hens.

The aim of this study was to examine whether Salmonella enteritidis (SE) vaccination affects innate immune function and histone modifications responsible for epigenetic reprogramming in the follicular theca of laying hens. White Leghorn laying hens were administered the SE vaccine or phosphate buffered saline (PBS; control) one week before sample collection. The largest follicles (F1) were collected for total RNA and histone protein extraction. Gene expression levels of immune molecules (Toll-like receptors [TLRs], cytokines, and avian ß-defensins [AvBDs]), and histone modifications in the follicular thecal tissues, were examined using real-time PCR and western blot, respectively. The results showed that the expression levels of TLR1-1, 2-1, 4, and 15 were upregulated by SE vaccination. Although vaccination caused no significant change in cytokine expression, AvBDl, 2, 4, and 7 expression levels were significantly upregulated in the vaccinated group. In addition, the relative density of histone H3-lysine9 dimethylation (H3K9me2) was increased by the vaccination. These results suggest that SE vaccination enhances innate immune functions in the ovary of laying hens, including upregulating TLR and AvBD expression, and is also associated with an increase in histone H3K9me2 in thecal cells.

Effects of colostrum whey on immune function in the digestive tract of goats.

The objective of the present study is to examine whether colostrum whey can have an effect on immune function in goats digestive tract. Two milliliters of colostrum whey (colostrum group) or water (control group) were administrated orally to goats every day for 3 weeks. Blood was collected twice a week for 3 weeks to measure immunoglobulin A (IgA), interleukin 8 (IL-8), and IL-10. At the end of the experimental period, the parotid glands, oral mucosa, lingua, esophagus, jejunum, and ileum were collected for immunohistochemical detection of IgA, cathelicidin-7, and S100A8. The ratio of the length of IgA-positive mucosal surface in the esophagus to the total esophageal length was significantly greater in the colostrum group than in the control group. The number of IgA-positive cells in the labial gland and ileum in the colostrum group was significantly higher than that in the control group. There were no significant differences between the colostrum and control groups in the number of cathelicidin-7-positive cells in the jejunum and ileum and in the number of S100A8-positive cells in the lingua, jejunum, and ileum. These results suggest that colostrum stimulates the recruitment of plasma cells into the labial gland, which then secrete more IgA into the saliva.

Microcystin-leucine-arginine Modulates the Expression Patterns of Proinflammatory Cytokines and an Apoptotic Gene in Chicken Liver.

Microcystins (MCs) are included in drinking water and a family of cyclic heptapeptide hepatotoxins that have been implicated in the impairment of liver function in various animals. There is scarce information on the effect of MCs on cytokines and apoptotic gene expression and on whether MCs can induce inflammation and apoptosis in avian hepatic tissue. This study investigated the expression of genes related to proinflammatory interleukins, apoptosis, and antioxidant function in chicken liver tissues cultured in the presence of different doses of microcystin-leucine-arginine (MC-LR). Livers were collected from five hens and liver slices were placed in sterile tubes containing Dulbecco's medium supplemented with 0, 1, 10, or 100 ng/mL of MC-LR. After 6 h of cultivation, total RNA was extracted and quantitative PCR analysis was performed for interleukin genes (IL-1ß, IL-6, and IL-8), TNF sf15, an apoptotic gene (caspase-3), and genes involved in antioxidant function ([catalase [CAT ], glutathione peroxidase [GSH-PX ], and superoxide dismutase [SOD]). Liver tissues in each group were fixed for histopathology. MC-LR downregulated the mRNA levels of IL-1ß, IL-8, and TNF sf15 as compared to the control (0 ng/mL) in dose-dependent patterns; however, the differences were not significant. The expression of IL-6 in liver tissues exposed to 100 ng/mL of MC-LR was significantly (P<0.05) lower than that in tissues exposed to 1 ng/mL. In contrast, MC-LR upregulated the mRNA expression of caspase-3 and genes involved in antioxidant function in the liver tissues after 6 h, without the difference reaching statistical significance. Hepatocytes showed vacuolar degeneration and focal necrosis according to the dose of MC-LR. This study highlighted the risk of low doses of MC-LR in chicken liver. Moreover, MC-LR could modulate the transcriptional patterns of at least IL-6 in liver-tissue culture of chicken after 6 h of exposure.

Effects of Interleukin-1ß and -6 on the Expression of Ion Transporters Involved in Eggshell Mineralization in Cultured Hen Uterine Mucosal Tissue.

This study determined the effects of pro-inflammatory cytokines (interleukin (IL)-1ß and IL-6) on the expression of eggshell mineralization-related ion transporters in the hen uterus mucosa. Uterine mucosal tissues collected from White Leghorn laying hens were cultured for 1.5 or 3 h in TCM-199 medium with or without 100 ng/mL recombinant chicken IL-1ß or IL-6. Total RNA and protein were extracted from the cultured tissues for real-time polymerase chain reaction (PCR) and western blot analyses and some tissues were processed into paraffin sections for immunostaining with calcium-binding protein D28K (CaBP-D28K) antibody. The gene expression of CaBP-D28K, PMCA1, PMCA2 (plasma membrane calcium-transporting ATPase 1 and 2; calcium pumps), CA2 (carbonic anhydrase 2), and SLC26A9 (solute carrier family 26 member 9; HCO3 - transporter) was analyzed by real-time PCR and protein density of CaBP-D28K by western blotting. Expression of CaBP-D28K, PMCA1, PMCA2, CA2, and SLC26A9 was significantly higher in the tissues treated with IL-1ß and IL-6 than in the control group at 1.5 h of incubation. Immunoreactive CaBP-D28K was localized in the uterine tubular gland cells in all groups, but its level was significantly lower in the tissues incubated for 1.5 h with IL-1ß and IL-6 than in the control group. No significant differences were observed in the expression of all tested genes and CaBP-D28k content between the cytokine-treated and control groups at 3 h of incubation. These results suggest that IL-1ß and IL-6 may not suppress the expression of genes related to Ca2+ and HCO3- transportation for eggshell formation, while CaBP-D28K protein content in uterine glandular cells was reduced by these cytokines during the early exposure phase. Thus, IL-1ß and IL-6 induced by infections may disrupt the transportation of Ca2+ for eggshell formation through decreased CaBP-D28K content in the uterus.

Changes in the Expression of Avian ß-defensins (AvBDs) and Proinflammatory Cytokines and Localization of AvBD2 in the Intestine of Broiler Embryos and Chicks during Growth.

The aim of this study was to determine the changes in the expression of avian ß-defensins (AvBDs) and proinflammatory cytokines and localization of AvBD2 in the intestine of broiler embryos and chicks during growth. The ileum and cecum of embryonic day 19 (ED19) and of day-old (D0) and 7-day-old (D7) chicks were collected. Gene expression levels of 10 AvBDs (AvBD1-8, 10, and 12) and proinflammatory cytokines (IL-1ß, -6, and -8) were analyzed using real-time PCR, and the localization of AvBD2 was examined by immunohistochemistry. Gene expression levels of AvBD1, 2, 6, and 7 in the ileum and of AvBD1 and 4 in the cecum were higher on ED19 than on D7. The expression of AvBD10 in the ileum was higher on D0 than on ED19, whereas the expression levels of AvBD8 and 10 in the cecum were higher on D0 than on ED19, and that of AvBD10 decreased on D7. The expression levels of IL-1ß, -6, and -8 in the ileum were higher on D7 than on ED19. The expression levels of IL-1ß, -6, and -8 in the cecum were higher on D0 than on ED19, and that of IL-1ß and -6 declined on D7. AvBD2-positive cells were localized in the lamina propria beneath epithelial cells of villi and crypts. The number of positive cells in the cecum mucosa was greater on D0 than on ED19 and D7. In conclusion, we suggest that AvBDs are expressed in the ileum and cecum of embryos and chicks at high levels before or just after hatching and decrease by D7. The expression of proinflammatory cytokines in the ileum increases with growth until D7, but is the highest in the cecum around hatching. These AvBDs and proinflammatory cytokines may play roles in host defense in the intestinal mucosa of embryos and neonatal chicks.