Addition of a protected complex of biofactors and antioxidants to breeder hen diets confers transgenerational protection against Salmonella enterica serovar Enteritidis in progeny chicks.

Addition of vitamins and antioxidants has been long associated with increased immunity and are commonly used in the poultry industry; however, less is known regarding their use in broiler breeder hens. The objective of this study was to determine if feeding a complex of protected biofactors and antioxidants composed of vitamins and fermentation extracts to broiler breeder hens conferred resistance against Salmonella enterica serovar Enteritidis (S. Enteritidis) in the progeny chicks. Three-day-old chicks from control- and supplement-fed hens were challenged with S. Enteritidis and necropsied 4- and 11-days postchallenge (dpc) to determine if there were differences in invasion and colonization. Serum and jejunum were evaluated for various cytokine and chemokine production. Fewer (P = 0.002) chicks from supplement-fed hens had detectable S. Enteritidis in the ceca (32.6%) compared to chicks from control-fed hens (64%). By 11 dpc, significantly (P < 0.001) fewer chicks from supplement-fed hens were positive for S. Enteritidis (liver [36%]; ceca [16%]) compared to chicks from the control hens (liver [76%]; ceca [76%]). The recoverable S. Enteritidis in the cecal content was also lower (P = 0.01) at 11 dpc. In additional to the differences in invasion and colonization, cytokine and chemokine production were distinct between the 2 groups of chicks. Chicks from supplement-fed hens had increased production of IL-16, IL-6, MIP-3α, and RANTES in the jejunum while IL-16 and MIP-1ß were higher in the serum of chicks from the control-fed hens. By 11 dpc, production of IFN-γ was decreased in the jejunum of chicks from supplement-fed hens. Collectively, these data demonstrate adding a protected complex of biofactors and antioxidants to the diet of broiler breeder hens offers a measure of transgenerational protection to the progeny against S. Enteritidis infection and reduces colonization that is mediated, in part, by a robust and distinct cytokine and chemokine response locally at the intestine and systemically in the blood.

Heat Stress and Feed Restriction Distinctly Affect Performance, Carcass and Meat Yield, Intestinal Integrity, and Inflammatory (Chemo)Cytokines in Broiler Chickens.

This study was conducted to distinguish the effects of heat stress (HS) and feed intake (FI) on broiler chicken's physiological responses. Day-old male Cobb 500 broilers (n = 672) were allocated to three treatments: (1) control (CTL): birds raised under normal temperature (23°C) from day 29 to 42; (2) cyclic heat stress (CHS): birds exposed to high temperatures (8 h/day at 35°C; from 9:30 am to 5:30 pm) from day 29 to 42; (3) pair-fed (PF): birds raised under thermoneutral condition but fed the same amount of feed as CHS from day 29 to 42. On day 42, 15 birds/pen were processed, to measure carcass and meat yields. To measure blood parameters and gut integrity (using fluorescein isothiocyanate-dextran), on day 42, CHS birds were sampled before (Pre-CHS) and 2 h after (Post-CHS) the temperature increased. Furthermore, after sampling CTL birds, they were exposed to 2h heat and sampled (acute heat stress, AHS). Data were analyzed using one-way ANOVA (JMP Pro15) and significance between treatments identified by LSD (P < 0.05). BW and relative carcass yield were significantly higher in CTL compared to CHS and PF. Compared to CHS, PF had significantly higher BW and lower relative carcass yield. Breast yield was significantly higher for CTL and PF, while leg quarters and wings yield were significantly lower compared to CHS. Gut barrier integrity was significantly altered in Post-CHS and AHS compared to CTL. mRNA abundances of tumor necrosis factor-α, C-C motif chemokine ligand-20, heat shock protein (HSP)-27, and HSP70 were significantly higher in Post-CHS and AHS compared to CTL. AHS had significantly higher mRNA abundances of CARD domain containing (NLRC)-3 and NLRC5 inflammasomes, and lower superoxide dismutase (SOD)-1 and SOD2 abundance compared with CTL. PF had significantly higher liver weight (% BW) compared to all other groups; while abdominal fat was significantly higher in Pre-CHS compared to CTL, PF, and AHS. Together, these data indicate that the negative effects of HS are partially due to reduced FI. However, the negative effect of HS on gut integrity, average daily gain, feed conversion ratio, and meat yield are direct and independent of the reduced FI during the HS. Thus, warrant investigating the underlying mechanisms in future research.

Novel Models for Chronic Intestinal Inflammation in Chickens: Intestinal Inflammation Pattern and Biomarkers.

For poultry producers, chronic low-grade intestinal inflammation has a negative impact on productivity by impairing nutrient absorption and allocation of nutrients for growth. Understanding the triggers of chronic intestinal inflammation and developing a non-invasive measurement is crucial to managing gut health in poultry. In this study, we developed two novel models of low-grade chronic intestinal inflammation in broiler chickens: a chemical model using dextran sodium sulfate (DSS) and a dietary model using a high non-starch polysaccharide diet (NSP). Further, we evaluated the potential of several proteins as biomarkers of gut inflammation. For these experiments, the chemical induction of inflammation consisted of two 5-day cycles of oral gavage of either 0.25mg DSS/ml or 0.35mg DSS/ml; whereas the NSP diet (30% rice bran) was fed throughout the experiment. At four times (14, 22, 28 and 36-d post-hatch), necropsies were performed to collect intestinal samples for histology, and feces and serum for biomarkers quantification. Neither DSS nor NSP treatments affected feed intake or livability. NSP-fed birds exhibited intestinal inflammation through 14-d, which stabilized by 36-d. On the other hand, the cyclic DSS-treatment produced inflammation throughout the entire experimental period. Histological examination of the intestine revealed that the inflammation induced by both models exhibited similar spatial and temporal patterns with the duodenum and jejunum affected early (at 14-d) whereas the ileum was compromised by 28-d. Calprotectin (CALP) was the only serum protein found to be increased due to inflammation. However, fecal CALP and Lipocalin-2 (LCN-2) concentrations were significantly greater in the induced inflammation groups at 28-d. This experiment demonstrated for the first time, two in vivo models of chronic gut inflammation in chickens, a DSS and a nutritional NSP protocols. Based on these models we observed that intestinal inflammation begins in the upper segments of small intestine and moved to the lower region over time. In the searching for a fecal biomarker for intestinal inflammation, LCN-2 showed promising results. More importantly, calprotectin has a great potential as a novel biomarker for poultry measured both in serum and feces.

The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota.

BACKGROUND: Microencapsulated organic acids and botanicals have the potential to develop into important tools for the poultry industry. A blend of organic acids and botanicals (AviPlus®P) has previously shown to reduce Salmonella and Campylobacter in chickens; however, changes to the microbiota of the jejunum and ileum have not been evaluated. Microbiota diversity is linked to, but not correlated with, the efficacy of natural products; therefore, understanding the effects on the microbiota is necessary for evaluating their potential as an antibiotic alternative. RESULTS: Ileal and jejunal segments from control and supplement-fed chickens (300 and 500 g/metric ton [MT]) were subjected to alpha diversity analysis including Shannon's diversity and Pielou's Evenness. In both analytics, the diversity in the ileum was significantly decreased compared to the jejunum irrespective of treatment. Similarly, beta diversity metrics including Bray-Curtis dissimilarity index and Weighted Unifrac Distance Matrix, were significant (Q < 0.05) for both tissue and treatments comparisons. Alpha and beta diversity analytics indicated compartmentalization effects between the ileum and jejunum. Additionally, analysis of communities in the microbiota (ANCOM) analysis showed Lactobacilliaceae predominated the total operational taxonomic units (OTU), with a stepwise increase from 53% in the no treatment control (NTC) to 56% in the 300 g/MT and 67% in the 500 g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500 g/MT groups. Enterobacteriaceae decreased in the 500 g/MT (31%) and increased in the 300 g/MT (37%) compared to the NTC (35%). Aerococcaceae was 0% for both doses and 7% in NTC. Ruminococcaceae were 0% in NTC and 2 and 1% in the 300 and 500 g/MT. These changes in the microbial consortia were statistically (Q < 0.05) associated with treatment groups in the jejunum that were not observed in the ileum. Least discriminant analysis effect size (LEfSE) indicated different changes directly corresponding to treatment. Enterobacteriaceae demonstrated a stepwise decrease (from NTC onward) while Clostridiaceae, were significantly increased in the 500 g/MT compared to NTC and 300 g/MT (P < 0.05). CONCLUSION: The bioactive site for the microencapsulated blend of organic acids and botanicals was the jejunum, and dietary inclusion enhanced the GIT microbiota and may be a viable antibiotic alternative for the poultry industry.

Dietary l-arginine supplementation influences growth performance and B-cell secretion of immunoglobulin in broiler chickens.

Our previous study has shown that high levels of l-arginine (ARG) have reduced serum and mucosal antibody concentrations. In order to provide a better understanding in the application of ARG supplementation in the poultry industry, the study was conducted to investigate the effect of high levels of ARG on performance and B-cell secretion of immunoglobulin M (IgM) and IgG development in broiler chickens. A total of 192 1-day-old male Arbor Acres Plus broilers were randomly allocated into 4 groups (8 replicates per group, 6 birds per replicate) fed diets containing one of four ARG concentrations (analysed): 9.8, 14.7, 19.1 and 23.4 g/kg respectively. Growth performance was measured based on body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR). Increasing ARG quadratically increased (p < 0.05) BWG and FI with reaching plateau at 14.7 g/kg, while linearly decreased (p < 0.05) FCR, indicating that maximal performance required ARG no more than 14.7 g/kg in diets. Serum IgG and IgM concentrations were linearly reduced (p < 0.05) with increasing ARG. Chickens fed 19.1 g/kg or 23.4 g/kg ARG had lower (p < 0.05) serum IgG or IgM than chickens fed 9.8 g/kg ARG. As for the mRNA expression of bursal IgG and IgM, they were significantly downregulated with increasing ARG (p < 0.05). Chickens on ARG (>19.1 g/kg) had a lower (p < 0.05) IgG and IgM mRNA expression than chickens fed 9.8 g/kg. Activator of transcription 3 (STAT3) mRNA expression was linearly reduced with increasing ARG (p < 0.05), the transcriptional repressor B-cell lymphoma 6 (BCL6) mRNA expression was quadratically (p < 0.05) responded, and these cytokines had the lowest expression at 19.1 g/kg. ARG supplementation (>14.7 g/kg) did not significantly improve the growth performance, while it may have a potential negative regulatory effect on B-cell-mediated humoral immunity in chickens associated with suppression of the STAT3 expression associated with the JAK/STAT3 pathway.

Inhibition of calmodulin increases intracellular survival of Salmonella in chicken macrophage cells.

Calcium (Ca2+) is a pivotal intracellular second messenger and calmodulin (CaM) acts as a multifunctional Ca2+-binding protein that regulates downstream Ca2+ dependent signaling. Together they play an important role in regulating various cellular functions, including gene expression, maturation of phagolysosome, apoptosis, and immune response. Intracellular Ca2+ has been shown to play a critical role in Toll-like receptor-mediated immune response to microbial agonists in the HD11 chicken macrophage cell line. The role of that the Ca2+/CaM pathway plays in the intracellular survival of Salmonella in chicken macrophages has not been reported. In this study, kinome peptide array analysis indicated that the Ca2+/CaM pathway was significantly activated when chicken macrophage HD11 cells were infected with S. Enteritidis or S. Heidelberg. Further study demonstrated that treating cells with a pharmaceutical CaM inhibitor W-7, which disrupts the formation of Ca2+/CaM, significantly inhibited macrophages to produce nitric oxide and weaken the control of intracellular Salmonella replication. These results strongly indicate that CaM plays an important role in the innate immune response of chicken macrophages and that the Ca2+/CaM mediated signaling pathway is critically involved in the host cell response to Salmonella infection.

Chicken macrophages infected with Salmonella (S.) Enteritidis or S. Heidelberg produce differential responses in immune and metabolic signaling pathways.

Protein kinases act in coordination with phosphatases to control protein phosphorylation and regulate signaling pathways and cellular processes involved in nearly every functions of cell life. Salmonella are known to manipulate the host kinase network to gain entrance and survive inside host cells. The effect of Salmonella infection on the host kinase network has been studied in mammalian cells, but information is largely lacking in chicken immune cells. Our previous study indicated that chicken macrophage cells respond differentially to different Salmonella strains. In order to better understand the interaction between chicken macrophages and Salmonella, we used a peptide array-based kinome analysis to identify cellular process and signaling pathways that may play a critical role in the outcome of Salmonella infection. The kinome assay was performed on chicken HD11 macrophages collected at 1.5, 3, and 7h post-infection (hpi) with either S. Heidelberg or S. Enteritidis. A large number of peptides show significantly changed phosphorylation (p≤0.05) during the infection: 390, 449, and 575 peptides for S. Enteritidis and 185, 470, and 442 for S. Heidelberg at 1.5, 3, and 7 hpi, respectively. Many pathways involved in immunity, signal transduction, cellular process, and metabolism were significantly altered, in some case differentially, during the infection by the two Salmonella strains. Particularly, effects on lysosome process, iNOS, CARD9, NLRP3, and MAPK pathway provide significant insight to the inter play between pathogens and chicken macrophage cells during the infection.

Combined CpG and poly I:C stimulation of monocytes results in unique signaling activation not observed with the individual ligands.

Toll-like receptors (TLRs) bind to components of microbes, activate cellular signal transduction pathways and stimulate innate immune responses. Previously, we have shown in chicken monocytes that the combination of CpG, the ligand for TLR21 (the chicken equivalent of TLR9), and poly I:C, the ligand for TLR3, results in a synergistic immune response. In order to further characterize this synergy, kinome analysis was performed on chicken monocytes stimulated with either unmethylated CpG oligodeoxynucleotides (CpG) and polyinosinic-polycytidylic acid (poly I:C) individually or in combination for either 1h or 4h. The analysis was carried out using chicken species-specific peptide arrays to study the kinase activity induced by the two ligands. The arrays are comprised of kinase target sequences immobilized on an array surface. Active kinases phosphorylate their respective target sequences, and these phosphorylated peptides are then visualized and quantified. A significant number of peptides exhibited altered phosphorylation when CpG and poly I:C were given together, that was not observed when either CpG or poly I:C was given separately. The unique, synergistic TLR agonist affected peptides represent protein members of signaling pathways including calcium signaling pathway, cytokine-cytokine receptor interaction and Endocytosis at the 1h time point. At the 4h time point, TLR agonist synergy influenced pathways included Adipocytokine signaling pathway, cell cycle, calcium signaling pathway, NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway. Using nitric oxide (NO) production as the readout, TLR ligand synergy was also investigated using signaling protein inhibitors. A number of inhibitors were able to inhibit NO response in cells given CpG alone but not in cells given both CpG and poly I:C, as poly I:C alone does not elicit a significant NO response. The unique peptide phosphorylation induced by the combination of CpG and poly I:C and the unique signaling protein requirements for synergy determined by inhibitor assays both show that synergistic signaling is not a simple addition of TLR pathways. A set of secondary pathways activated by the ligand combination are proposed, leading to the activation of cAMP response element-binding protein (CREB), nuclear factor κB (NFκB) and ultimately of inducible nitric oxide synthase (iNOS). Since many microbes can stimulate more than one TLR, this synergistic influence on cellular signaling may be an important consideration for the study of immune response and what we consider to be the canonical TLR signaling pathways.

Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection of chickens that are more than a few days old results in asymptomatic cecal colonization with persistent shedding of bacteria. We hypothesized that while the bacterium colonizes and persists locally in the cecum it has systemic effects, including changes to metabolic pathways of skeletal muscle, influencing the physiology of the avian host. Using species-specific peptide arrays to perform kinome analysis on metabolic signaling pathways in skeletal muscle of Salmonella Typhimurium infected chickens, we have observed key metabolic changes that affected fatty acid and glucose metabolism through the 5'-adenosine monophosphate-activated protein kinase (AMPK) and the insulin/mammalian target of rapamycin (mTOR) signaling pathway. Over a three week time course of infection, we observed changes in the phosphorylation state of the AMPK protein, and proteins up and down the pathway. In addition, changes to a large subset of the protein intermediates of the insulin/mTOR pathway in the skeletal muscle were altered by infection. These changes occur in pathways with direct effects on fatty acid and glucose metabolism. This is the first report of significant cellular metabolic changes occurring systemically in chicken due to a Salmonella infection. These results have implications not only for animal production and health but also for the understanding of how Salmonella infection in the intestine can have widespread, systemic effects on the metabolism of chickens without disease-like symptoms.

Nitric oxide as a biomarker of intracellular Salmonella viability and identification of the bacteriostatic activity of protein kinase A inhibitor H-89.

Salmonella enterica serovar Enteritidis is one of the most prevalent Salmonella serovars in poultry and is often associated with human salmonellosis. S. Enteritidis is known to suppress nitric oxide (NO) production in infected chicken macrophage HD11 cells, while dead S. Enteritidis stimulates a high level of NO production, suggesting a bacterial inhibitory effect on NO production. Based on these observations, the present study was conducted to evaluate whether NO production in S. Enteritidis-infected HD11 cells can be used as a biomarker to identify molecules that kill intracellular Salmonella. Since Salmonella are known to manipulate the host cell kinase network to facilitate intracellular survival, we screened a group of pharmaceutical inhibitors of various kinases to test our hypothesis. A protein kinase A inhibitor, H-89, was found to reverse the suppression of NO production in S. Enteritidis-infected HD11 cells. Production of NO in S. Enteritidis-infected HD11 cells increased significantly following treatment with H-89 at or above 20 µM. Inversely, the number of viable intracellular Salmonella decreased significantly in cells treated with H-89 at or above 30 µM. Furthermore, the growth rate of S. Enteritidis in culture was significantly inhibited by H-89 at concentrations from 20 to 100 µM. Our results demonstrate that NO-based screening using S. Enteritidis-infected HD11 cells is a viable tool to identify chemicals with anti-intracellular Salmonella activity. Using this method, we have shown H-89 has bacteriostatic activity against Salmonella, independent of host cell protein kinase A or Akt1 activity.

A comparative study on invasion, survival, modulation of oxidative burst, and nitric oxide responses of macrophages (HD11), and systemic infection in chickens by prevalent poultry Salmonella serovars.

Poultry is a major reservoir for foodborne Salmonella serovars. Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg are the most prevalent serovars in U.S. poultry. Information concerning the interactions between different Salmonella species and host cells in poultry is lacking. In the present study, the above mentioned Salmonella serovars were examined for invasion, intracellular survival, and their ability to modulate oxidative burst and nitric oxide (NO) responses in chicken macrophage HD11 cells. All Salmonella serovars demonstrated similar capacity to invade HD11 cells. At 24 h post-infection, a 36-43% reduction of intracellular bacteria, in log(10)(CFU), was observed for Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg, whereas a significantly lower reduction (16%) was observed for Salmonella Enteritidis, indicating its higher resistance to the killing by HD11 cells. Production of NO was completely diminished in HD11 cells infected with Salmonella Typhimurium and Salmonella Enteritidis, but remained intact when infected with Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg. Phorbol myristate acetate-stimulated oxidative burst in HD11 cells was greatly impaired after infection by each of the five serovars. When newly hatched chickens were challenged orally, a high rate (86-98%) of systemic infection (Salmonella positive in liver/spleen) was observed in birds challenged with Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, and Salmonella Kentucky, while only 14% of the birds were Salmonella Senftenberg positive. However, there was no direct correlation between systemic infection and in vitro differential intracellular survival and modulation of NO response among the tested serovars.

Gene Expression Analysis of Toll-Like Receptor Pathways in Heterophils from Genetic Chicken Lines that Differ in Their Susceptibility to Salmonella enteritidis.

Previously conducted studies using two chicken lines (A and B) show that line A birds have increased resistance to a number of bacterial and protozoan challenges and that heterophils isolated from line A birds are functionally more responsive. Furthermore, when stimulated with Toll-like receptor (TLR) agonists, heterophils from line A expressed a totally different cytokine and chemokine mRNA expression pattern than heterophils from line B. A large-scale gene expression profile using an Agilent 44K microarray on heterophils isolated from line A and line B also revealed significantly differential expression in many immune-related genes following Salmonella enteritidis (SE) stimulation, which included genes involved in the TLR pathway. Therefore, we hypothesize the differences between the lines result from distinctive TLR pathway signaling cascades that mediate heterophil function and, thus, innate immune responsiveness to SE. Using quantitative RT-PCR on mRNA from heterophils isolated from control and SE-stimulated heterophils of each line, we profiled the expression of all chicken homologous genes identified in a reference TLR pathway. Several differentially expressed genes found were involved in the TLR-induced My88-dependent pathway, showing higher gene expression in line A than line B heterophils following SE stimulation. These genes included the TLR genes TLR4, TLR15, TLR21, MD-2, the adaptor proteins Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP), Tumor necrosis factor-receptor associated factor 3 (TRAF3), the IκB kinases transforming growth factor-ß-activating kinase 1 (TAK1), IKKε and IKKα, the transcription factors NFkB2 and interferon regulatory factor 7, phosphatidylinositol-3 kinase (PI-3K), and the mitogen-activated protein kinase p38. These results indicate that higher expression of TLR signaling activation of both MyD88-dependent and TRIF-dependent pathways are more beneficial to avian heterophil-mediated innate immunity and a complicated regulation of downstream adaptors is involved in stronger induction of a TLR-mediated innate response in the resistant line A. These findings identify new targets for genetic selection of chickens to increase resistance to bacterial infections.

Neutrophil function of neonatal foals is enhanced in vitro by CpG oligodeoxynucleotide stimulation.

Rhodococcus equi is an intracellular bacterium that causes pneumonia in foals and immunocompromised adult horses. Evidence exists that foals become infected with R. equi early in life, a period when innate immune responses are critically important for protection against infection. Neutrophils are innate immune cells that play a key role in defense against this bacterium. Enhancing neutrophil function during early life could thus help to protect foals against R. equi infection. The objective of our study was to determine whether in vitro incubation with the TLR9 agonist CpG 2142 would enhance degranulation and gene expression of cytokines and Toll-like receptor 9 (TLR9) by neutrophils collected from foals at 2, 14, and 56 days of life, and to determine whether these stimulated responses varied among ages. Neutrophil degranulation was enhanced at all ages by in vitro stimulation with either CpG alone, R. equi alone, or in combination with either R. equi or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (P<0.05), but not by in vitro stimulation with fMLP alone. There were no significant differences among ages in CpG-induced cytokine expression, except for IL-12p40, which was induced more at 56 days of age than on days 2 or 14. Collapsing data across ages, CpG 2142 significantly (P<0.05) increased IL-6 and IL-17 mRNA expression. We concluded that in vitro stimulation of foal neutrophils with CpG enhances their function by promoting degranulation and inducing mRNA expression of IL-6 and IL-17, regardless of age.

Bacterial toll-like receptor agonists induce sequential NF-κB-mediated leukotriene B4 and prostaglandin E2 production in chicken heterophils.

Studies of the response of the primary avian polymorphonuclear leukocyte, the heterophil, to microbe associated molecular patterns (MAMPs) through toll-like receptors (TLR) has concentrated on the activation of the respiratory burst, release of intracellular granules, and the induction of cytokine and chemokine expression. Virtually no studies have been described on the role of lipid mediators, leukotrienes and prostaglandins, as effectors of the avian inflammatory response. We have previously shown that flagellin (FLG), the bacterial lipoprotein mimic palmitoly-3-cysteine-serine-lysine-4 (PAM), and unmethylated CpG motifs of bacteria DNA (CpG) are all potent activators of the avian innate immune system. In the present studies, we hypothesized that FLG, PAM, and CpG are also capable of eliciting the production of these lipid mediators of inflammation by avian heterophils. Compared to non-stimulated control heterophils, all three TLR agonists were potent inducers (3-5-fold increase) of a rapid production (30 min) of leukotriene B(4) (LTB(4)) followed by a later release (60-120 min) of prostaglandin (PGE(2)) by the heterophils. LTB(4) and PGE(2) production were derived from lipoxygenase-5 (5-LO) and cyclooxygenase-2 (COX-2) enzymatic activities, respectively, as the selective 5-LO (caffeic acid) and COX-2 (NS-398) inhibitors eliminated LTB(4) and PGE(2) production from the MAMP-stimulated heterophils. These results demonstrate that both the lipoxygenase and cycloxygenase pathways are operational in avian heterophils in response to bacterial MAMPs. Treatment of heterophils with either FLG, PAM, or CpG also induced a significant increase in DNA binding by NF-κB family members' p50, c-Rel, and RelB. Additionally, the production of LTB(4) and PGE(2) were inhibited following treatment of heterophils with the specific pharmacologic inhibitor of NF-κB (Bay 11-7086), thus suggesting that TLR pathway activation of NF-κB controls LTB(4) and PGE(2) production. This the first report of the production of lipid mediators of inflammation by avian heterophils in response to PAMPs. Since FLG, lipoproteins, and bacterial CpG DNA are abundant during bacterial infections, these data support their role in the inflammatory response mediated by avian heterophils.

Modulation of chicken macrophage effector function by T(H)1/T(H)2 cytokines.

Regulation of macrophage activity by T(H)1/2 cytokines is important to maintain the balance of immunity to provide adequate protective immunity while avoiding excessive inflammation. IFN-γ and IL-4 are the hallmark T(H)1 and T(H)2 cytokines, respectively. In avian species, information concerning regulation of macrophage activity by T(H)1/2 cytokines is limited. Here, we investigated the regulatory function of chicken T(H)1 cytokines IFN-γ, IL-18 and T(H)2 cytokines IL-4, IL-10 on the HD11 macrophage cell line. Chicken IFN-γ stimulated nitric oxide (NO) synthesis in HD11 cells and primed the cells to produce significantly greater amounts of NO when exposed to microbial agonists, lipopolysaccharide, lipoteichoic acid, peptidoglycan, CpG-ODN, and poly I:C. In contrast, chicken IL-4 exhibited bi-directional immune regulatory activity: it activated macrophage NO synthesis in the absence of inflammatory agonists, but inhibited NO production by macrophages in response to microbial agonists. Both IFN-γ and IL-4, however, enhanced oxidative burst activity of the HD11 cells when exposed to Salmonella enteritidis. IL-18 and IL-10 did not affect NO production nor oxidative burst in HD11 cells. Phagocytosis and bacterial killing by the HD11 cells were not affected by the treatments of these cytokines. Infection of HD11 cells with S.enteritidis was shown to completely abolish NO production regardless of IFN-γ treatment. This study has demonstrated that IFN-γ and IL-4 are important T(H)1 and T(H)2 cytokines that regulate macrophage function in chickens.

Chicken scavenger receptors and their ligand-induced cellular immune responses.

The scavenger receptors (SRs) comprise structurally and functionally divergent groups of cell surface and secreted proteins that play an important role in innate immune defenses. Searching translated chicken genomic databases revealed many proteins homologous to mammalian SRs. SR mediated immune functions (oxidative burst, degranulation, phagocytosis, nitric oxide (NO) production, and cytokine expression) were evaluated in chicken heterophils, peripheral blood mononuclear cells (PBMC), and a chicken macrophage cell line (HD11) using various SR class A and B ligands. Results showed that the SR-A ligands, fucoidan, poly(I) and poly(G), but not SR-B ligands, phosphatidylserine and LDL, stimulated dose-dependent NO production in HD11 cells. However, SR-A ligands failed to induce NO in chicken monocytes. Quantitative RT-PCR indicated that SR ligands differentially regulated the gene expression of cytokines and chemokine in HD11 cells with a strong up-regulation of the cytokines IL-1 beta and IL-6 and the chemokine MIP-1 beta, but had no effect on IL-4, IL-12, IFN-gamma, and IFN-beta. SR-B ligands did not alter expression of these genes. SR-A ligands had no stimulatory effect on functional response in heterophils. However, LDL, a SR-B ligand stimulated oxidative burst in both heterophils and PBMC. Additionally, results indicate that SRs are involved in bacterial binding in macrophages.

Flagellin and lipopolysaccharide up-regulation of IL-6 and CXCLi2 gene expression in chicken heterophils is mediated by ERK1/2-dependent activation of AP-1 and NF-kappaB signaling pathways.

The Toll-like receptor agonists, flagellin (FLG) and lipopolysaccharide (LPS), stimulate chicken heterophils to induce the expression and secretion of pro-inflammatory cytokines by a mechanism involving the triggering of differential MEK-ERK signaling cascades. However, the translocation and activation of transcription factors potentially involved in the control of cytokine gene expression remains unknown. Herein, we examined the effects of FLG and LPS on the activation of the transcription factors NF-kappaB and AP-1 and their role in regulating heterophil activation leading to cytokine gene expression. Treatment of heterophils with either FLG or LPS induced a significant increase in DNA binding by the NF-kappaB family members p50, c-Rel, and RelB. Likewise, FLG and LPS induced a significant increase in DNA binding by the AP-1 family members c-Jun and JunD. The activation of both NF-kappaB and AP-1 was inhibited following treatment of heterophils with specific inhibitors of ERK1/2 (U0126 and PD098059), NF-kappaB (Bay 11-7086 and the cell-permeable NF-kappaB peptide, SN50), and AP-1 (Tanshinone IIA). Likewise, the up-regulation of gene expression of the pro-inflammatory cytokine, IL-6, and the inflammatory chemokine, CXCLi2, were inhibited when heterophils were treated with the same specific inhibitors. Taken together these data demonstrate that FLG and LPS stimulate the up-regulation of expression of IL-6 and CXCLi2 through an ERK1/2-dependent activation of both NF-kappaB and AP-1.

Phospholipase C, phosphatidylinositol 3-kinase, and intracellular [Ca(2+)] mediate the activation of chicken HD11 macrophage cells by CpG oligodeoxynucleotide.

The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules. Oligodeoxynucleotides containing CpG motifs (CpG ODN) mimic microbial DNA and are immunostimulatory to most vertebrate species. In the present study, we used the production of nitric oxide (NO) as an indicator to evaluate the involvement of the signaling cascades of phospholipases and phosphatidylinositol 3-kinase (PI3K) in the activation of chicken HD11 macrophage cells by CpG ODN. Using selective inhibitors, we have identified the involvement of phosphatidylinositol (PI)-phospholipase C (PI-PLC), but not phosphatidylcholine (PC)-phospholipase C (PC-PLC) and PC-phospholipase D (PC-PLD), in CpG ODN-induced NO production in HD11 cells. Preincubation with PI-PLC selective inhibitors (U-73122) completely abrogated CpG ODN-induced NO production in HD11 cells, whereas PC-PLC inhibitor (D609) and PC-PLD inhibitor (n-butanol) had no inhibitory effects. Additionally, inhibition of PI3K and protein kinase C (PKC) with selective inhibitors and chelation of intracellular [Ca(2+)] also significantly attenuated NO production in CpG ODN-activated HD11 cells. Our results demonstrate that PI-PLC, PI3 K, PKC, and intracellular [Ca(2+)] are important components of the CpG ODN-induced signaling pathway that leads to the production of NO in avian macrophage cells.

In vivo priming heterophil innate immune functions and increasing resistance to Salmonella enteritidis infection in neonatal chickens by immune stimulatory CpG oligodeoxynucleotides.

Oligodeoxynucleotides (ODN) containing CpG dinucleotides (CpG-ODN) mimic bacterial DNA and stimulate the innate immune system of vertebrates. Here, we investigated the effects of intraperitoneal (ip) administered CpG-ODN on the innate immune functions of chicken heterophils. Our results demonstrated CpG-ODN-dependent priming of chicken heterophil degranulation and oxidative burst. Heterophils from chickens treated with CpG-ODN exhibited significantly higher (p<0.05) degranulation activity compared to PBS and control ODN (ODN containing no CpG motif) treated groups when stimulated with opsonized Salmonella enterica serovar enteritidis. Similarly, oxidative burst activity, which generates bactericidal reactive oxygen species, was significantly higher (p<0.05) in heterophils from the CpG-ODN treated group than from PBS and control ODN groups when stimulated with formalin-killed S. enteritidis. The priming effects of CpG-ODN on heterophil immune functions continued at least 4 days post-treatment. In the infection study, newly hatched chickens were treated with CpG-ODN, control ODN or PBS for 24h then challenged with oral inoculation of S. enteritidis. A significant reduction (p<0.05) in colonization by S. enteritidis was observed in chickens treated with CpG-ODN. Our study provides evidence that immunostimulatory CpG-ODN potentiates the innate immune responses of heterophils and enhances resistance to infectious pathogens in neonatal chickens.

Involvement of phosphatidylinositol-phospholipase C in immune response to Salmonella lipopolysacharide in chicken macrophage cells (HD11).

The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules. Lipopolysaccharide (LPS) is a principle component of the outer membrane of Gram-negative bacteria and a prime target for recognition by the innate immune system. In the present study, we evaluated the role of specific phospholipase in the activation of a chicken macrophage cell line HD11 by LPS. Activation of HD11 cells by LPS results in induction of nitric oxide (NO). Using selective inhibitors, we have identified that phosphatidylinositol (PI)-phospholipase C (PI-PLC), but not phosphatidylcholine (PC)-phospholipase C (PC-PLC) nor PC-phospholipase D (PC-PLD), was required for LPS-induced NO production. Preincubation with PI-PLC selective inhibitors (U-73122 and ET-18-OCH3) abrogated LPS-induced NO production in HD11 cells, whereas PC-PLC inhibitor (D609), phosphatide phosphohydrolase inhibitor (propranolol), and PC-PLD inhibitor (n-butanol) had no inhibitory effects. We also showed that inhibition of protein kinase C (PKC) by selective inhibitors Ro 31-8220 and calphostin C and chelating intracellular Ca2+ by BAPTA-AM significantly reduced NO production in LPS-stimulated HD11 cells. Our results demonstrate that PI-PLC plays a critical role, most likely through activation of PKC pathway, in TLR4 mediated immune responses of avian macrophage cells to LPS.