Michael H. Kogut

Differential cytokine mRNA expression in heterophils isolated from Salmonella-resistant and -susceptible chickens

We recently showed that increased in vitro heterophil functional efficiency translates to increased in vivo resistance to a systemic Salmonella enteritidis (SE) infection utilizing a parental pair of broiler chickens (lines A and B) and the F1 reciprocal crosses (C and D). Heterophils produce cytokines and modulate acute protection against Salmonella in young poultry. Therefore, we hypothesize that heterophils from SE‐resistant chickens (A and D) have the ability to produce an up‐regulated pro‐inflammatory cytokine response compared to that of heterophils from SE‐susceptible chickens (B and C). In this study, heterophils were isolated from day‐old chickens and treated with either RPMI‐1640 (as the control), or phagocytic agonists (SE, or SE opsonized with either normal chicken serum or immune serum against SE) and cytokine mRNA expression assessed using real‐time quantitative reverse transcription–polymerase chain reaction. Heterophils from SE‐resistant chickens (A and D) had significantly higher levels of pro‐inflammatory cytokine (interleukin (IL)‐6, IL‐8, and IL‐18) mRNA expression upon treatment with all agonists compared to heterophils from SE‐susceptible lines (B and C). Further, heterophils from SE‐resistant chickens had significantly decreased mRNA expression levels of transforming growth factor‐β4, an anti‐inflammatory cytokine, when compared to heterophils from SE‐susceptible chickens. These data indicate cytokine gene expression in heterophils may be a useful parameter in determining resistance to Salmonella, as indicated by our previous in vivo SE studies. Therefore, heterophil functional efficiency and cytokine production may be useful biomarkers for poultry breeders to consider when developing new immunocompetent lines of birds.

In vivo activation of heterophil function in chickens following injection with Salmonella enteritidis-immune lymphokines.

We have previously shown that increased resistance to Salmonella enteritidis organ infectivity in day‐old chicks was conferred by the immunoprophylactic administration of S. enteritidis‐immunc lymphokines (ILK). This resistance was associated with a significant increase in the number of circulating heterophils 4 h after ILK injection. The objective of the present study was to evaluate heterophil function following the administration of ILK in day‐old chicks. Significant increases (P < 0.001) in adherence, chemotaxis, and phagocytosis of S. enteritidis were found with heterophils isolated from ILK‐injected chickens compared to the heterophils isolated from birds injected with either pyrogen‐free saline or lymphokines from non‐immune T cells. After phagocytosis, the heterophils from the ILK‐injected chickens were also able to kill significantly greater numbers of S. enteritidis more rapidly than did the heterophils from the saline‐injected control birds (within 30 min, control cells killed 21.89% of the bacteria whereas ILK‐treated cells killed 88.22%). We also found that the heterophils from the ILK‐injected birds were more efficient killers of S. typhimurium, S. gallinarum, and E. coli These results strongly suggest that the protection against S. enteritidis organ invasion induced by the prophylactic treatment of day‐old chicks with ILK involves activated heterophils which migrate rapidly to the inflammatory stimulus where they phagocytize and kill the bacteria. J. Leukoc. Biol. 57: 56–62; 1995.

Identification of CpG oligodeoxynucleotide motifs that stimulate nitric oxide and cytokine production in avian macrophage and peripheral blood mononuclear cells.

Unmethylated CpG dinucleotides within specific flanking bases (referred to as CpG motif) are relatively abundant in bacterial DNA and are known to stimulate innate immune responses. In this study, synthetic CpG containing oligodeoxydinucleotides (CpG-ODNs) were evaluated for their ability to stimulate nitric oxide (NO), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma) production using an avian macrophage cell line (HD11) and peripheral blood mononuclear cells (PBMC). Results showed ODNs containing the CpG motif can activate the HD11cells and induce NO production. The optimal CpG-ODN motif for NO induction was GTCGTT. Increasing GTCGTT motifs in CpG-ODN significantly enhanced the stimulatory effect. Deviation of flanking bases of the CpG dinucleotide diminished the stimulatory activity. We also found CpG-ODN differentially stimulated expression of cytokine genes. The most active CpG motif for NO induction was also a strong stimulant for the IL-1beta gene expression in the HD11 cells, whereas different CpG motifs were found to induce IFN-gamma gene expression in PBMC.

Differential Regulation of Cytokine Gene Expression by Avian Heterophils During Receptor-Mediated Phagocytosis of Opsonized and Nonopsonized Salmonella enteritidis

Internalization of pathogens by phagocytic cells triggers the innate immune response, which in turn regulates the acquired response. Phagocytes express a variety of receptors that are involved in recognition of pathogens, including (1) pattern recognition receptors (PRR), which recognize conserved motifs, (2) complement receptors (CR), which recognize complement-opsonized pathogens, and (3) Fc receptors (FcR), which recognize antibody-opsonized pathogens. Recognition of microbes is accompanied by the induction of multiple cell processes, including the production of proinflammatory and anti-inflammatory cytokines and chemokines. The objective of the present experiments was to use probes to known avian proinflammatory and anti-inflammatory cytokines and TaqMan technology to ascertain levels of cytokine gene expression in avian heterophils following receptor-mediated phagocytosis of either nonopsonized Salmonella enteritidis (SE), serum-opsonized SE, or IgG-opsonized SE. Expression of interleukin-6 (IL-6) and IL-8, considered in mammals as a proinflammatory chemokine, were upregulated following exposure to the nonopsonized or the opsonized SE. However, mRNA expression for IL-18 and interferon-gamma (IFN-gamma) was downregulated, and the expression of mRNA for the anti-inflammatory cytokine transforming growth factor-beta4 (TGF-beta 4) was upregulated. Interestingly, IL-1beta mRNA expression was significantly upregulated in heterophils that phagocytized either the nonopsonized SE via PRRs or IgG-opsonized SE via FcRs, whereas serum-opsonized SE phagocytized by CRs induced a downregulation of IL-1beta mRNA. These results suggest that signaling interactions initiated by receptor recognition of the microbe surface differentially regulate the induction of inflammatory cytokines in avian heterophils.

The chicken gastrointestinal microbiome

The domestic chicken is a common model organism for human biological research and of course also forms the basis of a global protein industry. Recent methodological advances have spurred the recognition of microbiomes as complex communities with important influences on the health and disease status of the host. In this minireview, we provide an overview of the current state of knowledge of the chicken gastrointestinal microbiome focusing on spatial and temporal variability, the presence and importance of human pathogens, the influence of the microbiota on the immune system, and the importance of the microbiome for poultry nutrition. Review and meta-analysis of public data showed cecal communities dominated by Firmicutes and Bacteroides at the phylum level, while at finer levels of taxonomic resolution, a phylogenetically diverse assemblage of microorganisms appears to have similar metabolic functions that provide important benefits to the host as inferred from metagenomic data. This observation of functional redundancy may have important implications for management of the microbiome. We foresee advances in strategies to improve gut health in commercial operations through management of the intestinal microbiota as an alternative to in-feed subtherapeutic antibiotics, improvements in pre- and probiotics, improved management of polymicrobial poultry diseases, and better control of human pathogens via colonization reduction or competitive exclusion strategies.

Oxidative burst mediated by toll like receptors (TLR) and CD14 on avian heterophils stimulated with bacterial toll agonists.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), which are found in the cell walls of gram-negative and gram-positive bacteria, respectively. This study was conducted to determine if TLRs are present on chicken heterophils and if these receptors mediate oxidative burst. Heterophils isolated from neonatal chicks were exposed to gram-negative Salmonella enteritidis (SE), gram-positive Staphylococcus aureus (SA), SE-LPS, and SA-LTA and the oxidative burst quantitated by luminol-dependent chemiluminescence. SE, SA, SE-LPS, and SA-LTA stimulated a significant increase in oxidative burst from heterophils. Furthermore, we measured the inhibitory effects of polyclonal antibodies on rat CD14, human TLR2 and TLR4 on the oxidative burst of heterophils when stimulated with LPS and LTA. The data suggest that TLR2 and TLR4 mediate LPS-stimulated oxidative burst while CD14 and TLR2 mediate LTA-stimulated oxidative burst in heterophils. This is the first report of PAMPs from gram-positive and gram-negative bacteria interacting with TLRs of avian heterophils.

Heterophils isolated from chickens resistant to extra-intestinal Salmonella enteritidis infection express higher levels of pro-inflammatory cytokine mRNA following infection than heterophils from susceptible chickens

Previous studies showed differences in in vitro heterophil function between parental (A > B) broilers and F1 reciprocal crosses (D > C). Our objectives were to (1) determine if in vitro variations translate to differences in resistance to Salmonella enteritidis (SE) and (2) quantitate cytokine mRNA in heterophils from SE-infected chicks. One-day-old chicks were challenged and organs were cultured for SE. Chicks with efficient heterophils (A and D) were less susceptible to SE compared to chicks with inefficient heterophils (B and C). Heterophils were isolated from SE-infected chicks and cytokine mRNA expression was evaluated using quantitative real-time RT-PCR. Pro-inflammatory cytokine mRNA was up-regulated in heterophils from SE-resistant chicks compared to susceptible chicks. This is the first report to quantitate cytokine mRNA in heterophils from SE-infected chicks. These data show a relationship between in vitro heterophil function, increased pro-inflammatory cytokine mRNA expression, and increased resistance to SE in 1-day-old chicks.

CpG-ODN-induced nitric oxide production is mediated through clathrin-dependent endocytosis, endosomal maturation, and activation of PKC, MEK1/2 and p38 MAPK, and NF-κB pathways in avian macrophage cells (HD11)

We have characterized the nitric oxide (NO) induction by CpG oligodeoxydinucleotide (CpG-ODN) and lipopolysaccharide (LPS) in an avian macrophage cell line (HD11) and evaluated signal transduction pathways by using selective inhibitors. Our results indicate that while CpG-ODN and LPS both stimulate inducible NO synthase (iNOS) to produce NO through common signalling pathways involving activation of protein kinase C (PKC), mitogen-activated protein kinases (p38 MAPK and MEK1/2) and transcription factor NF-kappaB; CpG-ODN inducing NO production distinctively requires a clathrin-dependent endocytosis and subsequent endosomal maturation. Inhibitors of clathrin-dependent endocytosis such as monodansylcadaverine and hyperosmolar sucrose completely abolished CpG-ODN stimulated NO production by HD11 cells, but have no or less effect on LPS-induced NO production. The endosomal maturation is also critical for stimulation of NO induction by CpG-ODN, but not by LPS. Our findings are the first to demonstrate cellular signalling pathways that mediate CpG-ODN immunostimulatory activity in cells from non-mammalian species.

Gene expression profiling in chicken heterophils with Salmonella enteritidis stimulation using a chicken 44 K Agilent microarray

BackgroundSalmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis and usually results from the consumption of contaminated poultry products. The mechanism of SE resistance in chickens remains largely unknown. Previously, heterophils isolated from broilers with different genetic backgrounds (SE-resistant [line A] and -susceptible [line B]) have been shown to be important in defending against SE infections. To dissect the interplay between heterophils and SE infection, we utilized large-scale gene expression profiling.ResultsThe results showed more differentially expressed genes were found between different lines than between infection (SE-treated) and non-infection (control) samples within line. However, the numbers of expressed immune-related genes between these two comparisons were dramatically different. More genes related to immune function were down-regulated in line B than line A. The analysis of the immune-related genes indicated that SE infection induced a stronger, up-regulated gene expression of line heterophils A than line B, and these genes include several components in the Toll-like receptor (TLR) signaling pathway, and genes involved in T-helper cell activation.ConclusionWe found: (1) A divergent expression pattern of immune-related genes between lines of different genetic backgrounds. The higher expression of immune-related genes might be more beneficial to enhance host immunity in the resistant line; (2) a similar TLR regulatory network might exist in both lines, where a possible MyD88-independent pathway may participate in the regulation of host innate immunity; (3) the genes exclusively differentially expressed in line A or line B with SE infection provided strong candidates for further investigating SE resistance and susceptibility. These findings have laid the foundation for future studies of TLR pathway regulation and cellular modulation of SE infection in chickens.

Differential Activation of Signal Transduction Pathways Mediating Phagocytosis, Oxidative Burst, and Degranulation by Chicken Heterophils in Response to Stimulation with Opsonized Salmonella enteritidis

The activation of signal transduction pathways is required for the expression of functional enhancement of cellular activities. In the present studies, initial attempts were made to identify the signal transduction factors involved in activating phagocytosis, generation of an oxidative burst, and degranulation by heterophils isolated from neonatal chickens in response to opsonized Salmonella enteritidis (opsonized SE). Peripheral blood heterophils were isolated and exposed to known inhibitors of signal transduction pathways for either 20 min (staurosporin, genistein, or verapamil) or 120 min (pertussis toxin) at 39°C. The cells were then stimulated for 30 min at 39°C with opsonized SE. Phagocytosis, luminol-dependent chemoluminescence (LDCL), and β-D glucuronidase release were then evaluated in vitro. The G-protein inhibitor pertussin toxin markedly inhibited (>80%) phagocytosis of opsonized SE. Both the protein kinase inhibitor (staurosporin) and calcium channel inhibitor (verapamil) reduced phagocytosis in a dose response manner. Genistein, a tyrosine kinase inhibitor, had no effect on phagocytosis. Staurosporin had a marked inhibitory effect on LDCL (>90%) while genistein had a dose responsive inhibition on LDCL. Both verapamil (40–45%) and pertussin toxin (50–55%) had a statistically significant, but less biologically significant effect on LDCL. Genistein significantly reduced the degranulation (78–81%) of heterophils by opsonized SE. Staurosporin also reduced degranulation by 43–50%, but neither verapamil nor pertussis toxin had a significant effect on degranulation. These findings demonstrate that distinct signal transduction pathways differentially regulate the stimulation of the functional activities of avian heterophils. Pertussin toxin-sensitive, Ca++-dependent G-proteins appear to regulate phagocytosis of opsonized SE, protein kinase C-dependent, tyrosine kinase-dependent protein phosphorylation plays a major role in LDCL, and tyrosine kinase(s)-dependent phosphorylation regulates primary granule release.