Haiqi He

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.

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.

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.

Lipopolysaccharide Binding Protein/CD14/TLR4-Dependent Recognition of Salmonella LPS Induces the Functional Activation of Chicken Heterophils and Up-Regulation of Pro-Inflammatory Cytokine and Chemokine Gene Expression in These Cells

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern (PAMP) found in the cell wall of gram-negative bacteria and, in mammals, is recognized by the Toll-like receptor 4 (TLR4) in conjunction with the serum protein, lipopolysaccharide-binding protein (LBP), and the CD14 co-receptor. We have found that chicken heterophils constitutively express multiple TLRs including TLR4. Interestingly, ultrapure LPS from Salmonella minnesota directly induced the functional activation of heterophils without the presence of LBP. However, the role of LBP and CD14 in the recognition of LPS and the induction of innate immunity, including cell functional activation and the transcription of cytokine and chemokine genes in chicken heterophils, is not known. As previously seen, in the absence of chicken serum, heterophil exposure to ultrapure LPS from Salmonella minnesota stimulated an increased degranulation response. However, the presence of 5% chicken serum, presumed to be a source of LBP, increased heterophil degranulation by 84%. In addition, the presence of either soluble recombinant human LBP (rhLBP, 68%) or CD14 (39%) also induced the up-regulation of the heterophil degranulation response. Incubation of heterophils with either chicken serum or rhLBP also significantly induced the up-regulation of pro-inflammatory cytokine (IL-1β, IL-6, and IL-18) and chemokine (CCLi4, CXCLi1, CXCLi2, and the CXC receptor 1) mRNA expression. Moreover, polyclonal antibodies directed against rat CD14 and human TLR4, but not antibodies against human TLR2, blocked LPS-mediated degranulation and up-regulation of the pro-inflammatory cytokine and chemokine mRNA expression. These data clearly demonstrate that LBP and CD14/TLR4 engagement is directly involved in LPS-mediated functional activation and innate immune gene expression in chicken heterophils.

Expression profile of toll-like receptors within the gastrointestinal tract of 2-day-old Salmonella enteriditis-infected broiler chickens

Salmonella enterica serovar Enteriditis (SE) causes a majority of foodborne illness in the U.S. A more productive avian innate immune response could reduce bacterial colonization and the incidence of infection in humans. However, quantification and comparison of the toll-like receptors (TLR), a component of the innate immune system that recognize bacterial pathogens, and their response to SE colonization across the avian gastrointestinal (GI) tract has not been reported. Therefore, we assessed these changes using real-time qRT-PCR to measure expression of TLR 1LA, 2A, 2B, 3, 4, 5, 7, 15, and 21 in the duodenum, jejunum, ileum, cecal tonsil, ceca, and large intestine of uninfected and SE-infected 2-day-old broiler chickens. Samples were collected soon after hatch to approximate natural SE exposure and to measure initial changes in the immune response to infection. All TLRs had measurable expression within the duodenum, jejunum, ileum, cecal tonsil, ceca, and large intestine. The general expression pattern, with the exception of TLR 21, showed distal GI segments had higher TLR mRNA expression than proximal segments. Infected chickens had increased expression of TLR 1LA, 2A, 4, and 15 in distal GI segments and upregulation of TLR 2B, 3, and 15 in proximal segments, including the duodenum. Interestingly, SE-infection caused downregulation of TLR 5, with no change in TLR 7 or 21. Overall, we provide a comprehensive report of mRNA expression profiles for the TLR family of innate immune receptors in the GI tract of 2-day-old broilers and their differential response to SE colonization.

Gene Expression Profiling of the Local Cecal Response of Genetic Chicken Lines That Differ in Their Susceptibility to Campylobacter jejuni Colonization

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen.

Modulation of chicken macrophage effector function by TH1/TH2 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.

Expression of the avian-specific toll-like receptor 15 in chicken heterophils is mediated by Gram-negative and Gram-positive bacteria, but not TLR agonists.

Toll-like receptors (TLRs) are a critical component of the innate immune response of mammalian and avian species. While most mammalian TLRs have been well characterized, the chicken-specific TLR15 has not been extensively studied. We recently demonstrated that TLR15 is differentially expressed between Salmonella-susceptible-and-resistant chickens, indicating a potential role in the innate immune response to infection with Salmonella. The aim of the present study was to gain better insight into the nature of the ligand for TLR15 by characterizing gene expression patterns of TLR15 by heterophils in response to numerous bacterial-derived TLR agonists LPS, flagellin, CpG oligodeoxynucleotides, lipotechoic acid (LTA), peptidoglycan (PGN), and Pam3CSK4 (PAM), stimulation with live Salmonella enterica serovar Enteritidis (SE-used as a positive control), chicken isolates of Escherichia coli (EC) and Enterococcus gallinarum (EG), the equine-specific pathogen Rhodococcus equi, and stimulation with heat-killed, and formalin-killed SE, EC, and EG. TLR15 expression increased significantly in response to stimulation with live, heat-killed and formalin-killed SE, EC, and EG, but was unaffected by stimulation with known TLR agonists and R. equi. Overall, these observations demonstrate that the individual TLR agonists are not the ligand for TLR15, and that TLR15 recognizes a unique, non-secreted, heat-stabile component of both Gram-negative and Gram-positive bacteria commonly found in and/or capable of causing disease in chickens.

Inflammatory agonist stimulation and signal pathway of oxidative burst in neonatal chicken heterophils

Heterophils are the predominant polymorphonuclear leukocytes (PMNs) in poultry. The oxidative burst of activated heterophils, which generates reactive oxygen species (ROS), is one of the first line cellular defenses against invading microorganisms. In this report, the oxidative response of heterophils from neonatal chicks to in vitro stimulation by various inflammatory agonists was investigated using a fluorescence microplate assay. Both non-opsonized formalin-killed Salmonella enteritidis and Staphylococcus aureus were able to stimulate heterophil oxidative burst. The phorbol myristate acetate (PMA) was the most potent stimulant for the chicken heterophil oxidative response, whereas, the bacterial cell surface components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were less effective. Protein kinase C (PKC) is an essential signaling component regulating heterophil oxidative response to stimulation by PMA, LPS, LTA and S. enteritidis. However, inhibition of PKC did not affect the oxidative response to stimulation by S. aureus, suggesting differential signaling pathway responsible for the activation of oxidative burst by Gram-negative S. enteritidis and Gram-positive S. aureus. Inhibition of mitogen activated protein (MAP) kinase p38 and extracellular response kinase (ERK) by SB 203580 and PD 098059, respectively, did not inhibit activated oxidative burst.

Differential mRNA expression of the avian-specific toll-like receptor 15 between heterophils from Salmonella-susceptible and -resistant chickens

Pattern recognition receptors (PRRs) are essential for recognition of conserved molecular constituents found on infectious microbes. Toll-like receptors (TLRs) are a critical component of the PRR repertoire and are coupled to downstream production of cytokines, chemokines, and antimicrobial peptides by TLR adaptor proteins. Our laboratory previously demonstrated a role for TLR function in the differential innate response of two lines of chickens to bacterial infections. The aim of the present study was to elucidate the role of TLRs in the differential innate responsiveness by measuring differences between lines A (resistant) and B (susceptible) in heterophil mRNA expression of selected TLRs (TLRs 4, 5, and 15) and TLR adaptor proteins (MyD88, TRIF, and TIRAP) in response to stimulation with Salmonella enterica serovar Enteritidis (SE). Although heterophils from both lines had significantly increased expression of TLR 15 mRNA in response to stimulation with SE, heterophils from chickens resistant to infection with SE had significantly greater levels of TLR 15 mRNA expression prior to and following stimulation with SE than heterophils from chickens susceptible to infection with SE. No significant differences were noted between lines in nonstimulated levels of TIRAP, but upon SE stimulation, line A birds had higher levels of expression than B birds. No significant differences were found in heterophils between lines for mRNA expression of TLRs 4 and 5 nor MyD88 and TRIF. These data indicate that differences in the gene expression of TLR 15 by heterophils likely accounts for some of the observed differences between the lines in their susceptibility to infection.