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.

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.

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.

Age-related changes in cytokine expression by neutrophils of foals stimulated with virulent Rhodococcus equi in vitro.

Although evidence exists that neutrophils play a vital role in resistance to infection with Rhodococcus equi, the means by which neutrophils exert their effects have not been clearly defined. In the present study we evaluated differences in cytokine expression by unstimulated and R. equi-stimulated neutrophils obtained from newborn foals and subsequently at 2-, 4-, and 8-weeks of age. Stimulation with virulent R. equi induced significantly (P<0.05) greater expression of IFNgamma, TNFalpha, IL-6, IL-8, IL-12p40, IL-12p35, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, and there were significant effects of age on expression of IL-6, IL-8, IL-12p40 and IL-23p19. Neutrophil expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. Expression of IL-12p40 by R. equi-stimulated neutrophils from newborn and 2-week-old foals did not differ from that of unstimulated neutrophils; however, expression of IL-12p40 by neutrophils from 4- and 8-week-old foals was significantly greater when stimulated by R. equi than without stimulation. These results demonstrate that foal neutrophils increase mRNA expression of many pro-inflammatory cytokines, including IFNgamma, in response to in vitro stimulation with R. equi, and that the magnitude of this expression with respect to IL-6, IL-8, IL-12p40 and IL-23p19 is influenced by age. The clinical importance of the age-related difference in R. equi-induced expression of IL-12p40 to susceptibility to R. equi pneumonia remains to be determined.

Cytokine expression by neutrophils of adult horses stimulated with virulent and avirulent Rhodococcus equi in vitro.

Rhodococcus equi is an intracellular pathogen of macrophages that causes rhodococcal pneumonia in foals and immunocompromised people. Evidence exists that neutrophils play a vital role in resistance to infection with R. equi; however, the means by which neutrophils exert their effects have not been clearly defined. In addition to directly killing bacteria, neutrophils also may exert a protective effect by linking innate and adaptive immune responses. In the present study we evaluated the cytokine expression profiles of adult equine neutrophils in response to stimulation with isogenic strains of virulent and avirulent R. equi in vitro. After 2 and 4h incubation with virulent or avirulent R. equi, adult equine neutrophils expressed significantly (P<0.05) greater tumor necrosis factor alpha (TNFalpha), interleukin (IL)-12p40, IL-6, IL-8 and IL-23p19 mRNA, but not interferon gamma (IFNgamma) or IL-12p35 mRNA than unstimulated neutrophils. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA than avirulent R. equi. These results demonstrate that R. equi-stimulated neutrophils are a source of many proinflammatory cytokines. Furthermore, these results suggest that IL-23 may be preferentially expressed over IL-12 in response to exposure with R. equi, and that this response may be more strongly induced by virulent R. equi than avirulent R. equi. Collectively, the data presented herein suggest a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

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.

The selective Dectin-1 agonist, curdlan, induces an oxidative burst response in chicken heterophils and peripheral blood mononuclear cells.

A critical component of host innate immunity is recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Dectin-1 is the primary PRR for exogenous beta-glucan, a component of fungal and bacterial cell walls. A previous study conducted in our laboratory demonstrated that administration of beta-glucan as a feed additive resulted in increased innate immune function of neonatal chickens, suggesting that chickens possess a Dectin-1-like beta-glucan receptor. In the present study, we demonstrated that heterophils and peripheral blood mononuclear cells (PBMCs) from day-old chicks had a significant increase in the generation of reactive oxygen species (ROS) following stimulation with the Dectin-1 specific agonist, curdlan. Pretreatment of heterophils and PBMCs with laminarin, a beta-glucan receptor blocking agent and specific inhibitor of Dectin-1 activity, significantly reduced the curdlan-induced ROS production. Together these data provide evidence for the first time of the presence of a functional Dectin-1-like beta-glucan receptor in chicken heterophils and PBMCs.