Michael St. Paul

Characterization of Chicken Thrombocyte Responses to Toll-Like Receptor Ligands

Thrombocytes are the avian equivalent to mammalian platelets. In addition to their hemostatic effects, mammalian platelets rely in part on pattern recognition receptors, such as the Toll-like receptors (TLR), to detect the presence of pathogens and signal the release of certain cytokines. Ligands for TLRs include lipopolysaccharide (LPS), which is bound by TLR4, as well as unmethylated CpG DNA motifs, which are bound by TLR9 in mammals and TLR21 in chickens. Similar to mammalian platelets, avian thrombocytes have been shown to express TLR4 and secrete some pro-inflammatory cytokines in response to LPS treatment. However, the full extent of the contributions made by thrombocytes to host immunity has yet to be elucidated. Importantly, the mechanisms by which TLR stimulation may modulate thrombocyte effector functions have not been well characterized. As such, the objective of the present study was to gain further insight into the immunological role of thrombocytes by analyzing their responses to treatment with ligands for TLR4 and TLR21. To this end, we quantified the relative expression of several immune system genes at 1, 3, 8 and 18 hours post-treatment using real-time RT-PCR. Furthermore, production of nitric oxide and phagocytic activity of thrombocytes was measured after their activation with TLR ligands. We found that thrombocytes constitutively express transcripts for both pro- and anti-inflammatory cytokines, in addition to those associated with anti-viral responses and antigen presentation. Moreover, we found that both LPS and CpG oligodeoxynucleotides (ODN) induced robust pro-inflammatory responses in thrombocytes, as characterized by more than 100 fold increase in interleukin (IL)-1β, IL-6 and IL-8 transcripts, while only LPS enhanced nitric oxide production and phagocytic capabilities. Future studies may be aimed at examining the responses of thrombocytes to other TLR ligands.

Immunostimulatory properties of Toll-like receptor ligands in chickens.

Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that have been identified in mammals and avian species. Ligands for TLRs are typically conserved structural motifs of microorganisms termed pathogen-associated molecular patterns (PAMPs). Several TLRs have been detected in many cell subsets, such as in macrophages, heterophils and B cells, where they mediate host-responses to pathogens by promoting cellular activation and the production of cytokines. Importantly, TLR ligands help prime a robust adaptive immune response by promoting the maturation of professional antigen presenting cells. These properties make TLR ligands an attractive approach to enhance host-immunity to pathogens by administering them either prophylactically or in the context of a vaccine adjuvant. In this review, we discuss what is known about the immunostimulatory properties of TLR ligands in chickens, both at the cellular level as well as in vivo. Furthermore, we highlight previous successes in exploiting TLR ligands to protect against several pathogens including avian influenza virus, Salmonella, Escherichia coli, and Newcastle disease Virus.

Prophylactic treatment with Toll-like receptor ligands enhances host immunity to avian influenza virus in chickens.

Avian influenza viruses (AIV) pose a threat towards the health of both poultry and humans. To interrupt the transmission of the virus, novel prophylactic strategies must be considered which may reduce the shedding of AIV. One potential is the prophylactic use of Toll-like receptor (TLR) ligands. Many cells of the immune system express TLRs, and cellular responses to TLR stimulation include activation and the production of cytokines. TLR ligands have been employed as prophylactic treatments to enhance host resistance to pathogens both in mammals and chickens. Therefore, the present study was conducted to determine whether TLR ligands may be used prophylactically in chickens to enhance host immunity to AIV. Chickens received intramuscular injections of either low or high doses of the TLR ligands poly I:C, lipopolysaccharide (LPS) and CpG ODN. Twenty-four hours post-treatment, chickens were infected with the low pathogenic avian influenza virus H4N6, and both oropharyngeal and cloacal virus shedding were assessed on days 4 and 7 post-infection. To identify potential correlates of immunity, spleen and lungs were collected on days 2, 4 and 7 post-infection for RNA extraction. The results suggested that all of the TLR ligand treatments induced a significant reduction in virus shedding, with the TLR3 ligand poly I:C conferring the greatest AIV immunity compared to control birds, followed by CpG ODN and LPS. Furthermore, transcriptional analysis of gene expression in the spleen and lungs suggest IFN-α and IL-8 as correlates of immunity conferred by poly I:C, and IFN-γ for CpG ODN and LPS. In conclusion, TLR ligands, have the ability to enhance host immunity against AIV, and future studies should consider exploring the combinatory effects of poly I:C and CpG ODN prophylaxis in conjunction with AIV vaccination.

In vivo administration of ligands for chicken toll-like receptors 4 and 21 induces the expression of immune system genes in the spleen

Toll-like receptors (TLRs) are a group of conserved proteins that play an important role in pathogen recognition in addition to the initiation and regulation of innate and adaptive immune responses. To date, several TLRs have been identified in chickens, each recognizing different ligands. TLR stimulation in chickens has been shown to play a role in host-responses to pathogens. However, the mechanisms through which TLRs modulate the chicken immune system have not been well examined. The present study was conducted to characterize the kinetics of responses to TLR4 and TLR21 stimulation in chickens following intramuscular injections of their corresponding ligands, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODNs), respectively. To this end, relative expression of cytokine genes in the spleen was determined at 2, 6, 12 and 24 h after injection of TLR ligands. The results indicated that LPS strongly induced the up-regulation of some immune system genes early on in the response to treatment, including interferon (IFN)-γ, interleukin (IL)-10, and IL-1β. Furthermore, treatment with CpG ODN promoted the up-regulation of major histocompatibility complex (MHC)-II, IFN-γ and IL-10. The response to CpG ODN appeared to be somewhat delayed compared to the response to LPS. Moreover, we found a significant increase in IFN-α gene expression in response to LPS but not CpG ODNs. Future studies may be aimed to further characterize the molecular mechanisms of TLR activation in chickens or to exploit TLR agonists as vaccine adjuvants.

Chicken erythrocytes respond to Toll-like receptor ligands by up-regulating cytokine transcripts

Avian erythrocytes are nucleated cells of myeloid origin that are able to actively transcribe and translate proteins. Although their role in gas exchange and transportation has been well described, it has recently been shown that chicken erythrocytes produce cytokines in response to Toll-like receptor (TLR) ligands, which raises the possibility that they also contribute to host immunity. To this end, the objective of the study was to gain some further insight into the immunological role of erythrocytes by identifying the repertoire of TLRs that they express and to elucidate their responses to the TLR3 and TLR21 ligands poly I:C and CpG oligodeoxynucleotide (ODN), respectively. The results suggest that erythrocytes constitutively express transcripts for TLRs 2, 3, 4, 5, and 21, as well as for many immunological genes including type I interferons (IFN) and interleukin (IL)-8. Moreover, it was found that treatment with both poly I:C and CpG ODN up-regulated transcripts for type I IFNs, while only poly I:C up-regulated IL-8 transcripts and enhanced the production of nitrite. Future studies may be aimed at further characterizing the immunological role of erythrocytes.

A Toll-Like Receptor 3 Ligand Enhances Protective Effects of Vaccination Against Marek's Disease Virus and Hinders Tumor Development in Chickens

Mareks disease (MD) is caused by Mareks disease virus (MDV). Various vaccines including herpesvirus of turkeys (HVT) have been used to control this disease. However, HVT is not able to completely protect against very virulent strains of MDV. The objective of this study was to determine whether a vaccination protocol consisting of HVT and a Toll-like receptor (TLR) ligand could enhance protective efficacy of vaccination against MD. Hence, chickens were immunized with HVT and subsequently treated with synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], a TLR3 ligand, before or after being infected with a very virulent strain of MDV. Among the groups that were HVT-vaccinated and challenged with MDV, the lowest incidence of tumors was observed in the group that received poly(I:C) before and after MDV infection. Moreover, the groups that received a single poly(I:C) treatment either before or after MDV infection were better protected against MD tumors compared to the group that only received HVT. No association was observed between viral load, as determined by MDV genome copy number, and the reduction in tumor formation. Overall, the results presented here indicate that poly(I:C) treatment, especially when it is administered prior to and after HVT vaccination, enhances the efficacy of HVT vaccine and improves protection against MDV.

Treatment with Ligands for Toll-Like Receptors 2 and 5 Induces a Mixed T-helper 1- and 2-Like Response in Chicken Splenocytes

Toll-like receptors (TLRs) play an important role in the induction of host responses to pathogens. Interactions between TLRs and their ligands result in the production of cytokines that modulate the adaptive immune response through polarizing CD4+ T cells into either T-helper (T(H))1 or T(H)2 phenotypes. In this regard, TLR2 and TLR5 ligands have been shown to induce responses in mammals that are biased toward T(H)1 or T(H)2 phenotypes. However, whether a similar phenomenon occurs in chickens remains to be elucidated. To this end, chicken splenocytes were stimulated with the TLR2 ligand Pam3CSK4 and the TLR5 ligand flagellin, and the relative expression of several cytokines and transcription factors was quantified at 1, 3, 8, and 18 h poststimulation. The results suggest that both TLR ligands induce a mixed T(H)1- and T(H)2-like response, as characterized by the upregulation of both the T(H)1-associated cytokine interferon-γ and the T(H)1-inducing cytokine interleukin (IL)-12, in addition to the T(H)2-associated cytokine IL-4, and in the case of flagellin, IL-13 as well. Future studies may be aimed at assessing the adjuvant potential of these ligands.

Characterization of responses elicited by Toll-like receptor agonists in cells of the bursa of Fabricius in chickens.

Toll-like receptors (TLRs) are an evolutionarily conserved group of pattern recognition receptors that play an important role in mediating host-responses to pathogens. Several TLRs have been identified in chickens and their expression has been detected in many immune cell subsets including in B cells. However, the mechanisms through which TLRs modulate B cell responses have not been well characterized in chickens. The aim of the present study was to elucidate the responses mounted by cells of the bursa of Fabricius to treatment with the TLR 3, 4 and 21 ligands, poly I:C, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODN), respectively. The relative expression of several immune system genes was quantified at 1, 3, 8 and 18 h post-treatment. The results show that all three ligands induced the up-regulation of interferon (IFN)-γ and interleukin (IL)-10 transcripts and promoted the up-regulation of transcripts associated with antigen presentation, namely CD80 and major histocompatibility complex (MHC) class II. Furthermore, the results indicated that LPS and poly I:C induced the greatest IFN-γ and IL-10 responses, respectively, while CpG ODN was the most efficacious inducer of CD80 and MHC-II expression. Future studies may be aimed at elucidating the mechanisms of TLR-mediated activation of chicken B cells. These mechanisms may be then exploited for the development of adjuvants with enhanced ability to stimulate B cell responses.

Vaccination with CpG-Adjuvanted Avian Influenza Virosomes Promotes Antiviral Immune Responses and Reduces Virus Shedding in Chickens

The use of virosomes as a vaccine platform has proven successful against several viruses. Here we examined the protective efficacy of a virosome-based vaccine consisting of avian influenza virus (AIV) A/Duck/Czech/56/H4N6 in chickens against a homologous AIV challenge. Virosomes adjuvanted with CpG-ODN or recombinant chicken interferon (IFN)-γ significantly reduced virus shedding after virus challenge. Furthermore, immunization with virosomes adjuvanted with CpG-ODN increased hemagglutination inhibition (HI) and virus-specific neutralizing serum antibodies, as well as virus-specific serum IgG and mucosal IgA responses. We also found a significant increase in the expression of type I and II interferon genes in the protected birds following virus challenge. In summary, this study demonstrated the ability of virosomes adjuvanted with CpG-ODN to reduce AIV shedding, and elicit virus-specific protective antibody responses in vaccinated birds.

Characterization of responses initiated by different Toll-like receptor 2 ligands in chicken spleen cells

Toll-like receptors (TLRs) are pattern recognition receptors that mediate host responses to pathogens by promoting cellular activation and the production of cytokines. Ligands for TLRs are conserved structural motifs of pathogens termed pathogen-associated molecular patterns. In the case of TLR2, these ligands include peptidoglycan, lipomannan and lipopeptides. In mammals, it has been shown that different TLR2 ligands induce distinct cytokine responses. However, whether a similar phenomenon occurs in chickens remains to be determined. To this end, chicken splenocytes were stimulated with three different TLR2 ligands: Pam3CSK4, FSL-1 and lipomannan, and the relative gene expression of several cytokines was quantified at 2, 6 and 18h post-stimulation. The results suggest that Pam3 and FSL-1 modulate the kinetics of the pro-inflammatory cytokine response differently, as Pam3 induced a robust interleukin (IL)-1β response, while FSL-1 induced an early and prolonged up-regulation of IL-8. Furthermore, it appears that all three TLR2 ligands induce a mixed T-helper (TH) 1 and 2-like response, as characterized by the up-regulation of IFN-γ, IL-12, IL-4 and IL-13. In conclusion, we have demonstrated that different TLR2 ligands may induce different cytokine responses in chicken splenocytes. Future studies may be aimed at examining the immunomodulating effects of these ligands in vivo.