Characterization of Chicken Thrombocyte Responses to Toll-Like Receptor Ligands

Abstract

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.

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands

“…These results are similar to responses from avian macrophages treated with the same class of CpG-ODN or LPS (Haddadi et al, 2013). In contrast, avian thrombocytes showed significant up-regulation of IFN-␣, but marginal increase in IFN-␤ transcripts after high dose LPS or low dose CpG-ODN treatment (St Paul et al, 2012a). The lack of type I IFN responses may be related to the class of CpG-ODN used, as generally class A CpG-ODN robustly induce IFN-␣ compared to class B CpG-ODN, at least in mammals (Krug et al, 2001).…”

“…Recent TLR expression profiling studies in chickens have revealed that certain TLRs are expressed not only in the classical innate immune system cells, but also in thrombocytes (St Paul et al, 2012a) and various adaptive immune system cells, such as B cells (St Paul et al, 2012b). As a consequence of TLR-PAMP interactions, cells undergo changes in expression of an array of antiviral substances, such as type I and II interferons (IFNs) (St Paul et al, 2012b).…”

Phagocytosis by Thrombocytes is a Conserved Innate Immune Mechanism in Lower Vertebrates

“…Our functional assessment of the phagocytic responses of thrombocytes complements recent molecular analyses, which showed that thrombocytes express genes involved in antigen presentation and the inflammatory response (40, 41). The functional significance and evolutionary implications of the phagocytic thrombocytes are discussed in relation with the immune functions of mammalian platelets.…”

Reduction of avian influenza virus shedding by administration of Toll-like receptor ligands to chickens

“…Peak responses of chicken thrombocytes have been reported with high doses of LPS and CpG [29], but a low dose of LPS induces higher nitric oxide production in chicken macrophages [16]. Moreover, the dose of TLR2 ligands can affect chicken splenocyte responses [30].…”