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Thrombocytes express Toll-like receptor 4 and apparently use both mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathways for nuclear signaling. However, it is not well known if the same enzyme systems found in mammalian cells are fully functional in chickens. Therefore, kinase inhibitors were used with thrombocytes to block kinases in lipopolysaccharide (LPS) stimulated cells to determine if interleukin (IL)-6 expression and production would be diminished. Results demonstrated that extracellular-signal-regulated kinase (ERK)1/2 and p38 MAPK pathways influence gene expression of IL-6 through treatment with either ERK or p38 MAPK inhibitor. In addition, thrombocyte lysates from cells treated with ERK, p38, mitogen-activated protein kinase kinase (MEK)1/2 and inhibitor of nuclear factor kappa-B kinase (IKK) inhibitor showed different levels of the phosphorylated form of ERK1/2, p38 and NFκB. Furthermore, IL-6 gene expression and production were significantly upregulated in LPS stimulated thrombocytes relative to all inhibitor-treated cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Thrombocytes are nucleated platelets involved in immune functions such as pathogen recognition and release of pro-inflammatory bioactive compounds when exposed to bacterial and viral molecules. However, the complete role of these cells in innate and adaptive immune responses is not understood, and little is known about their biology at the molecular-genetic level. Highly sensitive RNA-sequencing technologies were used to analyze the complete transcriptome of thrombocytes for the first time with analytical resolution focused on cell-based components of the immune system/response. Amongst all the genes listed in the current chicken genome assembly, 10,041 gene transcripts were found in the chicken thrombocyte. After 1-hour in vitro stimulation with lipopolysaccharide (LPS, Salmonella minnesota), 490 genes were upregulated and 359 genes were downregulated, respectively, with at least a 1-fold change relative to unexposed thrombocytes. Additionally, by constructing a de novo assembly, we were able to identify a total of 3,030 novel genes in the thrombocyte transcriptome. The information generated here is useful in development of novel solutions to lower the economic burden and zoonotic threat that accompanies infectious diseases for birds and fish. In addition, the resources created here have translational utility as a model system to find orthologous genes and genes related to its enucleated counterpart, the platelet., Competing Interests: The authors have declared that no competing interests exist.

Thrombocytes express Toll-like receptors (TLRs) that detect bacterial or viral pathogens to signal the release of cytokines and mediators. We examined inflammatory responses when thrombocytes were exposed to four TLR ligands. Treatment of thrombocytes with TLR ligands demonstrates differential effects on gene expression of interleukin (IL)-6. Among the TLR ligands examined, lipopolysaccharide stimulation led to the most significant up-regulation of the IL-6 gene and a significant amount of active IL-6 in thrombocyte culture media. Lipoteichoic acid stimulation led to only marginal up-regulation of IL-6 gene expression. Although gene expression of inducible nitric oxide synthase (iNOS) did not increase due to different ligand exposure, a low level constitutive expression of iNOS was observed in all cases. Only thrombocytes treated with polyinosinic-polycytidylic acid and thymidine homopolymer phosphorothioate oligodeoxynucleotides induced rapid, significant production of nitric oxide. We also observed that thrombocytes are able to respond faster upon TLR ligand exposure compared to MQ.NCSU macrophages., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The effect of lipopolysaccharide (LPS) on the expression of proinflammatory cytokines, interleukin (IL)-1beta, IL-6, and IL-12 in broiler chick thrombocytes was investigated. At 4 wk of age, blood samples were collected, and isolated thrombocytes were incubated with LPS for 1 h. Ribonucleic acid was extracted from the cells to examine the expression of the proinflammatory cytokines using real-time reverse transcription PCR. It was found that expressions of IL-1beta, IL-6, and IL-12 in thrombocytes were unaffected by diets containing corticosterone and vitamin C fed to chicks. However, LPS exposure did increase the expressions of these cytokines. The fact that thrombocytes are so abundant and can be stimulated by LPS makes them primary effector cells in innate host defenses against bacterial infections in chickens.