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  • br Materials and methods br


    Materials and methods
    Discussion Chicken thrombocyte stimulation with LPS leads to a number of inflammatory responses through TLR4-linked pathways (Scott and Owens, 2008). In mammals, LPS binds to LPS-binding protein (LBP) in the blood serum and this complex is subsequently recognized by CD14, a protein that exists both in soluble form and as a glycosylphosphatidylinositol-anchored molecule preferentially expressed in monocytes, macrophages and neutrophils (Aderem and Ulevitch, 2000, Takeda et al., 2003). Activation of TLR4 by LPS triggers signal transduction via the cytoplasmic domain called the Toll/IL-1 Receptor (TIR) (Akira and Takeda, 2004, Lu et al., 2008). TLR4 uses several adaptor molecules including myeloid differentiation primary response gene (MyD)88, TIR domain-containing adaptor protein (TIRAP), TIR domain-containing adaptor inducing interferon-β (TRIF), TRAM (TRIF-related adaptor molecule), sterile α and HEAT-Armadillo motifs-containing protein (SARM) to affect downstream signaling (O'Neill and Bowie, 2007). Based on the published thrombocyte transcriptomic database (NCBI GenBank Sequence read archive accession numbers SAMN05818716-SAMN05818721 under the BioProject Accession: PRJNA34407), several interleukins in the TLR4 signaling pathway were present in unstimulated and LPS stimulated thrombocytes including myeloid differentiation factor (MD)2, cluster of differentiation (CD)14, TIRAP, Toll interacting protein (TOLLIP), MYD88, IL-1 receptor-associated kinase (IRAK)4, tumor necrosis factor receptor-associated factor (TRAF)6, transforming growth factor-β-activated kinase (TAK)1, TAK1 binding protein (TAB)1, TAB2, mitogen-activated protein kinase kinase (MKK)4/7, MKK3/6, mitogen-activated protein kinase kinase (MEK)1/2, NFΚB1, TpI2, IKKBIKB, NFΚB, ERK, p38MAPK, JNK, and AP1 (Ferdous et al., 2016) (Fig. 3). However, we have not detected LBP in the thrombocyte transcriptome (Ferdous et al., 2016). Based on published thrombocyte transcriptome and what is reported here, we propose the top part of Fig. 3 as the TLR4 signaling pathway in chicken thrombocytes. LPS binding through CD14 activates TLR4. Activated TLR4 complexes with MD2 and engages proteins of the adaptor family such as MyD88, TIRAP and TOLLIP and activates IRAK4. IRAK activates TRAF6, which leads to the activation of TAB1, TAB2 and TAK1. TAK1 then activates IκB kinase (IKKB) and MAPKs (MKK4/7, MKK3/6, and MEK1/2). The IKK complex phosphorylates IκB that results in nuclear translocation of NFκB while activation of three major MAPK (ERK, c-Jun NH2 terminal kinase (JNK) and p38) leads to translocation of activator protein-1 (AP)-1, to induce expression of pro-inflammatory genes such as IL-6. The cytoplasmic signaling pathways in thrombocytes would appear to be primarily devoted to immunologic responses in support of inflammation since these cells express Toll-like receptors (Scott and Owens, 2008) and are poised to activate upon exposure to various pathogens. The first experiment examined the effect of inhibiting two different MAPK (ERK and p38) pathways involved in the innate response to bacterial LPS on production of IL-6. This experiment demonstrated that expression of the IL-6 gene is mediated by two of these MAPK signaling pathways (i.e. ERK and p38). Previous work in our laboratory (Scott and Owens, 2008) has shown that LPS stimulates the NFκB pathway of thrombocytes, resulting in increased expression of IL-6 and COX-2 mRNA and PGE2 production. It was also demonstrated that MEK1/2 inhibition negatively affected expression of COX-2, but not IL-6 mRNA. In contrast, downstream ERK inhibition in the current experiments negatively affected expression of IL-6. Several other studies have shown involvement of different MAPK resulting in inflammatory responses (Chen et al., 1999, Khatri and Sharma, 2006). Khatri and Sharma showed stimulation of another type of chicken immune cell (macrophage) resulting in upregulation of COX-2 and IL-8 mRNA (Khatri and Sharma, 2006), and inhibition of p38 and NFκB in these cells suppressed the COX-2 mRNA response. In J774 macrophages, inhibition of p38 attenuates LPS-induced COX-2 activity, and consequently inhibits the release of PGE2 (Chen et al., 1999).