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    • The current study focused on the effects

    2020-07-30

    The current study focused on the effects of inhibiting specific signaling molecules in two mitogen-activated protein kinase (MAPK) pathways that are implicated in the SCR 7 of IL-6 in response to LPS. IL-6 initiates an inflammatory response to pathogens, activates both T and B lymphocytes, and promotes the differentiation of monocytes into macrophages (Burdin et al., 1995, Gessani et al., 1993, Rincon et al., 1997). In this study, SB203580 was used to inhibit p38, which is directly downstream of MEK3/6 in one of the MAPK pathways. An inhibitor was used to suppress the activity of extracellular-signal-regulated kinase (ERK), which is directly downstream of MEK1/2 in another MAPK pathway. These signal molecules were chosen for inhibition because previous studies in our laboratory have demonstrated the involvement of MEK in pro-inflammatory gene expression via ERK activation, but it was undetermined what role the p38 MAPK pathway would have in chicken thrombocyte gene expression. Previously, Scott and Owens were able to inhibit COX-2 gene expression if MEK1/2 was blocked by PD98059 (Scott and Owens, 2008). IL-6 gene expression was unaffected by this inhibitor at the concentration used. Also, there was no prior information regarding direct ERK or p38 MAPK inhibition on innate responses initiated by LPS. It was supposed, but unknown, that blocking ERK instead of MEK1/2 would lead to the same altered gene expression previously observed (Scott and Owens, 2008). This particular pathway, unlike the p38 MAPK pathway, has usually been associated with cell growth and differentiation rather than initiation of pro-inflammatory responses (Chae et al., 2005). The p38 MAPK pathway, on the other hand, has been linked to induction of pro-inflammatory gene expression (Widmann et al., 1999). In addition to inhibitors for ERK and p38 MAPK, we also used the two inhibitors employed by Scott and Owens for analysis of activated and released proteins in the MAPK and NFκB pathways (Scott and Owens, 2008). Experiments were conducted to gather more information on TLR4-linked pathways, gene expression and production of bioactive mediators in chicken thrombocytes.
    Materials and methods
    Results
    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 genes 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.