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Title:   Author  item
                                            Arsenault, Ryanitem
                                            NAPPER, SCOTT - University Of Saskatchewanitem
                                            Kogut, Michael

                                                         - *Mike*
  Submitted to: Veterinary Research
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 4/19/2013
                                    Publication  Date: 5/17/2013
                                    Publication URL: http://handle.nal.usda.gov/10113/58131Citation: Arsenault, R.J., Napper, S., Kogut, M.H. 2013. Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling. Veterinary Research. 44:35-50.Interpretive Summary: Immune cells recognize components of bacteria and viruses called ligands by binding to them.  These ligands bind to immune cell surface proteins called Toll-like receptors (TLRs).  Two of these ligands are CpG and poly I:C.  Individually, CpG and poly I:C induce an immune response.  When combined, the immune response of cells is significantly increased.  This is referred to as a synergistic effect.  We used peptide arrays and protein inhibitors to study the synergistic effect of the combination of ligands.  We found that the combination induced unique events within the cells not observed when cells are given CpG or poly I:C individually.  We determined the mechanism of this synergy involves calcium release within cells and the activation of transcription factors known as NFkappaB and CREB.
                                Technical Abstract: Toll-like receptors (TLRs) bind to components of microbes, activate cellular signal transduction pathways, and stimulate innate immune responses.  Previously, we have shown in chicken monocytes that the combination of CpG, the ligand for TLR21 (the chicken equivalent of TLR9), and poly I:C, the ligand for TLR3, results in a synergistic immune response.  In order to further characterize this synergy, kinome analysis was performed on chicken monocytes stimulated with either unmethylated CpG oligodeoxynucleotides (CpG) and polyinosinic-polycytidylic acid (poly I:C), individually or in combination for either 1 h or 4 h.  The analysis was carried out using chicken species-specific peptide arrays to study the kinase activity induced by the two ligands.  The arrays are comprised of kinase target sequences immobilized on an array surface.  Active kinases phosphorylate their respective target sequences, and these phosphorylated peptides are then visualized and quantified.  A significant number of peptides exhibited altered phosphorylation when given CpG and poly I:C together.  That was not observed when either CpG or poly I:C were given separately.  The unique, synergistic TLR agonist-affected peptides represent protein members of signaling pathways including Calcium signaling pathway, Cytokine-cytokine receptor interaction, and Endocytosis at the 1 h time point.  At the 4 h time point, TLR agonist synergy-influenced pathways included Adipocytokine signaling pathway, Cell cycle, Calcium signaling pathway, NOD-like receptor signaling pathway, and RIG-I-like receptor signaling pathway.  Using nitric oxide (NO) production as the readout, TLR ligand synergy was also investigated using signaling protein inhibitors.  A number of inhibitors were able to inhibit NO response in cells given CpG alone but not in cells given both CpG and poly I:C, as poly I:C alone does not elicit a significant NO response.  The unique peptide phosphorylation induced by the combination of CpG and poly I:C and the unique signaling protein requirements for synergy determined by inhibitor assays both show synergistic signaling is not a simple addition of TLR pathways.  A set of secondary pathways activated by the ligand combination are proposed, leading to the activation of cAMP response element-binding protein (CREB), nuclear factor kappaB (NFkappaB), and ultimately inducible nitric oxide synthase (iNOS).  Since many microbes can stimulate more than one TLR, this synergistic influence on cellular signaling may be an important consideration for the study of immune response and what we consider to be the canonical TLR signaling pathways.


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