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Title:   Author  item
                                            Arsenault, Ryanitem
                                            Kogut, Michael

                                                         - *Mike*item
                                            He, Louis
  Submitted to: Cellular Signaling
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 7/15/2013
                                    Publication  Date: 7/19/2013
                                    Publication URL: http://handle.nal.usda.gov/10113/58268Citation: Arsenault, R.J., Kogut, M.H., He, L.H. 2013. Combined CpG and poly I:C stimulation of monocytes results in unique signaling activation not observed with the individual ligands. Cellular Signaling. 25:2246-2254.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 NF'B 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, result in a synergistic immune response.  In order to further characterize this synergy, kinome analysis was performed on chicken monocytes stimulated with either CpG and poly I:C individually or in combination at 1 hour and 4 hour time points.  The analysis was carried out using 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 showed altered phosphorylation when given CpG and poly I:C together, which were not observed when either CpG or poly I:C were given separately.  The unique, synergistic peptides represent protein members of signaling pathways, including MAPK signaling, calcium signaling pathway, cytokine-cytokine receptor interaction, endocytosis and immune regulation at the 1 hour time point, and pathways including adipocytokine signaling pathway, calcium signaling pathway, cell cycle, NOD-like receptor signaling pathway, and RIG-I-like receptor signaling pathway at 4 hours.  Using nitric oxide (NO) production as a read out, TLR ligand synergy was also investigated using kinase 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 (poly I:C alone does not elicit a significant NO response).  These results confirmed that novel signaling events are induced by the combination of the two ligands, not a simple addition of signaling events which also occur separately.  Since many microbes can stimulate more than one TLR, this synergistic effect 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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