Combined CpG and poly I:C stimulation of monocytes results in unique signaling activation not observed with the individual ligands

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 1h or 4h. 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 CpG and poly I:C were given together, that was not observed when either CpG or poly I:C was 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 1h time point. At the 4h 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 that 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 κB (NFκB) and ultimately of 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.

Keywords: AKT; AP-1; ASK1; B cell linker protein; BLNK; BTK; Bruton tyrosine kinase; C/EBP; CCAAT/enhancer binding protein; CREB; CaM; CaMK2; CpG; DCs; Erk; GO; Geneontology; JNK; Jak-STAT; Janus kinase–signal transducers and activators of transcription; Jun N-terminal kinase; KEGG; Kinase; Kyoto Encyclopedia of Genes and Genomes; MAPK; MK2; MKK3; MKK6; NFκB; NO; PAMPs; PBMCs; PI3K; PKB; PKC; PLCγ; PRRs; Peptide array; Poly I:C; RAC-alpha serine/threonine-protein kinase 1/2; RAC1; RAF; RAS; RSK1 also referred to as p90RSK; Ras-related C3 botulinum toxin substrate 1; Rat sarcoma; STRING; Search Tool for the Retrieval of Interacting Genes; Signaling; TAK1; TANK-binding kinase 1; TBK1; TGF-beta activated kinase 1; TLR synergy; TLRs; Toll-like receptors; activator protein 1; apoptosis signal-regulating kinase 1; cAMP response element-binding protein; calcium/calmodulin-dependent protein kinase 2; calmodulin; dendritic cells; extracellular signal-regulated kinases; iNOS; inducible nitric oxide synthase; mitogen-activated protein kinase; mitogen-activated protein kinase kinase 3; mitogen-activated protein kinase kinase 6; mitogen-activated protein kinase-activated protein kinase 2; nitric oxide; nuclear factor κB; pathogen-associated molecular patterns; pattern recognition receptors; peripheral blood mononuclear cells; phosphoinositide 3-kinase; phospholipase C gamma; poly I:C; polyinosinic–polycytidylic acid; protein kinase B; protein kinase C; rapidly accelerated fibrosarcoma; ribosomal S6 kinase 1; unmethylated CpG oligodeoxynucleotides.