The synergy in cytokine production through MyD88-TRIF pathways is co-ordinated with ERK phosphorylation in macrophages.

Although specific single Toll-like receptor (TLR) ligands are known to drive the development of Th1 or Th2 immunity, the outcome of different combinations of TLR ligands on innate immunity is not well defined. Spatiotemporal dynamics are critical in determining the specificity of the immune response, but the mechanisms underlying combinatorial TLR stimulation remain unclear. Here, we tested pairwise combinations of TLR ligands separated by different time intervals for their effect on cytokine production in macrophages. We observed that stimulation via a combination of MyD88- and TRIF-utilizing adaptors leads to a highly synergistic cytokine response. On a timescale of 4-24 h, macrophages pretreated with poly(I:C) (TLR3 ligand) are cross-primed to a second stimulation with R848 (TLR7 ligand) and vice versa, and each condition exhibits different optimal time windows of synergistic response for each cytokine. We show that the synergy resulting from combinatorial stimuli (poly(I:C) and R848 is also regulated by the order and dosage of the TLR agonists. Secondary response genes, which depend on new protein synthesis for transcription, show greater synergy than primary response genes, and such enhancement is abolished when new protein synthesis is inhibited. Synergistic cytokine production appears concordant with sustained ERK phosphorylation, suggesting that the de novo factors act via inhibition of ERK dephosphorylation, for example, by the downregulation of dual specificity phosphatase 6. Taken together, our findings illustrate a checkpoint in the innate immune system, where the synchronization of timing of both MyD88 and TRIF pathways is required for a maximal cytokine response and potential memory effect in macrophages.