Dual stimulation of MyD88-dependent Toll-like receptors induces synergistically enhanced production of inflammatory cytokines in murine bone marrow-derived dendritic cells.

Triggering Toll-like receptors (TLRs) on dendritic cells (DCs) induces inflammatory cytokine production necessary for T helper type 1 immunity. The present study investigated whether simultaneous stimulation of two TLRs that signal through the same or different pathway(s) enhances cytokine production in DCs. Fms-like tyrosine kinase-3 ligand-generated murine DCs were used in stimulation assays with TLR agonists with or without pharmacological inhibitors of cell signaling pathways. Cytokine levels were evaluated by enzyme-linked immunosorbent assay or cytometric bead array. There was synergistic enhancement of interleukin (IL)-6 and IL-12, which were significantly inhibited by inhibitors of nuclear factor-kappaB and phosphatidylinositol 3-kinase. IL-12p40 was significantly inhibited by both p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase inhibitors, whereas IL-12p70 was inhibited by p38 MAPK inhibitor alone. IL-6 was significantly inhibited by extracellular signal-regulated kinase and, variably, by p38 MAPK and c-Jun N-terminal kinase inhibitors. Production of cytokines in DCs after simultaneous stimulation of TLRs that signal through the same or different pathway(s) showed differential use of MAPK signaling pathways, yet both nuclear factor-kappaB and the phosphatidylinositol 3-kinase pathway as a positive regulator of TLR signaling were important. Our data suggest an important role for MyD88-dependent signaling pathways in TLR-mediated synergistic enhancement of inflammatory cytokine production in DCs.