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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.