Regulation of Toll-like receptor-induced chemokine production in murine dendritic cells by mitogen-activated protein kinases.

Production of chemokines in dendritic cells (DCs) may be crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbial products. We evaluated chemokine production in DCs induced by TLR agonists and investigated the role of signaling pathways. DCs were generated from mouse bone marrow cells cultured with Fms-like tyrosine kinase-3 ligand and stimulated with a wide array of individual TLR agonists or simultaneously with pairs of combinations. Production of monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1 (MIP-1/CCL3) and regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), were determined in cell culture supernatants by ELISA or cytokine cytometric bead array. Pharmacological inhibitors of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), nuclear factor-kappaB (NF-kB) and phosphatidylinositol 3-kinase (PI3K), were used to investigate the role of signaling pathways. TLR agonists induced significantly elevated MCP-1, RANTES, and MIP-1. Production of RANTES and MIP-1 was particularly prominent after stimulation of DCs with TLR3 (Poly(I:C)), and TLR7/8 (R848) or TLR9 (CpG ODN) agonists, respectively. However, down-modulation of chemokine production was observed in simultaneously TLR-stimulated DCs. A positive role was identified for NF-kB, PI3K and ERK, whereas JNK had a negative regulatory effect on chemokine production in DCs. Positive and negative regulatory roles for the p38 MAPK pathway were observed. Thus, chemokine levels differed and most notably there was down-modulation of chemokines in DCs stimulated with combined TLR agonists. Furthermore, analysis of signaling pathways revealed a role for MAPKs in positive and negative regulation of chemokine production in DCs. The chemokine response of DCs induced by TLR agonists appears complex and could have important implications for vaccine design.