The cooperative interaction of TLR4-, TLR9- and NOD2-signaling pathways in mouse macrophages

Pattern recognition receptors (PRR) recognize conserved pathogen associated molecular patterns (PAMPs) and function as primary sensors of infection. As many pathogens carry multiple PAMPs, they are able to simultaneously activate not one but two, three or more types of sensory receptors. We reported previously a synergistic enhancement of response of innate immunity cells after their simultaneous activation with paired combinations of PRR agonists, in particular, TLR4+NOD2, TLR4+TLR9 or TLR9+NOD2 [1-3]. In this paper, we complicated the external signal up to three PRR-agonists. The responses of macrophages to triple activation with TLR4+TLR9+NOD2 agonists were compared with the ones to paired combinations or the same agonists used alone. The response of macrophages to agonists was evaluated according to activation of intracellular signaling pathways. We tracked activation of cellular protein kinases and transcription factors (TAK1, IKKα/β, NF-κB (NF-κB pathway), ERK1/2, c-Fos (MAPK pathway) TBK1 (IRF -way)) in macrophages treated with different PRR agonistic compositions. Along with the amplitude, the activation kinetics was studied. The highest activation response of macrophages was observed to paired TLR4+TLR9, and TLR4+NOD2 combinations of agonists or to TLR4+TLR9+NOD2 triple combination. These combinations exceeded the response to the TLR4, TLR9 or NOD2 agonists used alone according to the activation of TAK1, IKKα β, NF-κB, ERK1/2, c-Fos and TBK1. The combination of TLR9+NOD2 agonists showed weak or inhibitory effect compared to the same agonists used alone. The TLR4+TLR9+NOD2 triple combination of agonists induced more intensive activation of IKKαβ, NF-κB and ERK1/2 in macrophages as compared to the TLR4+TLR9 and TLR4+NOD2 double combinations. Our results evidence for a synergistic cooperation of intracellular NF-κB, MAPK and IRF-signal cascades in macrophages upon exposure to triple and paired combinations of TLR4, TLR9 and NOD2 agonists. As a consequence of NF-κB, MAPK and IRF pathways? cooperation the concentration of active transcription factors NF-κB and AP-1 (c-Fos) is increased. Probably, the enhanced transcription activity of these factors provides synergistic production of pro-inflammatory cytokines in macrophages activated with the combination of TLR4, TLR9 and NOD2 agonists [1, 2].