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Synergism of Toll-like receptor-induced interleukin-12p70 secretion by monocyte-derived dendritic cells is mediated through p38 MAPK and lowers the threshold of T-helper cell type I responses

Cancer Research UK, Breast Cancer Biology Group, Thomas Guy House, 3rd floor, Guy's Hospital, London SE1 9RT, UK.
Cellular Immunology (Impact Factor: 1.87). 07/2007; 247(2):72-84. DOI: 10.1016/j.cellimm.2007.07.008
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ABSTRACT Toll-like receptors (TLRs) recognise specific molecular signatures of pathogens and trigger antimicrobial defence responses. Thereby, two independent signalling pathways can be distinguished: The inflammatory signalling pathway acting via the adapter molecule MyD88, leading to the activation of nuclear factor-kappaB (NF-kappaB) and mitogen activated protein kinases (MAPK) such as SAPK/JNK and p38 MAPK and the interferon (IFN) dependent pathway that signals via TRIF and results in the production of IFN-alpha/beta. Several evolutionarily conserved molecular patterns are expressed by pathogens, leading to the question if concerted targeting of different TLRs may induce exaggerated immune responses by signalling via both TLR pathways. Here we report that monocyte-derived dendritic cells (MoDCs) combine and integrate signals received via the IFN-dependent pathway by engagement of TLR3 (poly I:C) and activation of TRIF with the MyD88-dependent pathway by ligation of TLR2 (PGN), TLR2/TLR6 (zymosan) and TLR5 (flagellin). The generally low IL-12p70 inducers resulted in combination of both pathways in cytokine levels similar to LPS, which acts via TLR4 and induces recruitment of MyD88/Tirap and TRIF/TRAM adapter proteins. The combination of TLR3 (poly I:C) or TLR4 (LPS) engagement with TLR8 (R848) ligation induced synergistic effects on cytokine production with a boost especially in IL-12p70 secretion. SB203580, a specific p38 MAPK inhibitor, completely blocked TLR ligand mediated IL-12p70 secretion, whereby specific inhibitors for SAPK/JNK (SP600125) and NF-kappaB (PDTC) only repressed partially the IL-12p70 secretion. Enhanced phosphorylation in poly I:C and R848 activated MoDCs revealed the critical contribution of p38 MAPK in synergistically induced IL-12p70 induction. Further investigation of primary and recall CD8+ T cell responses to the MUC(12-20) M1.2 peptide LLLLTVLTV and the influenza A virus matrix(58-66) peptide GILGFVFTL proved that synergistically activated MoDCs were superior compared with LPS or R848 alone. The results indicate that dendritic cells process, combine and integrate signals delivered by pathogens to launch effective adaptive immune responses.

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    • "The production of IL-12p70 by MDDCs can be induced by various individual TLR ligands; however, a simultaneous stimulation with selected TLR ligands has been shown to prime the induction of this cytokine, since MDDCs are able to integrate signals from different TLRs (Napolitani et al., 2005; Bohnenkamp et al., 2007). Similarly , we observed that stimulation with a combination of a TLR7/8 ligand (R848) and a TLR4 ligand (LPS) led to increased production of IL-12p70 by MDDCs (data not shown). "
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    • "It is now thought that to induce an effective immune response, microorganisms must stimulate complex sets of PRRs, both within and outside of the TLR family. Combined TLR ligation has been generally reported to synergistically activate APCs, leading to enhanced production of immune mediators and T cell responses both in vitro and in vivo (Bohnenkamp et al. 2007; Gautier et al. 2005; He et al. 2011; Hirata et al. 2008; Makela et al. 2009, 2011; Napolitani et al. 2005; Warger et al. 2006; Zhu et al. 2008). However, few studies showed that combined activation of TLRs can also give rise to antagonistic effects resulting in reduced chemokine secretion (Del Corno et al. 2009) or cross-tolerance (Bagchi et al. 2007; Re and Strominger 2004). "
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    • "Both in mouse and human DCs TLR7/8 and TLR3 or TLR4 ligands have been shown to induce synergistic expression of pro-inflammatory IL-6, IL-12 and TNF-␣ genes (Gautier et al., 2005; Makela et al., 2009; Napolitani et al., 2005). Several mechanisms have been suggested to be responsible for this synergism; simultaneous activation of TRIF and MyD88 adaptor molecules (Bagchi et al., 2007), IFN-mediated positive feedback mechanism (Gautier et al., 2005), sustained MAPK activation (Bohnenkamp et al., 2007; Napolitani et al., 2005), cooperation of different signaling pathways (Makela et al., 2009) and inhibition of negative regulation by the co-stimulatory TLR ligand (Petricevic et al., 2009). We observed that IFN genes, especially IFN-␤ and IFN-␭1 genes were expressed synergistically in human moDCs that were stimulated with TLR7/8 ligand R848 together with TLR3 or TLR4 ligand, polyI:C or LPS, respectively. "
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