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
Source: PubMed

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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    ABSTRACT: Secondary infections with Streptococcus pneumoniae (SP) are frequently observed following influenza A virus (IAV) infection and have a substantial impact on global health. Despite this, the basis for the disease progression is incompletely understood. To investigate the effect of co-infection on human monocyte-derived dendritic cells (MDDCs) we analyzed the expression of clinically important pro-inflammatory and immune-modulatory cytokines. IAV infection or treatment with supernatants from IAV-infected cell cultures resulted in priming of the DCs which subsequently influenced the production of IL-12p70, as well as IL-6, following SP infection. Co-infection of the same cell was not required but this effect was dependent on the time, dose and duration of the infections, as well as pathogen viability, bacterial uptake and endosome acidification. Bacterially infected cells were characterized as the main producers of IL-12p70. Finally, we showed that type I interferons were primarily responsible for the priming of IL-12p70 that was observed by infection with IAV. These results provide a probable mechanism for the elevated levels of particular cytokines observed in IAV and SP co-infected cell cultures with implications for the pathogenic outcome observed during in vivo infection.
    Cellular Microbiology 02/2013; 15(8). DOI:10.1111/cmi.12122 · 4.82 Impact Factor
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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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    ABSTRACT: Chemokines production in monocytes/macrophages is crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbes. During microbial onset, multiple pathogen-associated structures can be present at infection sites, and simultaneously trigger different TLRs. We report here that TLR3, TLR4 and TLR8 engagement induce CCL1, CCL2 and CCL4 production in freshly isolated monocytes. While differentiating cells maintain the capacity to secrete CCL2 and CCL4, CCL1 is no longer induced at later differentiation stages. Although different pairs of TLR agonists have been described to synergistically induce cytokine production in different cell types, agonist combinations cooperate in reducing CCL1 and CCL2, but not CCL4 secretion in freshly isolated monocytes, and fail to rescue CCL1 production at later differentiation stages. The effects of single, but not combined, TLR engagement on chemokine expression mostly occur at transcriptional level, and are IL-10 independent. Conversely, inhibition of CCL1 secretion upon combined TLR engagement is partially rescued by blocking IL-23. A different chemotactic activity of monocyte-conditioned medium on blood mononuclear cells as well as antigen uptake capacity of TLR agonist activated monocytes parallel the regulated production of chemokines. Overall, these findings indicate that simultaneous engagement of TLRs may lead to different patterns of chemokine expression depending on cellular differentiation state, chemokine, and TLR agonist combination. These different responses may be relevant for the distinct but complementary functions of monocytes and macrophages in the immune response, and may have important implications for the therapeutic manipulation of the innate immune system.
    Immunobiology 04/2011; 216(10):1135-42. DOI:10.1016/j.imbio.2011.04.005 · 3.18 Impact Factor
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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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    ABSTRACT: Toll-like receptors (TLRs) are pattern-recognition receptors of the innate immune system that recognize various pathogen-associated molecules. TLR ligands are potent activators of immune cells and certain TLR ligands have a synergistic ability to induce the production of pro-inflammatory cytokines. In the present study we have analyzed the potential synergy between TLR3, TLR4 and TLR7/8 ligands in type I and type III interferon (IFN) gene expression in human monocyte-derived dendritic cells (moDCs). We show that stimulation of moDCs with TLR7/8 ligand R848 together with TLR3 or TLR4 ligands, polyI:C or LPS, respectively, leads to a synergistic expression of IFN-β and IFN-λ1 mRNAs. Neutralization of type I IFNs as well as IFN priming prior to stimulation suggest that IFN-dependent positive feedback loop is at least partly responsible for the mechanism of synergy. Enhanced expression of TLR3 and especially TLR7, which are both under the regulation of type I IFNs, correlated to synergistic TLR ligand-dependent induction of IFN-β and IFN-λ1 genes. NF-κB, PI3 kinase and MAP kinase pathways were involved in TLR ligand-induced IFN gene expression as evidenced by pharmacological signaling inhibitors. The data indicates that IFNs contribute to TLR-dependent gene activation in human DCs stimulated with multiple TLR ligands.
    Molecular Immunology 10/2010; 48(4):505-15. DOI:10.1016/j.molimm.2010.10.005 · 3.00 Impact Factor
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