Bohnenkamp HR, Papazisis KT, Burchell JM et al.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 1 responses. Cell Immunol 247:72-84
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.
"As professional APCs, DCs secrete high levels of interleukin-12 (IL-12), a key Th1 polarizing cytokine that promotes CTL responses . Therefore, we next examined the effect of Tim-3 on IL-12p70 expression by M/MФ and DC induced from HCV-infected versus uninfected individuals following stimulation with TLR4 ligand-LPS and TLR7/8 ligand-R848, which are essential for IL-12 expression –. In contrast to the higher levels of Tim-3 expression, M/MФ as well as iDC and mDC from chronically HCV-infected patients exhibit lower levels of IL-12 production compared to uninfected individuals, including spontaneously HCV-resolved, HCV-treated SVR subjects, and HS. "
[Show abstract][Hide abstract] ABSTRACT: In this study, we engineered Listeria monocytogens (Lm) by deleting the LmΔactA/ΔinlB virulence determinants and inserting HCV-NS5B consensus antigens to develop a therapeutic vaccine against hepatitis C virus (HCV) infection. We tested this recombinant Lm-HCV vaccine in triggering of innate and adaptive immune responses in vitro using immune cells from HCV-infected and uninfected individuals. This live-attenuated Lm-HCV vaccine could naturally infect human dendritic cells (DC), thereby driving DC maturation and antigen presentation, producing Th1 cytokines, and triggering CTL responses in uninfected individuals. However, vaccine responses were diminished when using DC and T cells derived from chronically HCV-infected individuals, who express higher levels of inhibitory molecule Tim-3 on immune cells. Notably, blocking Tim-3 signaling significantly improved the innate and adaptive immune responses in chronically HCV-infected patients, indicating that novel strategies to enhance the potential of antigen presentation and cellular responses are essential for developing an effective therapeutic vaccine against HCV infection.
PLoS ONE 01/2014; 9(1):e87821. DOI:10.1371/journal.pone.0087821 · 3.23 Impact Factor
"MyD88 was necessary for the increased CD8+ T cell response seen with peptide immunization adjuvanted with MALP-2 and polyI:C . In vitro activation of dendritic cells with combinations of TLR3 and TLR9, TLR4 and TLR7, or TLR2 and TLR3 agonists enhanced their ability to induce CD8+ T cell responses when transferred in vivo , , . Despite the plethora of data showing the efficacy of single PRR agonists as vaccine adjuvants, there is surprisingly little data assessing the effects of combining PRR agonists for in vivo immunization. "
[Show abstract][Hide abstract] ABSTRACT: With over eight million cases of tuberculosis each year there is a pressing need for the development of new vaccines against Mycobacterium tuberculosis. Subunit vaccines consisting of recombinant proteins are an attractive vaccine approach due to their inherent safety compared to attenuated live vaccines and the uniformity of manufacture. Addition of properly formulated TLR agonist-containing adjuvants to recombinant protein vaccines enhances the antigen-specific CD4(+) T cell response characterized by IFN-γ and TNF, both of which are critical for the control of TB. We have developed a clinical stage vaccine candidate consisting of a recombinant fusion protein ID93 adjuvanted with the TLR4 agonist GLA-SE. Here we examine whether ID93+GLA-SE can be improved by the addition of a second TLR agonist. Addition of CpG containing DNA to ID93+GLA-SE enhanced the magnitude of the multi-functional TH1 response against ID93 characterized by co-production of IFN-γ, TNF, and IL-2. Addition of CpG also improved the protective efficacy of ID93+GLA-SE. Finally we demonstrate that this adjuvant synergy between GLA and CpG is independent of TRIF signaling, whereas TRIF is necessary for the adjuvant activity of GLA-SE in the absence of CpG.
PLoS ONE 01/2014; 9(1):e83884. DOI:10.1371/journal.pone.0083884 · 3.23 Impact Factor
"It has also been demonstrated that specific antigens and different TLRs can synergistically interact with DCs to induce a more efficient T-cell stimulation leading to an increase in the proinflammatory response as well as T-cell polarization [19,20]. In this sense, interesting data have been published about TLR2 and 4 in hapten-induced T-cell responses, as in contact dermatitis . "
[Show abstract][Hide abstract] ABSTRACT: Amoxicillin, a low-molecular-weight compound, is able to interact with dendritic cells inducing semi-maturation in vitro. Specific antigens and TLR ligands can synergistically interact with dendritic cells (DC), leading to complete maturation and more efficient T-cell stimulation. The aim of the study was to evaluate the synergistic effect of amoxicillin and the TLR2, 4 and 7/8 agonists (PAM, LPS and R848, respectively) in TLR expression, DC maturation and specific T-cell response in patients with delayed-type hypersensitivity (DTH) reactions to amoxicillin. Monocyte-derived DC from 15 patients with DTH to amoxicillin and 15 controls were cultured with amoxicillin in the presence or absence of TLR2, 4 and 7/8 agonists (PAM, LPS and R848, respectively). We studied TLR1-9 gene expression by RT-qPCR, and DC maturation, lymphocyte proliferation and cytokine production by flow cytometry. DC from both patients and controls expressed all TLRs except TLR9. The amoxicillin plus TLR2/4 or TLR7/8 ligands showed significant differences, mainly in patients: AX+PAM+LPS induced a decrease in TLR2 and AX+R848 in TLR2, 4, 7 and 8 mRNA levels. AX+PAM+LPS significantly increased the percentage of maturation in patients (75%) vs. controls (40%) (p=0.036) and T-cell proliferation (80.7% vs. 27.3% of cases; p=0.001). Moreover, the combinations AX+PAM+LPS and AX+R848 produced a significant increase in IL-12p70 during both DC maturation and T-cell proliferation. These results indicate that in amoxicillin-induced maculopapular exanthema, the presence of different TLR agonists could be critical for the induction of the innate and adaptive immune responses and this should be taken into account when evaluating allergic reactions to these drugs.
PLoS ONE 09/2013; 8(9):e74198. DOI:10.1371/journal.pone.0074198 · 3.23 Impact Factor
Pallavi Subramanian, Ioannis Mitroulis, George Hajishengallis, Triantafyllos Chavakis
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