Abstract
The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-β expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.
Highlights
As activation of Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) is critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained
Among 1,320 compounds and natural products from a commercially available compound library and pharmaceutical companies screened for their abilities to activate nuclear factor-B (NF-B), we identified only one sample, termed T⌱⌲-139, that was as active as lipid A and taxol
To confirm that membranous CD14 is dispensable for FNC-RED-induced responses (Fig. 5B), we studied the formation of TLR4/MD-2 dimerization using Ba/F3 cells expressing murine TLR4-GFP, murine TLR4-FLAG, murine MD-2-FLAG, and murine CD14 [42]
Summary
To identify non-endotoxin-derived TLR4/MD-2 agonists, we prepared Ba/F3 transfectant cells expressing murine TLR4/ MD-2, murine CD14, and an NF-B-GFP reporter construct. In contrast to lipid A stimulation, low concentrations of FNC-RED (0.1 or 0.5 g/ml) induced NF-B activation in the absence but not in the presence of FBS (supplemental Fig. 8A). This NF-B activation was significantly blocked by MTS510 mAb (supplemental Fig. 8B). The CD14 mAb blocked NF-B activation induced by a low dose of lipid A (0.3 ng/ml) (Fig. 5B) This was not seen in the FNC-RED-stimulated cells (Fig. 5B), suggesting that membranous CD14 may be dispensable for FNC-RED-induced NF-B activation. Lipid A stimulation induced NF-B activation in Ba/F3 transfectant cells, such a response was not observed with FNC-RED even at a high concentration (50 g/ml) (supplemental Fig. 10). The differences between FNC-RED and FNC-REDP01 in the binding affinity for human or murine TLR4/MD-2 were consistent with our findings in their stimulatory effects and the docking models
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