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Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity
Published on: January 7, 2019
Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor
Naoki Okamoto1,2, Keisuke Mizote3, Hiroe Honda1,4
1From the Department of Immunobiology and Pharmacological Genetics, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194.
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.
Insights
A novel funiculosin (FNC) variant, FNC-RED, acts as a Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) agonist in mice and humans. This discovery offers potential for new vaccine adjuvants targeting innate and adaptive immunity.
Area of Science:
- Immunology
- Molecular Biology
- Drug Discovery
Background:
- The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is crucial for recognizing lipopolysaccharide (LPS) and initiating innate immune responses against Gram-negative bacteria.
- TLR4/MD-2 activation is also vital for adaptive immunity, leading to the development of TLR4/MD-2 agonists as potential vaccine adjuvants, though their efficacy requires further validation.
Purpose of the Study:
- To investigate the potential of a funiculosin (FNC) variant, FNC-RED, and its derivatives as agonists for the TLR4/MD-2 complex in both murine and human systems.
- To elucidate the mechanism of action and species-specific activation of TLR4/MD-2 by FNC-RED and its derivatives.
Main Methods:
- Assessed TLR4/MD-2 agonistic activity using nuclear factor-κB (NF-κB) activation assays in murine and human cell systems.
- Employed Biacore analysis to determine binding kinetics of FNC-RED to TLR4/MD-2 and other related receptors.
- Investigated cytokine expression, co-stimulatory molecule induction, and TLR4/MD-2 complex dynamics (dimerization, internalization) in macrophages and dendritic cells.
- Utilized computational analysis to explore structural basis for species-specific receptor activation.
Main Results:
- FNC-RED demonstrated potent TLR4/MD-2 agonistic activity in murine systems, inducing NF-κB activation and pro-inflammatory cytokine production.
- FNC-RED exhibited species-specific activation, with reduced activity in CD14-dependent LPS responses and differential effects compared to lipid A.
- Synthetic derivatives, FNC-RED-P01 and FNC-P01, were identified as activators of human TLR4/MD-2, unlike FNC-RED, attributed to electrostatic surface potential differences.
Conclusions:
- FNC-RED and its synthetic derivatives represent a novel class of TLR4/MD-2 agonists with potential applications as vaccine adjuvants.
- The findings highlight the species-specific nature of TLR4/MD-2 activation and provide insights into the structural determinants of agonist binding and function.
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