Mustafa Sanam, Evans Samuel G, Hutchinson Mark R
School of Biomedicine, The University of Adelaide, Adelaide, SA, Australia.
Australian Research Council Centre of Excellence for Nanoscale BioPhotonics, The University of Adelaide, Adelaide, SA, Australia.
Front Immunol. 2025 Sep 1;16:1580070. doi: 10.3389/fimmu.2025.1580070. eCollection 2025.
Toll-like receptor 4 (TLR4) plays a pivotal role in the innate immune system by recognizing pathogens and initiating immune responses. Despite extensive research over three decades, current methods lack the resolution to measure ligand-induced TLR4 receptor dynamics at the earliest stages of signaling, relying instead on downstream outputs such as gene expression and cytokine secretion. Here, we present the illuminating TLR4 (iTLR4) assay, a novel Bioluminescence Resonance Energy Transfer (BRET)-based platform that provides real-time insights into TLR4 receptor-level events in live cells. The iTLR4 assay demonstrates, for the first time, that lipopolysaccharide (LPS) induces stable interactions between intracellular domains of TLR4 monomers, with an EC50 of 660 EU/mL. Kinetic analysis revealed a gradual, sustained increase in the BRET signal over time. Additionally, the assay uncovered subtle mechanistic differences among functional antagonists. While all antagonists completely abolished LPS-induced IL-8 secretion, the assay demonstrated that at the receptor level LPS-RS completely inhibited the LPS-induced BRET signal, TAK-242 partially inhibited it and (+)-naloxone potentiated it. The assay also identified potential regulatory roles for CD14 and MD2 in naloxone stereoisomer activity, marking the first report of such mechanistic differences. These findings highlight the unique capabilities of the iTLR4 assay to track nuanced TLR4 receptor dynamics, enabling high-throughput screening of TLR4-specific modulators. This platform provides critical insights into ligand-induced signaling, paving the way for the development of novel therapeutics targeting TLR4-related diseases and advancing our understanding of innate immune responses.
Toll样受体4（TLR4）通过识别病原体并启动免疫反应，在先天免疫系统中发挥关键作用。尽管经过三十多年的广泛研究，但目前的方法缺乏在信号传导最早阶段测量配体诱导的TLR4受体动态变化的分辨率，而是依赖于下游输出，如基因表达和细胞因子分泌。在这里，我们介绍了发光TLR4（iTLR4）检测方法，这是一种基于生物发光共振能量转移（BRET）的新型平台，可实时洞察活细胞中TLR4受体水平的事件。iTLR4检测方法首次证明，脂多糖（LPS）可诱导TLR4单体细胞内结构域之间的稳定相互作用，半数有效浓度（EC50）为660 EU/mL。动力学分析显示，BRET信号随时间逐渐持续增加。此外，该检测方法还揭示了功能拮抗剂之间细微的机制差异。虽然所有拮抗剂都完全消除了LPS诱导的IL-8分泌，但该检测方法表明，在受体水平上，LPS-RS完全抑制了LPS诱导的BRET信号，TAK-242部分抑制了该信号，而（+）-纳洛酮则增强了该信号。该检测方法还确定了CD14和MD2在纳洛酮立体异构体活性中的潜在调节作用，这是此类机制差异的首次报道。这些发现突出了iTLR4检测方法追踪细微TLR4受体动态变化的独特能力，能够对TLR4特异性调节剂进行高通量筛选。该平台为配体诱导的信号传导提供了关键见解，为开发针对TLR4相关疾病的新型疗法以及增进我们对先天免疫反应的理解铺平了道路。
