Liposomal Lipopolysaccharide Initiates TRIF-Dependent Signaling Pathway Independent of CD14

Lipopolysaccharide (LPS) is recognized by CD14 with Toll-like receptor 4 (TLR4), and initiates 2 major pathways of TLR4 signaling, the MyD88-dependent and TRIF-dependent signaling pathways. The MyD88-dependent pathway induces inflammatory responses such as the production of TNF-α, IL-6, and IL-12 via the activation of NFκB and MAPK. The TRIF-dependent pathway induces the production of type-I IFN, and RANTES via the activation of IRF-3 and NFκB, and is also important for the induction of adaptive immune responses. CD14 plays a critical role in initiating the TRIF-dependent signaling pathway response to LPS, to support the internalization of LPS via endocytosis. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) initiate only TRIF-dependent signaling via clathrin-mediated endocytosis, independent of CD14. In fact, LPS-liposomes do not induce the production of TNF-α and IL-6 but induce RANTES production in peritoneal macrophages. Additionally, LPS-liposomes could induce adaptive immune responses effectively in CD14-deficient mice. Collectively, our results strongly suggest that LPS-liposomes are useful as a TRIF-dependent signaling-based immune adjuvant without inducing unnecessary inflammation.脂多糖（Lipopolysaccharide, LPS）可被CD14与Toll样受体4（Toll-like receptor 4, TLR4）识别，并启动TLR4信号通路的两大经典分支：髓系分化因子88依赖型（MyD88-dependent）通路与TIR结构域衔接蛋白依赖型（TRIF-dependent）通路。其中，MyD88依赖型通路可通过激活核因子κB（NFκB）与丝裂原活化蛋白激酶（MAPK），诱导肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）及白细胞介素-12（IL-12）等炎症因子的产生。TRIF依赖型通路则通过激活干扰素调节因子3（IRF-3）与NFκB，诱导I型干扰素（type-I IFN）及趋化因子RANTES的生成，同时在适应性免疫应答的诱导过程中发挥关键作用。CD14在启动LPS诱导的TRIF依赖型信号通路应答中扮演核心角色，可通过内吞作用介导LPS的胞内摄取。本研究明确证实：经LPS配制的脂质体（LPS-liposomes）介导的LPS胞内递送，可通过网格蛋白介导的内吞作用仅启动TRIF依赖型信号通路，且该过程不依赖CD14。实验结果显示，LPS-脂质体在腹腔巨噬细胞中不会诱导TNF-α与IL-6的产生，却可有效促进RANTES的生成。此外，LPS-脂质体可在CD14缺陷型小鼠体内高效诱导适应性免疫应答。综上，本研究结果充分表明，LPS-脂质体可作为基于TRIF依赖型信号通路的免疫佐剂，且不会引发不必要的炎症反应。