Molecular Mechanisms of Recongnition of Lipopolysaccharide (LPS) via TLR4 and Activation in Innate Immunity

已知，在哺乳动物中，以脂多糖(LPS)为代表的各种微生物或病毒成分通过Toll样受体(TLR)激活天然免疫细胞。然而，细胞对真菌的反应仍有待分析，真菌是另一种重要的病原体。在本研究中，我们分析了真菌表面β-葡聚糖的巨噬细胞激活活性，以揭示通过TLR识别病原体和激活细胞的分子机制。当各种β-葡聚糖刺激MF-κβ时，线性(1→3)-β-_D-葡聚糖制剂Curdlan显示出最强的活性。在50-300 mM氢氧化钠溶液中，不溶于中性pH的Cordlan的活性显著增强。可得兰激活NF-κB，诱导肿瘤坏死因子-α、巨噬细胞炎性蛋白-2和诱导型一氧化氮合酶的表达。多粘菌素B不抑制柯尔德兰的活性，但可中和内毒素的活性。反之，β-葡聚糖结合区的因子G或短(1→3)-β-_D-葡聚糖则呈剂量依赖性地抑制Curdlan的活性。此外，突变的MyD88过表达抑制了细胞对Curdlan的反应，MyD88是TLR/IL-1受体信号通路所必需的接头分子。这些结果表明，LINEAR(1→3)-β-_D-葡聚糖对巨噬细胞的刺激依赖于其三级结构，并强烈提示该葡聚糖的受体是一种Toll/IL-1受体样(TIR)结构域膜蛋白(TIR)，如TLRs。

It has been known that, in mammals, various microbial or viral components, represented by lipopolysaccharide (LPS), activate innate immune cells via Toll-like receptor (TLR). However, cellular responses against fungi, another types of important pathogens, remain to be analyzed. In this study, we analyzed macrophage activating activities of β-glucans on the surface of fungi to reveal molecular mechanisms for the recognition of pathogens via TLRs and cellular activation.When the activities of various β-glucans to stimulate MF-κβ, a linear (1→3) - β-_D-glucan preparation, curdlan, exhibited the strongest activity. The activity cordlan, which is insoluble at neutral pH, was markedly enhanced by dissolving it in 50-300 mM NaOH. Curdlan activated NF-κB and induced the expression of tumor necrosis factor-α, macrophage inflammatory protein-2, and inducible nitric-oxide synthase. The activity of curdlan was not inhibited by polymyxin B, which neutralizes the activity of LPS. In contrast, the activity of curdlan was dose-dependently inhibited by β-glucan-binding domain of horseshoe crab factor G or short (1→3)-β-_D-glucans. Furthermore, the cellular response to curdlan was inhibited by overexpression of a mutant MyD88, an adaptor molecule essential for TLR/IL-1 receptor signaling pathways. These results indicate that linear (1→3)-β-_D-glucans stimulate macrophage depending on their tertiary structures and strongly suggest that the receptor for the glucans is a Toll/IL-1 receptor-like (TIR) domain-containing membrane protein(s), such as TLRs.