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组分通过Toll样受体（TLR）激活先天免疫细胞。然而，针对真菌（另一种类型的重要病原体）的细胞反应仍有待分析。在本研究中，我们分析了真菌表面β-葡聚糖的巨噬细胞活化活性，以揭示通过TLR和细胞活化识别病原体的分子机制，当各种β-葡聚糖刺激MF-κβ时，线性（1→3）- β-D-葡聚糖制剂，可得兰，表现出最强的活性。在中性pH下不溶的cordlan的活性通过将其溶解在50-300 mM NaOH中而显著增强。Curdlan激活NF-κB并诱导肿瘤坏死因子-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可中和LPS的活性，但不抑制凝胶多糖的活性。而鲎G因子β-葡聚糖结合域或短（1→3）-β-D-葡聚糖则呈剂量依赖性抑制凝胶多糖的活性。此外，对可德兰的细胞反应被突变体MyD 88（TLR/IL-1受体信号传导途径所必需的衔接分子）的过表达抑制。这些结果表明，线性（1→3）-β-D-葡聚糖依赖于其三级结构刺激巨噬细胞，并强烈表明葡聚糖的受体是一种含有Toll/IL-1受体样（TIR）结构域的膜蛋白，如TLR。

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.