Mechanisms of innate immune surveillance by pulmonary surfactant proteins and their clinical application to respiratory infection.

本课题的目的是研究肺表面活性物质蛋白(SP-A和SP-D)和Toll样受体识别配体的分子机制，以及巨噬细胞介导的集合素介导的细菌吞噬作用的分子机制，为临床应用于呼吸道感染奠定分子基础。1.我们已经证明，鸟分枝杆菌是肺集合素的配基，并且SP-D与来源于禽分枝杆菌的阿拉伯甘露聚糖结合。此外，我们还发现SP-A和SP-D通过增加巨噬细胞上甘露糖受体的活性来增强对禽类支原体的吞噬作用。2.SP-A而不是SP-D能够增强肺泡巨噬细胞对肺炎链球菌和金黄色葡萄球菌的吞噬作用。SP-A激活酪蛋白激酶2，增加清道夫受体A(SR-A)在细胞表面的定位。因此，提示SP-A通过酪蛋白激酶2依赖的机制增强SR-A介导的吞噬功能。SP-…协议分析更多的A/SP-D嵌合体表明，C端的Cys204-Cys218在SP-A的吞噬刺激作用中起重要作用。3.我们构建了由Glu24-Leu631区域(STLR4)组成的重组胞外TLR4结构域的可溶性形式，并分析了其与内毒素和MD-2的相互作用。MD-2与sTLR4的N-末端区域(Glu24-Pro34)结合。STLR4本身不与脂质A结合，但sTLR4增加了MD-2与脂质A的结合亲和力和与MD-2结合的脂质A的量。STLR4-MD-2复合体抑制内毒素与表达TLR4/MD-2的细胞的结合。此外，气管内滴注sTLR4和MD-2可抑制脂多糖引起的小鼠肺部炎症。4.已证明SP-A和SP-D可直接与TLR2和TLR4的胞外区结合。它们的结合依赖于钙离子，但被认为是通过蛋白质之间的相互作用，因为两种集合素都与脱糖的sTLR蛋白结合。抗SP-D单抗的表位定位及其对SP-D与sTLR结合的影响表明，SP-D通过糖识别结构域(CRD)与sTLR蛋白结合。胶原酶抗性的SP-A片段(CRF)与sTLR蛋白的结合亲和力显著低于野生型十八聚体SP-A。此外，CRF抑制脂多糖诱导的TLR4/MD-2细胞中的NF-κB活化的能力显著减弱。这些结果证明了N端胶原结构域在与TLR相互作用中形成房上性低聚物，以及在肺集合素调节天然免疫功能中的重要性。较少

The purposes of this project are to investigate the molecular mechanisms of ligand recognition by pulmonary surfactant proteins (SP-A and SP-D ; pulmonary collectins) and Toll-like receptors and of the collectin-mediated bacterial phagocytosis by macrophages, and to establish the molecular basis for the clinical application to respiratory infections.1.We have shown that Mycobacterium avium is a ligand for pulmonary collectins, and that SP-D binds to lipoarabinomannan derived from M.avium. In addition, we have found that SP-A and SP-D enhance the phagocytosis of M.avium by increased activity of mannose receptor on macrophages.2.SP-A but not SP-D has been shown to augment the phagocytosis of Streptococcus pneumoniae and Staphylococcus aureus by alveolar macrophages. SP-A activates casein kinase 2 and increases cell surface localization of scavenger receptor A (SR-A). Thus, it is suggested that SP-A augment SR-A-mediated phagocytosis by casein kinase 2-dependent machanism. Analysis of SP- … More A/SP-D chimeras has revealed that the C-terminal Cys204-Cys218 is important for the SP-A's stimulatory effect on phagocytosis.3.We constructed a soluble form of recombinant extracellular TLR4 domain consisting of the region Glu24-Leu631 (sTLR4) and analysed the interactions with LPS and MD-2. MD-2 binds to the N-termnal region (Glu24-Pro34) of sTLR4. sTLR4 alone by itself does not bind to lipid A, but sTLR4 increases the binding affinity of MD-2 to lipid A and the amount of lipid A bound to MD-2. The sTLR4-MD-2 complex inhibits the binding of LPS to the cells expressing TLR4/MD-2. In addition, intratracheal instillation of sTLR4 and MD-2 dampens pulmonary inflammation caused by LPS in mice.4.SP-A and SP-D have been shown to directly bind to the extracellular domains of TLR2 and TLR4. Their bindings are Ca2+-dependent but are thought to be via protein-protein interaction because both collectins bind to deglycosylated sTLR proteins. Epitope mapping for anti-SP-D monoclonal antibodies and their effects on the SP-D binding to sTLRs have revealed that SP-D binds to sTLR proteins through the carbohydrate recognition domain (CRD). Collagenase-resistant fragment of SP-A (CRF), which has been shown to form trimer consisting of the CRD and the neck, binds to sTLR proteins with significantly lower affinity than that of wild type octadecameric SP-A. In addition, the ability of CRF to inhibit smooth LPS-induced NF-κB activation in TLR4/MD-2-tranfected cells was significantly attenuated. The results demonstrate the importance of supratrimeic oligomer formation by the N-terminal collagenous domain in interaction with TLR and in modulation of innate immune function by pulmonary collectins. Less