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

本研究的目的是探讨肺表面活性蛋白识别配体的分子机制（SP-A和SP-D ;肺聚集素）和Toll样受体以及巨噬细胞介导的聚集素介导的细菌吞噬作用，并建立临床应用于呼吸道感染的分子基础。1.我们已经证明鸟分支杆菌是肺聚集素的配体，并且SP-D与来源于鸟分枝杆菌的脂阿拉伯甘露聚糖结合。2.SP-A能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，而SP-D无此作用。SP-A激活酪蛋白激酶2并增加清道夫受体A（SR-A）的细胞表面定位。因此，SP-A通过酪蛋白激酶2依赖性机制增强SR-A介导的吞噬作用。SP分析- 关于我们 A/SP-D嵌合体的研究表明，C端Cys 204-Cys 218是SP-A刺激吞噬功能的重要位点。3.构建了可溶性的重组胞外TLR 4结构域（sTLR 4），并分析了其与LPS和MD-2的相互作用。MD-2与sTLR 4的N端区域（Glu 24-Pro34）结合。sTLR 4本身不与脂质A结合，但sTLR 4增加MD-2与脂质A的结合亲和力和与MD-2结合的脂质A的量。sTLR 4-MD-2复合物抑制LPS与表达TLR 4/MD-2的细胞的结合。另外，肺内滴注sTLR 4和MD-2可减轻LPS引起的小鼠肺部炎症。4.SP-A和SP-D已被证明直接与TLR 2和TLR 4的胞外结构域结合。它们的结合是Ca 2+依赖性的，但被认为是通过蛋白质-蛋白质相互作用，因为两种凝集素都与去糖基化的sTLR蛋白结合。抗SP-D单克隆抗体的表位定位及其对SP-D与sTLR结合的影响揭示了SP-D通过碳水化合物识别结构域（CRD）与sTLR蛋白结合。SP-A的胶原酶抗性片段（CRF）已被证明形成由CRD和颈部组成的三聚体，其与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LPS诱导的NF-κB活化的能力在TLR 4/MD-2转染细胞中显著减弱。结果表明，在与TLR的相互作用中，由N-末端胶原结构域形成的supratrimeic寡聚体以及由肺胶原凝集素调节先天免疫功能的重要性。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