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

本课题旨在探讨肺表面活性蛋白（SP-A和SP-D；肺集合素）和toll样受体识别配体的分子机制，以及巨噬细胞对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。我们已经证明鸟分枝杆菌是肺集合素的配体，并且SP-D与从鸟分枝杆菌中提取的脂阿拉伯甘露聚糖结合。此外，我们发现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m.a avium的吞噬作用。SP-A可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则不能。SP-A激活酪蛋白激酶2，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。对SP-…More A/SP- d嵌合体的分析表明，c端Cys204-Cys218对SP-A的促吞噬作用起重要作用。我们构建了一个由gl24 - leu631 （sTLR4）区域组成的可溶重组细胞外TLR4结构域，并分析了其与LPS和MD-2的相互作用。MD-2结合sTLR4的n端区域（Glu24-Pro34）。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的数量。sTLR4-MD-2复合物抑制LPS与表达TLR4/MD-2的细胞的结合。此外，气管内灌注sTLR4和MD-2对LPS引起的小鼠肺部炎症有一定的抑制作用。SP-A和SP-D已被证明直接结合TLR2和TLR4的细胞外结构域。它们的结合依赖于Ca2+，但被认为是通过蛋白质-蛋白质相互作用，因为两种集合素结合去糖基化的sTLR蛋白。抗SP-D单克隆抗体的表位定位及其对SP-D与sTLR结合的影响表明，SP-D通过碳水化合物识别结构域（CRD）与sTLR蛋白结合。SP-A的抗胶原酶片段（CRF）形成由CRD和颈部组成的三聚体，与野生型十八聚体SP-A相比，与sTLR蛋白结合的亲和力明显降低。此外，在TLR4/ md -2转染的细胞中，CRF抑制lps诱导的NF-κB活化的能力显著减弱。结果表明，n端胶原结构域形成的基质上低聚物在与TLR相互作用和肺集合素调节先天免疫功能中的重要性。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