Role of the TLR-pp65-integrin system in host defense against bacterial infections

我们先前在脂多糖刺激的巨噬细胞中发现了p65/L-纤溶酶原蛋白被磷酸化。目前已阐明该蛋白具有钙结合、钙调蛋白结合和肌动蛋白结合结构域，并调节整合素介导的细胞黏附。已知的C3H/HeJ小鼠由于其TLR4的突变而对脂多糖无反应。脂多糖刺激的小鼠巨噬细胞中p65/L调节的黏附没有增强，这可能是小鼠对细菌感染易感性的原因。为了研究TLR-p65磷酸化细胞运动级联在宿主抵抗感染中的重要作用，我们进行了以下实验：(1)TLR KO小鼠：TLR4、TLR2、TLR9和MyD88 KO小鼠。(2)蛋白质：制备p65/L-纤溶酶原蛋白的重组蛋白。同时制备了抗p65/L-纤溶酶原激活素的单抗。为了评估单抗的特异性，重组T-纤溶酶是Plas…的异构体制备了更多的TIN蛋白家族，并在除白细胞外的其他组织中表达。还制备了重组形式的格兰卡菌素，它可能在细胞内与p65/L-纤溶酶形成复合体。(3)体外感染实验：用细菌或细菌成分体外刺激不同来源的小鼠巨噬细胞和树突状细胞。用特异性抗体免疫组织化学方法检测p65/L-纤溶酶原激活素或格兰卡霉素在细胞内的定位与细胞黏附增加的关系。(4)体内感染实验：用沙门氏菌活体感染小鼠，采集感染部位的白细胞。结果发现，细胞内p65/L-纤溶酶原蛋白的排列发生了很大的变化，格兰卡菌素被转移到包括p65/L-纤溶酶在内的肌动蛋白细胞骨架上。这些观察结果有力地表明TLR-pp65-整合素系统在宿主抵抗细菌感染的防御中发挥着至关重要的作用。较少

We previously identified p65/L-plastin that was phosphorylated in LPS-stimulated macrophages. It has been clarified that the protein has Ca-binding, calmodulin-binding and actin-binding domains., and that it regulates irtegrin-mediated cell adhesion. The C3H/HeJ mouse is known to be unresponsive to LPS due to the mutation of its TLR4. The p65/L-plastin-regulated adhesion is not enhanced in LPS-stimulated macrophages of the mouse, which may be the cause of the susceptibility of the mouse to bacterial infections. In order to investigate an important role of the TLR-p65 phosphorylation-cell locomotion cascade in host defense against infections, we have carried out the following experiments.(1)TLR KO mice : TLR4,TLR2,TLR9 and MyD88 KO mice were bled and used for experiments. (2)Proteins : Recombinant protein of p65/L-plastin was prepared. Monoclonal antibody to p65/L-plastin was also prepared. In order to assess the specificity of the mAb, recombinant T-plastin that is an isoform of a plas … More tin protein family and expressed in many other tissue except leukocytes was prepared. Recombinant form of grancalcin that is supposed to form a complex with p65/L-plastin in cells was also prepared. (3)In vitro infection experiments : Macrophages and dendritic cells from various mice were stimulated in vitro with bacteria or bacterial components. The relationship between intracellular localization of p65/L-plastin or grancalcin that was immunohistochemically detected with specific antibodies and the increase in cell adhesion was assessed. (4)In vivo infection experiments : Various mice were in vivo infected with Salmonella organisms and leukocytes recruited into the infected site were obtained. It was found that intracellular arrangemert of p65/L-plastin was greatly changed in the cells, and that grancalcin was translocated to actin-cytoskeleton including p65/L-plastin. These observations strongly suggest that TLR-pp65-integrin system plays a vital role in host defense against bacterial infections. Less