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

我们以前确定p65/L-plastin是磷酸化的LPS刺激的巨噬细胞。已阐明该蛋白具有Ca结合、钙调素结合和肌动蛋白结合结构域。并且它调节伊替格林介导的细胞粘附。已知C3 H/HeJ小鼠由于其TLR 4的突变而对LPS无反应。在LPS刺激的小鼠巨噬细胞中，p65/L-plastin调节的粘附没有增强，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防御感染中的重要作用，我们进行了以下实验。（1）TLR KO小鼠：取TLR 4、TLR 2、TLR 9和MyD 88 KO小鼠的血并用于实验。（2）蛋白质：制备p65/L-plastin重组蛋白。制备了抗p65/L-plastin的单克隆抗体。为了评估mAb的特异性，使用了重组T-plastin，它是一种plastin的同种型， 关于我们 tin蛋白家族，并在除白细胞外的许多其他组织中表达。还制备了粒钙蛋白的重组形式，其被认为在细胞中与p65/L-质体蛋白形成复合物。(3)In体外感染实验：在体外用细菌或细菌组分刺激来自各种小鼠的巨噬细胞和树突细胞。用特异性抗体免疫化学检测的p65/L-纤维蛋白酶或grancalcin的细胞内定位与细胞粘附增加之间的关系进行了评估。(4)In体内感染实验：用沙门氏菌生物体体内感染各种小鼠，并获得募集到感染部位的白细胞。结果发现，p65/L-plastin在细胞内的排列发生了很大的变化，grancacin移位到包括p65/L-plastin在内的肌动蛋白骨架上。这些观察结果有力地表明TLR-pp 65-整合素系统在宿主防御细菌感染中起着至关重要的作用。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