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

TLR-pp65-整合素系统在宿主防御细菌感染中的作用

基本信息

批准号：
15590390
负责人：
SHINOMIYA Hiroto
金额：
$ 2.3万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590390/
关键词：
p65 L-plastin Toll-like receptor integrin actin cytoskeleton cell adhesion host dfense macrophage

项目摘要

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

我们之前发现 p65/L-plastin 在 LPS 刺激的巨噬细胞中被磷酸化。已阐明该蛋白具有 Ca 结合、钙调蛋白结合和肌动蛋白结合结构域，并且调节整合素介导的细胞粘附。已知 C3H/HeJ 小鼠由于其 TLR4 突变而对 LPS 无反应。 LPS刺激的小鼠巨噬细胞中p65/L-plastin调节的粘附并未增强，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：将TLR4、TLR2、TLR9和MyD88 KO小鼠放血用于实验。 (2)蛋白质：制备p65/L-plastin的重组蛋白。还制备了针对 p65/L-plastin 的单克隆抗体。为了评估 mAb 的特异性，制备了重组 T-plastin，它是 plastin 蛋白家族的同种型，在除白细胞外的许多其他组织中表达。还制备了重组形式的大钙蛋白，其被认为与细胞中的p65/L-塑蛋白形成复合物。 (3)体外感染实验：用细菌或细菌成分在体外刺激各种小鼠的巨噬细胞和树突状细胞。评估了用特异性抗体免疫组织化学检测到的 p65/L-plastin 或 grancalcin 的细胞内定位与细胞粘附增加之间的关系。 (4)体内感染实验：用沙门氏菌体内感染各种小鼠，并获得募集到感染部位的白细胞。结果发现细胞内p65/L-plastin的胞内排列发生了很大的改变，grancalcin易位到包含p65/L-plastin的肌动蛋白细胞骨架中。这些观察结果强烈表明，TLR-pp65-整合素系统在宿主防御细菌感染方面发挥着至关重要的作用。较少的