Myeloid-cell mediated mechanisms driving muscle growth and regeneration

描述(申请人提供)：我们研究的一个目标是了解免疫系统和骨骼肌之间的相互作用，这些相互作用在调节肌肉修复、生长和再生方面非常重要。通过更好地了解这些关系，免疫系统或其产品的治疗性操作可以设计为在急性创伤、停用或慢性疾病后改善肌肉生长和再生。在过去的几年里，这方面的知识有了很大的进步，表明被称为M2巨噬细胞的免疫细胞在肌肉生长和再生中发挥着重要作用。然而，驱动再生的特定M2巨噬细胞衍生分子的特性仍不清楚。我们建议检验这一新的假设，即M2巨噬细胞产生的Klotho蛋白对于肌肉的正常生长和损伤后的再生是必不可少的。此外，我们还将开发一种新颖的、基于细胞的机制，“按需”将Klotho靶向受损肌肉，以便在肌肉再生的位置和时间特异性地提高治疗性蛋白质的表达。我们建议使用小鼠急性肌肉损伤模型来解决以下目标：目的1：验证调节Klotho水平的遗传或药物干预影响肌肉再生的假设。我们将通过表达Klotho转基因、去除Klotho基因、移植Klotho转基因小鼠的骨髓来源细胞(BMC)和注射Klotho蛋白来操纵肌肉损伤小鼠的Klotho水平。将通过分子、生理和形态分析对小鼠进行评估，以确定改良Klotho水平对肌肉生长和再生的影响。目的2：验证Klotho调节炎性细胞在肌肉损伤后从促炎的M1巨噬细胞向促再生的M2巨噬细胞转变的假设。我们将测试Klotho的遗传或药物操作是否会影响肌肉损伤后的巨噬细胞表型或功能。目的3.验证髓系细胞可以被设计为在再生过程中针对受损肌肉高表达Klotho的假设。我们将建立一个转基因小鼠系，其中Klotho的表达是由一个启动子驱动的，该启动子在炎症背景下在髓系细胞中最高激活，然后分析移植这些小鼠的骨髓细胞是否促进受者受损肌肉的生长和再生。我们预计，这些发现将确定Klotho在推动受伤肌肉的生长和再生方面的重要性。我们还相信，这项工作可以为新的、基于细胞的治疗分子输送到损伤和修复部位提供基础，这可能具有超越肌肉损伤治疗的意义。

DESCRIPTION (provided by applicant): A goal of our research is to understand interactions between the immune system and skeletal muscle that are important in regulating muscle repair, growth and regeneration. By better understanding these relationships, therapeutic manipulations of the immune system or its products can be designed to improve muscle growth and regeneration following acute trauma, disuse or chronic disease. Over the last several years, knowledge in this area has advanced substantially, showing that immune cells called M2 macrophages play a major role in muscle growth and regeneration. However, the identity of the specific, M2 macrophage-derived molecules that drive regeneration remains unknown. We propose to test the novel hypothesis that the protein Klotho generated by M2 macrophages is essential for normal growth and regeneration of muscle following injury. In addition, we will develop a novel, cell-based mechanism for targeting Klotho to injured muscle "on demand," so that expression of the therapeutic protein is elevated specifically at the location and time of muscle regeneration. We propose to use an acute, muscle-injury model in mice to address the following aims: Aim 1: Test the hypothesis that genetic or pharmacological interventions that modulate Klotho levels affect muscle regeneration. We will manipulate Klotho levels in mice that experience muscle injury by expressing a Klotho transgene, by ablating the Klotho gene, by transplanting bone marrow-derived cells (BMCs) from Klotho transgenic mice and by administering Klotho protein. Mice will be assessed by molecular, physiological and morphological assays to determine the effects of modified Klotho levels on muscle growth and regeneration. Aim 2: Test the hypothesis that Klotho modulates shifts in inflammatory cells from pro-inflammatory M1 macrophages to pro-regenerative M2 macrophages following muscle injury. We will test whether genetic or pharmacological manipulations of Klotho affect macrophage phenotype or function following muscle injury. Aim 3. Test the hypothesis that myeloid cells can be designed to target elevated Klotho expression to injured muscle during regeneration. We will generate a transgenic mouse line in which Klotho expression is driven by a promoter that is most-highly activated in myeloid cells in the context of inflammation and then assay whether transplanting BMCs from those mice promotes growth and regeneration of injured muscle in the recipients. We anticipate that these findings will establish the importance of Klotho in driving growth and regeneration of injured muscle. We also believe that this work can provide a basis for a new, cell-based delivery of therapeutic molecules to sites of injury and repair that may have significance beyond the treatment of muscle injuries.