Innate immune regulation of stem cells in bone formation

 描述(申请人提供)：包括骨折和骨质疏松症在内的肌肉骨骼疾病是全球第二大致残原因。不幸的是，我们治疗或修复受损骨骼的能力极其有限。这与人类的异位骨化疾病形成了鲜明的对比，后者清楚地表明，成年人可以形成大量的骨骼，特别是在急性创伤后。先天免疫系统是损伤反应的关键介质，在调节骨折修复和对激素的合成代谢反应方面具有关键作用。巨噬细胞等先天免疫细胞对异位骨的形成也很重要。在这项建议中，我们使用一种异位骨化的遗传性疾病--进行性骨化纤维发育不良(FOP)作为一个模型，以了解免疫系统如何影响出生后的骨形成。FOP的特点是损伤后可发生大量异位骨化。FOP是由ACVR1突变引起的，该突变增加了受体对骨形态发生蛋白(BMPs)的敏感性，但将损伤与成骨联系起来的机制尚不清楚。我们的中心假设是，先天免疫系统的激活可以促进FOP中骨骼前体的招募和分化。我们之前从FOP患者中创造了人类诱导多能干细胞(iPS细胞)。这些iPS细胞表现为软骨生成增加和矿物质沉积。我们的培养还显示内皮细胞(ECs)数量增加，当转染FOP ACVR1突变时，内皮细胞可以具有MSC样特性。我们还发现FOP iPS细胞来源的内皮细胞前体细胞(IECP)表达高水平的OSTERIX、RUNX2和SOX9，这三种骨形成的主要调节因子。我们将追求三个目标，以确定在FOP中ACVR1活性如何影响先天性免疫系统。在目标1中，我们将创建一组新的iPS细胞系，标记为OSX/SP7-mCherry报告程序来检测成骨前体细胞。然后我们将测试暴露在炎症信号下的FOP iECP是否比对照组形成更多的成骨前体。在目标2中，我们将测试FOP iECP细胞是否对我们初步实验中确定的候选细胞因子具有趋化作用，以及FOP iPS细胞是否对激活触发器做出异常反应。最后，在目标3中，我们将测试Q207D突变对巨噬细胞特异性激活ACVR1是否足以在体内启动损伤后的异位成骨。综上所述，我们的研究使用新开发的技术来了解ACVR1激活的BMP信号通路如何导致异位骨化。这些工具和发现直接适用于FOP，也可以用于研究骨骼内外的疾病。最后，了解免疫系统如何促进骨骼生长将有助于治疗骨丢失疾病，防止异常骨生长，并改善同种异体移植物的存活和功能。

 DESCRIPTION (provided by applicant): Musculoskeletal diseases including fractures and osteoporosis are the second-greatest cause of disability worldwide. Unfortunately, our ability to treat or repair damaged bone is extremely limited. This contrasts sharply with human diseases of heterotopic ossification which clearly show that adult humans can form large amounts of bone, particularly after acute trauma. The innate immune system is a key mediator of injury responses and has critical functions in regulating fracture repair and anabolic responses to hormones. Innate immune cells such as macrophages are also important for the formation of heterotopic bone. In this proposal we use a genetic disease of heterotopic ossification, fibrodysplasia ossificans progressiva (FOP), as a model to understand how the immune system affects post-natal bone formation. FOP is characterized by massive heterotopic ossification that can occur after injury. FOP is caused by a mutation in ACVR1 which increases receptor sensitivity to bone morphogenetic proteins (BMPs), but the mechanisms that link injury to ossification are unclear. Our central hypothesis is that activation of the innate immune system can enhance the recruitment and differentiation of skeletal precursors in FOP. We previously created human induced pluripotent stem cells (iPS cells) from patients with FOP. These iPS cells showed increased chondrogenesis and mineral deposition. Our cultures also showed increased numbers of endothelial cells (ECs), which can adopt MSC-like properties when transfected with the FOP ACVR1 mutation. We also found that FOP iPS cell-derived endothelial cell progenitors (iECPs) expressed high levels of OSTERIX, RUNX2, and SOX9, three master regulators of bone formation. We will pursue three aims to determine how the innate immune system is affected by ACVR1 activity in FOP. In Aim 1, we will create a new set of iPS cell lines marked with an OSX/SP7-mCherry reporter to detect osteoprogenitors. We will then test if FOP iECPs exposed to inflammatory cues form more osteogenic precursors than control lines. In Aim 2, we will test if FOP iECP cells are chemotactic to candidate cytokines we identified in our preliminary experiments, and if FOP iPS cell derived macrophages to respond abnormally to activation triggers. Finally, in Aim 3, we will test if macrophage-specific activation of ACVR1 by the Q207D mutation is sufficient to initiate heterotopic ossification after injury in vivo. Togethe, our studies use newly-developed techniques to understand how the BMP signaling pathway activated by ACVR1 leads to heterotopic ossification. The tools and findings are directly applicable to FOP and can also be used to study diseases in and out of the skeleton. Finally, understanding how the immune system can enhance skeletal growth will be useful for treating diseases of bone loss, preventing abnormal bone gain, and improving allograft survival and function.