Innate immune regulation of stem cells in bone formation

 描述（由申请人提供）：包括骨折和骨质疏松症在内的肌肉骨骼疾病是全球残疾的第二大原因。不幸的是，我们治疗或修复受损骨骼的能力非常有限。这与人类异位骨化疾病形成鲜明对比，异位骨化清楚地表明成年人可以形成大量的骨，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，并且在调节骨折修复和对激素的合成代谢反应中具有关键功能。先天性免疫细胞如巨噬细胞对于异位骨的形成也很重要。在这个建议中，我们使用异位骨化的遗传性疾病，进行性骨化纤维发育不良（FOP）作为模型来了解免疫系统如何影响出生后的骨形成。FOP的特征是损伤后可发生大量异位骨化。FOP是由ACVR 1突变引起的，ACVR 1突变增加了受体对骨形态发生蛋白（BMP）的敏感性，但将损伤与骨化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增强FOP中骨骼前体的募集和分化。我们之前从FOP患者中创建了人类诱导多能干细胞（iPS细胞）。这些iPS细胞显示出增加的软骨形成和矿物质沉积。我们的培养物还显示了内皮细胞（EC）数量的增加，当用FOP ACVR 1突变转染时，这些细胞可以采用MSC样特性。我们还发现，FOP iPS细胞衍生的内皮祖细胞（iECP）表达高水平的OSTERIX、RUNX 2和SOX 9，这三种主要的骨形成调节因子。我们将追求三个目标，以确定先天免疫系统是如何受到ACVR 1活性的影响。在目标1中，我们将创建一组新的用OSX/SP 7-mCherry报告基因标记的iPS细胞系以检测骨祖细胞。然后，我们将测试暴露于炎症线索的FOP iECP是否比对照线形成更多的成骨前体。在目标2中，我们将测试FOP iECP细胞是否对我们在初步实验中鉴定的候选细胞因子具有趋化性，以及FOP iPS细胞衍生的巨噬细胞是否对激活触发物产生异常反应。最后，在目标3中，我们将测试通过Q207 D突变的ACVR 1的巨噬细胞特异性激活是否足以在体内损伤后启动异位骨化。因此，我们的研究使用新开发的技术来了解ACVR 1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，预防异常骨获得，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.