Origins and Functions of Intramuscular Macrophages in Duchenne Muscular Dystrophy

项目摘要 Duchenne肌营养不良症(DMD)是最常见的遗传性肌肉疾病，是致命的，无法治愈。 指向。DMD及其小鼠模型mdx5Cv的肌肉病理特征为慢性炎症，以 巨噬细胞(MP)浸润。我们实验室和其他实验室的临床前研究表明，改善肌肉 炎症可改善肌营养不良表型。还可改善局部组织环境，促进 肌肉再生以及基因和细胞植入。组织巨噬细胞在功能上是不同的 不同的起源。根据组织的不同，它们可以是促炎、促纤维化或促再生的。 环境和起源。虽然组织炎性MPS来自血单核细胞(MOS)，但组织 常驻MPS可来源于血液、MOS和/或胚胎。小鼠外周血MOS由两种分子组成 Ly6chi和Ly6Clo细胞亚群，以及人类中相应的亚群。Ly6chi细胞(CCR2+/CX3CR1low)是 炎性MOS，通过CC趋化因子受体2(CCR2)进入组织，对损伤做出反应，然后 分化为炎症性MPS。在受损组织中，Ly6chi MPS可转换为Ly6Clo MPS。Ly6chi Mos 也可有助于组织驻留在稳定状态的MPS。Ly6Clo Mos(CCR2-/CX3CR1hi)在 血管内皮细胞表面并可能通过趋化因子受体CX3CR1进入正常组织进行补充 下院议员。胚胎衍生组织驻留的MPS也是Ly6Clo，它们通过 扩散式的自我更新。我们的初步数据显示，MPS的Ly6chi和Ly6Clo亚集都在 Mdx5Cv骨骼肌，CCR2对Ly6chi炎症性MOS的肌肉募集是必不可少的。 敲除CCR2在所有阶段都会减少肌肉内的Ly6chi MPS，但只在早期减少Ly6Clo MPS 各阶段。早期肌肉内MPS的减少伴随着肌肉损伤的减少， 减少肌肉纤维化，改善肌肉功能，这支持了肌肉内的致病作用 Ly6chi MPS。然而，在mdx5Cv/CCR2-/-小鼠中，这种有益作用在注射后的后期就消失了。 肌肉内扩张Ly6Clo MPS。仅以Ly6chi MP为目标并不能提供持续的好处。我们 从而得出我们的中心假设，即Ly6Clo MPS在mdx5Cv横隔膜中也起致病作用 营养不良，来自不同来源的Ly6Clo MPS可能有不同的贡献，而针对单核细胞来源的是 在治疗上很有用。我们将通过三个具体目标来检验我们的假设。目标1将研究 骨骼肌在正常稳定状态下的MPS。目标2将定义肌肉内MPS的起源 Mdx5cv隔膜。目标3将确定肌内MPS的效应器和调节功能 来自不同来源的mdx5Cv横隔膜，并测试靶向单核细胞来源的治疗潜力。 这个项目将解决与肌肉内MPS相关的关键问题，所获得的知识将是 对未来基于单核/巨噬细胞治疗DMD的新疗法的发展至关重要。

Project Summary Duchenne muscular dystrophy (DMD), the most common genetic muscle disease, is lethal with no cure at this point. Muscle pathology of DMD and its mouse model mdx5cv features chronic inflammation with predominant macrophage (MP) infiltration. Preclinical studies by our lab and others demonstrate that ameliorating muscle inflammation improves muscular dystrophy phenotype. It also improves local tissue environment to promote muscle regeneration and gene and cell engraftment. Tissue macrophages are functionally heterogeneous with diverse origins. They can be pro-inflammatory, pro-fibrotic, or pro-regenerative depending on the tissue environment and origins. While tissue inflammatory MPs are derived from blood monocytes (MOs), tissue resident MPs can originate from blood MOs and/or embryo. Murine peripheral blood MOs consist of two subsets, Ly6Chi and Ly6Clo cells, with corresponding subsets in humans. Ly6Chi cells (CCR2+/CX3CR1low) are inflammatory MOs, which enter tissues in response to injury via CC chemokine receptor 2 (CCR2) and then differentiate into inflammatory MPs. Within injured tissues, Ly6Chi MPs can switch into Ly6Clo MPs. Ly6Chi MOs may also contribute to tissue resident MPs at the steady state. Ly6Clo MOs (CCR2-/CX3CR1hi) patrol the vascular endothelial surface and may enter normal tissue via chemokine receptor CX3CR1 to replenish resident MPs. Embryo-derived tissue resident MPs are also Ly6Clo, and they persist into adult tissues through proliferative self-renewal. Our preliminary data show that both Ly6Chi and Ly6Clo subsets of MPs accumulate in mdx5cv skeletal muscle, and that CCR2 is essential to the muscle recruitment of Ly6Chi inflammatory MOs. Knockout of CCR2 diminishes intramuscular Ly6Chi MPs at all stages, but it only reduces Ly6Clo MPs at early stages. The reduction of intramuscular MPs at the early stages is accompanied by decreased muscle damage, reduced muscle fibrosis, and improved muscle function, which supports a pathogenic role for the intramuscular Ly6Chi MPs. However, the beneficial effects are lost at the late stage in the mdx5cv/Ccr2-/- mice after the expansion of intramuscular Ly6Clo MPs. Targeting Ly6Chi MP alone does not provide sustained benefits. We thus generate our central hypothesis that Ly6Clo MPs also play a pathogenic role in the mdx5cv diaphragm dystrophy, Ly6Clo MPs from different origins may contribute differently, and targeting the monocytic origins is therapeutically useful. We will test our hypothesis by three Specific Aims. Aim 1 will study the origins of skeletal muscle resident MPs at the normal steady state. Aim 2 will define the origins of intramuscular MPs in the mdx5cv diaphragm. Aim 3 will determine the effector and regulatory functions of intramuscular MPs derived from different origins in the mdx5cv diaphragm, and test the therapeutic potential of targeting monocytic origins. This project will address the key questions related to the intramuscular MPs, and the knowledge gained will be critical to the future development of novel monocyte/macrophage-based therapies for DMD.