Novel macrophage and regulatory T cell interactions that promote the pathogenesis of Duchenne muscular dystrophy

项目总结 我们研究的长期目标是确定免疫系统如何与体内的细胞相互作用。 为影响Duchenne肌营养不良症(DMD)的发病机制提供基础 为这种毁灭性的疾病开发新的治疗方法。目前这项研究的目标是界定 调节性T细胞(Treg)如何调节肌肉中巨噬细胞-基质的相互作用，以及这些相互作用是如何 可以通过药物操作来改变疾病的进展。我们提供了强有力的科学前提 提出Tregs通过抑制一种独特的Galectin-3来调节DMD严重程度的中心假设 促进纤维化的肌肉巨噬细胞群。这一假设得到了我们初步数据的支持，而且 先前的研究表明，1)营养不良的肌肉中存在一种可能导致纤维化的Galectin-3/Spp1 我们通过单细胞RNA测序(ScRNAseq)鉴定的巨噬细胞；2)先前的研究表明spp1 促进mdx鼠的纤维化；3)mdx鼠巨噬细胞特异性的Spp1缺失引起显著变化 通过单链RNA序列分析，在PGFRa基质细胞的转录谱中；4)Galectin-3巨噬细胞 在MDX小鼠中，当Tregs被耗尽时，Tregs会扩张，这表明Tregs抑制了纤维化的巨噬细胞 5)mdx小鼠Tregs缺失导致促纤维化基因表达增加。我们将测试我们的 通过解决两个具体目标来实现中心假设。特定目标1将定义Galectin-3肌肉 巨噬细胞促进纤维化。两个子目标将在具体目标2下处理，确定分子 Galectin-3肌肉巨噬细胞Treg抑制的基础。在次级目标2a中，我们将确定 Treg介导的对产生IFNG的细胞的抑制依赖于IL-10；在SubAim 2b中，我们将检查 Tregs的治疗性扩张是否抑制纤维化。尽管Spp1在促进纤维化中起到了作用 在mdx小鼠中，这种病理过程的机制在很大程度上是未知的。因此，我们的 拟议的研究意义重大，因为它将定义Spp1促进肌肉纤维化的细胞基础，以及 增进我们对Tregs如何改善肌肉营养不良的严重程度的理解。建议数 这项研究具有创新性，因为它将定义促进营养不良患者纤维化的细胞串扰机制 肌肉。建议的研究使用创新技术(例如scRNAseq)和实验疗法(例如： IL-2c)，这将为开发抑制纤维化发展的新方法提供框架。 这些潜在的发现将通过建立淋巴系和髓系之间的相互作用来推动这一领域的发展 免疫系统的隔室，在DMD中工作，以抑制或限制纤维化反应。此外， 拟议研究的结果预计将对DMD具有重要的临床意义，因为 Tregs的免疫抑制功能可以通过以下方法在治疗上增强以减轻疾病的严重程度 抑制促纤维化炎症反应。

PROJECT SUMMARY The long-term goal of our research is to determine how the immune system interfaces with cells in the dystrophic niche to impact the pathogenesis of Duchenne muscular dystrophy (DMD), to provide a foundation for developing novel treatments for this devastating disease. The objective of the current study is to define how regulatory T cells (Treg) regulate macrophage-stromal interactions in muscle, and how these interactions can be pharmacologically manipulated to alter disease progression. We provide strong scientific premise for proposing the central hypothesis that Tregs regulate the severity of DMD by suppressing a distinct galectin-3+ muscle macrophage population that promotes fibrosis. This hypothesis is supported by our preliminary data and prior research, showing that 1) dystrophic muscle is populated by a putative fibrogenic galectin-3+/Spp1+ macrophage that we identified by single-cell RNA sequencing (scRNAseq); 2) prior research showing that spp1 promotes fibrosis in mdx mice; 3) macrophage-specific deletion of Spp1 in mdx mice causes remarkable changes in the transcriptional profile of PGFRa+ stromal cells, as assessed by scRNAseq; 4) galectin-3+ macrophages are expanded when Tregs are depleted in mdx mice, suggesting that Tregs suppress fibrogenic macrophages and 5) depletion of Tregs in mdx mice causes an increased expression of pro-fibrotic genes. We will test our central hypothesis by addressing two specific aims. Specific aim 1 will define how galectin-3+ muscle macrophages promote fibrosis. Two subaims will be addressed under specific aim 2, identify the molecular basis for Treg suppression of galectin-3+ muscle macrophages. In subaim 2a we will determine whether the Treg-mediated suppression of IFNg-producing cells is IL-10 dependent; and in subaim 2b we will examine whether the therapeutic expansion of Tregs suppresses fibrosis. Although a role for Spp1 in promoting fibrosis in mdx mice has been documented, a mechanism for this pathological process is largely unknown. Thus, our proposed study is significant as it will define the cellular basis by which Spp1 promotes muscle fibrosis, and advance our understanding of how Tregs ameliorate the severity of muscular dystrophy. The proposed research is innovative because it will define mechanisms of cellular cross talk that promote fibrosis in dystrophic muscles. The proposed studies use innovative technologies (e.g. scRNAseq) and experimental therapeutics (e.g. IL-2c) that will provide the framework for developing novel approaches to inhibit the development of fibrosis. These potential discoveries will advance the field by establishing interactions between the lymphoid and myeloid compartment of the immune system that operate in DMD to suppress or limit fibrogenic responses. Further, findings from the proposed studies are expected to have significant clinical implications for DMD, as the immunosuppressive function of Tregs can be therapeutically augmented to ameliorate disease severity by inhibiting pro-fibrotic inflammatory responses.