Local modulation of S1P receptor signaling with nanofibrous hyaluronic acid scaffolds as a regenerative immunotherapy following critical volumetric muscle loss injury

项目摘要 创伤是平民和军事人群中日益重要的临床挑战，特别是 在导致肌肉体积损失（VML）的情况下。目前VML的治疗标准未能成功恢复 损伤后的肌肉功能，并导致纤维化，而不是新形成的肌肉纤维。许多方法旨在治疗 VML未能注意到宿主的局部内源性免疫反应，这是异常慢性免疫的基础。 VML病理学特征性炎症和纤维化信号传导。VML损伤迅速导致变性和坏死 损伤的肌纤维和广泛的免疫细胞的侵入和激活，包括单核细胞和 巨噬细胞这创造了一个富含促炎和抗炎信号的环境，这通常会导致病理性炎症。 纤维化设计抗炎策略以减少总体巨噬细胞负荷并促进其从部位清除 对恢复功能至关重要。这项研究的假设是，鞘氨醇-1-磷酸（S1 P），一种生物活性的信号传导， 在炎症时在组织中产生的鞘脂在延长免疫细胞滞留中起着至关重要的作用 在VML之后，因为S1 P是针对损伤的有效化学引诱物。S1 P信号通过5个已知的G蛋白偶联的 受体（S1 PR 1 -5），因此S1 P依赖性免疫细胞应答依赖于它们的S1 PR谱。S1 P有 通过S1 P/S1 PR 3信号轴参与组织纤维化的传播，我们以前的研究揭示了一个关键的 S1 PR 3在促进免疫细胞小生境占据或排出中的作用。目的1：S1 P在异常免疫细胞中的作用 通过脂质组学分析，在鼠股四头肌VML模型中评估巨噬细胞介导的纤维化和尿潴留。 损伤肌肉和来自损伤肌肉组织及其引流淋巴液的单细胞飞行时间质谱仪（CyTOF） node.鞘氨醇激酶1敲除（SPHK 1-/-）小鼠将用于直接评估S1 P在损害有效的细胞凋亡中的作用。 免疫细胞外出并介导纤维脂肪生成祖细胞（FAP）上的促纤维化巨噬细胞信号传导， 病理性纤维化在目的2中，研究了S1 PR 3拮抗作用对促进免疫细胞排出和消除免疫抑制的作用。 将评估巨噬细胞诱导的纤维化对增强VML损伤后的总体肌肉恢复的作用。这将是 通过在C57/BL 6小鼠和S1 PR 3-/-小鼠之间产生骨髓嵌合体来完成，以确定贡献 在慢性炎症刺激的微环境中，S1 PR 3信号对免疫细胞募集与流出的影响。 此外，通过递送来自新的， 将评估纳米纤维透明质酸支架对临界尺寸的VML缺陷的损伤环境的影响。脂质组学和 将进行单细胞CyTOF分析，以分析S1 PR 3拮抗作用如何影响局部脂质代谢， 损伤后的炎症。此外，通过再生肌肉的组织学染色进行的结构肌肉评估 标记将被评估。将等长扭矩产生量化为功能结局指标，以确定 我们的治疗策略增强功能性肌肉恢复。这项研究将证明S1 P受体调节剂 可以被重新用于局部靶向宿主中的内源性修复细胞，作为再生免疫疗法的新形式。

PROJECT SUMMARY Extremity trauma is an increasingly significant clinical challenge among both civilian and military populations, particularly in cases that result in volumetric muscle loss (VML). Current standards of treatment for VML fail to successfully restore muscle function after injury and result in fibrosis rather than newly formed muscle fibers. Many approaches aimed to treat VML fail to pay attention to the local endogenous immune response of the host which underlies the aberrant chronic inflammation and fibrotic signaling characteristic of VML pathology. VML injury rapidly leads to degeneration and necrosis of damaged myofibers and the invasion and activation of a broad range of immune cells, including monocytes and macrophages. This creates an environment rich in both pro- and anti-inflammatory cues that most often leads to pathological fibrosis. Designing anti-inflammatory strategies to reduce overall macrophage burden and promote their removal from sites of injury is critical to restore function. The study's hypothesis is that sphingosine-1-phophate (S1P), a bioactive signaling sphingolipid that is produced in tissue upon inflammation, plays a crucial role in the pro-longed immune cell retention following VML, as S1P is a potent chemoattractant towards injury. S1P signals through 5 known G protein-coupled receptors (S1PR1-5) and therefore S1P-dependent immune cell responses are dependent on their S1PR profile. S1P has been implicated in propagating tissue fibrosis via the S1P/S1PR3 signaling axis and our previous studies reveal a crucial role for S1PR3 in promoting immune cell niche occupancy or egress. In Aim 1, the role of S1P on aberrant immune cell retention and macrophage-mediated fibrosis will be evaluated in a murine quadriceps VML model via lipidomic analysis of injured muscle and single-cell time-of-flight mass cytometry (CyTOF) from injured muscle tissue and its draining lymph node. Sphingosine kinase 1 knockout (SPHK1-/-) mice will be utilized to directly assess the role of S1P in impairing efficient immune cell egress and mediating pro-fibrotic macrophage signaling on fibroadipogenic progenitors (FAPs) which drives pathological fibrosis. In Aim 2, the effect of S1PR3 antagonism on promoting immune cell egress and abrogating macrophage-induced fibrosis to enhance overall muscle recovery after VML injury will be assessed. This will be accomplished by creating bone marrow chimeras between C57/BL6 mice and S1PR3-/- mice to determine the contribution of S1PR3 signaling on immune cell recruitment vs egress in a microenvironment of chronic inflammatory stimuli. Moreover, local, pharmacological antagonism of S1PR3 by delivery of VPC01091 (S1PR3 antagonist) from novel, nanofibrous hyaluronic acid scaffolds to the injury milieu of critically sized VML defects will be evaluated. Lipidomic and single-cell CyTOF analysis will be performed to analyze how S1PR3 antagonism affects local lipid metabolism and inflammation following injury. In addition, structural muscle assessments via histological staining for regenerative muscle markers will be assessed. Isometric torque production will be quantified as a functional outcome measure to determine if our therapeutic strategy enhances functional muscle recovery. This study will demonstrate how S1P receptor modulators can be re-purposed to locally target endogenous repair cells in the host as a novel form of regenerative immunotherapy.