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

项目总结 四肢创伤在平民和军人中都是一个日益严重的临床挑战，尤其是 在导致容积性肌肉丧失(VML)的情况下。目前治疗VML的标准未能成功恢复 损伤后的肌肉功能，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没有注意到宿主的局部内源性免疫反应，这是异常慢性疾病的基础 VML病理的炎症和纤维化信号特征。VML损伤迅速导致变性和坏死 损伤的肌纤维和多种免疫细胞的入侵和激活，包括单核细胞和 巨噬细胞。这创造了一个充斥着促炎和抗炎线索的环境，这些线索通常会导致病理 纤维化症。设计抗炎策略以减少总体巨噬细胞负荷并促进其从部位移除 损伤的恢复是恢复功能的关键。这项研究的假设是神经鞘氨醇-1-磷酸(S1P)，一种生物活性信号 鞘磷脂是炎症时在组织中产生的，在持久的免疫细胞滞留中起着至关重要的作用。 在VML之后，因为S1P是一种强大的损伤趋化剂。S1P信号通过5个已知的G蛋白偶联 受体(S1PR1-5)和S1P依赖的免疫细胞反应依赖于它们的S1PR谱。S1P具有 通过S1P/S1PR3信号轴参与了组织纤维化的传播，我们之前的研究揭示了一个关键的 S1PR3在促进免疫细胞占据或退出中的作用。在目标1中，S1P在异常免疫细胞中的作用 在小鼠股四头肌VML模型中，将通过脂组学分析评估滞留和巨噬细胞介导的纤维化 损伤肌肉和损伤肌肉组织及其引流淋巴的单细胞飞行时间质谱仪 节点。鞘氨醇激酶1基因敲除(SPHK1-/-)小鼠将被用来直接评估S1P在降低效率中的作用 免疫细胞外流和介导纤维脂肪生成前体细胞(FAP)上的促纤维化巨噬细胞信号 病理性纤维化。目的2、S1PR3拮抗剂促进免疫细胞外流和清除的作用 将评估巨噬细胞诱导的纤维化促进VML损伤后整体肌肉恢复的作用。这将是 通过在C57/BL6小鼠和S1PR3-/-小鼠之间建立骨髓嵌合体来确定其贡献 在慢性炎症刺激的微环境中，S1PR3信号对免疫细胞募集和出口的影响。 此外，VPC01091(S1PR3拮抗剂)从新的， 纳米纤维透明质酸支架对严重VML缺陷的损伤环境将进行评估。脂肪组学和 单细胞细胞TOF分析将分析S1PR3拮抗如何影响局部脂质代谢和 受伤后的炎症。此外，通过组织学染色对再生肌肉进行结构性肌肉评估 记分员将被评估。等长扭矩产生将被量化为功能结果衡量标准，以确定 我们的治疗策略促进了功能性肌肉的恢复。这项研究将展示S1P受体调节剂如何 作为一种新的再生免疫疗法，可用于局部靶向宿主内的内源性修复细胞。

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.