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

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.