Modulation of Sphingosine-1-Phosphate Receptor Signaling as a Regenerative Immunotherapy for Volumetric Muscle Loss

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Hymel, Lauren Alexandra
Botchwey, Edward A.
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Extremity trauma is a significant clinical challenge among both civilian and military populations, particularly in cases that result in volumetric muscle loss (VML). Many approaches aimed to treat VML fail to pay attention to the local endogenous immune response of the host which underlies the chronic inflammation and fibrotic signaling characteristic of VML pathology and results in loss of function. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid locally produced during inflammation through the activity of endogenous sphingosine kinases and is increasingly recognized as a crucial regulator of the immune response. S1P signals through its 5 G protein-coupled receptors (S1PR1-5) to elicit a range of cellular responses governing complex functions such as immune cell trafficking, vascular integrity, and fibrosis. Using a pre-clinical model of VML, we found that critically sized injuries are characterized by a marked increase in the local production of S1P in injured tissue, a sustained presence of M2 macrophages with aberrant TNFa and TGFb co-expression, and significantly increased pro-fibrotic FAPs which highly express b1-integrin and are predisposed towards fibrotic differentiation. We demonstrated that by mediating S1P synthesis, S1P spatial gradients are perturbed which allows for regulation of immune cell trafficking into the injured tissue. We next delivered an S1P chaperone-based protein therapeutic to activate S1PR1 signaling on the damaged endothelium to increase its barrier function following injury. Our findings showed that sustained S1P/S1PR1 vascular signaling reduced pro-fibrotic FAP accumulation in injured muscle and abrogated fibrotic deposition. Finally, we revealed that small molecule antagonism of S1PR3 leads to enhanced skeletal muscle regeneration. To achieve a localized strategy for S1PR3 antagonism, we delivered the pharmacological inhibitor from nanofibrous poly(ethylene glycol)/hyaluronic acid hydrogels which facilitated macrophage egress from the local milieu and improved muscle repair. Taken together, these findings represent an improved understanding of the role of S1P/S1PR signaling in the pathogenesis of VML. Additionally, this work establishes that modulation of S1PR signaling provides an effective therapeutic strategy for shifting the local microenvironment from pro-fibrotic to pro-regenerative following traumatic injury.
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