Identifying dysregulated immune cell subsets following critical volumetric muscle loss with pseudo-time trajectories

Abstract

Volumetric muscle loss (VML) results in permanent functional deficits and remains a substantial regenerative medicine challenge. A coordinated immune response is crucial for timely myofiber regeneration, however the immune response following VML has yet to be fully characterized. Here, we leveraged dimensionality reduction and pseudo-time analysis techniques to elucidate the cellular players underlying a functional or pathological outcome as a result of subcritical or critical VML in the murine quadriceps, respectively. We found that critical VML presented with a sustained presence of M2-like and CD206hiLy6Chi "hybrid" macrophages whereas subcritical defects resolved these populations. These macrophage subsets may contribute to fibrogenesis in critical VML, especially in the presence of TGF-β. Furthermore, several T cell populations were significantly elevated in critical VML compared to subcritical injuries. Specifically, there was a significant increase of CD127+ T cells at days 3 and 7, and upregulated CD127 expression may indicate aberrant IL-7 signaling in critical VML. These results demonstrate a dysregulated immune response in critical VML that is unable to resolve the chronic inflammatory state and transition to a pro-regenerative microenvironment. These data provide important insights into potential therapeutic strategies which could reduce the immune cell burden and pro-fibrotic signaling characteristic of VML.

Harnessing Bilayer Biomaterial Delivery of FTY720 as an Immunotherapy to Accelerate Oral Wound Healing

“…Although we did not see a significant difference in FTY720-mediated macrophage polarization in our flow cytometric analyses, our SPADE dendrogram illustrate that by D7, macrophages indeed polarized to M1, M2, or hybrid M1/M2 phenotypes. It is possible that the hybrid macrophage phenotype helped to resolve inflammation which in turn helped to drive ONF repair, similar to a study where S1PR modulators induced hybrid macrophage polarization in a murine volumetric muscle loss model (18). In follow-up studies, we can continue to explore the role by which FTY720 modulates macrophages in the context of ONF healing and modifies their role toward a pro-regenerative phenotype.…”

“…Macrophages were identified as CD64 + MerTK + , a receptor tyrosine kinase, and polarization state was characterized further using markers CD86 + for pro-inflammatory M1 polarization or CD206 + for alternatively activated M2 macrophages( 17 ). The MerTK − Ly6C + antigen differentiates between classical Ly6C hi or non-classical Ly6C lo monocytes present in murine palatal tissue( 18 ).…”

Ectopic adipogenesis in response to injury and material implantation in an autoimmune mouse model

“…Surprisingly, as CD206+ macrophages have previously been associated with a pro-regenerative phenotype, while we hypothesized a more robust M2-like macrophage polarization, we found a decrease in M2-related genes as detected by RNA sequencing and confirmed via flow cytometry. This may relate to the persistence of CD206+ macrophages associated with pathologic outcomes in tissue regeneration and wound healing [37, 38]. As type-1 immune signals are needed for myoblast proliferation, this type-2 switch, if occurring too early, may stunt regeneration by signaling the transition to myotube fusion before sufficient precursor cell development [39, 40].…”