Neutrophil and natural killer cell imbalances prevent muscle stem cell mediated regeneration following murine volumetric muscle loss

Abstract

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell and progenitor mediated myogenic repair. However, how immune cell infiltration and inter-cellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observe heightened infiltration of natural killer (NK) cells as well as persistence of neutrophils beyond two weeks post injury. Functional validation of NK cells revealed an antagonistic role on neutrophil accumulation in part via inducing apoptosis and CCR1 mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFb1). Blocking TGFb signaling reduced neutrophil accumulation and fibrosis, as well as improved muscle specific force. Collectively, these results enhance our understanding of immune cell-stem cell crosstalk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

Spatiotemporal mapping of immune and stem cell dysregulation after volumetric muscle loss

“…To understand the fibrotic response that develops after VML, we administered 2mm full-thickness punch biopsies to the tibialis anterior (TA) muscles of young adult mice. Consistent with previous results 9 , we observed increased fibrosis by staining with picrosirius red at 7- and 28-days post injury (dpi) (Supp. Figure 1A).…”

“…In line with these observations, we observed increases in expression of NK cell-related transcripts with ITD1 treatment. Prior work from our group has shown that NK cell infiltration into VML injuries reduces neutrophil abundance, and that the neutrophil secretome impairs myogenesis in vitro 9 . Thus, enhanced NK cell activity and restrained inflammation from neutrophils as a result of TGFBR2 inhibition could be one mechanism through which this intervention contributes to a more favorable biochemical environment for muscle regeneration.…”

“…Within the transition zone, ITD-1 treatment reduced expression of several of the pro-fibrotic ligands and receptors predicted by CellChat ( Cd44, Tnfrsf1a, Gas6 ). Interestingly, among the upregulated genes were Nkg7 and Ccl5 , which are associated with NK cells 26 and cytolytic NK cell signaling post VML 9 . Overall, ITD1 treatment reduced many of the pro-fibrotic signaling ligands and receptors predicted to be activated post VML by CellChat (Figure 4E), including reduced mø ligands in all zones, reduced mø receptors in the intact zone, and reduced MDC ligands and receptors in the transition zone.…”

“…VML has consistently demonstrated a failure of regeneration 27 , fibrotic scarring 5,8 and reductions in muscle function 2 , but the cellular and molecular mechanisms that confer this behavior and their spatial context remain poorly understood. As a result, many therapeutics for VML have displayed limited improvements in muscle regeneration and functional output 3,4 . Our results address this need and demonstrate a conserved a signaling circuit between møs and MPCs in murine and canine models of VML that contributes to fibrotic development and impinges on MuSC-mediated regeneration.…”

“…Currently, the cellular and molecular factors that drive the pathological VML response and prevent healing remain poorly understood. The frank loss of tissue after VML manifests in immune cell infiltration that lasts for days to months 5,6 and is accompanied by fibrotic scarring 7,8 , which is in contrast with skeletal muscle injury that results in regeneration 9–11 . Previous studies have demonstrated increases in infiltration and accrual of neutrophils 9 , macrophages (mϕs) 6,8,12,13 , and T helper 2 14 cells in VML injury and the adverse effects of the sustained inflammation from these cells range from exacerbated tissue damage to prevention of muscle stem cell mediated repair.…”