The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury

Nicole J Edwards, Charles Hwang, Simone Marini, Chase A Pagani, Philip J Spreadborough, Cassie J Rowe, Pauline Yu, Annie Mei, Noelle Visser, Shuli Li, Geoffrey E Hespe, Amanda K Huber, Amy L Strong, Miriam A Shelef, Jason S Knight, Thomas A Davis, Benjamin Levi

Research output: Contribution to journalArticlepeer-review


Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.

Original languageEnglish
Pages (from-to)15753-15770
Number of pages18
JournalFASEB Journal
Issue number12
StatePublished - Dec 2020


  • Animals
  • Cell Proliferation/physiology
  • Disease Models, Animal
  • Extracellular Traps/metabolism
  • Fibrosis/metabolism
  • Interleukin-10/metabolism
  • MAP Kinase Signaling System/physiology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Muscle, Skeletal/metabolism
  • Muscular Diseases/metabolism
  • Neutrophils/metabolism
  • Protein-Arginine Deiminase Type 4/metabolism
  • Reperfusion Injury/metabolism
  • Signal Transduction/physiology
  • Toll-Like Receptors/metabolism
  • Tumor Necrosis Factor-alpha/metabolism


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