Formation of neutrophil extracellular traps in the early stages exacerbate the healing process by regulating macrophage polarization in Achilles tendon-bone injury.

Abstract

The influence of neutrophils and of neutrophil extracellular traps (NETs) on post-traumatic tendon-to-bone healing was studied in a murine model. The impact of neutrophil infiltration on macrophage polarization and peritendinous fibrosis in early-stage Achilles tendon injury is reported. Mice underwent Achilles tendon-bone injury and divided into four groups: sham operation, tendon injury (TI) treated with acetylcellulose (vehicle control), TI treated with a Protein arginine deiminase-4 (PAD4) inhibitor GSK484, and TI treated with a neutrophil elastase inhibitor Sivelestat. Each group was monitored for 21 days. Post-traumatic neutrophil infiltration and NET formation were assessed using flow cytometry and immunofluorescence. Immunohistochemistry, Western blot, and qPCR were used to evaluate macrophage polarization. Peritendinous fibrosis was assessed using Masson staining and Western blot. Neutrophil infiltration and NET formation increased significantly in the tendon following injury. A significant increase in M1-related markers and a decrease in M2-related markers were associated with NET formation. NET Inhibition using GSK484 or sivelestat reduced M1 markers and increased M2 markers. Furthermore, NET inhibition during the early stage suppressed peritendinous fibrosis and reduced inflammation during the healing process. In co-culture experiments, NETs induced proinflammatory cytokine secretion and upregulated M1 markers in bone marrow-derived macrophages while downregulating M2 markers. nlsNETs promote early-phase tendon-bone injury by inducing M1 macrophage polarization and peritendinous fibrosis. Targeting NETs during the initial phase of tendon injury could potentially facilitate the healing process.