Fig. 3.

Perturbations in inflammatory cell signaling after degenerative muscle injury. (A) Schematic of experiment design whereby mice received 3-mm defects on one limb and 2-mm defects on the contralateral limb, then neutrophils were quantified by flow cytometry (PI⁻CD11b⁺Ly6g⁺) at 3, 7, and 14 dpi. (B) Time course of neutrophil infiltration in 2-mm and 3-mm defects as quantified by flow cytometry. Bars show mean ± SEM. n = 5 to 6 defects from 5 to 6 mice per time point. *P < 0.05, **P < 0.01 by two-sided, paired t test. Cohen’s d = 2.1, 1.17, and 0.94 for 3, 7, and 14 dpi, respectively. (C) Top-ranked NicheNet ligand-receptor pairs based on prior literature and their prediction of downstream target gene expression. Ccl5 is one of the top ligands predicted to influence neutrophils following 3-mm defects. (D) UMAP overlays of Ccr1 and Ccl5 expression showing neutrophils with the highest Ccr1 mRNA expression and T and NK cells with the highest Ccl5 mRNA expression. (E) Representative flow cytometry scatter plots showing T cell and NK cell abundance in 2-mm vs. 3-mm VML defects at 7 dpi. (F) Flow cytometry quantification of NK cell abundance in 2-mm and 3-mm defects 7 dpi and in 3-mm defects 7 dpi following whole-body saline perfusion prior to dissection. Graph shows mean ± SEM. **P < 0.001, ****P < 0.0001 by one-way ANOVA and Bonferroni posthoc analysis. n = 6 injuries per group. n = 9 mice. Effect size = 2.21. (G) Immunofluorescent stains qualitatively show NK cell localization primarily within the defects and increased numbers in 3-mm defects. (Scale bars, 200 μm; Inset, 10 μm.)