Anti-inflammatory macrophages improve skeletal muscle recovery from ischemia-reperfusion

Abstract

The presence of macrophages (MPs) is essential for skeletal muscle to properly regenerate following injury. The aim of this study was the evaluation of MP profiles and their importance in skeletal muscle recovering from tourniquet-induced ischemia-reperfusion (I/R). Using flow cytometry, we identified two distinct CD11b(+) MP populations that differ in expression of the surface markers Ly-6C and F4/80. These populations are prominent at 3 and 5 days of reperfusion and molecularly correspond to inflammatory and anti-inflammatory MP phenotypes. Sorted MP populations demonstrated high levels of IGF-I expression, and whole muscle post-I/R IGF-I expression strongly correlates with F4/80 expression. This suggests MPs largely influence postinjury IGF-I upregulation. We additionally demonstrate that direct intramuscular injection of FACS-isolated CD11b(+)Ly-6C(lo)F4/80(hi) MPs improves the functional and histological recovery of I/R-affected muscle. Taken together, these data further support the substantial influence of the innate immune system on muscle regeneration and suggest MP-focused therapeutic approaches may greatly facilitate skeletal muscle recovery from substantial injury.

Keywords: flow cytometry; macrophage; myogenesis; regenerative medicine; reperfusion.

Fig. 1.

Flow cytometry was used to analyze macrophage (MP) infiltrate profiles following 2-h tourniquet-induced ischemia-reperfusion (I/R) injury of mouse lateral gastrocnemius (LG) muscle. By gating for CD11b⁺ cells, 3 distinct subpopulations were evident based on expression of Ly-6C and F4/80, including Ly-6C^hiF4/80^lo (Q4) and Ly-6C^loF4/80^hi (Q2) MP populations (A). The time course of recovery from I/R displayed distinctive MP profiles, shifting from predominantly Ly-6C^hiF4/80^lo MPs at 1 day of reperfusion to Ly-6C^loF4/80^hi by day 7 (B). Total cell numbers (C) in the LG peak following 3 days of reperfusion and decline during the course of recovery are shown. Analysis of individual cell populations (D) from days 3 and 5 show the selective decline in Ly-6C^hiF4/80lo cells. Values are expressed as means (B) or means ± SE (C and D); *P < 0.05 vs. previous day population size.

Fig. 2.

Gene expression analysis of CD11b⁺F4/80⁺ cells sorted from 1-, 3-, 5-, and 7-day reperfusion muscles (A) and the Ly-6C^hiF4/80^lo and Ly-6C^loF4/80^hi subsets of CD11b⁺ cells isolated from 3-day reperfusion muscles (B) reveals show divergent inflammatory and anti-inflammatory phenotypes. AU, arbitrary units. Values are expressed as means ± SD; *P < 0.05 vs. previous day (A) or Ly-6C^hiF4/80^lo 14 (B) value; n = 3 for each experiment.

Fig. 3.

Igf1 gene expression was analyzed in CD11b⁺F4/80⁺ cell populations from 1-, 3-, 5-, and 7-day reperfusion muscles (A), and Ly-6C^hiF4/80^lo and Ly-6C^loF4/80^hi populations from 3-day reperfusion muscles (B). Whole muscle Igf1 expression levels from 1-, 3-, 5-, and 7-day reperfusion gastrocnemius muscles show strong correlation to Emr1 (F4/80) expression (C; n = 3–5). Values are expressed as means ± SD; *P < 0.05 vs. previous day value.

Fig. 4.

Intramuscular injection of FACS-isolated CD11b⁺Ly-6C^loF4/80^hi 21 MPs into the 3-day reperfusion gastrocnemius muscles improves histological morphology, as determined by hematoxylin and eosin (H&E) staining (A), myofiber size (B), and functional recovery (C) at 14 days of reperfusion (n = 5–6). Values are expressed as means ± SE; *P < 0.05 vs. control muscle; †P < 0.05 vs. saline-treated muscle.