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. 2014 Apr;184(4):1167-1184.
doi: 10.1016/j.ajpath.2013.12.020. Epub 2014 Feb 11.

Altered macrophage phenotype transition impairs skeletal muscle regeneration

Hanzhou Wang  1 , David W Melton  2 , Laurel Porter  1 , Zaheer U Sarwar  1 , Linda M McManus  3 , Paula K Shireman  4
Affiliations

Altered macrophage phenotype transition impairs skeletal muscle regeneration

Hanzhou Wang et al. Am J Pathol. 2014 Apr.

Abstract

Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11b-diphtheria toxin receptor transgenic mice to transiently deplete monocytes/macrophages at multiple stages before and after muscle injury induced by cardiotoxin. Fat accumulation within regenerated muscle was maximal when ablation occurred at the same time as cardiotoxin-induced injury. Early ablation (day 1 after cardiotoxin) resulted in the smallest regenerated myofiber size together with increased residual necrotic myofibers and fat accumulation. However, muscle regeneration after late (day 4) ablation was similar to controls. Levels of inflammatory cells in injured muscle following early ablation and associated with impaired muscle regeneration were determined by flow cytometry. Delayed, but exaggerated, monocyte [CD11b(+)(CD90/B220/CD49b/NK1.1/Ly6G)(-)(F4/80/I-Ab/CD11c)(-)Ly6C(+/-)] accumulation occurred; interestingly, Ly6C(+) and Ly6C(-) monocytes were present concurrently in ablated animals and control mice. In addition to monocytes, proinflammatory, Ly6C(+) macrophage accumulation following early ablation was delayed compared to controls. In both groups, CD11b(+)F4/80(+) cells exhibited minimal expression of the M2 markers CD206 and CD301. Nevertheless, early ablation delayed and decreased the transient accumulation of CD11b(+)F4/80(+)Ly6C(-)CD301(-) macrophages; in control animals, the later tissue accumulation of these cells appeared to correspond to that of anti-inflammatory macrophages, determined by cytokine production and arginase activity. In summary, impairments in muscle regeneration were associated with exaggerated monocyte recruitment and reduced Ly6C(-) macrophages; the switch of macrophage/monocyte subsets is critical to muscle regeneration.

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Figures

Figure 1
Figure 1
Time-dependent consequences of CD11b cell depletion in skeletal muscle regeneration. TA muscle regeneration was studied in CD11b-DTR mice and analyzed 7 days after CTX-induced injury. Timing of DT [day −0.5 (d−0.5) to day 4 (d4)] or control DTm (day 1) administration was in reference to CTX injection (A). Although the area of muscle injury was similar in the six groups (average of 86% to 95%), residual necrosis (B), cross-sectional area of regenerated myofibers (C), and fat area (%) (D) at day 7 after CTX were dependent on the timing of DT administration. Data are presented as means ± SEM. n = 7 to 10 mice/group. ∗P ≤ 0.04 versus mice treated with DTm at day 1 after injury (B–D).
Figure 2
Figure 2
Impaired muscle regeneration with early (day 1) DT administration compared to control DTm and late (day 4) DT. TA muscle injury was induced in CD11b-DTR mice and analyzed after CTX-induced injury. Timing of DT or DTm administration was relative to CTX injection (A). Regenerated myofiber cross-sectional area (B), fat area (%) (C), capillaries/mm2 (D), and capillaries/fiber (E) measurements were performed in the TA muscle at day 0 (no injury) and after CTX-induced injury at indicated time points. Myofiber size at day 0 (no injury) was 2687 ± 101 mm2, and intermuscular fat was not detected in uninjured muscle. Data are presented as means ± SEM. n = 7 to 11 mice/group/time point. ∗P ≤ 0.003 versus mice treated with DTm at day 1 (B–D).
Figure 3
Figure 3
Inflammation, myofiber necrosis, and tissue regeneration in TA muscle following CTX-induced injury in CD11b-DTR mice after administration of DT or DTm control. Images were derived from TA muscle of CD11b-DTR mice. Control mice received DTm (A, D, and G) and were sacrificed at the indicated time point after CTX injection [day 2 (2d), 7, or 21 CTX]. DT was administered at day 1 after CTX (B, E, and H) or day 4 after CTX (F and I). Thus, specimens were derived at day 0 (no injury, baseline) (C), 2 (A and B), 7 (D–F), or 21 (G–I) days after CTX-injury. Asterisks identify necrotic muscle fibers; arrows indicate neutrophils, paraffin sections (3–4 μm), hematoxylin and eosin stain.
Figure 4
Figure 4
Diverse effects of early DT treatment on total cells and monocytes in BM, blood, and spleen. Timeline of CTX and DT/DTm injections and flow cytometry (A). Hatched bars (B–G) are cell counts per gram of tissue from uninjured (day 0) and injured (day 1) mice that did not receive DT or DTm (control) injections. BM (B, E, and H), blood (C, F, and I), and spleen (D, G, and J) were collected for flow cytometry analysis. Results are provided as total cells (B–D), monocytes (CD11b+(CD90/B220/CD49b/NK1.1/Ly6G)lo(F4/80/I-Ab/CD11c)loLy6Chi/lo) (E–G), and monocyte subsets (Ly6Chi or Ly6Clo) (H–J). Data are means ± SEM. n = 4 to 6 mice/group/time point. ∗P ≤ 0.01 versus DTm controls at each corresponding time point (B, C, and E–G); †P ≤ 0.003 Ly6Chi monocytes in DT- versus DTm-treated animals at each corresponding time point (H–J); and ‡P ≤ 0.03 Ly6Clo monocytes in DT- versus DTm-treated animals at each corresponding time point (H and I).
Figure 5
Figure 5
Effect of ablation on accumulation of inflammatory cells in injured muscle. Muscle was injured with CTX on day 0 in CD11b-DTR mice followed by DTm or DT treatment on day 1. Inflammatory cells were analyzed by flow cytometry at day 0 (no injury) and each day after CTX injury (A). Hatched bars (B–E) and the first two data sets in (F and G) are cell counts per gram of tissue from uninjured (day 0) and injured (day 1) mice that did not receive DT or DTm (control) injections. Results are provided as total cells (B), neutrophils (CD11b+/Ly6G+) (C), monocytes (CD11b+(CD90/B220/CD49/NK1.1/Ly6G)−(F4/80/I-Ab/CD11c)−Ly6C+/−) (D), monocyte subsets (F and H), macrophages (CD11b+F4/80+) (E), and macrophage subsets (G and I). Data are presented as means ± SEM. n = 4 to 6 mice/group/time point. ∗P ≤ 0.04 versus DTm animals at each corresponding time point (B–E); †P ≤ 0.02 Ly6C+ monocytes versus DTm animals at each corresponding time point (H); ‡P < 0.001 Ly6C+CD301− macrophages versus DTm animals at each corresponding time point; and §P ≤ 0.02 Ly6C−CD301− macrophages versus DTm animals at each corresponding time point (I).
Figure 6
Figure 6
Characterization of CD11b+F4/80+ macrophage subsets. Ly6C+CD301− and Ly6C−CD301− macrophages (CD11b+F4/80+) were sorted from CTX-injured muscle at day 3. Sorted cells were lysed with lysate buffer, and the lysates were used for measuring the concentrations of cytokines (A–J) using a bead-based multiplexing immunoassay. Measured analytes that were not significantly different between the macrophage subsets or below the level of detection included GM-CSF, KC-GRO, IFNγ, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-11, IL-12p70, IL-17A, IP-10, MCP-5, MIP-1β, RANTES, and TIMP-1. Lysates were also used to determine arginase activity (K). Data are presented as means ± SEM. n = 4 to 5; cells isolated from the skeletal muscles of six mice were pooled for each replicate. ∗P ≤ 0.05 cytokines in Ly6C+CD301− versus Ly6C−CD301− cells (A–I), for arginase activity; †P = 0.02 Ly6C+CD301− versus Ly6C−CD301− cells (K).
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References

    1. Summan M., Warren G.L., Mercer R.R., Chapman R., Hulderman T., Van Rooijen N., Simeonova P.P. Macrophages and skeletal muscle regeneration: a clodronate-containing liposome depletion study. Am J Physiol Regul Integr Comp Physiol. 2006;290:R1488–R1495. - PubMed
    1. Tidball J.G., Villalta S.A. Regulatory interactions between muscle and the immune system during muscle regeneration. Am J Physiol Regul Integr Comp Physiol. 2010;298:R1173–R1187. - PMC - PubMed
    1. Lin S.L., Castano A.P., Nowlin B.T., Lupher M.L., Jr., Duffield J.S. Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations. J Immunol. 2009;183:6733–6743. - PubMed
    1. Geissmann F., Jung S., Littman D. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003;19:71–82. - PubMed
    1. Swirski F., Nahrendorf M., Etzrodt M., Wildgruber M., Cortez-Retamozo V., Panizzi P., Figueiredo J.L., Kohler R., Chudnovskiy A., Waterman P., Aikawa E., Mempel T., Libby P., Weissleder R., Pittet M. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 2009;325:612–616. - PMC - PubMed

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