Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is the most common, lethal, muscle-wasting disease of childhood. Previous investigations have shown that muscle macrophages may play an important role in promoting the pathology in the mdx mouse model of DMD. In the present study, we investigate the mechanism through which macrophages promote mdx dystrophy and assess whether the phenotype of the macrophages changes between the stage of peak muscle necrosis (4 weeks of age) and muscle regeneration (12 weeks). We find that 4-week-old mdx muscles contain a population of pro-inflammatory, classically activated M1 macrophages that lyse muscle in vitro by NO-mediated mechanisms. Genetic ablation of the iNOS gene in mdx mice also significantly reduces muscle membrane lysis in 4-week-old mdx mice in vivo. However, 4-week mdx muscles also contain a population of alternatively activated, M2a macrophages that express arginase. In vitro assays show that M2a macrophages reduce lysis of muscle cells by M1 macrophages through the competition of arginase in M2a cells with iNOS in M1 cells for their common, enzymatic substrate, arginine. During the transition from the acute peak of mdx pathology to the regenerative stage, expression of IL-4 and IL-10 increases, either of which can deactivate the M1 phenotype and promote activation of a CD163+, M2c phenotype that can increase tissue repair. Our findings further show that IL-10 stimulation of macrophages activates their ability to promote satellite cell proliferation. Deactivation of the M1 phenotype is also associated with a reduced expression of iNOS, IL-6, MCP-1 and IP-10. Thus, these results show that distinct subpopulations of macrophages can promote muscle injury or repair in muscular dystrophy, and that therapeutic interventions that affect the balance between M1 and M2 macrophage populations may influence the course of muscular dystrophy.

Figure 1.

Th1 and Th2 responsive macrophages that express markers of M1 or M2 phenotype are present in mdx muscle. Cross-sections of 4-week-old mdx mouse quadriceps were immunolabeled with rat anti-mouse F4/80 to identify macrophage populations (A and D). The expression of iNOS (B) and CD206 (E), markers of classical and alternative activation, respectively, were simultaneously assayed by double immunofluorescence staining to determine the activation state of macrophages. Superimposed images demonstrate that macrophages undergo classical, M1 (C) and alternative, M2 activation (F) in mdx dystrophic muscle. Scale bar: 50 µm. Macrophages in mdx muscle express receptors for IFNγ and IL-4, suggesting that their activation may occur through IFNγ and IL-4-mediated signaling. Cross-sections of 4-week-old C57 (G, J) and mdx (H, I, K and L) quadriceps were immunohistochemically stained to determine the tissue distribution of IFNγR1 and IL-4R. Red reaction products, reflecting IFNγR1 (H) and IL-4R (K) expression, were noted in inflammatory cells present in necrotic lesions of mdx quadriceps, but absent in C57 quadriceps (G, J). Labeling of adjacent cross-sections with antibodies against F4/80 indicated that IFNγR1 and IL-4R-expressing cells were macrophages (I, L). Scale bar: 50 µm.

Figure 2.

Macrophages isolated from mdx muscle express iNOS and arginase-1, further supporting the conclusion that M1 and M2a macrophages are present in mdx muscle. Western blot shows that IFNγ induces iNOS expression in mdx muscle macrophages, which is further increased with the addition of TNFα (A, top panel). However, IL-4 reduced the expression of iNOS protein by mdx muscle macrophages (A, top panel). IFNγ reduced arginase-1 expression, while IL-4 or IL-10 increased arginase-1 expressed by mdx muscle macrophages (A, bottom panel). Representative blots from 3 to 4 independent experiments are shown. The treatment of muscle macrophages with IL-4 further decreased iNOS expression with time, and a greater repression was observed when IL-4 was used together with IL-10 (B, top panel). IL-4 treatment of muscle macrophages maximally induced arginase-1 expression by 24 h (B, bottom panel). However, arginase-1 expression was further induced by IL-4 in the presence of IL-10 with levels peaking by 48 h (B, bottom panel).

Figure 3.

Classically activated macrophages mediate muscle cell lysis via an NO-dependent mechanism. Cytotoxicity assays showed that M1 peritoneal (left panel, PMΦ) and mdx muscle macrophages (right panel, MMΦ) had the highest cytotoxic activity (A). *, P < 0.05 when compared with non-stimulated control at the same concentration. #, P < 0.05 when compared with macrophages cultured at 6250 MΦ/mm² within the same treatment condition. τ, P < 0.05 when compared with macrophages cultured at 6250 and 12 500 MΦ/mm² within the same treatment condition. The increase in muscle cells lysis mediated by M1 muscle macrophages paralleled increases in iNOS expression when they were cocultured with myotubes (B). Cytotoxicity assays performed in the presence of L-NAME showed that inhibition of NO synthesis resulted in a dose-dependent decrease in muscle cell lysis mediated by M1 peritoneal and muscle macrophages (C). *, P < 0.05 when compared with M1. #, P<0.05 when compared with M1 treated with 50 µM L-NAME. Non, non-stimulated macrophages; M1, M1 macrophages; M2a, M2a macrophages; PR, nitrocellulose membranes stained with ponceau red to ensure equal loading of total protein. Representative histograms or blots of 2–3 independent experiments are shown.

Figure 4.

Null mutation of iNOS reduces muscle membrane lysis in mdx muscles. (A) Intracellular fluorescence of procion orange treated muscles was used as an index of muscle fiber membrane damage. The peaks show the aggregate data for all fibers in cross-sections of the entire soleus muscle from all mice in each treatment group. The leftward shift of peaks on the abscissa indicates a decrease in fibers with membrane lesions in the iNOS null/mdx muscles, compared with iNOS expressing mdx. Background fluorescence set at intensity=0 was determined by measuring fluorescence at a region of the section where there was no tissue. (B and C) Null mutation of iNOS did not significantly affect the numbers of macrophages or neutrophils in mdx muscles at 4 or 12 weeks of age. However, ablation of iNOS caused a significant reduction in the percentage of muscle fibers that were regenerative, central-nucleated fibers. Bars=sem. *, P < 0.05 when compared with age-matched, iNOS-expressing mdx. #, P < 0.05 when compared with 4-week-old mice of the same genotype. (D) Western blotting for iNOS confirmed that iNOS was expressed in mdx muscle extracts, and that no iNOS protein expression was detectble in the double-mutant mouse muscles.

Figure 5.

M2 macrophages inhibit muscle cell lysis by M1 macrophages via an arginase-1 dependent mechanism. Cytotoxicity assays showed that M1 and M2 peritoneal macrophage cocultures resulted in a 17% reduction in myotube lysis when compared with M1 only cultures (A). In contrast, coculturing M1 macrophages with non-stimulated peritoneal macrophages resulted in a 17% increase in myotube lysis when compared with M1 only cultures (A). Measurements of nitrite showed that the reduction of muscle cell lysis observed in M1:M2 cocultures was attributed to reduced nitrite levels (B) and occurred without a reduction in iNOS protein (C). Cytotoxicity assays performed in the presence of BEC showed that inhibition of arginase activity resulted in a dose-dependent increase in nitrite formation (D) and muscle cell lysis (E) in M1:M2 cocultures compared with non-treated macrophage:M2 cocultures. *, P < 0.05 when compared with M1 only cultures. ♦, P < 0.01 when compared with non-treated macrophage:M1 cocultures. §, P < 0.05 when compared with non-treated M1:non-coculture. #, P < 0.01 when compared with same coculture conditions not treated with BEC. £, P < 0.01 when compared with same coculture condition treated with 1 µM BEC. τ, P < 0.05 when compared with same coculture condition treated with 10 µM BEC. Representative histograms of 2–5 independent experiments are shown.

Figure 6.

M1 macrophages in mdx muscle undergo deactivation as the disease progresses from the acute peak of pathology (4-weeks-old) to the regenerative phase of the disease (12 weeks). Cytotoxicity assays showed that macrophages isolated from 4-week-old mdx muscle are more cytolytic in vitro compared with macrophages isolated at 12 weeks (A). A representative of three independent experiments is shown. The greater cytotoxicity of 4-week-old macrophages correlated with higher expression levels of iNOS protein (B). The elevated levels of iNOS at 4 weeks are partly attributed to increased expression of STAT-1, which leads to increased levels of STAT-1 activation (phosphorylated STAT-1, B). Measurements of IFNγ by real-time PCR showed that it was elevated in mdx hamstrings but did not differ significantly between 4 and 12 weeks (C, left panel). Transcript levels of IL-4 were upregulated in 12-week-old mdx hamstrings (C, right panel). Representative histograms of two independent experiments are shown. Consistent with the increased expression of IL-4, genes associated with alternative activation increased in 12-week-old mdx hamstrings relative to 4-week-old mdx hamstrings (D). Measurements of genes associated with classical activation showed a downward trend of expression in 12-week-old mdx hamstrings relative to 4-week-old mdx hamstrings (D, top panel). *, P < 0.05, relative to age-matched control. #, P < 0.05, relative to 4-week-old mdx. τ, P < 0.01, relative to 4-week-old mdx. ♦, P < 0.01, relative to C₂C₁₂ cultures grown without macrophages (C₂C₁₂ only).

Figure 7.

Summary diagram of the macrophage phenotypes that dominate the acute necrotic phase and the regenerative phase of mdx muscular dystrophy. Muscle in the acute necrotic phase of the disease contains iNOS-expressing, M1 macrophages that are capable of lysing muscle fibers by NO-mediated cytotoxicity, and arginase-expressing M2a macrophages that compete with iNOS for arginine, which can thereby reduce M1 macrophage cytotoxicity. As mdx mice enter the regenerative phase, there is an increase in IL-10 expression that accompanies deactivation of the M1 and M2a phenotypes, reflected in a reduction in iNOS and arginase expression. Macrophages in regenerative muscle resemble an M2c population in which cytotoxicity is low, and the production of anti-inflammatory cytokines (IL-10, IL-1, IL-1Ra) are elevated, but arginase expression is greatly reduced.