IFN-γ promotes muscle damage in the mdx mouse model of Duchenne muscular dystrophy by suppressing M2 macrophage activation and inhibiting muscle cell proliferation

Abstract

Duchenne muscular dystrophy is a degenerative disorder that leads to death by the third decade of life. Previous investigations have shown that macrophages that invade dystrophic muscle are a heterogeneous population consisting of M1 and M2 macrophages that promote injury and repair, respectively. In the present investigation, we tested whether IFN-γ worsens the severity of mdx dystrophy by activating macrophages to a cytolytic M1 phenotype and by suppressing the activation of proregenerative macrophages to an M2 phenotype. IFN-γ is a strong inducer of the M1 phenotype and is elevated in mdx dystrophy. Contrary to our expectations, null mutation of IFN-γ caused no reduction of cytotoxicity of macrophages isolated from mdx muscle and did not reduce muscle fiber damage in vivo or improve gross motor function of mdx mice at the early, acute peak of pathology. In contrast, ablation of IFN-γ reduced muscle damage in vivo during the regenerative stage of the disease and increased activation of the M2 phenotype and improved motor function of mdx mice at that later stage of the disease. IFN-γ also inhibited muscle cell proliferation and differentiation in vitro, and IFN-γ mutation increased MyoD expression in mdx muscle in vivo, showing that IFN-γ can have direct effects on muscle cells that could impair repair. Taken together, the findings show that suppression of IFN-γ signaling in muscular dystrophy reduces muscle damage and improves motor performance by promoting the M2 macrophage phenotype and by direct actions on muscle cells.

Figure 1

Schematic of time course of mdx muscle pathology and changes of predominant macrophage phenotype (4, 10, 11, 15, 31, 40).

Figure 2

Diagram of breeding strategy used to generate IFNγ^−/−/mdx mice and background strain controls.

Figure 3

IFNγ promotes muscle fiber membrane damage in 12-week-old mdx mice. A, B: The intracellular fluorescence intensity of muscle fibers in muscle incubated in a fluorescent, extracellular marker dye, procion orange, was used as an index of muscle fiber membrane damage. Five mice were analyzed in each data set. At 4-weeks of age (A), there is no difference between the mean, cytosolic fluorescent intensity of mdx fibers (blue) and IFNγ^−/−/mdx fibers (red). At 12-weeks of age (B), intracellular fluorescence intensity of muscle fibers in IFNγ^−/−/mdx muscles was less than in mdx muscles indicating that IFNγ promotes muscle fiber injury during the regenerative stage of dystrophinopathy. Wild-type C57 muscle fibers showed no intracellular fluorescence above background levels, which were set at the value “0” (4). C–H: representative images of soleus muscles used to quantify muscle fiber injury are shown for each group. Bar = 100 μm. I: No significant differences in the numbers of F4/80+ macrophages occurred between IFNγ^−/−/mdx and mdx muscles at either 4-weeks or 12-weeks of age. Each sample is a muscle from a separate mouse for each genotype and age-group: 4-week-old wild-type (n = 5), 4-week-old mdx (n = 5), 4-week old IFNγ^−/−/mdx (n = 5), 12-week-old wild-type (n = 5), 12-week-old mdx (n = 5), 12-week old IFNγ^−/−/mdx (n = 5). Error bars = standard error of the mean (sem). J: Cytotoxicity assays to assess macrophage-mediated lysis of muscle cells in vitro show that macrophages isolated from muscles of 12-week-old mdx mice are unaffected by IFNγ^−/− mutation. Each bar represents the mean of 3 samples.

Figure 4

IFNγ null mutation decreases iNOS expression of muscle macrophages isolated from 4-week-old and 12-week-old mdx mice. A: Hindlimb muscles were used for RNA isolation and qPCR for iNOS expression. Data show that iNOS mRNA levels do not change significantly between 4 and 12-weeks-of-age in either mdx or IFNγ^−/−/mdx muscles and that ablation of IFNγ expression has no effect on iNOS mRNA levels at either age. Each bar represents the mean and sem for the muscles collected from 5 mice in each data set. B, C: Macrophages that were isolated from hindlimb muscles of mdx mice or IFNγ^−/−/mdx mice were assayed by western blotting for relative levels of iNOS expression. Membranes were stained with Ponceau red before antibody-binding to confirm equal loading of the gel (“Loading”). Densitometry of the western blots shows that iNOS concentration declines in mdx mice between 4 weeks and 12 weeks of age, and that ablation of IFNγ^−/− reduces iNOS in muscle at both ages. Each sample consists of total muscle macrophages isolated from the hindlimbs of one mouse. * indicates significantly different from age-matched sample at p < 0.05. # indicates significantly different from mice of same genotype at 4-weeks of age. Each bar represents the mean and sem of the western blot for 3 samples per data set. D: Q-PCR data for transcripts associated with M1 macrophage activation in RNA isolated from muscle samples. Each bar represents the mean and sem for the muscles collected from 5 mice in each data set. * indicates significantly different from age-matched sample at p < 0.05. # indicates significantly different from mice of same genotype at 4-weeks of age.

Figure 5

IFNγ represses the expression of genes associated with M2 macrophage activation during regeneration. A: Transcript levels of M2-associated genes were measured by qPCR for RNA isolated from hindlimb muscles. Each bar represents the mean and sem for the muscles collected from 5 mice in each data set. * indicates significantly different from age-matched sample at p < 0.05. # indicates significantly different from mice of same genotype at 4-weeks of age. B. Density of CD206+ macrophages in quadriceps muscles. Data show that the number of CD206+ macrophages is elevated in dystrophic muscles and that ablation of IFNγ in mdx muscles further elevates CD206+ cells in 12-week-old mdx muscles, but not in 4-week-old mdx muscles. Each bar represents the mean and sem for the CD206+ cell counts for the quadriceps of 5 mice. * indicates significantly different from age-matched, wild-type sample at p < 0.05. # indicates significantly different from mice of same genotype at 4-weeks of age. § indicates significantly different from mdx mice at same age.

Figure 6

Mdx dystrophy causes increases in the size of M2 macrophages and the level of activation of M2 macrophages. A: Section of a 4-week-old wild-type quadriceps muscle showing the presence of few, small CD206+ macrophages (arrows) within the endomysium between muscle fibers. B, C. The numbers and size of CD206+ macrophages (red) in muscle increases greatly in muscular dystrophy in 4-week-old mdx mice (B) and 4-week-old IFNγ^−/−/mdx mice (C). D. Section of 12-week-old, wild-type muscle. CD206+ macrophages are indicated by arrows. E, F. Sections of 12-week-old, mdx muscle (E) and 12-week-old IFNγ^−/−/mdx muscle (F) showing large CD206+ macrophages (red) within a region of muscle regeneration. All micrographs are shown at the same magnification and all sections were labeled under identical conditions. Bars = 50 μm.

Figure 7

IFNγ inhibits proliferation and differentiation of myogenic cells. The IFNγ receptor was expressed on the surfaces of muscle cells at higher levels in mdx mice at 4-weeks (B) and 12-weeks of age (D) compared to age-matched, wild-type controls (A and C). E: Flow cytometric analysis of muscle cell proliferation showed that IFNγ inhibits muscle cell proliferation of GFP-expressing C₂C₁₂ cells. C₂C₁₂ cells that did not express GFP and were not labeled with CellVue Claret (left panel in E) were used to set quadrant markers. F: Western blots of muscle cells treated with IFNγ or media only (“Control”) in vitro show that IFNγ reduces the level of myogenin expression without affecting the level of MyoD expression. G. Phase contrast images of C₂C₁₂ cultures show myotube growth is inhibited by IFNγ. IFNγ was added at the indicated concentrations following induction of cell differentiation and then muscle cells were incubated for 5-days to allow muscle cell differentiation and myotube growth. Bar = 160 μm. H, I: Relative transcript levels of MyoD and myogenin in whole muscle extracts were measured by qPCR. Each bar represents the mean and sem for the qPCR of RNA isolated from the muscles collected from 5 mice in each data set. H: MyoD expression in mdx muscle declined between 4 and 12-weeks-of-age. However, muscle from IFNγ^−/−/mdx mice showed no change in MyoD expression between these ages. I: Muscles from IFNγ^−/−/mdx mice showed an insignificant trend for higher levels of myogenin expression compared to mdx mice. J: The proportion of central-nucleated, regenerating fibers was counted and expressed as a percentage of total fibers counted in entire cross sections of soleus muscles. Each bar represents the mean and sem for the central-nucleated fiber counts from muscles collected from 5 mice in each data set. The proportion of regenerating fibers increased at 12 weeks, but no difference was observed between mdx and IFNγ^−/−/mdx mice at either 4 or 12 weeks of age. # indicates significantly different from 4-week-old mdx at p < 0.01. § indicates significantly different from 12-week-old mdx at p < 0.01. * indicates significantly different from 4-week old mice of same genotype at p < 0.05.

Figure 8

Null mutation of IFNγ in mdx mice improves muscle function in 12-week-old mice. A: The mean maximum run time is shown for mice that ran uphill on a treadmill at a speed of 8 m/min at a 5° incline. Each sample is a separate mouse for each genotype and age-group: 4-week-old mdx (n = 19), 4-week old IFNγ^−/−/mdx (n = 18), 12-week-old mdx (n = 15), 12-week old IFNγ^−/−/mdx (n = 20). Error bars = sem. B: Muscle function was assessed using the wire hang test to measure front paw grip strength. Each sample is a separate mouse for each genotype and age-group: 4-week-old mdx (n = 20), 4-week old IFNγ^−/−/mdx (n = 20), 12-week-old mdx (n = 20), 12-week old IFNγ^−/−/mdx (n = 20). * indicates significantly different from 12-week-old mdx at p < 0.05. # indicates significantly different from 4-week-old mdx at p < 0.01.