Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype - PubMed (original) (raw)

. 2011 Feb 15;20(4):790-805.

doi: 10.1093/hmg/ddq523. Epub 2010 Nov 30.

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Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype

S Armando Villalta et al. Hum Mol Genet. 2011.

Abstract

M1 macrophages play a major role in worsening muscle injury in the mdx mouse model of Duchenne muscular dystrophy. However, mdx muscle also contains M2c macrophages that can promote tissue repair, indicating that factors regulating the balance between M1 and M2c phenotypes could influence the severity of the disease. Because interleukin-10 (IL-10) modulates macrophage activation in vitro and its expression is elevated in mdx muscles, we tested whether IL-10 influenced the macrophage phenotype in mdx muscle and whether changes in IL-10 expression affected the pathology of muscular dystrophy. Ablation of IL-10 expression in mdx mice increased muscle damage in vivo and reduced mouse strength. Treating mdx muscle macrophages with IL-10 reduced activation of the M1 phenotype, assessed by iNOS expression, and macrophages from IL-10 null mutant mice were more cytolytic than macrophages isolated from wild-type mice. Our data also showed that muscle cells in mdx muscle expressed the IL-10 receptor, suggesting that IL-10 could have direct effects on muscle cells. We assayed whether ablation of IL-10 in mdx mice affected satellite cell numbers, using Pax7 expression as an index, but found no effect. However, IL-10 mutation significantly increased myogenin expression in vivo during the acute and the regenerative phase of mdx pathology. Together, the results show that IL-10 plays a significant regulatory role in muscular dystrophy that may be caused by reducing M1 macrophage activation and cytotoxicity, increasing M2c macrophage activation and modulating muscle differentiation.

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Figures

Figure 1.

Figure 1.

IL-10-mediated signaling between macrophages and muscle cells is enhanced in muscular dystrophy. (A) Inflamed lesions in 4-week-old mdx muscle contain M1 macrophages, M2 macrophages and satellite cells. Cross-section of quadriceps muscle was labeled with anti-F4/80 (red), which binds all macrophage phenotypes, anti-CD206 (green), which binds M2 macrophages and satellite cells, and DAPI reagent (blue), which binds DNA to show the position of nuclei. M1 macrophages are CD206−/F4/80+ (red). M2 macrophages are CD206+/F4/80+ (orange). Satellite cells are CD206+/F4/80− (green). Bar = 50 µm. (B) Quantitative, real-time PCR results for relative mRNA levels for IL-10 in quadriceps muscles of 4-week-old or 12-week-old wild-type or mdx mice. Expression levels are relative to 4-week-old wild-type muscles, for which the expression level is set at one unit. Asterisks indicate significantly different from 4-week-old wild-type quadriceps at P< 0.001. Hash symbol indicates significantly different from 4-week-old quadriceps from mice that are the same genotype at P< 0.001. Each experimental group included quadriceps from five mice. Error bars are too small to appear in the figure. (C) Quantitative, real-time PCR results for relative mRNA levels for IL-10R1 in quadriceps muscles of 4-week-old or 12-week-old mdx mice. Expression levels are relative to 4-week-old muscles, for which the expression level is set at one unit. Asterisks indicate significantly different from 4-week-old quadriceps at P< 0.05. Each experimental group included quadriceps from five mice. Bars represent sem. (D–G) Cross-sections of quadriceps muscle immunolabeled for IL-10R1 or antibody control preparations. Bars = 50 µm. (D) Section of 4-week-old mdx muscle showing an inflamed lesion containing cells expressing IL-10R1 (red). (E) Section of 4-week-old mdx muscle showing regenerative muscle fibers (labeled 1 or 2) that express IL-10R1 (red). (F) Section adjacent to the section shown in (E). The section was incubated with anti-IL-10R1 from which immunoglobins specific for IL-10R1 were depleted from the antibody solution by incubation with mouse IL-10R1 prior to labeling the section. The fibers labeled 1 or 2 are the same regenerative fibers as those labeled 1 or 2 in (E). (G) Section from 4-week-old wild-type muscle immunolabeled for IL-10R1 using treatment conditions that were identical to those used to label the mdx muscle section shown in (E).

Figure 2.

Figure 2.

IL-10 deactivates M1 macrophages and reduces muscle damage in mdx mice. (A) Macrophages isolated from 4-week-old mdx mice were stimulated with a range of IL-10 concentrations (1–100 ng/ml) and lyzed after 24 h of stimulation. Macrophage lysates were separated by SDS–PAGE and relative levels of iNOS were assayed by western blotting. The membrane used for immunoblotting was stained with Ponceau red (‘Loading’) before application of the antibody to verify equal protein loading in each lane. (B) Macrophages purified from 4-week-old mdx muscles were stimulated with IL-10 (10 ng/ml) for 24 h prior to co-culturing with C2C12 myotubes. After 24 h of co-culture, cell lysis was decreased 26% by IL-10. Asterisks indicate P< 0.05 compared with non-stimulated control. (C and D) Macrophages isolated from 4-week-old (C) or 12-week-old (D) IL-10−/−/mdx mice displayed increased cytotoxic potential compared with IL-10+/+/mdx controls (30–50% increase in IL-10−/−/mdx versus IL-10+/+/mdx). Asterisks indicates significant difference at P< 0.05 compared with IL-10+/+/mdx. (E) Quantification of the number of central-nucleated fibers in cross-sections of quadriceps muscles from 4-week-old or 12-week-old mdx mice that expressed IL-10 (+/+) or were IL-10 null mutants (−/−). Asterisk indicates significantly different from 4-week-old IL-10+/+/mdx quadriceps at P< 0.05. Hash symbol indicates significantly different from age-matched IL-10+/+/mdx mice quadriceps. Each experimental group included quadriceps from six mice. Bars represent sem. (F and G) Measurements of fluorescence intensity in muscle fibers of 4-week-old (F) and 12-week-old mice (G) indicated increases in myofiber injury in IL-10−/−/mdx mice compared with IL-10+/+/mdx controls. Blue, striped peaks represent data from IL-10+/+/mdx. Orange peaks represent data from IL-10−/−/mdx mice.

Figure 3.

Figure 3.

Null mutation of IL-10 reduces strength and endurance of mdx mice. Mouse strength was assessed in the wire-hang test (A) and endurance was measured by time-to-fatigue in treadmill running (B). Asterisk indicates significant difference at P< 0.05 compared with IL-10+/+/mdx at same age. Bars indicate sem.

Figure 4.

Figure 4.

IL-10 induces M2c activation of macrophages. (A) Mdx muscle macrophages were stimulated with a range of concentrations of IL-10 for 24 h. Western analysis showed that maximal induction of CD163 occurred in response to 10 ng/ml of IL-10. The membrane used for immunoblotting was stained with Ponceau red (‘Loading') to verify equal protein loading in each lane. (B) CD206 expression was measured by quantitative, real-time PCR in peritonial macrophages cultured for 24 h in presence of 10 ng/ml of IFN-γ or IL-10. Unstimulated macrophages were used as controls. Asterisk indicates significantly different from unstimulated cells, P< 0.001. Each experimental group included five plates containing 6×106 cells. (C–G) M2c macrophages are present in dystrophic muscle. Cross-sections of 4-week-old mdx quadriceps were labeled with antibodies against CD163 (green) and F4/80 (red) to determine the presence of M2c macrophages in dystrophic muscle. Nuclei were labeled with DAPI reagent (blue). (C) F4/80+ macrophage. Bar = 12 µm. (D) Macrophage shown in (C) double-labeled with anti-CD163. Bar = 12 µm. (E) Merged image of (C) and (D). Bar = 12 µm. (F) C57 muscle contains resident macrophages that express CD163. Bar = 15 µm. (G) M2c macrophages represent a minor population of macrophages present in inflamed lesions (arrow). Bar = 20 µm. (H) Quantitative immunohistochemistry shows that null mutation of IL-10 in mdx mice reduces CD163+ macrophages in 12-week-old IL-10−/−/mdx mice quadriceps muscles (striped bar; 10 mice analyzed) compared with IL-10+/+/mdx mice quadriceps (black bar; 11 mice analyzed), to levels that do not differ from wild-type mice (white bar; nine mice analyzed). Asterisks indicate significant difference from IL-10+/+/mdx at P< 0.05. Bars represent sem.

Figure 5.

Figure 5.

IL-10 induces phagocytosis by macrophages and clodronate depletion of phagocytes from mdx mice selectively reduces M2c macrophages in skeletal muscle. (A–D) Flow cytometric data showing that IL-10 stimulation of macrophages increases macrophage phagocytosis. Macrophages were untreated with exogenous cytokines (A), treated with IFN-γ (B), IL-4 (C) or IL-10 (D) prior to incubation with flourescent microspheres for phagocytosis. Cells were then analyzed by flow cytometry to determine the proportion of cells that contained fluorescent microspheres, as an index of phagocytosis. Neither IFN-γ (68.1%) or IL-4 (61.2%) increased the proportion of cells that were phagocytic, compared with untreated controls (69.4%). However, IL-10 treatments increased the proportion of cells that were phagocytic (79.5%). (E and F) Intraperitoneal injections of clodronate-containing liposomes significantly reduced the numbers of macrophages (E) and MHC-2-presenting cells in the quadriceps muscles of 4-week-old mdx mice. Each experimental group included muscles from six mice. Asterisks indicate a significant difference from PBS-treated controls. Bars represent sem. (G–J) Quantitative, real-time PCR data show that clodronate-mediated depletions of phagocytes did not cause a significant change in the expression of CD68 in the quadriceps muscles of 4-week-old mdx mice (G), but caused large, significant reductions in the expression of CD206 (H), CD163 (I) and IL-10 (J). Each experimental group included muscles from five mice. Asterisks indicate a significant difference from PBS-treated controls. ‘n.s.' indicates no significant effect of the clodronate treatment. Bars represent sem, which is too small to appear for graphs of some data sets.

Figure 6.

Figure 6.

IL-10 modulates muscle cell differentiation in vitro and in vivo. (A) Myoblasts were cultured without addition of exogenous factors (‘none') or in the presence of IL-10, LPS, LPS and IL-10 together or bFGF, and then assayed for cell number at the end of 3 days of stimulation. Neither IL-10, LPS or IL-10 combined with LPS affected proliferation, although bFGF, which was used as a positive control, increased cell number by nearly 50% under the same culture conditions. (B) Pre-treatment of macrophages with IFN-γ and TNF-α prior to co-culture with myoblasts decreased myoblast numbers at the end of a 3-day period of co-culture, compared with myoblasts cultured alone (‘none'). Pre-treatment of macrophages with IL-10 or IL-4 combined with IL-13 to induce M2c phenotype generated macrophages that promoted myoblast proliferation in co-cultures. n = 6 for each treatment group. Asterisks indicate significant difference from the ‘none' group at P< 0.05. Bars represent sem. (C) Quantitative, real-time PCR results for relative mRNA levels for Pax7 in quadriceps muscles of 4-week-old or 12-week-old mdx mice that expressed IL-10 (+/+) or were IL-10 null mutants (−/−). Expression levels are relative to 4-week-old IL-10+/+/mdx muscles, for which the expression level is set at one unit. Each experimental group included quadriceps from five mice. Bars represent sem. (D) Western blots of extracts of myoblast cultures collected 0–7 days after initiating cultures in the presence of IL-10 or addition of no exogenous cytokine (‘none'). Blots were probed with antibodies to MyoD or myogenin. The membrane used for immunoblotting was stained with Ponceau red (‘Loading') before application of the antibody to verify equal protein loading in each lane. (E and F) Quantitative, real-time PCR results for relative mRNA levels for MyoD (E) or myogenin (F) in quadriceps muscles of 4-week-old or 12-week-old mdx mice that expressed IL-10 (+/+) or were IL-10 null mutants (−/−). Expression levels are relative to 4-week-old IL-10+/+/mdx muscles, for which the expression level is set at one unit. Each experimental group included quadriceps from five mice. Hash symbol indicates significant difference from age-matched IL-10+/+/mdx muscles. Asterisks indicate significant difference from 4-week-old IL-10+/+/mdx muscles. Bars represent sem.

Figure 7.

Figure 7.

Model of the influence of IL-10 on macrophage phenotype shift and the progression of muscular dystrophy. The acute onset of pathology in mdx muscle prior to 4 weeks of age involves muscle inflammation, with a bias toward M1 macrophages (6,24). M1 macrophages contribute to oxidative stress and muscle fiber lysis through the production of iNOS-derived NO, and promote inflammation and myoblast proliferation through the production of Th1 cytokines (24). Elevated IL-10 production shifts the macrophage population toward an M2c phenotype and deactivates M1 macrophages, causing reductions in iNOS expression and attenuating muscle damage (present study). IL-10, possibly derived from CD163+ M2c macrophages, can also affect the differentiation of myogenic cells (present study). As mdx muscle reaches the later, progressive stage of the mdx pathology, arginase expression by M2 macrophages is increased (41), which may reflect a shift toward an M2a phenotype. Arginase activity can increase fibrosis in mdx muscle (41) by driving the production of ornithine and thereby increasing collagen deposition (–67). Green bar, healthy/regenerating muscular tissue; red bar, inflamed tissue; thin red arrow, stimulating cytokines; thick red arrow, macrophage-produced factors.

Figure 8.

Figure 8.

Selection of reference genes for real-time, quantitative RT–PCR. (A) The most stable reference genes within and between two different groups of muscle samples were determined using geNorm 3.5 software. The variability in the average expression (M) of each gene was set at 0.5. Between 4-week-old or 12-week-old mdx and C57 quadriceps, RNSP1 and SRP14 had the least variability of the nine genes tested. The expression of EEF1A was recorded as the most unstable relative to the other genes. (B) GeNorm was also used to determine the optimal number of genes required to calculate a reliable normalization factor that was applied to normalized each gene of interest. The chart reports the pairwise variation (V) between two sequential normalization factors (Vn/Vn+1). When the value of variation is below the selected threshold, the addition of additional reference genes is unnecessary for data normalization. The pairwise variation was analyzed starting from the normalization factor generated by the two most stable genes, RNSP1 and SRP14, and adding the following normalization factor generated by adding TPT1, and so on. The cut-off value suggested by Vandesomple et al. (59) was 0.15. Since we reported a very low variation between _V_2/3, we decided that using RNSP1 and SRP14 was sufficient to generate a reliable normalization factor. (C and D) To confirm the minimum number of genes to use, normalization factors generated by the two most stable genes in C57 (C) and mdx (D) groups were correlated with the normalization factors obtained using the five best genes resulting from picture (A). Correlation between three and five top normalization factors was also tested but not reported. Each point is the mean of five values. Pearson coefficient shows that there is a tight correlation between the two groups of normalization factors and that using two genes for normalization of C57 and mdx 4-week-old and 12-week-old quadriceps data is adequate.

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References

    1. Hoffman E.P., Brown R.H., Jr., Kunkel L.M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51:919–928. - PubMed
    1. Petrof B.J., Shrager J.B., Stedman H.H., Kelly A.M., Sweeney H.L. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc. Natl Acad. Sci. USA. 1993;90:3710–3714. - PMC - PubMed
    1. Tidball J.G., Wehling-Henricks M. Evolving therapeutic strategies for Duchenne muscular dystrophy: targeting downstream events. Pediatr. Res. 2004;56:831–841. - PubMed
    1. Gorospe J.R., Tharp M., Demitsu T., Hoffman E.P. Dystrophin-deficient myofibers are vulnerable to mast cell granule-induced necrosis. Neuromuscul. Disord. 1994;4:325–333. - PubMed
    1. Hodgetts S., Radley H., Davies M., Grounds M.D. Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFalpha function with Etanercept in mdx mice. Neuromuscul. Disord. 2006;16:591–602. - PubMed

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