Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors

Journal name:
Nature Medicine
Volume:
21,
Pages:
786–794
Year published:
DOI:
doi:10.1038/nm.3869
Received
Accepted
Published online

Abstract

Depending on the inflammatory milieu, injury can result either in a tissue's complete regeneration or in its degeneration and fibrosis, the latter of which could potentially lead to permanent organ failure. Yet how inflammatory cells regulate matrix-producing cells involved in the reparative process is unknown. Here we show that in acutely damaged skeletal muscle, sequential interactions between multipotent mesenchymal progenitors and infiltrating inflammatory cells determine the outcome of the reparative process. We found that infiltrating inflammatory macrophages, through their expression of tumor necrosis factor (TNF), directly induce apoptosis of fibro/adipogenic progenitors (FAPs). In states of chronic damage, however, such as those in mdx mice, macrophages express high levels of transforming growth factor β1 (TGF-β1), which prevents the apoptosis of FAPs and induces their differentiation into matrix-producing cells. Treatment with nilotinib, a kinase inhibitor with proposed anti-fibrotic activity, can block the effect of TGF-β1 and reduce muscle fibrosis in mdx mice. Our findings reveal an unexpected anti-fibrotic role of TNF and suggest that disruption of the precisely timed progression from a TNF-rich to a TGF-β−rich environment favors fibrotic degeneration of the muscle during chronic injury.

At a glance

Figures

  1. After their damage-induced expansion, excess FAPs undergo apoptosis.
    Figure 1: After their damage-induced expansion, excess FAPs undergo apoptosis.

    (a) Quantification of the total number of FAPs per TA muscle in WT mice after NTX damage. *P < 0.05; one-way analysis of variance (ANOVA); n = 4. The gating strategy is described in Supplementary Figure 1a. (b) Representative immunofluorescence images of TA sections from PDGFR-α–H2B::eGFP mice stained for laminin and nuclei (TOTO3). Day 0, n = 8 sections; day 3, n = 12 sections; day 7, n = 9 sections. Scale bar, 10 μm. (c) Flow cytometry plots (left) and quantification (right) of TUNEL-positive FAPs at the indicated time points following damage. *P < 0.05; one-way ANOVA; n = 3. (d) TUNEL assay performed on TA muscle sections from PDGFR-α–H2B::eGFP mice 5 d after NTX injection. Arrowheads point to TUNEL-positive nuclei. Scale bar, 10 μm. Five sections were analyzed. All experiments were performed at least twice; Error bars, mean ± s.d.

  2. FAP clearance is impaired during skeletal muscle regeneration in Ccr2-/- mice.
    Figure 2: FAP clearance is impaired during skeletal muscle regeneration in Ccr2−/− mice.

    (a) Quantification by flow cytometry of the total number of FAPs per TA muscle in Ccr2−/− mice at the indicated time points after NTX injection. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus WT; two-way ANOVA; n = 3–4 per time point. Data from WT mice (from Fig. 1a, gray dotted line) is shown for comparison. (b) Representative images of TA muscle sections from PDGFR-α–H2B::eGFP (WT) and PDGFR::H2B-eGFP/Ccr2−/− mice at the indicated time points following damage. n = 5 at day 0, 8 at day 7 and 6 at day 10 for PDGFR-α–H2B::eGFP mice; n = 7 sections at day 0, 5 at day 7 and 8 at day 10 for PDGFR::H2B-eGFP/Ccr2−/− mice. Scale bar, 20 μm. (c) Quantification of Casp3 activity in TA muscles from WT and Ccr2−/− mice measured by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA; n = 3–4 per time point. The complete gating strategy is described in Supplementary Figure 1b. (d) qPCR analysis of genes associated with matrix deposition in FAPs sorted from TA muscles from WT and Ccr2−/− mice at day 7 p.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. two-way ANOVA. All experiments were performed at least two times; Error bars, mean ± s.d.

  3. Macrophages induce FAP death via TNF signaling.
    Figure 3: Macrophages induce FAP death via TNF signaling.

    (a) Effect of TNF on FAP survival in vitro. FAPs were sorted from the TA muscles of PDGFR-α–H2B::eGFP mice at day 3 p.d. and incubated with 100 ng/ml TNF for 48 h in the presence or absence of either a monoclonal rat antibody against TNF or etanercept. Control, untreated. Scale bar, 10 μm. ***P < 0.001, ****P < 0.0001; one-way ANOVA, n = 4 wells per treatment. (b) Detection of TNF in CD45+F4/80+ cells from WT and Ccr2−/− mice during the FAP clearance phase. **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA, n = 3. (c) Representative images (n = 4 at day 0, 7 at day 3) showing immunofluorescence of TNF-expressing macrophages and FAPs in damaged muscle. Sections stained for TNF and the macrophage marker CD68. Arrowheads indicate points of contact between FAPs and TNF-expressing macrophages. Scale bars, 7 μm for lower-magnification images and 10 μm for higher-magnification images. (d) Representative images (left, n = 12 for all groups) and quantification of apoptosis (right) in co-cultures of FAPs from PDGFR-α–H2B::eGFP mice and Ly6Chigh inflammatory macrophages. Ctl, control represents FAP cultured in the absence of macrophages. *P < 0.05, ***P < 0.001; one-way ANOVA; n = 4 wells per time point. Scale bar, 10 μm. (e) Quantification of FAP apoptosis under the indicated tissue culture conditions. ***P < 0.001, ****P < 0.0001; one-way ANOVA; n = 4 wells per treatment. (f) Representative images (n = 6) of activated Casp3 in FAPs after 30 h of co-culture. Scale bar, 5 μm. All experiments were performed at least two times. Error bars, mean ± s.d.

  4. TNF blockade leads to increased FAP survival and collagen deposition.
    Figure 4: TNF blockade leads to increased FAP survival and collagen deposition.

    (a) Top, a schematic showing the experimental setup: anti-TNF antibody was delivered on days 3–6 p.d. and the TAs were collected on day 7 p.d. Bottom, flow cytometry analysis (left) and its quantification (right) of the total number of FAPs in damaged TA muscles of mice treated with anti-TNF antibody. SSC, side scatter. *P < 0.05; two-tailed Student's t-test; n = 4. (b) Frequency of apoptotic (Casp3+) FAPs 5 d after damage to the TA muscles of mice treated with anti-TNF antibody. ***P < 0.001, ****P < 0.0001; one-way ANOVA; n = 4. (c) Top, a schematic representation of the experimental setup: anti-TNF antibody was delivered on days 3–6 p.d. and the TAs were collected on day 10 p.d. Middle and bottom, collagen expression (top) and deposition (bottom) during tissue regeneration following TNF blockade in Col1a1*3.6-eGFP transgenic mice. Scale bars, 20 μm for middle images and 10 μm for bottom images. (d) Representative images (n = 16 for both groups) of Picrosirius red staining (left) and quantification (right) of TA muscle sections from WT mice treated with either vehicle or anti-TNF antibody and killed on day 14 p.d. **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA; n = 4. Scale bar, 200 μm. (e) Quantification of the total number of FAPs in the damaged TA muscles of WT and LysM-Cre/TNFfl/fl mice 7 d after damage. **P < 0.01, ***P < 0.001; two-way ANOVA; n = 5. (f) Representative images (n = 16 for both groups), of Picrosirius red staining (left) and quantitation (right) of TA muscles of WT and LysM-Cre/TNFfl/fl mice at day 21 p.d. *P < 0.05; two-tailed Student's t-test. Scale bar, 50 μm. All experiments were performed at least two times; Error bars, mean ± s.d.

  5. Tgf-[beta]1 expression during muscle regeneration.
    Figure 5: Tgf-β1 expression during muscle regeneration.

    (a) Tgfb1 mRNA expression in specific macrophage subsets purified from damaged muscle at different time points. *P < 0.05, **P < 0.01, ***P < 0.001; Tukey's multiple comparison test. n = 6 at day 1 and 3, n = 4 at day 2 and 5, n = 2 at day 7. Hprt, hypoxanthine-guanine phosphoribosyltransferase. (b) Detection of the macrophage phenotype switch by flow cytometry. (c) Representative immunofluorescence images (n = 6 at day 0 and 5, n = 9 at day 2) depicting mature TGF-β1 and the macrophage marker CD68 in the TA muscles of PDGFR-α–H2B::eGFP mice at the indicated time points after damage. Scale bar, 5 μm. (d) Detection of TGF-β1 precursors in whole muscle extracts at the indicated days p.d. GADPH was used as a loading control. (e) Detection of mature TGF-β1 by ELISA in whole TA muscle extracts from WT mice. n = 3. All experiments were performed at least two times. (f) Cell counts of sorted FAPs after culturing with the indicated doses of TNF (left), or in the presence of a lethal concentration of TNF (0.1 μg/ml) and the indicated doses of TGF-β1 (right). n = 3 wells. (g) Top, a schematic representation of the experimental setup: TGF-β1 was delivered on day 3 and 4 p.d. and the TAs were collected on day 7 p.d. Bottom, quantification of FAPs after intramuscular TGF-β1 delivery at days 3 and 4 p.d. *P < 0.05; two-tailed Student's t-test; n = 4. (h) A schematic representation of the experimental setup: Alk5i was delivered on days 3–6 p.d. and the TAs were collected on day 7 p.d. Flow cytometry and quantification (below) of FAPs after treatment with Alk5i (10 mg/kg/d). *P < 0.05, determined by two-tailed Student's t-test; n = 3. (i) Top, a schematic representation of the experimental setup: Alk5i was delivered on day 3 and 4 p.d. and the TAs were collected on day 5 p.d. Bottom, flow cytometry (left) and quantification (right) of FAP apoptosis following Alk5i treatment. **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA; n = 5. Error bars, mean ± s.d.

  6. Inhibition of Tgf-[beta]1 signaling by nilotinib restores FAP apoptosis in mdx mice.
    Figure 6: Inhibition of Tgf-β1 signaling by nilotinib restores FAP apoptosis in mdx mice.

    (a) Effect of nilotinib (nilo; 0.5 μM) on TGF-β1–induced p38 MAPK phosphorylation (p-p38) and Smad3 phosphorylation (p-Smad3) in cultured purified FAPs. (b) Effect of nilotinib (0.5 μM) on FAP survival in vitro. ****P < 0.0001, one-way ANOVA; n = 4 wells. (c) Effect of nilotinib (20 mg/kg/d i.p.) on FAP apoptosis at day 5 p.d. in PDGFR-α–H2B::eGFP mice. *P < 0.05, two-tailed Student's t-test; n = 4. Scale bar, 30 μm; inset, 10 μm. (d) Quantification of FAPs at day 7 p.d. in the TA muscles of WT mice treated with nilotinib. *P < 0.05; two-tailed Student's t-test; n = 3. (e) Analysis of the macrophage population composition in skeletal muscles from aged mdx mice. (f) Single-cell PCR analysis of TNF and Tgfb1 mRNAs on CD45+Ly6GCD11b+ cells from diaphragm muscle of an mdx mouse. Percentages of cells that express either one of the cytokines or both are indicated. (g) Representative images (n = 4 for both groups) (left) and quantification (right) of apoptosis in PDGFR-α–H2B::eGFP FAPs co-cultured with macrophages (Mac) sorted from muscles from aged mdx mice in the presence or absence of nilotinib. Scale bar, 10 μm. ***P < 0.001; one-way ANOVA; n = 4 wells per treatment. (h) Top, a schematic representation of the experimental setup: daily microdamage was inflicted for 2 weeks and TAs were dissected 1 week after the end of the microdamage protocol. Bottom, a composition analysis of macrophages in microdamaged muscles from adult mdx mice. **P < 0.01; one-way ANOVA; n = 4. (i) Top, a schematic representation of the experimental setup: the 2-week microdamage protocol was followed by a third week in which nilotinib was delivered on a daily basis. TAs were dissected at the end of week 3. Bottom, representative images (n = 3 for no damage (ND) and microdamage, n = 4 for microdamage + nilotinib) of FAP apoptosis in microdamaged muscles of mdx mice treated with nilotinib. Scale bar, 10 μm. (j) Top, a schematic representation of the experimental setup: same as in i, except TAs were dissected 1 week after the end of the nilotinib treatment. Bottom, flow cytometry (left) and its quantification (middle) of FAPs, as well as representative images (right, n = 3 for ND, 4 for microdamage, 5 for microdamage + nilotinib) of collagen deposition in muscles from mdx mice 2 weeks after the end of the microdamage protocol. A group of mice received daily nilotinib treatment as indicated in i. Percentages indicate the fraction of red (collagen-positive) pixels. **P < 0.01, ****P < 0.0001; two-way ANOVA and one-way ANOVA (mdx samples). n = 3 or 4 per data point. Error bars, mean ± s.d.

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Author information

Affiliations

  1. Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

    • Dario R Lemos,
    • Farshad Babaeijandaghi,
    • Marcela Low,
    • Chih-Kai Chang,
    • Sunny T Lee,
    • Regan-Heng Zhang,
    • Anuradha Natarajan &
    • Fabio M V Rossi
  2. Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada.

    • Dario R Lemos,
    • Farshad Babaeijandaghi,
    • Marcela Low,
    • Chih-Kai Chang,
    • Regan-Heng Zhang,
    • Anuradha Natarajan &
    • Fabio M V Rossi
  3. Department of Experimental Medicine, Section of Medical Pathophysiology Food Science and Endocrinology, Sapienza University of Rome, Rome, Italy.

    • Daniela Fiore
  4. Engelhardt Institute of Molecular Biology, Moscow, Russia.

    • Sergei A Nedospasov
  5. Lomonosov Moscow State University, Moscow, Russia.

    • Sergei A Nedospasov
  6. German Rheumatism Research Center, Berlin, Germany.

    • Sergei A Nedospasov

Contributions

D.R.L. designed, directed and carried out experiments; analyzed data; and wrote the manuscript. F.B., M.L., C.-K.C., S.T.L., D.F., R.-H.Z. and A.N. carried out experiments and analyzed data. S.A.N. provided important advice on experimental design. F.M.V.R. designed experiments, directed the project and wrote the manuscript.

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The authors declare no competing financial interests.

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