Denervation-activated STAT3–IL-6 signalling in fibro-adipogenic progenitors promotes myofibres atrophy and fibrosis

Abstract

Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3–IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3–IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SODG93A mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases.

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Fig. 1: Abnormal accumulation of FAPs during muscle atrophy by acute denervation.
Fig. 2: FAPs from denervated muscles exhibit distinct functional properties and gene expression profiles.
Fig. 3: Persistent activation of IL-6–STAT3 pathway in FAPs and myofibres from denervated muscles and prevention of myofibre atrophy and fibrosis by IL-6 blockade.
Fig. 4: STAT3 inhibition prevents the atrophic effect of denervation-derived FAPs.
Fig. 5: Selective STAT3 inhibition in FAPs is sufficient to counter myofibre atrophy.
Fig. 6: FAPs with aberrant IL-6–STAT3 activation accumulate in skeletal muscles from SCI.
Fig. 7: FAPs with aberrant IL-6–STAT3 activation accumulate in ALS skeletal muscles.

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Acknowledgements

This work was supported by the Italian Ministry of Health (grant no. GR-2013-02356592) to L.M., NIH grants R01AR056712, R01AR052779, P30 AR061303, an MDA grant and EPIGEN grant to P.L.P., NIH grants R01 AR064873 and P30 AR061303 and MDA grant to A.S., California Institute for Regenerative Medicine (CIRM) training grant TG2-01162 and AFM-Téléthon Postdoctoral fellowship (no. 21084) to D.S. The authors thank E. Aleo at the Institute of Applied Genomics in Udine, Italy, for RNA–seq library preparation and sequencing, L. Battistini and G. Borsellino for flow cytometry related discussions and advice, L. Berghella for preliminary analysis, and R. Rizzi for support in in vivo treatments. The authors also thank D. Guttridge, S. Akira and S. Schenk for sharing mice generated in their laboratories, and C. Heil for help with graphic formatting. D.S. and U.E. contributed equally to this work.

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Project conception and design was carried out by L.M. and P.L.P., denervation procedures and related experiments in vivo and ex vivo, data generation and analysis by L.M., M.P., F.L., D.P. and M.V.A., RNA–seq data analysis and statistics by C.N. and S.G., experiments with STAT3 fl/fl mice, ex vivo STAT3 deletion/blockade and related transplantation assays by D.S., U.E., L.M. and A.S., flow cytometry by L.M. and M.d.B., human ALS sample sourcing by R.R.-G., CD90 expression analysis in mouse and human FAPs by L.G. and L.M., experiments with SODG93A mice and ALS sample analysis by L.M. and M.P., experiments with SCI by S.M. and L.M., and experiments with SMA by V.P., C.S. and L.M. The manuscript was written by P.L.P., and funding was sourced by P.L.P. and L.M.

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Correspondence to Pier Lorenzo Puri.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Collagen deposition but not regeneration characterize denervated muscles.

(a) Representative immunofluorescence of Collagen1 (Green) in TA muscle cryosection. Collagen area values represent mean ± s.d. **P < 0.01; by one-way ANOVA (n=3 animals/group), 3 independent experiments were performed. Scale bar 100 µm. (b) Representative immunofluorescence of embryonic Myosin Heavy Chain (eMyHC) (Red) in TA muscle cryosections. eMyHC-positive fibers were detected at 7days post-injury only. Scale bar 200 µm. Data shown represent results from 3 animals/group, 3 independent experiments were performed. (c) General gating strategy is shown (d) Representative EdU (Red), CD90 (Green), Laminin (Cyan) and Dapi (White) immunofluorescence in muscle cryosections of control (Ct) and 15 days denervated (Den) TA muscle. Quantification is shown in the graph (left panel). Values represent mean ± s.d. **P<0.01; by student t-test (n=3 animals/group), 3 independent experiments were performed.

Supplementary Figure 2 Increased number of FAPs but not Satellite Cells and Macrophages in denervated muscles.

(a) Pax7 (Red) immunofluorescence in control, injured and denervated TA muscle cryosections at the indicated time-point. Laminin (green) and Dapi (white) counterstain is shown in the bottom merged panel. (b) F4/80 (Green) immunofluorescence analysis is shown in control (CTR), injured (CTX) and denervated (DEN) TA muscle cryosections at the indicated time-points. Laminin (Cyan) and Dapi (white) counterstain is shown in the bottom merged panel. (c) Sca-1 (Red) immunofluorescence analysis is shown in control (CTR), injured (CTX) and denervated (DEN) TA muscle cryosections at the indicated time-points. Laminin (Cyan) and Dapi (white) counterstain is shown in the bottom merged panel. In a-c, Scale bar 100 µm. Data shown in a-c represent results from 3 animals/group, 3 independent experiments were performed. Quantification was shown in (d-e-f). Values represent mean ±s.d. *P<0.05, **P<0.01; by one-way ANOVA (n=3 animals/group).

Supplementary Figure 3 Pathway analysis of RNA-seq from FAP DEN and FAP CTX.

(a) MA-plots showing the fold change and normalized counts for FAPs from denervated (DEN 7 and 15 days) and injured (CTX) muscles, compared to control mice. Data show results from the mean of 2 animals/group (b) Top enriched pathways identified with IPA software, for differentially expressed genes unique to injury (CTX; left), denervation day 15 (DEN15d; right) or common to the two conditions (down). For each plot, x-axis represents the –log of the P-value (right-tailed Fisher’s exact test) and the orange dots on each pathway bar correspond to the ratio of the number of genes in a given pathway that meets the cutoff criteria, divided by the total number of genes belonging to that pathway.

Supplementary Figure 4 RNA-seq analysis from denervated myofibres vs control.

(a) MA-plots showing the fold change and normalized counts for myofibers from 7 days denervated (DEN) muscles compared to control myofibers. Data show results from the mean of 2 animals/group (b) Expression heatmap of genes differentially expressed in myofibers derived from denervated (DEN) muscle compared to myofibers derived from Control (CTR) mice. Gene expression is represented as z-score calculated across the rows. Data show results from the mean of 2 animals/group (c) Upstream Regulators predicted to be differentially activated or inhibited in myofibers from denervated (DEN) myofibers compared to control (CTR) myofibers, by IPA analysis. (d) Top Canonical Pathways altered in myofibers from 7 days Denervated (DEN) muscles, compared to control myofibers. For each plot, x-axis represents the –log of the P-value (right-tailed Fisher’s exact test) and the orange dots on each pathway bar correspond to the ratio of the number of genes in a given pathway that meet the cutoff criteria, divided by the total number of genes belonging to that pathway. (e) Top Metabolic Pathways altered in Myofibers from 7 days Denervated (DEN) muscle compared to control myofibers (see Supp.Fig.3b for further details). For each plot, x-axis represents the –log of the P-value (right-tailed Fisher’s exact test) and the orange dots on each pathway bar correspond to the ratio of the number of genes in a given pathway that meets the cutoff criteria, divided by the total number of genes belonging to that pathway.

Supplementary Figure 5 Myofiber- or satellite cell-specific genetic deletion of STAT3 does not prevent denervation induced muscle atrophy.

(a) Representative immunofluorescence of Laminin (Red) in TA sections from WT, MCK-STAT3-/-, Denervated (WT Den) and MCK_STAT3-/- mice, either unperturbed or 15 days after denervation. (n=4 WT and 5 KO animals per group, from two independent experiments). Scale bar 100 µm (b) Normalized TA muscle weight. (n=4 WT and 5 KO animals per group, from two independent experiments). (c) Frequency distribution of fiber CSA in denervated (WT-DEN) and MCK-STAT3-/- TA muscles. Values represent mean ± S.E.M. (n=4 WT and 5 KO animals per group, from two independent experiments). (d) Western Blot analysis of phopsho- and total-STAT3 with whole muscle extracts. (e) Representative Western Blot of total STAT3, from extract of muscle fibers. (2 independent experiments were performed) (f) Representative immunofluorescence of Laminin (Red) in TA muscle from WT and Pax7-STAT3-/-, mice, either unperturbed or 15 days after denervation. (n=4–5 animals per group, two independent experiments). Scale bar 100 µm (g) Frequency distribution of fiber CSA in WT and Pax7-STAT3-/-, mice, either unperturbed or 15 days after denervation. Values represent mean ± S.E.M. (n=4 WT and 5 KO animals per group, from two independent experiments). (h) Left panel: Western Blot analysis of phosphor-STAT3 from protein extracts of freshly isolated FAPs pooled from from denervated TA muscles of mice (n=3 animals) treated or not with aIL6 neutralizing antibodies or STAT3i. Right panel: Summary of ELISA quantification of secreted IL-6 by FAPs from denervated TA muscles of mice treated or not with aIL6 neutralizing antibodies or STAT3i. Values represent mean of two independent assays.

Supplementary Figure 6 Alteration of FAPs during aging and in a mouse model of SMA.

(a) Frequency distribution of fiber CSA in TA of 2month (Young) and ~ 24 month-old mice (Old). Values represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001; by two side student t-test (n=4 animals/group). (b) Representative Sirius red staining in Young and Old muscle cryosections. Scale bar 200 μm. (c) Quantification of collagen staining shown in j. Values represent mean ± s.d., **P = 0.006; by two side student t-test (n=4 animals/group). (d) Cyto-fluorimetric count of FAPs in Young and Old mice. Values represent mean ± s.d. (n=3 CTR and n=4 Old animals) (e) Relative expression of indicated genes in freshly isolated FAPs from Young or Old mice. Values represent mean ± s.d. (n=3 CTR and n=4 Old animals) (f) Representative images of 9-day old WT and SMAD7 mice. (g) Muscle weight of 9-day old WT and SMAΔ7 mice. Values represent mean ± s.d. ***P = 0.0001; by two side student t-test (n=5 animals). (h) Cyto-fluorimetric counts of FAPs from 9-day old WT or SMAΔ7 mice. Values represent mean ± s.d., ***P = 0.0001; by two side student t-test. (n=4 animals/group) (i) FAP’s number normalization over muscle weight. Values represent mean ± s.d., **P = 0.001; by two side student t-test. (n=4 animals/group) (j) qPCR analysis of indicated genes in WT and SMAΔ7 freshly isolated FAPs. Values represent mean ± s.d. *P / 0.05; by two side student t-test (n=4 animals/group).

Supplementary Figure 7 Activation of STAT3 in CD90 positive FAPs from human ALS muscles.

(a) Graph showing the Cytofluorimetric profile of surface markers, CD90 and SCA1, expressed in FAPs derived from WT mice. Data shown represent 2 independent experiments) (b) Relative expression of the indicated genes in SC, FAPs Sca1 and FAPs CD90 isolated from WT muscle by qPCR. (n=2 independent experiments) (c) Representative Images of Laminin (Cyan), DAPI (White), STAT3tyr705(Green) and CD90 (purple) immunolocalization in ALS muscle cryosection from SODG93A. Arrows indicate STAT3tyr705 myonuclei while stars indicate STAT3tyr705 positive CD90 interstitial cells. Data shown represent 3 human samples.

Supplementary Figure 8 Unprocessed scan of blots: Part1.

Unprocessed scan of blots was shown.

Supplementary Figure 8 Unprocessed scan of blots: Part2.

Unprocessed scan of blots was shown.

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Madaro, L., Passafaro, M., Sala, D. et al. Denervation-activated STAT3–IL-6 signalling in fibro-adipogenic progenitors promotes myofibres atrophy and fibrosis. Nat Cell Biol 20, 917–927 (2018). https://doi.org/10.1038/s41556-018-0151-y

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