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 (SOD^G93A 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.

Figure 1:. Abnormal accumulation of FAPs during muscle atrophy by acute denervation

(a) Hematoxylin-Eosin staining of control (CTR), injured (CTX) or denervated (DEN) TA muscle cryosections at the indicated time points. Scale bar 200 μm. Data shown represent 3 independent experiments, (b) Quantification of normalized TA muscle weight. Values represent mean ± s.d. **P = 0.001; by one-way ANOVA (n=3 animals/group) (c) Representative images of Sirius red staining of control (CTR), injured (CTX) and denervated (DEN) TA muscle cryosection. Scale bar 200 μm. Data shown represent 3 independent experiments) (d) Quantification of collagen staining shown in c. Values represent mean ± s.d. **P < 0.01; by one-way ANOVA (n=3 animals/group), (e) Frequency distribution of fiber Cross-Sectional Area (CSA) in injured (CTX) (left panel) and denervated (DEN) (right panel) TA muscles. Values represent mean ± s.d. (n=3 animals/group), (f) Cyto-fluorimetric analysis of indicated cell types in injured (CTX) or denervated (DEN) muscles at the indicated time points. Values represent mean ± s.d. **P < 0.01, *P<0.05; by one-way ANOVA (n=12 CTR, n=20 DEN15d, n=6 DEN3d, CTX7d, n=4 CTX3d, CTX15d, n=3 DEN7d, DEN30d, CTX30d; “n” refers to animal numbers).

Figure 2:. FAPs from denervated muscles exhibit distinct functional properties and gene expression profiles

(a) Experimental setting and MyHC (Green) and DAPI (White) immunofluorescence of C2C12 myoblasts cultured alone (CTR) or in co-culture with FAP NT, FAP CTX3d or FAP DEN15d. Scale bar 200 μm. Data shown represent 3 biological independent experiments. (b) Fusion index of C2C12 myoblasts as described in a. “<2”: less than 2 nuclei in MyHC− or in MyHC+ mononucleated cells; “2<5”: MyHC+ myotubes with 2 to 5 nuclei; “> 5”: MyHC+ myotubes with than 5 nuclei. Values represent mean ±s.d. *P=0.021 **P=0.007; by one-way ANOVA (n=6 biological independent experiments). (c) MyHC (Green) and DAPI (White) immunofluorescence of myotubes cultured for 72h alone (CTR) or co-cultured with FAP NT, FAP CTX3d and FAP DEN15d. Scale bar 200 μm. Data shown represent 3 independent experiments. (d) Quantification of C2C12 myotube diameter. Values represent mean ±s.d., *P<0.05; by one-way ANOVA (n=4 independent experiments. CTR, FAP NT, FAP DEN; n=3 FAP CTX. (e) Heatmap of DE genes in FAPs DEN7d and DEN15d, or FAPs CTX3d, as compared to FAPs NT. Gene expression is represented as z-score calculated across the rows. Data shown are from two RNA-seq experiments. (f) PCA from RNA-seq in FAPs NT, FAP CTX3d and FAP DEN7 and DEN 15). Data from two independent RNA-seq experiments. (g) Venn diagram of genes common or uniquely in FAPs CTX3d and FAPs DEN7d and DEN15d, compared with FAPs NT. Statistical method was Deseq2. (h) qPCR analysis of IL-6 transcripts in FAPs CTX or FAP DEN. Values represent mean ±s.d. *P=0.021 **P=0.075; by one-way ANOVA (n=4 biologically independent samples). (i) ELISA quantification of secreted IL-6 by MuSCs, FAPs and MPs from the described conditions. (n=2 biologically independent samples (j) Representative Immunoblot of phosphoSTAT3 in the described conditions out of 3 independent experiments. See Supplementary Table 1 for source data shown in panels b, I and d.

Figure 3:. Persistent activation of IL6-STAT3 pathway in FAPs and myofibers from denervated muscles and prevention of myofiber atrophy and fibrosis by IL6 blockade

(a) Caveolin-3 (red), phospho-STAT3 (green) and DAPI (white) immunofluorescence in TA muscles from CTR or DEN muscle. Scale bar 200 μm. Data shown represent 3 independent experiments. (b) Immunoblot of phospho-STAT3 in FAPs and MuSCs from CTR or DEN muscles. Data shown represent 2 independent experiments. (c) Caveolin-3 (red), phospho-STAT3 (green) and DAPI (white) immunofluorescence in single myofibers from CTR or DEN muscles. Scale bar 200 μm. Data shown represent data from 3 animals/group. (d) Representative Immunoblot of phospho-STAT3 from single myofiber out of 2 independent experiments. (e) MyHC (Green) and DAPI (White) immunofluorescence of myotubes co-cultured with FAPs DEN, treated or not with alL6 antibodies (72h). Scale bar 200 μm. (f) Quantification of myotube diameter. Values represent mean ±s.d.***P = 0.0002; by two side student t-test (n=4 biologically independent samples). (g) MyHC (Green) and DAPI (White) immunofluorescence of myotubes cultured with FAP DEN-conditioned media, with or without alL6 antibodies (72h). Scale bar 200 μm. (h) Quantification of myotube diameter. Values represent mean ± s.d. **P = 0.003; by two side student t-test. (n=6 supFAP DEN, n=3 supFAP DENalL6 independent samples) (i) Laminin (red) immunofluorescence from DEN or contralateral (CTL) TA muscles, treated or not with alL6 antibody. Scale bar 100 μm. Data from 4 animals/group. (j) Frequency distribution of fiber CSA in denervated (DEN) TA muscle treated or not with alL6 antibodies. Line graph represents relative frequencies as percentage (n=4 animals). Values represent mean ± s.d. *P < 0.05, **P < 0.01; by two side student t-test. (k) Normalized TA muscle weight in the indicated conditions. Values represent mean ±s.d. *P<0.05, **P<0.01; by one-way ANOVA (n=3 animals/group).(I) FAP quantification by FACS. Values represent mean n=2 animals. (m) Immunoblot of phospho-STAT3 from whole TA muscles. Data shown represent data from 4 animals/group. (n) Sirius-red of TA muscle from contralateral (CTL) or DEN muscle treated or not with alL6 antibody. Scale bar 100 μm. (o) Quantification of collagen area. Values represent mean ±s.d. **P=0.002; by one-way ANOVA (n=5 animals/group.

Figure 4:. STAT3 inhibition prevents the atrophic effect of denervation-derived FAPs:

(a) Immunofluorescence of Laminin (red) of TA muscle derived from DEN muscle with or without STAT3i treatment. Scale bar 100 μm. Data shown represent 4 independent experiments. (b) Frequency distribution of fiber Cross-Sectional Area (CSA) TA muscle. Values represent mean ±s.d. *P<0.05 **P<0.01 ***P<0.001; by one-way ANOVA (n=4 animals/group). (c) Normalized TA muscle weight for the indicated conditions. Values represent mean ± s.d. *P = 0.018; by two side student t-test (n=6 CTR, n=3 DEN, n=4 DEN STAT3i animals). (d) Representative Sirius-red stained DEN muscles with or without STAT3i treatment. Scale bar 100 μm. Data shown represent 3 independent experiments. (e) Quantification of collagen staining. Values represent mean ±s.d. *P=0.017; by student two side t-test (n=3 DEN15d n=5 DEN15d+STAT3i animals). (f) Immunofluorescence of MyHC (Green) and DAPI (White) of C2C12 myotubes co-cultured for 72h with FAP DEN15d pretreated or not with STAT3i for 24h. Scale bar 200 μm. (g) Quantification of myotubes diameter from experiments shown in f. Values represent mean ±s.d. **P=0.003; by student two side t-test (n=5 biologically independent samples). (h) Immunofluorescence of MyHC (Green) and DAPI (White) of C2C12 myotubes cultured alone (control) or with FAP-conditioned media with or without STAT3 inhibitor for 72h. Scale bar 200 μm. (i) Quantification of myotubes diameter from experiments shown in h. Values represent mean ± s.d.(n=6 supFAP DEN; n=3 supFAP DEN STAT3i biological independent samples).

Figure 5:. Selective STAT3 inhibition in FAPs is sufficient to counter myofiber atrophy:

FAPs were isolated from denervated muscles 15 days after transection of the sciatic nerve. Donor EGFP mice were used. (a) Representative Images of GFP (green), Laminin (red) immunofluorescence staining of muscle derived from mice transplanted with FAP DEN incubated for 3 hours with DMSO or STAT3 inhibitor ex vivo. Scale bar 100 μm. Data shown represent results from n=5 untreated, n=3 DMSO and STAT3i animals. (b-c) Mean CSA and frequency distribution of myofiber CSA from untreated mice or mice transplanted with FAP DEN incubated for 3 hours with DMSO or STAT3 inhibitor. Values represent mean ± S.E.M. **P<0.01 (p=0.008) vs DMSO; Two-tail student’s t-test (Data shown represent results from n=5 untreated, n=3 DMSO and STAT3i animals). (d) qPCR analysis of STAT3 transcripts in FAP DEN treated with the STAT3 inhibitor for 3 or 24 hours in vitro. Values represent mean. (n=2 technical replicates). (e-h) Donor R26R^TdTSTAT3^flox/floxTdT^f/f mice and control R26R^TdTSTAT3^+/+animals were used. (e) Representative Images of TdT (red), Laminin (green) immunofluorescence staining of muscle derived from mice transplanted with FAP DEN infected O/N with adenoviruses expressing Cre Recombinase. Scale bar 100 μm (f-g) Mean CSA (f) and frequency distribution (g) of myofiber CSA from untreated mice or mice transplanted with FAP DEN infected 0/N with adenoviruses expressing Cre Recombinase (KO) or Adeno control (CTR). Values represent mean ± S.E.M. **P<0.01 (p=0.003) vs CT; #P<0.05 (p=0.033) vs Untreated; Two-tail student’s t-test (n=5 animals/group). (h) qPCR analysis of STAT3 transcripts in FAP DEN 24 hours after infection. Values represent mean ±s.d. *P<0.05 (p=0.036) vs CT; Two-tail student’s t-test (n=2 technical replicates).

Figure 6:. FAPs with aberrant IL6-STAT3 activation accumulate in skeletal muscles from SCI

(a) Frequency distribution of fiber CSA in Quadriceps of control (CTR) or Spinal Cord Injury (SCI) animal model. Values represent mean ±s.d. *P<0.05, **P<0.01, ***P<0.001; by one-way ANOVA (n=3 CTR and n=5 SCI animals; two independent experiments). (b) Sirius-red staining of CTR or SCI muscles. Scale bar 200 μm. (n=3 CTR and n=5 SCI animals; Data shown represent 2 independent experiments). (c) Quantification of data in b. Values represent mean ±s.d., **P<0.01; by student t-test. (n=3 CTR and n=5 SCI animals)(d) Cyto-fluorimetric count of FAPs from CTR or 15days SCI mice. Values represent mean ±s.d., *P<0.05; by t-test. (n=3 CTR and n=5 SCI animals). (e) Immunofluorescence of Laminin (Green), DAPI (White) and CD90 (Red) in CTR and SCI muscle sections (n=3 CTR and n=5 SCI animals). (f) CD90-positive cells quantification. Values represent mean ±s.d., **P=0.003; by two side student t-test (n=3 CTR and n=5 SCI animals). (g) Immunoblot of phospho- and total-STAT3 from whole muscles (n=3 animals/group) of CTR or SCI mice. Samples are loaded on the same gel; intermediate time points were removed. Representative blot out of 2 independent experiments. (h) qPCR analysis of indicated genes in freshly isolated FAPs from WT and SCI. Values represent mean ±s.d. *P<0.05, **P<0.01; by t-test (n=3 CTR and n=4 SCI animals).

Figure 7:. FAPs with aberrant IL6-STAT3 activation accumulate in ALS skeletal muscles:

a) Hematoxylin-Eosin staining of TA muscles from WT and SOD^G93A mice. Scale bar 200 μm. Data represent 3 independent experiments. (b) Normalized TA muscle weight of WT and SOD^G93Amice. Values represent mean ±s.d. **P<0.01; by one-way ANOVA (n=4 animals for all experiments, but SOD140 n=10). (c) Mean CSA of fibers from WT and SOD^G93A mice. Values represent mean ±s.d. *P<0.05, **P<0.01; by one-way ANOVA (n=3 animals/group)(d) Frequency distribution of fiber CSA in WT and SOD^G93A mice. Values represent mean ±s.d. *P<0.05, **P<0.01, ***P<0.001; by one-way ANOVA (n=3 animals/group). (e) Sirius-red staining of TA muscles from WT and SOD^G93A mice. Scale bar 200 μm. Data represent results from 3 independent experiment. (f) Quantification of data in e. Values represent mean ±s.d. *P<0.05, **P<0.01; by one-way ANOVA. (n=3 animals/group). (g) FACS quantification of FAPs from WT and SOD^g93a mice. Values represent mean ±s.d. *P<0.05, **P <0.01; by one-way ANOVA. (n=4 WT90d, n=5 SOD90d, n=6 WT140d and SOD140d animals). (h) qPCR analysis of IL6 transcripts in FAPs from WT and SOD^G93A muscle. Values represent mean ±s.d. **P<0.01; by one-way ANOVA (n=3 WT n=4 SOD animals). (i) Immunoblot of phospho-STAT3 from TA muscles. Data shown represent 2 independent experiments. (j) Immunofluorescence of Laminin (Red), DAPI (White) and phospho-STAT3 (Green) in TA sections from SOD^G93A mice. Scale bar 200 μm. (k) Masson-Trichrome staining in control or 3 ALS patient muscles. (3 section/condition analyzed). Scale bar200 μm(I) Quantification of data in k. Values represent mean ±s.d. ***P<0.0007; by two side student t-test. (n=3 independent samples/groups)(m) CD90 (Green) immunofluorescence of control or ALS patient muscles. Scale bar 200 μm (n) phospho-STAT3 (Green) immunofluorescence of control and ALS patient muscles. Scale bar 200 μm. Representative images out of 3 independet experiment. (o) Top: Scheme of treatment. Bottom: Frequency distribution of fiber CSA of TA muscle. Values represent mean ±s.d. *P<0.05, **P<0.01; by two-side t-test. n=4 animals/group.. (p) Sirius-red staining of TA muscles from Control and STAT3i-treated mice. (q). Quantification of data in p. Values represent mean ±s.d. *P<−0.05; by t-test. n=4 animals/group.