Combinatorial activation of the WNT‐dependent fibrogenic program by distinct complement subunits in dystrophic muscle

Figures

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  Figure 1. C1 is locally produced by skeletal muscles and is enhanced in dystrophy A–F Axin2 (A), TGFβ2 (B), LGR5 (C), collagen 1a1 (D), collagen 3a1 (E), and fibronectin (F) mRNA expression in FACS‐isolated FAPs from ~1‐year‐old WT and MDX hindlimb muscles. N (biological replicates) = 3.G Axin2 mRNA expression in C2C12 cultured for 6 h in DMEM with 5% serum collected from ~7‐months‐old WT, ~7‐months‐old MDX, and ~2.5‐years‐old WT (OLD) mice or with 50 ng/ml WNT3A (CNTR+ve). N (biological replicates) = 8 (WT), 7 (MDX), 6 (OLD), and 4 (CNTR+ve).HSchematic representation of C1 protein complex. Created with BioRender.com.I, J C1qa, C1qb, C1qc, C1r, and C1s mRNA expression in hindlimb muscles of ~1‐year‐old WT and MDX mice (I) and biceps muscles of healthy controls (CNTR) and DMD patients (DMD) (J). N (biological replicates) = 3 (WT, MDX, CNTR) and 4 (DMD).KRepresentative immunofluorescence (left) and quantification (right) of C1q in the gastrocnemius of ~1‐year‐old WT and MDX stained with anti‐C1q (green), anti‐Laminin2α (gray) antibodies, and Hoechst (blue). Scale bar: 20 μm. N (biological replicates) = 3.LWestern blot (left) and quantification (right) of C1s in human healthy (CNTR) and…

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  Figure EV1. Canonical WNT signaling is increased in dystrophic FAPs A, BRepresentative immunofluorescence (left) and quantification (right) of Axin2 (A) and TGFβ2 (B) signal intensities in the PDGFRα+ve cells (i.e., FAPs) of the gastrocnemius of ~1‐year‐old WT and MDX stained with anti‐Axin2 (yellow in A), anti‐PDGFRα (green), anti‐TGFβ2 (red in B), anti‐laminin 2α (white) antibodies, and Hoechst (blue). High‐magnification images in the left panels show representative PDGFRα+ve cells. N (biological replicates) = 3 for all samples (except for WT TGFβ2 analysis, N = 2). Scale bar: 20 μm (low‐magnification images), scale bar: 2 μm (high‐magnification images).C–H Axin2 (C), TGFβ2 (D), LGR5 (E), collagen 1a1 (F), collagen 3a1 (G), and fibronectin (H) mRNA expression in FACS‐isolated FAPs from ~3‐months‐old WT and MDX hindlimb muscles. N (biological replicates) = 3. Data information: Data are presented as mean with interquartile range in (A and B) and as mean ± SEM in (C–H). In (A and B), dots represent single cells' measurements (131 for WT and 130 for MDX in Axin2 analysis, 126 for WT and 215 for MDX in TGFβ2 analysis). Statistical differences were calculated by unpaired two‐tailed Mann–Whitney…

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  Figure EV2. C1 and C4 complement levels are increased in dystrophic muscles Representative immunofluorescence of ~1‐year‐old WT and MDX gastrocnemius stained with anti‐C1q (red), anti‐C4 (yellow) antibodies, and Hoechst (blue). High‐magnification images represent different zoomed areas of the muscle. Note the expression of both C4 and C1q in the same muscle areas. Scale bar: 100 μm (low‐magnification images), scale bar: 20 μm (high‐magnification images). Source data are available online for this figure.

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  Figure 2. Distinct cell types express the C1 complex's components in the skeletal muscles A–E C1qa (A), C1qb (B), C1qc (C), C1r (D), and C1s (E) mRNA expression in MuSCs, macrophages (MAC), FAPs, Lin−veSca1−veVCAM−ve, Lin+veF4/80−ve, and myofibers isolated from hindlimb muscles of ~1‐year‐old WT and MDX mice and muscles of WT mice 2.5 days after acute injury (WT INJ). N (biological samples) = 3 for all samples except for C1qb (N = 5) and fibers (N = 4).F, GNumber of macrophages (F) and FAPs (G) per mg of tissue in hindlimb (HL) and diaphragm (DIA) muscles of ~1‐year‐old WT and MDX mice. N (biological samples) = 10 (HL) and 9 (DIA). Data information: Data are presented as mean ± SEM. In (A–E), statistical differences between two groups were calculated by unpaired two‐tailed Student's t‐test, and the corresponding P‐values are reported on the graphs. Statistical differences between three or more groups were calculated by one‐way ANOVA test, Tukey's multiple‐comparison test was used as a post hoc test, and all the corresponding P‐values are enclosed in Appendix Tables S2 and S3. In (F, G) statistical differences between two groups were calculated by unpaired two‐tailed Student's t‐test.…

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  Figure EV3. Complement expression in different subsets of macrophages in dystrophic muscles ARepresentative gating and sorting strategy used to FACS isolate different macrophage populations from MDX mice. Macrophages were gated as CD45+veF4/80+ve cells. Within the macrophage population, CD206−ve, CD206Low, and CD206High subpopulations were isolated.B, CNumber of CD206−ve, CD206Low, and CD206High macrophages from MDX mice FACS‐isolated as in (A). Data are expressed as the percentage of F4/80+ve cells (B) and the number of cells per mg of tissue (C). N (biological samples) = 4.D–H CD206 (D), CD163 (E), C1qa (F), C1qb (G), and C1qc (H) mRNA expression in CD206−ve, CD206Low, and CD206High macrophages from MDX mice FACS isolated as in (A). N (biological samples) = 4.I, J IL‐10/TNFα (I) and IL‐10/TGFβ1 (J) mRNA expression in CD206−ve, CD206Low, and CD206High macrophages from MDX mice FACS isolated as in (A). N (biological samples) = 4. Data information: Data are presented as mean ± SEM. Statistical differences between two groups were calculated by unpaired two‐tailed Student's t‐test. P‐values are as indicated. Source data are available online for this figure.

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  Figure 3. C1 activates the canonical WNT signaling in murine fibroblasts, myoblasts, and FAPs A–C C1qa (A), C1qb (B), and C1qc (C) mRNA expression in cell lines as indicated (Mac: macrophages). N (independent experiments) = 3.DRepresentative immunofluorescence image of cells as indicated stained with an anti‐C1q (green) antibody and Hoechst (blue). C1q protein is expressed selectively by macrophages. Scale bar: 10 μm.E, F C1r (E) and C1s (F) mRNA expression in cell lines as indicated. N (independent experiments) = 3.G–I Axin2 mRNA expression in STO (G, H) and C2C12 (I) cells cultured with STO‐conditioned medium (STOm) or STOm+C1q for 6 (G) or 24 (H, I) hours. N (biological samples) = 3 (G, H), 6 (CNTR−ve and STOm in I), 4 (STOm + C1q in I), and 2 (CNTR+ve in I). CNTR+ve: WNT3A 25 ng/ml in (G, H) and 100 ng/ml in (I). CNTR−ve: cell medium plus reagents' solvents.JScheme of the experiments shown in (K–M).K–N Axin2 mRNA expression in C2C12 cells cultured with cell media (CNTR−ve) or reagents as indicated for 24 (K, M), 6 (L), or 18 (N) hours. N (biological replicates) = 3 (K, M, STOm + RAWm, and STOm + RAWm + XAV in L), 4 (CNTR−ve in L, C1q + C1r + C1s, and CNTR+ve in N), 5 (CNTR−ve in N),…

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  Figure 4. Enhanced C1/WNT axis in the dystrophic regenerating foci ADistance (μm) between macrophages (MAC) and FAPs in gastrocnemius of ~12‐months‐old male PDGFRα eGFP/WT (CNTR WT), ~18‐months‐old female PDGFRα eGFP/WT ;MDX +/− (CNTR HET), and ~18‐months‐old male PDGFRα eGFP/WT ;MDX (MDX) mice. The analysis was performed by measuring the distance of each macrophage in the image field from its closest FAP. N (biological replicates) = 3 (CNTR HET, MDX) and 1 (CNTR WT).BRepresentative immunofluorescence of a bona fide regenerating area in the gastrocnemius of a ~18‐months‐old male PDGFRα eGFP/WT ;MDX mouse stained with anti‐F4/80 (red), anti‐GFP (green) antibodies, and Hoechst (blue). White arrows indicate contacts between macrophages (F4/80+ve) and FAPs (eGFP+ve). Scale bar: 20 μm.CPercentage of contacts (i.e., distance of 3 μm or less) between macrophages and FAPs calculated in same muscles as in (A). N (biological replicates) = 3 (CNTR HET, MDX) and 1 (CNTR WT).D, ERepresentative immunofluorescence of gastrocnemius of ~1‐year‐old MDX stained with anti‐C1q (red), anti‐Axin2 (green in D), anti‐TGFβ2 (green in E) antibodies, and DAPI (blue). Scale bar: 100 μm (low‐magnification images) and…

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  Figure 5. The fib‐MDX mouse is a valuable model for studying FAPs in dystrophy AScheme of the generation of the fib‐MDX mouse model.BRepresentative immunofluorescence image of a gastrocnemius of a ~1‐year‐old MDX and fib‐MDX stained with anti‐collagen 1 (yellow), anti‐laminin 2α (red) antibodies, and Hoechst (blue). Scale bar: 50 μm.C, DNumber of FAPs (C) and macrophages (D) per mg of tissue in the hindlimb of fib‐MDX or uninjured MDX muscles. N (biological samples) = 3.E–I C1r (E), C1s (F), C1qa (G), C1qb (H), and C1qc (I), mRNA expression in FAPs (E, F) and macrophages (G–I) FACS isolated from fib‐MDX or uninjured MDX hindlimb muscles. N (biological samples) = 4 (for all samples except MDX C1qa and MDX C1qb, N = 5).J–N Axin2 (J), LGR5 (K), TGFβ2 (L), collagen 1a1 (M), and collagen 3a1 (N) mRNA expression in FAPs FACS isolated from fib‐MDX or uninjured MDX hindlimb muscles. N (biological samples) = 3. Data information: In (C–N) data are presented as mean ± SEM. Statistical differences were calculated by unpaired two‐tailed Student's t‐test. P‐values are as indicated. Source data are available online for this figure.

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  Figure EV4. The fib‐MDX skeletal muscle: macrophages/FAPs interplay ARepresentative immunofluorescence of gastrocnemius of ~1‐year‐old WT 4 days after cardiotoxin injury (WT INJ), MDX, and fib‐MDX mice stained with anti‐PDGFrα (green), anti‐F4/80 (orange) antibodies, and Hoechst (blue). White dollar symbols indicate macrophages (i.e., F4/80+ve cells), red asterisks indicate FAPs (i.e., PDGFRα+ve cells), and dotted lines indicate examples of distance measured between macrophages and FAPs. Scale bar: 10 μm.BDistance (μm) between macrophages (MAC) and FAPs measured in the gastrocnemius of ~1‐year‐old WT 4 days after cardiotoxin injury (WT INJ), MDX, and fib‐MDX mice stained as in (A). N (biological samples) = 3.CExample of a contact between a macrophage and a FAP in muscles represented in (A). Scale bar: 10 μm.DPercentage of contacts (i.e., a distance of 3 μm or less) between macrophages and FAPs calculated in the same muscles as in (A). N (biological samples) = 3.E, F TNFα/TGFβ1 (E) and TNFα/IL‐10 (F) mRNA expression in F4/80+ve macrophages FACS isolated from ~3‐months‐old MDX, ~1‐year‐old MDX, and ~1‐year‐old fib‐MDX hindlimb muscles. N (biological replicates) = 4.G, H CD206 (G) and CD163 (H)…

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  Figure 6. In vivo inhibition of C1r/s rescues the dystrophic FAPs phenotype AScheme of the C1r/s inhibition experiment in the fib‐MDX mice.B–F Axin2 (B), LGR5 (C), TGFβ2 (D), collagen 1a1 (E), and collagen 3a1 (F) mRNA expression in FACS‐isolated FAPs from ~1‐year‐old MDX mice processed as in (A). N (biological samples) = 3.GRepresentative immunofluorescence of gastrocnemius muscles from ~1‐year‐old MDX mice processed as in (A). Muscles were stained with anti‐collagen 1 (red), anti‐laminin 2α (white) antibodies, and Hoechst (blue). Scale bar: 50 μm.HFibrotic area quantification of the same muscles as in (G). N (biological samples) = 3.ICross‐sectional area (CSA) quantification of the muscle fibers of the same muscles as in (G). N (biological samples) = 3.JCollagen 1 pixel intensity quantification in the interstitial space between myofibers of the same muscles as in (G). N (biological samples) = 3.KModel of the mechanism through which the C1/WNT axis affects FAPs in dystrophic muscles. C1r/s subunits are indicated as pharmacological targets for the effective inhibition of fibrosis. Created with BioRender.com. Data information: In (B–F) and (H–J), data are presented as mean ± SEM. In (H and…

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  Figure EV5. In vivo inhibition of C1r/s in WT muscles after acute injury: FAPs, macrophages, and tissue analysis AScheme of the C1r/s inhibition experiment in WT muscles after acute cardiotoxin injury. Note that the length of the treatment with the C1r/s inhibitor corresponds to the time between the last needle injury and the moment of muscle dissection in the fib‐MDX experiments (Fig 6A).B–F C1r (B), C1s (C), C1qa (D), C1qb (E), and C1qc (F), mRNA expression in FACS‐isolated FAPs (C1r and C1s), and macrophages (C1qa, C1qb, and C1qc) from fib‐MDX and WT mice processed as in (A). N (biological samples) = 3. Fib‐MDX samples are the same as used in the analysis shown in Fig 5E–I.G–K Axin2 (G), LGR5 (H), TGFβ2 (I), collagen 1a1 (J), and collagen 3a1 (K) mRNA expression in FACS‐isolated FAPs from WT mice processed as in (A). N (biological samples) = 3.LRepresentative immunofluorescence of gastrocnemius muscles from WT mice processed as in (A). Muscles were stained with anti‐collagen 1 (green), anti‐laminin 2α (red) antibodies, and Hoechst (blue). Scale bar: 50 μm.MFibrotic area quantification of the same muscles as in (L). N (biological samples) = 3.NCross‐sectional area (CSA) quantification of…