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Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration

Nature Aging volume 3, pages 585–599 (2023)Cite this article

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Abstract

Age-related decline in skeletal muscle regenerative capacity is multifactorial, yet the contribution of immune dysfunction to regenerative failure is unknown. Macrophages are essential for effective debris clearance and muscle stem cell activity during muscle regeneration, but the regulatory mechanisms governing macrophage function during muscle repair are largely unexplored. Here, we uncover a new mechanism of immune modulation operating during skeletal muscle regeneration that is disrupted in aged animals and relies on the regulation of macrophage function. The immune modulator mesencephalic astrocyte-derived neurotrophic factor (MANF) is induced following muscle injury in young mice but not in aged animals, and its expression is essential for regenerative success. Regenerative impairments in aged muscle are associated with defects in the repair-associated myeloid response similar to those found in MANF-deficient models and could be improved through MANF delivery. We propose that restoring MANF levels is a viable strategy to improve myeloid response and regenerative capacity in aged muscle.

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Fig. 1: MANF is essential for skeletal muscle regeneration.
Fig. 2: Aging and MANF deficiency affect the myeloid response during muscle regeneration.
Fig. 3: MANF is expressed in prorepair macrophages.
Fig. 4: MANF derived from prorepair macrophages is essential for skeletal muscle regeneration.
Fig. 5: MANF-deficient macrophages have a delayed phenotypic transition.
Fig. 6: MANF-deficient macrophages have defective lysosomal activity.
Fig. 7: MANF therapy restores regenerative success in aged skeletal muscles.

Data availability

All the data generated or analyzed during this study are included in the published article and its Supplementary Information and Source Data files and are available from the corresponding author upon reasonable request. RNA sequencing data generated in this study are available under accession numbers GSE224982 and GSE224983 from the NCBI Gene Expression Omnibus database. Correspondence and requests for materials should be addressed to P.S.-V. and J.N.

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Acknowledgements

We thank the Flow Cytometry, Comparative Pathology, Bioimaging and Rodent facilities of Instituto de Medicina Molecular João Lobo Antunes for technical support. We thank A. S. Pacheco and E. M. Tranfield from the Electron Microscopy Facility at the Instituto Gulbenkian de Ciência for sample processing, data collection and discussion of the results. This work was supported by EMBO (IG4448 to P.S.V.) and FCT (PTDC/MED-OUT/8010/2020 and EXPL/MED-OUT/1601/2021 to P.S.V. and J.N.). P.S.V. was supported by ‘la caixa’ Foundation Junior Leader Fellowship (LCF/BQ/PI19/11690006). J.N. was supported by an assistant research contract from FCT (2021.03843.CEECIND). P.L. was supported by the Academy of Finland (grant 343299) and by the Jane and Aatos Erkko Foundation.

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Authors and Affiliations

  1. Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal

    Neuza S. Sousa, Margarida F. Brás, Inês B. Antunes, Joana Neves & Pedro Sousa-Victor

  2. Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland

    Päivi Lindholm

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  1. Neuza S. Sousa
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  2. Margarida F. Brás
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  3. Inês B. Antunes
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Contributions

J.N. and P.S.V. conceived the study, designed experiments, analyzed and interpreted data, and wrote the manuscript. N.S.S. and M.F.B. designed and performed experiments, and analyzed and interpreted data. I.B.A. performed experiments and analyzed data. P.L. performed the ELISA analysis of muscle extracts. All authors revised the manuscript.

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Correspondence to Joana Neves or Pedro Sousa-Victor.

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Nature Aging thanks Matthias Wiens and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Regulation of muscle regeneration by MANF.

a, Relative levels of MANF mRNA, detected by RT-qPCR, in TA muscles of yg wt (C57BL/6) mice non-injured and at different time points following injury (2, 3, 4, 5, 6, 10 dpi) (n = 3 for 3,4,6,10dpi; n = 4 for n.i.,2,5dpi). b, Illustrative Western blot analysis of MANF levels in protein extracts of TA muscles from yg (2–6 mo) and old (22–25 mo) wt (C57BL/6) mice at 3dpi. Ponceau S-staining of the membrane was used to verify equal protein loading in each sample. c, MANF protein levels, quantified by ELISA, in extracts of TA muscles from yg (2–6 mo) and old (22–25 mo) wt (C57BL/6) mice, at 2 and 3dpi (n = 5 for yg 3dpi; n = 3 for all other conditions). d, Western blot analysis of MANF levels in protein extracts from ManfR26WT and ManfR26Δ mice at 3dpi. Ponceau S-staining of the membrane was used to verify equal protein loading in each sample. In a, p values are from one-way ANOVA with Bonferroni’s multiple comparison post-test. n.i., non-injured; dpi, days post-injury; yg, young.

Source data

Extended Data Fig. 2 Effects of aging and MANF-deficiency in the cellular response during muscle regeneration.

a, d, Gating strategy used in flow cytometry analysis of (a) CD45p°s immune cell population, endothelial cells, FAPs and MuSCs; and (d) myeloid cells (CD11bp°s), pro-repair macrophages (F4/80posLy6CLow), pro-inflammatory macrophages (LyC6High), and neutrophils (F4/80negLy6Gpos). b, c, e, f, Quantification, by flow cytometry, of FAPS (b, n = 3/condition), endothelial cells (c, n = 3/condition) and neutrophils (e, n = 7 for ManfR26WT; n = 6 for ManfR26Δ) in regenerating muscles of ManfR26WT and ManfR26Δ mice at 3dpi; and neutrophils (f, n = 4/condition) in regenerating muscles of yg (2–6 mo) and old (22–24 mo) wt (C57BL/6) mice at 3dpi. g–i, Quantification, by flow cytometry, of myeloid cells (CD11bpos, g), pro-repair macrophages (F4/80posLy6CLow, h) and pro-inflammatory macrophages (Ly6CHigh, i) in regenerating muscles of yg (2–6 mo) and old (22–25 mo) wt (C57BL/6) mice at 2 and 3dpi (n = 4 for yg and old 3dpi; n = 5 for old 2dpi; n = 6 for yg 2dpi). Data are represented as average ± s.e.m. and each n represents one animal. p values are from two-tailed Student’s t-test. FAPs, Fibroadipogenic progenitors; MuSCs; Muscle Stem Cells; yg, young.

Source data

Extended Data Fig. 3 Macrophage-derived MANF in muscle regeneration.

a, d Quantification, by flow cytometry, of macrophages (a), pro-repair macrophages (d, top; F4/80posLy6CLow) and pro-inflammatory macrophages (d, bottom; Ly6CHigh) in regenerating muscles of wt (C57BL/6) mice at different time points following injury (n = 6 for macrophages 2dpi; n = 8 for F4/80posLy6CLow and Ly6CHigh at 2dpi; n = 3 for all other conditions). b, Representative images of cryosections from Tibialis anterior (TA) muscles immunostained against F4/80 (green) and MANF (red). DAPI is used to identify nuclei. Arrowheads indicate cells with high MANF expression co-localized with F4/80. Scale bar: 10μm c, Quantification, by flow cytometry, of macrophages and neutrophils in regenerating quadriceps (QC) muscles of wt (C57BL6/J) mice at 3dpi, treated with clodronate liposomes or control PBS liposomes (n = 6/condition). Data are represented as average ± s.e.m. and each n represents one animal. p values are from two-tailed Student’s t-test. dpi, days post-injury; PBS-Lipo, PBS Liposomes; Clo-Lipo, Clodronate Liposomes.

Source data

Extended Data Fig. 4 Macrophage-derived MANF in muscle regeneration.

a–c, Quantification, by flow cytometry, of myeloid cells (CD11bpos, a), pro-repair macrophages (F4/80posLy6CLow, b), and ratio of pro-repair to pro-inflammatory macrophages (LyC6Low/LyC6High, c) in regenerating quadriceps (QC) muscles of tamoxifen treated Manffl/fl and ManfCx3cr1Δ mice at 3dpi (n = 4/condition). d, Representative images of cryosections from Tibialis anterior (TA) muscles of Manffl/fl, ManfCx3cr1WT and ManfCx3cr1Δ mice, at 4 and 14dpi, stained with H&E and immunostained with mouse IgG. Asterisks indicate necrotic myofibres. Scale bars: 50 μm for H&E 4dpi; 20μm for IgG 4dpi; 100μm for H&E 14dpi. e, f, Quantification of the average cross-sectional area of central nucleated new myofibres (f) and frequency distribution of new myofibres by size (e), in regenerating TA muscles from Manffl/fl and ManfCx3cr1Δ mice at 14dpi (n = 4 for Manffl/fl; n = 3 for ManfCx3cr1Δ). g, Quantification of the average cross-sectional area of myofibers in non-injured TA muscles from ManfCx3cr1WT and ManfCx3cr1Δ mice (n = 5 for ManfCx3cr1WT; n = 3 for ManfCx3cr1Δ). Data are represented as average ± s.e.m. and each n represents one animal. p values are from two-tailed Student’s t-test. dpi, days post-injury; H&E, Hematoxylin and Eosin; msIgG, mouse Immunoglobulin; CSA, cross-sectional area.

Source data

Extended Data Fig. 5 Effects of MANF ablation in Cx3cr1pos cells.

a, Experimental timeline for analysis of animals with conditional ablation of MANF only in Cx3cr1-expressing cells existing prior to muscle injury. b–d, Quantification, by flow cytometry, of myeloid cells (CD11bpos, b), pro-repair macrophages (F4/80posLy6CLow, c), and pro-inflammatory macrophages (LyC6High, d) in 3dpi regenerating QC muscles of ManfCx3cr1WT and ManfCx3cr1Δ mice, treated with tamoxifen prior to the injury (n = 3/condition). e, f, Quantification, by flow cytometry, of macrophages (CD11bposF4/80pos, e) and neutrophils (CD11bposF4/80negLy6Gpos, f) non-injured QC muscles of tamoxifen treated Manffl/fl and ManfCx3cr1Δ mice (n = 4/condition). g, Representative density plots from flow cytometry analysis of myeloid blood cell populations gated on CD11bposF4/80neg population, identifying neutrophils (Ly6Gpos), Ly6Cpos classical monocytes (Ly6GnegLy6Cpos) and Ly6Cneg non-classical monocytes (Ly6GnegLy6CnegCx3cr1pos). h-i, Quantification, by flow cytometry, of myeloid cells (CD11bpos), Ly6Cpos classical monocytes, Ly6Cneg non-classical monocytes and neutrophils in the blood of ManfCx3cr1WT and ManfCx3cr1Δ mice prior to muscle injury (h, n = 6 for ManfCx3cr1WT; n = 4 for ManfCx3cr1Δ), and at 3dpi (i, n = 4/ condition). j, l, Representative density plots from flow cytometry analysis of macrophage populations from ManfCx3cr1WT and ManfCx3cr1Δ animals at 3dpi, showing EdU signal (j) and Apopxin-Green signal (l). For both stains the FMO density plot is shown to define the positive population. k, Quantification, by flow cytometry, of EdUpos macrophages as a percentage of pro-repair and pro-inflammatory populations, in ManfCx3cr1WT and ManfCx3cr1Δ animals at 3dpi (n = 3 for ManfCx3cr1WT; n = 4 for ManfCx3cr1Δ). m, Quantification, by flow cytometry, of Apopxinpos macrophages as a percentage of pro-repair the population, in ManfCx3cr1WT and ManfCx3cr1Δ animals at 3dpi (n = 7 for ManfCx3cr1WT; n = 5 for ManfCx3cr1Δ). Data are represented as average ± s.e.m. and each n represents one animal. p values are from two-tailed Student’s t-test. FSC, Forward Scatter; Edu, 5-ethynyl-2’-deoxyuridine; FMO, Fluorescence Minus One Control.

Source data

Extended Data Fig. 6 MANF-deficiency affects macrophage phenotypic transition and inflammatory status.

a, Experimental timeline for analysis of animals with ablation of MANF in macrophages. b, Western blot analysis of MANF levels in protein extracts from F4/80pos cells FACS-isolated from QC muscles of Manffl/fl and ManfLysMΔ mice at 3dpi. Vinculin was used to verify equal protein loading in each sample. c–e, Quantification, by flow cytometry, of myeloid cells (CD11bpos, c), pro-repair macrophages (F4/80posLy6CLow, d) and pro-inflammatory macrophages (LyC6High, e) in regenerating QC muscles of Manffl/fl and ManfLysMΔ mice at 3dpi (n = 4/condition). f, Relative levels of Manf, Il1β and TNFα mRNA, detected by RT-qPCR, in BMDMs generated from Manffl/fl and ManfR26Δ mice in control conditions or 3 h after stimulation with Fibrinogen (n = 6 for ManfWT and n = 8 for ManfKO). Data are represented as average ± s.e.m. and each n represents one animal or one cell culture derived from one independent animal. p values are from two-tailed Student’s t-test. BMDMs, Bone marrow-derived macrophages; Fgn, Fibrinogen.

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Extended Data Fig. 7 Defects of MANF-deficient macrophages.

a, GO categories of biological processes showing significant enrichment in the dataset of genes differentially expressed in macrophages (CD45posF4/80pos) FACS-isolated at 3dpi from quadriceps muscles of ManfCx3cr1Δ mice compared to ManfCx3cr1WT mice (fold change < 0.75 or >1.5 and p ≤ 0.05, p values from two-tailed Student’s t-test, n = 3/condition). b, Representative histogram of the Fluoresbrite® 641 signal in BMDMs generated from Manffl/fl (grey) and ManfR26Δ (pink) mice, 3 h after stimulation with opsonized Fluoresbrite® 641 Carboxylate beads. Signal in non-stimulated BMDMs is shown in blue. BMDMs, Bone marrow-derived macrophages.

Extended Data Fig. 8 Transmission electron microscopy analysis MANF-deficient and macrophages.

Representative images of pro-repair macrophages (F4/80posLy6CLow) FACS-isolated at 3dpi from QC muscles of ManfCx3cr1WT and ManfCx3cr1Δ mice, analyzed by TEM. Scale bars: 2 μm Quantifications of these images, for independent cells, are shown in Fig. 6c–e. The quantification includes 54 macrophages isolated from ManfCx3cr1WT mice and 42 macrophages isolated from ManfCx3cr1Δ mice. Macrophages were sorted from n = 3 animals/condition in 2 independent experiments (experiment 1: n = 1/condition; experiment 2: n = 2/condition, pooled). Samples obtained in each experiment were processed independently for analysis.

Extended Data Fig. 9 Defects of aged macrophages.

a, GO categories of biological processes with relevance within the context of tissue regeneration showing significant enrichment in the dataset of genes down-regulated in pro-repair macrophages (CD11bposF4/80posLy6CLow) FACS-isolated at 3dpi from quadriceps muscles of old (22–24 mo) mice compared to yg (2–6 mo) mice (fold change < 0.75 and p ≤ 0.05, p values from two-tailed Student’s t-test, n = 3/condition). e, Representative histograms of the FITC signal derived from lysosomal hydrolysis of a self-quenched substrate in basal conditions (light) and after stimulation with Apop-necro debris (dark), in BMDMs generated from yg (grey) and old (brown) mice, and in old BMDMs cultured in the presence of rMANF (blue), 3 h after stimulation. GO, Gene Ontology; BP, Biological process; BMDMs, Bone marrow-derived macrophages.

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Sousa, N.S., Brás, M.F., Antunes, I.B. et al. Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration. Nat Aging 3, 585–599 (2023). https://doi.org/10.1038/s43587-023-00382-5

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