Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Neves, J., Sousa-Victor, P. & Jasper, H. Rejuvenating strategies for stem cell-based therapies in aging. Cell Stem Cell 20, 161–175 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Schultz, M. B. & Sinclair, D. A. When stem cells grow old: phenotypes and mechanisms of stem cell aging. Development 143, 3–14 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Conboy, I. M. et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433, 760–764 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Conboy, I. M., Conboy, M. J. & Rebo, J. Systemic problems: a perspective on stem cell aging and rejuvenation. Aging 7, 754–765 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ruckh, J. M. et al. Rejuvenation of regeneration in the aging central nervous system. Cell Stem Cell 10, 96–103 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Neves, J. & Sousa-Victor, P. Regulation of inflammation as an anti-aging intervention. FEBS J. 287, 43–52 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Yousefzadeh, M. J. et al. An aged immune system drives senescence and ageing of solid organs. Nature 594, 100–105 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wosczyna, M. N. & Rando, T. A. A muscle stem cell support group: coordinated cellular responses in muscle regeneration. Dev. Cell 46, 135–143 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sousa-Victor, P., Garcia-Prat, L. & Munoz-Canoves, P. Control of satellite cell function in muscle regeneration and its disruption in ageing. Nat. Rev. Mol. Cell Biol. 23, 204–226 (2022).

    Article  CAS  PubMed  Google Scholar 

  10. Munoz-Canoves, P., Neves, J. & Sousa-Victor, P. Understanding muscle regenerative decline with aging: new approaches to bring back youthfulness to aged stem cells. FEBS J. 287, 406–416 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Tidball, J. G. Regulation of muscle growth and regeneration by the immune system. Nat. Rev. Immunol. 17, 165–178 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chazaud, B. Inflammation and skeletal muscle regeneration: leave it to the macrophages! Trends Immunol. 41, 481–492 (2020).

    Article  CAS  PubMed  Google Scholar 

  13. Arnold, L. et al. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J. Exp. Med. 204, 1057–1069 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Perdiguero, E. et al. p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair. J. Cell Biol. 195, 307–322 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Neves, J. et al. Immune modulation by MANF promotes tissue repair and regenerative success in the retina. Science 353, aaf3646 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Jntti, M. & Harvey, B. K. Trophic activities of endoplasmic reticulum proteins CDNF and MANF. Cell Tissue Res. 382, 83–100 (2020).

    Article  PubMed  Google Scholar 

  17. Sousa-Victor, P., Jasper, H. & Neves, J. Trophic factors in inflammation and regeneration: the role of MANF and CDNF. Front. Physiol. 9, 1629 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Lindahl, M., Saarma, M. & Lindholm, P. Unconventional neurotrophic factors CDNF and MANF: structure, physiological functions and therapeutic potential. Neurobiol. Dis. 97, 90–102 (2017).

    Article  CAS  PubMed  Google Scholar 

  19. Tang, Q., Li, Y. & He, J. MANF: an emerging therapeutic target for metabolic diseases. Trends Endocrinol. Metab. 33, 236–246 (2022).

    Article  CAS  PubMed  Google Scholar 

  20. Sousa-Victor, P. et al. MANF regulates metabolic and immune homeostasis in ageing and protects against liver damage. Nat. Metab. 1, 276–290 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Neves, J. et al. MANF delivery improves retinal homeostasis and cell replacement therapies in ageing mice. Exp. Gerontol. 134, 110893 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sousa-Victor, P. et al. Geriatric muscle stem cells switch reversible quiescence into senescence. Nature 506, 316–321 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Rahman, F. A., Angus, S. A., Stokes, K., Karpowicz, P. & Krause, M. P. Impaired ECM remodeling and macrophage activity define necrosis and regeneration following damage in aged skeletal muscle. Int. J. Mol. Sci. 21, 4575 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mircescu, M. M., Lipuma, L., van Rooijen, N., Pamer, E. G. & Hohl, T. M. Essential role for neutrophils but not alveolar macrophages at early time points following Aspergillus fumigatus infection. J. Infect. Dis. 200, 647–656 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Kowalski, E. A. et al. Monocyte proinflammatory phenotypic control by ephrin type A receptor 4 mediates neural tissue damage. JCI Insight 7, e156319 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Lehmann, M. L. et al. CCR2 monocytes repair cerebrovascular damage caused by chronic social defeat stress. Brain Behav. Immun. 101, 346–358 (2022).

    Article  CAS  PubMed  Google Scholar 

  27. Varga, T. et al. Highly dynamic transcriptional signature of distinct macrophage subsets during sterile inflammation, resolution, and tissue repair. J. Immunol. 196, 4771–4782 (2016).

    Article  CAS  PubMed  Google Scholar 

  28. Wang, Y., Welc, S. S., Wehling-Henricks, M. & Tidball, J. G. Myeloid cell-derived tumor necrosis factor-alpha promotes sarcopenia and regulates muscle cell fusion with aging muscle fibers. Aging Cell 17, e12828 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Wang, Y. et al. Aging of the immune system causes reductions in muscle stem cell populations, promotes their shift to a fibrogenic phenotype, and modulates sarcopenia. FASEB J. 33, 1415–1427 (2019).

    Article  CAS  PubMed  Google Scholar 

  30. Runyan, C. E. et al. Impaired phagocytic function in CX3CR1+ tissue-resident skeletal muscle macrophages prevents muscle recovery after influenza A virus-induced pneumonia in old mice. Aging Cell 19, e13180 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Tobin, S. W. et al. Delineating the relationship between immune system aging and myogenesis in muscle repair. Aging Cell 20, e13312 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Patsalos, A. et al. In vivo GDF3 administration abrogates aging related muscle regeneration delay following acute sterile injury. Aging Cell 17, e12815 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Summan, M. et al. Macrophages and skeletal muscle regeneration: a clodronate-containing liposome depletion study. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1488–R1495 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Sahu, A. et al. Age-related declines in alpha-Klotho drive progenitor cell mitochondrial dysfunction and impaired muscle regeneration. Nat. Commun. 9, 4859 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wehling-Henricks, M. et al. Macrophages escape Klotho gene silencing in the mdx mouse model of Duchenne muscular dystrophy and promote muscle growth and increase satellite cell numbers through a Klotho-mediated pathway. Hum. Mol. Genet. 27, 14–29 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Zhang, C. et al. Age-related decline of interferon-gamma responses in macrophage impairs satellite cell proliferation and regeneration. J. Cachexia Sarcopenia Muscle 11, 1291–1305 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Paliwal, P., Pishesha, N., Wijaya, D. & Conboy, I. M. Age dependent increase in the levels of osteopontin inhibits skeletal muscle regeneration. Aging 4, 553–566 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tidball, J. G., Flores, I., Welc, S. S., Wehling-Henricks, M. & Ochi, E. Aging of the immune system and impaired muscle regeneration: a failure of immunomodulation of adult myogenesis. Exp. Gerontol. 145, 111200 (2021).

    Article  CAS  PubMed  Google Scholar 

  39. Al-Zaeed, N., Budai, Z., Szondy, Z. & Sarang, Z. TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice. Cell Death Dis. 12, 611 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Petrova, P. et al. MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J. Mol. Neurosci. 20, 173–188 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Voutilainen, M. H. et al. Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson’s disease. J. Neurosci. 29, 9651–9659 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lu, J. et al. Photoreceptor protection by mesencephalic astrocyte-derived neurotrophic factor (MANF). eNeuro 5, ENEURO.0109-18.2018 (2018).

    Article  PubMed  Google Scholar 

  43. Glembotski, C. C. et al. Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion. J. Biol. Chem. 287, 25893–25904 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhang, Y. et al. Hormonal therapies up-regulate MANF and overcome female susceptibility to immune checkpoint inhibitor myocarditis. Sci. Transl. Med. 14, eabo1981 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. He, M. et al. Mesencephalic astrocyte-derived neurotrophic factor ameliorates steatosis in HepG2 cells by regulating hepatic lipid metabolism. World J. Gastroenterol. 26, 1029–1041 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lindahl, M. et al. MANF is indispensable for the proliferation and survival of pancreatic beta cells. Cell Rep. 7, 366–375 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Herranen, A. et al. Deficiency of the ER-stress-regulator MANF triggers progressive outer hair cell death and hearing loss. Cell Death Dis. 11, 100 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Han, D. et al. Mesencephalic astrocyte-derived neurotrophic factor restores blood-brain barrier integrity of aged mice after ischaemic stroke/reperfusion through anti-inflammation via TLR4/MyD88/NF-κB pathway. J. Drug Target. 30, 430–441 (2022).

    Article  CAS  PubMed  Google Scholar 

  49. Yang, F. et al. Bone marrow mesenchymal stem cells induce M2 microglia polarization through PDGF-AA/MANF signaling. World J. Stem Cells 12, 633–658 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Zhang, J. X. et al. Mesencephalic astrocyte-derived neurotrophic factor (MANF) prevents the neuroinflammation induced dopaminergic neurodegeneration. Exp. Gerontol. 171, 112037 (2023).

    Article  CAS  PubMed  Google Scholar 

  51. Tonkin, J. et al. Monocyte/macrophage-derived IGF-1 orchestrates murine skeletal muscle regeneration and modulates autocrine polarization. Mol. Ther. 23, 1189–1200 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhao, W., Lu, H., Wang, X., Ransohoff, R. M. & Zhou, L. CX3CR1 deficiency delays acute skeletal muscle injury repair by impairing macrophage functions. FASEB J. 30, 380–393 (2016).

    Article  CAS  PubMed  Google Scholar 

  53. Pakarinen, E. et al. MANF ablation causes prolonged activation of the UPR without neurodegeneration in the mouse midbrain dopamine system. eNeuro 7, ENEURO.0477-19.2019 (2020).

    Article  PubMed  Google Scholar 

  54. Pakarinen, E., Lindholm, P., Saarma, M. & Lindahl, M. CDNF and MANF regulate ER stress in a tissue-specific manner. Cell. Mol. Life Sci. 79, 124 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Roy, A. et al. The IRE1/XBP1 signaling axis promotes skeletal muscle regeneration through a cell non-autonomous mechanism. eLife 10, e73215 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Chen, L. et al. Mesencephalic astrocyte-derived neurotrophic factor is involved in inflammation by negatively regulating the NF-κB pathway. Sci. Rep. 5, 8133 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Oh, J. et al. Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging 8, 2871–2896 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Yagi, T. et al. Neuroplastin modulates anti-inflammatory effects of MANF. iScience 23, 101810 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Ren, H., Xia, X., Dai, X. & Dai, Y. The role of neuroplastin65 in macrophage against E. coli infection in mice. Mol. Immunol. 150, 78–89 (2022).

    Article  CAS  PubMed  Google Scholar 

  60. Sereno, D. et al. An evolutionary perspective on the role of mesencephalic astrocyte-derived neurotrophic factor (MANF): at the crossroads of poriferan innate immune and apoptotic pathways. Biochem. Biophys. Rep. 11, 161–173 (2017).

    PubMed  PubMed Central  Google Scholar 

  61. Doyle, S. E. et al. Toll-like receptors induce a phagocytic gene program through p38. J. Exp. Med. 199, 81–90 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Ortuste Quiroga, H. P., Goto, K. & Zammit, P. S. Isolation, cryosection and immunostaining of skeletal muscle. Methods Mol. Biol. 1460, 85–100 (2016).

    Article  PubMed  Google Scholar 

  63. Bencze, M., Periou, B., Baba-Amer, Y. & Authier, F. J. Immunolabelling myofiber degeneration in muscle biopsies. J. Vis. Exp. https://doi.org/10.3791/59754 (2019).

  64. Galli, E. et al. Mesencephalic astrocyte-derived neurotrophic factor is upregulated with therapeutic fasting in humans and diet fat withdrawal in obese mice. Sci. Rep. 9, 14318 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Neuza S. Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Margarida F. Brás
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Inês B. Antunes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Päivi Lindholm
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joana Neves
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pedro Sousa-Victor
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Joana Neves or Pedro Sousa-Victor.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

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

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

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.

Supplementary information

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 6

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s43587-023-00382-5

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing