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Restoration of SIRT3 gene expression by airway delivery resolves age-associated persistent lung fibrosis in mice

Nature Aging volume 1, pages 205–217 (2021)Cite this article

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Abstract

Aging is a risk factor for progressive fibrotic disorders involving diverse organ systems, including the lung. Idiopathic pulmonary fibrosis, an age-associated degenerative lung disorder, is characterized by persistence of apoptosis-resistant myofibroblasts. Here we demonstrate that sirtuin 3 (SIRT3), a mitochondrial deacetylase, is downregulated in the lungs of humans with idiopathic pulmonary fibrosis and in mice subjected to lung injury. Overexpression of Sirt3 cDNA via airway delivery restored the capacity for fibrosis resolution in aged mice, in association with activation of the forkhead box transcription factor FoxO3a in fibroblasts, upregulation of pro-apoptotic members of the Bcl2 family and recovery of apoptosis susceptibility. While transforming growth factor-β1 reduced levels of SIRT3 and FOXO3A in lung fibroblasts, cell non-autonomous effects involving macrophage-secreted products were necessary for SIRT3-mediated activation of FOXO3A. Together, these findings reveal a novel role of SIRT3 in pro-resolution macrophage functions that restore susceptibility to apoptosis in fibroblasts via a FOXO3A-dependent mechanism.

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Fig. 1: SIRT3 is decreased in lungs of individuals with IPF.
Fig. 2: SIRT3 is suppressed during the fibrogenic phase of lung injury, and its restitution resolves age-associated persistent fibrosis.
Fig. 3: Mitochondrial SIRT3 is decreased in activated myofibroblasts.
Fig. 4: SIRT3 in vivo overexpression sensitizes myofibroblasts to undergo apoptosis.
Fig. 5: SIRT3 or FoxO3a overexpression promotes apoptosis in murine fibroblasts.
Fig. 6: SIRT3 deficiency induces fibroblast senescence and resistance to apoptosis and upregulates pro-fibrotic markers.
Fig. 7: SIRT3 overexpression in macrophages activates FoxO3a in fibroblasts via a paracrine signaling mechanism.
Fig. 8: Airway delivery of Sirt3 cDNA resolves age-associated persistent lung fibrosis in mice.

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Data availability

Microarray data have been deposited in the Gene Expression Omnibus with the accession code number GSE17518. The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information. Source data are provided with this paper. Extra data are available from the corresponding author upon request.

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Acknowledgements

This work was supported by the NIH grants P01 HL114470 (to V.J.T.), R01 AG046210 (to V.J.T.) and R01 HL139617 (to J.W.Z. and V.J.T.); the US Department of Defense grant W81XWH-17-1-0577 (to J.W.Z.); and the US Department of Veterans Affairs Merit Award I01BX003056 (to V.J.T.). We thank Y. Wang for her assistance in the FACS experiment.

Author information

Author notes

  1. Mohammad Rehan & Victor J. Thannickal

    Present address: Department of Medicine, Tulane University School of Medicine, New Orleans, LA, USA

Authors and Affiliations

  1. Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

    Mohammad Rehan, Deepali Kurundkar, Naomi J. Logsdon, Samuel R. Smith, Diptiman Chanda, Karen Bernard, Yan Y. Sanders, Jessy S. Deshane, Kevin G. Dsouza, Jaroslaw W. Zmijewski & Victor J. Thannickal

  2. Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA

    Ashish R. Kurundkar

  3. Division of Pulmonary Sciences and Critical Care, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Sunad Rangarajan

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Contributions

M.R. and V.J.T. conceived and designed the study. M.R., D.K., A.R.K., K.B., D.C., S.R.S. and N.J.L. performed experiments. M.R., D.K., A.R.K., S.R., Y.Y.S., J.S.D., K.G.D. and V.J.T. analyzed and interpreted the data. M.R., D.K., J.W.Z. and V.J.T. drafted the manuscript.

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Correspondence to Victor J. Thannickal.

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Competing interests

V.J.T. has served as a consultant for Mistral Therapeutics, Inc., Boehringer Ingelheim, United Therapeutics, Blade Therapeutics, Versant Venture and Translate Bio. All other authors have no competing interests.

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Peer review informationNature Aging thanks David Lagares, David Sinclair and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 SIRT3 antibody specifically recognizes SIRT3 protein.

(a) Western blot showing over expression of HA tagged SIRT3 in human fibroblasts transfected with control or SIRT3 cDNA plasmid. (b) Western blot showing expression levels of SIRT3 in human fibroblasts transfected with non-targeting or SIRT3 siRNA. SIRT3 and HA antibodies were used to probe SIRT3 or SIRT3-HA expression levels.

Source data

Extended Data Fig. 2 Exogenous SIRT3 cDNA is preferentially expressed in lung macrophages.

(a) Schematic of experiment design. (b) RT-PCR analysis of total SIRT3 or vector derived SIRT3-HA mRNA levels in bronchoalveolar lavage (BAL) from mice subjected to bleomycin injury followed by treatment with control vector (n = 3) or SIRT3 cDNA plasmid (n = 5), as indicated in the Methods section. Data presented as means ± s.e.m., *P = 0.0357 (unpaired t-test, non-parametric, two-tailed). (c) Flow cytometric analysis of macrophages (CD45+ F4 80+ MerTK+) sorted from collagenase lung digests of naïve mice and mice injured with bleomycin followed by treatment with control vector or SIRT3 cDNA are shown. (d) Gating strategy: (i) cells were gated for FSC-A against SSC-A; (ii) doublets were excluded using FSC-H against FSC-A; (iii) singlets were gated for CD45 positive/negative population; (iv) within the CD45- population, epithelial cells were gated as EPCAM+CD31-; and (v) within the CD45+ population, macrophages were gated as F480+MerTK+. (e–g) RT-PCR analysis of total SIRT3 or vector derived SIRT3-HA mRNA levels in FACS sorted macrophages (e) and epithelial cells (f), and adherence-purified fibroblasts (g) from whole lung collagenase digest from mice subjected to bleomycin injury followed by treatment with control vector (n = 3) or SIRT3 cDNA plasmid (n = 5). Data presented as means ± s.e.m., *P = 0.0357 (macrophages;unpaired t-test, non-parametric, two-tailed), P = 0.0052 (fibroblasts; unpaired t-test, two-tailed statistical analysis), n.s. = not significant.

Extended Data Fig. 3 TGF-β1 downregulates SIRT3 and FoxO3a levels in human fibroblasts.

Top panel, schematic diagram showing experiment design. Bottom panel, western blots demonstrating early and late downregulation of SIRT3 and FoxO3a, respectively, and upregulation of α-SMA at indicated time points in TGF-β1 treatment of human fibroblasts.

Source data

Extended Data Fig. 4 FoxO3a overexpression in IPF fibroblasts induces Noxa and inhibits Col1a1.

Western blots and quantitative analysis of FoxO3a, cleaved caspase-3, cleaved PARP, Noxa and Col1a1 protein levels in IPF fibroblasts transfected with control plasmid or FoxO3a cDNA. n = 3 per group; Data presented as means ± s.e.m., P values as indicated by unpaired t-test, two-tailed statistical analysis.

Source data

Extended Data Fig. 5 The effects of SIRT3 overexpression on FoxO3a levels in control and IPF fibroblasts.

(a) Left panel, schematic of experiment design. Right panel, western blot showing levels of SIRT3 and FoxO3a in IMR-90 fibroblasts and IPF fibroblasts overexpressing control or SIRT3 plasmid. (b) Left panel, schematic diagram of experiment design. Right panel, western blot showing SIRT3 and FoxO3a levels in cytoplasmic and nuclear extracts of human fibroblasts overexpressing SIRT3 at 24 and 48 hours after transfection.

Source data

Extended Data Fig. 6 The effects of SIRT3 overexpression on FoxO3a recovery in TGF-β1 treated human lung fibroblasts.

(a) Left panel, schematic of the experiment design. Right panel, representative western blot showing levels of HA tag, SIRT3, FoxO3a and α-SMA in human fibroblasts cells transfected with SIRT3-HA plasmid followed by treatment with TGF-β1 (2.5 ng/ml) for 48 hours. (b) Left panel, schematic diagram of the experiment design. Right panel, representative western blot analyses of HA, SIRT3, FoxO3a and α-SMA in IMR-90 cells treated with TGF-β1 for 48 hours followed by SIRT3 overexpression.

Source data

Extended Data Fig. 7 Effects of conditioned media from SIRT3-overexpressing human and mouse lung epithelial cells on FoxO3a levels in human lung fibroblasts.

(a) Schematic of the experimental design. (b) Left panel, representative western blot showing overexpression of SIRT3 in A549 cells. Right panel, western blot showing levels of FoxO3a in human fibroblasts incubated with the conditioned media from SIRT3-overexpressing A549 cells. Cond. Med. = conditioned media (c) Left panel, western blot showing overexpression of SIRT3 in L2 mouse epithelial cells. Right panel, representative western blot showing levels of FoxO3a in IMR-90 fibroblasts incubated with conditioned media from SIRT3-overexpressing L2 cells at 48 hours. Cond. Med. = conditioned media.

Source data

Extended Data Fig. 8 Effects of conditioned media from SIRT3-overexpressing macrophages (THP1 or RAW264.7 cells) on FoxO3a levels in human fibroblasts.

(a) Schematic of the experimental design. (b) Left panel, representative western blot showing overexpression of SIRT3 in human macrophage line THP1 cells. Right panel, western blot showing expression of FoxO3a in human fibroblasts incubated with conditioned media of SIRT3 overexpressing THP1 cells. Cond. Med. = conditioned media. (c) Left panel, western blot showing overexpression of SIRT3 in RAW264.7 mouse macrophages. Right panel, western blot showing levels of FoxO3a in fibroblasts incubated with conditioned media of SIRT3 overexpressing RAW264.7 cells at 48 hours. Cond. Med. = conditioned media.

Source data

Extended Data Fig. 9 FoxO3a levels in young and aged mouse fibroblasts co-cultured with SIRT3 overexpressing L2 cells.

(a) Schematic of the experiment design; FB = fibroblasts. (b) Representative western blots indicate SIRT3 levels in whole cell lysates of L2 cells (b), FoxO3a levels in the cytoplasm (c), and in nuclear fractions (d) from young and old mouse fibroblasts. Fibroblasts were co-cultured with L2 cells that overexpress SIRT3 or control plasmid.

Source data

Extended Data Fig. 10 Cytokine array of secreted factors by SIRT3 overexpressing macrophages.

Mouse XL cytokine array and quantitative analysis performed on cell supernatants of co-cultured SIRT3-overexpressing macrophages and mouse fibroblasts.

Supplementary information

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Rehan, M., Kurundkar, D., Kurundkar, A.R. et al. Restoration of SIRT3 gene expression by airway delivery resolves age-associated persistent lung fibrosis in mice. Nat Aging 1, 205–217 (2021). https://doi.org/10.1038/s43587-021-00027-5

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