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Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages

Nature Aging volume 3, pages 1251–1268 (2023)Cite this article

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Abstract

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.

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Fig. 1: Lamin A/C protects alveolar macrophages from depletion.
Fig. 2: Loss of alveolar macrophages is caused by a p53-dependent response to DNA damage.
Fig. 3: Constricted movement, nuclear envelope rupture and DNA damage in alveolar macrophages.
Fig. 4: A lysosomal/CD63 response is induced by DNA damage.
Fig. 5: CD63 is required for clearance of damaged DNA in macrophages.
Fig. 6: A signature of aging in lamin A/C-deficient alveolar macrophages.
Fig. 7: Lamin A/C protects against the acquisition of hallmarks of aging in alveolar macrophages.
Fig. 8: Lamin A/C protects against influenza virus infection and lung cancer growth.

Data availability

Gene expression data are deposited at the Gene Expression Omnibus under accession numbers GSE146080 and GSE211713.

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Acknowledgements

We thank G. Bonne, B. Ryffel, X. Lahaye, O. Lantz, A. Darbois Delahousse, J. Helft, H. Salmon, L. Perie, A. -M. Lennon, A. Yatim, I. Simeonova and G. Almouzni for discussions and technical help, TMS consortium for early access to data and the Institut Curie Institut Curie flow cytometry, next-generation sequencing and animal facilities. This work was supported by Institut Curie, INSERM, CNRS, Agence Nationale de la Recherche grant LUSTRA (to A.L.), Agence Nationale de la Recherche grants ANR-17-CE15-0025-01, ANR-19-CE15-0018-01, ANR-18-CE92-0022-01, 11-LABX-0043 (to N.M.), ANR-19-CE14-0015 (to A.L.-V. and C.F.), Institut National du Cancer grant 2019-1-PL BIO-07-ICR-1 (to N.M. and M.P.), Institut Thématique Multi-Organisme Cancer grant 19CS007-00 (to N.M. and M.P.), Fondation Chercher et Trouver (to N.M.), Mutuelle Bleue (to N.M.), Fondation ARC pour la recherche sur le cancer grants ARCAGEING2022050004956 (to N.M.), PGA1 RF20190208474 (to G.v.N.), Fondation pour la Recherche Médicale grant EQU202103012774 (to N.M.), EDF grants ORG20190038 and CT9818 (to A.L.-V), Instituto de Salud Carlos III grant PI20/00306, Fondo Europeo de Desarrollo Regional « A way to build Europe » (J.M.G.-G.), Deutsche Forschungsgemeinschaft 369799452-TRR237 B19 (to R.B.), EMBO ALTF grant 1298-2016 (to N.S.D.S.), European Commission Horizon 2020 grant H2020-MSCA-IF-2016 DCBIO (to N.S.D.S.), Région Ile-de-France DIM Longévité et Vieillissement (to N.S.D.S.) and MCNU FPU program grant FPU18/00895 (to B.H.F.).

Author information

Author notes

  1. These authors contributed equally: Nilushi S. De Silva, Johan Siewiera.

  2. These authors jointly supervised this work: Chantal Alkhoury, Guilherme P. F. Nader, Francesca Nadalin.

Authors and Affiliations

  1. Institut Curie, PSL Research University, INSERM U932, Paris, France

    Nilushi S. De Silva, Johan Siewiera, Chantal Alkhoury, Francesca Nadalin, Kevin de Azevedo, Helena M. Izquierdo, Anvita Bhargava, Cécile Conrad, Mathieu Maurin, Konstantina Antoniadou & Nicolas Manel

  2. Institut Curie, PSL Research University, CNRS UMR144, Paris, France

    Guilherme P. F. Nader & Matthieu Piel

  3. Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Team van Niel, Paris, France

    Mickaël Couty & Guillaume van Niel

  4. Institut Curie, PSL Research University, Université Paris-Saclay, CNRS, INSERM, UMR3347, U1021, Orsay, France

    Charles Fouillade

  5. Institut Curie, PSL Research University, CNRS UMR3244, Paris, France

    Arturo Londono-Vallejo

  6. Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany

    Rayk Behrendt

  7. Biocellvia, Marseille, France

    Karine Bertotti & Cindy Serdjebi

  8. Sorbonne Université, Inserm, CNRS, Centre d’Immunologie et des Maladies Infectieuses, Cimi-Paris, Paris, France

    François Lanthiez & Alexandre Boissonnas

  9. Biochemical Institute, Christian-Albrechts-University Kiel, Kiel, Germany

    Lisa Gallwitz & Paul Saftig

  10. Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid (UAM), Madrid, Spain

    Beatriz Herrero-Fernández

  11. Facultad de Ciencias Experimentales, Universidad Francisco de Vitoria (UFV), Pozuelo de Alarcón, Spain

    Angela Saez

  12. LamImSys Lab, Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12). Department of Immunology, Ophthalmology and ENT, School of Medicine, Universidad Complutense de Madrid. CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain

    José María González-Granado

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Contributions

N.S.D.S., J.S., C.A., G.N., F.N., K.A., M.C., H.I., A. Bhargava, C.C., M.M., K.A., C.F., A.L.-V., R.B., K.B., C.S., F.L., L.G., P.S., B.H., A.S., J.G., G.v.N., A. Boissonnas, M.P. and N.M. carried out experimental design and studies, analyzed data and/or contributed to the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Nicolas Manel.

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Nature Aging thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Analysis of immune cells in Lamin A/C CKO mice.

(A) Flow cytometric analysis of WT vs Lamin A/C CKO macrophage and dendritic cell (DC) populations in spleen, with gating strategies for each population represented by colored gating. Left, representative example of the gating strategy. Right, percentage of each population of live spleen cells and absolute counts (n = 4 mice, combined from 2 independent experiments, bar indicates mean ± SD, one-way ANOVA with Šidák test). (B) Flow cytometric analysis of WT vs Lamin A/C CKO Natural killer, T and B cell populations in spleen and lymph nodes, with gating strategies for each population represented by colored gating. Top, representative example of the gating strategy. Bottom, percentage of each population of live spleen or lymph node cells and absolute counts (spleen, n = 6 WT and 5 Lamin A/C CKO mice for NK and T cells analysis, 4 WT and 4 Lamin A/C CKO mice for DCs, macrophages and B cells; lymph nodes: n = 8 WT and 6 Lamin A/C CKO mice for NK and T cells analysis, 6 WT and 5 Lamin A/C CKO mice for B cells combined from 4 independent experiments, bar indicates mean ± SD, one-way ANOVA with Šidák test). (C) Flow cytometric analysis of WT vs Lamin A/C CKO to analyze hematopoietic stem cell (HSC) and plasmacytoid dendritic cell populations in bone marrow, with gating strategies for each population represented by colored gating. Top, representative example of the gating strategy. Bottom, percentage of each population of live bone marrow cells and the absolute counts (n = 4 WT and 4 Lamin A/C CKO mice for pDC analysis, n = 6 WT and 6 Lamin A/C CKO mice for all the other populations, combined from 3 independent experiments, bar indicates mean ± SD, one-way ANOVA with Šidák test). Acronyms used: Short term HSC (ST-HSC), Multipotent hematopoietic progenitor (MPP), common myeloid progenitor (CMP), common lymphoid progenitor (CLP), granulocyte macrophage progenitor (GMP), megakaryocyte-erythroid progenitor (MEP), plasmacytoid dendritic cell (pDC).

Source data

Extended Data Fig. 2 Analysis of immune cells in lungs of Lamin A/C CKO mice.

(A) Representative flow cytometric analysis of WT vs Lamin A/C CKO lung immune populations including AM, eosinophils, interstitial macrophages, CD103+ DC and CD11b+ DC with gating strategies for each population represented by colored gating. (B) Percentages and absolute counts of AM, eosinophils, interstitial macrophages, CD103 + DC and CD11b+ DC of live WT vs Lamin A/C CKO lung cells (n = 17 WT and 14 Lamin A/C CKO mice, combined from 9 independent experiments, bar indicates mean ± SD, one-way ANOVA with Šidák test). (C) Percentage of AM (CD45.2+SSChiMHCintSiglecF+) following flow cytometric analysis of WT (Lmnafl/fl LysM-Cre-/-) vs Lmnafl/fl LysM-Cre+/- lung (n = 4 mice combined from 2 independent experiments, bar indicates mean ± SD, two-tailed unpaired t test).

Source data

Extended Data Fig. 3 scRNAseq analysis of lung immune cells in WT and Lamin A/C CKO mice.

(A) Heatmap showing the expression of cluster-specific gene markers. Differential expression analysis is done for each cluster against all the other cells (including the contaminants). Among the top 50 significant DEGs, the 10 DEGs with the top fold change are shown. Common DEGs between clusters are reported only once. Entries are the z-scores of the normalized expression level, clipped to fit the interval. (B) tSNE representation showing WT (left) and Lamin A/C CKO (right) cells only, and colored by replicate. In WT, replicate 1 contains 4075 cells and replicate 2 contains 3980. In Lamin A/C CKO, replicate 1 contains 4000 cells and replicate 2 contains 3677. (C) tSNE representation showing WT cells (top) and Lamin A/C CKO cells (bottom). Cells are colored with a gradient representing the normalized expression level of immune and alveolar macrophage markers.

Extended Data Fig. 4 DNA damage and related markers in alveolar macrophages of Lamin A/C CKO mice.

(A) Gene ontology enrichment analysis of DEGs upregulated in Lamin A/C CKO, specific to the AM cluster. Top 10 terms (p-values computed with clusterProfiler) from the biological process (BP) ontology are shown, and the DNA-damage-related pathway is highlighted (red). (B) Violin plot showing the normalized expression of Cdkn1a in the AM cluster, with values for individual WT and Lamin A/C CKO replicates shown (p-value computed with MAST with Bonferroni correction). (C) Expression of cGAS and Vinculin in BAL cells from mice of the indicated genotypes (n = 2 Lmnafl/fl CgasKO/KOVav1-Cre+/-, n = 1 Lmnafl/fl CgasKO/KOVav1-Cre-/-, n = 2 Lmnafl/fl p53fl/flVav1-Cre-/- and n = 2 Lmnafl/fl p53fl/flVav1-Cre+/- from one experiment). (D) Detection of Tp53 floxed (flox) and deletion (del) alleles by PCR in BAL cells from mice of the indicated genotypes (n = 2 WT (Lmnafl/fl p53fl/flVav1-Cre-/-) vs Lamin A/C p53 DKO (Lmnafl/fl p53fl/flVav1-Cre+/-) from one experiment). (E) Representative intracellular flow cytometric analysis of mice from WT (Lmnafl/fl Vav1-Cre-/-) vs Lamin A/C CKO (Lmnafl/fl Vav1-Cre+/-) and WT (Lmnafl/fl p53fl/flVav1-Cre-/-) vs Lamin A/C p53 DKO (Lmnafl/fl p53fl/flVav1-Cre+/-) to identify AM (CD45.2+SSChiMHCintSiglecF+) and assess gH2AX (fill) and isotype control (open) levels. (F) Representative flow cytometry analysis and quantification of Caspase 3/7 activity in live (SYTOX–) and dead (SYTOX+) cells in the indicated populations (n = 4 mice combined from 2 experiments, bar indicates mean ± SD, Kruskal-Wallis H-test with Dunn’s correction). In the SYTOX channel, based on the control condition omitting the SYTOX reagent, Caspase 3/7-negative AM are SYTOX– but show higher auto-fluorescence than other cells.

Source data

Extended Data Fig. 5 Live imaging of alveolar macrophages in lungs.

(A) AM from WT mouse lung (female) undergoing constricted migration in vivo. Top left, XY and XZ planes. Bottom left, contour representation. Right, measurement of the cell width at the most acute point of squeezing. (B) BAL from Lamin A/C CKO mice confined at a height of 2 µm for 1.5 hours and subsequently stained for DAPI, BAF and SiglecF (representative of n = 4 independent experiments, each time BAL was pooled from 2 mice aged 8-26 weeks). (C) BAL from WT and Lamin A/C CKO mice confined at 2 µm and 10 µm for 1.5 hours and subsequently stained for DAPI, Lamin A/C and SiglecF. Left, representative field. Right, quantification of mean intensity of the Lamin A/C intensity (violin plot with lines indicating the median and quartile values, representative of 2 independent experiments, 6-7 images fields per group were used, one-way ANOVA with Tukey test, each time BAL was pooled from 2 mice of each genotype). (D) Staining for Lamin A/C, SiglecF, γH2AX and DAPI in BAL form Lamin A/C CKO mice after confinement at 2 µm (scale bar 10 µm).

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Extended Data Fig. 6 Analysis of markers in Lamin A/C CKO mice.

(A) Left, intracellular flow cytometric analysis of WT vs Lamin A/C CKO lung for CD88. levels within AM (CD45.2+SSChiMHCintSiglecF+). Right, mean fluorescence intensity (MFI) of CD88 signal normalized to isotype control (n = 5 WT and 8 Lamin A/C CKO mice, combined from 3 independent experiments, mice aged 28-39 weeks, bar indicates mean ± SD, two-tailed unpaired t test). (B) Cathepsin L activity in BAL extracted from WT vs Lamin A/C CKO mice (n = 10 WT and 11 Lamin A/C CKO mice from 8 independent experiments, bar indicates mean ± SD, aged 13-34 weeks, two-tailed unpaired t-test). (C) Flow cytometric analysis of BAL obtained from WT vs Lamin A/C CKO mice to analyze neutrophils, T and B cells with gating strategies for each population represented by colored gating. Left, representative example. Right, percentage of each population of live BAL cells and absolute counts of each population in extracted BAL (n = 8 WT and 7 Lamin A/C CKO mice, combined from 4 independent experiments, mice aged 24-34 weeks, bars indicate mean ± SD, one-way ANOVA with Šidák test). (D) Protein concentration in the BAL of WT and Lamin A/C CKO mice (n = 9 mice, bars indicate mean ± SD, two-tailed unpaired t-test). (E) Quantification of alveolar density, collagen content and fibrotic foci in lung sections of WT and Lamin A/C CKO mice. Top, representative images of staining and corresponding automated mapping. Bottom, quantification (n = 4 WT and 5 CKO mice, 4-10 images fields per group were used, bars indicate mean ± SD, two-tailed unpaired t-test).

Source data

Extended Data Fig. 7 scRNAseq of irradiated mice and CD63 expression in knock-out mice.

(A) Heatmap showing single cell gene expression (z-score) of DEGs in AM 5 months post-17Gy irradiation and non-irradiated controls. Cells are grouped by condition. DEGs are divided among up- and down-regulated in case vs control, clustered with a ward.D2 method computed on euclidean distances and shown as dendrogram. DEGs are annotated with log2 fold change (FC) values. (B) Number of AMs and analysis of intracellular levels of CD63 and γH2AX in p53fl/fl Vav1-Cre+/– and p53fl/fl Vav1-Cre–/– mice, normalized median fluorescent intensity (MFI) (n = 3 mice combined from 2 experiments, bars indicate mean ± SD, two-tailed Welch t-test). (C) Number of AMs and analysis of intracellular levels of CD63 and γH2AX in Lmnafl/fl Vav1-Cre–/– (WT), LmnaWT/WT Vav1-Cre+/– and Lmnafl/fl Vav1-Cre+/– (Lamin A/C CKO) mice, normalized median fluorescent intensity (MFI) (n = 5 mice combined from 3 experiments, bars indicate mean ± SD, one-way ANOVA with Sidak test). (D) CD63 and γH2AX in fresh AMs and AM maintained in vitro for 5 and 20 hours. Open line, isotype; filled line, specific antibody. (E) Immunofluorescence analysis of CD63 (red) and BAF (green) or CD63 (red) and dsDNA (green) in WT (right panels) and Lamin A/C CKO (left panels) BAL cells following confinement at 2 μm for 1.5 hours (z-projection, max intensity; images representative of n = 4 mice from 2 independent experiments). Bar, 5 μm. (F) Intracellular flow cytometric analysis for CD63 levels within AM (CD45.2+SSChiMHCintSiglecF+) of WT vs CD63 KO lung single cell suspension, treated as indicated. MFI of CD63 normalized to isotype control (n = 3 mice in one experiment, mice were 19 weeks of age, bars indicate mean ± SD, one-way ANOVA with Šidák test).

Source data

Extended Data Fig. 8 Hallmarks of aging in alveolar macrophages.

(A) Percentage AM (CD45.2+SSChiMHCintSiglecF+) of live lung cells in Young (14 weeks) vs Old (63 weeks) mice (n = 4 mice combined from 2 independent experiments, bars indicate mean ± SD, unpaired t test). (B) Normalized enrichment scores (NES) and adjusted p-values (statistical test from GSEA) following gene set enrichment analysis of the indicated upregulated or downregulated genes. Left, TMS gene sets enrichment in Lamin A/C CKO vs WT transcriptome. Geneset obtained after individual pairwise time-point comparisons and overall aging signature were tested. Right, Lamin A/C CKO gene sets enrichment in TMS transcriptome, ranked by the indicated time-point comparisons. n genes in sets indicates the number of genes in given gene set detected in the tested transcriptome. (C) Young (14 weeks) vs Old (63 weeks) lung single cell suspension was directly analyzed or incubated with DMSO or 50 mM etoposide for 1 hour. Intracellular flow cytometric analysis for gH2AX levels within AM (CD45.2+SSChiMHCintSiglecF+) was then performed. The geoMFI of gH2AX signal is shown normalized to isotype control (n = 4 mice from 2 independent experiments, bars indicate mean ± SD, one-way ANOVA with Šidák test). (D) Beta-galactosidase staining in RPE-1 cells, control, or after confinement at 2 µM that induces senescence as shown previously18, and WT and Lamin A/C CKO cells from BAL (scale bar 90 µm).

Source data

Extended Data Fig. 9 Influenza infections in Lamin A/C CKO mice.

(A) Percentage of day 0 weight observed each day post infection with 50 pfu influenza A virus via the intranasal route in WT and Lamin A/C CKO mice. Top, individual curves, bottom, bar plot (n = female 12 mice per genotype, combined from 2 independent experiments, two-way ANOVA with Tukey test, bars indicate mean ± SD). (B) Survival of WT vs Lamin A/C CKO mice following infected with 100 pfu of influenza A virus PR8 via the intranasal route (n = 6 mice per genotype in one experiment, Log-rank Mantel-Cox test). (C) Percentage of day 0 weight observed each day post infection described in (B). Curves of average ± SEM weights are shown and continued until a first death occurs in the group. (D) Percentage of day 0 weight observed each day post infection described in (B) (two-way ANOVA with Tukey test, bars indicate mean ± SD). (E) Percentage of day 0 weight in Lmnafl/fl LysM-cre+/- and Lmnafl/fl LysM-cre-/- mice in 4 weeks and 8 weeks mice, each post infection with 35 pfu influenza virus via the intranasal route. Top, individual curves, bottom, bar plots (n = 7 WT and 8 Lamin A/C CKO mice 4 weeks old and 8 WT and 9 Lamin A/C CKO mice 8 weeks old in one experiment per age group, two-way ANOVA with Sidak test, bars indicate mean + SD).

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Supplementary information

Reporting Summary

Supplementary Table 1

DEGs for cluster assignment. P values were computed with MAST with Bonferroni correction.

Supplementary Table 2

Expression of Lmna in the Lung Connectome dataset.

Supplementary Table 3

DEGs identified in clusters. P values were computed with MAST with Bonferroni correction.

Supplementary Table 4

DEGs in the alveolar macrophage cluster of irradiated mice. P values were computed with MAST with Bonferroni correction.

Supplementary Table 5

DEGs in the alveolar macrophage cluster of TMS. P values were computed with MAST with Bonferroni correction.

Supplementary Table 6

Sex of mice used in the study.

Supplementary Table 7

Antibodies used in the study.

Supplementary Video 1

Live imaging of a WT mouse lung. The lung was imaged after administration of Hoechst and an anti-SiglecF antibody. Left is a broad field of a lung region, right features individual AMs (SiglecF+) demonstrating constricted migration (white boxes).

Supplementary Video 2

A single AM undergoing constricted migration in vivo. Left, contour representation. Right, XY and XZ planes.

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Extended Data Fig. 4C uncropped western blots.

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De Silva, N.S., Siewiera, J., Alkhoury, C. et al. Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages. Nat Aging 3, 1251–1268 (2023). https://doi.org/10.1038/s43587-023-00488-w

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