Mitochondrial iron chelation ameliorates cigarette smoke–induced bronchitis and emphysema in mice


Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element–binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Irp2 is pathogenic in experimental COPD.
Figure 2: Novel targets of IRP2 in the lung.
Figure 3: Irp2−/− mice resist CS-induced mitochondrial dysfunction.
Figure 4: IRP2-associated mitochondrial-iron loading and CS exposure.
Figure 5: COX is pathogenic in experimental COPD.
Figure 6: Targeting mitochondrial iron in experimental COPD.

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus


  1. 1

    Barnes, P.J., Shapiro, S.D. & Pauwels, R.A. Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur. Respir. J. 22, 672–688 (2003).

  2. 2

    Hogg, J.C. et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N. Engl. J. Med. 350, 2645–2653 (2004).

  3. 3

    Siedlinski, M. et al. Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility. Hum. Genet. 132, 431–441 (2013).

  4. 4

    DeMeo, D.L. et al. Integration of genomic and genetic approaches implicates IREB2 as a COPD susceptibility gene. Am. J. Hum. Genet. 85, 493–502 (2009).

  5. 5

    Qiu, W. et al. ECLIPSE Investigators. Genetics of sputum gene expression in chronic obstructive pulmonary disease. PLoS One 6, e24395 (2011).

  6. 6

    Hardin, M. et al. CHRNA3/5, IREB2 and ADCY2 are associated with severe chronic obstructive pulmonary disease in Poland. Am. J. Respir. Cell Mol. Biol. 47, 203–208 (2012).

  7. 7

    Pillai, S.G. et al. ICGN Investigators. A genome-wide association study in chronic obstructive pulmonary disease (COPD): identification of two major susceptibility loci. PLoS Genet. 5, e1000421 (2009).

  8. 8

    Saccone, N.L. et al. Multiple independent loci at chromosome 15q25.1 affect smoking quantity: a meta-analysis and comparison with lung cancer and COPD. PLoS Genet. 6, e1001053 (2010).

  9. 9

    Thorgeirsson, T.E. et al. A variant associated with nicotine dependence, lung cancer and peripheral arterial disease. Nature 452, 638–642 (2008).

  10. 10

    Lee, J.H. et al. IREB2 and GALC are associated with pulmonary artery enlargement in chronic obstructive pulmonary disease. Am. J. Respir. Cell Mol. Biol. 52, 365–376 (2015).

  11. 11

    Meyron-Holtz, E.G. et al. Genetic ablations of iron-regulatory proteins 1 and 2 reveal why iron-regulatory protein 2 dominates iron homeostasis. EMBO J. 23, 386–395 (2004).

  12. 12

    Ghosh, M.C. et al. Deletion of iron-regulatory protein 1 causes polycythemia and pulmonary hypertension in mice through translational derepression of HIF2α. Cell Metab. 17, 271–281 (2013).

  13. 13

    LaVaute, T. et al. Targeted deletion of the gene encoding iron-regulatory protein 2 causes misregulation of iron metabolism and neurodegenerative disease in mice. Nat. Genet. 27, 209–214 (2001).

  14. 14

    Jeong, S.Y. et al. Iron insufficiency compromises motor neurons and their mitochondrial function in Irp2-null mice. PLoS One 6, e25404 (2011).

  15. 15

    Cooperman, S.S. et al. Microcytic anemia, erythropoietic protoporphyria and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2. Blood 106, 1084–1091 (2005).

  16. 16

    Yoshida, T. et al. Rtp801, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke–induced pulmonary injury and emphysema. Nat. Med. 16, 767–773 (2010).

  17. 17

    Mizumura, K. et al. Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD. J. Clin. Invest. 124, 3987–4003 (2014).

  18. 18

    Lam, H.C. et al. Histone deacetylase 6–mediated selective autophagy regulates COPD-associated cilia dysfunction. J. Clin. Invest. 123, 5212–5230 (2013).

  19. 19

    Leopold, P.L. et al. Smoking is associated with shortened airway cilia. PLoS One 4, e8157 (2009).

  20. 20

    Thorley, A.J. & Tetley, T.D. Pulmonary epithelium, cigarette smoke and chronic obstructive pulmonary disease. Int. J. Chron. Obstruct. Pulmon. Dis. 2, 409–428 (2007).

  21. 21

    Moroishi, T., Nishiyama, M., Takeda, Y., Iwai, K. & Nakayama, K.I. The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo. Cell Metab. 14, 339–351 (2011).

  22. 22

    An, C.H. et al. TLR4 deficiency promotes autophagy during cigarette smoke–induced pulmonary emphysema. Am. J. Physiol. Lung Cell. Mol. Physiol. 303, L748–L757 (2012).

  23. 23

    Braber, S. et al. Cigarette smoke–induced lung emphysema in mice is associated with prolyl endopeptidase, an enzyme involved in collagen breakdown. Am. J. Physiol. Lung Cell. Mol. Physiol. 300, L255–L265 (2011).

  24. 24

    Kantrow, S.P., Shen, Z., Jagneaux, T., Zhang, P. & Nelson, S. Neutrophil-mediated lung permeability and host defense proteins. Am. J. Physiol. Lung Cell. Mol. Physiol. 297, L738–L745 (2009).

  25. 25

    Qiu, C. et al. Anti–interleukin-33 inhibits cigarette smoke–induced lung inflammation in mice. Immunology 138, 76–82 (2013).

  26. 26

    Hubeau, C., Kubera, J.E., Masek-Hammerman, K. & Williams, C.M. Interleukin-6 neutralization alleviates pulmonary inflammation in mice exposed to cigarette smoke and poly(I:C). Clin. Sci. 125, 483–493 (2013).

  27. 27

    Galy, B. et al. Iron-regulatory proteins secure mitochondrial iron sufficiency and function. Cell Metab. 12, 194–201 (2010).

  28. 28

    Fujimura, M., Morita-Fujimura, Y., Murakami, K., Kawase, M. & Chan, P.H. Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J. Cereb. Blood Flow Metab. 18, 1239–1247 (1998).

  29. 29

    Huang, M.L. et al. Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant. Proc. Natl. Acad. Sci. USA 106, 16381–16386 (2009).

  30. 30

    Hentze, M.W., Muckenthaler, M.U., Galy, B. & Camaschella, C. Two to tango: regulation of mammalian iron metabolism. Cell 142, 24–38 (2010).

  31. 31

    Shaw, G.C. et al. Mitoferrin is essential for erythroid iron assimilation. Nature 440, 96–100 (2006).

  32. 32

    Whitnall, M. et al. Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich's ataxia. Proc. Natl. Acad. Sci. USA 109, 20590–20595 (2012).

  33. 33

    Slebos, D.J. et al. Mitochondrial localization and function of heme oxygenase–1 in cigarette smoke–induced cell death. Am. J. Respir. Cell Mol. Biol. 36, 409–417 (2007).

  34. 34

    Clemente, P. et al. hCOA3 stabilizes cytochrome c oxidase 1 (COX1) and promotes cytochrome c oxidase assembly in human mitochondria. J. Biol. Chem. 288, 8321–8331 (2013).

  35. 35

    Gattermann, N. et al. Heteroplasmic point mutations of mitochondrial DNA affecting subunit I of cytochrome c oxidase in two patients with acquired idiopathic sideroblastic anemia. Blood 90, 4961–4972 (1997).

  36. 36

    Huttemann, M. et al. Cytochrome c oxidase subunit 4 isoform 2–knockout mice show reduced enzyme activity, airway hyporeactivity and lung pathology. FASEB J. 26, 3916–3930 (2012).

  37. 37

    Yang, H. et al. Analysis of mouse models of cytochrome c oxidase deficiency owing to mutations in Sco2. Hum. Mol. Genet. 19, 170–180 (2010).

  38. 38

    Ning, W. et al. Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease. Proc. Natl. Acad. Sci. USA 101, 14895–14900 (2004).

  39. 39

    Sohn, Y.S., Breuer, W., Munnich, A. & Cabantchik, Z.I. Redistribution of accumulated cell iron: a modality of chelation with therapeutic implications. Blood 111, 1690–1699 (2008).

  40. 40

    Filosa, A. et al. Long-term treatment with deferiprone enhances left ventricular ejection function when compared to deferoxamine in patients with thalassemia major. Blood Cells Mol. Dis. 51, 85–88 (2013).

  41. 41

    Hancock, D.B. et al. Meta-analyses of genome-wide association studies identify multiple loci associated with pulmonary function. Nat. Genet. 42, 45–52 (2010).

  42. 42

    Repapi, E. et al. Genome-wide association study identifies five loci associated with lung function. Nat. Genet. 42, 36–44 (2010).

  43. 43

    Lambrechts, D. et al. The 15q24/25 susceptibility variant for lung cancer and chronic obstructive pulmonary disease is associated with emphysema. Am. J. Respir. Crit. Care Med. 181, 486–493 (2010).

  44. 44

    Cho, M.H. et al. A genome-wide association study of COPD identifies a susceptibility locus on chromosome 19q13. Hum. Mol. Genet. 21, 947–957 (2012).

  45. 45

    Cho, M.H. et al. Risk loci for chronic obstructive pulmonary disease: a genome-wide association study and meta-analysis. Lancet Respir. Med. 2, 214–225 (2014).

  46. 46

    Cho, M.H. et al. Variants in FAM13A are associated with chronic obstructive pulmonary disease. Nat. Genet. 42, 200–202 (2010).

  47. 47

    Wilk, J.B. et al. A genome-wide association study of pulmonary function measures in the Framingham Heart Study. PLoS Genet. 5, e1000429 (2009).

  48. 48

    Rensvold, J.W. et al. Complementary RNA and protein profiling identifies iron as a key regulator of mitochondrial biogenesis. Cell Reports 3, 237–245 (2013).

  49. 49

    Agarwal, A.R., Yin, F. & Cadenas, E. Short-term cigarette smoke exposure leads to metabolic alterations in lung alveolar cells. Am. J. Respir. Cell Mol. Biol. 51, 284–293 (2014).

  50. 50

    Caron, M.A., Debigaré, R., Dekhuijzen, P.N. & Maltais, F. Comparative assessment of the quadriceps and the diaphragm in patients with COPD. J. Appl. Physiol. 107, 952–961 (2009).

  51. 51

    Crul, T. et al. Gene expression profiling in vastus lateralis muscle during an acute exacerbation of COPD. Cell. Physiol. Biochem. 25, 491–500 (2010).

  52. 52

    Ghio, A.J. et al. Particulate matter in cigarette smoke alters iron homeostasis to produce a biological effect. Am. J. Respir. Crit. Care Med. 178, 1130–1138 (2008).

  53. 53

    Philippot, Q. et al. Increased iron sequestration in alveolar macrophages in chronic obstructive pulmonary disease. PLoS One 9, e96285 (2014).

  54. 54

    Silverberg, D.S. et al. Anemia and iron deficiency in COPD patients: prevalence and the effects of correction of the anemia with erythropoiesis stimulating agents and intravenous iron. BMC Pulm. Med. 14, 24 (2014).

  55. 55

    Schneckenpointner, R. et al. The clinical significance of anemia and disturbed iron homeostasis in chronic respiratory failure. Int. J. Clin. Pract. 68, 130–138 (2014).

  56. 56

    Nickol, A.H. & Frise, M.C. A cross-sectional study of the prevalence and associations of iron deficiency in a cohort of patients with chronic obstructive pulmonary disease. BMJ Open 5, e007911 (2015).

  57. 57

    Tandara, L. et al. Systemic inflammation upregulates serum hepcidin in exacerbations and stabile chronic obstructive pulmonary disease. Clin. Biochem. 48, 1252–1257 (2015).

  58. 58

    Sauleda, J. et al. Cytochrome oxidase activity and mitochondrial gene expression in skeletal muscle of patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 157, 1413–1417 (1998).

  59. 59

    Antonicka, H. et al. Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency. Hum. Mol. Genet. 12, 2693–2702 (2003).

  60. 60

    Castaldi, P.J. et al. Genetic control of gene expression at novel and established chronic obstructive pulmonary disease loci. Hum. Mol. Genet. 24, 1200–1210 (2015).

  61. 61

    Lee, P.J. et al. Regulation of heme oxygenase–1 expression in vivo and in vitro in hyperoxic lung injury. Am. J. Respir. Cell Mol. Biol. 14, 556–568 (1996).

  62. 62

    Siempos, I.I. et al. Cecal ligation and puncture–induced sepsis as a model to study autophagy in mice. J. Vis. Exp. 84, e51066 (2014).

  63. 63

    Chen, Z.H. et al. Autophagy protein microtubule-associated protein 1 light chain–3B (LC3B) activates extrinsic apoptosis during cigarette smoke–induced emphysema. Proc. Natl. Acad. Sci. USA 107, 18880–18885 (2010).

  64. 64

    Parameswaran, H., Majumdar, A., Ito, S., Alencar, A.M. & Suki, B. Quantitative characterization of airspace enlargement in emphysema. J. Appl. Physiol. 100, 186–193 (2006).

  65. 65

    Laucho-Contreras, M.E., Taylor, K.L., Mahadeva, R., Boukedes, S.S. & Owen, C.A. Automated measurement of pulmonary emphysema and small-airway remodeling in cigarette smoke–exposed mice. J. Vis. Exp. 95, e52236 (2015).

  66. 66

    Jacob, R.E. et al. Comparison of two quantitative methods of discerning airspace enlargement in smoke-exposed mice. PLoS One 4, e6670 (2009).

  67. 67

    Jacob, R.E., Minard, K.R., Laicher, G. & Timchalk, C. 3D 3He diffusion MRI as a local in vivo morphometric tool to evaluate emphysematous rat lungs. J. Appl. Physiol. 105, 1291–1300 (2008).

  68. 68

    Wilson, A.A. et al. Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages. J. Clin. Invest. 120, 379–389 (2010).

  69. 69

    Hamakawa, H. et al. Structure-function relations in an elastase-induced mouse model of emphysema. Am. J. Respir. Cell Mol. Biol. 45, 517–524 (2011).

  70. 70

    Summer, R. et al. Alveolar macrophage activation and an emphysema-like phenotype in adiponectin-deficient mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L1035–L1042 (2008).

  71. 71

    Nobuyuki, O. A threshold selection method from gray-level histograms. IEEE Trans. Syst. Man Cybern. Syst. 9, 62–66 (1979).

  72. 72

    Soille, P. in Morphological Image Analysis: Principles and Applications (Springer-Verlag, Berlin, Heidelberg) 170–171 (1999).

  73. 73

    Meyer, F. Topographic distance and watershed lines. Signal Processing 38, 113–125 (1994).

  74. 74

    Kuhn, C. 3rd et al. Airway hyper-responsiveness and airway obstruction in transgenic mice. Morphologic correlates in mice overexpressing interleukin (IL)-11 and IL-6 in the lung. Am. J. Respir. Cell Mol. Biol. 22, 289–295 (2000).

  75. 75

    Bhashyam, A.R. et al. A pilot study to examine the effect of chronic treatment with immunosuppressive drugs on mucociliary clearance in a vagotomized murine model. PLoS One 7, e45312 (2012).

  76. 76

    Mortensen, J., Lange, P., Nyboe, J. & Groth, S. Lung mucociliary clearance. Eur. J. Nucl. Med. 21, 953–961 (1994).

  77. 77

    Goforth, J.B., Anderson, S.A., Nizzi, C.P. & Eisenstein, R.S. Multiple determinants within iron-responsive elements dictate iron-regulatory protein binding and regulatory hierarchy. RNA 16, 154–169 (2010).

  78. 78

    Dai, M. et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 33, e175 (2005).

  79. 79

    Dennis, G. Jr. et al. DAVID: Database for annotation, visualization and integrated discovery. Genome Biol. 4, 3 (2003).

  80. 80

    Huang, W., Sherman, B.T. & Lempicki, R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

  81. 81

    Clauset, A., Newman, M.E. & Moore, C. Finding community structure in very large networks. Phys. Rev. E 70, 066111 (2004).

  82. 82

    Oron, A.P., Jiang, Z. & Gentleman, R. Gene set–enrichment analysis using linear models and diagnostics. Bioinformatics 24, 2586–2591 (2008).

  83. 83

    Blake, J.A. et al. Gene Ontology Consortium. Gene Ontology annotations and resources. Nucleic Acids Res. 41, D530–D535 (2013).

  84. 84

    Smyth, G.K. Bioinformatics and Computational Biology Solutions Using {R} and Bioconductor (Springer, New York, 2005).

  85. 85

    Johnson, W.E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118–127 (2007).

  86. 86

    Vestbo, J. et al. ECLIPSE investigators. Evaluation of COPD longitudinally to identify predictive surrogate end points (ECLIPSE). Eur. Respir. J. 31, 869–873 (2008).

  87. 87

    Campbell, J.D. et al. A gene-expression signature of emphysema-related lung destruction and its reversal by the tripeptide GHK. Genome Med. 4, 67 (2012).

  88. 88

    Chen, Z.H. et al. Egr-1 regulates autophagy in cigarette smoke–induced chronic obstructive pulmonary disease. PLoS One 3, e3316 (2008).

  89. 89

    Epsztejn, S., Kakhlon, O., Glickstein, H., Breuer, W. & Cabantchik, I. Fluorescence analysis of the labile iron pool of mammalian cells. Anal. Biochem. 248, 31–40 (1997).

  90. 90

    Hoff, K.G. et al. In vivo fluorescence detection of Fe-S clusters coordinated by human GRX2. Chem. Biol. 16, 1299–1308 (2009).

Download references


The authors thank J.S. Moon, H.C. Lam, K. Taylor and B. Ding for technical assistance. The authors also acknowledge S. Chan (Harvard Medical School) for the cyto-GRX2 and mito-GRX2 plasmids, Y. Hua (Columbia University) for the breeding of the Sco2ki/ki and Sco2ki/ko mice and J. Connelly (ApoPharma Inc.) for providing Ferriprox. The authors also thank R. Rubio for assistance with RNA-seq, Y. Shao for assistance with the microarray study and M. Ericsson for assistance with transmission electron microscopy. The authors also acknowledge discussion and input from S.W. Ryter, C.A. MacRae and P.Y. Sips. This work was supported by US National Institutes of Health (NIH) grants P01-HL114501 (A.M.K.C.), R01-HL055330 (A.M.K.C.), R01-HL079904 (A.M.K.C.), R01-AI111475-01 (C.A.O.), R01-HL86814 (C.A.O.), R21-HL111835 (C.A.O.), HL122513 (H.P.), R01-HL086936 (to J.M.D'A.) and P01-HD080642 (Project 2 to E.A.S.), NIH–National Heart, Lung and Blood Institute grant K99-HL125899 (S.M.C.), American Lung Association Biomedical Research grant RG-348928 (S.M.C.), a Flight Attendants Medical Research Institute (FAMRI) clinical innovator award (A.M.K.C.), clinical innovator FAMRI grant CIA#123046 (C.A.O.), FAMRI Young Clinical Scientist awards YFEL141004 (F.P.) and YFEL103236 (M.P.G.), and US Department of Defense grant W911F-15-1-0169 (E.A.S.). S.M.C., A.M.K.C., J.Q. and E.K.S. were also supported by NIH grant P01-HL105339 (to E.K.S.). K.G. was supported by NIH grant R01-HL111759 (to J.Q., G.C.Y. and E.K.S.). C.A.O. was also supported by NIH grants R21-ES025379-01 (to A. Fedulov), P01-HL105339 (to E.K.S.) and P01-HL114501 (to A.M.K.C.) and by Brigham and Women's Hospital–Lovelace Respiratory Research institute Research Consortium grants. G.M. and C.K. were supported by NIH grant R01-GM088999 (to G.M.). M.C.G. and T.A.R. acknowledge support from the intramural research program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH. Additional support was provided by the Muscular Dystrophy Association (E.A.S.) and the J. Willard and Alice S. Marriott Foundation (E.A.S.).

Author information

S.M.C. and A.M.K.C. conceived and designed the study. S.M.C., K.G., M.E.L.-C., M.A.P., I.I.S., E.P., C.K., K.M., Z.-H.C., N.C.W., K.T.R., M.C.G. and A.M. performed experiments. K.G. analyzed RIP-seq, gene expression and human expression data and performed functional clustering analysis. A.R.B. and M.C. reconstructed and analyzed MCC images. S.C.M. provided technical support for the MCC experiments. C.A.O., F.P. and H.P. analyzed morphometric data. M.C.G. and T.A.R. provided the Irp2−/− mice. E.A.S. provided the Sco2ki/ki and Sco2ki/ko mice, and M.P.G. and J.M.D'A. provided technical support. D.L.D. helped with the LGRC human data set. S.M.C., K.G., G.-C.Y., J.Q., E.K.S., G.M., C.A.O. and A.M.K.C. provided critical analysis and discussions. S.M.C. and A.M.K.C. wrote the paper with significant input and contributions from K.G. and C.A.O. All coauthors reviewed and approved the final manuscript.

Correspondence to Augustine M K Choi.

Ethics declarations

Competing interests

In the past three years, E.K.S. received honoraria and consulting fees from Merck and grant support and consulting fees from GlaxoSmithKline.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–4 (PDF 4840 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cloonan, S., Glass, K., Laucho-Contreras, M. et al. Mitochondrial iron chelation ameliorates cigarette smoke–induced bronchitis and emphysema in mice. Nat Med 22, 163–174 (2016).

Download citation

Further reading