© 2015 Nature America, Inc. All rights reserved. potential potential therapeutic intervention for the axis mitochondrial-iron in COPD. pulmonary inflammation and lung CS-associated injury in mice with established COPD, suggesting a critical functional role and were protected from CS-induced COPD. iron Mitochondrial chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a iron mitochondrial chelator or mice fed a low-iron diet whereas mice deficient in the synthesis of cytochrome iron mitochondrial loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, (COX), which led to dysfunction mitochondrial and subsequent experimental COPD. mice, Frataxin-deficient which had higher of function mitochondrial in the lungs of mice. Irp2 increased iron mitochondrial loading and levels of cytochrome RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator cigarette smoke (CS)-induced experimental COPD. By integrating RNA followed by immunoprecipitation sequencing (RIP-seq), protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from addressed addressed to A.M.K.C. ( New York, New York, USA. 16 China. Boston, USA. Massachusetts, Shriver National Institute of Child Health and Human Development (NICHD), Bethesda, Maryland, USA. 10 New York, New York, USA. Brigham and Women’s Hospital, Harvard Medical School, Boston, USA. Massachusetts, USA. School of Public Health, Boston, USA. Massachusetts, and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA. Massachusetts, 1 gene as known (also susceptibility COPD influence also factors that genetic have suggested however, studies COPD; multiple the of exposure CS to lung responses cellular dysregulated and inflammatory aberrant involves it but understood, poorly remains COPD of pathogenesis of ranging destruction to phenotypes lung and of (emphysema) tissue remodeling the airways small bronchitis) pathologic (chronic and inflammation airway clinical from of variety a passes encom that disease lung debilitating complex, a as presents COPD protein identified element–binding iron-responsive 2 ( Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic Wedeterminants. have previously Ashfaq Mahmood Tracey A Rouault Zhi-Hua Chen Kenji Mizumura Morgan Cervo Suzanne M Cloonan smoke–induced bronchitis and emphysema in mice Mitochondrial iron chelation ameliorates cigarette nature medicine nature Received 7 October 2015; accepted 1 December 2015; published online 11 January 2016; Joan Joan and Sanford I. Weill Department of Medicine, New York–Presbyterian Hospital, Weill Cornell Medical College, New York, New York, USA. Department Department of Physiology & Cellular Biophysics, Columbia University, New York, New York, USA. First Department of Critical Care Medicine and Pulmonary Services, Evangelismos Hospital, University of Athens Medical School, Athens, Greece. CS exposure remains the greatest environmental risk factor for for factor risk environmental greatest the remains exposure CS 4– 5 Channing Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA. Massachusetts, 14 6 in humans on the basis of genome-wide association studies studies association genome-wide of basis the on humans in Department Department of Anesthesiology, Columbia University, New York, New York, USA. IREB2) 2 6

, , , Maria A Pabón 13 [email protected] 1 1 advance online publication online advance . 11 , , , Monica P Goldklang 2 6 18 8 , , Manik C Ghosh as a leading candidate COPD susceptibility susceptibility COPD candidate leading a as Lovelace Lovelace Respiratory Research Institute, Albuquerque, New Mexico, USA. , Edwin , K Edwin Silverman , Jeanine M D’Armiento Department Department of Genetics and Development, Columbia University Medical Center, New York, New York, USA. should Correspondence be 1 13 , 2 Department Department of Respiratory and Critical Care Medicine, Second Hospital of Zhejiang University School of Medicine, Hangzhou, , , Kimberly Glass 3 . We previously identified identified We previously . 1 , , Csaba Konrad ). 11 4 , , Harikrishnan Parameswaran Department Department of and Biostatistics Biology,Computational Dana-Farber Cancer Institute, Boston, Massachusetts, 3 14 2 – , 5 5 , 15 , , Caroline A Owen , , Maria E Laucho-Contreras 14

, , Guo-Cheng Yuan – 16 IRP2 , Eric A Schon c 7 oxidase, which have reduced COX, were protected from CS-induced , , Francesca Polverino 1 , 2 . . The IRP2 ) ) as an important COPD gene susceptibility and have shown that IRP2 -

tive diseases tive and neurodegenera hypertension of pulmonary pathogenesis in the and system, nervous central and cord spinal duodenum, the in roles cells mammalian regulatory in major protein the as serving IRP2 with homeostasis, iron lular GWAS in addiction nicotine and disease arterial peripheral cancer, lung with associated been has 15q25 COPD, to addition In receptor. acetylcholine nicotinic the of components several encode that genes includes which 15q25, chromosome human on genes of in COPD with increased subjects of is lungs the protein IRP2 that demonstrated we and (GWAS), 7 The iron-regulatory proteins (IRPs) IRP1 and IRP2 regulate cel regulate IRP2 and IRP1 (IRPs) proteins iron-regulatory The 15 Brain Brain and Mind Research Institute, Weill Cornell Medical College, 2 17 , Department Department of Medicine, Columbia University, New York, New York, USA. 8 3 & Augustine M K Choi , doi:10.1038/nm.402 , 18 4 , , Stephen C Moore , Giovanni Manfredi 17 12 12– Department Department of Neurology, Columbia University Medical Center, 9 , , Niamh C Williams 2 1 Pulmonary Pulmonary Department, University of Parma, Parma, Italy. 2 5 , , Abhiram R Bhashyam 12 , . In the setting of iron depletion, IRPs decrease iron IRPs decrease of iron depletion, . In setting the 8 Department Department of Biomedical Engineering, Boston University, , 9 , , Ilias I Siempos 1 3 Department Department of Harvard Biostatistics, T.H. Chan 1 1 . IRPs have important physiological physiological important have IRPs . 6 , , Dawn L Demeo 4 7 . . 1 , John Quackenbush IRP2 , 2 1 1 , , Kristen T Rooney , is located within a cluster cluster a within located is 10 6 Department Department of Radiology, , Elizabeth , PerezElizabeth 2 ,

2 Division Division of Pulmonary s e l c i t r a c 11 2 oxidase , Eunice Eunice Kennedy 5 ,

7– 3 – 1 5 0 . , 1 1 ,

,  - -

© 2015 Nature America, Inc. All rights reserved. diameter diameter and were protected from remodeling ( small-airway mice resisted CS-induced changes in mean chord length and air-space COPD of of thickness indices the (all established airways experimental small ( diameters to CS for 4–6 months had increased mean chord lengths and air-space COPD. exposed mice WT of in Irp2 experimental study the function mice in lethality ( controls RA-exposed to compared and cells in as the cell line human Beas2B, epithelial bronchial airway of model human in CS epithelial primary bronchial exposure) airway (an treatment (CSE) extract cigarette-smoke aqueous after 1a Fig. Supplementary ( controls RA to compared as exposure), (6-month CS to response in macrophages alveolar infiltrating in as well as cells, lial epithe and ciliated in I, II, secretory type type was higher expression basal in (cytokeratin 5–positive) staining epithelial cells ( negligible with vessels, intermediate-size of walls the to localize to seemed also expression Irp2 analyses. tochemical ( (acetylated airway ciliated and positive) (prosurfactant protein C (SPC)-positive), secretory airway II type alveolar (uteroglobin- I (podoplanin-positive), type including alveolar cells, ( controls to exposed CS for 1–6 months as compared to room air (RA)-exposed mice (WT) wild-type of lungs the in activity and expression) Irp1 in COPD with bronchitis chronic and MCC leads to excessive airway mucus, recurrent pulmonary infection dys airway-cell by impaired be function can motion, ciliary by mucus of exposure) exposure) of models experimental 6-month to (4- emphysema CS-induced COPD, namely CS-induced pathogenesis well-established the two in used IRP2 we of COPD, of role functional the characterize To Irp2-deficient mice resist experimental COPD RESULTS therapeutic novel COPD. for approach potential a highlighting COPD), (experimental injury and inflammation pulmonary CS-induced of model a in ease chelation using the siderophore deferiprone alleviates established dis cytochrome COPD by iron in and experimental mitochondrial loading regulating dysfunction mitochondrial promotes Irp2 wherein lung, the in Irp2 for role functional a characterize to first the are studies Our COPD. of models mouse experimental with data expression COPD human gene COPD. CS-induced of pathogenesis the in or exposure CS to lung the of response the in role a has IRP2 whether clear it is nor known, well not are by IRP2 targeted mRNA transcripts the and scripts tran the of stabilization or repression translational in results which homeostasis, iron in involved genes of mRNAs the in located (IREs) storage and increase iron uptake by binding to iron-response elements A  and Fig. 1 Fig.

Overexpression of Irp2 has been associated with embryonic embryonic with associated been has Irp2 of Overexpression Consistent with our from observations the lungs of human subjects Here we sought to delineate the function of the COPD susceptibility s e l c i t r Supplementary Fig. 2a Fig. Supplementary IRP2 12 d 2 , and and 1 , 1 1 7 9 Fig. 1a Fig. 17 5 ), as compared to RA-exposed WT mice ( mice WT RA-exposed to compared as ), 16 in the pathogenesis of CS-induced COPD by integrating integrating by COPD CS-induced of pathogenesis the in or infiltration of immune cells into the lung the into cells immune of infiltration or . The critical physiological function of IRP2 in the lung lung the in IRP2 of function physiological critical The . Fig. 1 Fig. , 1 4 , c 1 8 , we observed higher Irp2 expression (with no change change no (with expression Irp2 higher observed we , Supplementary Fig. 1a Fig. Supplementary oxidase. Notably,oxidase. we iron show also that mitochondrial 7 . MCC, which is characterized by the upward movement and CS-induced impairment of MCC (1-month (1-month MCC of impairment CS-induced and 2 – d 1 c ; we therefore used a loss-of-function approach to to approach loss-of-function a used therefore we ; and ). Irp2 expression was localized in lung epithelial epithelial lung in localized was expression Irp2 ). 1 Supplementary Fig. 2a Fig. Supplementary , . b ). We also observed higher Irp2 expression expression Irp2 higher We). observed also ). ). Irp2 −/− Supplementary Fig. 1c Fig. Supplementary , mice were also protected from from protected also were mice b ), as assessed by immunohis by assessed as ), Supplementary Supplementary Fig. 1b α -tubulin–positive) cells cells -tubulin–positive) ), and showed greater greater ), and showed Fig. 1 Fig. 2 Fig. 1 Fig. 0 . Impaired Impaired . e ). ). , d Fig. Fig. 1e in vitro in Irp2 ). ). Irp2 d and −/− , - - - - ­ f

Supplementary Fig. 2b Fig. Supplementary lated lated with deferoxamine (DFO) (to stabilize IRP2 expression stimu either Beas2B, line cell epithelial airway human the used we of RNA of in IRP2 targets the lung. Foridentification IRP2, unbiased of targets downstream the identifying first by COPD experimental therefore sought to delineate the mechanism by which Irp2 promoted We models. COPD experimental two in COPD of pathogenesis the in impacts functional important conferred Irp2 above, described As Identification of novel target pathways of IRP2 in the lung COPD. with associated injury lung CS-induced in IRP2 of role functional the of specificity the lighting failed to protect against lung injury (with increased numbers of BAL numbers increased (with injury lung against to protect failed IL-6 and WT mice ( IL-33 than CS-exposed CS-impaired MCC, and CS-exposed ( controls that of than lower RA-exposed to CS was significantly exposed lungs ( mice WT RA-exposed function lung in decline and exacerbations COPD acute of naive mice in inflammation airway induces that tissue lung in expressed nantly trations of BALF interleukin (IL)-33 (a pleiotropic predomi cytokine ( and permeability injury cell epithelial counts and greater total BALF protein levels (an indicator of increased (BAL) fluid (BALF) from WT mice CS-exposed had greater leukocyte MCC measuring and exposure) (1-month CS to acutely mice exposing by bronchitis CS-exposed and expression the markers LC3B autophagy apoptosis) increased (indi cating caspase-3 cleaved including emphysema, with to associated be shown previously markers injury CS-induced of expression the RIP-seq–derived communities for differential expression patterns patterns using a microarray studyexpression of lungs from WT and differential for communities RIP-seq–derived of ment each of the five of we the lung in IRP2, evaluated the absence of pathogenesis or ( COPD progression the in involved be to known were and COPD with subjects in expression altered had communities five ( shown is also ring), (black to controls as that of compared healthy (LGRC)), Consortium indi Research Lung the (from COPD with Genomics of viduals tissue lung the in genes of expression differential the plot, in plot Circos the by demonstrated word as as clouds), (depicted pathways or ‘communities’ functional core five analysis clustering and Methods enrichment Online (see functional used we genes, target system. model the validating ( (TfR) receptor transferrin the and ferritin as such targets, IRP2 known for transcripts included which 4a Fig. ( (RIP-seq) sequencing whole-transcriptome formed per and complexes IRP2-RNA immunoprecipitated unstimulated, polymicrobial sepsis mouse model ( puncture–induced lung and ligation acute cecal a in and hyperoxia-induced model mouse a injury in mortality and protein) IL-33 and IL-6 BAL and protein BALF of levels increased and leukocytes Supplementary Fig. Supplementary 2e We of model CS-induced an have experimental developed recently To human this data associate to the heterogeneous IRP2 the behind meaning biological the understand better To – Fig. 2 Fig. Supplementary Table 1 Supplementary c ). This analysis identified 1,806 IRP2-related target genes, genes, target IRP2-related 1,806 identified analysis This ). P 2 5 00) ( 0.05) < ) and BALF IL-6 (a cytokine associated with the severity severity the with ) associated and BALF (a IL-6 cytokine Irp2 a ). We observed that many of the genes in each of the the of each in genes the of many that We ). observed advance online publication online advance 17 −/− , 1 mice ( mice 8 ( Fig. Fig. 1 i. 1 Fig. , , Fig. 1 Fig. c f ). CS-exposed ). mice CS-exposed also had higher concen ). Irp1 expression did not change in RA-in not change or did expression Irp1 ). Supplementary Fig. 2d Fig. Supplementary 2 Supplementary Fig. 4g Fig. Supplementary h 2 h , matrix metalloproteinase 9 (MMP9) , metalloproteinase matrix ). ). In lavage bronchoalveolar this model, ). ). i ). Using this model, the MCC of WT WT of MCC the model, this Using ). ). Irp2 Supplementary Fig. Supplementary 3a Supplementary Fig. 4d Fig. Supplementary Irp2 −/− 2 4 ) than RA-exposed WT mice mice WT RA-exposed ) than mice were protected from from protected were mice −/− Fig. Fig. 1h mice exhibited less BALF 1 7 and Atg7 ( Irp2

Figure 2 Figure nature medicine nature , ). i −/− ). Irp2 deficiency ). Irp2 deficiency ) and identified identified and ) Supplementary Supplementary in in vivo mice ( Fig. Fig. 1 a – . On this this On . environ 2 – g Fig. 2 7 2 ), ), high f ) ) or left 6 ), thus thus ), ) than than ) g and b 2 ). 3 ------

© 2015 Nature America, Inc. All rights reserved. blood) cohorts. A strong association between the differential expres the differential between A strong association cohorts. blood) dinally to identify predictive surrogate end points’; gene expression in expression in lung tissue) and ECLIPSE (‘evaluation of COPD longitu expression using two human COPD namely cohorts, the LGRC (gene or low high with and individuals in COPD control with viduals indi in genes these of pattern expression the evaluated we COPD, chondrial genes ( mito to related was emerge to pathways chondrial main the of one that ( associated 2 genes community expressed with differentially the of analysis database the between genes of Irp2 expression differential having significant as most 2, the community community, one identified analysis This correction; Bonferroni with (ANOVA) variance of analysis ELISA, ( (right), clearance ( replicates). technical n ( tissue) lung of milligram per milliliter per caspase-3 cleaved of (picograms caspase-3 cleaved and WT in airways small the around protein (ECM) matrix extracellular of thickness and (top) WT from sections lung stained and WT months) (6 CS-exposed or RA- of (right) diameters 50 bars, Scale 6 months. for (right) CS or (left) RA to 50 bars, Scale shown. are (right) areas boxed of images higher-magnification and (left) images Lower-magnification months. 1–6 for (bottom) CS or (top) RA to exposed lungs ( months) (1–6 CS or RA to exposed lungs (middle; Irp total of right) and (middle quantification and (left) 1 Figure nature medicine nature in and differences genes of sion mitochondrial g e a = 3; = 3;

To assess the relationship between the differentially expressed mito Irp2 (normalized to –/– −/− Irp2 WT actin and RA)

Cleaved caspase-3 0 1 2 Irp2 Time ofCSexposure n and WT mice ( mice WT and

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(months) 0 function. ## : RA, : RA, mRNA (at 6 months; right) levels ( levels right) 6 months; (at mRNA ## WT # n.s. ** Fig. Fig. 3 i n ) BALF IL-33 (left, ELISA; WTRA WTRA ELISA; (left, IL-33 ) BALF Irp2 ** = 6; CS, CS, = 6; 6

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−/− Mean chord Irp2 Irp2 Biotin-IRE b n.s. n IRE-Ctl CS 6 Protein mice exposed to RA or CS (1 month). Throughout, data are mean mean are data Throughout, month). (1 CS or RA to exposed mice length Lm (µm) IRE:Irp = 3 per group). ( group). = 3 per 10 20 20 25 30 35 –/ 0 – −/− CS RA

m Free (bottom) mice exposed to RA (left) or CS (right). Scale bars, 50 50 bars, Scale (right). CS or (left) RA to exposed mice (bottom) biotin-IRE ** WT n + + + – – – = 8 per group). ( group). = 8 per h IRP2 a −8 + + Clearance – – n n ) Quantification of Irp2 protein in WT mouse lungs exposed to room air (RA) or CS (1–6 months) months) (1–6 CS or (RA) air room to exposed lungs mouse WT in protein Irp2 of ) Quantification µ

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** Irp2 CS ## −/− that CS-induced mitochondrial damage localized to alveolar and and alveolar to localized damage mitochondrial CS-induced that cytochrome abnormal ( with morphology mitochondria fewer developed cells airway mouse at to as and to compared CS, in controls, WT response baseline, morphology mitochondrial abnormal of levels higher had mice ( swelling and loss mitochondrial of cristae evidence with morphology, mitochondrial abnormal had CS to exposed mice WT from COPD. Wecells epithelial lung that found in experimental we humans, and mice role the assessed functional of IRP2 in responses to mitochondrial CS from analyses differential-expression and RIP-seq the in highlighted pathways functional the of basis the On Irp2 ( COPD with subjects in observed association stronger overall an with observed, CS RA n RA RA −/−

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n (fold change to RA) – = 5; = 5; < 0.005 by Student’s unpaired unpaired Student’s by < 0.005 0 1 2 3 4 mice resist CS-induced mitochondrial dysfunction = 3 per group) activity. ( activity. group) = 3 per n −/− = 4) electrophoretic mobility shift assay (EMSA) (left) and and (left) (EMSA) assay shift mobility electrophoretic = 4) f RA WT ** mice exposed to RA or CS for 6 months. ( 6 months. for CS or RA to exposed mice Irp2 Irp2–/– WT c CS Fig. 3b Fig. ## * , a marker of mitochondrial damage mitochondrial of marker a , Fig. 3 Fig. n −/− = 7 per group) (left) and MMP-9 (WT: MMP-9 RA, and (left) group) = 7 per c * n Irp2

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–/ to actin and RA) , = c ). Immunohistochemical staining for cytosolic cytosolic for staining Immunohistochemical ). – 0 1 2 3 and and exposure (months) CS RA 6 4 2 1 0 ) and BALF IL-6 protein concentrations (right, (right, concentrations protein IL-6 BALF ) and Time ofCS Supplementary Fig. 6 Fig. Supplementary i ±

s.e.m. * s.e.m. IL-33 (pg/ml BAL) n n 100 150 = 2 technical replicates) of trichrome- of replicates) = 2 technical c 50 = 10) for 1 month. 3 h 1 month. for = 10) µ ) Irp1 protein expression in WT mouse mouse WT in expression protein ) Irp1 n.s. 0 L m. Right, quantification of the the of quantification Right, m. m 6 (middle) and weighted mean mean weighted and (middle) WT ## Irp2 t P -test; n.s., not significant. not n.s., -test; d < 0.05, ** < 0.05, Irp2 ** CS RA n.s. −/− –/ (bottom) mice exposed exposed mice (bottom) – CS RA Thickness of ECM protein (µm) around small airways P IL-6 (pg/ml BAL) ). < 0.01 by one-way one-way by < 0.01 g 300–699 m in diameter µ s e l c i t r a 30 40 50 10 20 ) Quantification of ) Quantification 20 40 60 80 2 0 0 8 Fig. 3 Fig. 99m , demonstrated demonstrated , n WT # = 2 WT n Tc- ** ** = 5: CS, CS, = 5: d Irp2 sc CS RA ). ). *

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© 2015 Nature America, Inc. All rights reserved. consumption ( consumption oxygen inhibit thus and III complex through electrons of flux the inhibit to injected A was antimycin and respiration, ATP-linked the differentiate to used and WT of (right) ( (top). TMRE of (MFI) intensity fluorescent mean ( replicates). ( ( 4 months for (right) CS or (left) ( TEM. by assessed as mitochondria, abnormal of quantification Right, mitochondria. m, nuclei; n, mitochondria; ‘abnormal’ indicate arrowheads Red nm. 500 bars, scale fields; EM (15 4 months for (right) CS or (left) RA to exposed airways ( expression. ( non-smokers) ( red. in are genes OxPhos Mitochondrial 3 Figure RA-exposed from those did than mitochondria, healthy in mulation accu by cated of uptake a JC1, potential-dependent dye that exhibits ( potential membrane lower had mice WT CS-exposed ( exposure CS after cells thelial negligible staining for cytosolic cytochrome mice. WT in cells epithelial airway Irp2 ( pathways. metabolism purine and nucleotide in involved genes for enrichment 5 shows Community translation. and transcription proteins, of metabolism the in involved genes for enrichment 4 shows Community transduction. signal and cycle cell the repair, apoptosis, DNA in involved genes for enrichment 3 shows Community trafficking. membrane and mitochondria metabolism, in involved genes for enrichment 2 shows Community system. immune the and proteasome the RNA, of metabolism the cycle, cell the in involved genes for enrichment 1 shows Community community. the in pathways the of names the among frequency its reflecting word each of size the with community, each in terms GO represent Word clouds COPD. with individuals in higher, expression denotes magenta lower, and denotes Green controls. and COPD with subjects between levels expression gene in (FC) change fold COPD; with subjects ( score peak the to corresponding bars the of height the with rings, individual by represented are sets data peak (purple) common and (red) DFO-specific (blue), (Ctl)-specific Control set. data RIP-seq the in altered and/or 2 Figure A  unpaired Student’s by b f

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Novel targets of IRP2 in the lung. ( lung. the in IRP2 of targets Novel Irp2 d g −/− ) Representative plots of TMRE staining (bottom; (bottom; staining TMRE of plots ) Representative ) Left, representative transmission electron microscopy (TEM) images ( images (TEM) microscopy electron transmission representative ) Left, i b ) Schematic of the role of IRP2 in mitochondrial responses to CS exposure. Throughout, data are mean mean are data Throughout, exposure. CS to responses mitochondrial in IRP2 of role the of ) Schematic ) and ECLIPSE ( ECLIPSE ) and mice resist CS-induced mitochondrial dysfunction. ( dysfunction. mitochondrial CS-induced resist mice Irp2 ity 3 mun n om C −/− e C o = 20 non-smokers; = non-smokers; 20

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= 84 smokers, smokers, = 84 C ) Quantification of OCR (left) and ECAR (middle), normalized to Hoechst, and of the OCR:ECAR ratio ratio OCR:ECAR the of and Hoechst, to normalized (middle), ECAR and (left) OCR of ) Quantification ∆ Ψ n ), as indi as ), = 3 experiments; = 3 experiments; n n = 6 non-smokers) ( = 6 non-smokers) = 2 biological replicates). Gene expression data (mRNA) from the LGRC ( LGRC the from (mRNA) data expression Gene replicates). = 2 biological c a immunostaining of WT (top) or or (top) WT of immunostaining n ) Community 2 genes that annotated to ‘mitochondria’ GO categories. categories. GO ‘mitochondria’ to annotated that 2 genes ) Community ). = 1 per group). Insets show expanded images of single mitochondria. All All mitochondria. single of images expanded show Insets group). = 1 per

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© 2015 Nature America, Inc. All rights reserved. nature medicine nature d Slc25a24 a Timmdc1 g Sec23B Nup133 Abhd10 Sec11a Sec22a Ndufb9

Count Relative MFI Ndufa6 Psma6 Nucb2 Cyb5b Lrpprc Ssbp1 Vdac2 Cox8a Cox18 Sptlc2 –/– Mtmr Anxa7 20 40 60 Uqcrh Insig2 Hars2 Chst3 Crks1 Mon2 Pi4kb Sdh Coa3 Cdk7 Sun2 lrp2 WT Nck1 Mfn1 Myof 0 Mcl1 Txn2 (normalized to WT Ctl) Glg1 Ssr1 Cnih Stx5 Ipo8 10 0.5 1.0 1.5 b 0 6 0 –2.0 m 2 1 10 m

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–/ –4 0 –/ – – h h Percentage abnormal 4 45 0

mitochondria per unit area of High h h FCCP Time (min) 63 cytoplasm

10 20 30 40 50 IRP2 CS RA 81 0 –2 + rotenone Antimycin 99 WT 135 * n.s. t -statistic

135 ** lrp2 2 0 n.s. A 153 –/ –

ECAR (mpH/min/µg DNA) e –/– Low

100 150 lrp2 WT controls Non-smoker Smoker COPD subjects 50 0 IRP2 Oligomycin 9 Irp2 Irp2 WT 4 WT 0 27

–/ –/ 4 – – h h 45 4 0 h h Time (min)

63 FCCP

81 SLC25A24 c TIMMDC1 + rotenone Antimycin LRPPRC 99 NDUFB9 NDUFA6 ABHD10 SEC23B SEC11A SEC22A NUP133 SPTLC2 MTMR PSMA6 NUCB2 HARS2 COX8A SSBP1 INSIG2 CRLS1 COX18 CHST3 ANXA7 MON2 MYOF PI4KB SDHB MFN1 COA3 MCL1 GLG1 NCK1 CDK7 135 SUN2 SSR1 TXN2 CNIH STX5 IPO8 135 6

153 A –10

OCR:ECAR pmol/mpH High 0.5 1.0 1.5 2.0 2.5 0 CS RA IRP2 –5 WT * n.s. ** Irp2 Percentage JC1 uptake f ** t -statistic –/ (compared to WT RA) – 100 120 5 0 20 40 60 80 i 0 Irp2 WT CS CS s e l c i t r a –/ WT – # Low Glycolysis n.s. Glycolysis controls Non-smoker Smoker COPD subjects Phos lrp2 Phos Fatty acids IRP2 Ox Ox n.s. Fatty acids –/ – 10 CS RA ∆ψ ∆ψ  © 2015 Nature America, Inc. All rights reserved. controls ( controls RA-exposed from those to compared as CS, to exposed lungs mouse WT of fractions mitochondrial in higher were levels iron heme and Non-heme mitochon biosynthesis. into cluster (Fe-S) iron-sulfur imported or heme for be dria may it or proteins, iron-containing targeting (shRNA) RNA hairpin short a with treatment (by IRP2 in deficient cells epithelial airway human CSE-exposed in change not did levels ( lungs CS-exposed in iron less deposition revealed iron deposits, ferric detects Blue, which WT CS-exposed mice ( similar total non-heme iron levels in whole lung tissue, as compared to CS-exposed of mice. airways the in iron deposition of increased COPD, models CS exposure that in gested experimental ( RA to compared as CSE was in higher human cells after to epithelial exposure airway aqueous and ferritin, respectively ( TfR proteins target Irp2 the of expression lower and higher had also ( mice RA-exposed than homogenates whole-lung in and sections lung inflated in iron non-heme had higher mice WT mice, WT in CS-exposed expression IX) protoporphyrin with complexed (Fe ferric the in iron in the lung. In body tissues, iron (Fe) is found either as non-heme free lung. the in pathways iron mitochondrial in responses to CS ( mitochondrial IRP2-mediated observed the whether investigate to dysfunction mitochondrial to lead can which of overload, both iron mitochondrial excessive or loss iron mitochondrial regulation iron a mitochondrial rely on precise and processes cellular of vital number iron, intracellular of consumers main the are Mitochondria IRP2 promotes CS-induced mitochondrial iron loading ( dysfunction mitochondrial to response adaptive an as metabolism to anaerobic (OxPhos) aerobic from switching regulates glycolysis ( on reliance less with metabolism mitochondrial aerobic of enhanced indicative to of relative glycolysis, OxPhos levels greater by characterized was which ratio, OCR:ECAR in response opposite CSE-exposed glycolysis. to OxPhos from olism ratios than RA-exposed cells, indicative of cells shifting energy metab epithelial cells ( and WT in CSE-exposed activity and glycolytic for OxPhos of capacity reserve and surrogate rates the a determined we glycolysis), (ECAR; acidification phosphorylation extracellular oxidative and of (OxPhos)) marker a (OCR; rate consumption oxygen of measurements real-time through function mitochondrial CS-induced of COPD. experimental regulator in dysfunction a mitochondrial as IRP2 implicate data these together, (mtROS) species oxygen tive reac mitochondrial of CSE-induced production the from and death for RNA specific interfering (siRNA) short with treatment of result a (as IRP2 in cient charge ( negative relative A  transporter iron membrane mitochondrial inner the of use the on Fig. 3 Fig.

We first investigated whether CS regulated general iron metabolism on information provides which apparatus, XF96 Using Seahorse a Once inside the cell, free iron may be delivered to ferritin to delivered be may iron free cell, the inside Once s e l c i t r Supplementary Fig. 8b Fig. Supplementary h ). These results suggest that, in lung epithelial cells, IRP2 IRP2 cells, epithelial lung in that, suggest results These ). IRP2 Fig. 4c Fig. 2 9 . Failure to control mitochondrial iron levels leads to to leads levels iron mitochondrial control to Failure . ) ( ) Fig. 3 Irp2 Fig. 3d , Supplementary Fig. 8a Fig. Supplementary d ). Increased mitochondrial iron import is reliant reliant is import iron mitochondrial Increased ). −/− Fig. 4 Fig. h 3+ IRP2 ). CSE-exposed WT cells had lower OCR:ECAR mouse lungs than in CS-exposed WT mouse mouse WT CS-exposed in than lungs mouse ) or ferrous (Fe ferrous or ) Fig. Fig. 4 – Fig. Fig. 3 h Fig. Fig. 4 a Supplementary Fig. 8a Fig. Supplementary ) ) were related to IRP2-mediated alterations ) were protected from CSE-induced cell cell CSE-induced from protected were ) ). Whole-lung homogenates of WT mice mice WT of homogenates Whole-lung ). c ); ); however, staining with Perls’ Prussian 1 g ). Furthermore, intracellular free iron free intracellular Furthermore, ). 7 b ). Human airway epithelial cells defi cells ). Human epithelial airway ( ). ). Additionally, intracellular free iron Supplementary Fig. 8a Fig. Supplementary 3 0 2+ . Consistent with greater Irp2 Irp2 greater with Consistent . ) state or as heme iron (Fe iron heme as or state ) ). Irp2 Irp2 ). These data sug data These ). Irp2 −/− −/− 2 primary lung primary 9 cells had the the had cells Fig. 3 Fig. −/− . We wished Wewished . , b mice had mice ). Taken ). i ). 3 0 and and 2+ - - - - -

ously ously shown that heme oxygenase 1 (HO-1), an enzyme that catalyzes previ Wehave loading. iron mitochondrial CS-induced promoted ( levels iron heme mitochondrial and iron non-heme mitochondrial higher CS-associated from protected ( mice WT CS-exposed to compared as expression, 2 mitoferrin and COPD. experimental in loading iron mitochondrial induced CS to exposure that demonstrate data airway epithelial cells ( impaired and mitochondria Fe-S cytosolic cluster assembly in human ( unchanged repeat protein 5 a also (Fbxl5, negative regulator of Irp2 (ref. iron and protein of iron-binding F-box the and cytosolic leucine-rich RA-exposed mice, but the expression from of cytosolic tissue non-heme in and than heme mice CS-exposed from tissue lung in lower was ( CSE with treated cells epithelial airway human in as well as mice homogenates WT of whole-lung in exposure CS frataxin with regulator associated Fe-S also was mitochondrial the of loss loading, increased of iron indicator Another mitochondria. into iron of import ( controls RA-exposed in than mice (ref. 2 mitoferrin function ( function non-heme and heme iron levels, impaired significantly MCC baseline ko); (knockout, frataxin of deletion a heterozygous and ki) (knock-in, frataxin of mutated sion) (GAA insertion transgenic heterozygous a with mice (i.e., ( point time and WT between loading iron mitochondrial ( differences significant observed we as esis, hypoth our test to model exposure 1-month acute this use to chose We model. mouse (bronchitis) of MCC impairment CS-induced our expression frataxin whether evaluated mice with iron (i.e., reduced loading we mitochondrial mice with higher COPD, experimental in pathogenic is load death. cell mitochondria-mediated from protection baseline mice WT ( CS-exposed did than exposure, CS after change not did of lungs the regulating in role death cell protective mitochondria-mediated a CS-induced have may it that and lungs mouse CS-exposed in elevated is iron, produce to heme of degradation the the mitochondrial electron transport chain and is responsible for and chain the is responsible transport electron the mitochondrial activity and COX protein subunit I that stabilizes drial and promotes its assembly mitochon a (CCDC56), 56 protein domain–containing coiled-coil in ( 3 factor assembly COX expressed gene differentially ( significant ( analysis expression gene community-based and study RIP-seq our in identified genes mitochondrial the to attention our we focused dysfunction, mitochondrial and CS-induced loading iron Tomitochondrial of the investigate mechanism IRP2-associated IRP2 and CS increased lung COX activity and expression ( models COPD mental CS-induced mitochondrial iron loading may be pathogenic in experi ( protein Supplementary Fig. 8h Supplementary i. 4d Fig. Irp2 To assess whether abnormally increased mitochondrial iron over iron mitochondrial increased abnormally To whether assess Irp2 i. 4 Fig. −/− −/− , Fig. 4g Fig. e mice were protected from the effects of CS on ferritin ferritin on CS of effects the from protected were mice mice than in WT mice ( mice WT in than mice and and P < 0.05) and higher baseline levels of whole-lung IL-6 IL-6 whole-lung of levels baseline higher and 0.05) < 3 Fig. 4 Fig. Supplementary Fig. 8d Supplementary Irp2 f 4 and and ( advance online publication online advance , Fig. 5 Fig. h upeetr Fg 8d Fig. Supplementary −/− 3 ) as compared to WT mice. These data suggest that suggest data ) These to as mice. WT compared d 1 3 ). Here we found that mice deficient in frataxin frataxin in deficient mice that found we Here ). mice had higher baseline levels of HO-, which which HO-, of levels baseline higher had mice ). Mitoferrin 2 levels were higher in CS-exposed CS-exposed in higher were levels 2 Mitoferrin ). upeetr Fg 8c Fig. Supplementary 2 ) had differential responses to CS exposure in in exposure CS to responses differential had ) a Supplementary Fig. Supplementary 8g ). COX is the terminal electron acceptor of of acceptor electron terminal the is COX ). Fig. 4 Fig. ), suggesting ), that suggesting the Coa3 Fxn i ), which was expressed at lower levels levels lower at expressed was which ), ). ki/ko mice) had higher mitochondrial mitochondrial higher had mice) Figs. 3 Figs. – f Fig. 4 Fig. Fig. 4 Fig. ). ). Additionally, CSE treatment P ). ). < 0.01) in this analysis was a e d and and Irp2 P ), suggesting increased increased suggesting ), ), suggesting that IRP2 IRP2 that suggesting ), Irp2 ). Ferritin expression expression Ferritin ). < 0.05) in CS-induced CS-induced in 0.05) < ). ). Taken together, these 3

3 Irp2 −/− . . Here that we show nature medicine nature 5 −/− Fig. 3 Fig. a mice were also also were mice ). ). mice have more −/− Coa3 mice at this this at mice a ). The most most The ). 230 encodes encodes 2 expan 1 )) )) was 3 2 ------,

© 2015 Nature America, Inc. All rights reserved. heme and is the site of iron reduction and transport into mitochon into transport and reduction iron of site the is and heme metabolism iron drial mito for ( apoptosis and integrity important chondrial proteins other to addition in analysis, this to in expressed differentially also cytochrome ( sulfur iron B, subunit complex, ( 9 subcomplex, beta 6 ( subcomplex, alpha 1 (Nduf) (ubiquinone) nase Cox8A subunits and factors important for COX assembly (such as other encoding genes of water. number A into oxygen of conversion 4 Figure nature medicine nature dria one-way ANOVA followed by Bonferroni correction; correction; Bonferroni by followed ANOVA one-way mean are data Throughout, Irp2. by loading iron mitochondrial of regulation of ( concentrations protein IL-6 and mean) ( iron heme mitochondrial (WT: 6 months RA, months), (4 CS ( replicates). technical and WT (for iron and WT (for iron non-heme in changes n (RA, ( control. ( CS or RA to exposed lungs mouse WT in (FT-H) expression chain heavy ferritin ( control. negative NC, (right; levels iron non-heme and (middle) group) 50 bars, Scale staining. indicate group; per mice = 3; CS (6 months), months), (6 CS = 3; f c a It has that proposed been COX acts as a key regulator of mitochon Frataxin expression Non-heme iron 3

n (normalized to actin (µg/ml/g lung tissue) 5 = 11; CS (1 month), month), (1 CS = 11; 10 15 , where it supplies iron for heme biosynthesis heme for iron supplies it where , and WT RA) 50 ) and mitochondrial OxPhos (including NADH dehydroge NADH (including OxPhos mitochondrial and ) 0.5 1.0 1.5 0 0 0

c 0 IRP2-associated mitochondrial-iron loading and CS exposure. ( exposure. CS and loading mitochondrial-iron IRP2-associated ) Quantification of total non-heme iron ( iron non-heme total of ) Quantification ## WT ** WT * c reductase hinge protein ( protein hinge reductase n n.s. n Irp2 = 6) after exposure to RA or CS. ( CS. or RA to exposure after = 6) * Irp2 = 2 technical replicates) from WT mice exposed to RA (left) or CS (right) for 1–6 months (6-month time point is shown). Arrowheads Arrowheads shown). is point time (6-month months 1–6 for (right) CS or (left) RA to exposed mice WT from replicates) = 2 technical n.s. Irp n * ** –/

= 5; CS, CS, = 5; 2 −/− – –/– b n e advance online publication online advance CS RA Nduf9 ; RA, ; RA, ) Quantification of mitoferrin 2 (Mfn2) expression in whole lungs of mice (for WT and and WT (for mice of lungs whole in expression 2 (Mfn2) mitoferrin of ) Quantification ) Representative immunoblot analysis ( analysis immunoblot ) Representative = 5) non-heme iron (right) in WT mouse lungs exposed to RA or CS ( CS or RA to exposed lungs mouse WT in (right) iron non-heme = 5) 3 CS 6months CS 1month RA 5 n . Specifically, COX subunit I is regulated by regulated is I subunit COX Specifically, . = 4 per group) ( group) = 4 per g n Mitochondrial non-heme iron n = 6; CS (4 months), months), (4 CS = 6; ) (complex I), succinate dehydrogenase dehydrogenase succinate I), (complex ) n = 10, CS-exposed, CS-exposed, = 10, (µg/ml/g mitochondrial protein) = 3; = 3; 0 1 2 3 4 5 µ m. Middle and right, quantification of Perls’-stained lung areas at 6 months ( 6 months at areas lung Perls’-stained of quantification right, and Middle m.

Irp2 WT Sdhb

Irp2 Fxn Fold change in non-heme # Fig. 3 Fig. ki/ko Irp2 iron (normalized to WT RA) n −/− 0.5 1.0 1.5 2.0 2.5 = 4 per group) ( group) = 4 per −/− ) (complex II) and ubiquinol– and II) (complex ) 0 −/− versus WT mice according according mice WT versus

g Percentage lung area : RA, : RA, Uqcrh Time ofCSexposure a , right), 3-h 3-h , right), 1 0

mice: RA, RA, mice: Perls’ stained ). Mitochondrial heme iron 0 1 2 3 4 (µM/mg) Cyto Mito n (months) 0 2 4 6 NC n = 4 mice per group; group; per = 4 mice n n n ) (complex III)) were were III)) (complex ) = 6; CS, CS, = 6; # f = 5 mice per group; group; per = 5 mice = 5 per group) (right) in mitochondrial fractions from mouse lungs exposed to RA or CS ( CS or RA to exposed lungs mouse from fractions mitochondrial in (right) group) = 5 per = 8; CS (6 months), months), (6 CS = 8; ) Quantification of frataxin protein expression in whole lungs of mice exposed to RA or CS for for CS or RA to exposed mice of lungs whole in expression protein frataxin of ) Quantification P

WT n.s. RA ** 4 < 0.05, < 0.05, Fxn # Nduf66

ki/ko ** n.s. *** CS n 99m

6 h = 13; CS (1 month), month), (1 CS = 13; , right) in the lungs of WT and and WT of lungs the in , right) 3 5 n Tc- h . Consistent Consistent . = 6). ( = 6). d ##

) ) and Nduf Percentage of 3-h n

Cox18 Non-heme iron

sc 99m Fold change = 3 experiments) (left) and quantification (right) of transferrin receptor (TFRC) and and (TFRC) receptor transferrin of (right) quantification and (left) = 3 experiments) P clearance of Tc–SC mitochondrial non-heme (µg/mg lung tissue) clearance ( clearance –10 < 0.01, < 0.01, 100 150 –5 10 5 0 iron (normalized to WT RA) 50 0 0 1 2 3 g , and h Time ofCSexposur n ) Quantification of mitochondrial non-heme iron ( iron non-heme mitochondrial of ) Quantification #### 6 4 1 0 = 4 technical replicates) in WT mice exposed to RA or CS for 1–6 months. months. 1–6 for CS or RA to exposed mice WT in replicates) = 4 technical a n n **** ) Left, representative micrographs of Perls’-stained lung sections ( sections lung Perls’-stained of micrographs representative ) Left, - - - ­ = 4 technical replicates) (left) and fold changes in mitochondrial (mito) (mito) mitochondrial in changes fold and (left) replicates) = 4 technical WT *** = 9) and cytosolic (cyto) (RA, (RA, (cyto) cytosolic and = 9)

WT #### n.s. *** (months) h ** CS-exposed CS-exposed WT mice. (ref. Cox4i2 lack that tissues other in COX than active more twofold COX lung renders which (Cox4i2), COX II and ( I complexes of expression the in reduction CS-induced less CS-exposed from lungs in than mice WT expression CS-exposed COX from lungs that in higher was observed we chain, transport electron drial control from ( those subjects in than COPD with of individuals from biopsies expression altered with ( and expression COX-related mitochondrial genes in WT Irp2 as compared to higher with , left; each circle represents a single mouse, and horizontal lines denote denote lines horizontal and mouse, a single represents circle each , left; Supplementary Fig. 9a Fig. Supplementary Figs. 3 Figs. ± P n *** n s.e.m. * s.e.m. Using an antibody that detects each complex of the mitochon the of complex each detects that antibody an Using < 0.001 by Student’s unpaired unpaired Student’s by < 0.001 = 5 per group; group; = 5 per = 5; CS (4 months), months), (4 CS = 5; n.s. Irp2 *** CS 6months CS 4months CS 1mont RA Fxn n.s. e –/– a ki/k and and Fig. 5 Fig. TFRC P o FT- b Acti Acti Fxn CS RA < 0.05, ** < 0.05, Irp2 h H n n n ki/ko 5 = 2 technical replicates). ( replicates). = 2 technical a a −/−

). IL-6 (pg/ml)/mg) ), we found that COX expression is higher in lung lung in higher is expression COX that found we ), n Fold change mice exposed to RA or CS for 1 month. ( 1 month. for CS or RA to exposed mice 300 400 100 200 = 2 technical replicates). Actin was used as a loading a loading as used was Actin replicates). = 2 technical

mitochondrial heme Iron RA mice ( 0 (normalized to WT RA) P 0. 1. 1. 2.

n WT Irp2 < 0.01, *** < 0.01, 5 0 5 0 0 = 3; CS (6 months), months), (6 CS = 3; Fxn ). Expression of the lung-specific isoform of isoform lung-specific of the ). Expression # n ki/ko Fig. 5 n.s. = 6; CS (1 month), month), (1 CS = 6; −/− WT CS # Irp2 mice had lower Cox4i2 expression than i n b t CS 6months CS 1mont RA = 10 images per mouse; mouse; per images = 10 -test; n.s., not significant. not n.s., -test; Irp2 −/− ). ). iron loading Mitochondria n.s. P Irp2 : RA, : RA, –/–

< 0.005, **** < 0.005, Fold change CS (normalized to actin and RA) 0. 1. 1. 2. 2. −/− h d n 0 5 0 5 0 5 n ) Quantification of fold fold of ) Quantification = 4 per group) ( group) = 4 per = 8; CS (1 month), month), (1 CS = 8; mouse lungs also exhibited Irp2 l e Time ofCSexposure n Mitoferrin 2 expression 4 2 1 0 = 5) (left) and heme heme and (left) = 5) n 3

= 11; CS (4 months), months), (4 CS = 11; (normalized to 6 (months

), was also higher in in higher also was ), actin and WT RA) 0 1 2 3 4 Mfn2 P Heme s e l c i t r a < 0.001 by < 0.001 Non-heme iron ) * * * *** WT Fx i ) Schematic ) Schematic 6 * * n g n Irp2 n = 3 per = 3 per , left), , left), *** n Irp2 = 3 CS 4months CS 1mont RA n.s. = 2 TFRC FT- n −/− –/– = 3; H

mice

h

 -

© 2015 Nature America, Inc. All rights reserved. we used mice with impaired COX (activity and expression) in our our CS-induced in bronchitis model. COX expression) is a and multi-subunit complex whose (activity COX impaired with mice used we from cells than epithelial airway CSE-exposed activity and expression COX greater had cells epithelial lung Irp2 mouse lungs, but it did not change in from mitochondria CS-exposed WT from CS-exposed in mitochondria higher was also COX activity 4 months, as compared to those from RA-exposed controltions isolated from miceWT mice that were ( to exposed CS for 1 month and 9b Fig. WT mice, which did not change after exposure to CS ( correction; mean are data Throughout, loading. iron mitochondrial ( iron heme ( iron non-heme lung total (WT: IL-6 RA, ( mean. denote lines horizontal and mouse, one and WT in (right) levels protein BALF total and (middle) leukocytes BALF total or WT from cells ( analysis and WT of fractions mitochondrial in exposure CS of 4 months after activity COX total and (left) time over activity COX in changes of ( expression IV complex of quantification and WT from fractions ( ( (right) analysis COPD with individuals from tissue lung in expression COX and (middle) assembly COX of schematic normalized), ( 5 Figure A  by encoded that including factors ancillary >20 requires assembly n n e c a

= 3 experiments) of OxPhos complexes I–V (CI–CV) in mitochondria-enriched mitochondria-enriched in (CI–CV) I–V complexes OxPhos of = 3 experiments) and = 6)

To examine the function of COX in the response of of lung to Tothe COX CS, in response the function the examine Percent 3-h clearance Coa3 s e l c i t r 99m Fold change COX 10 11 0 6 8 9 −/− of Tc–SC 10 15 activity (nmol/min/mg 0 5 os lns ( lungs mouse ). Similarly, COX activity was higher in mitochondrial frac mitochondrial in higher was activity COX Similarly, ).

normalized to WT RA) WT n COX is pathogenic in experimental COPD. ( COPD. experimental in pathogenic is COX 0 1 2 3 = 3 experiments) as in in as = 3 experiments) # n Irp2 exposure (months) WT *** P = 3 per group) ( group) = 3 per < 0.05, < 0.05, 1 0 n Irp2 Time ofCS = 4; CS, CS, = 4; −/− n # Irp2 = 2 technical replicates). ( replicates). = 2 technical –/ ( * – Sco2 * n −/− = 5) mouse lungs (left) (robust multiarray average (RMA) (RMA) average multiarray (robust (left) lungs mouse = 5) CS RA ## Irp2 ki/k ** mice exposed to 20% CSE ( CSE 20% to exposed mice 4 P n o n < 0.01, < 0.01, i. 5 Fig. = 5; = 5;

−/− Sco2 = 4 per group) ( group) = 4 per Coa3 h mouse lungs exposed to RA or CS for 4 months (left) and and (left) 4 months for CS or RA to exposed lungs mouse , right) levels in WT and and WT in levels , right) 4 COXII COX activity COXI Total cells × 10 /ml BAL Sco2

10 15 20 25 (normalized to WT RA) 0 5 c 0 1 2 3 b b ( assembly (n = (n ). Additionally, CSE-exposed WT WT CSE-exposed Additionally, ). ### COX ki/ko WT left), time course of COX activity (middle) and COX activity after 4 h of exposure (right) in primary lung epithelial epithelial lung primary in (right) exposure 4 h of after activity COX and (middle) activity COX of course time left), Irp2 ** P n.s. 5) and controls ( controls and 5) *** WT < 0.005 by unpaired Student’s Student’s unpaired by < 0.005 : RA, : RA, Irp2 n −/− n.s. = 3 technical replicates) (right). ( (right). replicates) = 3 technical COXII g –/ mice exposed to RA or CS ( CS or RA to exposed mice b ; ; – Irp2 ) Representative immunoblot analysis analysis immunoblot ) Representative CS RA n n COXI Cytochrome f c oxidase = 5; CS, CS, = 5; = 3 technical replicates), mitochondrial non-heme iron ( iron non-heme mitochondrial replicates), = 3 technical ) Quantification of BALF IL-33 (WT: IL-33 RA, BALF of ) Quantification Sco2 −/− CSE (h) n.s. Tom20 d n CS RA a Actin ki/k mice ( mice = 2 per group; group; = 2 per ) ) CIV CIII CV CII COXIII o CI Sco2 Coa3 ± n s.e.m. * s.e.m. n Supplementary Supplementary = 5) by immunoblot immunoblot by = 5) = 4) levels for 1 month, using ELISA ( ELISA using 1 month, for levels = 4) ki/ko expression in WT in expression Total BAL protein (mg) 0 Fig. 5 Fig. WT

0.1 0.2 0.3 0.4 0.5 0

mice exposed to RA or CS for 1 month. ( 1 month. for CS or RA to exposed mice 2.0 COX expression

P 4.0

Fig. 5 (normalized to TOM20 < 0.05, ** < 0.05, d and Ctl) Irp2 WT

). 0 n 0 1 2 3 #

= 2 technical replicates). ( replicates). = 2 technical 0.5 –/ n c t 2.0 – -test; n.s., not significant. not n.s., -test; ). = 3 per group; group; = 3 per Ctl - 4.0

12 kDa 30 kDa 35 kDa 58 kDa c COPD Sco2 ) Quantification ) Quantification ### P Conversely, Conversely, ( levels protein BALF total WT mice had impaired MCC, higher BAL leukocyte counts and higher of MCC than deficiency COX severe more (with activity COX impaired and defects bly of a either boring of deletion non-smokers and smokers healthy of lungs the to in COPD with as is the lungs of compared higher expression subjects of analysis gene (SAGE), expression we showed previously that ‘synthesis of cytochrome CS RA

Sco2 n.s. < 0.01, *** < 0.01,

Sco2 ki/k o ki/ko COX activity (nmol b Tom20 on the other allele, or two ki mutations, have COX assem COX have mutations, ki two or allele, other the on i cyt. c oxidase/min/mg) Actin Sco2 mice exposed to RA or CS for 1 month. Each circle represents represents circle Each 1 month. for CS or RA to exposed mice

IL-33 (pg/ml BAL) CIV 0 2 4 6 CIII CV CS CII n 40 50 10 20 30 n CI exposure (hours) f 0 = 3 technical replicates). ( replicates). = 3 technical = 4; CS, CS, = 4; Sco2 0 CS Time ofCS P ki/ko iron loading Mitochondrial Sco2 < 0.005 by one-way ANOVA followed by Bonferroni Bonferroni by followed ANOVA one-way by < 0.005 0.5 ### WT advance online publication online advance WT

Sco2 is embryonic lethal in mice in lethal embryonic is 2.0 and and n # Sco2 e ki/ko + + = 3 technical replicates). ( replicates). = 3 technical ) Quantification of 3-h 3-h of ) Quantification 4.0 n.s. n # ki mutation on one allele and a deletion (ko) (ko) deletion a and allele one on mutation ki CS Irp2 RA = 5; = 5; ki/k Sco2 and and o n i Irp2 c ) Schematic of the role of COX Irp2–associated Irp2–associated COX of role the of ) Schematic COX activity (nmol = 3 per group) ( group) = 3 per oxidase’ ( Sco2 ki/ki IL-6 (pg/ml BAL) cyt. c oxidase/min/mg) + + –/ 20 25 10 15 Fig. 5 Fig. 0 2 4 6 0 5 – Sco2 mice that were exposed acutely to CS. to CS. acutely exposed were that mice Non-heme iron ki/ko + COX *** WT WT ## Sco2 ki/ki e : RA, : RA, # 12 kDa 30 kDa 35 kDa 58 kDa Sco2 Irp2 ** and and Sco2 SCO2 ## d ki/ko ) Representative immunoblot immunoblot ) Representative mice were protected from from protected were mice ## –/– ki/ko ki/ki n CSE Ctl 99m = 5; CS, CS, = 5; h Supplementary Fig. 9c Fig. Supplementary CS RA ) , left) and mitochondrial mitochondrial and , left) 3 mice 7 Tc- g ( g , Sco2 Fig. Fig. 5

h COX expression (normalized h 3 Fold change Non-heme iron sc 7 ) Quantification of ) Quantification to Tom20 and WT RA) ; however, mice har mice however, ; mitochondrial non-heme (µg/ml/g lung tissue) clearance (left), (left), clearance 0.5 1.0 1.5 2.0 2.5 3 Mitochondrial heme iron 100 150

7 iron (compared to WT RA) n nature medicine nature 50 0 ki/ko ). We assessed the the We ). assessed 1.5 0.5 1.0 0 = 4) and BALF BALF and = 4) (µM/mg protein) a 0

2 4 6

0 8 3 ). ). By using serial 8 . Homozygous Homozygous . mice showing showing mice WT WT ** WT *** WT *

# n.s. n.s. # n.s. ** Sco2

n.s. Sco2 Sco2 Irp2 * n.s.

n.s. * n.s. ki/ko –/– ki/ko ki/ko SCO2 , CS CS CS CS RA RA RA RA d ). ). - -

© 2015 Nature America, Inc. All rights reserved. in mitochondrial non-heme iron ( iron non-heme in mitochondrial mice, ( levels mice iron WT heme and RA-exposed non-heme than mitochondrial higher as well levels iron as non-heme total-lung mice, higher had WT mice in WT obtained CS-exposed results the with Consistent month). 1 (for and WT in heme) levels, and (non-heme iron mitochondrial and levels, iron cytosolic as non-heme well as tissue total-lung measured we Irp2, of loss the with observed that to similar manner a in iron mitochondrial of COPD. experimental in pathogenicity confers activity COX 9d Fig. Supplementary ( mice WT CS-exposed than counts BAL leukocyte lower Sco2 than (with MCC of loss CS-induced correction. Bonferroni by followed ANOVA one-way ( addition. DFP of time in described strategy ( replicates) technical control DFP n 4 weeks, + DFP: weeks) (4 CS line; (blue exposure) CS of duration the for continued and arrow) blue by (indicated exposure CS 4-week 4 weeks, only: CS line; dashed ( addition). DFP of time indicate arrows (blue strategy dosing a therapeutic as or strategy dosing a prophylactic as either DFP with treated and ( mean. denote lines horizontal and a mouse, represents circle Each diet. iron) carbonyl (2% high-iron or iron) p.p.m. (6 low-iron iron), (300-p.p.m. a control on and 1 month for CS or RA to exposed 6 Figure nature medicine nature iron heme and non-heme cytosolic in or levels heme mitochondrial d e (weeks) exposur CS c a = 11; 6 weeks, 6 weeks, = 11; Iron diet ) Quantification of weight gain in WT mice over time after CS exposure (relative to initial weight at the start of CS exposure) in the absence (black (black absence the in exposure) CS of start the at weight initial to (relative exposure CS after time over mice WT in gain weight of ) Quantification

To determine whether loss of COX resulted in higher levels levels higher in resulted COX of loss whether determine To Fold change mitochondrial Percentage of 3-h clearance Percentage of 3-h clearance 99m 99m non-heme iron (weeks) exposur CS Irp ki/ko of Tc–SC of Tc–SC Sco2 CS 10 15 20 10 15 20 DFP

(normalized to WT RA) Irp 0 5 DFP e 2 0 5 CS Sco2 CS 0. 1. 1. 2.

2 0 5 0 5 0 DFP mice exhibited lower total BALF levels of IL-6 and IL-33 and Targeting mitochondrial iron in experimental COPD. ( COPD. experimental in iron mitochondrial Targeting Control + + + – e ki/ki 0 2 0 + + + – – – **** ** + – – – ki/ko n = 8; CS (1 month), month), (1 CS = 8; mice) ( + – 4 2 DFP * mice were protected from CS-associated changes changes CS-associated from protected were mice + + n + + + – 6 4 = 11; 7 weeks, 7 weeks, = 11; – + + + + + – High

+ – ** c e Fig. 5 Sco2 but continued for an additional 2 or 4 weeks of CS exposure. Black arrows indicate time of CS exposure and blue arrows indicate indicate arrows blue and exposure CS of time indicate arrows Black exposure. CS of 4 weeks 2 or additional an for continued but advance online publication online advance h ), total numbers of BAL leukocytes ( leukocytes BAL of numbers total ), * DFP ) Schematic of the major findings of this study. Throughout, data are mean mean are data study. Throughout, this of findings major the of ) Schematic 2 0 – – + – n.s. ), supporting the hypothesis that increased increased that hypothesis the supporting ), * 8 # ki/ko + + – + – – e – – and Low * n + – f n.s. mouse lungs exposed to RA or CS CS or RA to exposed lungs mouse = 17; 5 weeks, 5 weeks, = 17; 4 + + + + – DFP n (weeks) exposur CS i. 5g Fig. Total cells ( 10 /ml BALF) 4

Supplementary Supplementary Fig. 9c × Sco2 CS n = 10; CS (6 weeks), weeks), (6 CS = 10; 10 15 20 0 5 = 5; 8 weeks, 8 weeks, = 5; 6 4 2 0 b Figs. 4d 4d Figs. Percentage weight DFP + – – – ki/ko e Iron diet 5 4 gain at 6 weeks + + + + + + ** ,

DFP (relative to initial) Irp h ** CS showing greater protection protection greater showing 80 20 40 60 + + + – ). Similarly to the the to Similarly ). c 0 2 ** 6 ) Quantification of 3-h 3-h of ) Quantification and + + + – – ** 7 + + – n Control = 12; 6 weeks, 6 weeks, = 12; ** # 5 n P = 4). ( = 4). 8 + – h ** ** < 0.05, < 0.05, d + ). No difference in ). No difference + + Percentage weight gain n

(relative to weight at = 4; CS + DFP (6 weeks) weeks) (6 + DFP CS = 4; g + + – f start of CS exposure) e ). ). CS-exposed ) and total BALF protein levels in WT mice treated with DFP using the therapeutic dosing dosing therapeutic the using DFP with treated mice WT in levels protein BALF total ) and Total BALF protein (mg) DFP 10 15 High – ** (weeks) exposur CS CS 0. 0. 1. 0 5 g ## a Fig. Fig. 0 4 8 2 ) Quantification of changes in lung mitochondrial non-heme iron levels (control, (control, levels iron non-heme mitochondrial lung in changes of ) Quantification , DFP + 6 4 2 0 b P 8 7 6 5 4 Time ofCSexposure ) Quantification of 3-h 3-h of ) Quantification < 0.01 by Student’s unpaired unpaired Student’s by < 0.01 + – – – n Irp2 e CS +DFP CS 5 = 12; 7 weeks, 7 weeks, = 12; + – f 99m Low and (weeks n.s. DFP + + −/− + – * Tc- + + + –

) ## sc clearance in WT and and WT in clearance weight gain at baseline but exhibited similar mucociliary responses responses mucociliary similar exhibited but impaired baseline at gain showed weight they and diet, iron-replete control a fed mice to compared as levels, iron non-heme lung and serum higher had diet ( diet no (with a fed iron-replete to control mice as compared gain) on weight effect impairment MCC CS-induced from protected were and levels iron non-heme lung and serum lower had diet low-iron a fed Mice model. bronchitis in our CS-induced to CS exposure responses first assessed whether modulating iron in the diets of WT mice altered which leads to in dysfunction COPD,mitochondrial experimental we To test the iron that hypothesis mitochondrial Irp2 loading, increases Targeting mitochondrial iron in experimental COPD ( COPD experimental in iron non-heme mitochondrial altered with ciated asso be may which COX, increased Irp2 that suggested data These pared to those from WT mice ( CS-exposed or RA- in observed was levels * + – 8 + + n n Fig. 6a Fig. = 4; CS (8 weeks) weeks) (8 CS = 4; = 4; 8 weeks, 8 weeks, = 4; h Non-heme iron Fig. 5 Fig. 99m DF , WT Gene expressio Tc- b i P and and ).

versus Mitochondrial dysfunction Immune cellin ltration Impaired MC sc Epithelial celldysfunction LGRC COPD Chronic bronchiti iron loading Mitochondrial t clearance ( clearance -test; n.s., not significant. not n.s., -test; subjects Healthy Fx Irp2 Supplementary Fig. 10a Fig. Supplementary ±

Fx n Irp n s.e.m. * s.e.m. = 4) or presence of DFP ( administered after after ( administered DFP of presence or = 4) ki/ko n 2 –/ −/− n – n C Sco2

mice = 4; CS + DFP (8 weeks), weeks), (8 + DFP CS = 4; Inflammation and excess mucu mice exposed to RA or CS for 1 month 1 month for CS or RA to exposed mice CO ki/ko a s P ) and weight gain in WT mice that were were that mice WT in gain weight ) and X < 0.05, ** < 0.05, Fig. Fig. 5 dysfunction Mitochondrial gene signatur Mitochondrial Functional clustering COPD analysis CS s Phos h Ox Irp and 2 Healthy e P E mphysema < 0.01, **** < 0.01, Epithelial celldeath Mitochondrial loss Small-airway remodelin Airspace enlargement Supplementary Fig. 9e Supplementary ∆Ψ Sco2 COPD subjects ECLIPSE ). Mice fed a high-iron high-iron a fed Mice ). epithelial cells in human RIP-seq ofIRP2targetRNA Alveolar breakdow ki/ko n s e l c i t r a = 4; = 4; n n mice, as com as mice, = 11; 5 weeks, 5 weeks, = 11; dysfunction Mitochondrial Lung epithelialcell P < 0.001 by < 0.001 n = 2

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). ).  - - © 2015 Nature America, Inc. All rights reserved. treated with DFP ( DFP with treated not but duration same the for CS to exposed mice versus treatment and acute lung injury (total BALF protein) after 2 and 4 weeks of DFP concentrations) protein IL-6 and IL-33 BALF and counts, leukocyte also exposure CS of weeks showed 4 significant reductions in pulmonary or inflammation (BAL total 2 additional an during DFP with treated then were that exposure) CS of weeks 4 after concentration IL-33 protein concentration and higher whole-lung tissue IL-6 protein total counts, BALF BAL higher BALF protein higher leukocyte levels, (higher injury and inflammation pulmonary CS-induced with mice ( DFP with treated not were that but weeks 8 or 6 for CS to exposed were that mice than loading iron mitochondrial lung and gain lower had weight improved showed of 4 CS weeks exposure to CS for DFP 6 after exposed mice or WT 8 and weeks administered ( treatment DFP without but time of period same ( clearance ary mucocili improved additional significantly had an exposure CS of during weeks 3 or 1.5 DFP with treated were weeks that 4 exposure) after CS develops of (which MCC impaired existing with mice WT strategy. dosing a as therapeutic administered when injury) and inflammation (pulmonary COPD experimental established alleviate Irp2 or mice WT DFP-treated than impairment MCC CS-induced from synergistic effects, i.e., DFP-treated showed background Irp2-deficient an in DFP Administering mice. afforded by DFP treatment was to in similar that the observed ( impairment MCC CS-induced from protected mice exposure CS of duration the for DFP of Administration to promote cellular homeostasis may be affected by increased IRP2 IRP2 increased by affected be may homeostasis cellular promote to ably the case that cells in the lung that rely on function mitochondrial COPD with associated destruction emphysematous and ( COPD in strate experimental that dysfunction Irp2 promotes mitochondrial expression in subjects with COPD ( and genes of mitochondrial expression differential the between human well-established COPD cohorts and show a strong association related pathways in the lung. We validate this critical regulation in two mitochondria- regulates IRP2 that demonstrate we data, expression IRP2 target identification in cells and mouse lungs with human COPD COPD. of models mouse experimental two in COPD with subjects human of pathogenesis COPD. IRP2 Consistent expression with in increased genes, these of one of role functional the edge, cated ( protein hedgehog-interacting and ( A member 13, similarity sequence with family (15q25), loci—including most of COPD; with these associated loci bility suscepti multiple for associations GWAS compelling provided have DISCUSSION loading iron mitochondrial prevent to elsewhere them cates relo and deposits iron non-heme that mitochondrial targets chelator iron specifically an is ferriprox) or (DFP Deferiprone model. bronchi tis CS-induced our in exposure CS to responses alter would ( diet iron-replete control and a fed mice as exposure CS to A 1 of cells of types may epithelial cells These ciliated include expression. 0 By using novel experimental approaches and integrating unbiased unbiased and integrating approaches experimental novel By using could chelation iron mitochondrial whether evaluated we Finally, iron mitochondrial excess alleviating whether examined We next

s e l c i t r −/− Supplementary Fig. 10a Fig. Supplementary 3 , 7 mice ( mice , 10 Fig. 6 Fig. , 41– 4 Fig. 6 Fig. 7 h P . Here we characterize, . for to time Here the first our knowl we characterize, ), which may ultimately lead to epithelial cell death death cell epithelial to lead ultimately may which ), < 0.05), as compared to mice exposed to CS for the the for CS to exposed mice to compared as 0.05), < Fig. 6f Fig. c ). , g and and 4 , we confirm that Irp2 promotes COPD COPD promotes Irp2 that , we confirm ). Supplementary Fig. 10c Fig. Supplementary Fig. 6 Irp2 HHIP −/− h ). ). Furthermore, we demon mice had better protection —ae en el repli well been )—have Fig. 6 Fig. IRP2 Fig. 6 Fig. c ). The protection protection The ). ( Fig. 6d Fig. IREB2 c 1 ). Similarly, Similarly, ). – . It is prob It is . e FAM13A) i. 6a Fig. ). ), in the the in ), , e Irp2 ). WT WT ). IRP2 IRP2 39 , −/− 4 , 0 b ------.

the pathogenesis of COPD of pathogenesis the stress oxidative and death cell mation, inflam promote that radicals hydroxyl generate may iron chondrial hepcidin serum upregulates COPD in inflammation systemic that reports with and in outcomes COPD and functional clinical poor with associated deposition may also be consistent with recent reports COPD of anemia being with subjects human from macrophages lar and in alveo systemically iron that CS increases deposition exposure observations previous with is COPD consistent in experimental load lung. the in function chondrial mito role the of in elucidate IRP2 regulating to fully needed insights additional important provide will biosynthesis heme mitochondrial Fe (i.e., iron non-heme mitochondrial accumulating the of valency the into dynamics investigations mitochondrial altered and dysfunction cells to mice detrimental and is IRP2 increasing that with consistent observations are earlier data These production. surfactant regulate to of mitochondria stocks abundant on their rely which cells, epithelial II alveolar type and parenchymal frequency, ciliary-beat for efficient rely which for on tract, ATPthe mitochondria respiratory generation multiple genetic variants simultaneously associated with COPD and and COPD with associated simultaneously variants genetic multiple are loci GWAS-identified the that the indicate analyses Recent needed. in variants genetic functional and genes key of identification the for approaches integrative improved COPD, as such diseases complex of pathogenesis the in roles tional func important play that targets identify us help to clues generate by GWAS.GWAS Although identified 15q25 of chromosome region COPD-associated in the located genes susceptibility COPD multiple COPD. Our results strongly suggest that mechanistic role of the COPD susceptibility gene model. COPD the synergistic effects of DFP with Irp2 deficiency in our experimental of DFP may effects for account therefore synthesis Mitochondria-independent hemoglobin for cells pre-erythroid to it donate cells and iron-loaded from iron mobilize also can DFP extracellular apotransferrin. to endosomes, and nuclei as other such between organelles, also cellular but mitochondria of out only not iron shuttle beta-thalassemia of ment treat the in use human for DFP of availability the given intriguing is DFP of effect cytoprotective This COPD. experimental alleviated ironmitochondrial using the membrane-permeant iron chelatorexcessive DFP redistributing Specifically, COPD. in iron mitochondrial anemia lastic (siderob iron of regulation abnormal have COX of subunits encode mutations in with that genes individuals notion, this with Consistent lung exposure. CS to the response in iron in of mitochondrial regulation the for determinant central a be may COX that illustrate also COPD with subjects human from muscle skeletal in increased responsiveness way study COPD ( experimental in destruction emphysematous and inflammation damage, cell lial epithe dysfunction, mitochondrial to leading and preceding genic, IRP2, COX increases in airway epithelial cells and may become patho Our data showing that IRP2 promotes mitochondrial iron over iron mitochondrial promotes IRP2 that showing data Our Taken together, these studies provide valuable new insights into the targeting therapeutically of efficacy the highlight we Finally, of expression sustained of consequence a as that Wehypothesize 3 6 reported that high COX activity is required for maximal air maximal for required is activity COX high that reported 2 1 and that COPD itself is associated with mitochondrial mitochondrial with associated is itself COPD that and 3 5 ) and defects in mitochondrial heme biosynthesis heme mitochondrial in defects and ) advance online publication online advance 5 7 . Excess tissue or, more specifically, excess mito excess specifically, more or, tissue Excess . 3 6 , and others have shown that COX activity is is activity COX that shown have others and , Fig. Fig. 6 2+ or Fe or 4 1 0 h . and in various clinical trials. DFP can can DFP trials. clinical various in and ). ). In a line recent with our observations, 3+ ) and the role of IRP2 in regulating regulating in IRP2 of role the and ) 2 IRP2 1 , all of which contribute to to contribute which of all , IRP2 is one of the potentially -15q25 locus includes includes locus -15q25

Irp2 nature medicine nature 17 in experimental 52 , 49– , 5 3 . Such iron iron Such . 5 1 . Further Further . 5 8 39 54– . . We 5 , 9 4 . 4 5 0 6 8 ------.

© 2015 Nature America, Inc. All rights reserved. the the paper with significant input and contributions from K.G. and C.A.O. All A.M.K.C. provided analysis critical and discussions. S.M.C. and A.M.K.C. wrote the LGRC human data set. S.M.C., K.G., G.-C.Y., J.Q., E.K.S., G.M., C.A.O. and mice, and M.P.G. and J.M.D’A. provided technical support. D.L.D. helped with and T.A.R. provided the the MCC experiments. C.A.O., F.P. and H.P. analyzed morphometric data. M.C.G. reconstructed and analyzed MCC images. S.C.M. provided technical support for expression data and clusteringperformed functional analysis. A.R.B. and M.C. performed experiments. K.G. analyzed RIP-seq, gene expression and human 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. S.M.C. and A.M.K.C. conceived and the designed study. S.M.C., K.G., M.E.L.-C., and the J. Willard and Alice S. Marriott Foundation (E.A.S.). Additional support was provided by the Muscular Dystrophy Association (E.A.S.) Shriver National Institute of Child Health and Human Development, NIH. acknowledge support from the intramural research program of the Eunice Kennedy were supported by NIH grant R01-GM088999 (to G.M.). M.C.G. and T.A.R. Respiratory Research institute Research Consortium grants. G.M. and C.K. P01-HL114501 (to A.M.K.C.) and by Brigham and Women’s Hospital–Lovelace by NIH grants R21-ES025379-01 (to A. Fedulov), P01-HL105339 (to E.K.S.) and NIH grant R01-HL111759 (to J.Q., G.C.Y. and E.K.S.). C.A.O. was also supported also supported by NIH grant P01-HL105339 (to E.K.S.). K.G. was supported by Defense grant W911F-15-1-0169 (E.A.S.). S.M.C., A.M.K.C., J.Q. and E.K.S. were awards YFEL141004 (F.P.) and YFEL103236 (M.P.G.), and US Department of innovator FAMRI grant CIA#123046 (C.A.O.), FAMRI Young Clinical Scientist Medical Research Institute (FAMRI) clinical innovator award (A.M.K.C.), clinical Association Biomedical Research grant RG-348928 (S.M.C.), a Flight Attendants Heart, Lung and Blood Institute grant K99-HL125899 (S.M.C.), American Lung HL086936 (to J.M.D’A.) and P01-HD080642 (Project 2 to E.A.S.), NIH–National (C.A.O.), R01-HL86814 (C.A.O.), R21-HL111835 (C.A.O.), HL122513 (H.P.), R01- R01-HL055330 (A.M.K.C.), R01-HL079904 (A.M.K.C.), R01-AI111475-01 by US National Institutes of Health (NIH) grants P01-HL114501 (A.M.K.C.), and input from S.W. Ryter, C.A. MacRae and P.Y. Sips. This work was supported with transmission electron microscopy. The authors also acknowledge discussion Y. Shao for assistance with the microarray study and M. Ericsson for assistance providing Ferriprox. The authors also thank R. Rubio for assistance with RNA-seq, breeding of the the andcyto-GRX2 mito-GRX2 plasmids, Y. Hua (Columbia University) for the assistance. The authors also acknowledge S. Chan (Harvard Medical School) for The authors thank J.S. Moon, H.C. Lam, K. Taylor and B. Ding for technical online ve Note: Any Supplementary Information and Source Data files are available in the versus code accession under able codes. Accession the t of version in available are references associated any and Methods M COPD. for approaches therapeutic novel as chelators mitochondrial-iron of use the supports strongly study our Finally, COPD. of development the in result ultimately that inflammation of initiation the and death cell subsequent and dysfunction mito to chondrial leading levels, tissue and cellular molecular, the deleterious at have effects may time, of periods long for sustained if which, processing iron systemic possibly and cellular in act responders first as mitochondria, the on converging those particularly responses, susceptibility. COPD influence or 3 alpha nicotinic, receptor, ergic to proximity close tive trait loci)) quantita (expression eQTLs (COPD genes nearby of expression the nature medicine nature coauthors reviewed and approved the manuscript.final AUTHOR CONTRIBUTIONS A cknowledgments ethods Genes, such as as such Genes, Irp2 rsion of the pape −/− he pape he Sco2 mice are available under accession code code accession under available are mice 6 0 ; ; it is that therefore possible multiple genes in located ki/ki Gene Expression Omnibus: RIP-seq data are data RIP-seq avail Omnibus: Expression Gene IRP2

r and IRP2 . Irp2 r advance online publication online advance . Sco2 −/− (for example, the nicotinic receptors cholin receptors nicotinic the example, (for , that are involved in CS-induced stress stress CS-induced in involved are that , mice. E.A.S. provided the ki/ko GSE5707 mice and J. Connelly (ApoPharma Inc.) for 3 CHRNA3 , and microarray data for WT WT for data microarray and , and and Sco2 ki/ki

CHRNA5 and GSE5704 Sco2

ki/ko online online ) may may )

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- - - - 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints version of t The authors declare competing interests:financial details are available in the 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 29. 28. 27. 26. 25. 24. COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING

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© 2015 Nature America, Inc. All rights reserved. (129S4/SvJae and C57Bl/6 background), starting at 10 weeks of age and selected the abdominal muscles of age- and sex-matched Briefly, conditions, aseptic under and skin abdominal the dissected we incised model. sepsis (CLP) ligation-puncture Cecal overintervals 0–120 h. water and O chamber (>99% exposure Plexiglas 3.70-ft3 hyperoxia a in liters/min 12 to random, at selected and age of weeks 10 at starting background), C57Bl/6 and (129S4/SvJae mice littermate exposures. Hyperoxia to that for control mice. Water solutions were changed twice weekly. Animals were checked daily, and the quantity of water consumed was compared 4 weeks of smoke exposure and continuing therapy for an additional 2–4 weeks. deferiprone therapeutically in the animals’ drinking water, We exposure. administered smoke initiating the also ofduration the forwater drinking therapy after Ferriprox solution (1 mg/ml) (ApoPharma Inc.) prophylactically in the animals’ paring it to that for mice on a normal-iron diet. We administered deferiprone or animal on a weekly basis, and we each We recordedweighed exposures. thesmoke quantitythe throughout ofused and chowage of consumed, weeks 3 com 2% carbonyl iron. Iron diets were exchanged for normal diets upon weaning at contained which (TD.10213), Laboratories Harlan from diet) (iron-replete 3 p.p.m.Diet iron. 2–6 contained which (TD.10210), HarlanLaboratories from which contained 300 parts per million (p.p.m.) iron. Diet 2 (iron-deficient diet) AIN-93G), (TD.94045 Laboratories Harlanfrom diet) (control 1 diet follows: microscopy (TEM) analysis. electron transmission for fixed was only) exposures (4-month lung left the of portion proximal A overnight. °C 4 at formalin 4% in lung Weright the fixed off tied the left lung with a suture, it dissected and place it into liquid nitrogen. lated the tracheas and inflated the lungs with PBS at 25 cm of H the end of the exposure regimen, we euthanized mice by CO to cigarette smoke exposure, early as andeath was used exclusion criterion. At an average total particulate matter (TPM) of 150 mg/m smoke) from 100 3R4F cigarettes (University of Kentucky), which correlated to used for all CS exposures. Mice were exposed to CS (mainstream and sidestream per week for 4 weeks or 4–6 months. Age-matched male and female mice were smoke (Model exposure device TeagueTE-10, Enterprises) for 2 h day,per 5 d them exposed to total body CS in a stainless steel chamber using a whole-body selected age- and sex-matched mice, starting at 6–12 weeks of vivo age, at In random and Women’s and Brigham of Hospital. for Animal Welfare and by the Institutional Animal Care and Use Committees Committee Standing Harvard the by approved were protocols experimental and smell color,a distinctive and were obvious to All animal the investigators. displayed CS to exposed cages mouse during In addition, exposure. smoke monitored after and be to needed mice as exposure, CS blinded the a during use approach not did We exposures. RA or CS for random at chosen Cages were experiments. prior on based chosen experiment. typically each were for mice of Numbers calculated explicitly not was Power (mg/kg). weight kg body mg 200 per was diet iron of content standard The the cycle. and dark light a 12-h under kept and room same the in housed were animals All some. ( mutation 4–deleted exon frataxin ( mutation expansion Laboratory. and (C57BL/6) and have on tion one allele and alleles, on both mutation University). (Columbia Schon E. Development). Human and (C57BL/6), Health Child of Institute (National and were and from T. C57Bl/6 of 129S4/SvJae consisting background Rouault Animals. ONLINE METHODS doi: Mice exposed to CS for 4 weeks had modifications to their dietary iron as as iron dietary their to modifications had weeks 4 for CS to exposed Mice 10.1038/nm.4021 S xoue, hmcl ramns n de modifications. diet and treatments chemical exposures, CS

Irp2 ad libitum ad Sco2 Fxn −/− ki/ko Fxn ki/ko and WT littermate control mice were from a mixed genetic genetic mixed a from were mice control littermate WT and and (C57BL/6) WT (C57BL/6) littermate mice were from ki/ko mice harbor one allele of the frataxin (GAA) 230 (GAA) frataxin the of allele one harbor mice . We assessed survival by checking the mice at regular regular at mice the checking by survival assessed We . Fxn We exposed age- and sex-matched sex-matched and age- exposed We (C57/BL6) mice were purchased from the Jackson Jackson the from purchased were mice (C57/BL6) tm1.1Pand Sco2 Sco2 deleted on the other allele (KO) allele on other the deleted ) on one chromosome, and one allele of the of the allele one and ) on chromosome, one ki/ko Sco2 Fxn mice have a mice ki/ki tm1Mkn mice have a a have mice ) on the homologous chromo homologous the on ) CLP was done as described as done was CLP Irp2 −/− Sco2 and WT littermate mice 3 6 . . For animals subjected 1 knock-in (KI) muta (KI) knock-in , with access to chow to access with , 2 Sco2 a a lw ae of rate flow a at ) 2 narcosis, cannu Irp2 2 knock-in (KI) (KI) knock-in O O pressure. We 3 −/− 7 . Wild-type . Wild-type and WT WT and Sco2 ∆ We neo neo ki/ki 6 2 - - - - .

monoclonal IgG to cytochrome c EPR1327 (D00355 Abcam) at a 1:50 dilution, rabbit with dilution, 1:250 a at Biosciences) BD (556433, c, anti–cytochrome with a rabbit primary IgG to Irp2 (LS-B48, LS Bio) at a 1:50 dilution, with mouse ing by incubating in 10% normal serum for 45 min. Tissues were immunostained(DAKO Cytomation), and the slides were blocked for nonspecific protein bind blocker peroxidase a using blocked were peroxidases Endogenous min. for10 microwave oven using a citrate buffer (Target Retrieval Solution, S1699, DAKO) alcohols and washed in PBS. Heat-activated antigen retrieval was performed in a 5- using standard procedures. Briefly, paraffin-embedded sections were deparaffinized formalin-fixed, through graded stained and staining. immunofluorescence and Immunohistochemistry Biologicals). 2.5 using out carried were assays with a biotinylated IRE, according to the manufacturer’s Scientific) (Thermo Kit instructions. EMSA RNA Supershift Chemiluminescent LightShift the using (EMSA). assay shift mobility Electrophoretic of the operated mice. injected 1 ml of prewarmed (37 °C) normal saline to restore heat and hydration (i.p.) intraperitoneally surgery,we After cecum. exposed the of puncture and ligation. Sham-operated same the procedure underwent mice without ligation of area the to near holes) (two puncture through-and-through one by needle G 21 a with it perforated and suture silk 6-0 a with it of 75% ligated we and at random, to gain access to the peritoneal cavity. We located the mouse cecum in all in cases. all sample area image forthe acquisition to choose coverslip were used gridded a acquiring before images and before data identifiers analysis. Random-number generations using excel and slide the taping by blinded were sections Lung magnification). 20× (at lung the of areas under-inflated avoiding condition, mouse in a randomized manner, with the observer blinded to the experimental per captured were magnification) (200× images 20–30 images. digital quality a microscope, 20× objective, and a camera and software that can acquire high- HRc; Carl Axiocam MicroImaging) Zeiss as black and white using TIFF files a camera digital a with equipped MicroImaging Zeiss Carl (Axiophot; acquired or diameter mean area-weighted the calculates that algorithm image-processing automated published, viously the mean linear intercept (chord)-length (MLI or previously as described staining Gill’s Modified performed We paraffin. in embedded and parasagittally cut sections. lung of analysis Morphometric at a 1:300 dilution. Technologies) at a 1:1,000 dilution or Hoechst (33342, ThermoFisher Scientific) 1:500 dilution. Nuclei were counter-stained using TO-PRO-3 Iodide (T3605, secondaryLife antibody–Alexa Fluor 488 conjugate (A-11055, Life technologies) at a (R-6394, Life Technologies) at a 1:500 dilution or donkey anti–goat conjugateIgG red (H rhodamine + L) L) + (H IgG anti–rabbit goat using out carried was staining Secondary dilution. 1:100 a at Abcam) (ab40873, uteroglobin to IgG for non-ciliated secretory epithelial cells was conducted using a rabbit polyclonal (Lys40) Signaling Technology)(D20G3, Cell at a 1:50 dilution, and co staining acetyl- for IgG rabbit a using conducted was cells epithelial airway ciliated of markers and Irp2 for Co-staining dilution. 1:500 a at Affymetrix) (53-5381-80, IgG 488–conjugated Fluor Alexa podoplanin anti–mouse using cells epithelial I type for and dilution 1:50 a at Abcam) (ab90716, (Pro-SPC) C protein prosurfactant to IgG polyclonal rabbit a using out carried was cells to Abcam)Irp2 (ab181153, at a fordilution. 1:50 II epithelial Co-staining type IgG to Irp2 (ab106926, Abcam) at a 1:50 dilution or with rabbit monoclonal IgG Epifluorescence microscopy. PBS for the antibody.primary manufacturer’sthe The negative instructions. control consisted of substituting stains were purchased from Sigma-Aldrich, and staining was carried out as per Laboratories), according to the manufacturer’s protocol. Hematoxylin and eosin VECTASTAIN usingthe were nals developed Vector Kit (PK-6101; Elite ABC sig and dilution 1:400 a at Sigma-Aldrich) (L7543, anti-LC3B rabbit with or Briefly, tissues were stained with goat polyclonal 6 3 D . . We air-space enlargement using quantified µ 2 index g of anti–Irp2 IgG (NB100-1798, Novus (NB100-1798, IgG anti–Irp2 of g 6 Formalin-fixed lung samples were samples lung Formalin-fixed 4 . . Briefly, wereimages randomized Irp2 EMSAs were carried out carried were EMSAs Irp2 L m ) ) method µ m-thick lung sections sections lung m-thick nature medicine nature 6 3 or using a pre We prepared prepared We α -tubulin -tubulin - - -

© 2015 Nature America, Inc. All rights reserved. eter. Specifically, 20 resuspended in 500 forice followed15 min by centrifugation at 600 on left and pellet cell the to added was (Sigma-Aldrich) buffer lysis cell blood BALFPBS total). eight(4 ml attimes was centrifuged 500 the tracheas cannulated and the lungs lavaged with 0.5-ml increments of ice-coldBALF isolation, cell counts and ELISA. with arteries or other airways were not included in the analysis. diameter between 300 and 699 sub-epithelial layer in microns was calculated for airways having a mean internal by a different blinded, reader.experienced The mean ( stained blue by Masson’s Trichrome stain at 12 separate sites aroundlayer the fibrosis airway sub-epithelial the of thickness the and diameter luminal airway mean the measure to used was LLC) Device (Molecular MetaMorph software 200× magnification by a blinded, experienced reader as previously described at MicroImaging) Zeiss Carl HRc; (Axiocam camera digital a with equipped MicroImaging) Zeiss Carl (Axiophot; microscope light a using acquired were tions were stained with Masson’s Trichrome stain (Sigma-Aldrich), and images from WT and method Small-airway remodeling. the area-weighted mean diameter diameter, alveolar factor. The area of each alveolar A was section then converted to an equivalent ( (O units real the number of pixels in each region. The measured area was convertedthen to alveolus. The area occupied by each alveolar section was measured by counting rithm resulting modified distance map was then segmented using the watershed algo transform minima H the called operator morphological a by removed were minima shallow The walls. alveolar the of curvature the in fluctuations local small by created walls alveolar the to close minima local shallow many also and section alveolar each of center the near minima deep a had function negative of the distance to the the assigned nearest is blackpixel (tissue) each pixel. where Thefunction resulting distance distancesigned a calculate to used variance of the black and white pixels intra-class the minimize to threshold optimum the calculates which method, lapping blocks, and the threshold for each block was calculated using the Otsu’s associated with non-uniform illumination, each image was split into non-over To tissue. to correspond pixels black avoidand errors air to correspond pixels white where image black-and-white a create to thresholded first were images histological The above. described as acquisition image randomized with stain D for discerning airspace enlargement in smoke-exposed mice enlargement in many studies heterogeneity in airspace sizes and has been used in the past to quantify airspace mean diameter or Measurement of alveolar diameters. average mean chord length able in NIH Image. Alveolar chord length for each image was calculated avail and macro the the from adapted was macro alveolar-chord-length The images. the from removed manually were structures non-alveolar other and vessels, and customized macros to analyze airspace enlargement Airspace enlargement was quantified using the MLI method using Scion Imagemanner automated relatively and standardized a in lung the of parts all in airspace as software the by identified areas within chord each of length the quantifies (chords), lines of horizontal and softwareuniform a then grid and the vertical images tissue is (in which white and airspace is black), and superimposes then binary to sections Gill’slungof images converts software Morphometry stain. chord length and alveolar area on paraffin-embedded lung sections stained with measurements. Mean-chord-length nature medicine nature 2 index was calculated on paraffin-embedded lung sections stained with Gill’s 7 3 into distinct regions, with each region corresponding to a section of an 65 6 5 , 7 . Using this method, it is possible to measure the size of the alveoli alveoli the of size the measure to possible is it method, this Using . 4 to quantify small-airway remodeling in formalin-fixed lung sections Irp2 µ m 2 D −/− ) for mouse alveolar sections) by multiplying with a scaling scaling a bymultiplying with sections) alveolar for mouse ) d 2 as as µ µ index. Compared to the mice exposed to RA or CS for 6 months. Briefly, lung sec l PBS, and leukocytes were l PBS, andcounted leukocytes using a hemocytom l l was removed for counting cell in triplicate) (performed d = = We used a modification of a published morphometry 2 ± 66– s.e.m. for each mouse was calculated. A 7 µ 0 / m. m. Sections of airways sharing their adventitia , where it has a higher sensitivity and specificity p D . From the measured equivalent diameters diameters equivalent measured the From . 2 This protocol measures the area-weighted as 7 This protocol measures mean alveolar alveolar mean measures protocol This 1 . . The black-and-white images were then Mice were euthanized by CO D 2 = Lm ) ( ) ( d d g , the 2 3 for 3 min. The cell pellet was for pellet The 3 cell min. was calculated. D ± 2 6 index characterizes the s.e.m.) s.e.m.) thickness of the 5 . Large airways, blood g for red5 1 min. ml 6 6 . In this study, the 2 narcosis, 7 2 . The The . 6 6 5 5 - - - - - . . lung deposition of Regional clearance. mucociliary by removed percentage the as expressed and analyzed blindly reconstructed, obtained, were points time 3-h and 1-, 0-, at single-photon micro emission computed tomography ( (3D) dimensional three Whole-mouse h. 3 and h 1 at aspiration.We aspiration ( after lungs mouse imaged by immediately oropharynx the of part distal the Women’sinto and Hospital) (Brigham taining approximately 0.3–0.5 mCi of ously previ described procedure aspiration oropharyngeal non-invasive, a using clearance. mucociliary of Assessment DY401-05). kitSystems) R&D ELISA 7625, and IL-1 (IL-18/IL1F4 IL-18 DY3626-05), Systems R&D DuoSet, IL-33 (mouse IL-33 DY406), Systems R&D DuoSet, IL-6 (mouse IL-6 DYC835-2), Systems R&D IC, DuoSet (Asp175) Caspase-3 Cleaved (Human/Mouse caspase-3 cleaved Systems), R&D TotalMMP-9DY6718,(Mouse MMP-9instructions: DuoSet, in homogenates or BALF of lung samples from mice following the manufacturer’s absolute numbers of subset each wereleukocyte calculated. polymorphonuclear leukocytes (PMNs) were counted in a total of 300 cells, and and lymphocytes macrophages,of percentages The Scientific). (Fisher system (Shandon Cytospin3, 300 r.p.m. for 5 min) and stained using the Hema3 using staininga hemocytometer, and 80 cccb ( MA Boston, Institute, Cancer Dana-Farber Biology, Computational Sample preparation out andbywas carried sequencing The Center for Cancer (Illumina), and sequencing was performed on the Illumina HiSeq2000 platform. cates) were prepared for RNA-seq using the TruSeq RNA-seq Lib Prep Reagent ( Samples Trizolextraction. by beads RIP Magna from RNA extracted We °C. 4 at overnight incubated and supernatants to added Santa Cruz) or 4 Immunoprecipitation Kit (Millipore). 4 Protein RNA-Binding RIP Magna the using immunoprecipitation RNA out carried We scraping. by tubes microcentrifuge 2-ml into collected and PBS ATCC), treated with or without 10 RIP-seq. linear regression multi-variable standard using differences C:P for corrected were rates MCC previously described for, as to the trachea) to peripheral airway distributions ratio (C:P ratio), was corrected counts) greater than 5. Poisson a sample, to input the relative tag the of enrichment twofold a had peak that if region genomic ( sample DFO or Ctl the only in bound uniquely is IRP2 where highlight sons compari two latter the whereas samples, DFO or Ctl either in bound is IRP2 where comparisonsidentified two first The (DFO/Ctl). Ctl comparedto DFO DFO compared to DFO-IgG (DFO/IgG), Ctl compared to DFO (Ctl/DFO) and binding motif (CAGWGH) 4e Fig. ( TfR and ferritin including targets, IRP2 known in peaks of detection the included data our Validationof peaks. DFO-specific and peaks were sharedthe control between (Ctl) and DFO samples as well as Ctl-specific that genes) 1,806 to (mapping ‘common’ peaks 3,497 of identification the in resulting reads, aligned the on performed was another.Peak-calling to pared identify and annotate ‘peaks’, or regions enriched for reads in one sample com the and replicates from HOMER algorithm combined ( then were reads Aligned genome. hg19 the to map to allowed then were reads tophat- unmapped and Any 2.0.7.Linux_x84_64. bowtie2-2.0.6 using transcriptome known hg19 human the to in each replicates biological control (Ctl) and DFO two samples, as well as Ctl-IgG and DFO-IgG, were aligned for reads Sequenced follows. as analyzed Supplementary Fig. 4d Fig. Supplementary Commercial ELISAs were used to measure the following analytes in duplicate Four primary comparisons were made: Ctl compared to Ctl-IgG (Ctl/IgG),Ctl-IgG comparedto Ctl made: werecomparisons primary Four .dfci.harvard.ed 1 8 . Briefly, we anesthetized mice and introduced 50 , f ). We also estimated the percentage of peaks with the known IRE- known the with peaks of percentage the estimated also We ). Three 15-cm 7 µ 6 99m g g of IgG mouse control antibody Santa(sc-2749, Cruz) were . u http://homer. Tc- ). RIP-seq data (GEO accession number accession (GEO RIP-seq data ). 2 dishes (1.5 × 10 sc ). In each comparison a peak was associated with a with associated was peak a comparison each In ). , characterized by calculating the central (area closest 7 7 7 P 5 . . To correct for any abnormality in distribution, distribution, in abnormality any for To . correct < 0.1 and a peak-score (position-adjusted read read (position-adjusted peak-score a and 0.1 < µ l was removed for cytocentrifuge preparations salk.edu/homer/ngs/i µ µ -SPECT) images fromacquisitions 10-min M M DFO (16 h), were washed with ice-cold 99m 6 Mucociliary clearance was quantified quantified was clearance Mucociliary µ cells) of Beas2B cells (purchased from β Technetium sulfur colloid ( g g of mouse Irp2 IgG sc-33682, (7H6: (Mouse IL-1 β µ ndex.htm n /IL-1F2 R&D Systems/IL-1F2 R&D l of normal saline con = 2 biological repli biological 2 = doi: 10.1038/nm.4021 Supplementary Supplementary GS l E5707 ) was used to t = 0 h) and h) 0 = 99m Tc- 3 http:// ) was was ) sc - - - - - )

© 2015 Nature America, Inc. All rights reserved. on whether they are annotated to mitochondrial functions ( functions mitochondrial to annotated are they whether on annotated and made three heat maps ( We examined to which GO categories (Gene Ontology terms ( expressed differentially differentially expressed genes and genes in community 2. seq communities Irp2 P compared to those not in the community, and a ‘meta’- in the RIP-seq data, the values of the ( terms and genes of analysis resulted in the identification of five communities containing related sets (FDR) < 1 × 10 RIP-seqrate analysis and (false-discovery as the GO terms enriched identified the in identified genes the between annotations GO the by defined network a algorithm community-structure fast-greedy the analysis, this common), both independently and combined. To better interpret the results of was carried out on the genes in each of three RIP-seq–defined sets (Ctl, DFO Discovery (DAVID)and ment analysis using the Database for Annotation, Visualization, and Integrated enrich outfunctional carried we lung, the pathways in cellular of activity the clustering Functional enrichment analysis. the human homolog For identified. genes expressionthe these difference between gous group ( 16,573 genes on the expression array that belong to a single conserved homolo Files (CDF) and probe sets were mapped to EntrezGene IDs using a custom Chip Definition analyzed as follows. Kit (Qiagen). The gene expression data GEO accession number and and3 females) ( age-matched WT from harvested lungs from isolated was RNA the in expression gene assess to used was (Affymetrix) Gene expression microarray analysis. gene transcripts, respectively ( unique 2,135 and 2,203 to mapped peaks DFO-specific and Ctl-specific and to their target genes. Common mapped peaks to 1,806 unique gene transcripts peak regions for the known IRE-binding motif (CAGWGH) we used HOMER’s ‘annotatePeaks’ function to scan the genomic sequence of thetively ( to the Ctl/IgG and Ctl/DFO or the DFO/IgG and DFO/Ctl comparisons, respec common by werepeaks determining identified peaks and DFO-specific peaks Ctl-specific comparisons. DFO/IgG and Ctl/IgG the between shared as tified iden were DFO) and Ctl both in bound is IRP2 (where peaks common 3,497 function. ‘mergePeaks’ HOMER’s using comparisons different the between doi: DFO-specific (red) and (blue), common (purple) Ctl-specific data our sets. The in inner altered ring of and/or the Circos enriched pathways and transcripts gene the demonstrate graphically to generated was plot Circos a analysis, set Circos plot generation. genes were in identified the DFO-only set. peak (so they were in the common peaks, the CTRL-only peaks, or both). Only seven in results are related to IRP2 only. Specifically, 83% (34 of 41) of the genes we show enrichment the functional clustering analysisout carried in this study, and the in WT versus ground) and then was followed by a comparison to the baseline gene expression tated IRP2-mRNA complexes only (subtracting IgG-mRNA complexes as back score–normalized to aid in visualizing differences in expression values. tocommunity ( 2 localization functions ( value was computed, representing the significance of the association of the the of association the of significance the representing computed, was value Figure 3 To build a robust set of peaks for further analysis, we identified peaks common Because Because RIP-seq was based on a specific IRP2 antibody that immunoprecipi genes belonging to the communityNext that2 we selected arestrongly also 10.1038/nm.4021 Irp −/− −/− Supplementary Supplementary Fig. 4d versus WT differentially expressed genes with genes in each of the RIP- and mice WT was quantified using an unpaired two-tailed a 7 http://www.ncbi.nl were identified as a target of IRP2 in the Ctl versus IgG comparison 8 . . To compare the expression data to the RIP-seq data, we selected Irp2 −3 ) in the DAVID analysis on the combined gene set. This network 8 Supplementary Fig. 5b Fig. Supplementary 2 −/− ( GSE5704 Irp2 Fig. 2 7 Supplementary Fig. 4g Fig. Supplementary lungs, the effects of DFO are therefore not confounded on 9 tool Supplementary Fig. Supplementary 5a −/− By using the DAVID output from the combined gene- P b 79 < 0.01) in in 0.01) < ( ). This analysis revealed a strong association between n 8 , 8 was robust multiarray average (RMA)-normalized, = 3 males and= 3 males using3 RNeasyfemales) the Mini 0 Supplementary Supplementary Fig. 4d . . DAVID analysis ( m.nih.gov/homologene ). ). For belonging peaks to one of these three sets, t -statistic -statistic for genes in the community was Fig. 3 Irp2 The GeneChip Mouse Gene 1.0 ST Array ). In heat ). map,each rows the were −/− a and ). For each community identified identified community each For ). To evaluate how may IRP2 affect mice, as compared to WT mice. mice. WT to compared as mice, ) ) or other functions that belong ht Supplementary Fig. 5 tp://david.abcc.ncifcrf.go / ). t ) with the corresponding -statistic and associated Irp2 7 7 −/− 8 and to map peaks 3 8 Fig. 3 Fig. ) ) these genes are 1 and WT mice. mice. WT and was appliedto was GSE5704 n a = 3 males males 3 = ), protein ), t -test. ) based 8 was v z ------)

peak sets andpeak human COPD RNA-seq (black). RIP-seq (purple) common and (red) DFO (blue), Ctl the between differences COPD). Circos-plot generation allowed for the visualization of similarities and were highlighted (green denotes lower than COPD, magenta denotes higher in identified IRP2 peak. Genes with fold changes of an morecontaining genes forthan change fold 1.5 the orshows plot less Circos the than of 1/1.5ring) (black ring inner the samples;control versus COPD the in gene each for values sion n publicly the contained data from from downloaded was set data LGRC data available expression mRNA tissue Lung Consortium and was byfunded the National Heart, Lung, and Institute.Blood Research Consortium used lung tissue samples from the Lung Tissue Research (LGRC; COPD and those from controls from the Lung Genomics Research Consortium ( (mRNA) data expression gene included we COPD, with subjects in IRP2 by dysregulated be may that ways LGRC gene expression (mRNA) plots. Circos scores, was then on visualized different ‘rings’ overaligned genes.these atedgenes, such as with these COPD expressiondifferential and RIP-seq peak circlethe community. their located within genomic Other information associ each gene belongs to only one community, genes were to assigned segments of plot is colored to represent the five identified functional communities. Because emphysema-predominant COPD was defined by >10% of lung voxels voxels lung of >10% by defined with attenuation was Surrogate study COPD observational Predictive 3-year emphysema-predominant a Identify was study to This study. Longitudinally End-points) COPD of (Evaluation had a DLCO greater than 80%. non-smokers)andsmokers controls (both all and 80%, than less DLCO a had non-smokers and 159-subject mRNA gene-expression data set contained ( a with us leaving control, quality on based removed subsequently were jects status (219 and 107 individuals, respectively). Approximately half of these sub publicly available data, we identified those with a COPD diagnosis or a control array-specific batch effects using ComBat for corrected then was set data merged This name. gene same the shared that duplicate probes and then merged the two array platforms by matching probes corrected and then normalized between arrays using limma Data collected using each of the two array platforms included was background- ( LGRC the of gene expression. expression that we identified, we used two independent COPD cohort data sets low or high with cohorts. tissue lung COPD human COPD in genes mitochondrial the two in genes mitochondrial of Validation Table 3 presented is in diagnosis pathological alongwith lowerlobe), pathologic diagnosis (CPAL1) is presented in and from data contained set the publicly available the DLCO was less than 80%. we included only individuals with a DLCO greater than 80%, and for test cases controls, For available. were data monoxide) carbon for lungs the of capacity ardized series of questions. In addition to standard spirometry, DLCO (diffusing were collected as part of the routine care of patients and submitted with a stand ( biorepository (LTRC) criteria, for human subjects in the NHLBI’s Lung Tissue Research Consortium provides genetic, molecular, and quantitative phenotype data as well as exclusionPhenotypic characterization of emphysema and source of tissue. = 18 smokers. 18 = Weexpres the fold-changethe between difference calculated COPD data set 2. set data COPD 1. set data COPD or upper left, or (right resection tissue lung the of location Distributional used set data gene-expression mRNA LGRC the mentioned, previously As n = 18 smokers with phenotypic information along with LTRC overall final . h ttp://www.lung-genom http://www.ncbi ≤ n −950 HU (Hounsfield units) on inspiratory chest CT scans. = 18 smokers). In this final data set, all subjects with COPD We also used gene expression data from the ECLIPSE ECLIPSE the from data expression gene used also We n We used publicly available gene expression data from from data expression gene available publicly used We GSE4746 = 121 individuals with COPD, http://www.lung n GSE4746 = 121 individuals with COPD, with individuals 121 = .nlm.nih.gov/geo/que 0 data set. The 159-mRNA gene expression data ics.or Fig. 2 Fig. 0 . This 159-mRNA gene expression data set set data expression gene 159-mRNA This . g ) on the Circos plot. The Lung Genomics a ) from lung tissue of individuals with with individuals of tissue lung from ) -genomics.or 8 5 . Of the 580 subjects included in this Supplementary Supplementary Table 2 To anyidentify common path ry/acc.cgi?acc=GSE47 n g = 20 non-smokers and ). Lung tissue samples samples tissue Lung ). n nature medicine nature = 121 COPD, n 8 = 20 non-smokers 20 = 4 . Next we removed Supplementary Supplementary To compare compare To The LGRC 8 6 . , where where , n 46 IRP2 = 20 0 ). ). - - - - -

© 2015 Nature America, Inc. All rights reserved. samples in the National Heart, Lung and Blood Institute LTRC Institutebiorepository Blood and Lung Heart, National the in samples (LGRC) provides genetic, molecular, and quantitative phenotype data for humanTable 4 tissues obtained from open lung biopsies and are in described Research Consortium (LTRC) used in described previously as (GOLD2) Disease Lung Obstructive for Initiative Global the of approval.(IRB) Board Human on samples guidelines the based were classified Women’sand Review Brigham Institutional of Hospital guidelines the under approval. study and samples Human with increased emphysema severity. gene-expression signature that the had were a DFO peak found to have expression decreased in levels genes 12 all experiment; DFO the in calls peak with genes by driven highly was This signature). expression full the in genes all of expression emphysema with increased severity, as compared toor 73/114 64% an have that signature expression the in genes of 78% or (21/27 severity emphysema increased with expression decreased with that also had an identified IRP2 127 genes in the expression signature and the genes that had common ofpeaks genes, (18 overlap significant strong a was there common), and DFO, (CTRL, separately peaks of sets three the investigating ( significant marginally is that overlap an identified an had 27 genes, 114 these Of calls. peak our mapping when HOMER, by used annotations the in included were ture signa expression this in genes the of 114 thatstudy, RIP-seqnoted our we to Campbell from signatureexpression (out of 17,879), based on the 2,282 and value, change fold the on based 17,879), of (out 2,475 ranked was it had an FDR of and <1 (0.63) a While from microCT information of COPD subjects as reported by Campbell defined a 127-gene expression signature associated directly with increases in corresponding to more green on the left of the heat maps. low in genes these of expression decreased expression is consistent with observations in the 10 × 1.6108 × 10 1.4088 subjects: overall datathe meta set, 0.9564; non-smoker controls: 0.5303 ( 10 × 1.4 subjects: COPD were:map) heat the in genes the ( 3 Figure as same the ordered are Rows subjects). all across median the than (greater high or subjects) all across median the than (less expression low having as identified were individuals which delimiting bar white a ordered are subjects of values increasing on the based right) map, to left (from heat each In populations. subject different ( LGRC and ECLIPSE in levels genes mitochondrial selected our ( of which identified We CDF. custom a ( ECLIPSE from ( LGRC from (.TAR) files data raw ing RMA-normalized using a custom CDF. n ?acc=GSE5483 ( GEO from downloaded was tion, ( ECLIPSE from files expression CEL distance walking 6-min and saturation oxygen resting impedance, body outcomes, health condensate), breath exhaled and urine sputum, blood, (in measurement tomography,biomarker computed chest oscillometry, impulse (spirometry, measurements function pulmonary include assessments Other nature medicine nature Supplementary Supplementary Fig. 6 Fig. 3 Fig. = 84 smoker controls, smoker 84 = Finally, we also compared our results with a study published earlier that that earlier published study a with results our compared also we Finally, ‘meta’ overall the set, data gene-expression LGRC the For expressionRaw data from includ two the COPD cohorts was downloaded, . . Furthermore, ofwithin the set 27 genes in the gene expression signature IRP2 a (adapted from Lam ) were measured in each of these cohorts and showed their expression andshowed cohorts their of these each werein ) measured a 17 and each row is is row each and was not enriched in the 127-gene expression list in Campbell , 18 −5 , ( 2 7 2 Supplementary Fig. 6b Fig. Supplementary ) . Human samples obtained from the Lung Genomics Tissue Genomics Lung the from obtained samples Human . GSE5483 8 6 . This particular data . contained set This particular −7 ; ; smoker-controls: × 10 6.1838 ). n P 7 IRP2 = 6 non-smoker controls. The ECLIPSE data was was data ECLIPSE The controls. non-smoker 6 = values (for genesthe in heatthe map) were: COPD et al. ); the ECLIPSE data was RMA-normalized using using RMA-normalized was data ECLIPSE the ); t z -statistic -statistic value. When we examined the 127-gene -score–normalized for visualization purposes purposes visualization for -score–normalized peak, there was a relative enrichment for genes 1 Fig. 3b Fig. t 8 -statistic of −1.44. On this list, -statistic of On this −1.44. ). The Lung Genomics Research Consortium Figure 5 Supplementary Fig. 6a http:// et al. et GSE5483 , GSE4746 c Human lung tissues were analyzed analyzed were tissues lung Human ). In all cases the overall differential differential overall the cases all In ). and and P for gene targets that were common were that targets gene for www.ncbi.nlm.nih.gov = 0.06, Fisher’s Exact test). When test). Fisher’s Exact 0.06, = IRP2 a were homogenates from GOLD2 Supplementary Fig. 6 Fig. Supplementary Irp2 7 0 ), with phenotype informa phenotype with ), P as compared to high high to compared as IRP2 ), and CEL expression files files expression CEL and ), = 1.6 × 10 × 1.6 = −/− IRP2 −3 versus WT mice (overall n IRP2 ; ; non-smoker controls: gene expression, with with expression, gene = 136 COPD subjects, peak have decreased decreased have peak −3 peak in our data, data, our in peak ). For the ECLIPSE ; smoker controls:smoker ; IRP2 Supplementary Supplementary −3 ) between the the between ) IRP2 P aus (for values /geo/geo2r/ expression ( ) within within ) IREB2 et al. et al. IRP2 IRP2 8 L 8 6 8 m 7 7 ). ). - - - ) , . ,

glutamine and 1 mM pyruvate for 1 h before the measurement. After the the After 1 of concentrations final measurement. OCR, and ECAR the of rates basal the before of recording h 1 for pyruvate mM 1 and glutamine assay medium (Seahorse Bioscience), supplemented with 5 mM glucose, 4 mM the experiment, cells were treated with CSE for 4 h. Cells were incubated in XF (3400-010-01, Trevigen) for 4 d (with medium change on day 2). On the day of ng/ 50 with coated Bioscience) microplates(Seahorse culture cell Primary airway epithelial cells were isolated as described above and plated onto rates were determined by the Seahorse XF 96 flux analyzer (Seahorseanalysis. Seahorse Bioscience). ferricytochrome ferrocytochrome of tions using an optimized colorimetric assay based on the decrease in frac absorbance mitochondria-enriched in measured was activity oxidase c Cytochrome (BD Biosciences) and analyzed with FlowJo analytical software (Tree Star Inc.). for 20 min at 37 °C with 150 nM TMRE. Data were acquired with p a FACSCanto II (TMRE) (Abcam). Briefly, cells ester were ethyl treatedtetramethylrhodamine using with cells CSE orepithelial 20 primary in measured chondrial isolationmito kitMITOISO1 (Sigma-Aldrich). the using Mitochondrialby measured membrane was uptake potential JC1 was and mice of lungs c activity. cytochrome oxidase and potential membrane mitochondrial of Measurement 100% at be to considered strength and was diluted in complete was DMEM medium for cell treatment. fashion this in generated CSE The thawing. upon immediately used and °C, storedat−80 filter-sterilized, was extract The Each cigarette was smoked within 6 min until approximately 17 mm remained. DMEM. ml 50 through removed filters with cigarettes 3R4F five from smoke Briefly, a peristaltic pump (VWR International) was used to bubble mainstream and HBE cell lines were authenticatedlines cell and HBE andfor checked mycoplasma infection. (100 gentamicin and FBS 10% containing (DMEM) medium Eagle’s Dulbecco’smodified in maintained and ATCC from purchased were cells Beas-2B epithelial bronchial lung Human instructions. ATCC’s to accord ing cultured were and (ATCC) Collection Culture Type American the passage before experiments for used and described previously as lungs mouse from CSE. and shRNAsiRNA, culture, Cell unit area of cytoplasm were counted using ImageJ (NIH). characteristics (defined as mitochondria with swollen or abnormal cristae) per ‘abnormal’ with mitochondria of number the and calculated, was cytoplasm the of area total The key. blinded a using slides analyzed S.M.C. technician. facility core experienced an by acquired were images and facility core TEM ( sample or mouse each for reader blinded a by random at selected were fields described previously as protocolstandard a using performed was embedding Sample processing. until °C 4 at kept and pH 7.4. The samples were washed and stored in 0.1 M sodium cacodylate buffer 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer,sodium of solution fixative TEM-grade using °C 4 at overnight fixed were CS chronic (TEM). microscopy electron Transmission subjects included in studies.these ( positive positive shRNA clones were forselected using puromycin. containing cells and procedures,standard using weregenerated particles viral coli (pLKO.1-puro) were purchased as frozen bacterial glycerol plasmids stocks lentiviral in shRNA in clones shRNA MISSION shRNA). MISSION 2 protein element–binding iron-responsive (NM_004136, Sigma-Aldrich from shRNAhuman to techniques. targeted standardtransfection using 48 h using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies)for Healthcare) GE Dharmacon, L-022281, siRNA ON-TARGETplus IREB2 human to targeted siRNA with treated were cells Beas2B n http:/ -trifluoromethoxyphenylhydrazone (FCCP) for the indicated times and stained = 1 mouse per group). Specifically, a blinded code was generated by the the by generated was code blinded a Specifically, group). per mouse 1 = CSE was prepared and added to culture media as previously described previously as media culture to added andprepared was CSE for propagation and downstream purification of the shRNA clones. shRNA /www.lung-genomics.or 1 7 . . Primary human bronchial epithelial (HBE) cells were obtained from c Mitochondria-enriched fractions were isolated from the the from isolated were fractions Mitochondria-enriched by cytochrome Extracellular acidification rates and oxygen consumption oxygen and rates acidification Extracellular c measured at 550 nm, which is caused by its oxidationto itsby caused is which nm, at550 measured g ). Informed consent was obtained from all all from obtained was consent Informed ). c oxidase (Sigma-Aldrich). Primary epithelial cells were isolated isolated were cells epithelial Primary 1 8 . For EM quantification, 15 image 15 quantification, ForEM . Tissues from mice exposed to to exposed mice from Tissues doi: µ IRP2 M carbonylcyanide 10.1038/nm.4021 µ (SMARTpool: g/ml). Beas2B Beas2B g/ml). µ Escherichia l laminin 1 laminin l IREB2 63 was was µ , 8 M 8 - - - .

© 2015 Nature America, Inc. All rights reserved. extraction kit (74104, Qiagen) and reverse-transcribed with SuperScript III III SuperScript with reverse-transcribed and Qiagen) (74104, kit extraction Real-time qPCR. (1:1,000 H00084701-M01 Abnova) and HO-1 (1:1,000 sc-1797, Santa Cruz). NeocloneBiotechnology)N0039,(1:1,000Cox4i2Fbxl5 Cruz), Santa sc-7382, Cruz), cleaved caspase-3 (1:500 Asp175, Cell Signaling Technology),(1:1,000 Bcl2 H-155:(1:1,000 sc-25820, Santa Cruz), mitoferrin sc-25617,2 Santa Cruz),Tom20 (1:1,000 (1:2,000 FL-145, sc-11415, SantaP-12: Cruz),frataxin sc-138430, Santa MitoProfileTotal OxPhosantibody, ab110413,Abcam), ferritin(1:1,000 H-53: Aldrich),electron transport chaincomponents including COXMTCO1(1:500 1 (1:1,000 CD71 (H-300): sc-9099, Santa Cruz), actin (1:10,000 A00158, Sigma- Aldrich), Atg 7 (1:2,000 APG7 (H-300): sc-33211,antibody, aconitase19412,1 Santa Novus Sigma-Biologicals),L7543,(1:2,000 LC3B Cruz), transferrin receptor Biologicals and Irp2 antibody from T.A.R.ing proteins: at Irp2a 1:1,000(1:1,000 7H6:dilution), sc-33682, 1:1,000Irp1 Santastandard(1:1,000 Cruz, immunoblotting NB100-1798,NBP1- techniquesNovus with the following antibodies to natesthe or mitochondria-enriched follow fractions isolated from thelungs of mice using Immunoblotting. to calculated construct a versuscalcein of increasing concentrations of calcein were added sequentially and the series a blue, trypan without cells In sample. each for calculated was addition hyde isonicotinoyl hydrazine (SIH). The change in fluorescence ( excitation, 488-nm measuring the blank rate before and the addition of 100 emission 517-nm at measured was Fluorescence fluorescence. (25 blue trypan with incubated and HBS in pended Hanks’ Balanced Salts (HBS) solution (containing phenol red). Cells were resus 0.5 with incubated were Cells DMEM. serum-free in resuspended and counted trypsinization, by harvested overnight, left dish, 10-cm a in seeded were cells pool of Beas2B cells was measured as previously described at 400 nm against a standard of curve known concentrations of heme. 10 according to the manufacturer’s instructions. Briefly, heme iron was measuredcytosolic fractionsin using the QuantiChrom Heme Assay Kit (BioAssay Systems) were fromcalculated a standard curve. unknowns and nm, 535 at read were Samples solution). NaAc saturated 1/2 in sulfonate bathophenanthroline 0.02% acid, thioglycolic (0.2% BATBuffer using NHI Acid) were incubated in a 1-ml cuvette for 5–10 min at atRT 2,000with r.p.m.800 for 15 s. Equal volumes of sample or iron standard in 3 (25–0.39 M HCl) overnight at 65 °C. Samples were cooled, vortexed and centrifuged fractions were incubated with 100–500 acquisition in all cases. for area image sample the choose to used were coverslip gridded a and excel acquiring images and before data analysis. Random-number generations using ture in ImageJ. Lung sections were blinded by taping the slide identifiers before fea Threshold the using quantified staining and reader experienced blinded in quantified Perls’nification. with iron stain reacts to form a blue-black color. Staining was mag MicroImaging)at 200× Zeiss Carl HRc; (Axiocam camera digital a with reader using a light microscope (Axiophot; Carl Zeiss MicroImaging) equipped dehydrated using graded alcohols and samples viewed by a blinded experienced were Slides min. for 4 incubated and slide each to added was solution fast-red RT. at500 min 30 for acid) hydrochloric 5% with ferrocyanide potassium (5% Perls’stain in incubated were Slides PBS. in washed and hols embedded 5- measurements. iron non-heme and stain Perls’ concentrations of DNA. known with standards against 33342 Hoechst with DNAby normalized were mal respiratory capacity and non-mitochondrial respiration, respectively. Rates maxi protoninstrument’sobtain leak, the to through ports (Sigma) injection oligomycin, 2 doi: Measurementlabileof iron using(CA-AM). calcein-AMpool assay.iron Heme Non-hemeironassay. µ µ 10.1038/nm.4021 l of mitochondrial or cytosolic fractions by measuring the intensity obtained M M CA-AM at 37 °C for 8 min with constant agitation before washing with µ µ n m-thick m-thick lung sections were through deparaffinized graded alco = 3 or 4 mice per group ( group per mice 4 or 3 = M FCCP and 0.5–0.5 mRNA was extracted from lung tissue using the Qiagen RNA Immunoblot analyses were performed in whole-lung homoge Heme iron was measured in isolated mitochondrial and mitochondrial isolated in measured was iron Heme Lung tissue (50–200 mg) or 50- to 200- to 50- or mg) (50–200 tissue Lung µ M rotenone and antimycin A were added µ ∆ l of NHI Acid (10% trichloroacetic acid F n standard curve. = 10 20× images per mouse) by a by mouse) per images 20× 10 = omlnfxd paraffin- Formalin-fixed, µ g) to block extracellular extracellular block to g) 8 9 . Briefly, 10 µ The labile iron labile The M M salicylalde ∆ µ µ F l subcellular subcellular l l of nuclear of l ) ) upon SIH 6 Beas2B ∆ µ F g/ml was µ ------l

in PBS twice and 0.5% bovine serum albumin (BSA) in PBS twice. The cells cells The twice. PBS in (BSA) albumin serum bovine 0.5% and twice PBS in washed then Triton-X 100 0.01% times. in min 15 indicated for the permeabilized were cells for The CSE with treated were plate 6-well a in slips cover imaging. Confocal software (Tree Star Inc.). analytical FlowJo with analyzed and Biosciences) (BD II FACSCanto a with (M36008, Invitrogen) MitoSOXstaining by(2.5 cells in measured was mtROS Probes). Molecular (L-3224, Kit loss of plasma membrane integrity using the LIVE/DEAD Viability/Cytotoxicity cellular esterase activity and red fluorescent intra ethidium homodimer–1 indicate to to indicate calcein-AM fluorescent green with stained simultaneously cytometry. Flow using TaqMan PCR Master Mix (Life Technologies). System Detection Sequence 7300 PRISM ABI an with out carried was qPCR Technologies. Life fromReal-time purchased were assays gene-expressionfor ferrin receptor 1, human ferritin heavy chain, human frataxin and was humanused from RIP-seq library preparations. TaqMan primers for human trans in mRNA The Technologies). (Life Transcriptase extraction kit (74104, Qiagen) and reverse-transcribedMix (Life Technologies).with mRNASuperScript was extracted from IIIcells using Reversethe Qiagen RNA with an ABI PRISM 7300 Sequence Detection System using TaqMan assaysPCR were Masterpurchased from Life Technologies. Real-time qPCR was carried out Reverse Transcriptase (Life Technologies). TaqMan primers for gene-expression 62. 61. in CS-exposure studies. an exclusion criterion. However, only one mouse died out of all of those exposed test. For animals subjected to cigarette smoke exposure, early death was used as appropriate an statistical documenting figure each with distributed, normally at accepted was significance statistical ( diets iron and DFP, CS, ratio, C:P IRP2, STATA/ICof using conducted effects was the isolate to (v13) significance level was made for multiple comparisons. Multivariable regression two-sample a significant a assays, way ANOVAsFor tests. MCC pairwise subsequent with Bonferroni-corrected t Student’s unpaired the using assessed were groups control and experimental least two independent experiments. Differences in measured variables between software (GraphPad Software). Data are presented as the mean analysis. Statistical associate (data not shown). transcriptional activator were used as a control for Venus fragments that stably Venus fragments to fused the leucine-zipper regionself-associating of the of yeast Gcn4 fluorescence The fluorescence). FITC (measuring cytometry flow and drial Venuscytosolic fragment pairs to fused was GRX2 determined using mitochon the express that vectors with transfected transiently cells of cence for clusters, 2Fe-2S coordinates 24 h. Transfected which cells were treated with 20% CSE for 0.5, 1 (GRX2), or 2 h. The fluores 2 glutaredoxin human to conjugated and cytosol or mitochondrion the to targeted fragments protein (0.8 constructs with transfected were cells cells. Beas2B were in measured in Beas2B cells assembly as previously described cluster Fe-S of Measurement using DAPI. stained wereNuclei (1:400). anti-rabbitantibody 488 Alexa IgG secondary in washed five were times samples with The 0.5% Cruz). BSA Santa and thensc-33682, incubated(1:100, IRP2 forhuman 1 to h IgG atrabbit room temperature primary a with temperature room at once h 1 for incubated washed then and were BSA 0.5% in cells The PBS. in BSA 2% with min 45 for blocked were -test and between multiple groups and conditions using one-way and two- and one-way using conditions and groups multiple between and -test

Siempos, I.I. Siempos, P.J.Lee, autophagy in mice. in autophagy hyperoxic lung injury. lung hyperoxic et al. et t -test for a priori hypotheses only. Thus, no adjustment in the the in adjustment no Thus, only. hypotheses priori a for -test et al. et Regulation of heme oxygenase–1 expression oxygenase–1 heme of Regulation To discriminate between live and dead cells, cells were were cells cells, dead and live between discriminate To Statistical analysis was conducted using GraphPad Prism using conducted was analysis Statistical 000 esB el see ot poly- onto seeded cells Beas2B 80,000 Cecal ligation and puncture–induced sepsis as a model to study to model a as sepsis puncture–induced and ligation Cecal P J. Vis. Exp. Vis. J. value was followed by a pair-wise comparison using using comparison pair-wise a by followed was value Am. J. Respir. Cell Mol. Biol. Mol. Cell Respir. J. Am. Fig. 6a Fig.

84 , c µ ). ). , e51066 (2014). e51066 , M for 10 min at 37 °C). Data were acquired P values were calculated, and minimum minimum and calculated, were values P < 0.05. Mean values appeared to be be to appeared values Mean 0.05. < µ g) containing Venus fluorescent Venusfluorescent containing g)

14 Supplementary Figure 4f Figure Supplementary 9 , 556–568 (1996). 556–568 , 0 . Briefly, 8 × 10 nature medicine nature in vivo in 2Fe-2S clusters clusters 2Fe-2S l ± -lysine–coated -lysine–coated s.e.m. from at and in vitro in 5 Beas2B β -actin in ­ - - -

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