Fig 2: Irp2 is pathogenic in experimental COPD. (a) Irp2 protein (left), mRNA (right) (n = 8 per group), (b) representative (n = 4) EMSA (left) with quantification of total-Irp (n = 5 per group) and specific-Irp2 activity (n = 3 per group), (c) Irp1 protein expression (n = 3 per group) and (d) representative Irp2 immunostaining (arrows indicate Irp2) in WT mouse lungs exposed to room air (RA) or CS (1–6 months). (e) Representative Hematoxylin-Eosin stained lung-sections (left), (n= 3 mice per group) mean chord length (middle), weighted mean diameters (right) and (f) representative trichrome stained lung-sections (left) and ECM protein thickness around small airways (right) in WT and Irp2-/- mice exposed to RA or CS (6 months), staining; n = 2 technical replicates. (g) Cleaved caspase 3 (left) (ELISA, n = 7 per group) and MMP-9 (right) (ELISA, WTRA n = 5; WTCS n = 3; Irp2-/- RA, CS n = 6) levels in whole lung of WT and Irp2-/- mice exposed to RA or CS (6 months), n = 2 technical replicates. (h) 99mTc-SC clearance over 1–3 hours (left) in WT mouse lungs exposed to RA (n = 9) or CS (n = 10) (1 month). 3 hour 99mTc-SC clearance (right), (i) BALF IL-33 (left, ELISA; WTRA n = 3; WTCS n = 5; Irp2-/- RA n = 5; Irp2-/-C n = 6) and BALF IL-6 protein concentrations (right, ELISA, n = 3 per group) in WT and Irp2-/- mice exposed to RA or CS (1 month). (d–f) Scale bars, 50 µm. All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.005 by one-way ANOVA with Bonferroni correction. #P < 0.05, ##P < 0.01, ###P < 0.005 by student’s unpaired t-test. n.s., not significant.

Fig 3: IRP2-associated mitochondrial iron loading and CS. (a) Representative Perls’ stained lung sections (n=3 mice per group) (left) (n = 2 technical replicates) with quantification (middle) (n = 10 images per mouse, n = 3 per group) and non-heme iron levels (right, n = 4 mice per group, n = 4 technical replicates) in WT mice exposed to RA or CS (1–6 months). Scale bars, 50 µm. Arrows indicate staining. n.c.; negative control. (b) Representative immunoblot (n=3 experiments) (left) with quantification (right) of transferrin and ferritin expression (n = 5 per group, n = 2 technical replicates), (c) total non-heme iron (left, n = 5 mice per group, n = 4 technical replicates) and fold change mitochondrial (RA n = 11; CS 1 month n = 6, 4 months n = 8, 6 months n = 9) and cytosolic non-heme (RA n = 6; CS 1 month n = 11, 4 months n = 3, 6 months n = 5) iron (right) in WT mouse lungs exposed to RA or CS, n = 2 technical replicates. (d) Fold change non-heme iron (left, WT and Irp2-/-; RA n = 13, CS 1 and 6 months n =5, CS 4 months n =3) and heme-iron (right, WT and Irp2-/-; RA n = 10, CS 1–6 months n =5) in mitochondrial fractions from WT mouse lungs exposed to RA or CS, n = 2 technical replicates. (e) Mitoferrin 2 (WT and Irp2-/-; RA n = 8; CS 1 month n = 3, 4 months n = 6), (f) frataxin expression (WT RA n = 5; CS n = 3; Irp2-/- RA n = 6; CS n = 6) in whole lung of mice exposed to RA or CS. (g) Mitochondrial non-heme (left, n = 4 per group), mitochondrial heme iron (right, n = 4 per group), (h) 3 hour 99mTc-SC clearance (left) and IL-6 protein concentrations (right, n = 4 per group) in the lungs of WT and Fxnki/ko mice exposed to RA or CS (1 month). (i) Schematic of mitochondrial iron loading regulated by Irp2. All data are mean ± s.e.m. *P < 0.05. **P < 0.01, ***P < 0.005 by one-way ANOVA followed by Bonferroni correction. #P < 0.05 by student’s unpaired t-test.

Fig 4: Novel targets of IRP2 in the lung. (a) Circos plot of communities of genes and GO terms to demonstrate gene transcripts and pathways enriched and/or altered in the RIP-Seq data set. CTL-specific (blue), DFO-specific (red) and common (purple) peak data sets are represented by individual rings, with the height of bars corresponding to the peak score (n = 2 biological replicates). Gene expression data (mRNA) from the LGRC (n = 121 COPD subjects, n = 20 non-smokers and n = 18 smokers) represented on inner black ring. The height of these bars corresponds to the log2 fold-change (FC) in gene expression levels between subjects with COPD and controls. Green denotes lower expression in COPD and magenta denotes higher expression in COPD. Word Clouds representing GO terms in each community with the size of each word reflecting its frequency among the names of the pathways in the community. Community 1 shows enrichment for the cell cycle, metabolism of RNA, the proteasome and immune system; community 2 for metabolism, mitochondria and membrane trafficking; community 3 for DNA repair, apoptosis, the cell cycle and signal transduction; community 4 for metabolism of proteins, transcription and translation; community 5 for nucleotide/purine metabolism pathways. (b) Functional enrichment clustering analysis workflow to evaluate collective differential expression of genes in “communities” of RIP-Seq and in Irp2-/- versus WT gene expression data. (c) Results of functional enrichment clustering analysis. ***P = 1.08 x10-8 by a two-tailed unpaired t-test.

Fig 5: Targeting mitochondrial iron in experimental COPD. (a) 3 hour 99mTc-SC clearance and (b) percentage weight gain of WT mice exposed to RA or CS (1 month) on a control (300 ppm iron), low iron (6 ppm iron) or high iron (2% carbonyl iron) diet. (c) 3 hour 99mTc-SC clearance in WT and Irp2-/- mice exposed to RA or CS (1 month) and treated with DFP as a prophylactic dosing strategy or as a therapeutic dosing strategy; blue arrows indicate point of DFP addition. (d) Percentage weight gain (relative to initial weight at the start of smoke exposure) (4 weeks CS n = 17; 5 weeks CS n = 12; 6 weeks CS n = 12; 7 weeks CS n = 4; 8 weeks CS n = 4; 4 weeks + DFP n = 11; 5 weeks + DFP n = 11; 6 weeks + DFP n = 11; 7 weeks + DFP n = 5; 8 weeks + DFP n = 4), (e) fold change in lung mitochondrial non-heme iron (control n = 8;DFP control n = 8; 1 month CS n = 10; 6 weeks CS n = 4; 6 weeks +DFP n = 4; 8 weeks CS n = 4; 8 weeks CS +DFP n = 4; n = 2 technical replicates), (f) total BAL leukocytes and (g) total BALF protein in WT mice treated with DFP using a therapeutic dosing strategy as in (c), but continued for 2 or 4 weeks of CS exposures. Black arrows indicate time of CS exposure and blue arrows indicate time of DFP addition. (h) Schematic of the major findings of this study. All data are mean ± s.e.m. *P < 0.05. **P < 0.01, ***P < 0.005 by one-way ANOVA followed by Bonferroni correction.