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. 2019 Jul 17;10(1):3145.
doi: 10.1038/s41467-019-10991-7.

Involvement of cigarette smoke-induced epithelial cell ferroptosis in COPD pathogenesis

Masahiro Yoshida  1 , Shunsuke Minagawa  2 , Jun Araya  1 , Taro Sakamoto  3 , Hiromichi Hara  1 , Kazuya Tsubouchi  1 , Yusuke Hosaka  1 , Akihiro Ichikawa  1 , Nayuta Saito  1 , Tsukasa Kadota  1 , Nahoko Sato  1 , Yusuke Kurita  1 , Kenji Kobayashi  1 , Saburo Ito  1 , Hirohumi Utsumi  1 , Hiroshi Wakui  1 , Takanori Numata  1 , Yumi Kaneko  4 , Shohei Mori  4 , Hisatoshi Asano  4 , Makoto Yamashita  4 , Makoto Odaka  4 , Toshiaki Morikawa  4 , Katsutoshi Nakayama  1 , Takeo Iwamoto  5 , Hirotaka Imai  3 , Kazuyoshi Kuwano  1
Affiliations

Involvement of cigarette smoke-induced epithelial cell ferroptosis in COPD pathogenesis (VSports)

Masahiro Yoshida et al. Nat Commun. .

Abstract

Ferroptosis is a necrotic form of regulated cell death (RCD) mediated by phospholipid peroxidation in association with free iron-mediated Fenton reactions. Disrupted iron homeostasis resulting in excessive oxidative stress has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we demonstrate the involvement of ferroptosis in COPD pathogenesis. Our in vivo and in vitro models show labile iron accumulation and enhanced lipid peroxidation with concomitant non-apoptotic cell death during cigarette smoke (CS) exposure, which are negatively regulated by GPx4 activity. Treatment with deferoxamine and ferrostatin-1, in addition to GPx4 knockdown, illuminate the role of ferroptosis in CS-treated lung epithelial cells. NCOA4-mediated ferritin selective autophagy (ferritinophagy) is initiated during ferritin degradation in response to CS treatment. CS exposure models, using both GPx4-deficient and overexpressing mice, clarify the pivotal role of GPx4-regulated cell death during COPD. These findings support a role for cigarette smoke-induced ferroptosis in the pathogenesis of COPD. VSports手机版.

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Conflict of interest statement (VSports注册入口)

The authors declare no competing interests.

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Fig. 1
Fig. 1
CSE induces ferroptosis in Human bronchial epithelial cells (HBECs). HBECs were treated with 5% Cigarette smoke extract (CSE) for 24 h. Lipid peroxidation assay in HBECs using the lipophilic redox-sensitive dye BODIPY 581/591, which shifts its fluorescence from red to green in response to oxidation. Scale bar = 100 µm.Cell death was assessed by cytotoxicity using LDH assay and cell viability using MTT assay. a, b Saline or Deferoxamine (DFO) (100 µM) was added to HBECs 1 hr before control or 5% CSE treatment. a Representative phase-contrast microscopy images of BODIPY581/591 staining are shown. Scale bar = 100 µm. n = 8 independent experiments. b Representative phase-contrast microscopy images of HBECs, LDH assay (n = 4 independent experiments), and MTT assay (n = 3 independent experiments) are shown. c Saline or Ferrostatin-1 (10 µM) was added to HBECs 1 h before control or 5% CSE treatment. Representative phase-contrast microscopy images of BODIPY581/591 staining are shown. n = 3 independent experiments. d Saline or Ferrostatin-1 (10 µM) or Necrostatin-1 (50 µM) or zVAD-fmk (20 µM) was added to HBECs 1 h before control or 5% CSE treatment. Representative phase-contrast microscopy images of HBECs, LDH assay, and MTT assay are shown. e Western blotting (WB) showing expression levels of GPx4 and β-actin in HBECs transfected with control siRNA or GPx4 siRNA. f, g. HBECs were transfected with control siRNA or GPx4 siRNA 48 h before control or 5% CSE treatment. f Representative phase-contrast microscopy images of BODIPY581/591staining are shown. Scale bar = 100 µm. n = 4 in each group. g. Representative phase-contrast microscopy images of HBECs, LDH assay, and MTT assay are shown. a, c, f Each panel shown represents the mean ± SEM of FITC/Texas Red ratio, taken from at least 3 independent experiments. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. b, d, g Each panel shown represents the mean ± SEM taken from at least 3 independent experiments. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. h Representative transmission electron microscopy(TEM) images of human bronchial epithelial cells transfected with control siRNA or GPx4 siRNA (top row). HBECs were treated with saline or 5% CSE for 24 h. Mitochondria are shown enlarged in bottom rows. Scale bars; 2 µm (top row), 500 nm (bottom row)
Fig. 2
Fig. 2
Ferritinophagy is involved in CS-induced cell death. BEAS-2B cells were treated with 5% CSE for 24 h. a Time course of ferritin, NCOA4, and β-actin expression in response to 5% CSE were assessed by western blotting (WB). b Data shown represent the mean ± SD (n = 3 independent experiments). c Labile iron pool was measured using calcein-AM method in control or 5% CSE treated BEAS-2B. Cells were transfected with control siRNA or NCOA4 siRNA 48 h before control or CSE treatment. control siRNA (n = 5 biologically independent samples). NCOA4 siRNA (n = 4 independent experiments). d WB showing expression levels of NCOA4, ferritin, and β-actin in control or 5% CSE treated BEAS-2B. Cells were transfected with control siRNA or NCOA4 siRNA 48 h before control or CSE treatment. (n = 4 independent experiments.) e–g WB showing expression levels of ferritin and β-actin in control or 5% CSE treated BEAS-2B. (n = 3 independent experiments.) e BEAS-2B cells were transfected with control siRNA or ATG5 siRNA. f BEAS-2B cells were treated with control or PE (Pepstatin A 10 µg/ml and E64 10 µg/ml). g BEAS-2B cells were treated with control or MG132. h Representative images of BEAS-2B show colocalization (yellow) of ferritin (red) with LC3-GFP (green). BEAS-2B were treated with 5% CSE for 6 h. Scale bars = 20 µm. i–k BEAS-2B were transfected with control siRNA or NCOA4 siRNA 48 h before control or CSE treatment. i BODIPY staining (n = 5 independent experiments), j LDH assay (n = 4 independent experiments) and k MTT assay (n = 3 independent experiments) are shown. All bar charts shown represent the mean ± SEM *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test
Fig. 3
Fig. 3
CS induces ferroptosis in mouse models. a, c, d WT mice were exposed to Room Air (RA) or cigarette smoke (CS) for 6 months. Ferric iron deposits were stained with Perls’ DAB staining in lung samples from RA (upper) or CS (lower) exposed mice. Counter staining is Fast red. Image (original magnification, ×400) is representative of 5 images/mouse. 3 biologically independent mice/group were analyzed. Scale bar = 100 µm. b The amount of non-heme iron was measured by means of inductively coupled plasma mass spectrometry (ICP-MS) (n = 4 biologically independent mice). *P < 0.05 by student’s t-test. c WB showing expression levels of ferritin and β-actin in lung homogenate of WT mice. *P < 0.05 by student’s t-test. (n = 7 biologically independent mice). d WB showing expression levels of NCOA4 and β-actin in lung homogenates from RA (n = 4) or CS (n = 4) exposed mice. (n = biologically independent mice.) *P < 0.05 by student’s t-test. e–j Heterozygous GPx4-deficient mice (GPx4+/−), GPx4+/+ (WT), and TG(loxP-GPx4):GPx4+/+ (GPx4TG) mice were exposed to RA or CS for 4 weeks. e LC-MS analysis of PC and their oxidation products in lung homogenates. PC-OOH/PC (16:0/18:2), (16:0/20:4) were shown. f LC-MS analysis of PE and their oxidation products in lung homogenates. PE-OOH/PE (16:0/18:2), (16:0/20:4) were shown. e, f TG RA n = 5,TG CS n = 6, WT RA n = 4,WT CS n = 4, +/− RA n = 4, +/− CS n = 6. (n = biologically independent mice) *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. g Immunohistochemical(IHC) staining of 4-HNE in mouse lung airway (upper panels) and parenchyma (lower panels). Original magnification ×400. Bar = 100 µm. h TUNEL assay staining (green) in GPx4 +/−, WT, and GPx4 TG mice lung sections. Nuclei were counterstained with DAPI (blue). TG RA n = 3,TG CS n = 4, WT RA n = 3,WT CS n = 3, +/− RA n = 3, +/− CS n = 5. (n = biologically independent mice)**P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. i Immunofluorescence staining of cleaved caspase3 (red). Nuclei were counterstained with DAPI (blue). Original magnification ×200. Bar = 100 µm. j WB showing expression levels of caspase 3, cleaved caspase3, and β-actin in lung homogenate of GPx4+/−, WT, and GPx4 TG mice. All bar charts shown represent the mean ± SEM
Fig. 4
Fig. 4
GPx4 modulates COPD phenotype in smoking mouse models. a–e GPx4+/−, GPx4+/+ (WT), and TG(loxP-GPx4):GPx4+/+ (GPx4TG) mice were exposed to RA or CS for 4weeks. a Cell counts of total cells, macrophages, lymphocytes, neutrophils in bronchoalveolar lavage fluid (BALF) from RA or CS exposed mice. n = 6(mice) in TG-RA(TR), n = 10 TG-CS(TC), n = 4 in WT-RA(WR), n = 6 in WT-CS(WC), n = 5 in GPx4+/− RA (GR), n = 9 in GPx4+/− CS(GC). b ELISA assay of IL-33 (n = 8 in TR, n = 12 TC, n = 8 in WR, n = 8 in WC n = 8 in GR, n = 14 in GC), c IL-1α (n = 8 in TR, n = 12 TC, n = 8 in WR, n = 8 in WC, n = 8 in GR, n = 14 in GC), e TNF-α (n = 4 in TR, n = 5 TC, n = 4 in WR, n = 4 in WC, n = 4 in GR, n = 6 in GC), d in lung homogenate in BALF and d HMGB-1 in BALF(n = 4 in TR, n = 9 in TC, n = 4 in WR, n = 7 in WC, n = 4 in GR, n = 9 in GC), from RA or CS exposed mice. f, g Mice were exposed to RA or CS for 6 months. Lung sections were stained with haematoxylin and eosin. Representative histological images of GPx4+/−, WT, and GPx4 TG mice lung parenchyma. Original magnification ×200. Bar = 100 µm. f Alveolar size was estimated by means of the mean linear intercept (MLI) method. n = 3 in TR, n = 5 TC, n = 3 in WR, n = 6 in WC, n = 3 in GR, n = 4 in GC. g Representative histological images of GPx4+/−, WT, and GPx4 TG mice airways. Original magnification ×200. Bar = 100 µm. Alveolar wall thickness determined by calculating the airway wall/basement membrane length using Image J. n = 3 in TR, n = 5 TC, n = 3 in WR, n = 6 in WC, n = 3 in GR, n = 4 in GC h Representative TEM image of bronchial epithelial cell of Room Air exposed WT mice (left, top), CS exposed WT mice (middle, top), and CS exposed GPx4+/− mice (right, top). Mitochondria are shown enlarged in bottom rows. Scale bars: 2 µm (top row), 500 nm (bottom row). (n = 4 biologically independent mice). All data shown represent the mean ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. Throughout, n = biologically independent mice
Fig. 5
Fig. 5
Involvement of ferroptosis in human COPD lung. a WB showing expression levels of GPx4 and β-actin in HBECs isolated from never smokers (n = 6) or non COPD smokers (n = 6) or COPD patients (n = 7). The bar chart shown represent the mean ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. The correlation analysis between GPx4 expression level and the percentages of FEV1/FVC (n = 19) in HBECs from never smokers, non-COPD smokers, and COPD patients. b IHC of GPx4 in never smoker, healthy smoker and COPD lung. Original magnification ×400. Bar = 100 µm. c WB showing expression levels of NCOA4 and β-actin in lung homogenates from never smokers (n = 5) or non COPD smokers (n−6) or COPD patients (n = 15). The bar chart shown represent the mean ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. The correlation analysis between NCOA4 expression level and the percentages of FEV1/FVC (n = 31) in human lung homogenates from never smokers (n = 10), non-COPD smokers (n = 6), and COPD patients (n = 15). d IHC of NCOA4 in human never smoker, healthy smoker and COPD lung. Original magnification ×400. Bar = 100 µm. e LC-MS analysis of PC and their oxidation products in never smokers, smoker non-COPD, and COPD lung homogenates. MS of precursor ion scanning of m/z 184 in the positive ion mode were analyzed. PC-OOH/PC (18:0/18:2), (18:1/18:2), (16:0/20:4) shown in upper panel. non-COPD, n = 6; COPD, n = 6. LC-MS analysis of PE and their oxidation products in never smokers, smoker non-COPD, and COPD lung homogenates. *P < 0.05 by student’s t-test. MS of neutral loss scanning of 141 Da in the positive ion mode were analyzed. PE-OOH/PE (18:0/18:2), (18:1/18:2), (16:0/20:4) were shown in lower panel. non-COPD, n = 6; COPD, n = 7. *P < 0.05 by by student’s t-test. f Perls’ DAB and 4-HNE staining in never smoker, healthy smoker and COPD lung tissues (original magnification, ×400). Bar = 100 µm. g Representative TEM images of bronchial epithelial cells in never smoker lung and COPD lung (Brinkman Index = 2100, FEV1% = 62.5%). Scale bars ;2 µm. Throughout, n = independent patients
Fig. 6
Fig. 6
Schematic representation of smoking induced ferritinophagy and ferroptosis. Cigarette smoke increases intracellular ferritin and decreases intracellular glutathione transiently. Ferritin is delivered to autophagosomes by NCOA4 and is degraded to non-heme iron via ferritinophagy. The excess of free iron leads to generation of reactive oxygen species and lipid peroxidation through Fenton reactions, which can be regulated by deferoxamine, ferrostatin-1, and GPx4. Lipid peroxidation leads to cell membrane disruption, including in mitochondoria, resulting in ferroptosis. DAMPs and ROS released from ferroptotic cells are implicated in COPD pathogenesis
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