Involvement of GPx4-Regulated Peroxidation in Idiopathic Pulmonary Fibrosis Pathogenesis

This information is current as Kazuya Tsubouchi, Jun Araya, Masahiro Yoshida, Taro of September 28, 2021. Sakamoto, Tomoko Koumura, Shunsuke Minagawa, Hiromichi Hara, Yusuke Hosaka, Akihiro Ichikawa, Nayuta Saito, Tsukasa Kadota, Yusuke Kurita, Kenji Kobayashi, Saburo Ito, Yu Fujita, Hirofumi Utsumi, Mitsuo Hashimoto, Hiroshi Wakui, Takanori Numata, Yumi Kaneko, Shohei Mori, Hisatoshi Asano, Hideki Matsudaira, Takashi Downloaded from Ohtsuka, Katsutoshi Nakayama, Yoichi Nakanishi, Hirotaka Imai and Kazuyoshi Kuwano J Immunol published online 18 September 2019 http://www.jimmunol.org/content/early/2019/09/17/jimmun ol.1801232 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 18, 2019, doi:10.4049/jimmunol.1801232 The Journal of Immunology

Involvement of GPx4-Regulated Lipid Peroxidation in Idiopathic Pulmonary Fibrosis Pathogenesis

Kazuya Tsubouchi,*,†,1 Jun Araya,*,1 Masahiro Yoshida,* Taro Sakamoto,‡ Tomoko Koumura,‡ Shunsuke Minagawa,* Hiromichi Hara,* Yusuke Hosaka,* Akihiro Ichikawa,* Nayuta Saito,* Tsukasa Kadota,* Yusuke Kurita,* Kenji Kobayashi,* Saburo Ito,* Yu Fujita,* Hirofumi Utsumi,* Mitsuo Hashimoto,* Hiroshi Wakui,* Takanori Numata,* Yumi Kaneko,* Shohei Mori,x Hisatoshi Asano,x Hideki Matsudaira,x Takashi Ohtsuka,x Katsutoshi Nakayama,* Yoichi Nakanishi,† Hirotaka Imai,‡,{ and Kazuyoshi Kuwano* Downloaded from The imbalanced redox status in lung has been widely implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis. To regulate redox status, must be adequately reduced to water by (GPx). Among GPx isoforms, GPx4 is a unique that can directly reduce phospholipid hydroperoxide. Increased lipid peroxidation products have been demonstrated in IPF lungs, suggesting the participation of imbalanced lipid peroxidation in IPF pathogenesis, which can be modulated by GPx4. In this study, we sought to examine the involvement of GPx4-modulated lipid peroxidation in regulating TGF-b–induced myofibroblast differentiation. Bleomycin-induced lung fibrosis development in mouse models with genetic ma- http://www.jimmunol.org/ nipulation of GPx4 were examined. Immunohistochemical evaluations for GPx4 and lipid peroxidation were performed in IPF lung tissues. Immunohistochemical evaluations showed reduced GPx4 expression levels accompanied by increased 4-hydroxy-2- nonenal in fibroblastic focus in IPF lungs. TGF-b–induced myofibroblast differentiation was enhanced by GPx4 knockdown with concomitantly enhanced lipid peroxidation and SMAD2/SMAD3 signaling. Heterozygous GPx4-deficient mice showed enhance- ment of bleomycin-induced lung fibrosis, which was attenuated in GPx4-transgenic mice in association with lipid peroxidation and SMAD signaling. Regulating lipid peroxidation by Trolox showed efficient attenuation of bleomycin-induced lung fibrosis devel- opment. These findings suggest that increased lipid peroxidation resulting from reduced GPx4 expression levels may be causally associated with lung fibrosis development through enhanced TGF-b signaling linked to myofibroblast accumulation of fibroblastic focus formation during IPF pathogenesis. It is likely that regulating lipid peroxidation caused by reduced GPx4 can be a promising by guest on September 28, 2021 target for an antifibrotic modality of treatment for IPF. The Journal of Immunology, 2019, 203: 000–000.

diopathic pulmonary fibrosis (IPF) is characterized by pro- and antioxidant expression levels in IPF lungs (3). Oxidative gressive and devastating lung parenchymal fibrosis with stresses, including hydrogen peroxide, have increased in IPF I unknown etiology (1). Although detailed molecular mecha- lungs (4). In addition, downregulation of such as nisms for IPF development remains uncertain, alveolar epithelial glutathione (GSH) and NF erythroid 2–related factor 2 (Nrf2) cell damages followed by aberrant wound healing processes rep- has been shown in the lungs from IPF patients (5–8). In con- resented by the formation of fibroblastic focus (FF) are respon- trast, several antioxidant , including heme oxygenase sible for fibrotic remodeling (2). Among a variety of mechanisms, 1 (HO-1) and thioredoxin, are also upregulated in IPF lungs, the imbalanced redox status in lung has been widely impli- which can be a part of adaptive mechanisms for excessive oxidative cated in IPF pathogenesis based on the analyses of oxidant stress (9–11).

*Division of Respiratory Diseases, Department of Internal Medicine, Jikei University (to H.H.), JP15K09232 (to K. Kuwano), a GlaxoSmithKline Japan Research School of Medicine, Tokyo 105-8461, Japan; †Research Institute for Diseases of the Grant 2017 (to K. Kobayashi), and Japan Agency for Medical Research and Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Development Grant JP18gm0910013 (to H.I.). Japan; ‡Laboratory of Hygienic Chemistry and Medicinal Research Laboratories, Address correspondence and reprint requests to Dr. Kazuya Tsubouchi, Research School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-0072, Japan; x Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu Division of Chest Diseases, Department of Surgery, Jikei University School of { University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address: Medicine, Tokyo 105-8461, Japan; and Advanced Research and Development Pro- [email protected] grams for Medical Innovation, Japan Agency for Medical Research and Develop- ment, Tokyo 100-0004, Japan The online version of this article contains supplemental material. 1K.T. and J.A. contributed equally to this work. Abbreviations used in this article: BLM, bleomycin; FF, fibroblastic focus; GPx, GSH ; GSH, glutathione; HETE, hydroxyeicosatetraenoic acid; 4-HNE, ORCIDs: 0000-0002-9314-4745 (N.S.); 0000-0002-9533-7241 (T. Kadota); 0000-0002- 4-hydroxy-2-nonenal; HODE, hydroxyoctadecadienoic acid; IPF, idiopathic pulmonary 8916-7303 (Y.F.); 0000-0003-2784-2331 (T.N.); 0000-0002-8521-0175 (H.A.); 0000- fibrosis; LF, lung fibroblast; MDA, malondialdehyde; NAC, N-acetylcysteine; ROS, 0002-8817-275X (H.M.); 0000-0002-8039-9159 (T.O.); 0000-0002-0141-7081 (K.N.); reactive oxygen species; siRNA, small interfering RNA; a-SMA, a–smooth muscle 0000-0003-0551-7386 (K. Kuwano). actin; WB, Western blot. Received for publication September 10, 2018. Accepted for publication August 8, 2019. Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 This work was supported by grants from the Japan Society for the Promotion of Science KAKENHI (JP15K09231 and JP18K08158 to J.A.), JP15K09194

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801232 2 GPx4-REGULATED LIPID PEROXIDATION IN IPF PATHOGENESIS

Hydrogen peroxide has an important role in imbalanced redox Technology), rabbit anti–phospho-SMAD2 (3101; Cell Signaling Tech- status and can generate highly reactive hydroxyl radicals through nology), rabbit anti–phospho-SMAD3 (8769; Cell Signaling Technology), Fenton reaction. Hence, hydrogen peroxide must be adequately rabbit anti–phospho-SMAD3 (phospho-S423+S425) (no. 52903; Abcam), and mouse anti-ACTB/b-actin (A5316; Sigma-Aldrich). Human rTGF-b1/ reduced to water mostly by GSH peroxidases (GPx) for protecting TGF-b1 (100-B; R&D Systems), N-acetylcysteine (NAC; 017-05131; cells, which uses the reducing equivalents from its substrate Wako Chemicals), Trolox (202-17891; Wako Chemicals), CM-H2DCFDA GSH. In this enzymatic reaction, GSH becomes oxidized to GSH (C6827; Life Technologies), Hoechst 33258 (B2883; Sigma-Aldrich), disulfide, which is recycled back to GSH by the NADPH-dependent C11-BODIPY 581/591 probe (C10445; Invitrogen), MitoSOX Red (M36008; Molecular Probes/Life Technologies), and BLM (Nippon GSH disulfide reductase. Mammals express eight isoforms of GPx. Kayaku, Tokyo, Japan) were purchased. GPx1 to GPx4 and GPx6 are in humans, whereas the latter is a Cys-containing variant in mice (12). GPx5, GPx7, and Small interfering RNA and transfection GPx8 are Cys-containing GPx, and GPx7 and GPx8 share signifi- Small interfering RNA (siRNA)–targeting GPx4 (s6112; Applied Bio- cant structural similarities to GPx4. GPx4 is frequently referred to systems Life Technologies), and negative control siRNAs (AM4635 and as phospholipid hydroperoxide GPx because of its unique bio- AM4641; Applied Biosystems, Life Technologies) were purchased. Transfections of LF were performed using the Neon Transfection Sys- chemical functions toward phospholipid hydroperoxides. GPx4 tem (MPK5000; Invitrogen Life Technologies), using matched opti- regulates oxidative modifications of phospholipids, including cho- mized transfection kits (MPK10096; Invitrogen Life Technologies). lesteryl esters and cardiolipin. Intriguingly, GPx4 prevents apoptosis through maintaining the integrities of electron transport chain and Measurement of ROS production oxidative phosphorylation in mitochondria (13–15). Increased lipid LF, at a density of 1 3 104 per well, were seeded in a 96-well microplate peroxidation products have been reported in the exhaled breath (237105; Thermo Fisher Scientific). CM-H2DCFDA was used to measure Downloaded from condensate and bronchoalveolar lavage fluid from patients with IPF total cellular ROS according to the manufacturer’s instructions. After in- cubation with CM-H2DCFDA (10 mM) for 30 min at 37˚C, fluorescence of (16–19), suggesting the involvement of imbalanced lipid peroxidation DCF was measured at an excitation wavelength of 485 nm and an emission in IPF pathogenesis, which can be modulated by GPx4. wavelength of 535 nm by a fluorescence microplate reader (Infinite F200; TGF-b, a major profibrotic cytokine, has been widely implicated Tecan Japan, Kanagawa, Japan). Mitochondrial ROS production was an- in IPF pathogenesis (20). TGF-b–mediated biological activities are alyzed by MitoSOX Red staining according to the manufacturer’s in- structions, which was evaluated by fluorescence microscopy (Olympus, regulated via intracellular signaling pathways composed of canon- Tokyo, Japan and BZ-X700; Keyence). http://www.jimmunol.org/ ical SMADs and SMAD-independent noncanonical pathways, in- cluding mitogen-activated protein (MAP) kinases and PI3K (21). Measurement of lipid peroxidation in vitro Reactive oxygen species (ROS) modulate TGF-b–induced cell LF, at a density of 1 3 104 per well, were seeded in a 96-well microplate signaling pathways via activating tyrosine kinases and inactivating (237105; Thermo Fisher Scientific). Lipid peroxidation was measured protein tyrosine phosphatases (22). TGF-b is expressed in a latent using a C11-BODIPY 581/591 probe (C10445; Invitrogen) as described previously (26). Briefly, cells were incubated for 30 min with C11-BODIPY form that must be activated to function and ROS is involved in the m b 581/591 (1 M) in growth medium. Fluorescence of C11-BODIPY was mechanisms for TGF- activation (23). In addition to activating measured by simultaneous acquisition of the green (484/510 nm) and latent TGF-b, ROS can also stimulate the expression and secretion red signals (581/610 nm), providing a ratiometric indication of lipid of TGF-b in many types of cells (22). In macrophages, the lipid peroxidation. by guest on September 28, 2021 peroxidation product 4-hydroxy-2-nonenal (4-HNE) can upregu- Western blotting late TGF-b expression (24), suggesting the existence of vicious cycle between -mediated lipid peroxidation and LF grown on six-well culture plates were lysed in radioimmunoprecipitation b assay buffer (89900; Thermo Fisher Scientific) containing a protease in- excessive TGF- function. However, the involvement of GPx4 hibitor mixture (11697498001; Roche Diagnostics) and 1 mM sodium in regulating lipid peroxidation and TGF-b action with respect to orthovanadate (13721-39-6; Wako Chemicals), or lysed with Laemmli phenotypic alterations of fibroblast remains to be elucidated dur- sample buffer. Western blotting was performed as previously described ing lung fibrosis development in IPF pathogenesis. (25). For each experiment, equal amounts of total protein were resolved In this study, we show the involvement of GPx4-regulated lipid by 7.5–15% SDS-PAGE. After SDS-PAGE, proteins were transferred to polyvinylidene difluoride membrane (ISEQ00010; MerckMillipore), and b peroxidation in TGF- –induced myofibroblast differentiation in incubation with specific primary Ab was performed for 1 h at 37˚C, or 24 h human lung fibroblasts (LF). Using genetic manipulation, including at 4˚C. After washing several times with phosphate-buffered saline with GPx4 transgenic mice and heterozygous GPx4-deficient mice, we Tween 20 (161-25521; Wako Chemicals), the membrane was incubated verify the crucial role of GPx4-regulated lipid peroxidation in bleo- with anti-rabbit IgG, HRP-linked secondary Ab (7074; Cell Signaling Technology), anti-mouse IgG, HRP-linked secondary Ab, 7076) or anti- mycin (BLM)–induced lung fibrosis models. goat IgG (H+I), and HRP-linked secondary Ab (A50-100P; Bethyl Lab- oratories), followed by chemiluminescence detection (34080; Thermo Fisher Scientific, and 1705061; Bio-Rad Laboratories) with the ChemiDoc Materials and Methods Touch Imaging System (Bio-Rad Laboratories). Cell culture, Abs, and reagents Mouse models Normal lung tissues were obtained from pneumonectomy and lobectomy specimens from primary lung cancer. Informed consent was obtained from Heterozygous GPx4-deficient mice (GPx4+/2) and GPx4-transgenic mice all surgical participants as part of an approved ongoing research protocol by (TG [loxP-GPx4]+/+/GPx4+/+) on mixed background (TT2, ICR, and BDF1 the ethical committee of Jikei University School of Medicine. LF were strains) were provided by Prof. H. Imai, Kitasato University (27–29). Also, isolated and characterized as previously described (25). Briefly, LF out- we used GPx4+/+(wild-type) mice as negative control, which is on the same grown from lung fragments were cultured in fibroblast growth medium background. C57BL/6J mice were purchased (CLEA Japan, Tokyo, Japan). (DMEM with 10% FCS and penicillin/streptomycin). LF were serially Mice were maintained in the animal facility at the Jikei University School passaged and used for experiments until passage 6. LF demonstrated of Medicine. All experimental procedures were approved by the Jikei .95% positive staining with anti-VIM/vimentin Abs (V 6630; Sigma- University School of Medicine Animal Care Committee. A dosage Aldrich), and ,5% positive staining with the anti-KRT/cytokeratin Ab of 3 U/kg BLM (4234400D4032; Nippon Kayaku) was intratracheally (Lu-5; Biocare Medical; data not shown). Abs used were rabbit anti-GPx4 administered in 50 ml saline using the MicroSprayer Aerosolizer and a (125066; Abcam), rabbit anti–4-HNE (46545; Abcam), goat anti-COL1/ high-pressure syringe (Penn-Century, Philadelphia, PA). Intraesophageal type I collagen (1310-01; Southern Biotech), mouse anti-a–smooth muscle administrations of Trolox (50 mg/kg) were given daily from day 7 to actin (a-SMA) (A2547; Sigma-Aldrich), mouse anti-FN1/cellular fibro- day 20. On the 21st day, the lungs were removed, fixed overnight in 10% nectin containing extra domain A (ab6328; Abcam), rabbit anti-SMAD2 buffered formalin, embedded in paraffin, and the sections stained with (3122; Cell Signaling Technology), rabbit anti-SMAD3 (9513; Cell Signaling H&E and Masson trichrome according to conventional protocols for The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 1. GPx4 expression levels and the degree of myofibroblast differentiation in response to TGF-b in LF derived from IPF lung. (A) WB using anti-GPx4 and anti–b-actin of cell lysates from LF isolated from control normal lungs (lanes 1–3) and from IPF lungs (lanes 4–6). In the lower panel is the by guest on September 28, 2021 averages (6SEM) shown as relative expression (control normal lung [n = 5] and IPF lung [n = 5]). Open bar is control LF from normal lung, and filled bar is LF from IPF lung. *p , 0.05. (B) Immunohistochemical staining of GPx4 (n = 4) in IPF lung tissues. All photomicrographs of FF in IPF. Metaplastic epithelial cells covering FF (black arrow) and fibroblasts constituting FF (white arrow). Scale bar, 100 mm. (C) WB using anti-GPx4, anti-EDA/fibronectin, anti–type I collagen, anti–a-SMA, and anti–b-actin of cell lysates from control and TGF-b (2 ng/ml)–treated LF isolated from control normal lung and from IPF lung. Protein samples were collected after 24 h treatment with TGF-b. In the right panels are the average (6SEM) taken from five independent experiments shown as relative expressions. *p , 0.05. histopathological evaluation (25). Immunohistochemistry was performed centrifuged (10 min, 2500 rpm), and the lower phase was collected in other on the paraffin-embedded lung tissues as previously described with minor tube. Two milliliters of chloroform and 25 ml of 2 M HCl were added to modifications (25). the upper phase, and the mixture was stirred by a Voltex mixer for 10 min. After centrifugation (10 min, 2500 rpm), the lower phase was put together Sircol soluble collagen assay the first lower phase. The extracted were dried under a gentle stream of nitrogen, dissolved in 100 ml of methanol, and stored at 280˚C until For quantitatively measuring collagen in the left lungs of the mice, the Sircol further use. The liquid chromatography/electrospray ionization/tandem soluble collagen assay was performed according to the manufacturer’s instructions (S100; Biocolor Life Science Assay). mass spectrometry analysis was performed using QTRAP 4500 Quadru- pole Linear Ion Trap hybrid mass spectrometer (AB Sciex, Concord, ON, Measurement of lipid peroxidation in vivo Canada) coupled to a Nexera XR HPLC system (Shimadzu, Kyoto, Japan). The sample was subjected to liquid chromatography/electrospray ionization/ For quantitatively measuring lipid peroxidation in the right lungs of the tandem mass spectrometry analysis using the XBridge BEH C18 column mice, OxiSelect TBARS Assay Kit (MDA Quantitation) was performed (3.5 mm, 150 3 1.0 mm; Waters). Sample (10 ml) was injected by the according to the manufacturer’s instructions (STA-330; Cell Biolabs). autosampler and separated by a step gradient with mobile phase A (0.1% formate in water) and mobile phase B (acetonitrile/methanol, 4:1 v/v) in Liquid chromatography/electrospray ionization/tandem mass spectrometry analysis Table I. Patient characteristics Lung homogenates of the mice were extracted in Tissue Protein Extraction Reagent buffer (78510; Thermo Fisher Scientific) containing a protease inhibitor mixture (05892988001; Roche Diagnostics) and phosphatase Normal IPF inhibitor (4906845001; Roche Diagnostics), having protein at 4.0 mg/ml. Age 65.6 6 3.5 65.2 6 6.0 The sample contained lipids that were extracted from lung homogenates by 6 6 Bligh and Dyer method using 17:0-LysoPC (855676; Avanti Polar Lipids) Smoking index 0 0 1052.5 395.6 m FEV1/FVC (%) 75.7 6 1.7 79.3 6 3.2 as an internal standard (30). Briefly, 100 l of sample were mixed with 6 6 3.7 ml of chloroform/methanol/water (1:2:0.7 v/v/v). The mixture was VC (%) 110.36 6.1 84.48 9.7 stirred by a Voltex mixer for 10 min, and then 2 ml of chloroform/water All values expressed as the average (6 SEM). (1:1 v/v) was added. The mixture was restirred by a Voltex mixer for 10 min, FEV, forced expiratory volume; FVC, forced vital capacity; VC, vital capacity. 4 GPx4-REGULATED LIPID PEROXIDATION IN IPF PATHOGENESIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. GPx4 regulates TGF-b–induced myofibroblast differentiation via modulating ROS and SMAD2/3 signaling in LF. (A) WB using anti-GPx4, anti-EDA/fibronectin, anti–type I collagen, anti–a-SMA, and anti–b-actin of cell lysates from control siRNA (lanes 1 and 2) and GPx4 siRNA (lanes 3 and 4)–transfected LF. TGF-b (2 ng/ml) stimulation was started 48 h posttransfection, and protein samples were collected after 24 h treatment with TGF-b.In the right panels are the average (6SEM) taken from five independent experiments shown as relative expressions. *p , 0.05. (B) Fluorescence intensity of CM-H2DCFDA staining. TGF-b (2 ng/ml for 24 h) treatment was started 48 h after siRNA transfection. The fluorescence level in the control siRNA- transfected cells without TGF-b treatment was designated as 1.0. Shown panels are the average (SEM) taken from six (Figure legend continues) The Journal of Immunology 5 the following ratios: 70:30 (0 min), 50:50 (0–5 min), 20:80 (5–35 min), suggest that reduced GPx4 expression levels in IPF LF might be 0:100 (35–40 min), 0:100 (40–50 min), 70:30 (50–51 min), and 70:30 associated with enhanced myofibroblast differentiation by TGF-b (51–65 min) at a flow rate of 70 ml/min and a column temperature of 40˚C. Multiple-reaction monitoring was carried out to detect specific hydrox- through insufficient antioxidative activity. yoctadecadienoic acid (HODE) and hydroxyeicosatetraenoic acid (HETE) GPx4 regulates TGF-b–induced myofibroblast differentiation 2 in negative ion mode with the following settings: ion spray voltage: 4500 V; via modulating ROS and SMAD2/3 signaling in LF curtain gas (N2): 30 arbitrary units; collision gas (N2): medium; declustering potential: 2100 V; collision energy: 220 V; and temperature: 500˚C. The In line with other containing GPx enzymes, GPx4 shows transitions for the detection of HODE and HETE were as follows: 295.1/171.0 an antioxidative property and catalyzes GSH-mediated reduction of (9-HODE), 295.1/195.0 (13-HODE), and 319.0/219.0 (15-HETE). The standard lipids of 9-HODE (38410; Cayman Chemical), 13-HODE (38610; hydrogen peroxide (H2O2) and organic hydroperoxides (32). ROS Cayman Chemical), and 15-HETE (34720; Cayman Chemical) were used have been widely implicated in regulating TGF-b–evoked cell for preparing the standard curves. signaling and myofibroblast differentiation (25). To elucidate the b TUNEL assay involvement of GPx4 regulating ROS in TGF- –induced myofi- broblast differentiation, we performed knockdown experiments by TUNEL assay was performed using a DeadEnd Fluorometric TUNEL using GPx4 siRNA. GPx4 knockdown significantly enhanced System (G3250; Promega) according to manufacturer’s instructions. The b TUNEL-positive cells in lung were detected using fluorescence micros- TGF- –induced myofibroblast differentiation (Fig. 2A). Next, intra- copy (Olympus, Tokyo, Japan, and BZ-X700; Keyence). The average number cellular ROS production was examined by means of CM-H2DCFDA of dead cells was assessed by manual counting of TUNEL+ cellsineachhigh assay. TGF-b treatment solely induced intracellular ROS pro- power field (3200). duction and significant increase was detected in the setting of

Statistics GPx4 knockdown (Fig. 2B). In general, TGF-b–induced myofi- Downloaded from 6 broblast differentiation is regulated via activation of the SMAD, a Data are shown as the average ( SEM) taken from at least three inde- b b pendent experiments. Student t test was used for comparison of two canonical TGF- signaling pathway (33). TGF- significantly datasets and ANOVA for multiple datasets. Tukey or Dunn tests were used increased phosphorylated forms of SMAD2/3 (1 h after TGF-b for parametric and nonparametric data, respectively, to find where the treatment), and GPx4 knockdown obviously enhanced TGF-b– difference lay. Significance was defined as p , 0.05. Statistical software induced SMAD2/3 activation, suggesting that SMAD2/3 signaling used was Prism v.7 (GraphPad Software, San Diego, CA). is participated in enhanced myofibroblast differentiation in case of http://www.jimmunol.org/ GPx4 reduction. Results To confirm the involvement of ROS in enhanced TGF-b–induced Decreased GPx4 expression levels in IPF LF and GPx4 myofibroblast differentiation during reduced GPx4, NAC (a rep- b reduction enhances TGF- –induced resentative antioxidant) was used. Regardless of GPx4, NAC signifi- myofibroblast differentiation cantly suppressed TGF-b–induced ROS production and myofibroblast To elucidate the involvement of GPx4 in IPF pathogenesis, we differentiation of EDAFN1, type I collagen, and a-SMA at the examined the protein expression levels of GPx4 in LF derived from concentration of 10 mM (Fig. 2D, 2E). These findings support the normal (n = 5) and IPF patients (n = 5). Consistent with other notion that GPx4-modulated ROS production is involved in the reg- antioxidant protein levels, including Nrf2 and GSH in IPF lungs, ulation of TGF-b–induced myofibroblast differentiation in LF. by guest on September 28, 2021 GPx4 expression levels were significantly decreased in LF iso- GPx4-regulated lipid peroxidation is participated in lated from IPF patients (Fig. 1A) (5–7). Patient characteristics are TGF-b–induced myofibroblast differentiation in LF and presented in Table I. To further clarify the physiological relevance increased lipid peroxidation in IPF lungs of GPx4 expression levels in IPF pathogenesis, we performed immunohistochemical evaluations of GPx4 in IPF lungs. Inter- Because of its unique biochemical functions toward phospholipid estingly, in comparison with metaplastic epithelial cells covering hydroperoxides, GPx4 has been recognized as phospholipid hy- FF, fibroblasts constituting FF exhibited barely detectable GPx4 droperoxide GPx (34). Hence, we focused on GPx4-regulated lipid expression (Fig. 1B). peroxidation during TGF-b–induced myofibroblast differentiation. Several lines of evidence suggest that fibroblasts derived from To elucidate the participation of lipid peroxidation, Trolox (a IPF and normal lungs differ with respect to fibrogenic property water-soluble analogue of vitamin E) was used for experiments. (31). Thus, we compared the degree of myofibroblast differenti- Intriguingly, Trolox apparently suppressed TGF-b–induced myo- ation in response to TGF-b in LF from normal and IPF patients. fibroblast differentiation in a dosage-dependent manner, and sig- Compared with normal LF, TGF-b treatment remarkably induced nificant inhibition was observed at the concentration of 500 mM myofibroblast differentiation shown by enhanced extra domain A (Fig. 3A), suggesting the involvement of lipid peroxidation in the (EDA)–containing cellular fibronectin, collagen type I, and a-SMA mechanisms for TGF-b–induced myofibroblast differentiation. expression in IPF LF. Intriguingly, TGF-b clearly increased GPx4 Accordingly, a Trolox concentration of 500 mM was chosen for further protein levels in normal LF. In contrast, no increase in GPx4 was analysis of cell culturing models. The participation of GPx4-regulated demonstrated in TGF-b–treated IPF LF (Fig. 1C). These findings lipid peroxidation in TGF-b–induced myofibroblast differentiation

independent experiments shown as relative expressions. *p , 0.05. (C) WB using anti–phospho-SMAD2, anti-SMAD2, anti–phospho-SMAD3, anti- SMAD3, and anti–b-actin of cell lysates from control siRNA (lanes 1 and 2) and GPx4 siRNA (lanes 3 and 4)–transfected LF. TGF-b (2 ng/ml) stimulation was started 48 h posttransfection, and protein samples were collected after 1 h treatment with TGF-b. In the right panels are the average (6SEM) taken from three independent experiments shown as relative expressions. *p , 0.05. (D) WB using anti-EDA/fibronectin, anti–type I collagen, anti–a-SMA, and anti–b-actin of cell lysates from control siRNA (lanes 1–4) and GPx4 siRNA (lanes 5–8)–transfected LF. NAC (10 mM) treatment was started 48 h posttransfection and 1 h before TGF-b (2 ng/ml) stimulation, and protein samples were collected after 24 h treatment with TGF-b. In the lower panels are the average (6SEM) taken from five independent experiments shown as relative expressions. *p , 0.05. (E) Fluorescence intensity of CM-H2DCFDA staining. NAC (10 mM) treatment was started 48 h posttransfection and 1 h before TGF-b (2 ng/ml) stimulation, and fluorescence intensity was measured after 24 h treatment with TGF-b. The fluorescence level in the control siRNA transfected cells without TGF-b treatment was designated as 1.0. Shown panels are the average (SEM) taken from six independent experiments shown as relative expressions. *p , 0.05. 6 GPx4-REGULATED LIPID PEROXIDATION IN IPF PATHOGENESIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Participation of GPx4-regulated lipid peroxidation in TGF-b–induced myofibroblast differentiation in LF and increased lipid peroxidation in IPF lungs. (A) WB using anti-EDA/fibronectin, anti–type I collagen, anti–a-SMA, and anti–b-actin of cell lysates from control (lane 1) and TGF-b (2 ng/ml) and indicated concentrations of Trolox (lanes 2–6)–treated LF. Trolox treatment was started 1 h before TGF-b stimulation, and protein samples were collected after 24 h treatment with TGF-b. In the lower panels are the average (6SEM) taken from six independent experiments shown as relative expressions. *p , 0.05. (B) Fluorescence intensity ratio of C11-BODIPY 591/581. Trolox (500 mM) treatment was started 48 h posttransfection and 1 h before TGF-b (2 ng/ml) stimulation, and fluorescence intensity was measured after 24 h treatment with TGF-b. The fluorescence intensity ratio in the control siRNA-transfected cells without TGF-b treatment was designated as 1.0. Shown panels are the average (Figure legend continues) The Journal of Immunology 7 was further confirmed by means of C11-BODIPY assay detecting GPx4+/2 mice but clearly decreased in BLM-treated GPx4- intracellular lipid peroxidation. TGF-b treatment increased lipid transgenic mice (Fig. 4G). GPx4 has also been recognized as a peroxidation, which was significantly enhanced in the setting of key regulatory factor in , a recently proposed nonapoptotic GPx4 knockdown. Trolox inhibited TGF-b–induced lipid perox- and iron-dependent-regulated necrosis (35). Accordingly, cell death idation in both control and GPx4 siRNA-transfected LF (Fig. 3B). was evaluated by TUNEL staining in BLM-treated lungs on day 7. Consistent with lipid peroxidation, Trolox clearly suppressed Compared with BLM-treated wild type, significant increase in TGF-b–induced SMAD2/3 phosphorylation and myofibroblast number of TUNEL-positive dead cells was detected in BLM- differentiation in both control and GPx4 siRNA transfected LF treated GPx4+/2, and significant reduction was demonstrated in (Fig. 3C, 3D). BLM-treated GPx4-transgenic mice (Supplemental Fig. 2). Next, to demonstrate the physiological involvement of lipid Trolox attenuates BLM-induced lipid peroxidation and lung peroxidation in IPF pathogenesis, expression levels of 4-HNE, fibrosis development aldehydic products of lipid peroxidation, was assessed by means of immunohistochemical evaluations and Western blot (WB) using LF Finally, antifibrotic role of Trolox in BLM-induced lung fibrosis isolated from patient lungs. Increase in 4-HNE expression levels development was evaluated. In general, day 7 after BLM treatment were demonstrated in fibroblasts comprising FF in IPF lungs is recognized to be the beginning of fibrotic phase. To demonstrate (Fig. 3E). In line with previous report, significantly increased a potential clinical implication of Trolox treatment in IPF with 4-HNE expression levels were also detected in IPF LF com- progressive fibrosis, Trolox was orally administered to mice from pared with those in normal LF, which can be attributed to re- day 7 until day 20 following BLM treatment. BLM-induced body duced GPx4 (Figs. 1A, 3F) (5). weight loss was markedly prevented by Trolox treatment (Fig. 5A). Downloaded from Trolox treatment significantly attenuated lung fibrosis develop- GPx4 regulates BLM-induced lipid peroxidation and lung ment on day 21, as shown by Sircol collagen assay and Masson fibrosis development trichrome staining (Fig. 5B, 5C). Immunohistochemical evalu- To elucidate whether enhanced myofibroblast differentiation ations of 4-HNE also showed clear reduction of lipid perox- conferred by reduced GPx4 is responsible for lung fibrosis de- idation by Trolox treatment in BLM-treated lungs (Fig. 5D),

velopment or not, we employed a BLM-induced lung fibrosis indicating that regulating lipid peroxidation may be a plausible http://www.jimmunol.org/ model using heterozygous GPx4-deficient mice (GPx4+/2) (27), antifibrotic approach for lung fibrosis associated with reduced wild-type (GPx4+/+) mice, and GPx4-overexpressing transgenic GPx4,IPF(Fig.6). (GPx4+/+/Tg [LoxP-GPx4]+/+) mice (28). GPx4 is essential for development, and its ablation is embryonic lethality at 7.5 d in Discussion mice (27); thus, we used GPx4+/2 mice. GPx4 expression levels in In the current study, we demonstrated the involvement of lipid those mice were confirmed by means of WB in lung homogenates peroxidation regulated by GPx4 in lung fibrosis development as a (Fig. 4A). Compared with control-treated mice, BLM-treated mice part of IPF pathogenesis. Reduced GPx4 and increased lipid showed apparent body weight loss. Although no difference in body peroxidation related to 4-HNE were observed in IPF LF compared weight changes was demonstrated between BLM-treated wild-type with non-IPF LF. Although GPx4 was upregulated by TGF-b in by guest on September 28, 2021 and BLM-treated GPx4+/2 mice, BLM-treated GPx4-transgenic normal LF, a clear resistance to GPx4 upregulation with con- mice showed marked recovery on day 21 (Fig. 4B). BLM treat- comitantly enhanced myofibroblast differentiation by TGF-b was ment induced lung fibrosis development in wild type on day 21, observed in IPF LF. In in vitro GPx4, knockdown experiments which was significantly exaggerated in GPx4+/2 mice. In contrast, clarify that GPx4 regulates not only lipid peroxidation, but also GPx4 transgenic mice showed significant attenuation of lung fi- SMAD2/3 signaling in terms of modulating TGF-b–induced brosis by means of Sircol collagen assay and Masson trichrome myofibroblast differentiation in LF. Immunohistochemical evalu- staining (Fig. 4C, 4D). Lipid peroxidation in lungs was examined ations show reduced GPx4 expression levels accompanied by in- by immunohistochemical evaluations of 4-HNE and malondial- creased 4-HNE in FF in IPF lungs. In in vivo BLM-induced lung dehyde (MDA) assay, respectively. Consistent with lung fibrosis fibrosis models, GPx4 expression levels were found to be respon- development, BLM-treated GPx4+/2 mice demonstrated enhanced sible for determining the degree of lung fibrosis development lipid peroxidation, but GPx4 transgenic mice showed reduced mainly through regulating lipid peroxidation and SMAD signaling. lipid peroxidation (Fig. 4E, 4F). In addition, we evaluated HODE Trolox efficiently attenuated lipid peroxidation and lung fibrosis and HETE levels in BLM-treated mouse lungs, respectively. development. Taken together, increased lipid peroxidation resulting BLM treatment increased HODE and HETE levels and further from reduced GPx4 expression levels may be causally associated enhancement was detected in GPX42/+ mice (Supplemental Fig. 1). with lung fibrosis development through enhanced TGF-b–induced In comparison with BLM-treated wild-type mice, phosphory- myofibroblast differentiation linked to myofibroblast accumulation lated SMAD3 expression levels were increased in BLM-treated of FF formation during IPF pathogenesis (Fig. 6).

(6SEM) taken from four independent experiments shown as relative expressions. *p , 0.05. (C) WB using anti–phospho-SMAD2, anti-SMAD2, anti– phospho-SMAD3, anti-SMAD3, and anti–b-actin of cell lysates from control siRNA (lanes 1–4) and GPx4 siRNA (lanes 5–8)–transfected LF. Trolox (500 mM) treatment was started 48 h posttransfection and 1 h before TGF-b (2 ng/ml) stimulation, and protein samples were collected after 1 h treatment with TGF-b. In the lower panels are the average (6SEM) taken from five independent experiments shown as relative expressions. *p , 0.05. (D) WB using anti-EDA/fibronectin, anti–type I collagen, anti–a-SMA, and anti–b-actin of cell lysates from control siRNA (lanes 1–4) and GPx4 siRNA (lanes 5–8)– transfected LF. Trolox (500 mM) treatment was started 48 h posttransfection and 1 h before TGF-b (2 ng/ml) stimulation, and protein samples were collected after 24 h treatment with TGF-b. In the right panels are the average (6SEM) taken from five independent experiments shown as relative ex- pressions. *p , 0.05. (E) Immunohistochemical staining of 4-HNE in IPF lung tissues (n = 4). Fibroblasts constituting FF (white arrow). Scale bar, 100 mm. (F) WB using anti–4-HNE and anti–b-actin of cell lysates from LF isolated from control normal lung (lanes 1–3) and from IPF lung (lanes 4–6). In the lower panel is the averages (SEM) shown as relative expression (control normal lung [n = 5] and IPF lung [n = 5]). Open bar is control LF from normal lung, and filled bar is LF from IPF lung. *p , 0.05. 8 GPx4-REGULATED LIPID PEROXIDATION IN IPF PATHOGENESIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. GPx4 regulates BLM-induced lipid peroxidation and lung fibrosis development. (A) WB using anti-GPx4 and anti–b-actin of lung ho- mogenates from heterozygous GPx4-deficient mice (GPx4+/2), wild-type (GPx4+/+) mice, and GPx4-overexpressing transgenic (GPx4+/+/Tg [GPx4]) mice. (B) Body weight (BW) changes after BLM treatment. BW on day 0 before treatment was designated as 1.0. (C) Photomicrographs of Masson trichrome staining of mouse lungs on day 21. Original magnification 340. (D) Shown in the panel is the average (6SEM) soluble collagen measurement from Sircol assay using lung homogenates on day 21. Control groups (n = 4) and treatment groups (n = 9) were composed of the (Figure legend continues) The Journal of Immunology 9

FIGURE 5. Effect of Trolox on BLM-induced lipid peroxidation and lung fibrosis development in mice. (A) Body weight (BW) changes after BLM treatment. BW on day 0 before treatmentwasdesignatedas1.0. (B) Photomicrographs of Masson trichrome staining of mouse lungs on day 21. Original magnification 340. (C) Shown in the panel is the average (6SEM) soluble collagen measure- Downloaded from ment from Sircol assay in control (n =4), BLM-treated (n = 6), Trolox-treated (n = 4), and BLM-treated with sub- sequent Trolox injection mouse lungs (n = 3) on day 21. *p , 0.05. (D) Immunohistochemical staining of

4-HNE in mouse lungs on day 21. http://www.jimmunol.org/ Original magnification 3200. by guest on September 28, 2021

Increased oxidative stress resulting from excessive ROS pro- 3B, 3D). Trolox significantly suppressed not only TGF-b–induced duction and/or depleted antioxidant defenses has been widely lipid peroxidation, but also myofibroblast differentiation, indicat- implicated in molecular mechanisms underlying fibrosis devel- ing a regulatory role of GPx4-modulated lipid peroxidation in opment in a variety of organs, including lung (36–39). Oxidative TGF-b–induced myofibroblast differentiation. To further clarify modifications of cellular components resulting from oxidative the mechanistic link between GPx4-regulated lipid peroxidation stress have been recognized as the mechanisms for cell damage. and lung fibrosis development, we evaluated HODE and HETE Among oxidative modifications, lipid peroxidation has a pivotal levels, representative eicosanoid derivatives of polyunsaturated role during oxidative stress-mediated cell damage, as it produces fatty acids in mouse lungs, respectively. In comparison with wild- detrimental oxidized byproducts, represented by 4-HNE and MDA type mice, HODE and HETE levels were increased in GPx4+/2 (40). Consistent with previous reports, we showed that the ex- mice, which were significantly decreased in GPx4 transgenic mice pression levels of 4-HNE were significantly increased in LF iso- even without BLM treatment, suggesting the physiological role of lated from IPF lungs compared with normal LF. The increase in GPx4 in regulating HODE and HETE levels. Consistent with other 4-HNE expression levels was clearly demonstrated in fibroblasts oxidized byproduct assays, BLM treatment further increased comprising FF in IPF lungs. Intriguingly, Rahman et al. (41) HODE and HETE levels, and apparent worsening trend was de- showed that the levels of products of lipid peroxidation in bron- tected in GPX42/+ mice. Both enzymatic and nonenzymatic lipid choalveolar lavage fluid and plasma were higher in patients with peroxidation accompanied by production of eicosanoid derivatives IPF, further supporting the notion that lipid peroxidation has an have been implicated in a variety of pathological process via important role in IPF pathogenesis. Although TGF-b treatment modulating cellular redox signaling (42), including TGF-b (43), induced both GPx4 expression and lipid peroxidation, GPx4 further supporting the notion that enhanced lipid peroxidation knockdown clearly enhanced lipid peroxidation by TGF-b with caused by reduced GPx4 may have an important role in modu- concomitantly increased myofibroblast differentiation (Figs. 2D, lating lung fibrosis development via enhancing TGF-b signaling,

same number of mice. *p , 0.05. (E) Immunohistochemical staining of 4-HNE in mouse lungs on day 21. Original magnification 3200. (F) Shown in the panel is the average (6SEM) lipid peroxidation measurement from MDA assay using control lungs (n = 3) and BLM-treated lungs (n = 5) on day 21. Control and treatment groups were composed of the same number of mice. *p , 0.05. (G) Immunohistochemical staining of phospho-SMAD3 in mouse lungs on day 21. Original magnification 3400. Scale bar, 100 mm. Data shown represent the mean 6 SD of the percentage of phospho-SMAD3 positively stained area using ImageJ (mice/samples/sections in each group = 3/5/3). *p , 0.05. 10 GPx4-REGULATED LIPID PEROXIDATION IN IPF PATHOGENESIS

FIGURE 6. Hypothetical model of GPx4 reduction– induced myofibroblast differentiation and lung fibrosis development. Increased lipid peroxidation caused by reduced GPx4 is responsible for enhanced TGF-b signaling associated with myofibroblast differentiation. Lipid peroxidation may also induce TGF-b expression. FF formation of myofibroblast accumulation for lung fibrosis development can be attributed to increased lipid peroxidation-mediated enhanced TGF-b signaling dur- ing IPF pathogenesis.

especially in the setting of reduced GPx4 expression like IPF was involved in myofibroblast differentiation and proliferation Downloaded from lungs. Interestingly, previous paper showed that 4-HNE induces through activating platelet-derived growth factor receptor (PDGFR) synthesis of TGF-b, suggesting the existence of vicious cycle signaling in terms of IPF pathogenesis (49). In the current study, between increased lipid peroxidation and TGF-b production dur- GPx4 knockdown without TGF-b treatment induced intracellular ing pulmonary fibrosis development, especially in the setting of ROS production (Fig. 2B). Furthermore, GPx4 knockdown in- reduced GPx4 (24). creased not only lipid peroxidation (Fig. 3B), but also mitochondrial

GPx4 is a GPx that directly reduces peroxidized ROS, as shown by MitoSOX Red staining, which is an indicator of http://www.jimmunol.org/ phospholipids in cell membranes for redox homeostasis (44–46). cumulative mitochondrial damage (Supplemental Fig. 3). Car- GPx4 has a crucial role during development, and whole-body diolipin is a kind of diphosphatidylglycerol lipid and is normally ablation of the GPx4 in mice was found to be embryonic localized to the inner mitochondrial membrane. Interestingly, lethal between 7.5 and 8.5 d post coitum, with concomitantly Huang et al. (50) reported that lysocardiolipin acyltransferase, enhanced cell death (27). GPx4 has been proposed to be a central which is the mitochondrial cardiolipin remodeling enzyme, regulator of ferroptosis, a recently recognized programmed ne- regulates the development of pulmonary fibrosis. Human studies crosis characterized by the production of iron-dependent ROS revealed strong positive correlation between lysocardiolipin generation (47, 48). Programmed cell deaths, including apoptosis acyltransferase expression levels in PBMCs isolated from IPF and necroptosis in alveolar epithelial cells, have been widely patients and lung function parameters, such as the percentage- by guest on September 28, 2021 implicated in IPF pathogenesis, indicating that the difference in predicted diffusing capacity of the lung for carbon monoxide BLM-induced lung fibrosis in GPx4-modulated mice can be at- and the percentage-predicted forced vital capacity, which suggests tributed to altered ferroptosis. Actually, a significant reduction of a causal association between dysregulation of mitochondrial car- cell death by means of TUNEL assay was demonstrated in BLM- diolipin and lung fibrosis development during IPF pathogenesis. treated GPx4 transgenic mice and significant increase in number Galam et al. (51) demonstrated that 4-HNE induces an increase in of TUNEL-positive dead cells was detected in BLM-treated the production of mitochondrial ROS, followed by a reduction in GPx4+/2. Although we cannot exclude potential enhancement of mitochondrial oxygen consumption in human small airway epi- apoptotic cell clearance in GPx4 transgenic mice, we speculate thelial cells. Katunga et al. (52) showed obese GPx4 haploinsufficient that GPx4-regulated cell death may be partly involved in lung (GPx4+/2) mice exhibited increased levels of lipid peroxides and fibrosis development. In our in vitro models, GPx4 knockdown 4-HNE, mitochondrial dysfunction, and pronounced cardiac fibrosis. significantly enhanced TGF-b–induced myofibroblast differentia- Accordingly, it is likely that the functional cross-talk between tion via increasing activation of the SMAD2/3 signaling in LF. excessive lipid peroxidation and mitochondrial dysfunction can also BLM-treated GPx4+/2 mice also demonstrated enhanced lung be involved in the mechanisms underlying the development of lung fibrosis development accompanied by increased activation of fibrosis associated with GPx4 reduction. SMAD signaling, suggesting SMAD2/3 signaling modulation by We demonstrated that GPx4 expression levels were significantly GPx4 may have a key regulatory role for myofibroblast differ- reduced in LF from IPF patients, and fibroblasts constituting FF entiation and lung fibrosis development. Although the exact exhibited barely detectable GPx4 expression. Moreover, compared mechanisms for SMAD2/3 signaling modulation by GPx4 re- with normal LF, no increase in GPx4 was observed in TGF-b– main to be determined, the efficient inhibition by Trolox indi- treated IPF LF. These findings suggest that reduced GPx4 ex- cates that excessive lipid peroxidation is involved in the process pression levels could be associated with enhanced myofibroblast of enhanced SMAD2/3 signaling (Fig. 3). differentiation and lung fibrosis development in IPF. Actually, GPx4 has a protective role in the maintenance of electron GPx4-overexpressing TG mice showed a marked attenuation of transport chain and oxidative phosphorylation in the mitochondria BLM-induced lung fibrosis development, suggesting GPx4 and the by regulating the integrity of phospholipids, cholesteryl esters, and modulation of lipid peroxidation could be promising therapeutic cardiolipin (13–15). GPx4 has three isoforms, localized in the targets for IPF. Vitamin E (namely a-tocopherol, the most abun- mitochondria, nucleoli, and cytosol (44, 45). Mitochondrial GPx4 dant isoform) is a potent antioxidant and is associated with GPx4 could play an important role of maintenance of the mitochondrial via a chain-breaking electron donor mechanism. In line with membrane potential and in the inhibition of lipid peroxidation previous report, Trolox (a water-soluble analogue of vitamin E) in cardiolipin, a mitochondria-specific phospholipid (14, 15, 45). suppressed TGF-b–induced myofibroblast differentiation and In our previous report, we showed that mitochondrial ROS production BLM-induced lung fibrosis development (Figs. 3, 5) (53). It has The Journal of Immunology 11 been reported that Trolox prevented the increase in TGF-b of oxidative stress in idiopathic pulmonary fibrosis. Eur. J. Clin. Invest. 36: 362–367. production, and it also regulates the functional and morpholog- 19. Kanoh, S., H. Kobayashi, and K. Motoyoshi. 2005. Exhaled ethane: an ical mitochondrial integrity through modulating ROS (54, 55). in vivo biomarker of lipid peroxidation in interstitial lung diseases. Chest These findings suggest that the suppression lipid peroxidation 128: 2387–2392. 20. Fernandez, I. E., and O. Eickelberg. 2012. The impact of TGF-b on lung fibrosis: can be a reasonable and promising IPF treatment in the setting of from targeting to biomarkers. Proc. Am. Thorac. Soc. 9: 111–116. reduced GPx4. 21. Yeganeh, B., S. Mukherjee, L. M. Moir, K. Kumawat, H. H. Kashani, In conclusion, our findings indicate the participation of enhanced R. A. Bagchi, H. A. Baarsma, R. Gosens, and S. Ghavami. 2013. Novel non- canonical TGF-b signaling networks: emerging roles in airway smooth muscle lipid peroxidation regulated by reduced GPx4 in IPF pathogenesis. phenotype and function. Pulm. Pharmacol. Ther. 26: 50–63. To our knowledge, this study is the first report showing the causal 22. Liu, R. M., and K. A. Gaston Pravia. 2010. Oxidative stress and glutathione in link between reduced GPx4 and IPF. Lipid peroxidation induced by TGF-beta-mediated fibrogenesis. Free Radic. Biol. Med. 48: 1–15. 23. Pociask, D. A., P. J. Sime, and A. R. Brody. 2004. Asbestos-derived reactive GPx4 reduction can be responsible for myofibroblast differentia- oxygen species activate TGF-beta1. Lab. Invest. 84: 1013–1023. tion and lung fibrosis development, at least partly via enhancing 24. Leonarduzzi, G., A. Scavazza, F. Biasi, E. Chiarpotto, S. Camandola, S. Vogel, TGF-b signaling. The regulation of lipid peroxidation by reduced R. Dargel, and G. Poli. 1997. The lipid peroxidation end product 4-hydroxy- 2,3-nonenal up-regulates transforming growth factor beta1 expression in the GPx4 is likely a promising target for an antifibrotic treatment macrophage lineage: a link between oxidative injury and fibrosclerosis. FASEB J. strategy for IPF. 11: 851–857. 25. Tsubouchi, K., J. Araya, S. Minagawa, H. Hara, A. Ichikawa, N. Saito, T. Kadota, N. Sato, M. Yoshida, Y. Kurita, et al. 2017. Azithromycin attenuates Disclosures myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4. Autophagy 13: 1420–1434. The authors have no financial conflicts of interest.

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LC-MS analysis of 9-HODE, 13-HODE and 15-HETE in mouse lung homogenate using control lungs (n = 4) and BLM treated lungs (n = 5) at day 21. Data shown represent the average±SEM. *p < 0.05. Supple.Fig.2

GPx4 WT NS GPx4 TG BLM GPx4 WT BLM GPx4 +/- BLM

TUNEL assay staining (green) in BLM-treated GPx4+/-, wild type (WT) and GPx4 transgenic (TG) mice lung sections. Nuclei were counterstained with DAPI (blue). Scale bars, 100 mm. Data shown represent the average±SEM of TUNEL positive cell counts. (mice/section/sample = 3/5/3) *P<0.05. 6XSSOH)LJ

Images of Hoechast 33258 and MitoSOX Red fluorescence staining in control siRNA or GPx4 siRNA transfected LF. Scale bars, 100 μm. 3 independent experiments showed same result.