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

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.

is positively regulated by NCOA4-mediated autophagy targeting the iron-storage 23 protein ferritin (ferritinophagy). The authors further showed that ferroptosis 24 plays a crucial role in COPD pathogenesis in a mouse model. They also found that 25 increased lipid peroxidation and NCOA4 expression levels are associated with COPD.

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Overall, the authors provided convincing evidence to demonstrate the pathological 28 relevance of ferroptosis to human COPD. However, the in vivo (animal model) 29 evidence for the role of NCOA4-mediated ferritinophagy in CS-induced COPD is 30 lacking. If the authors can perform in vivo experiments to demonstrate that 31 blockage of NCOA4-mediated ferritinophagy is indeed able to decrease CS 32 induced-iron deposition, cell death and other pathological phenotypes, their central hypothesis will be more convincingly supported, and the significance of 34 this study will be greatly improved.

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We really appreciate your thorough review and supportive comments. We agree with your 37 comments. NCOA4 knockout mouse has been reported to show impaired systemic iron 38 homeostasis, including iron accumulation in the liver and spleen. (Bellrlli R et al. Cell Reports 39 14,411-421 Jan 2016   Using an in vivo model of smoke-induced COPD, the authors demonstrate that mice 67 with a heterozygous deletion of a negative regulator of ferroptosis, GPX-4 68 exacerbates smoke induced injury and inflammation whereas mice with transgenic 69 activation of GPX-4 are protected from smoke induced COPD. Finally, they end the 70 paper by examining the association of NCOA4 with clinical lung function decline 71 in patients with COPD as well as demonstrating the association between an increase 72 in lipid peroxidation products and presence of disease in these patients.

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We thank the reviewer for a thorough review.

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We really appreciate your thorough review. The comments were constructive and the merit of 90 the work was clearly appreciated and the data was obviously carefully inspected.

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While this is a nice study, the mechanistic link between NCOA4 and ferroptosis 93 presented by the authors is somewhat flawed. Of main concern is why, when the 94 authors focus on ferroptosis as the major concept for this paper, they fail to 95 demonstrate if GPX-4 is regulated by smoke in any of their model systems, in vitro 96 or in vivo.

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We appreciate this thoughtful comment. As pointed out by reviewer, we observed no significant 99 correlation between COPD severity and GPx4 expression levels in lung homogenates in human 100 lung samples and CSE treatment did not modulate GPx4 expression levels in HBEC, which 101 were added a new Supplementary Figure6. Furthermore, GPx4 expression levels appears to be 102 elevated in smoke exposed mice lung compared to room air control lungs, indicating that GPx4 103 levels may be upregulated in COPD lungs as a part of adaptive response to not only increased 104 lipid peroxidation but also reduced GSH(gluthatione). GPx4 utilizes the reducing equivalents 105 from its substrate GSH and it has been reported that GSH levels were clearly reduced in 106 response to CS (Exp Toxicol Pathol. 2013 Sep ;65(6)    to take up DFO-however not usually faster than 1hour. In this study the authors 290 use 100uM DFO pre-treated for one hour before the addition of CSE (5%) for 24hours.

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If we presume that DFO is not taken up into the cell that fast to prevent any

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We have added new panels of 4-HNE and Perl's staining by using healthy smoker's lung in Fig   374   5a. 4HNE was stained in healthy smoker ,stronger than never smoker but weaker than COPD.

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The resolution of the images is really bad.

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"Perl's DAB staining" is to be used consistently. We have added counter staining with fast red 446 on Figure 3a. Figure

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We have corrected "animal models" in the methods.

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We look forward to hearing from you regarding our submission. We would be glad to respond to 486 any further questions and comments that you may have.

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Dear reviewers, 1 2 We thank referees for careful reading our manuscript and for giving insightful comments. 3 We have incorporated referees and editors comments ,and revised our manuscript as 4 possible. 5 6 *In the revised manuscript, first revised place is highlighted yellow, and 2 nd revised 7 place is highlighted green. The authors have sufficiently addressed the concern of this reviewer, except that in the 19 revision they still have not established the in vivo function of NCOA4. On the other 20 hand, this reviewer does understand their reasoning of lack of the in vivo data (that to 21 demonstrate the in vivo relevance of NCOA4 is time-consuming and might be beyond 22 the scope of this study). Therefore, this reviewer will accept the lack of this important 23 data but request the authors to unambiguously state in the paper that while all 24 observations are consistent with the proposed role of NCOA4, the definite in vivo 25 causative evidence is lacking. 26

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Thank you for your suggestion. Associate Editor also concerned about lacking of 28 NCOA4 experiment in vivo, hence we have attempted to performe in vivo experiment by 29 using NCOA4 knockdown models.