NCX1 reverse mode promotes calcium-dependent Neutrophil Extracellular Trap formation and lung damage in chronic obstructive pulmonary disease

Domain: biology
Published: 2026-03-11
Editorial outcome: major_revision

Abstract
Neutrophil-driven inflammation is central to the pathogenesis of chronic obstructive pulmonary disease (COPD). Emerging evidence suggests that Ca²⁺ signaling is critical in regulating neutrophil activation, recruitment and tissue residency. In this study, we investigated the function of Na⁺/Ca²⁺ exchanger 1 (NCX1), a Ca²⁺/cation membrane transporter, in neutrophils during COPD pathogenesis. Analysis of human specimens show that NCX1 is primarily upregulated in neutrophils from patients with mixed chronic bronchitis and emphysema. Cigarette smoke exposure induces NCX1 upregulation and promotes its reverse-mode transport activity, leading to elevated intracellular Ca²⁺ levels and enhanced NETs formation. Neutrophil-specific genetic deletion of Slc8a1 or pharmacological inhibition of NCX1 reverse transport effectively suppresses Ca²⁺ influx, NETs release, and neutrophil accumulation and retention, thereby ameliorating chronic bronchitis and emphysematous changes. Collectively, our findings identify NCX1 as a regulator of Ca²⁺-dependent NETs release in neutrophils. Targeting NCX1-mediated Ca²⁺ influx or NETs formation represents a potential therapeutic strategy for neutrophilic inflammation in COPD.

Introduction
Chronic obstructive pulmonary disease (COPD) is a progressive and debilitating lung disorder characterized by chronic bronchitis, fibrosis, small airway remodeling, and emphysema.1 It is widely recognized that the development and progression of COPD are primarily driven by the inhaled noxious particles, particularly tobacco smoke and environmental pollutants, which promotes activation and recruitment of neutrophils in the lungs, triggering persistent inflammatory responses.2,3 Neutrophils are currently established as key players in the pathophysiology of COPD. Elevated sputum neutrophil counts have been shown to correlate with peripheral airway dysfunction, airway obstruction4 and impaired lung function.5 Clinical evidence suggests that therapeutic strategies targeting neutrophilic inflammation may improve lung function, as indicated by increases in forced expiratory volume in one second (FEV1).6 However, neutrophil-targeted therapies remain challenging in COPD due to the risk of drug-induced neutropenia and subsequent infection.7 Therefore, understanding the mechanisms underlying aberrant neutrophil activation and accumulation in the lungs is essential for developing safer and more effective treatments.

Disruption of intracellular Ca2+ homeostasis in airway smooth muscle has been implicated in the pathogenesis of various lung diseases.8,9 Airway neutrophilia is a hallmark feature of COPD, and further becomes pronounced during exacerbations.10 Compelling evidence highlights the critical role of Ca2+ signaling in regulating neutrophil functions, including NADPH oxidase activation, exocytosis, degranulation, and cytokines release.11-14 Recent studies also have shown that Ca2+ flux regulates neutrophil clustering and activation.15,16 The Na+/Ca2+ exchangers (NCXs) represent a family of bidirectional membrane transporters that mediate the exchange of Na+ and Ca2+ in either direction, depending on the electrochemical gradient across the plasma membrane.17 Elevation of Na+/Ca2+ exchanger 1 (NCX1, encoded by Slc8a1), in airway smooth muscle has been shown to contribute to airway hyperresponsiveness in asthma.9 Notably, data from Human Protein Atlas (https://www.proteinatlas.org/ENSG00000183023-Slc8a1/immune+cell), indicate that NCX1 (encoded by Slc8a1) is highly expressed in human neutrophils in contrast to other Ca2+/cation membrane transporters and NCX family members. Nevertheless, little is known about the role of NCX1 in neutrophil function and COPD pathogenesis.

In the present study, we demonstrate NCX1 is primarily expressed in human neutrophils, and cigarette smoke (CS) upregulated its expression, promoting the reverse mode of NCX1 transport and subsequently elevates intracellular Ca²⁺ flux. Using a neutrophil-specific Slc8a1 knockout mouse model, our work shows that NCX1 depletion or NCX1 reverse-mode inhibition ameliorates airway remodeling and preserves alveolar stem cells. This protective effect might be attributed to NCX1-mediated declined Ca²⁺ influx in neutrophils and reduced formation of neutrophil extracellular traps (NETs), which diminishes neutrophil accumulation during COPD and limits their retention during the stable phase, without impairing neutrophil differentiation, development, migration, or phagocytic activity. Our study identifies NCX1 as a regulator of Ca²⁺-dependent NETs formation in COPD pathology. Pharmacological inhibition of NETs and NCX1 reverse transport offer a promising therapeutic strategy for COPD management.

Results
Increased neutrophilic NCX1 expression in patients with mixed chronic bronchitis and emphysema
Human lung tissues were obtained from Clinical Biobank of Sichuan University West China Hospital, with clinical characteristics and demographics detailed in Supplementary Table 1. In the lungs (encompassing distal airways and bronchioles, and adjacent alveolar structures) of patients with mixed chronic bronchitis and emphysema (Mixed CBE), NCX1 was predominantly localized to neutrophils (Fig. 1a), as corroborated by immune cell-specific expression profiles from the Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000183023-Slc8a1/immune+cell). Notably, both mRNA and protein levels of NCX1 were significantly elevated in the lungs of patients with Mixed CBE compared to those with Controls or emphysema alone (Fig. 1b, c, Supplementary Fig. 1a, b). In parallel, the enhanced neutrophil infiltration and activity in the lung tissue of COPD patients was further validated by increased mRNA and protein levels of neutrophil activation markers, myeloperoxidase (MPO) and neutrophil elastase (NE) (Fig. 1d-h, Supplementary Fig. 1a, c, d). NCX1 mRNA levels were positively correlated with both MPO and NE expression levels (Fig.1i, j), reinforcing its association with neutrophilic activation. Immunofluorescence staining further confirmed the upregulation of NCX1 in Mixed CBE lung tissues, and its co-localization with CD66b+ neutrophils (Fig. 1k). To validate the neutrophil-specific NCX1 enrichment, we performed flow cytometry with the bronchoalveolar lavage fluid (BALF) from Control and Mixed CBE patients, exhibiting prominently augmented neutrophils along with higher NCX1 mean fluorescence intensity in neutrophils of Mixed CBE patients (Fig. 1l-n). Across immune cell types, NCLX1 expression was markedly enriched in BALF-derived neutrophils relative to other immune cell populations (macrophages, lymphocytes, etc.) in both Mixed CBE patients and controls (Supplementary Fig. 1e). Immunofluorescence of fluorescence-activated cell sorting (FACS)-sorted neutrophils further verified increased NCX1 expression in BALF-derived neutrophils from Mixed CBE patients (Fig. 1o). Importantly, this neutrophil-specific upregulation of NCX1 extended beyond the pulmonary compartment. Aligning with our lung tissue/BALF data, NCX1 protein levels were significantly elevated in peripheral blood neutrophils from Mixed CBE patients compared to controls (Supplementary Fig. 1f, g). These finding indicated NCX1 overexpression is a generalized neutrophil feature in COPD, detectable in both lung and circulatory compartments.

NCX1 deletion mitigates neutrophils expansion and pathological changes of COPD.
To functionally test the role of neutrophilic NCX1 in COPD pathogenesis, we next employed CS-induced COPD mouse model. Following 12 weeks of CS exposure, mice developed hallmark pathological features, including emphysema and airway inflammation (Supplementary Fig. 2a, b). Consistent with human data, NCX1 was co-localized with neutrophils and significantly upregulated in lung tissues of these COPD mice, accompanied by elevated MPO and NE levels (Supplementary Fig. 2c-i). To determine whether neutrophil-specific NCX1 contributes causally to disease progression, we generated conditional knockout (cKO) mice using the Cre-loxP technology, specifically targeting Slc8a1 deletion in neutrophils (Slc8a1f+/f+;Mrp8-Cre+), with genotyping confirmed by PCR (Supplementary Fig. 3a, b). Neutrophils were isolated from bone marrow, with purity verification by flow cytometry with 95%-98% (Supplementary Fig. 3c), and cell-type specificity of Slc8a1 deletion was demonstrated by unaltered NCX1 expression in other immune populations (monocytes, macrophages, lymphocytes, and dendritic cells), while neutrophils from Slc8a1 cKO mice exhibited nearly complete loss of NCX1 at both mRNA and protein levels (Supplementary Fig. 3d, e). Immunofluorescence further validated the effective ablation of NCX1 in neutrophils, showing absent NCX1 signal in Slc8a1 cKO mice relative to robust expression in control neutrophils (Supplementary Fig. 3f).

Both Slc8a1f+/f+;Mrp8-Cre- and Slc8a1f+/f+;Mrp8-Cre+ mice were exposed to CS or air for 12 weeks (5 days/week), followed by pulmonary function tests prior to lung tissue collection post-euthanasia (Fig. 2a). Under air-exposed conditions, Slc8a1 cKO and control mice showed comparable pulmonary function, indicating that basal respiratory mechanics were unaffected by NCX1 deletion. However, both genotypes showed significant impairment of lung function after CS exposure. Pressure–volume loop analysis revealed that CS exposure significantly decreased dynamic compliance (Cdyn) in both groups, while Slc8a1 cKO mice retained higher Cdyn compared with CS-exposed controls (Fig. 2b, c). These results further support that neutrophil-specific NCX1 deficiency partially preserves lung compliance and mitigates airway resistance under chronic CS exposure. Consistently, significant airflow limitation (decreased FEV20(ms)/FVC and FEV50(ms)/FVC) and pulmonary hyperinflation (increased FRC/TLC) were shown in CS-exposed control mice (Fig. 2d, e, Supplementary Fig. 4a. However, Slc8a1 cKO mice were partially protected from lung function decline relative to CS-exposed controls, improving airflow limitation (higher FEV20(ms)/FVC and FEV50(ms)/FVC ratios) and reducing hyperinflation (lower FRC/TLC) (Fig. 2d, e, Supplementary Fig. 4a). Meanwhile, histological and cytometric analyses revealed that under baseline conditions, both genotypes had similar neutrophil infiltration, lung morphology (MLI), collagen deposition area, airway smooth muscle thickness, and alveolar stem cells numbers (SFTPC+). In contrast, following CS exposure for 12 weeks, Slc8a1 cKO mice exhibited significant protection against CS-triggered neutrophilic inflammation, airspace enlargement, collagen deposition expansion, airway remodeling, and loss of alveolar stem cells (Fig. 2f-o). These results demonstrate that neutrophil-specific deletion of NCX1 confers significant protection against CS-induced COPD-characteristic airway dysfunctions and key pathological features, without adversely affecting normal lung structure and function.

Slc8a1 deletion impaired NETs formation without affecting neutrophil development, maturation, migration and phagocytosis.
To investigate the contribution of NCX1 in neutrophil function, we conducted a series of in vitro assays with CD45+CD11b+Ly6g+ neutrophils purified from bone marrow of non-CS exposed Slc8a1f+/f+;Mrp8-Cre- mice and Slc8a1f+/f+;Mrp8-Cre+ mice using FACS. Flow cytometry, Diff-quick staining and scanning electron microscopy revealed no obvious morphological differences between the two genotypes (Fig. 3a-c). Genetic deletion of Slc8a1 in neutrophils did not alter neutrophilic development and maturation (Fig. 3d-f). Moreover, total neutrophil counts in the bone marrow, peripheral blood, and lungs along with white blood cell count in peripheral blood remained comparable between cKO and control groups (Fig. 3g-j), suggesting that Slc8a1 deficiency did not impair neutrophil migration ability. Phagocytic function was also preserved using neutrophils sorted from bone marrow, as shown by immunohistochemical staining and quantified using pHrodo Red-conjugated Escherichia coli particles in flow cytometry assays, with no significant differences in phagocytic efficiency or index (Fig. 4a-f).

We next assessed the role of NCX1 in NETs formation. Following 4-hour PMA stimulation at 37°C, bone marrow neutrophils from Slc8a1f+/f+;Mrp8-Cre- neutrophils produced obvious NETs, which was decreased in Slc8a1f+/f+;Mrp8-Cre+ mice (Supplementary Fig. 4b). Similarly, bone marrow-derived neutrophils from Slc8a1 cKO mice exhibited markedly impaired NETs formation as evidenced by reduced NETs area and diminished levels of Cit-H3 (Citrullinated Histone H3) (Fig. 5a-c), indicating a specific role for NCX1 in neutrophil pathogenicity. Consistently, human lung specimens from Mixed CBE patients showed enhanced NETs compared to Controls (Fig. 5d, e). Notably, NETs were significantly reduced in Slc8a1f+/f+;Mrp8-Cre+ mice following CS exposure for 12 weeks, with decreased NETs area and Cit-H3 expression (Fig. 5f-h). Whereas no differences of Cit-H3 levels were observed under air-exposed conditions (Fig. 5h).

To investigate whether the persistence of neutrophils in the lung after CS exposure cessation correlates with NETs formation, we assessed the abundance of tissue-resident neutrophils (TRNs) in lungs of Slc8a1f+/f+;Mrp8-Cre- mice and Slc8a1f+/f+;Mrp8-Cre+ mice two weeks after cessation of CS (Fig. 5i). To distinguish between circulating and TRNs, we performed intravenous (i.v.) injection of Alexa Fluor 700-conjugated anti-CD45 labeling prior to the lung harvest enabled distinction between circulating (Alexa Fluor anti-CD45+ cells) and resident neutrophils (anti-APC-Cy7 CD45+/CD11b+F4/80-Gr1+Ly6c-Ly6g+, referred to as TRNs in murine lungs) (Fig. 5j). Flow cytometry analyses confirmed that Slc8a1 deletion in neutrophils significantly reduced the number of TRNs post-CS exposure (Fig. 5k). Our findings demonstrated that the accumulation of TRNs in the COPD lungs is closely linked to NCX1 which impeded the normal regression of neutrophils from the lung. We further explored whether NCX1 sustain a pro-inflammatory microenvironment that favors neutrophil retention. Indeed, CS exposure triggered significant upregulation of key pro-inflammatory mediators (TNF, CXCL-2, IL-6, IL-1β, and IL-17a)18,19 in Control mice, whereas their levels were markedly suppressed in Slc8a1 cKO mice (Fig. 5l-p), highlighting that NCX1 sustains neutrophil retention by creating a chemokine-rich microenvironment. Given prior studies elucidating that NETs stimulate chemokine production in lung epithelium20 and fibroblasts21, we speculate NCX1-driven NETs formation might be a key upstream regulator of chemokine-rich microenvironment favoring neutrophil retention.

NETs blockade depressed the neutrophils expansion and NETs formation to attenuate lung parenchymal cell loss and airway remodeling
To determine whether CS-induced NETs formation drives the pathological changes in COPD, we pharmacologically inhibited NETs formation using Cl-amidine in mice every two days during the course of CS exposure (Fig. 6a). Cl-amidine is used as a pan-peptidylarginine deiminase (PAD) inhibitor to broadly suppress NETs formation, given its demonstrated efficacy in reducing citrullination-dependent NETs release.22,23 Pulmonary function tests revealed a marked reduction of dynamic compliance after CS exposure indicated by pressure–volume loop data, which was alleviated by Cl-amidine treatment (Fig. 6b, c). Meanwhile, CS exposure significantly impaired respiratory mechanics compared to Control mice, manifesting as decreased FEV20(ms)/FVC and FEV50(ms)/FVC ratios, along with increased FRC/TLC (Fig. 6d, e, Supplementary Fig. 4c), indicative of airflow limitation and pulmonary hyperinflation. Remarkably, Cl-amidine administration substantially attenuated these CS-induced functional damage, with FEV20(ms)/FVC and FEV50(ms)/FVC ratios showing significant recovery and FRC/TLC returning toward baseline levels (Fig. 6d, e, Supplementary Fig. 4c). These improvements demonstrate that NETs inhibition depressed airflow limitation and increased residual volume in COPD pathogenesis. After 12 weeks of CS exposure, ELISA confirmed elevated Cit-H3 levels in mouse BALF and flow cytometry showed a marked expansion of pulmonary neutrophils compared to Control group, while immunofluorescence staining revealed increased NETs area in lung tissues (Fig. 6f-i). Treatment with Cl-amidine significantly decreased neutrophil accumulation, reduced Cit-H3 levels and NETs area compared to the CS+Vehicle and CS group (Fig. 6f-i), confirming its efficacy in suppressing NETs release in vivo. H&E staining displayed that CS exposure induced severe emphysematous destruction and airway inflammation, which was notably attenuated by Cl-amidine treatment (Fig. 6j, k). Furthermore, Masson's Trichrome staining and immunofluorescence for a-SMA and SFTPC revealed that Cl-amidine effectively suppressed airway remodeling and preserved alveolar epithelial progenitor cells. Specifically, CS exposure triggered pronounced peribronchial fibrosis, thickened airway smooth muscle and depleted alveolar stem cells, whereas Cl-amidine administration markedly reduced collagen deposition, diminished bronchial smooth muscle hyperplasia and restored alveolar stem cells (Fig. 6l-q). These findings suggested that inhibiting NETs formation reversed CS-induced pulmonary structural and functional damage, linking NETs inhibition to alveolar preservation, airway remodeling suppression, small airway function improvement.

Inhibition of NCX1 reverse mode activity decreased Ca2+ flux and NETs release
Multiple neutrophil-activating stimuli, including phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP) and cigarette smoke extract (CSE), were found to upregulate the expression of NCX1, with CSE showing the most robust effect, as confirmed by RT-qPCR, WB, and immunofluorescence staining (Fig. 7a-d). Importantly, NCX1 functions bidirectionally, operating in both forward and reverse transport modes. To elucidate the underlying mechanism by which NCX1 enhances NETs release, we examined the transport patterns of NCX1 after CSE exposure. In vitro Ca2+ flux detection showed that CSE induced a sharp increase of Ca2+ levels within neutrophils, but this Ca2+ influx was effectively suppressed by pretreatment with KB-R7943, an inhibitor of NCX1 reverse transport (Fig. 7e). This finding may suggest that CSE induced Ca2+ overload in neutrophils primarily through reverse transport of NCX1. Najder et al. reported the increase of intracellular Na+ concentration can reverse the activity of NCX1 in neutrophils.24 To dissect whether CSE acts directly on NCX1 or indirectly via Na+ dynamics, we simultaneously measured real-time changes in intracellular Na⁺ ([Na⁺]i) and Ca²⁺ ([Ca²⁺]i) in neutrophils stimulated with three agents (CSE, PMA, and fMLP) by ion-sensitive fluorescent probes. Consistent with the study of Najder et al., fMLP induced concurrent increases in both [Na⁺]i and [Ca²⁺]i, indicating indirect NCX1 reverse-mode activation secondary to Na+ rise (Fig. 7f, g). In contrast, CSE and PMA triggered a rapid [Na⁺]i decrease alongside a robust [Ca²⁺]i increase (Fig. 7f, g). This divergence demonstrated CSE and PMA might act via a distinct, direct mechanism favoring reverse-mode NCX1 operation (Ca²⁺ influx/Na⁺ efflux). Moreover, CSE-induced [Ca²⁺]i rise was nearly abolished in NCX1-deficient neutrophils (Fig. 7h), proving NCX1 is the primary route for CSE-triggered Ca²⁺ influx.

Considering the possibility that mechanical stimulation during drug application might transiently activate mechanosensitive ion channels such as Piezo, we simultaneously pretreated neutrophils with GsMTx4, a specific inhibitor of Piezo channels25, to rule out this potential artifact. Inhibition of Piezo channels delayed the time to reach peak Ca²⁺ and Na⁺ fluxes compared to untreated cells, yet the magnitude and stability of the sustained fluxes remained unchanged (Supplementary Fig. 4d, e). Similarly, GsMTx4 pretreatment did not alter the trend of CSE-stimulated Ca²⁺ flow in mice of different genotypes, indicating that the observed Ca²⁺ and Na⁺ fluxes primarily reflect true NCX1-dependent transport rather than mechanical artifacts. Furthermore, to further confirm that NCX1 is directly involved, we overexpressed NCX1 in CHO-K1 cells and observed the effects on intracellular ion dynamics (Supplementary Fig. 4g-k). CSE stimulation of NCX1-overexpressing CHO-K1 cells led to a pronounced increase in [Ca²⁺ ]i and a significant decrease in [Na⁺]i compared to control cells (Supplementary Fig. 4j, k). These results provide further evidence that enhanced NCX1 expression directly facilitates reverse-mode activity (Ca²⁺ influx coupled with Na⁺ efflux) during CSE exposure.

Consistently, NCX1 depletion significantly impaired CSE-induced NETs formation in neutrophils from Slc8a1f+/f+;Mrp8-Cre- and Slc8a1f+/f+;Mrp8-Cre+ mice, as shown by reduced Cit-H3 levels and NETs area (Fig. 7Ai, j). Based on these results, we hypothesized that CSE triggers a reverse transport pattern of NXC1, which increases Ca²⁺ inflow and subsequent NETs release, ultimately exacerbating COPD. To further validate this mechanism, we conducted in vitro experiments and observed that treatment with the NCX1 reverse-mode inhibitor KB-R7943 decreased NETs formation and Cit-H3 levels in CSE- or PMA- stimulated neutrophils (Fig. 7k, l, Supplementary Fig. 4l, m). We further assessed the functional association of NCX1 with Ca²⁺ influx, and found that the inhibitory effect of KB-R7943 administration on CSE-induced Ca²⁺ inflow was abolished in neutrophils from Slc8a1f+/f+;Mrp8-Cre+ mice (Supplementary Fig. 4n), supporting the specificity of KB-R7943 and crucial role of NCX1 in neutrophil Ca²⁺ dynamics. Pre-treatment with BAPTA-AM, a cytosolic Ca²⁺ chelator, significantly depressed CSE-triggered [Ca²⁺]i influx, Cit-H3 expression, and NETs release (Fig. 7k, l, Supplementary Fig. 4o, p). Conversely, treatment with Ca²⁺ ionophore A-23187 elevated [Ca²⁺]i, robustly inducing Cit-H3 expression and NETs formation (Fig. 7k, l; Supplementary Fig. 4o, p), confirming Ca²⁺ overload alone is sufficient to drive NETs formation. These findings underscore the critical role of NCX1-mediated Ca²⁺ influx in driving NETs formation and COPD pathogenesis.

NCX1 reverse mode inhibition preserved pulmonary function and mitigated CS-induced injury
To evaluate the therapeutic potential of targeting NCX1 reverse-mode activity in COPD, we administered KB-R7943 to mice subjected to CS exposure (Fig. 8a). Under normal air-exposed conditions, pressure–volume loop analysis demonstrated that CS exposure significantly reduced dynamic compliance (Cdyn), while KB-R7943 treatment largely restored Cdyn toward normal levels (Fig. 8b, c). These results provide functional evidence that pharmacological inhibition of NCX1 reverse-mode activity mitigates CS-induced decline in lung compliance. Additional pulmonary function tests revealed that KB-R7943 treatment alone does not alter baseline lung function, as Control and KB-R7943-alone groups exhibited no significant differences in FEV20(ms)/FVC, FEV50(ms)/FVC, FRC/TLC data (Fig. 8d, e, Supplementary Fig. 4q). In contrast, CS-exposed mice showed severe functional impairment, including significant airflow limitation and pulmonary hyperinflation, which were significantly attenuated by KB-R7943 treatment (Fig. 8 d, e). The protective effects of KB-R7943 treatment were evidenced by substantial reduction in NETs formation, indicated by decreased protein levels of Cit-H3 and shrunk NETs area compared to CS group (Fig. 8f-h). No obvious differences were observed in NETs release and area between Control and KB-R7943 treatment groups under air exposure (Fig. 8f-h), supporting the specificity and safety of the inhibitor under physiological conditions. Furthermore, CS exposure-induced emphysematous alveolar destruction, peribronchial fibrosis, airway smooth muscle hyperplasia, and depletion of alveolar epithelial progenitor cells were reversed with KB-R7943 treatment in CS-exposed mice. Specifically, H&E staining demonstrated that KB-R7943 preserved alveolar architecture (Fig. 8i, j), while Masson’s trichrome staining and immunofluorescence for α-SMA and SFTPC confirmed mitigation of fibrotic remodeling and restoration of alveolar stem cells (Fig. 8k–p). No significant alterations were shown between Control and KB-R7943 treatment groups under air exposure (Fig. 8k–p). These findings collectively demonstrated that pharmacological inhibition of NCX1 reverse mode activity protected against CS-induced COPD pathogenesis without affecting baseline pulmonary function.

Methods
Human samples
The BALF and blood sample collection protocol was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Approval No. KLL-2025-019). This study was conducted in strict accordance with the Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. The usage of clinical lung samples was approved by Science and Technology Review Board and Ethics Committee of Sichuan University (Sichuan, China, Ethics approval: IORG No. 20221582). Lung tissue samples were obtained from Clinical Biobank of Sichuan University West China Hospital (Sichuan, China). Lung specimens were collected from lung cancer patients, and only lung tissues from distal areas far from the cancer site were included in this study. The clinical characteristics and demographics of patients was shown in the Supplementary Table 1. Based on chest computed temography (CT) imaging, samples were stratified into 3 groups: Control group, normal lung tissue from individuals without chronic lung disease; Emphysema group, radiologically confirmed emphysema without chronic bronchitis features; Mixed CBE group, combined features of chronic bronchitis and emphysema. BALF samples were additionally collected from COPD patients and healthy controls. COPD patients were diagnosed based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines for the diagnosis, management, and prevention of COPD. Between April and May 2025, 5 Control individuals (patients with asymptomatic pulmonary nodules) and 5 Mixed CBE patients with clinically diagnosed COPD and CT imaging were recruited from the Affiliated Hospital of Zunyi Medical University (Guizhou, China). Among the five healthy controls, four were non-smokers and one was a smoker, while all five Mixed CBE patients had a history of smoking. BALF procedures were performed by experienced clinicians, and was processed immediately after collection for downstream analysis. A total of 50 mL of BALF was collected from each participant. In addition, 5 mL of peripheral venous blood was drawn from each individual for further analysis. The involved patients’ information was provided in the Supplementary Table 2. All participants provided written informed consent prior to sample collection.

Preparation of CSE
CSE was prepared using a standard protocol. Briefly, mainstream smoke from one cigarette was drawn using a peristaltic pump and bubbled through 10 mL of RPMI-1640 (Gibco, 11875101) at a constant flow rate. The resulting solution was adjusted to a neutral pH of 7.4, then filtered through a 0.22-μm filter to remove particulate matter and bacteria. The resulting solution was designated as 100% CSE stock. For experimental use, this stock solution was diluted to the indicated working concentrations (typically 10%) using fresh RPMI-1640 (Gibco, 11875101).

FACS analysis of NCX1 expression on neutrophils in human BALF
BALF cells were filtered through 100 μm and 70 μm cell strainers to obtain a single-cell suspension. Red blood cell lysis was performed by incubating the suspension with RBC lysis buffer for 5 min at room temperature. The resulting cell pellet was resuspended in 200 μL of FACS buffer. For the detection of NCX1 expression, cells were incubated with anti-NCX1 antibody (Abcam, ab2869) at room temperature in the dark for 30 min. After incubation, the cells were washed twice with FACS buffer. Subsequently, the cells were stained with a combination of the following antibodies: PerCP/Cyanine5.5 anti-human CD45 (Biolegend, 368503), PE anti-human CD66b (Biolegend, 392903), APC/Fire750 anti-human CD11b (Biolegend, 301351), Brilliant Violet605 anti-mouse IgD (Biolegend, 405727). After incubation, the cells were washed twice with FACS buffer and analyzed by flow cytometer (BD FACSCanto II), and data were processed using FlowJo (v.10.0) software.

Animal care
All mice used in the studies were housed under specific pathogen-free conditions at the Animal Center of Zunyi Medical University, with controlled temperature, humidity, and a 12-hour light/dark cycle, and were provided with food and water ad libitum. Slc8a1flox/flox (Slc8a1f+/f+) mice and Mrp8 (S100a8) Cre recombinase (Mrp8-Cre) mice, both on a C57BL/6 background, were purchased from Cyagen Biosciences (Beijing, China). To generate neutrophil-specific Slc8a1 knockout mice (Slc8a1f+/f+;Mrp8-Cre+), which carry a genomic DNA fragment deletion of exon 2 of Slc8a1, Slc8a1f+/f+ mice were crossed with Mrp8-Cre mice. The generated conditional knockout mice carried a loxP-flanked exon 2 of the Slc8a1 gene, which is selectively excised in neutrophils via Cre recombinase expression under the Mrp8 promoter. Genomic DNA was extracted from the tail biopsies of transgenic mice and amplified with specific primers to identify the mouse genotype. Primers used for genotyping were listed in Supplementary Table 3.
To establish COPD mouse models, 8-12- week-old male mice were passively exposed to CS generated from commercially available cigarettes (12 cigarettes/day, 4 times/day). Smoke exposure was carried out 5 days per week for consecutive 12 weeks. All animal experiments were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Zunyi Medical University (Approval No. ZMU20202409).

Drug administration
WT mice received intravenous (i.v.) injection of KB-R7943 (1.5 μL of 100 mg/mL KB-R7943 diluted in 98.5 μL 0.9% saline per mouse, MCE, HY-15415) or Vehicle (1.5 μL DMSO in 98.5 μL 0.9% saline per mouse) every 2 days for total 12 weeks. In the experiments of Cl-amidine injection, WT mice were intraperitoneally (i.p.) injected of Cl-amidine (30 μL of 50 mg/kg Cl-amidine in 600 μL 0.9% saline per mouse, Selleck, 1043444-18-3) or Vehicle (30 μL DMSO in 600 μL 0.9% saline per mouse) every 2 days for total 12 weeks.

Pulmonary function test
Pulmonary function was measured using a computer-controlled small animal ventilator system (DSI BuxcoPFT, America). Mice were deeply anesthetized with inhalation of isoflurane and placed in the supine position on a foam board. After tracheostomy and insertion of a cannula, mice were connected to the ventilator and respiratory functions were assessed. Standardized deep inflation maneuvers were performed to normalize lung volume history prior to measurements. Lung function parameters, including forced expiratory volume at 20 ms and 50 ms (FEV20(ms)), FEV50(ms)), forced vital capacity (FVC), functional residual capacity (FRC), and total lung capacity (TLC = inspiratory capacity + FRC), were measured. Data were analyzed using the manufacturer’s software, and FEV20(ms)/FVC, FEV50(ms)/FVC, and FRC/TLC ratios were calculated to assess airway obstruction and hyperinflation. For pressure–volume loop analysis55, the inflation and deflation maneuvers were performed by stepwise increasing airway pressure from 0 to 30 cmH2O, followed by gradual release to baseline. The corresponding lung volume changes were continuously recorded to generate pressure–volume loops. From these curves, dynamic compliance (Cdyn) was calculated as the slope of the line portion of the inflation curve using FinePointe software (DSI). The pressure–volume loops and Cdyn values were used to evaluate alterations in respiratory compliance under different experimental conditions.

ELISA detection of Cit-H3 in BALF and cultured neutrophils
For mouse BALF samples, 0.5 mL of cold PBS was instilled in fractions into the airways of mice. After allowing the PBS to stay in the lungs for 20 seconds, it was slowly withdrawn. This process was repeated three times to collect the lavage fluid. The collected BALF was centrifuged at 800-1000×g for 10 min at 4°C to separate the supernatants which was stored at -80°C for subsequent analysis. For neutrophils isolated from mouse bone marrow using flow cytometry, cells were firstly washed twice with BPS buffer, and 5 × 10⁵ neutrophils were seeded into 24-well plates, followed by indicated treatment (CSE, KB-R7943, BAPTA-AM, A-23187). The cells were incubated in a 37°C incubator for 4 hours. After incubation, both cells and supernatants were collected. The supernatant was separated by centrifugation at 300×g for 10 min at 4°C. The levels of Cit-H3 in both BALF and cell culture supernatants were measured using the Mouse Guanylated Histone H3 ELISA Kit (Jonlnbio, 48480), following the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader, and the concentration of Cit-H3 in the samples was determined from a standard curve.

Analysis of mean linear intercepts (MLI)
The right lung of mice was perfused with 1 mL of phosphate-buffered saline (PBS), followed by inflation with 4% paraformaldehyde (PFA) overnight at 4℃ for paraffin embedding. After fixation, the lungs were washed 3 times using cold PBS in 2 hours at 4℃, and were then dehydrated through a graded ethanol series (30%, 50%, 75%, 95%, and 100%). The dehydrated lungs were cleared with xylene for 1 hour at room temperature, followed by incubation in paraffin at 65°C for two 100-min cycles before being embedded in paraffin blocks.
To analyze the alveolar morphology, H&E staining was performed on paraffin-embedded lung sections. For each mouse, at least three randomly chosen sections were selected for examination. The MLI, an indicator of alveolar airspace enlargement, was calculated by dividing the total length of lines drawn across the lung section by the number of intercepts between the lines and alveolar walls, and analyzed using Image-Pro Plus software. The pulmonary vessels and large airways were excluded from the analysis.

Masson’s Trichrome staining
After dewaxing, the lung sections were stained with iron hematoxylin for 5 min, followed by differentiation with an acid ethanol solution to achieve a blue color. Then, they were washed with distilled water, and treated with Masson compound staining solution for 1 min, Ponceau magenta staining solution for 5 min, phosphomolyb date for 1 min, weak acid working solution for 5 min and aniline blue solution for 2 min. After each staining step, the sections were dehydrated in 95% ethanol, washed 3 times with xylene, and sealed completely with a neutral glue. Fibrotic area was visualized with a pathology Slide Viewing Software (Leica Biosystem) and analyzed using ImageJ_Fiji software.

Multiplex immunofluorescence staining
Lung specimens and neutrophils were immunostained with multiplex immunofluorescence kit (Afantibody, AFIHC037). The harvested mouse lungs were fixed in 4% PFA (Servicebio) for 24 hours, washed with PBS, embedded in paraffin, and sectioned at 4-μm thickness. For deparaffinization, slides were immersed with xylene (3 × 10 min), dehydrated with graded ethanol (100% for 5 min; 95% for 5 min; and 70% for 5 min), and rinsed in distilled water for 2 min. Antigen retrieval was performed using antigen repair solution (pH 8.0, Servicebio) in the microwave at 75% power for 8 min, followed by 25% power for 7 min. Non-specific binding was blocked with 5% BSA for 35 min at room temperature. Human lung specimens were processed using the same protocol. Neutrophils sorted from mouse bone marrow or human BALF were seeded on coverslips coated with poly-L-lysine (Sigma, P4707), fixed with 4% PFA, washed with PBS, permeabilized with 0.3% Triton X-100 in 5% BSA for 30 min at room temperature. The samples were then incubated overnight at 4°C with primary antibodies: CD11c (Servicebio, GB11059, 1:200), CD68 (Proteintech, 25747-1, 1:200), CD3 (Servicebio, GB11014, 1:100), Ly-6G (Invitrogen, 14-5931-82, 1:50), αSMA (Invitrogen, 50976082, 1:200), CD66b (Novus Biologicals, NB100-77808, 1:100), SFTPC (Proteintech, 10774-1-AP, 1:200), MPO (Proteintech, 22225-1-AP, 1:200), NE (Abclonal, A8953, 1:200), Cit-H3 (Abways, CY6587, 1:250) and NCX1 (Abclonal, A5583, 1:200). Detailed information of primary antibodies was provided in Supplementary Table 4. Subsequently, slides were washed with PBS (3 × 5 min) and incubated with secondary antibodies (Poly horseradish peroxidase (HRP), goat anti-mouse/anti-rabbit, 1:200) for 50 min at room temperature. Fluorescent dye reaction solution was added to amplify the signals, and were washed again 3 times with PBS. To enable multiplex staining, antibody stripping was performed by boiling tissue sections in antigen retrieval buffer (for tissue) or by incubating cell slides with antibody elution buffer (Afantibody, AFIHC038). Blocking and antibody incubation steps were repeated sequentially for each additional marker. Finally, nuclei were counterstained with DAPI (Beyotime, C1002). Three sections for each mouse were evaluated. Images were captured using Zeiss LSM 980 microscope and analyzed using ImageJ_Fiji software. The quantification of NETs area was determined based on the colocalized regions of MPO and Cit-H3 signals.

Immunohistochemistry (IHC)
Immunohistochemistry was performed to detect the expression of MPO and NE in mouse lung tissues. The prepared sections were deparaffinized in xylene (3 × 10 min), rehydrated through graded ethanol (100%, 95%, and 70%, each for 5 min), and rinsed in distilled water. Antigen retrieval, blockade of endogenous peroxidase activity and non-specific binding were followed. Sections were then incubated overnight at 4°C with the following primary antibodies: MPO (Proteintech, 22225-1-AP, 1:200) and NE (Abclonal, A8953, 1:200). Detailed information of primary antibodies was provided in Supplementary Table 4. After PBS washing, HRP-conjugated secondary antibody (Servicebio, 1:200, goat anti-rabbit) was applied for 50 min at room temperature. Signal was developed using DAB substrate (Servicebio, G1212), and nuclei were counterstained with hematoxylin. Slides were dehydrated, cleared, and mounted using neutral resin. Three sections per mouse were evaluated. Images were captured using Zeiss LSM 980 microscope and analyzed using ImageJ_Fiji software.

Neutrophil isolation for in vitro experiments
Bone marrow neutrophils were isolated from C57BL/6 mice aged 12 weeks with genotypes confirmed by PCR. Cells were washed with sterile Hanke’s Balanced Salt Solution (HBSS, Invitrogen, 14185052), and incubated with CD45-APC-Cy7 (BD, 557659), CD11b-BB515 (BD, 564454), and Ly6g-PE (BD, 551461) antibodies. Neutrophils (CD45+CD11b+Ly6g+ cells) were then isolated by flow cytometry. Detailed information of antibodies was provided in Supplementary Table 4. The isolated neutrophils were verified by flow cytometry, with 95-98% purity. Human neutrophils were isolated from peripheral blood of COPD patients and healthy controls using Human peripheral blood Neutrophil isolation Solution kit (Solarbio, P9040), following the manufacturer’s protocol. Neutrophils in human BALF were sorted as CD45+CD11b+CD66b+ cells by FACS.

Tissue dissociation and flow cytometry
A digestion cocktail including DNase (50 U/ml, Sigma), Dispase (15 U/ml, Beyotime), and Collagenase Type I (225 U/ml, Servicebio) was tracheally perfused into the mouse lung, followed by perfusion with warm PBS through the right ventricle. The lungs were then subsequently removed from the chest. To prepare single-cell suspensions for FACS analysis, the lungs were digested using a cocktail consisting of DNase (40 U/ml, Sigma) and Liberase TM (80 mg/mL, Roche), diced with tissue scissors, and further processed using the gentle MACS Octo Dissociator with Heaters (Miltenyibiotec, 130096427). The obtained suspensions were washed with FACS buffer and passed through 70 μm and 40 μm cell filters, respectively. After centrifugation, the precipitation was re-suspended with RBC lysis buffer (Thermo Fisher, NC9067514), incubated for 3 min, centrifuged again, and finally washed twice with FACS buffer to remove residual lysis buffer. For surface marker staining, cell suspensions were incubated with appropriate antibodies in FACS buffer for 45 min at 4℃, followed by washes with FACS buffer. The antibodies used for staining included: CD45-APC-Cy7 (BD, 557659), CD11b-BB515 (BD, 564454), Ly6g and Ly6C-APC (BD, 553129), Ly6g-PE (BD, 551461), Ly6C-BV605 (BD, 563011), F4/80-BV421 (BD, 565411). For FACS analysis of neutrophil development, antibodies Ly6g/Ly6C-FITC (Proteintech, 65140), CD11b-APC (Biolegend, 101211), CD117(c-kit)-PE (Biolegend, 161503) were used. Detailed information of antibodies was provided in Supplementary Table 4. For FACS analysis of neutrophil phagocytosis, neutrophils were incubated with pHrodo Red-conjugated Escherichia coli (Invitrogen, P35361). For distinguishing the circulating and resident neutrophils, 3 mg of Alexa Fluor 700-conjugated CD45 antibody (Biolegend, 147715) diluted in 100 μL PBS (1×) was intravenously injected via tail vein 3 min before euthanasia. The stained samples were analyzed using a flow cytometer (BD FACSCanto II), and data were processed using FlowJo (v.10.0) software.

Real time quantitative PCR (RT-qPCR)
Total RNA was extracted from whole lungs or neutrophils isolated by flow cytometry using PicoPure RNA Isolation Kit (Applied Biosystems, KIT0204), following the protocols provided by the manufacturer. The SuperScript RT Kit (Invitrogen, 18080044) was used to reverse-transcribe total RNA into cDNA. PCR amplification was performed using the qPCR SYBR Green Master Mix (Yeasen, 11201ES08). Gene-specific primers for NCX1, MPO, NE, TNF, CXCL-2, IL-6, IL-1β, and IL-17a were designed by Youkang Biotech and listed in Supplementary Table 3. GAPDH was used as the internal control. Relative expression levels were calculated using the 2−ΔΔCt method with normalization to GAPDH.

Western blot
Lung tissues and isolated neutrophils were homogenized in RIPA buffer containing a mixture of protease and phosphatase inhibitors Cocktail (Sigma-Aldrich, PPC1010). Total protein concentration was determined using BCA Protein Assay Kits (Thermofisher, 23227). Equal amounts of protein from each sample were loaded onto a PAGE Gel Quick Preparation Kit (Yeasen, 20325ES05), and subsequently transferred to PVDF membranes (Yeasen, 36125ES03, 36126ES03). After being blocked with QuickBlock Blocking Buffer (Beyotime, P0252FT), the membranes were incubated with primary and secondary antibodies successively. The following antibodies were used: anti–NE (Abclonal, A8953, 1:1000, rabbit), anti–MPO (Proteintech, 22225-1-AP, 1:5000, rabbit), anti–Cit-H3 (Abways, CY6587, 1:5000, rabbit), anti-Slc8a1 (Abclonal, A5583, 1:5000, rabbit), anti–β-actin (CST, 4967S, 1:1000, rabbit), and secondary antibodies (Goat Anti-Rabbit IgG, Abcam, ab6702, 1:5000). Detailed information of primary antibodies was provided in Supplementary Table 4. Protein bands were visualized using Amersham Imager 600 system, and blots images were imported into the ImageJ_Fiji software for densitometric analysis and quantification.

Measurement of real-time Ca2+ flux using non-invasive micro-test technology (NMT)
Neutrophils were isolated from bone marrow and resuspended in testing solution (0.4 mM Ca(NO3)2, 100 mM NaCl, 5.0 mM Na2HPO4, 5.0 mM KCl, 1.0 mM HEPES (pH 7.0)). The cells were incubated in this solution for 10 min before measurement. To measure Ca2+ flux, neutrophils were placed under a microscope and targeted cells were identified. A Ca2+ flux microsensor (NMT Physiolyzer, YoungerUSA) was positioned 5 μm from the target cell to begin the measurement. Baseline Ca2+ flux data were recorded for 5 min for each cell. Subsequently, CSE or CSE+KB-R7943 stock solutions were added to the testing environment until working concentrations achieved. Immediate post-treatment Ca2+ flux data were then recorded for an additional 5 min. Each experimental group consisted of 6 single-cell measurements (6 biological replicates). Ca2+ flux data were read and exported directly using imFluxes V3.0 software (Xuyue Company). The flux rate was expressed in units of pico mol • cm-2 • s-1. The sign of the flux value indicates the direction of Ca2+ transport: a positive value represents the efflux of Ca2+ from the cell, whereas a negative value represents the influx into the cell.

CHO-K1 cell culture and transfection
CHO-K1 cells (IMMOCELL, China) were cultured in F12-K media (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO₂ incubator. To overexpress NCX1, CHO-K1 cells were transfected with plasmids carrying the cDNA of Slc8a1 (mouse), purchased from Miaoling Plasmid (https://en.miaolingbio.com/). The plasmids were transfected using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. To select successfully transfected cells, G418 (Sigma-Aldrich, USA), an antibiotic for positive selection, was applied to the cells. The effective concentration of G418 was determined by a preliminary titration to inhibit 100% of the non-transfected cells. Transfected cells were cultured in the presence of G418 for approximately two weeks to allow the selection of stable clones. To confirm the successful transfection of NCX1 plasmids, WB and immunofluorescence were used to confirm NCX1 expression.

Measurements of [Ca²⁺]i and [Na⁺]i using fluorescent ion indicators
Mouse neutrophils were sorted from bone marrow using flow cytometry (CD45⁺CD11b⁺Ly6g⁺). Approximately 5 × 10⁵ neutrophils were used per experiment. Prior to measurement, the cells were washed twice with Ringer's solution (1.2 mM CaCl₂, 140 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl₂, 5.5 mM glucose, 10 mM HEPES, pH 7.4). Neutrophils were then loaded with 3 μM Fura-2 AM (Maokangbio, MX4502) or 5 μM NaTrium Green-2 AM (Maokangbio, MX4514), both containing 0.02% Pluronic F-127 (Maokangbio, MX4502), and incubated for 30 min at 37°C. After incubation, the cells were washed twice with Ringer's solution and further incubated at 37°C for an additional 30 min to ensure complete de-esterification of the AM esters. Intracellular [Ca²⁺]i and [Na⁺]i levels were measured using a Multifunctional Microplate Detector (Agilent, USA). Basal [Ca²⁺]i and [Na⁺]i concentrations were recorded from 0 to 60 seconds. Subsequently, the following reagents were sequentially added using an automatic drug sampling system: CSE (10%), PMA (50 nM), fMLP (10 μM), CSE+Vehicle, CSE+KB-R7943 (10 μM), BAPTA-AM (10 μM), A-23187 (10 μM). The [Ca²⁺]i and [Na⁺]i levels were measured from 61 to 360 seconds. Fura-2 AM emission ratios were used to quantify the integrated [Ca²⁺]i signal (F340/F380) and [Na⁺]I (F500/545). To exclude the potential involvement of mechanosensitive ion channels, an additional set of experiments was performed using GsMTx4 (MCE, HY-P1410), a specific inhibitor of Piezo channels. Neutrophils were preincubated with 2.5 μM GsMTx4 for 10 min at 37°C before stimulation with CSE, PMA, or fMLP, and subsequent [Ca²⁺]i and [Na⁺]i fluxes were recorded under the same experimental conditions as described above.

NETs formation in vitro and scanning electron microscope observations
To visualize NETs formation, neutrophils (5 × 105 cells) were resuspended in RPMI-1640 (Gibco, 11875101) and were seeded on cell culture slides (Biosharp, BS14RC) in 24-well plates. The cells were incubated at 37°C for 4 hours, and were then treated with PMA (50 nM, Selleck), CSE, and/or Cl-amidine (100 mM, Selleck) to induce NETs formation. Following the conventional dehydration and sputter coating procedures, the NETs were observed using a scanning electron microscopy (ZEISS, EVO10) at an accelerated voltage of 30 kV. Three sections per group were selected for quantification of NETs area.

Statistical analysis
Statistical analysis was performed using GraphPad Prism software (v.9.0). Data were assessed for normality using the Shapiro-Wilk test. Continuous variables were described as median (interquartile range, IQR) or mean (standard deviation, SD). Categorical variables are described as proportions. Student's t-test (for normally distributed data) or Mann-Whitney U-test (for non-normally distributed data) was used for comparisons of continuous quantitative variables between two groups. Proportional differences were compared using chi-square test (or Fisher's exact test). For comparisons among three or more groups, ANOVA was employed. Correlation of NCX1 expression with levels of NE and MPO was evaluated using Spearman's correlation analysis (nonparametric data). The significance of differences between two groups was determined using two-sided unpaired t-test. Among three or more groups, one-way ANOVA with Tukey’s multiple comparison test was used for comparisons. A p-value less than 0.05 was considered statistically significant for all analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Liao and coll. postulates that the reverse mode of NCX1 (Slc8a1) sodium/calcium exchanger is responsible for neutrophil-mediated lung damage in mice under conditions mimicking chronic obstructive pulmonary disease (COPD). Authors found enhanced expression of NCX1 in human samples from COPD individuals as well as from mice exposed to cigarette smoke. Authors propose that NCX1 acting in reverse mode, thus mediating calcium influx, is responsible for enhanced release of neutrophil extracellular traps (NETs). To support their claim, authors generate a conditional mouse model with selective ablation of NCX1 expression in neutrophils. This mouse model shows reduced formation of NETs upon stimulus and decreased COPD signs. However, the study lacks several control experiments) that are needed to support the conclusions, particularly regarding the direct involvement of NCX1-mediated calcium influx in promoting altered neutrophil function,\nSPECIFIC POINTS\nFigure 1 A. The specificity of the NCX1 antibody used for immunofluorescence in murine and human samples should be demonstrated by disappearance of the signal in neutrophils from KO animals.\nSupplementary Figure 2E. The expression of NCX1 detected by immunoblot in neutrophils from KO animals is markedly decreased but not absent. Is this a sign of a residual expression/function of the transporter?\nFigure 7. In my opinion, the claim that NCX1 is working in reversed mode and promoting high intracellular concentration is not adequately supported by experiments. In particular, the reverse mode of NCX1 is not an intrinsic characteristic of the transporter but depends on the ion gradients across the plasma membrane. An increase in intracellular sodium concentration can reverse the activity of NCX1 in neutrophils (Najder et al. Role of the intracellular sodium homeostasis in chemotaxis of activated murine neutrophils. Front Immunol 11:2124, 2020). Therefore, the effect of cigarette smoke may be indirect, increasing intracellular sodium. This issue has not been investigated. To study NCX1 function, authors use an unconventional method, based on a sensor that supposedly measures calcium flux direction. Looking at the vendor website, this instrument is mostly used for ion flux in plants. There are no tests in the paper to demonstrate that the method is really working compared to classical methods (e.g. measurement of intracellular calcium with ion-sensitive fluorescent probes). For example, there is no experiment showing that the calcium flux is abolished in neutrophils from NCX1 KO mice. Also, the NCX1 inhibitor should also be tested in neutrophils from KO animals to show that is does not alter flux when NCX1 expression/function is ablated. Additional experiments should have used methods to buffer or increase intracellular calcium by other means (e.g. calcium-chelating agents, calcium-mobilizing agonists, calcium ionophores) to check the effect on NET release and/detection of calcium flux by the sensor.", "reviewer_2": "Major strengths:\n1. Fascinating findings supporting a novel mechanism in which the NCX1 transporter reverses its mode in neutrophils resulting in altered intracellular calcium and triggering NETosis.\n2. The correlations in Figure 1I are very nice.\n3. Beautiful SEM pictures throughout the manuscript.\n4. The data shown in Figure 6 strengthened the manuscript.\n5. Strengthened by both and human and murine data.\n6. Further made stronger by using multiple approaches to prove their mechanism (gene deletion, pharmacologic inhibition).\nMajor critiques:\n1. The nomenclature used of \"simple emphysema\" vs “COPD” is confusing and not consistent with clinical nomenclature as emphysema is a form of COPD. Would suggest using the term \"mixed chronic bronchitis emphysema\" vs \"emphysema\" to differentiate the two, if that is in fact what they mean.\n2. Figure 1C lists mRNA changes between \"emphysema\" and their “COPD” group, but the protein results for NCX1 , MPO or NE in their “emphysema” group are not shown. Showing those results would strengthen their findings that the “simple emphysema” vs their \"COPD/mixed chronic bronchitis emphysema” groups are molecularly distinct.\n3. Figure 1A: where in the lung are these tissue samples from? Hard to discern underlying lung structure. Perhaps labels of alveoli, etc would improve this.\n4. Figure 1K: would be greatly strengthened if they could isolate neutrophils vs other immune cell subsets from patient BAL and run western blots for NCX1 expression. This may also be demonstrated by flow cytometry if NCX1 can be stained for by flow. The microscopy alone on lung tissue is insufficient to substantiate their statement that NCX1 is much higher in neutrophils vs other immune cells in control vs COPD patients.\n5. Are elevations in neutrophil NCX1 expression limited to lung neutrophils in COPD patients or is NCX1 expression increased in peripheral blood too?\n6. Demonstrate that Slc8a1 deletion was neutrophil specific by isolating other immune cell types (mono/macs, etc) from these animals and showing NCX1 protein remains present in similar concentrations to control animals.\n7. Figures 3/4: While demonstrating that neutrophil numbers in each compartment are similar in both genotypes and showing that neutrophil function of bone marrow neutrophils are similar in both genotypes are solid starts, particularly as it relates to #5 above, would suggest testing functions of lung neutrophils between both genotypes as well.\n8. Figure 5: In addition to microscopy, would be much stronger if use an additional method to quantify NETs to substantiate the authors conclusions. For example, could measure NE: DNA, MPO:DNA or CitH3: DNA complexes by duel-ELISAs or Cit-H3 ELISA in the murine BAL. Same critique applies to Figure 7F &H.\n9. Insufficient evidence to substantiate the proposed mechanism re NETs impeding the normal regression of tissue resident neutrophils from the lung, described in lines #191-193. There are several potential explanations including NETs promoting the release of chemokines. This has been previously demonstrated in lung epithelium by the authors of PMID # 36703990 and in fibroblasts by the authors of PMID # 23536012. Could measure the chemokine and cytokine levels in the BAL and/or whole lung to substantiate this as a mechanism.\n10. Figure 6: Need to confirm the Cl-amidine treatment decreased NET generation as they expect by microscopy or ELISA, etc.\n11. For Figure 7I: are there microscopy pictures that coincide with the graphs enumerating changes in the lungs?\n12. The authors use PMA to induce NETs for comparison. While PMA is a robust stimulator of NETs, would suggest they also compare to more physiologically relevant stimuli such as LPS or IL-8 (Figure 2,5 and 7).\n13. Likely, outside the scope of the current study,however, showing that the Slc8a1 deleted mice have improvement in their pulmonary function tests after CSE would be an outstanding demonstration of the functional impact of NCX1 transporter in a COPD model.\n14. Discussion needs to be strengthened by a deeper discussion of how these findings are consistent or inconsistent with prior studies, providing enhanced context for the current findings and how the results truly advance the field.\nMinor critiques:\n1. Abstract: several typos and minor grammatical errors need correcting\n2. Abstract: write out what MPO and NE are abbreviations for\n3. This statement as it currently reads, is incorrect, lines #63-64: “…COPD patients may suffer from drug induced neutropenia…” That is not a known lab finding in COPD unless they are referring to result of a specific therapy that is not widely used? Also a reference is needed for this statement.\n4. Figure 1A legend or the text: indicate what immune cell types that markers listed in green are meant to identify, e.g. CD3= T cells. Of note, both CD66b and CD11c are present on neutrophils.\n5. In section 2.2, indicate that the Slc8a1 is the gene for NCX1.\n6. Clarify if NET area is measured where there is overlap of at least two relevant markers (Figure 4).\n7. Section 2.4: Note Cl-amidine is a pan-PAD inhibitor and is not specific for PAD4.", "reviewer_3": "In the manuscript of Liao et al., the investigators characterized the role of NCX1 in neutrophils and its contribution to the pathogenesis of COPD.\nThey utilized in vitro and in vivo models including human samples showing significant upregulation of NCX11 in neutrophils, which correlated with MPO and NE. Furthermore, they showed attenuation of the neutrophil response in COPD including decreased emphysema in smoke-exposed mice with a neutrophil specific NCX1 deletion. Furthermore, cigarette smoke led to NCX1 to operate in reverse transport mode causing increased intracellular calcium and abnormal NET formation. Inhibition of these processes also attenuated CS-induced chronic bronchitis and development of emphysema like changes.\nOverall, this manuscript describes the involvement of neutrophil NCX1 in the COPD lung in addition to elucidation the downstream signaling pathways that are mediating the lung changes and neutrophil response thereby describing a potential novel pathway to target in the neutrophilic COPD phenotype. There are some moderate concerns that should be addressed.\n1) Supplemental figure 1A will need quantification of airway disease (MLI etc).\n2) Fig. 2D-J, control mice are missing, that are not exposed to cigarette smoke.\n3) Fig. 3 says that there was no difference in neutrophil number, but Fig. 2C shows the difference in number in the lung – this should be explained/it is not made clear whether these mice were exposed to cigarette smoke in Fig 3 (if not, it would be hard to measure migration to the lung if not stimulated). This is definitely a group that should be included.\n4) Fig. 6 G does not correlate with the images and should be shown with better representative images.\n5) The PAD4 inhibitor should be better described and should be validated. Neutrophils are not the only cells that express PAD4 and this should be taken into consideration.\n6) FlexiVent analysis of these mice would be helpful.\n7) Please comment on how many mice (male, female) were used and how many total donors and replicates in the Methods Statistics section.\nMinor:\n1) The PAD4 inhibitor and specificity for NETs should be introduced and validated.\n2) The neutrophil isolation should be described in more detail: in the methods section, it is said that they were isolated from BALB/c mice, but the models used are all C57BL/6\n3) Were neutrophils isolated via flowcytometry sorting?"}, "author_response": "A point-by-point response to referees’ comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by Liao and coll. postulates that the reverse mode of NCX1 (Slc8a1)\nsodium/calcium exchanger is responsible for neutrophil-mediated lung damage in mice\nunder conditions mimicking chronic obstructive pulmonary disease (COPD). Authors\nfound enhanced expression of NCX1 in human samples from COPD individuals as well as\nfrom mice exposed to cigarette smoke. Authors propose that NCX1 acting in reverse mode,\nthus mediating calcium influx, is responsible for enhanced release of neutrophil\nextracellular traps (NETs). To support their claim, authors generate a conditional mouse\nmodel with selective ablation of NCX1 expression in neutrophils. This mouse model shows\nreduced formation of NETs upon stimulus and decreased COPD signs. However, the study\nlacks several control experiments) that are needed to support the conclusions, particularly\nregarding the direct involvement of NCX1-mediated calcium influx in promoting altered\nneutrophil function,\nSPECIFIC POINTS\nFigure 1 A. The specificity of the NCX1 antibody used for immunofluorescence in murine\nand human samples should be demonstrated by disappearance of the signal in neutrophils\nfrom KO animals.\nResponse: We thank the reviewer for raising this important point regarding antibody\nvalidation. To address this concern,we have performed additional immunofluorescence\nexperiments using neutrophils isolated from our neutrophil-specific Slc8a1 cKO mice(as\ndescribed in the manuscript). Ourimmunofluorescence analysis clearly shows the absence\nof the NCX1 signal in neutrophils derived from Slc8a1 cKO mice, compared to robust\nsignal detection in control neutrophils. These new data are now shown asRevised\nSupplementary Figure 3f and incorporated into the revised manuscript (lines 128-130,\nlegends of Supplementary Fig. 3f).\nSupplementary Figure 2E. The expression of NCX1 detected by immunoblot in neutrophils\nfrom KO animals is markedly decreased but not absent. Is this a sign of a residual\nexpression/function of the transporter?\nResponse: We appreciate the reviewer’s keen observation regarding the residual NCX1\nsignal in immunoblots of Slc8a1 cKO neutrophils. We agree that this warrants clarification.\nIn our initial submission,we inadvertently omitted details regarding purity in the Methods\nsection. Actually, as we mentioned in the Methods section, neutrophils were isolated by\nfluorescence-activated cell sorting (FACS) with 95%-98% purity (lines 586-587).\nConsequently, about 2%-5% of sorted cells are non-neutrophils (e.g., monocytes,\nmacrophages, eosinophils), which express NCX1 endogenously. This small contaminating\n1\npopulation contributes detectably to the total protein lysate used for immunoblotting. We\nhave included the post-sort purity FACS plots confirming this level of purity in the Revised\nSupplementary Fig. 3c. Besides, we have performed additional immunofluorescence\nexperiments using neutrophils isolated from our neutrophil-specific Slc8a1 cKO mice\nwhich shows the absence of the NCX1 signal in neutrophils derived from Slc8a1 cKO mice\n(Revised Supplementary Figure 3f). We have updated the relevant content in figure\npanels accordingly (lines 123-124, 129-130, legends of Supplementary Fig. 3f).\nIn addition, our subsequent data also highlighted the substantially functional changes in\nthe absence of NCX1 activity in Slc8a1 cKO mice:\n1) Significant depletion of NCX1 expression in Slc8a1 cKO mice (Revised\nSupplementary Fig. 3f);\n2) Slc8a1 depletion showed significant protection against CS-induced NETs formation,\nlung damage and airway functions in COPD mice (Revised Fig. 2).\n3) NETs formation in lung neutrophils was profoundly inhibited in CS-exposed Slc8a1\ncKO mice (Revised Fig. 5f);\n4) Ca²⁺ influx was prominently depressed in Slc8a1 cKO neutrophils (Revised Fig. 7h).\nCollectively, our immunofluorescence and functional assays demonstrate that the genetic\nablation effectively eliminates NCX1-mediated Ca²⁺ influx and its downstream\npathological consequences (NETs formation and lung damage) in neutrophils. This\nconfirms that the residual immunoblot signal does not affect the reliability of our\nexperimental outcomes.\nFigure 7. In my opinion, the claim that NCX1 is working in reversed mode and promoting\nhigh intracellular concentration is not adequately supported by experiments. In particular,\nthe reverse mode of NCX1 is not an intrinsic characteristic of the transporter but depends\non the ion gradients across the plasma membrane. An increase in intracellular sodium\nconcentration can reverse the activity of NCX1 in neutrophils (Najder et al. Role of the\nintracellular sodium homeostasis in chemotaxis of activated murine neutrophils. Front\nImmunol 11:2124, 2020). Therefore, the effect of cigarette smoke may be indirect,\nincreasing intracellular sodium. This issue has not been investigated. To study NCX1\nfunction, authors use an unconventional method, based on a sensor that supposedly\nmeasures calcium flux direction. Looking at the vendor website, this instrument is mostly\nused for ion flux in plants. There are no tests in the paper to demonstrate that the method\nis really working compared to classical methods (e.g. measurement of intracellular calcium\nwith ion-sensitive fluorescent probes). For example, there is no experiment showing that\nthe calcium flux is abolished in neutrophils from NCX1 KO mice. Also, the NCX1 inhibitor\nshould also be tested in neutrophils from KO animals to show that is does not alter flux\nwhen NCX1 expression/function is ablated. Additional experiments should have used\nmethods to buffer or increase intracellular calcium by other means (e.g. calcium-chelating\nagents, calcium-mobilizing agonists, calcium ionophores) to check the effect on NET\n2\nrelease and/detection of calcium flux by the sensor.\nResponse: We sincerely thank the reviewer for these insightful critiques, which\nsignificantly strengthened our mechanistic investigation. We have now performed\nextensive new experiments to directly address these points.\n1) Clarifying direct vs. indirect activation of reverse-mode NCX1 by stimuli\nWe fully agree that NCX1 reverse-mode activation depends on transmembrane ion\ngradients (e.g., elevated intracellular Na⁺), commonly reported in references1. To\ndissect whether cigarette smoke extract (CSE) acts on NCX1 via Na⁺ dynamics, we\nsimultaneously measured real-time changes in intracellular Na⁺ ([Na⁺]i) and Ca²⁺\n([Ca²⁺]i) in neutrophils stimulated with three agents: CSE, PMA (phorbol ester), and\nfMLP (chemoattractant). Consistent with the reference mentioned by the reviewer1,\nwhich reports indirect NCX1 reverse-mode activation secondary to Na⁺ rise, our newly\nadded data (Ca²⁺ measurement by Fura-2 AM; Na²⁺ measurement by NaTrium Green-\n(Revised Fig. 7f-g), confirming this indirect pattern. In stark contrast, CSE and PMA\ntriggered rapid [Na⁺]i decrease alongside robust [Ca²⁺]i increase (Revised Fig. 7f-g).\nThis divergence demonstrates CSE and PMA might act via a distinct, direct mechanism\nfavoring reverse-mode NCX1 operation (Ca²⁺ influx/Na⁺ efflux), independent of prior\nmentioned [Na⁺]i elevation (lines 229-240).\n2) Addition of ion flux detection methodology and specificity validation of NCX1\ninhibitor (KB-R7943) in neutrophils from Slc8a1 cKO mice\nTo address concerns about the ion flux detection, we measured [Ca²⁺]i with the standard\nfluorescent Ca²⁺ indicator Fura-2 AM, and [Na²⁺]i measurement by NaTrium Green-2\nAM. CSE and PMA stimulation triggered rapid [Na⁺]i decrease alongside robust [Ca²⁺]i\nincrease (Revised Fig. 7f-g), and CSE-induced [Ca²⁺]i rise was near-abolished in\nSlc8a1 cKO neutrophils vs. Controls (Revised Fig. 7h), proving the critical role of\nNCX1 in CSE-triggered Ca²⁺ influx (results:lines 229-240; methods: lines 650-664).\nFurthermore, we also supplemented experiments to validate the specificity of NCX1\ninhibitor on Ca²⁺ influx, which revealed that treatment with the NCX1 reverse-mode\ninhibitor KB-R7943 had no obvious effect on CSE-induced [Ca²⁺]i in Slc8a1 cKO\nneutrophils (Revised Supplementary Fig. 4d, lines 248-251), demonstrating its\nspecificity and confirming successful NCX1 ablation.\n3) Validation of NCX1-mediated Ca²⁺ influx and NETosis using Ca²⁺ ionophore and\nCa²⁺ chelator\nAs requested, we applied Ca²⁺ ionophore (A-23187) and Ca²⁺ chelator (BAPTA-AM)\nto validate the causality between Ca²⁺ influx and NETs release. Pre-treatment with\nBAPTA-AM significantly reduced CSE-triggered [Ca²⁺]i rise, Cit-H3 expression, and\nNETs formation (Revised Fig. 7k, l; Revised Supplementary Fig. 4e, f; lines 245-\n257). Neutrophils showed reduced protrusions consistent with inhibited NCX1 reverse\nmode activation. Meanwhile, treatment with A-23187 elevated [Ca²⁺]i, potently\ninducing Cit-H3 expression and NETs release (Revised Supplementary Fig. 4e, f;\nlines 245-257), confirming Ca²⁺ overload is key factor to drive NETosis. Furthermore,\nwe conducted in vitro experiments using human peripheral blood neutrophils to\nvalidate that both the Ca²⁺ ionophore (A-23187) and the Ca²⁺ chelator (BAPTA-AM)\nmodulate NETs formation, consistent with our findings in murine cells.\nNCX1 (evidenced by Na⁺ efflux/Ca²⁺ influx); validate the NCX1-dependent Ca²⁺ influx via\nCa²⁺ measurement and genetic/pharmacologic controls; prove NCX1-mediated Ca²⁺ influx\nis necessary and sufficient for NETosis in this model. All new data have been incorporated\ninto revised manuscript. We thank the reviewer for prompting these critical experiments,\nwhich substantially reinforce our mechanistic conclusions.\nReferences\nof Activated Murine Neutrophils. Front Immunol 11, 2124,\ndoi:10.3389/fimmu.2020.02124 (2020).\nMajor strengths:\n1. Fascinating findings supporting a novel mechanism in which the NCX1 transporter\nreverses its mode in neutrophils resulting in altered intracellular calcium and triggering\nNETosis.\n2. The correlations in Figure 1I are very nice.\n3. Beautiful SEM pictures throughout the manuscript.\n4. The data shown in Figure 6 strengthened the manuscript.\n5. Strengthened by both and human and murine data.\n6. Further made stronger by using multiple approaches to prove their mechanism (gene\ndeletion, pharmacologic inhibition).\nResponse: We sincerely appreciate the reviewer’s thoughtful and positive comments. We\nare glad to hear that our findings were considered fascinating and that the proposed\nmechanism regarding NCX1-mediated Ca2+ influx and its role in NETs formation was well\nreceived. We also appreciate the acknowledgment of key research design and outcomes in\nour manuscript. Furthermore, we are grateful for the recognition of the strength derived\nfrom using both human and murine data, as well as the multiple experimental approaches\n(gene deletion, pharmacological inhibition) to validate our hypothesis. In this revision, we\nhave supplemented more data to make our conclusions more robust in accordance with\nyour constructive suggestions.\nMajor critiques:\n1. The nomenclature used of \"simple emphysema\" vs “COPD” is confusing and not\nconsistent with clinical nomenclature as emphysema is a form of COPD. Would suggest\nusing the term \"mixed chronic bronchitis emphysema\" vs \"emphysema\" to differentiate the\ntwo, if that is in fact what they mean.\nResponse: We sincerely appreciate the reviewer’s professional suggestion regarding the\nuse of clinically appropriate terminology. In the initial submission, we used the term\n“COPD” for simplicity and broader reader comprehension. However, we acknowledge that\n“emphysema” is indeed a subtype of COPD, and the terminology should be used precisely.\nFollowing your recommendation, we have revised the manuscript accordingly and now\ndifferentiate between “mixed chronic bronchitis and emphysema (mixed CBE)” and\n“emphysema” (Revised Fig. 1b-k, Revised Fig. 5d-e, Revised Supplementary Fig. 1a-\ng; lines 87-113, 167-168).\nIn addition, to enhance the clinical relevance of our findings, we further collected\nbronchoalveolar lavage fluid (BALF) and peripheral blood from five clinically diagnosed\nCOPD patients in this revision. These patients had confirmed airflow limitation by\npulmonary function tests and chest CT scans consistent with mixed CBE. Neutrophils\nisolated from these samples were used in functional assays, and in this context, we have\nadopted the term “mixed CBE” in accordance with your suggestion as well (Revised Fig.\n1l-o; lines 100-113).\n5\n2. Figure 1C lists mRNA changes between \"emphysema\" and their “COPD” group, but the\nprotein results for NCX1, MPO or NE in their “emphysema” group are not shown. Showing\nthose results would strengthen their findings that the “simple emphysema” vs their\n\"COPD/mixed chronic bronchitis emphysema” groups are molecularly distinct.\nResponse: Thank the reviewer for this excellent suggestion to strengthen our comparative\nanalysis of the clinical phenotypes. As requested, we have now performed Western blot\nanalyses of NCX1, MPO, and NE protein expression in samples from both emphysema\ngroup and Mixed CBE group. Protein levels of NCX1, MPO, and NE were significantly\nelevated in the Mixed CBE group compared to the emphysema group. This protein-level\nvalidation reinforces that neutrophil-associated pathology (NETosis drivers) is more\npronounced in Mixed CBE. These new data are presented in Revised Supplementary Fig.\n1a-d and described in the revised manuscript (lines 92-97).\nFigure 1A: where in the lung are these tissue samples from? Hard to discern underlying\nlung structure. Perhaps labels of alveoli, etc would improve this.\nResponse: We appreciate the reviewer’s request for histological clarity, and apologize for\nthe lack of explanation in our original manuscript. The human lung sections in Fig. 1a were\nobtained from distal airways and bronchioles and adjacent alveolar structures, representing\nregions most affected in COPD pathogenesis. This panel primarily focused on assessing\nNCX1 co-localization with immune cells (lymphocytes, macrophages, dendritic cells and\nneutrophils). The observed characteristic nuclear distribution and tissue morphology\n(flattened epithelial nuclei lining airspaces) strongly suggest these regions primarily\nrepresent alveolar structures. To improve interpretability, we have updated the Revised Fig.\n1a legend to explicitly state the tissue source, and supplemented more details in the revised\nmanuscript (lines 88, 838).\n4. Figure 1K: would be greatly strengthened if they could isolate neutrophils vs other\nimmune cell subsets from patient BAL and run western blots for NCX1 expression. This\nmay also be demonstrated by flow cytometry if NCX1 can be stained for by flow. The\nmicroscopy alone on lung tissue is insufficient to substantiate their statement that NCX1 is\nmuch higher in neutrophils vs other immune cells in control vs COPD patients.\nResponse: We sincerely thank the reviewer for this constructive suggestion. To directly\naddress this important point, we have performed flow cytometry analysis using\nbronchoalveolar lavage fluid (BALF) samples from both control subjects (primarily\nasymptomatic pulmonary nodule patients) and COPD patients in this revision.\nQuantification of neutrophil count and NCX1 expression via flow cytometry revealed\nsignificantly greater number of neutrophils and higher NCX1 mean fluorescence intensity\n(MFI) in COPD neutrophils compared to controls (Revised Fig. 1l-n). We further confirm\nthe neutrophil-specific NCX1 enrichment using flow cytometry, since NCX1 expression\nwas markedly higher in neutrophils than in other immune cell populations (Revised\nSupplementary Fig. 1e). These results provide multiple lines of evidence that NCX1 is\nprimarily upregulated in COPD neutrophils, and the increase is neutrophil-specific relative\nto other immune cells. These new data have been incorporated into Results, figure legends,\n6\nand Discussion sections, with corresponding methods updated (lines 100-113, 299-306,\n581-613).\n5. Are elevations in neutrophil NCX1 expression limited to lung neutrophils in COPD\npatients or is NCX1 expression increased in peripheral blood too?\nResponse: Thanks for raising this critical question about the systemic scope of NCX1\ndysregulation. To address this, we isolated peripheral blood neutrophils using neutrophil\ncell extraction kit from healthy volunteers and COPD patients and performed Western blot\nanalysis of NCX1 expression. As demonstrated, NCX1 protein levels were significantly\nelevated in peripheral blood neutrophils of COPD patients compared to healthy controls\n(Revised Supplementary Fig. 1f, g). This aligns with our lung tissue/BAL data,\nsuggesting NCX1 dysregulation is systemic, not confined to pulmonary neutrophils, which\nmay contribute to extrapulmonary COPD comorbidities. These new data have been\nincluded and discussed in the revised manuscript (lines 109-113, 299-303, 449-451, 586-\n587).\n6. Demonstrate that Slc8a1 deletion was neutrophil specific by isolating other immune cell\ntypes (mono/macs, etc) from these animals and showing NCX1 protein remains present in\nsimilar concentrations to control animals.\nResponse: Thanks for underscoring the importance of cell-type specificity in our genetic\nmodel. To validate the neutrophil-specific deletion of Slc8a1 (NCX1), we isolated and\nanalyzed non-neutrophil immune cells: flow-sorted non-neutrophils\n(monocytes/macrophages (Mono/Macs), lymphocytes, and dendritic cells) from both\nControl and neutrophil-specific Slc8a1 cKO mice. Quantification of NCX1 expression via\nRT-qPCR (mRNA) found no significant difference in non-neutrophil immune cells\nbetween Slc8a1 cKO and Control groups (Revised Supplementary Fig. 3d). Western blot\n(protein) exhibited comparable NCX1 protein expression in non-neutrophil immune\nsubsets between Slc8a1 cKO and Control groups (Revised Supplementary Fig. 3e). In\ncontrast, neutrophils from Slc8a1 cKO mice showed near-complete loss of NCX1 at both\nmRNA and protein levels (Revised Supplementary Fig. 3d, e). Immunofluorescence\nfurther validated the absence of NCX1 signal exclusively in neutrophils of Slca8a KO mice\n(Revised Supplementary Fig. 3f). These data unequivocally demonstrate that Slc8a1\nablation is primarily restricted to neutrophils. The results are now integrated into the\nrevised manuscript with more details updated (lines 123-130).\n7. Figures 3/4: While demonstrating that neutrophil numbers in each compartment are\nsimilar in both genotypes and showing that neutrophil function of bone marrow neutrophils\nare similar in both genotypes are solid starts, particularly as it relates to #5 above, would\nsuggest testing functions of lung neutrophils between both genotypes as well.\nResponse: Thank the reviewer for this insightful suggestion, which has strengthened our\ninvestigation of compartment-specific neutrophil dysfunction in COPD. As recommended,\nwe performed functional analyses of lung-infiltrating neutrophils in Control and\nneutrophil-specific Slc8a1 cKO mice exposed to CS. CS-exposed Slc8a1 cKO mice\nshowed significantly reduced Cit-H3 levels in mouse BALF and NETs area in lungs\ncompared to controls (Revised Fig. 5f-h). No obvious differences were observed in Cit-\nH3 levels between Control and Slc8a1 cKO mice under normal air exposure. Additionally,\nwe detected levels of several chemokines and cytokines secreted by neutrophils, which\nrevealed significantly diminished release of pro-inflammatory mediators in CS-exposed\ncKO mice relative to controls, including TNF-α, IL-8, IL-6, IL-1β, and IL-17a (Revised\nFig. 5i, l-p). This suggest that the critical role of NCX1 in neutrophil-mediated immune\nmicroenvironment. Moreover, CS-triggered pulmonary dysfunction and COPD\npathological changes exhibited improvement in Slc8a1 cKO mice (Revised Fig. 2).\nTogether, these new data demonstrate that NCX1 deletionspecifically improves\npathogenic functions of lung neutrophils (pulmonary function, Cit-H3 levels, NETs area,\ncytokine release, COPD pathological changes). The results are now integrated into the\nrevised maunscript, with more details updated in Methods section (lines 164-189, 131-147,\n324-371, 495-519).\n8. Figure 5: In addition to microscopy, would be much stronger if use an additional method\nto quantify NETs to substantiate the authors conclusions. For example, could measure NE:\nDNA, MPO:DNA or CitH3: DNA complexes by duel-ELISAs or Cit-H3 ELISA in the\nmurine BAL. Same critique applies to Figure 7F & H.\nResponse: We sincerely appreciate the reviewer’s suggestion to strengthen our NETs\nquantification through additional methods. To address this concern, we have performed\nELISA assays of Cit-H3 to quantify NETs formation across key figures in this revision.\nIn vitro, we isolated bone marrow neutrophils from mice (mice with two genotypes; WT mice),\nand treated the cell culture supernatants with CSE or PMA stimulation. Cit-H3 levels were\nsignificantly decreased either with NCX1 depletion or inhibition of NCX1 reverse mode\n(KB-R7943 treatment) (Revised Fig. 5c-h, Revised Fig. 7i, Revised Fig. 7l,\nSupplementary Fig. 4c). In vivo, we collected BALF from air or CS-exposed mice (Slc8a1\ncKO vs. Control), and found reduced Cit-H3 levels in CS-induced Slc8a1 cKO mice\n(Revised Fig. 5h). Besides, Cit-H3 expression in BALF from WT mice with/without Cl-\namidine (NETs inhibition) or KB-R7943 (NCX1 reverse mode inhibition) treatment have\nalso been detected, which exhibited prominent reduction in mice with NETs formation\nsuppressed (Revised Fig. 6e), or reverse mode of NCX1 inhibited (Revised Fig. 8e). These\nnew data have been integrated into Results, Discussion sections, and corresponding\nmethods have been supplemented in the Methods section (lines 164-171, 241-248,\n203-208, 266-271).\n9. Insufficient evidence to substantiate the proposed mechanism re NETs impeding the\nnormal regression of tissue resident neutrophils from the lung, described in lines #191-193.\nThere are several potential explanations including NETs promoting the release of\nchemokines. This has been previously demonstrated in lung epithelium by the authors of\nPMID # 36703990 and in fibroblasts by the authors of PMID # 23536012. Could measure\nthe chemokine and cytokine levels in the BAL and/or whole lung to substantiate this as a\nmechanism.\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the\nmechanistic link between NETs and neutrophil retention in the lung. As recommended,\nwe have now performed chemokine/cytokine (TNF-α, IL-8, IL-6, IL-1β, and IL-17a) profiling\n8\nin lung tissues from Control and neutrophil-specific Slc8a1 cKO mice exposed to CS. Our\nnewly added data revealed that CS exposure triggeredsignificant upregulation of pro-\ninflammatory mediators in Control neutrophils. Whereas Slc8a1 cKO mice exhibited\nmarkedly reduced levels of all measured cytokines/chemokines relative to Controls\n(Revised Fig.5l-p). This highlights that NXC1 help sustain neutrophil accumulation and\nretention to create a chemokine-rich microenvironment. Given prior reports that NETs\namplify chemokine production in lung epithelium2 and fibroblasts3, we speculate NCX1-\ndriven NETs formation is a key upstream regulator of chemokine-rich microenvironment\nfavoring neutrophil retention. These new data substantiate our proposed mechanism by\nhighlighting a self-amplifying loop (NCX1--chemokine release--neutrophil retention).\nNCX1 inhibition could break this cycle by reducing NETs release and downstream\nchemokine production. These data are presented and discussed in the revised manuscript\n(lines 172-189, 341-371), with mentioned references cited in Discussion section (line 356).\nWe thank the reviewer for prompting this critical validation, which significantly\nstrengthens the mechanistic depth of our study.\nReferences\nof airway epithelial barrier function. Front Immunol 13, 1023553,\ndoi:10.3389/fimmu.2022.1023553 (2022).\ninflammatory responses in rheumatoid arthritis. Sci Transl Med 5, 178ra140,\ndoi:10.1126/scitranslmed.3005580 (2013).\n10. Figure 6: Need to confirm the Cl-amidine treatment decreased NET generation as they\nexpect by microscopy or ELISA, etc.\nResponse: Thanks for this important suggestion to validate the effects of Cl-amidine on\nNETs formation. As requested, ELISA assay was performed to detect Cit-H3 levels in the\nBALF from CS-exposed mice. The results demonstrated that Cl-amidine treatment\nsignificantly reduced Cit-H3 levels in CS-exposed mice compared to control mice without\ntreatment (Revised Fig. 6e). In addition, we supplemented immunostaining of\nNE/MPO/Cit-H3 in mouse lung tissues and found that decreased NETs area in Cl-amidine-\ntreated mice (Revised Fig. 6g, h). These results have been incorporated into the revised\nmanuscript (lines 203-208, 506-519, 542-568).\n11. For Figure 7I: are there microscopy pictures that coincide with the graphs enumerating\nchanges in the lungs?\nResponse: Thank the reviewer for requesting this important visual validation. In the\nrevised manuscript, representative microscopy images corresponding to the mean linear\nintercept (MLI) quantification in Fig. 7i have been included as Revised Fig. 8h, j, l, n.\nThese histological analysis of lung sections clearly demonstrate enhanced-alveolar\n9\ndestruction and airway inflammation in CS-exposed mice, while these were reversed in the\nCS-exposed mice with inhibition of NCX1 reverse mode. These images and related\ndescription are now integrated in the revised manuscript (lines 271-280). We agree this\naddition strengthens the pathological evidence supporting our conclusions about NCX1's\nrole in COPD progression.\n12. The authors use PMA to induce NETs for comparison. While PMA is a robust\nstimulator of NETs, would suggest they also compare to more physiologically relevant\nstimuli such as LPS or IL-8 (Figure 2,5 and 7).\nResponse: We appreciate the reviewer's valuable suggestion regarding additional use of\nmore physiologically relevant NETs stimuli. Actually, we compared the effects of several\nstimuli—including LPS, fMLP, and Cigarette Smoke Extract (CSE)—on NCX1 expression\nin neutrophils. Among these, CSE most robustly upregulated both mRNA and protein\nlevels of NCX1(Revised Fig. 7a–d). Given that cigarette smoke is the dominant risk factor\nfor COPD, we prioritized Cigarette Smoke Extract (CSE) as our main stimulus to maximize\nclinical relevance. We also found that CSE stimulation elicited a comparable increase in\nCit-H3 expression to that induced by PMA, the widely used positive control (Revised Fig.\n5c, Revised Fig. 7i, Revised Fig. 7l, Supplementary Fig. 4c). In the cell culture\nsupernatants of bone marrow neutrophils from mice, with either CSE or PMA stimulation,\nCit-H3 levels were significantly decreased with NCX1 depletion or inhibition of NCX1\nreverse mode (Revised Fig. 5c, Revised Fig. 7i, Revised Fig. 7l, Revised Supplementary\nFig. 4c). This consists with our in vivo outcomes that CS exposure led to increased Cit-H3\nlevels and expanded NETs area in mouse BALF and lung tissue (Revised Fig. 5f–h,\nRevised Fig. 6e–g, Revised Fig. 8e–g). These data have been updated in the revised\nmanuscript (lines 220-223, 241-248).\n13. Likely, outside the scope of the current study,however, showing that the Slc8a1 deleted\nmice have improvement in their pulmonary function tests after CSE would be an\noutstanding demonstration of the functional impact of NCX1 transporter in a COPD model.\nResponse: Thank the reviewer for this exceptional suggestion to strengthen the\ntranslational impact of our findings. We recognized the importance of pulmonary function\ntests and have now performed comprehensive respiratory physiology measurements. Our\nnew results revealed striking improvements in CS-exposed Slc8a1 cKO mice, indicated by\nenhanced airway function (increased FEV₂₀ /FVC ratio and FEV₅₀ /FVC ratio) and\n(ms) (ms)\nreduced air trapping (decreased FRC/TLC ratio) compared to Controls (Revised Fig. 2b-\nd). These data suggested preserved expiratory flow rates at critical timepoints and\nimproved lung volume regulation in Slc8a1 deleted COPD mice. Similar improvements\nwere seen with NETs blockade (CI-amidine) (Revised Fig. 6b-d) and pharmacological\ninhibition of NCX1 reverse mode (KB-R7943) (Revised Fig. 8b-d) in vivo. We also\nsupplemented air controls in comparison with CSE induction, demonstrating no obvious\ndifferences of pulmonary functions between Slc8a1 cKO and Control mice under air\nexposure, but substantial dysfunctions in CS-exposed groups (Revised Fig. 2b-d). These\nresults provide the direct evidence that NCX1 ablation and NETs formation blockade could\nrestore airway functions of COPD mice. These data have been integrated into the Results,\nMethods, discussion of the revised manuscript (lines 133-139, 196-202, 261-266).\n10\n14. Discussion needs to be strengthened by a deeper discussion of how these findings are\nconsistent or inconsistent with prior studies, providing enhanced context for the current\nfindings and how the results truly advance the field.\nResponse: We sincerely appreciate this constructive suggestion. We have now significantly\nexpanded the Discussion section to provide deeper integration with prior literature and\nclarify the conceptual advances of our study. The revised discussion has been strengthened\nwith our newly added empirical data and more than 20 new references added. We believe\nthese changes better position our work within the field's continuum while highlighting its\ntransformative potential (lines 282-422).\nMinor critiques:\n1. Abstract: several typos and minor grammatical errors need correcting\nResponse: Thank the reviewer for pointing this out. We have thoroughly revised the\nAbstract to correct all typographical and grammatical errors. All corrections were marked\nin red in the revised manuscript (lines 28-41).\n2. Abstract: write out what MPO and NE are abbreviations for\nResponse: Thank you for the reviewer’s kind reminder. In the revised manuscript, we have\nmodified the Abstract and there’s no mention on these two words now due to content\nadjustment. We now use their full names at first mention—myeloperoxidase\n(MPO)andneutrophil elastase (NE) in the Results section (line 96). We have ensured\nconsistency in abbreviation usage throughout the manuscript.\n3. This statement as it currently reads, is incorrect, lines #63-64: “…COPD patients may\nsuffer from drug induced neutropenia…” That is not a known lab finding in COPD unless\nthey are referring to result of a specific therapy that is not widely used? Also a reference is\nneeded for this statement.\nResponse: We have revised the sentence accordingly to improve its accuracy and clarity,\nand we have added an appropriate reference to support the revised statement. The updated\nsentence now reads “However, neutrophil-targeted therapies remain challenging in COPD\ndue to the risk of drug-induced neutropenia and subsequent infection.”, and the citation has\nbeen included in the revised manuscript (lines 53-55).\n4. Figure 1A legend or the text: indicate what immune cell types that markers listed in\ngreen are meant to identify, e.g. CD3= T cells. Of note, both CD66b and CD11c are present\non neutrophils.\nResponse: Thank the reviewer for this helpful suggestion. We have revised the legend of\nFigure 1A and the relevant text to indicate the immune cell types identified by the labeled\nmarkers. Thank you for your reminder on both CD66b and CD11c present on neutrophils.\nTo address this concern, we performed triple immunostaining of NCX1+CD66b+/CD11c+\nto further robustly identify neutrophils and NCX1 expression in these cells. Indeed, we\nobserved major NCX1 co-localization with CD66b+/CD11c+ cells, highlighting the\npredominant expression of NCX1 in neutrophils. These data have been incorporated into\nthe revised manuscript (lines 89-90, 836-839).\n11\n5. In section 2.2, indicate that the Slc8a1 is the gene for NCX1.\nResponse: We have now indicated in Introduction section that Slc8a1 is the gene encoding\nNCX1 (Na2+-Ca2+ Exchanger 1) to provide clarity for readers (line 65).\n6. Clarify if NET area is measured where there is overlap of at least two relevant markers\nResponse: Thank the reviewer for this important methodological clarification. We confirm\nthat NETs area quantification was performed using co-localization criteria with NET-\nspecific markers (MPO, Cit-H3) to ensure accurate identification. Our stringent multi-\nmarker approach minimizes false positives and aligns with current NETs research\nstandards4. All NETs measurements required simultaneous positivity for Cit-H3 and MPO.\nRepresentative images are shown in Revised Fig. 5d (human lung tissues), Fig. 5f (mouse\nlung tissues), Revised Fig. 6g (Cl-amidine treatment), Fig. 8f (KB-R7943 treatment).\nMethodological and figure legend details have been added in the revised manuscript (lines\n567-568, 916-921, 942-945, 986-988). The reviewer's suggestion helped us improve\ntransparency in NETs identification criteria.\nReferences\nThromboinflammation. Circ Res 134, e93-e111, doi:10.1161/circresaha.124.324214\n(2024).\n7. Section 2.4: Note Cl-amidine is a pan-PAD inhibitor and is not specific for PAD4.\nResponse: We appreciate the reviewer's accurate observation regarding Cl-amidine's\npharmacological properties. As correctly noted by the reviewer, Cl-amidine inhibits\nmultiple PAD isoforms (PAD1-4). We sincerely apologize for any confusion caused by\nour initial wording. Actually, in this part, we aimed to test the effects of NETs blockade\non COPD pathology, without intent to dissect individual PAD isoform contributions. Thus,\nCl-amidine was employed as a NETs inhibitor rather than a PAD4-specific probe. This\naligns with its established use for broad NETs suppression5,6. Our data also consistently\ndemonstrated Cl-amidine treatment contributed to marked Cit-H3 decline and NETs area\ndecreased, along with subsequent attenuation of pulmonary function and COPD pathology.\nThis confirms the successful blockade of NETs release by Cl-amidine treatment. We\napologize for the inappropriate description about this inhibitor in the original manuscript,\nwhich has been corrected in the Results and Methods sections of the revised manuscript\n(lines 190-196).\nReferences\ncl-amidine prevents diabetes development in nonobese diabetic mice. Eur J Pharmacol\n916, 174623, doi:10.1016/j.ejphar.2021.174623 (2022).\ncl-amidine prevents diabetes development in nonobese diabetic mice. European Journal of\nPharmacology916, 174623, doi:https://doi.org/10.1016/j.ejphar.2021.174623 (2022).\nIn the manuscript of Liao et al., the investigators characterized the role of NCX1 in\nneutrophils and its contribution to the pathogenesis of COPD.\nThey utilized in vitro and in vivo models including human samples showing significant\nupregulation of NCX11 in neutrophils, which correlated with MPO and NE. Furthermore,\nthey showed attenuation of the neutrophil response in COPD including decreased\nemphysema in smoke-exposed mice with a neutrophil specific NCX1 deletion.\nFurthermore, cigarette smoke led to NCX1 to operate in reverse transport mode causing\nincreased intracellular calcium and abnormal NET formation. Inhibition of these processes\nalso attenuated CS-induced chronic bronchitis and development of emphysema like\nchanges.\nOverall, this manuscript describes the involvement of neutrophil NCX1 in the COPD lung\nin addition to elucidation the downstream signaling pathways that are mediating the lung\nchanges and neutrophil response thereby describing a potential novel pathway to target in\nthe neutrophilic COPD phenotype. There are some moderate concerns that should be\naddressed.\n1) Supplemental figure 1A will need quantification of airway disease (MLI etc).\nResponse: We thank the reviewer for this suggestion to strengthen our supplemental data.\nWe have now performed comprehensive quantitative analysis of airway and parenchymal\npathology in Supplemental Figure 1A (Revised Supplementary Fig. 2a, b).\nThe\nquantifications of mean linear intercept confirm that mice with CS exposure for 6 and 12\nweeks exhibited alveolar destruction. These data have been integrated into the revised\nResults section, and highlighted in the updated figure legends (lines 115-118, legends of\nRevised Supplementary Fig. 2a, b).\n2) Fig. 2D-J, control mice are missing, that are not exposed to cigarette smoke.\nResponse: Thank the reviewer for highlighting this important omission. We have now\nincluded comprehensive data from air-exposed control groups in pulmonary function tests\n(FEV /FVC, FEV /FVC, FRC/TLC), HE staining for MLI and collagen deposition\n20(ms) 50(ms)\nquantification, α-SMA immunofluorescence, Masson's trichrome, and SFTPC+\ncell immunofluorescence (Revised Fig. 2). No significant differences were found between\ntwo groups under air exposure, but CS-exposed mice exhibited prominently significant\npulmonary dysfunctions and pathological changes compared to air-exposed control groups.\nThe inclusion of air-exposed controls confirms that NCX1 deletion does not alter baseline\nlung structure and strengthens the injury-specific interpretation of our KO phenotype. We\nhave updated all relevant methodological details, Results and figure legends in the revised\nmanuscript (lines 114-147, 865-869).\n3) Fig. 3 says that there was no difference in neutrophil number, but Fig. 2C shows the\ndifference in number in the lung – this should be explained/it is not made clear whether\nthese mice were exposed to cigarette smoke in Fig 3 (if not, it would be hard to measure\nmigration to the lung if not stimulated). This is definitely a group that should be included.\nregret any misunderstanding due to our insufficient clarification in the initial submission.\nIn this revision, we have clarified the experimental design and added critical missing data\nto resolve any confusion. Fig. 2C (Revised Fig. 2f) showed significantly increased\nneutrophil number in CS-exposed Control mice (12-week exposure) compared to CS-\nexposed Slc8a1 cKO mice. Meanwhile, we analyzed air-exposed mice and confirmed no\ndifference in neutrophil numbers between Control and Slc8a1 cKO groups (Revised Fig.\n2f). Data analyzed in Fig. 3 were all from non-CS exposed mice, since this section aimed\nto investigate the contribution of NCX1 in neutrophil function. We concluded that Slc8a1\n(NCX1) deletion reduced NETs formation of neutrophils, without affecting development,\nmaturation, migration and phagocytosis of neutrophils. More details were provided in the\nResults and Figure legends of the revised manuscript (lines 139-147, 150-152, 865-872,\n896-897).\n4) Fig. 6 G does not correlate with the images and should be shown with better\nrepresentative images.\nResponse: Thank the reviewer for this important observation regarding image\nrepresentation. We have carefully addressed this concern by replacing the original Fig. 6G\n(Revised Fig. 6k) images with new, more representative Masson's trichrome staining ones.\nThe updated images now clearly correspond to the quantitative data in Fig. 6l.\n5) The PAD4 inhibitor should be better described and should be validated. Neutrophils are\nnot the only cells that express PAD4 and this should be taken into consideration.\nResponse: We appreciate the reviewer's accurate observation regarding Cl-amidine's\npharmacological properties. As correctly noted by the reviewer, Cl-amidine inhibits\nmultiple PAD isoforms (PAD1-4). We sincerely apologize for any confusion caused by\nour initial wording. Actually, in this part, we aimed to test the effects of NETs blockade\non COPD pathology, without intent to dissect individual PAD isoform contributions. Thus,\nCl-amidine was employed as a NETs inhibitor rather than a PAD4-specific probe.\nThis aligns with its established use for broad NETs suppression5,6. Our data also\nconsistently demonstrated Cl-amidine treatment contributed to marked Cit-H3 decline\nand NETs area decreased, along with subsequent attenuation of pulmonary function and\nCOPD pathology. This confirms the successful blockade of NETs release by Cl-amidine\ntreatment. We apologize for the inappropriate description about this inhibitor in the\noriginal manuscript, which has been corrected in the Results and Methods sections of the\nrevised manuscript (lines 190-196).\nReferences\ncl-amidine prevents diabetes development in nonobese diabetic mice. Eur J\nPharmacol 916, 174623, doi:10.1016/j.ejphar.2021.174623 (2022).\ncl-amidine prevents diabetes development in nonobese diabetic mice. European\nJournal of Pharmacology 916, 174623,\n14\ndoi:https://doi.org/10.1016/j.ejphar.2021.174623 (2022).\n6) FlexiVent analysis of these mice would be helpful.\nResponse: Thank the reviewer for this exceptional suggestion to strengthen the\ntranslational impact of our findings. We recognized the importance of pulmonary function\ntests and have now measure obstructive ventilation dysfunction (FEV /FVC ratio and\n20(ms)\nFEV /FVC ratio) and residual capacity (FRC/TLC) by Buxco/DSI system (DSI\n50(ms)\nBuxcoPFT, America). Our new results revealed striking pulmonary function improvements\nin CS-exposed Slc8a1 cKO mice, indicated by increased FEV /FVC ratio and\n20(ms)\nFEV /FVC ratio and decreased FRC/TLC ratio compared to Controls (Revised Fig.\n50(ms)\n2b-d). Similar improvements were demonstrated with NETs blockade (Cl-amidine)\n(Revised Fig. 6b-d) and pharmacological inhibition of NCX1 reverse mode (KB-R7943)\n(Revised Fig. 8b-d) in vivo. We also supplemented air controls in comparison with CSE\ninduction, demonstrating no obvious differences of pulmonary functions between Slc8a1\ncKO and Control mice under air exposure, but substantial dysfunctions in CS-exposed\ngroups (Revised Fig. 2b-d). These results provide the direct evidence that NCX1 ablation\nand NETs formation blockade could restore airway functions of COPD mice. These data\nhave been integrated into the Results, Methods, discussion of the revised manuscript (lines\n133-139, 196-203, 261-266).\n7) Please comment on how many mice (male, female) were used and how many total\ndonors and replicates in the Methods Statistics section.\nResponse: Thank the reviewer for emphasizing the importance of transparent experimental\nreporting. We have now included detailed information about sample used and replicates of\nmice and human donors in both the Methods and figure legends (lines 834-1011). All\noriginal data including biological and technical replicates were provided in the Source data\nfile uploaded with resubmission.\nMinor:\n1) The PAD4 inhibitor and specificity for NETs should be introduced and validated.\nResponse: We appreciate the reviewer's accurate observation regarding Cl-amidine's\npharmacological properties. As correctly noted by the reviewer, Cl-amidine inhibits\nmultiple PAD isoforms (PAD1-4). We sincerely apologize for any confusion caused by\nour initial wording. Actually, in this part, we aimed to test the effects of NETs blockade on\nCOPD pathology, without intent to dissect individual PAD isoform contributions. Thus,\nCl-amidine was employed as a NETs inhibitor rather than a PAD4-specific probe. This\naligns with its established use for broad NETs suppression5,6. Our data also consistently\ndemonstrated Cl-amidine treatment contributed to marked Cit-H3 decline and NETs area\ndecreased, along with subsequent attenuation of pulmonary function and COPD pathology.\nThis confirms the successful blockade of NETs release by Cl-amidine treatment. We\napologize for the inappropriate description about this inhibitor in the original manuscript,\n15\nwhich has been corrected in the Results and Methods sections of the revised manuscript\n(lines 190-196).\nReferences\ncl-amidine prevents diabetes development in nonobese diabetic mice. Eur J\nPharmacol 916, 174623, doi:10.1016/j.ejphar.2021.174623 (2022).\ncl-amidine prevents diabetes development in nonobese diabetic mice. European\nJournal of Pharmacology 916, 174623,\ndoi:https://doi.org/10.1016/j.ejphar.2021.174623 (2022).\n2) The neutrophil isolation should be described in more detail: in the methods section, it is\nsaid that they were isolated from BALB/c mice, but the models used are all C57BL/6\nResponse: We sincerely apologize for this oversight in our original submission. All mouse\nstudies were indeed performed using C57BL/6 mice. The erroneous reference to BALB/c\nhas been modified throughout the manuscript, and more methodological details about\nanimal strain and neutrophil isolation have been updated in Methods section (lines 472-\n476, 582). We deeply regret this error and have implemented additional verification steps\nin our manuscript preparation process to prevent such issues in future submissions. The\ncorrections have been highlighted in the revised manuscript with track changes for easy\nidentification.\n3) Were neutrophils isolated via flowcytometry sorting?\nResponse: We appreciate this opportunity to clarify our neutrophil isolation methodologies.\nOur approach differed by sample type to ensure optimal purity and viability. Human blood\nneutrophils were isolated using negative selection kits (EasySep™ Human Neutrophil\nEnrichment Kit, StemCell #19257). Murine neutrophils from bone marrow were isolated\nvia FACS sorting (gating strategy: CD45+CD11b+Ly6g+), with purity >95% (Revised\nSupplementary Fig. 3c). We have updated Methods section and all relevant figure legends\nto specify isolation methods (lines 581-613).\nMurine Neutrophils. Front Immunol11, 2124, doi:10.3389/fimmu.2020.02124 (2020).\nepithelial barrier function. Front Immunol 13, 1023553, doi:10.3389/fimmu.2022.1023553\n(2022).\nresponses in rheumatoid arthritis. Sci Transl Med 5, 178ra140,\ndoi:10.1126/scitranslmed.3005580 (2013).\nThromboinflammation. Circ Res134, e93-e111, doi:10.1161/circresaha.124.324214 (2024).\nprevents diabetes development in nonobese diabetic mice. Eur J Pharmacol916, 174623,\ndoi:10.1016/j.ejphar.2021.174623 (2022).\nprevents diabetes development in nonobese diabetic mice. European Journal of\nPharmacology916, 174623, doi:https://doi.org/10.1016/j.ejphar.2021.174623 (2022).\n17"}, {"round": 2, "reviewer_comments": {"reviewer_2": "Authors provide a thoughtful and thorough response addressing all of my critiques and concerns.\nOne important clarification needs to be made: The authors refer to measuring IL-8 in mice in Figure 5, but mice do not express IL-8, which is a bit concerning. The murine homologues are CXCL-1, CXCL-2 and CXCL-5 and are important neutrophil chemoattractants. What exactly did the authors measure? Did they use murine primers or human primers for this RT-PCR?\nI would strongly consider showing pressure-volume loops which they should be able to generate and would be visually more clear as the FEV1(20) and (50) are not used commonly used clinically. The authors likely also have compliance and elastance data too that would strengthen their findings as well of emphysema or lack thereof.", "reviewer_3": "All concerns have been appropriately addressed.", "reviewer_4": "Dear ladies and gentlemen,\nI was asked to re-review new data and comments given to address the concerns of reviewer #1 following the initial submission of the manuscript.\nReviewer #1 only had three specific points, and the authors have included several new data to address those:\nPoint 1 - Specificity of the NCX1 antibody - The authors now very clearly demonstrate the specificity of the NCX1 antibody, the new data are very convinsing.\nPoint 2 - Remaining expression of NCX1 in neutrophils from knockout animals - The authors now demonstrate that the purity of neutrophils following FACS is \"only\" 95%-98%. Thus, 2-5% of the cells are other immune cells that still express NCX1. This is convinsing and sufficient to address this concern.\nPoint 3 - This point is more \"major\" than point 1 and 2. Here the reviewer asked the authors to provide more solid evidence that, and how cigarette smoke induces a \"reverse mode\" of NCX1. Furthermore, the reviewer questioned the accuracy of the method used to examine intracellular calcium.\nThe authors have performed all experiments that were suggested by the reviewer. By performing classical calcium and sodium imaging, they demonstrate that cigarette smoke, PMA and fMLP induce a very rapid and sustained increase in intracellular calcium. For cigarette smoke, this effect was stronlgy reduced in NCX1-knockout cells, and the NCX1-blocker KB-R7943 failed to inhibit effects in knockout cells. Cigarette smoke and PMA (but not fMLP) also induce a rapid reduction in intracellular sodium. With these data, the authors argue that their hypothesis about a reverse mode of NCX1 is strongly supported.\nIt is clear that the authors have performed all (or more) experiments that were required or suggested by reviewer 1. The new data seems to be conclusive, and it perfectly fits into the story told in the manuscript. As such, the authors made a great job in reacting to the comments of the reviewer.\nMy personal view on this is a little different. Essentially, the exact mechanism(s) accounting for cigarette smoke-induced modification of NCX1 remains poorly defined. Is it a direct effect on NCX1, or an indirect effect due to shifts of Calcium and Sodium over other mechanisms? Given that the authors only work on primary neutrophils, this question remains to be addressed. A straight forward approach to investigate this however, would be to examine recombinant NCX1 in an expression system allowing celular imaging or electrophysiology specifically on NCX1.\n\nA surprising, or even a little irritating observation in figure 7f, g and h, is that CSE, PMA and fMLP induce very rapid calcium and sodium fluxes. I believe that signals were recorded at ~0.3 Hz, thus all three substances induce effects that saturated within 3 seconds. Given that fMLP and PMA most likely do not directly modify NCX1, how can these very rapid effects by all three compounds be explained? Is it even plausible? Can artifacts from the application as such be ruled out?, i.e. a role of mechano-sensitive mechanisms like Piezo channels?\n\nCigarette smoke is a fairly well defined mixture of several substances. Which of these substances can induce this rapid effect? Again, experiments on NCX1 in simplified cellular system would enable the authors to address these points.\nFor now, the authors seem to consider these open but relevant questions as outside the scope of this study. Importantly, the authors do mention \"Mechanistic understanding of NCX1 modes\" as a point with limitations."}, "author_response": "Response:\nWe sincerely thank the reviewer for this important reminder. After checking on the\nprimer design website, we confirmed that, the primers labeled as “IL-8” in our RT-PCR\nexperiments using tissues from the conditional knockout mice were actually designed\nto target CXCL-2, the murine homologue of human IL-8. We apologize for this\noversight and for not clearly distinguishing between human and mouse nomenclature\nin the manuscript. We have now corrected the gene name accordingly throughout the\ntext and figure panels in the revised version (Revised Fig. 5m, lines 190, 656, 987).\nRevised Fig. 5m RT-qPCR detected mRNA levels of TNF-α, CXCL-2, IL-6, IL-1β, and\nIL-17a in mouse lungs with different genotypes.\n\nResponse:\nThank you very much for your valuable and constructive suggestions. We fully agree\nwith your recommendation to include pressure-volume loops, as they provide a clearer\nand more intuitive visualization of the data, and serve as a more effective representation\nof pulmonary function. Upon further discussion with our technical team, we realized\nthat pressure–volume recordings were indeed part of the data we had previously\ncollected. Thanks to your insightful suggestion, we reanalyzed and visualized the\ncomplete pressure–volume loops in the revised manuscript (Revised Fig. 2b, Revised\nFig. 6b, Revised Fig. 8b).Additionally, we do have compliance data as well, which\nhave been incorporated into the revised manuscript (Revised Fig. 2c, Revised Fig. 6c,\nRevised Fig. 8c). Pressure–volume loops and compliance data demonstrated that\ndeletion of NCX1 significantly ameliorated CS-induced decrease of lung compliance\n(Revised Fig. 2c). Consistently, PAD4 inhibition by Cl-amidine (Revised Fig. 6c) and\npharmacological inhibition of NCX1 reverse-mode activity with KB-R7943 (Revised\nFig. 8c) improved dynamic compliance (Cydn). These additional data enhance the\nrobustness of our interpretation that NCX1 reverse-mode activity aggravates lung\nmechanical dysfunction in COPD. We have now incorporated the newly added data into\nthe text and figure panels in the revised manuscript (lines 133-140, 202-206,\n285-288, 536-542, 929-932, 998-1002, 1046-1050).\nRevised Fig. 2b-c The pressure-volume curves and dynamic compliance data of Slc8a1\ncKO mice and Control mice under normal air or CS exposure.\nRevised Fig. 6b-c The pressure-volume curves and dynamic compliance data of Control,\nCS, CS+Vehicle and CS+Cl-amidine mice.\nRevised Fig. 8b-c The pressure-volume curves and dynamic compliance data of Control,\nKB-R7943, CS, CS+KB-R7943 mice.\n\nResponse:\nThank you very much for your positive evaluation. We sincerely appreciate your careful\nreview and are grateful for your recognition that all concerns have been appropriately\naddressed.\n\nResponse: Thank you for this important suggestion. To directly address whether\ncigarette smoke–related stimuli act on NCX1 directly rather than indirectly, we\nemployed CHO-K1 cells in which NCX1 was not present based on literature reports1,2,\nwhich were also confirmed by our western blot and immunofluorescence data (Revised\nSupplementary Fig. 4h, i). After CHO-K1 cells transfected with recombinant NCX1,\nthe real-time [Ca²⁺]i and [Na⁺]i were monitored using ion-sensitive fluorescent probes.\nCompared with vector controls, NCX1-overexpressing CHO-K1 cells displayed a rapid\nrise in intracellular Ca²⁺ accompanied by a decrease in intracellular Na⁺ upon\nstimulation (Revised Supplementary Fig. 4j, k), consistent with our results\ndemonstrating Ca²⁺/Na⁺ fluxes stimulated by CSE in primary neutrophils. As you’ve\nconcerned about mechanosensitive artifacts in the next question below, this experiment\nincluded pre-incubation with GsMTx4 to block Piezo channels. The CHO-K1\noverexpression data, together with NCX1 depletion in Slc8a1 Cko and pharmacological\ninhibition of NCX1 reverse mode (KB-R7943), make a compelling case that NCX1\nmediates the CSE-triggered NCX1 reverse-mode Ca²⁺ influx we observed in primary\nneutrophils. The related text and figure panels have been updated in the revised\nmanuscript (lines 257-263, 418-420, 686-697, 712-717, legends of Supplementary\nFig. 4).\nRevised Supplementary Fig. 4h, i Western blot analysisand immunofluorescence\nvalidated the expression of NCX1 in NCX1-transfected CHO-K1 cells and vector\ncontrols.\nRevised Supplementary Fig. 4j, k Ca²⁺ and Na2+influx in NCX1-transfected CHO-K1\ncells and vector controls(pre-incubated with GsMTx4)upon CSE stimulation\n\nResponse:\nThank you for your thoughtful observation. We fully acknowledge your concern\nregarding the rapid calcium and sodium fluxes observed in Figure 7f, g, and h following\ntreatment with CSE, PMA, and fMLP, which may be caused by the mechanical\nstimulation during the drug application through activating mechanosensitive channels,\nleading to the transient ion fluxes that you have pointed out.\nTo address this concern, we performed additional cellular experiments using GsMTx4,\na specific inhibitor of Piezo channels3, to block mechanosensitive ion channels before\naddition of different stimuli. The results of these experiments revealed after inhibiting\nPiezo channels, the time required for Ca²⁺ and Na⁺ fluxes to reach their peak was\ndelayed compared to the untreated condition,while the trends of peak fluxes themselves\nremained unchanged in later several minutes (Revised Supplementary Fig. 4d-f).\nNotably, after the initial delay, the ion fluxes stabilized and continued at a consistent\nlevel. In our response to your first question, Piezo inhibition (GsMTx4 pretreatment)\nwas also performed in detecting Ca²⁺ and Na⁺ fluxes in NCX1-overexpressing CHO-\nK1 cells with CSE stimulation, revealing swift Ca²⁺ elevation and Na⁺ decrease in\nNCX1-transfected CHO-K1 cells (Revised Supplementary Fig. 4j, k). These findings\nstrongly suggest that mechanosensitive Piezo channels indeed influence\ntheinitial speed of Ca²⁺ and Na⁺ fluxes, but do not affect the sustained flux direction\nand the overall stability of the ion fluxes over time.\nWe believe this newly-added experiment successfully rules out mechanical stimulation\nas a significant artifact affecting the sustained calcium and sodium fluxes, as Piezo\ninhibition did not alter the long-term stability of the ion fluxes. This provides further\nconfidence that CSE, PMA, and fMLP triggered NCX1 activity rather than mechanical\nartifacts of Piezo channels.\nOnce again, we sincerely appreciate your thoughtful comments, which have prompted\nus to conduct these additional experiments. We believe the revised manuscript with\nresults and related details updated (lines 250-263, 686-697, 712-717, legends of\nSupplementary Fig. 4), now provides a more rigorous and scientific explanation for\nthe observed phenomena.\nRevised Supplementary Fig. 4d-f Simultaneous real-time measurements of\nintracellular Ca²⁺ (F340/380) and Na⁺ (F500/545) in GsMTx4pre-incubated\nneutrophils by fluorescent probes upon stimulation.\nRevised Supplementary Fig. 4j, k Ca²⁺and Na+influx in NCX1-transfected CHO-K1\ncells and vector controls(pre-incubated with GsMTx4)upon CSE stimulation.\n\nResponse: We greatly appreciate the reviewer’s insightful question. Cigarette smoke\n(CS) contains thousands of chemicals—including oxidants, reactive aldehydes, nicotine,\nparticulate matter, and various free radicals. The U.S. Food and Drug Administration\nestablished a list of 93 harmful and potentially harmful constituents in tobacco\nproducts4, highlighting the extreme complexity of CS composition. Given the vast\nnumber of components, verifying each component and their collaborative relationships\nwould be a massive research undertaking.\nOur study focused on investigating the key effector cell type (neutrophils) associated\ndisease mechanisms in a physiologically relevant setting—CS-induced COPD. The\nwhole-smoke exposure might better mimic the authentic pathological environment.\nNevertheless, we fully agree that determining which specific toxicants may trigger\nrapid NCX1-related responses is an important future direction. Studies using simplified\ncellular systems and selected CS constituents (e.g., acrolein, ROS donors, nicotine, or\nparticulate extracts) would indeed help clarify the direct molecular triggers. We plan to\nincorporate such mechanistic analyses in our future work to further elucidate the\ncomponent-specific effects. This has been mentioned as limitations in our Discussion\nsection (lines 447-448). Thanks again for your valuable suggestions on our work.\n\nResponse: We fully understand the reviewer’s concerns. We acknowledge that the\nmechanistic questions related to NCX1 modes are important and biologically relevant.\nWithin the current revision, we have made substantial efforts to strengthen the scientific\nrigor by adding multiple complementary experiments and analysis, includingpressure–\nvolume analysis, dynamic compliance data, ion fluxes monitoring with\nmechanosensitive channels depressed, and validation in recombinant NCX1 cellular\nexpression system. These additions reinforce the robustness of our conclusions, and\nprovide a more comprehensive picture of how NCX1 reverse mode contributes to\nCOPD pathogenesis. Nevertheless, this study did not investigate the molecular\nstructural basis underlying the transition between NCX1's reverse and forward modes.\nUnderstanding the mechanisms driving this mode switching remains an important open\nquestion in the field. We acknowledge that fully dissecting the molecular determinants\nthat drive NCX1 transition between forward and reverse modes—especially in response\nto complex CS stimuli—requires integrating more high-resolution structural biology\nwith real-time functional analyses and dedicated mechanistic approaches. This point\nhas been discussed in the limitations section of the revised manuscript (lines 447-453).\nYour suggestions have been invaluable in improving the quality and clarity of our\nmanuscript. We are greatly thankful for your time and effort in reviewing our work."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed all my concerns and provided informative new translational data to support their conclusions.", "reviewer_4": "The authors made a perfect job and addressed all my concerns with an astonishing accuracy. I recommend publication."}, "author_response": "Response: We would like to express our sincere gratitude to Reviewer #2 and Reviewer #4 for their\nthoughtful and constructive comments. We greatly appreciate the time and effort they dedicated to\nreviewing our manuscript. Their feedback was instrumental in improving the quality of our study,\nand we are pleased to hear that the revisions we made addressed their concerns. We are also grateful\nfor their positive evaluation and recommendation for publication.\nThank you again for your invaluable input."}]

Decision letter