Persistent Neutrophilic Inflammation is Associated with Delayed Toxicity of Phenylarsine Oxide in Lungs

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Sanchez, Gopikrishnan Mani, Carlin Jones, Jaroslaw W. Zmijewsli, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5100050/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Phenyl arsine oxide (PAO) is a vesicant, similar to Lewisite, a potential chemical warfare agent and an environmental contaminant. PAO-induced skin burns can trigger acute organ injury, including lungs. We have recently demonstrated that PAO burns can also has a delayed toxicity, although the specific mechanism/s remain to be determined. A single cutaneous exposure to PAO resulted in inflammatory acute lung injury at 6 and 24 hours. While acute injury subsiding by 1 week, we observed a significant airway remodeling at 10 weeks post-PAO exposure. The mechanism of prolonged PAO toxicity was associated with the influx of neutrophils that produced harmful neutrophil extracellular traps (NETs). We demonstrated that the crosstalk between NET deployments and expression of IL-33, a pro-remodeling mediator was associated with the development of peribronchial fibrosis. In summary, these results suggest that a single cutaneous exposure to PAO causes the acute inflammatory phase followed by NETs/IL-33 feed forward signaling implicated for the persistent neutrophil influx and NETs formation resulting in airway remodeling. Health sciences/Diseases/Respiratory tract diseases/Chronic obstructive pulmonary disease Health sciences/Medical research/Experimental models of disease Phenyl Arsine Oxide Arsenicals Neutrophil extracellular traps IL-33 persistent inflammation Airway remodeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lewisite, a chemical warfare agent containing organoarsenic, was developed and deployed during the World Wars I and II [ 1 ]. After the wars, more than 40 countries disposed of their Lewisite stockpiles in the sea [ 2 ]. According to a recent report, levels of lewisite and its degradation product; phenyl arsine oxide (PAO) are rising in the Baltic Sea at its munition sites [ 3 ], which is a significant concern for environmental scientists [ 4 , 5 ]. Also, the unmarked buried sites, accidental exposures and intentional unethical use of this chemical agent is among the risk factors for the exposure to lewisite, and its degradation product PAO [ 6 ]. PAO, a trivalent organic compound with vesicant properties similar to Lewisite, is a potential chemical warfare agent and an environmental contaminant. For the first time, our group published a mechanistic study in which we demonstrated that single cutaneous exposure to PAO led to acute lung injury [ 7 ]. In our follow up studies, we also found that a single skin exposure to a sub-lethal dose of PAO was sufficient to demonstrate delayed lung injury effects in 10 and 20 weeks after exposure and led to the pathogenesis of constrictive bronchiolitis (CB) in mice [ 7 ]. CB in the mice lungs was similar to the CB presented in the survivors of sulfur mustard, a similar organoarsenic vesicant CWA, attacks during the Iran-Iraq War [ 8 ]. However, the mechanism of the delayed pulmonary toxicity of cutaneous exposure to PAO is yet unknown. In this study, we aim to investigate the mechanisms of delayed pulmonary toxicity posed by cutaneous PAO exposure. In our PAO induced acute lung injury (PAO-ALI) model, we demonstrated that the acute yet the systemic injury to the lungs was triggered by neutrophil inflammation and netosis. Neutrophils have short life span, and specifically, netosis is the suicidal death of neutrophils, where the activated neutrophils spill out the chromatin DNA in the extracellular space [ 9 ]. These extracellular chromatin DNA strands are called neutrophil extracellular traps (NETs), which are rich in neutrophil elastase and MPO content and, in excess, cause tissue damage. CB is a chronic inflammatory disease of the airways, where repeated injury of airway epithelial cells leads to fibrotic constriction and/or pruning of small airways [ 10 ]. The molecular mechanism of this disease is not well studied. Interestingly, deployment-related constrictive bronchiolitis (DRCB), linked to toxic exposures such as burn pit fumes, is associated with sustained NET formation in the lungs of affected individuals [ 11 ]. Based on these important observations, we performed the time-dependent single cutaneous PAO exposure in mice to understand the inflammation and its role in delayed PAO toxicity. Results PAO exposure on the skin triggers ALI, and inflammation mitigates after a week. The histopathological analysis of lung tissues involved using hematoxylin and eosin (H&E) staining. The groups exposed to PAO at 6 hours and 24 hours post-exposure exhibited a significant increase in lung tissue cellularity. The lungs at these time points showed a higher presence of proteinaceous debris in the airspaces, indicating acute lung injury (Fig. 1 a, ALI insets). However, at 1 week mice demonstrated reduced inflammation (Fig. 1 a, DLI). The assessment of lung injury scores according to the American Thoracic Society guidelines [ 12 ] indicated significant lung injury in the 6-hour, 24-hour (p < 0.0001, control vs . PAO), and 1-week exposure samples (p < 0.001, control vs . PAO) (Fig. 1 b). However, the 10-week groups showed insignificant differences in the lung injury scores, compared to the control (Fig. 1 b). PAO exposure displays neutrophilic inflammation in the lungs. Acute lung injury is characterized by intense neutrophilic inflammation. In our study, we conducted immunohistochemistry of lung sections to detect the presence of neutrophils. Our analysis revealed a significant increase in neutrophil influx in the lungs of mice exposed to PAO at 6 hours and 24 hours post-exposure compared to the control group (Fig. 2 a). Additionally, the mice at 1 week and 10 weeks post-exposure; also displayed elevated numbers of neutrophils in the lung interstitium. We observed magnified areas of anti-NE stained neutrophils but did not find an increased number of neutrophils in the alveolar spaces upon close examination (Fig. 2 a, inset). Furthermore, whole lung lysate analysis for the expression of neutrophil elastase demonstrated increased expression in the PAO-exposed mice compared to the control group (Fig. 2 b). Densitometry analysis demonstrated that PAO exposed 6-hours and 10-weeks mice groups exhibited increased neutrophil elastase levels (Fig. 2 c), while the PAO exposed 24-hour and 1-week groups showed reduced expression levels of neutrophil elastase (Fig. 2 b-c). A single cutaneous exposure to PAO instigates persistent presence of NETs in the lungs. We analyzed the lungs of mice exposed to a single dose of PAO cutaneously for the increased neutrophilic inflammation in terms of netosis. Netosis is an inflammatory phenomenon where the activated neutrophils spill DNA traps in the extracellular space. Citrullinated histone 3 (Cit-H3) is attached to this extracellular DNA and is utilized as a netosis marker. The immunohistochemistry of the lung section for Cit-H3 demonstrated the presence of NETs in all the PAO-exposed exposure groups (Fig. 3 a). The control group had an absence of NETs. The 6-hour and 24-hour PAO-exposed mice groups demonstrated an approximately 3-fold increase in Cit-H3, as compared to controls (Fig. 3 b). The 1-week and 10-week PAO-exposed mice groups shown significantly increased levels of Cit-H3 (*p < 0.05, control vs. PAO-exposed groups) (Fig. 3 c). IL-33 is a pro-netotic 'alarmin' cytokine [ 13 ]. 6-hour, 24-hour, 1-week and 10-week PAO-exposed mice groups demonstrated significantly elevated levels of IL-33 compared to controls in their whole lung lysates (Fig. 3 d). Single cutaneous exposure to PAO induced airway remodeling The histological analysis demonstrated the presence of restrictive airways at 1-week and 10-weeks post-PAO exposure. There were changes observed in the collagen fibers, and α-SMA deposition around airways after 6 and 24 hours of cutaneous PAO exposure (Fig. 4 a,b). The 1-week PAO-exposed mice group showed increased collagen around the airways; however, no changes were observed in the thickness of α-SMA around the airway (denoted as AW in the image). The 10-week PAO-exposed mice group showed increased deposition of collagen fibers and the increased thickness of α-SMA around the airways (Fig. 4 a,b). PAO-induced IL-33; but not the pro-fibrotic SMAD3 expression in primary human airway epithelial cells To assess whether the aforementioned delayed lung injury effects directly depend on PAO toxicity, we treated human primary airway epithelial cells (BEAS-2B cells) with different concentrations of PAO (0, 20, 50, or 100 nM) for 24 hours. The representative immunoblot demonstrated concentration dependent gradual increase in the expression levels of IL-33 (Fig. 5 a-b), which were decreased at higher concentration of 100nM PAO. Furthermore, PAO treatment did not affect the expression levels of SAMD2/3 (Fig. 5 c-d). PAO-induced NETs trigger upregulation of SMAD2/3 and IL-33 in primary human airway epithelial cells. We performed dose-dependent and time-dependent studies on primary airway epithelial cells to determine the possible mechanism of induced inflammation and air-ways remodeling. The effects of NETs were assessed in the early (12 hours) and later (72 hours) time of injury, with low and high doses of NETs (100 and 200 ng/ml) on BEAS-2B cells. The treatment with NETs could induce the expression of IL-33 at 12 hours (Fig. 6 a-b). However, the 72-hours treatment demonstrated a robust increase in the expression of IL-33 (Fig. 6 c-d). Furthermore, we determined the effects of chronic treatment with NETs on the expression of the EMT signaling regulator SMAD2/3 (Fig. 6 e-f). The chronic treatment of NETs for 48 and 72 hours demonstrated increased expression levels of SMAD2/3 (Fig. 6 e-f). The EMT marker and vimentin expression were also upregulated in NET-treated BEAS-2B cells (Fig. 6 e and 6 g). Discussion The respiratory system is the most susceptible organ for arsenical-induced systemic injury. Though Lewisite was not used in the World Wars, its production, transportation, and military tests led to exposure in military personnel, and its delayed effects were manifested in years later [ 14 ]. Hence, the U.S. Department of Veterans Affairs offered healthcare aid to the veterans who were exposed to Lewisite during their service [ 14 ]. Sulfur mustard is an arsenic-based CWA which was used in Iran-Iraq War in 1980s. The follow-up clinical reports documented that 45% of the sulfur mustard survivors had delayed respiratory complications [ 15 ]. Hence, it is now established that the vesicant organoarsenicals cause delayed pulmonary injury in humans. Due to the paucity of studies in this field, we developed a novel murine model to further understand the pathological effects. Our study revealed that a single cutaneous exposure of PAO can cause ALI at 6 hours [ 16 ], and DLI at 10 weeks in mice [ 7 ]. In this chronological study, we aimed to understand single-cutaneous PAO-induced inflammation, its resolution, and the persistence of inflammation over the course of the acute and delayed effects in mice. We observed that all the PAO-exposed groups, i.e., 6, 24 hours, 1 and 10-week groups, demonstrated increased neutrophils and neutrophil elastase in the lung tissue. The role of neutrophils in the pathogenesis of ARDS/ALI is indisputable [ 17 ]. We and others have demonstrated the crucial role of NETs in different animal models of ALI [ 2 , 4 , 5 ]. It is recognized that the neutrophil numbers play a crucial role in predicting the survival of ARDS patients, as a decreased count at the 24-hours mark signifies survival. In contrast, a persistent high count indicates a grim prognosis [ 3 ]. The neutrophil-to-lymphocyte ratio is a predictor for the survival of ARDS patients. A clinical retrospective study showed that neutrophils-to-lymphocyte ratio (NLR) could predict fatal complications in ARDS patients with COVID-19. The lower NLR value was associated with a survival advantage in the ARDS patients with COVID-19 [ 18 ]. Our data demonstrate that even a non-lethal dose of PAO, when administered on the skin, led to acute lung injury within 6 hours with the increased presence of neutrophils. Interestingly, 24 hours later, there was a notable decrease in neutrophils and neutrophil elastase. These findings indicate that the reduced number of neutrophils at 24 hours post exposure aided in the survival of the mice following PAO-induced acute lung injury. NETs are generated by oxidative stress during inflammation. Our previous study demonstrated that PAO-induced netosis depended on the immediate Ca + 2 influx pathway [ 16 ], rather than the NADPH-oxidase-dependent pathway [ 19 ]. PAO is an organic arsenic compound which is cleared from the system in a few days (ATSDR)[ 20 ]. It is understood that PAO exposure-mediated netosis is short-lived and should have led to a depletion of neutrophils and NETs in the lungs after a week. However, we observed an increased number of neutrophils and NETs in the lungs of the PAO-exposed 10-week group compared to controls. Hence, we investigated the levels of IL-33, a known mediator of NETs-airway crosstalk [ 21 ]. In this chronological study, we observed the increased levels of IL-33 in the lungs throughout the 10-week post-PAO exposure. IL-33 activates NADPH oxidase to produce ROS, and this ROS production in IL-33-primed neutrophils leads to netosis [ 22 – 24 ]. These data suggest that the IL-33-mediated oxidative stress can maintain the sustained presence of NETs in cutaneous PAO-exposed mice. IL-33 is an ‘alarmin’ released due to tissue damage and lytic cell death [ 13 ]. The chromatin fibers and neutrophil elastase increase the activation and secretion of IL-33 [ 6 ]. The secretion of IL-33 in acute respiratory distress syndrome (ARDS) is associated with worse outcomes [ 25 ]. At the same time, chronic release is linked to diseases of autoimmunity [ 26 ] and airway hyperresponsiveness [ 22 , 27 ]. Given the close association between NETs and IL-33, we analyzed the expression levels of IL-33 in the lungs of mice exposed to PAO cutaneously and observed a significant increase in IL-33 levels in all the PAO-exposed groups as compared to controls, particularly highest at the 6-hour time point. PAO is a lipophilic and alkylating agent, which is very cytotoxic to the cells. PAO-mediated acute injury and cell death can cause an increase in IL-33 levels. IL-33 can perpetuate inflammation by increasing the infiltration of neutrophils and NETosis [ 22 , 26 , 28 ]. NETs exacerbated the inflammation through IL-33 [ 28 ]. At 24 hours, a decrease in PAO-mediated neutrophils, neutrophil elastase, and IL-33, was observed compared to the 6-hour time point, serving as biomarkers for the survival of mice from PAO-induced acute lung injury. However, the decreased levels of IL-33 and NETs remained significantly higher in PAO-exposed mice when compared to control mice, indicating the persistent presence of NETs leading to upregulated levels of IL-33 in the lungs, or vice-versa . It is previously known that IL-33 secretion from airway epithelial cells increases neutrophil recruitment and NETosis in airways [ 7 , 8 ]. NETs are an important factor in enhancing the bioactivity of IL-33 [ 29 ]. The proteases on NETs can cleave the full-length IL-33 into its more potent and bioactive form [ 29 ]. NETs and IL-33 complexes exert type I IFN-mediated autoimmune inflammation [ 26 ]. Additionally, TLR2, which DAMPs can activate, can induce the IL- mRNA of IL-33 in a dependent TRAF6 and IRF7-dependent manner [ 30 ]. IRF7 can be activated by self-DNA [ 31 ]. Based on these studies and our data, we postulate that NETs increase the IL-33 expression through IRF7 signaling. In addition, IL-33 induces the production of Th2 cytokines, promoting the development of Th2-related airway remodeling and inflammation [ 32 ]. As a future direction for this study, we will investigate the exact mechanisms of IL-33 and NETs interactions for the feedforward cycle of systemic and persistent inflammation in cutaneous arsenical induced airway disease. Airway remodeling includes increased airway epithelial cell injury, inflammation, increased ECM production, and pro-fibrotic EMT signaling. NETs promote pro-fibrotic signaling, and the transgenic mice model of NETs deficiency are protected from bleomycin-induced lung fibrosis [ 33 ]. Additionally, studies have demonstrated that in vitro NETs treatments trigger EMT, and TGFβ1 secretion [ 34 ]. Our results also demonstrated increased SMAD2/3 and increase in the expression of EMT markers vimentin in the BEAS-2B cells. IL-33 expression are elevated by cell damage, and chromatin binding, hence, as expected, PAO and NETs both were capable of increasing the IL-33 expression levels. Interestingly, direct PAO treatment did not increase the SMAD2/3 expression. It may be due to it’s ability to inhibit endocytosis. PAO reacts with vicinal sulfhydryl groups, forming stable ring structures, and is the inhibitor of receptor-mediated endocytosis. A report demonstrated that treatment with PAO inhibited the endocytosis of TGFβ1, which inhibited the activation of SMAD2/3 [ 9 ]. It is demonstrated that endocytosis of TGFβ1 and TGFβ1 receptors is essential for SMAD-mediated EMT signaling. Conclusions A sub-lethal dose of PAO triggers acute lung injury in the early phase through neutrophilic and NETs-mediated mechanisms. During the resolution of inflammation, levels of neutrophils and NETs are reduced but not completely eliminated. The continuous presence of the NETs-IL-33 axis may contribute to airway remodeling 10 weeks after PAO exposure. While PAO itself is unable to initiate pro-fibrotic EMT signaling in human primary airway epithelial cells, it does increase IL-33 levels. Overall, PAO-induced NETs have a direct effect on airway remodeling by increasing IL-33 and SMAD 2/3 signaling. Our data suggest that PAO and NETs-mediated increased IL-33 signaling perpetuate a feed-forward cycle of NETs generation. However, only NETs are responsible for the pro-fibrotic signaling in bronchial airway epithelial cells. The dysregulated NETs-IL-33 responses disrupt the immune response and sustain persistent neutrophil recruitment to the lungs, perpetuating the cycle of inflammation. Our model indicates that local and systemic damage to lung tissue by PAO and subsequently generated NETs are responsible for delayed lung injury. Our study suggests that NETs-targeting approaches mitigate airway remodeling in organoarsenical induced delayed lung injuries. Methods Animal Exposure to phenyl arsine oxide . All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham (UAB). PAO (MilliporeSigma, catalog P3057) exposures were performed on C57BL/6 at the University of Alabama animal facility. Mice were purchased from The Jackson Laboratory. PAO was applied once topically on the dorsal skin of mice on a 2.56 cm 2 skin area, as described [ 7 ]. Briefly, mice were anesthetized with 100 mg/kg of ketamine and 5–7 mg/kg of xylazine injection i.p., and 0.05–0.1 mg/kg buprenorphine as an analgesic. Shaved 8- to 12-week-old male and female C57BL/6 mice were treated with PAO topically (150 µg/mouse diluted in 30 µL ethanol and applied over 2.56 cm 2 skin area). Mice were sacrificed at 6 hours, 24 hours, 1 week, and 10-week time points. The lungs were harvested for further assessment as previously described [ 7 ]. ELISA for Cit-H3 and IL-33 measurements . Lungs homogenates obtained from animals of each group and assessed Cit-H3 (Cell Signal Technology,), and mouse IL-33 (Abcam) measurements in the lung tissue according to the manufacturers’ specifications. Immunoblot analysis . Whole-lung lysates were processed for immunoblotting according to standard procedures [ 16 ] using the following primary antibodies: neutrophil elastase (Cell Signaling Technology, Cat. # 90120), anti-GAPDH-HRP antibody (Proteintech, Cat. # HRP-60004), SMAD2/3 (Cell Signaling Technology, Cat. # 8685), and IL-33 (Cell Signaling Technology, Cat. # 88513S). Briefly, after electrophoresis on 4–20% gels (Bio-Rad), proteins were transferred to a PVDF membrane (Bio-Rad). Membranes were blocked, incubated with primary antibodies, and exposed to HRP-conjugated anti-rabbit or anti-mouse antibodies (MilliporeSigma, catalog AP187P, and AP130). KwickQuant-Pro imager system was used for the image acquisition, and band intensities were quantitated using ImageJ (NIH). Histopathological examination and immunohistochemistry . The H&E staining procedure was performed following the method described [ 16 ]. In summary, the lungs were fixed in 10% buffered formalin and then embedded in paraffin. Tissue sections of 5 µm thickness were obtained using a microtome (HM 325, Thermo Fisher Scientific). These sections were deparaffinized in xylene and then rehydrated. At least 3 independent tissue sections from each group underwent H&E staining and were examined for histological changes using a Keyence microscope. Immunohistochemistry of the lungs for anti-NE, anti-Cit-H3, anti-α-SMA, and anti-Collagen I was carried out as previously described. The lung section was immunostained for anti-NE (Proteintech, dilution 1:500), anti-Cit-H3 (Abcam, dilution 1:300), anti-α-SMA (Santa Cruz Technology, 1:250), and anti-Collagen I (Rockland Immunochemicals, dilution 1:500) overnight. Picro-Sirius Staining . Paraffin-embedded lung sections were deparaffinized [ 7 ] and stained for Picro-Sirius using a Picro Sirius staining kit (Abcam) as per the manufacturer's instructions. Treatments of Human Primary Airway Epithelial with NETs. The concentrated (5 times) NETs (100 ng/mL) from the supernatant of neutrophils were isolated as described [ 16 ] and incubated with BEAS-2B cells (Lonza). A total of 5 × 10 5 cells were seeded per well in the 6-well plate for various time points according to the experiments. Cell were maintained in the specialized media The cell lysate was prepared using RIPA buffer supplemented with a Protease Inhibitor cocktail (ThermoFisher) as described [ 16 ]. Statistical Analysis . Statistical analyses were performed using GraphPad Prism Software (Version 8.0). Data are presented as means ± SEM. Significance was determined using the unpaired Student's t-test (n ≥ 3 for cell culture studies; n = 3–6 mice/group). Statistical significance was as follows: (ns) non-significant, *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control group scores. Declarations Acknowledgements This work was supported by the National Institutes of Environmental Health grant R01ES035072 (RS), the U.S. Department of Defense HT9425-24-1-0304 (JWZ), Translational Program for ARDS (JWZ), and NIH/NHLBI 2R01HL139617 (JWZ). Author contributions Concept and Design: NS, RS; Development of Methodology: NS, RS; Performed Experiments: NS, RS, GM, CJ; Acquisition of Data: NS, RS, GM, CJ, JWZ; Analysis and Interpretation of Data: RS, NS. NS and RS wrote the manuscript. All authors read and reviewed the manuscript. Declaration of competing interest The authors declare that they have no competing interests. Declaration of competing interest The authors declare that they have no competing interests. Data availability Data is provided within the manuscript or supplementary information files. References Goldman L, C.G., The vesicant chemical warfare agents. . Arch Derm Syphilol, 1940. 42 : p. 123–136. J. Bełdowski, Z.K., M. Szubska, R. Turja, A.I. Bulczak, D. 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Front Immunol, 2021. 12 : p. 663303. Additional Declarations No competing interests reported. Supplementary Files WBoriginalsandmembranesuploadedversion10124.pdf Cite Share Download PDF Status: Published Journal Publication published 07 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 05 Nov, 2024 Reviews received at journal 24 Oct, 2024 Reviewers agreed at journal 24 Oct, 2024 Reviews received at journal 15 Oct, 2024 Reviewers agreed at journal 10 Oct, 2024 Reviewers invited by journal 09 Oct, 2024 Editor assigned by journal 09 Oct, 2024 Editor invited by journal 07 Oct, 2024 Submission checks completed at journal 07 Oct, 2024 First submitted to journal 16 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5100050","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":374215214,"identity":"ff1e8927-984c-4a2b-8928-b85a738af064","order_by":0,"name":"Nilda C. Sanchez","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Nilda","middleName":"C.","lastName":"Sanchez","suffix":""},{"id":374215215,"identity":"1ab1ac33-e5c5-4fc6-a47a-4075d92a4896","order_by":1,"name":"Gopikrishnan Mani","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Gopikrishnan","middleName":"","lastName":"Mani","suffix":""},{"id":374215216,"identity":"6af31675-af1b-4029-b523-9db26b42bec7","order_by":2,"name":"Carlin Jones","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Carlin","middleName":"","lastName":"Jones","suffix":""},{"id":374215217,"identity":"96c5858c-c46f-41fb-b62b-98ab20b1570f","order_by":3,"name":"Jaroslaw W. Zmijewsli","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Jaroslaw","middleName":"W.","lastName":"Zmijewsli","suffix":""},{"id":374215218,"identity":"09aba668-1a0a-4058-9c33-4e48bea5b0b6","order_by":4,"name":"Ranu Surolia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDADPmYGxgcMDAfAHAmitLAxMzAbkKgFiCSI0mLefvaY5M+2e3Js7MzPqnlq7kTzNzAfvM2DR4vMmbw0ad62YmM2Zjaz2zzHnuXOOMCWbI1PiwRDjpk0Y1tCYhszA1AL2+HcDQw8ZtJ4tfC/MQM6DKSF/Vsxzz+QFv5v+LVI5JhJ8IK18Jgx87aBbWEjoOWNsTXPuQSgX3iKJef2Af1ymM3Ycg5eh+UY3vxRliDHz39844c33+7k9rc3P7zxBo8WLICZNOWjYBSMglEwCrAAAA/vQfw5OoT3AAAAAElFTkSuQmCC","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":true,"prefix":"","firstName":"Ranu","middleName":"","lastName":"Surolia","suffix":""}],"badges":[],"createdAt":"2024-09-17 01:36:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5100050/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5100050/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-95645-z","type":"published","date":"2025-04-07T16:04:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73630263,"identity":"3c7ea622-5902-4a66-a8ca-a97c0cd75437","added_by":"auto","created_at":"2025-01-13 06:14:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1933161,"visible":true,"origin":"","legend":"\u003cp\u003eCutaneous exposure to PAO induces acute and delayed lung injury in mice. \u0026nbsp;Mice (\u003cem\u003en\u003c/em\u003e=3-5 per group) were exposed to PAO (0 or 150 mM) for 6, 24 hours, or 1 and 10-weeks. \u0026nbsp;(\u003cstrong\u003ea\u003c/strong\u003e) Upper panel indicates a representative H\u0026amp;E staining of the lung sections from indicated groups of mice, while lower panel shows enlarged regions of lungs indicated by dashed boxes. Scale bar 100 mm. (\u003cstrong\u003eb\u003c/strong\u003e) Lung Injury Score is shown. Data presented as mean ± SEM (\u003cem\u003en \u003c/em\u003e= 3-5 mice per group), ****p\u0026lt;0.0001, ANOVA, control \u003cem\u003evs\u003c/em\u003e. PAO exposure. \u003cem\u003eALI\u003c/em\u003e- acute lung injury; \u003cem\u003eDLI\u003c/em\u003e-delayed lung injury.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/25c5dc2b8fdd4255e6dd9d7b.png"},{"id":73629011,"identity":"fb754df3-b13b-4695-9b82-524ef6d9d67a","added_by":"auto","created_at":"2025-01-13 06:06:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4136304,"visible":true,"origin":"","legend":"\u003cp\u003ePAO-induced neutrophil-dependent inflammation in the lungs. (a) Representative images of lung sections show staining of neutrophil elastase (NE) in control and PAO exposure groups (n=3). Scale bar 100 μm. (b and c) Immunoblot analysis of the whole lung homogenates for NE and GAPDH. Data normalized to the loading control GAPDH. Data are presented as mean ± SEM (n = 3) *P \u0026lt;0.05, ANOVA, as compared PAO to the control group of mice.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/9bb64e1b98436b4b63b5ae04.png"},{"id":73629005,"identity":"1e312eba-c2de-4d38-a1fc-9f011eaf84af","added_by":"auto","created_at":"2025-01-13 06:06:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2225303,"visible":true,"origin":"","legend":"\u003cp\u003ePersistent NETosis in lungs of PAO-exposed mice. (\u003cstrong\u003ea\u003c/strong\u003e) Representative images shows NETosis primed neutrophils and diploid NETs using immunostaining for citrullinated histones 3 (Cit-H3) in lungs of control \u003cem\u003evs\u003c/em\u003e. PAO-treated groups of mice (\u003cem\u003en\u003c/em\u003e=3/group). Scale bar 100 mm. (\u003cstrong\u003eb-c\u003c/strong\u003e) Western blot analysis of Cit-H3 in lung homogenates from control and PAO-exposed mice \u0026nbsp;(\u003cem\u003en\u003c/em\u003e=3-5 per group). \u003cstrong\u003ed\u003c/strong\u003e) Western blot analysis of IL-33 in lung homogenates from indicated groups of mice (\u003cem\u003en\u003c/em\u003e=3-5). \u0026nbsp;All data are presented as mean ± SEM, (ns) non-significant, *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001, ANOVA, as compared to control.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/130d3f8c92caa867c482e737.png"},{"id":73629012,"identity":"ff087ebe-42fd-4e96-8877-a6150f7a1792","added_by":"auto","created_at":"2025-01-13 06:06:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6781453,"visible":true,"origin":"","legend":"\u003cp\u003eA single cutaneous exposure to PAO causes delayed airway remodeling after one or 10 weeks. (\u003cstrong\u003ea\u003c/strong\u003e) Representative images show lung sections stained with Picro Sirius, while (\u003cstrong\u003eb\u003c/strong\u003e) depicted the levels of a-SMA positive cells within the airways of PAO-treated mice. Data obtained from \u003cem\u003en\u003c/em\u003e=3 mice per group. Scale bar 100 mm. AW = airway.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/865270d7386ff92a2b9435e4.png"},{"id":73630411,"identity":"e1f6d6c7-d923-4fcb-aece-b70c74ac5899","added_by":"auto","created_at":"2025-01-13 06:22:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":596101,"visible":true,"origin":"","legend":"\u003cp\u003ePAO-induced production of IL-33 in BEAS-2B cells. (\u003cstrong\u003ea-b\u003c/strong\u003e) Immunoblot analysis of hIL-33/GAPDH in lysates from BEAS-2B cells that were treated with PAO (0, 20, 50, 100 nM) for 24 hours. (\u003cstrong\u003ec\u003c/strong\u003e) Immunoblot analysis of Smad2/3, and GAPDH in lysates from BEAS-2B cells that were treated with NETs for 24 hours. (\u003cstrong\u003ed\u003c/strong\u003e) Densitometry Smad2/3 analysis from cell treated as depicted in (c). \u0026nbsp;Data are presented as mean ± SEM (\u003cem\u003en\u003c/em\u003e=3), \u0026nbsp;*\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ANOVA, as compared control (vehicle) to PAO-treated cells.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/ffc812a799c0fc0806ea8cab.png"},{"id":73629007,"identity":"a61288ea-736a-407e-a630-19e02c4741fa","added_by":"auto","created_at":"2025-01-13 06:06:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":730182,"visible":true,"origin":"","legend":"\u003cp\u003eNETs-induced IL-33 secretion and EMT of BEAS-2B cells. (\u003cstrong\u003ea-d\u003c/strong\u003e) Immunoblot analysis of hIL-33 and GAPDH in lysates of BEAS-2B cells that were treated with NETs (0, 100 or 200 ng/ml) for 12 or 72 hours. The levels of hIL-33 is normalized to the loading control GAPDH (right panel). (\u003cstrong\u003ee-g\u003c/strong\u003e) Smad2/3, vimentin and GAPDH levels in cell lysates from untreated (control) BEAS-2B cells or after exposure to PAO for indicated time. All data are presented as mean ± SEM, (\u003cem\u003en\u003c/em\u003e=3). *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ANOVA.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/9b8abdfdbb984821a35fcd5e.png"},{"id":80559186,"identity":"e912ce04-f28c-4475-8dc3-3439a39f2316","added_by":"auto","created_at":"2025-04-14 16:18:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15667476,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/52c94b51-fc35-4a2a-b9c7-8da68605770b.pdf"},{"id":73629010,"identity":"8a03ca6c-bb84-46a7-9d10-94202da3d819","added_by":"auto","created_at":"2025-01-13 06:06:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":392514,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalsandmembranesuploadedversion10124.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5100050/v1/df633ca059bd79653ae81348.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Persistent Neutrophilic Inflammation is Associated with Delayed Toxicity of Phenylarsine Oxide in Lungs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLewisite, a chemical warfare agent containing organoarsenic, was developed and deployed during the World Wars I and II [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. After the wars, more than 40 countries disposed of their Lewisite stockpiles in the sea [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to a recent report, levels of lewisite and its degradation product; phenyl arsine oxide (PAO) are rising in the Baltic Sea at its munition sites [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which is a significant concern for environmental scientists [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Also, the unmarked buried sites, accidental exposures and intentional unethical use of this chemical agent is among the risk factors for the exposure to lewisite, and its degradation product PAO [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePAO, a trivalent organic compound with vesicant properties similar to Lewisite, is a potential chemical warfare agent and an environmental contaminant. For the first time, our group published a mechanistic study in which we demonstrated that single cutaneous exposure to PAO led to acute lung injury [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In our follow up studies, we also found that a single skin exposure to a sub-lethal dose of PAO was sufficient to demonstrate delayed lung injury effects in 10 and 20 weeks after exposure and led to the pathogenesis of constrictive bronchiolitis (CB) in mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. CB in the mice lungs was similar to the CB presented in the survivors of sulfur mustard, a similar organoarsenic vesicant CWA, attacks during the Iran-Iraq War [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the mechanism of the delayed pulmonary toxicity of cutaneous exposure to PAO is yet unknown. In this study, we aim to investigate the mechanisms of delayed pulmonary toxicity posed by cutaneous PAO exposure.\u003c/p\u003e \u003cp\u003eIn our PAO induced acute lung injury (PAO-ALI) model, we demonstrated that the acute yet the systemic injury to the lungs was triggered by neutrophil inflammation and netosis. Neutrophils have short life span, and specifically, netosis is the suicidal death of neutrophils, where the activated neutrophils spill out the chromatin DNA in the extracellular space [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These extracellular chromatin DNA strands are called neutrophil extracellular traps (NETs), which are rich in neutrophil elastase and MPO content and, in excess, cause tissue damage. CB is a chronic inflammatory disease of the airways, where repeated injury of airway epithelial cells leads to fibrotic constriction and/or pruning of small airways [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The molecular mechanism of this disease is not well studied. Interestingly, deployment-related constrictive bronchiolitis (DRCB), linked to toxic exposures such as burn pit fumes, is associated with sustained NET formation in the lungs of affected individuals [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Based on these important observations, we performed the time-dependent single cutaneous PAO exposure in mice to understand the inflammation and its role in delayed PAO toxicity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePAO exposure on the skin triggers ALI, and inflammation mitigates after a week.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe histopathological analysis of lung tissues involved using hematoxylin and eosin (H\u0026amp;E) staining. The groups exposed to PAO at 6 hours and 24 hours post-exposure exhibited a significant increase in lung tissue cellularity. The lungs at these time points showed a higher presence of proteinaceous debris in the airspaces, indicating acute lung injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, ALI insets). However, at 1 week mice demonstrated reduced inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, DLI). The assessment of lung injury scores according to the American Thoracic Society guidelines [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] indicated significant lung injury in the 6-hour, 24-hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, control \u003cem\u003evs\u003c/em\u003e. PAO), and 1-week exposure samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, control \u003cem\u003evs\u003c/em\u003e. PAO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). However, the 10-week groups showed insignificant differences in the lung injury scores, compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePAO exposure displays neutrophilic inflammation in the lungs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAcute lung injury is characterized by intense neutrophilic inflammation. In our study, we conducted immunohistochemistry of lung sections to detect the presence of neutrophils. Our analysis revealed a significant increase in neutrophil influx in the lungs of mice exposed to PAO at 6 hours and 24 hours post-exposure compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Additionally, the mice at 1 week and 10 weeks post-exposure; also displayed elevated numbers of neutrophils in the lung interstitium. We observed magnified areas of anti-NE stained neutrophils but did not find an increased number of neutrophils in the alveolar spaces upon close examination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, inset). Furthermore, whole lung lysate analysis for the expression of neutrophil elastase demonstrated increased expression in the PAO-exposed mice compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Densitometry analysis demonstrated that PAO exposed 6-hours and 10-weeks mice groups exhibited increased neutrophil elastase levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), while the PAO exposed 24-hour and 1-week groups showed reduced expression levels of neutrophil elastase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA single cutaneous exposure to PAO instigates persistent presence of NETs in the lungs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe analyzed the lungs of mice exposed to a single dose of PAO cutaneously for the increased neutrophilic inflammation in terms of netosis. Netosis is an inflammatory phenomenon where the activated neutrophils spill DNA traps in the extracellular space. Citrullinated histone 3 (Cit-H3) is attached to this extracellular DNA and is utilized as a netosis marker. The immunohistochemistry of the lung section for Cit-H3 demonstrated the presence of NETs in all the PAO-exposed exposure groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The control group had an absence of NETs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 6-hour and 24-hour PAO-exposed mice groups demonstrated an approximately 3-fold increase in Cit-H3, as compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The 1-week and 10-week PAO-exposed mice groups shown significantly increased levels of Cit-H3 (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. PAO-exposed groups) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eIL-33 is a pro-netotic 'alarmin' cytokine [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. 6-hour, 24-hour, 1-week and 10-week PAO-exposed mice groups demonstrated significantly elevated levels of IL-33 compared to controls in their whole lung lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSingle cutaneous exposure to PAO induced airway remodeling\u003c/h2\u003e \u003cp\u003eThe histological analysis demonstrated the presence of restrictive airways at 1-week and 10-weeks post-PAO exposure. There were changes observed in the collagen fibers, and α-SMA deposition around airways after 6 and 24 hours of cutaneous PAO exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). The 1-week PAO-exposed mice group showed increased collagen around the airways; however, no changes were observed in the thickness of α-SMA around the airway (denoted as AW in the image). The 10-week PAO-exposed mice group showed increased deposition of collagen fibers and the increased thickness of α-SMA around the airways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePAO-induced IL-33; but not the pro-fibrotic SMAD3 expression in primary human airway epithelial cells\u003c/h2\u003e \u003cp\u003eTo assess whether the aforementioned delayed lung injury effects directly depend on PAO toxicity, we treated human primary airway epithelial cells (BEAS-2B cells) with different concentrations of PAO (0, 20, 50, or 100 nM) for 24 hours. The representative immunoblot demonstrated concentration dependent gradual increase in the expression levels of IL-33 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b), which were decreased at higher concentration of 100nM PAO. Furthermore, PAO treatment did not affect the expression levels of SAMD2/3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePAO-induced NETs trigger upregulation of SMAD2/3 and IL-33 in primary human airway epithelial cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe performed dose-dependent and time-dependent studies on primary airway epithelial cells to determine the possible mechanism of induced inflammation and air-ways remodeling. The effects of NETs were assessed in the early (12 hours) and later (72 hours) time of injury, with low and high doses of NETs (100 and 200 ng/ml) on BEAS-2B cells. The treatment with NETs could induce the expression of IL-33 at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). However, the 72-hours treatment demonstrated a robust increase in the expression of IL-33 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we determined the effects of chronic treatment with NETs on the expression of the EMT signaling regulator SMAD2/3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-f). The chronic treatment of NETs for 48 and 72 hours demonstrated increased expression levels of SMAD2/3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-f). The EMT marker and vimentin expression were also upregulated in NET-treated BEAS-2B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe respiratory system is the most susceptible organ for arsenical-induced systemic injury. Though Lewisite was not used in the World Wars, its production, transportation, and military tests led to exposure in military personnel, and its delayed effects were manifested in years later [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Hence, the U.S. Department of Veterans Affairs offered healthcare aid to the veterans who were exposed to Lewisite during their service [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Sulfur mustard is an arsenic-based CWA which was used in Iran-Iraq War in 1980s. The follow-up clinical reports documented that 45% of the sulfur mustard survivors had delayed respiratory complications [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Hence, it is now established that the vesicant organoarsenicals cause delayed pulmonary injury in humans. Due to the paucity of studies in this field, we developed a novel murine model to further understand the pathological effects. Our study revealed that a single cutaneous exposure of PAO can cause ALI at 6 hours [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and DLI at 10 weeks in mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this chronological study, we aimed to understand single-cutaneous PAO-induced inflammation, its resolution, and the persistence of inflammation over the course of the acute and delayed effects in mice. We observed that all the PAO-exposed groups, i.e., 6, 24 hours, 1 and 10-week groups, demonstrated increased neutrophils and neutrophil elastase in the lung tissue.\u003c/p\u003e \u003cp\u003eThe role of neutrophils in the pathogenesis of ARDS/ALI is indisputable [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We and others have demonstrated the crucial role of NETs in different animal models of ALI [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is recognized that the neutrophil numbers play a crucial role in predicting the survival of ARDS patients, as a decreased count at the 24-hours mark signifies survival. In contrast, a persistent high count indicates a grim prognosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The neutrophil-to-lymphocyte ratio is a predictor for the survival of ARDS patients. A clinical retrospective study showed that neutrophils-to-lymphocyte ratio (NLR) could predict fatal complications in ARDS patients with COVID-19. The lower NLR value was associated with a survival advantage in the ARDS patients with COVID-19 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our data demonstrate that even a non-lethal dose of PAO, when administered on the skin, led to acute lung injury within 6 hours with the increased presence of neutrophils. Interestingly, 24 hours later, there was a notable decrease in neutrophils and neutrophil elastase. These findings indicate that the reduced number of neutrophils at 24 hours post exposure aided in the survival of the mice following PAO-induced acute lung injury.\u003c/p\u003e \u003cp\u003eNETs are generated by oxidative stress during inflammation. Our previous study demonstrated that PAO-induced netosis depended on the immediate Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e influx pathway [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], rather than the NADPH-oxidase-dependent pathway [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. PAO is an organic arsenic compound which is cleared from the system in a few days (ATSDR)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It is understood that PAO exposure-mediated netosis is short-lived and should have led to a depletion of neutrophils and NETs in the lungs after a week. However, we observed an increased number of neutrophils and NETs in the lungs of the PAO-exposed 10-week group compared to controls. Hence, we investigated the levels of IL-33, a known mediator of NETs-airway crosstalk [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this chronological study, we observed the increased levels of IL-33 in the lungs throughout the 10-week post-PAO exposure. IL-33 activates NADPH oxidase to produce ROS, and this ROS production in IL-33-primed neutrophils leads to netosis [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These data suggest that the IL-33-mediated oxidative stress can maintain the sustained presence of NETs in cutaneous PAO-exposed mice.\u003c/p\u003e \u003cp\u003eIL-33 is an \u0026lsquo;alarmin\u0026rsquo; released due to tissue damage and lytic cell death [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The chromatin fibers and neutrophil elastase increase the activation and secretion of IL-33 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The secretion of IL-33 in acute respiratory distress syndrome (ARDS) is associated with worse outcomes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. At the same time, chronic release is linked to diseases of autoimmunity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and airway hyperresponsiveness [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Given the close association between NETs and IL-33, we analyzed the expression levels of IL-33 in the lungs of mice exposed to PAO cutaneously and observed a significant increase in IL-33 levels in all the PAO-exposed groups as compared to controls, particularly highest at the 6-hour time point. PAO is a lipophilic and alkylating agent, which is very cytotoxic to the cells. PAO-mediated acute injury and cell death can cause an increase in IL-33 levels. IL-33 can perpetuate inflammation by increasing the infiltration of neutrophils and NETosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. NETs exacerbated the inflammation through IL-33 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. At 24 hours, a decrease in PAO-mediated neutrophils, neutrophil elastase, and IL-33, was observed compared to the 6-hour time point, serving as biomarkers for the survival of mice from PAO-induced acute lung injury. However, the decreased levels of IL-33 and NETs remained significantly higher in PAO-exposed mice when compared to control mice, indicating the persistent presence of NETs leading to upregulated levels of IL-33 in the lungs, or \u003cem\u003evice-versa\u003c/em\u003e. It is previously known that IL-33 secretion from airway epithelial cells increases neutrophil recruitment and NETosis in airways [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. NETs are an important factor in enhancing the bioactivity of IL-33 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The proteases on NETs can cleave the full-length IL-33 into its more potent and bioactive form [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. NETs and IL-33 complexes exert type I IFN-mediated autoimmune inflammation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, TLR2, which DAMPs can activate, can induce the IL- mRNA of IL-33 in a dependent TRAF6 and IRF7-dependent manner [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. IRF7 can be activated by self-DNA [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Based on these studies and our data, we postulate that NETs increase the IL-33 expression through IRF7 signaling. In addition, IL-33 induces the production of Th2 cytokines, promoting the development of Th2-related airway remodeling and inflammation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. As a future direction for this study, we will investigate the exact mechanisms of IL-33 and NETs interactions for the feedforward cycle of systemic and persistent inflammation in cutaneous arsenical induced airway disease.\u003c/p\u003e \u003cp\u003eAirway remodeling includes increased airway epithelial cell injury, inflammation, increased ECM production, and pro-fibrotic EMT signaling. NETs promote pro-fibrotic signaling, and the transgenic mice model of NETs deficiency are protected from bleomycin-induced lung fibrosis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, studies have demonstrated that in vitro NETs treatments trigger EMT, and TGFβ1 secretion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our results also demonstrated increased SMAD2/3 and increase in the expression of EMT markers vimentin in the BEAS-2B cells. IL-33 expression are elevated by cell damage, and chromatin binding, hence, as expected, PAO and NETs both were capable of increasing the IL-33 expression levels. Interestingly, direct PAO treatment did not increase the SMAD2/3 expression. It may be due to it\u0026rsquo;s ability to inhibit endocytosis. PAO reacts with vicinal sulfhydryl groups, forming stable ring structures, and is the inhibitor of receptor-mediated endocytosis. A report demonstrated that treatment with PAO inhibited the endocytosis of TGFβ1, which inhibited the activation of SMAD2/3 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is demonstrated that endocytosis of TGFβ1 and TGFβ1 receptors is essential for SMAD-mediated EMT signaling.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA sub-lethal dose of PAO triggers acute lung injury in the early phase through neutrophilic and NETs-mediated mechanisms. During the resolution of inflammation, levels of neutrophils and NETs are reduced but not completely eliminated. The continuous presence of the NETs-IL-33 axis may contribute to airway remodeling 10 weeks after PAO exposure. While PAO itself is unable to initiate pro-fibrotic EMT signaling in human primary airway epithelial cells, it does increase IL-33 levels. Overall, PAO-induced NETs have a direct effect on airway remodeling by increasing IL-33 and SMAD 2/3 signaling. Our data suggest that PAO and NETs-mediated increased IL-33 signaling perpetuate a feed-forward cycle of NETs generation. However, only NETs are responsible for the pro-fibrotic signaling in bronchial airway epithelial cells. The dysregulated NETs-IL-33 responses disrupt the immune response and sustain persistent neutrophil recruitment to the lungs, perpetuating the cycle of inflammation. Our model indicates that local and systemic damage to lung tissue by PAO and subsequently generated NETs are responsible for delayed lung injury. Our study suggests that NETs-targeting approaches mitigate airway remodeling in organoarsenical induced delayed lung injuries.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eAnimal Exposure to phenyl arsine oxide\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham (UAB). PAO (MilliporeSigma, catalog P3057) exposures were performed on C57BL/6 at the University of Alabama animal facility. Mice were purchased from The Jackson Laboratory. PAO was applied once topically on the dorsal skin of mice on a 2.56 cm\u003csup\u003e2\u003c/sup\u003e skin area, as described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, mice were anesthetized with 100 mg/kg of ketamine and 5\u0026ndash;7 mg/kg of xylazine injection i.p., and 0.05\u0026ndash;0.1 mg/kg buprenorphine as an analgesic. Shaved 8- to 12-week-old male and female C57BL/6 mice were treated with PAO topically (150 \u0026micro;g/mouse diluted in 30 \u0026micro;L ethanol and applied over 2.56 cm\u003csup\u003e2\u003c/sup\u003e skin area). Mice were sacrificed at 6 hours, 24 hours, 1 week, and 10-week time points. The lungs were harvested for further assessment as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eELISA for Cit-H3 and IL-33 measurements\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eLungs homogenates obtained from animals of each group and assessed Cit-H3 (Cell Signal Technology,), and mouse IL-33 (Abcam) measurements in the lung tissue according to the manufacturers\u0026rsquo; specifications.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoblot analysis\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWhole-lung lysates were processed for immunoblotting according to standard procedures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] using the following primary antibodies: neutrophil elastase (Cell Signaling Technology, Cat. # 90120), anti-GAPDH-HRP antibody (Proteintech, Cat. # HRP-60004), SMAD2/3 (Cell Signaling Technology, Cat. # 8685), and IL-33 (Cell Signaling Technology, Cat. # 88513S). Briefly, after electrophoresis on 4\u0026ndash;20% gels (Bio-Rad), proteins were transferred to a PVDF membrane (Bio-Rad). Membranes were blocked, incubated with primary antibodies, and exposed to HRP-conjugated anti-rabbit or anti-mouse antibodies (MilliporeSigma, catalog AP187P, and AP130). KwickQuant-Pro imager system was used for the image acquisition, and band intensities were quantitated using ImageJ (NIH).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistopathological examination and immunohistochemistry\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe H\u0026amp;E staining procedure was performed following the method described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In summary, the lungs were fixed in 10% buffered formalin and then embedded in paraffin. Tissue sections of 5 \u0026micro;m thickness were obtained using a microtome (HM 325, Thermo Fisher Scientific). These sections were deparaffinized in xylene and then rehydrated. At least 3 independent tissue sections from each group underwent H\u0026amp;E staining and were examined for histological changes using a Keyence microscope.\u003c/p\u003e \u003cp\u003eImmunohistochemistry of the lungs for anti-NE, anti-Cit-H3, anti-α-SMA, and anti-Collagen I was carried out as previously described. The lung section was immunostained for anti-NE (Proteintech, dilution 1:500), anti-Cit-H3 (Abcam, dilution 1:300), anti-α-SMA (Santa Cruz Technology, 1:250), and anti-Collagen I (Rockland Immunochemicals, dilution 1:500) overnight.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePicro-Sirius Staining\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eParaffin-embedded lung sections were deparaffinized [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and stained for Picro-Sirius using a Picro Sirius staining kit (Abcam) as per the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTreatments of Human Primary Airway Epithelial with NETs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe concentrated (5 times) NETs (100 ng/mL) from the supernatant of neutrophils were isolated as described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and incubated with BEAS-2B cells (Lonza). A total of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were seeded per well in the 6-well plate for various time points according to the experiments. Cell were maintained in the specialized media The cell lysate was prepared using RIPA buffer supplemented with a Protease Inhibitor cocktail (ThermoFisher) as described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism Software (Version 8.0). Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Significance was determined using the unpaired Student's t-test (n\u0026thinsp;\u0026ge;\u0026thinsp;3 for cell culture studies; n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6 mice/group). Statistical significance was as follows: (ns) non-significant, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to control group scores.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Institutes of Environmental Health grant R01ES035072 (RS),\u0026nbsp;the U.S. Department of Defense HT9425-24-1-0304 (JWZ), Translational Program for ARDS (JWZ), and NIH/NHLBI 2R01HL139617 (JWZ). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConcept and Design: NS, RS; Development of Methodology: NS, RS; Performed Experiments: NS, RS, GM, CJ; Acquisition of Data: NS, RS, GM, CJ, JWZ; Analysis and Interpretation of Data: RS, NS. NS and RS wrote the manuscript. All authors read and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGoldman L, C.G., \u003cem\u003eThe vesicant chemical warfare agents. .\u003c/em\u003e Arch Derm Syphilol, 1940. \u003cstrong\u003e42\u003c/strong\u003e: p. 123\u0026ndash;136.\u003c/li\u003e\n\u003cli\u003eJ. Bełdowski, Z.K., M. Szubska, R. Turja, A.I. Bulczak, D. Rak, M. Brenner, T. Lang, L. Kotwicki, K. 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4375-87.\u003c/li\u003e\n\u003cli\u003eSuzuki, M., et al., \u003cem\u003ePAD4 Deficiency Improves Bleomycin-induced Neutrophil Extracellular Traps and Fibrosis in Mouse Lung.\u003c/em\u003e Am J Respir Cell Mol Biol, 2020. \u003cstrong\u003e63\u003c/strong\u003e(6): p. 806-818.\u003c/li\u003e\n\u003cli\u003ePandolfi, L., et al., \u003cem\u003eNeutrophil Extracellular Traps Induce the Epithelial-Mesenchymal Transition: Implications in Post-COVID-19 Fibrosis.\u003c/em\u003e Front Immunol, 2021. \u003cstrong\u003e12\u003c/strong\u003e: p. 663303.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phenyl Arsine Oxide, Arsenicals, Neutrophil extracellular traps, IL-33, persistent inflammation, Airway remodeling","lastPublishedDoi":"10.21203/rs.3.rs-5100050/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5100050/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhenyl arsine oxide (PAO) is a vesicant, similar to Lewisite, a potential chemical warfare agent and an environmental contaminant. PAO-induced skin burns can trigger acute organ injury, including lungs. We have recently demonstrated that PAO burns can also has a delayed toxicity, although the specific mechanism/s remain to be determined. A single cutaneous exposure to PAO resulted in inflammatory acute lung injury at 6 and 24 hours. While acute injury subsiding by 1 week, we observed a significant airway remodeling at 10 weeks post-PAO exposure. The mechanism of prolonged PAO toxicity was associated with the influx of neutrophils that produced harmful neutrophil extracellular traps (NETs). We demonstrated that the crosstalk between NET deployments and expression of IL-33, a pro-remodeling mediator was associated with the development of peribronchial fibrosis. In summary, these results suggest that a single cutaneous exposure to PAO causes the acute inflammatory phase followed by NETs/IL-33 feed forward signaling implicated for the persistent neutrophil influx and NETs formation resulting in airway remodeling.\u003c/p\u003e","manuscriptTitle":"Persistent Neutrophilic Inflammation is Associated with Delayed Toxicity of Phenylarsine Oxide in Lungs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 06:06:15","doi":"10.21203/rs.3.rs-5100050/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-05T07:18:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-24T17:54:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106797625178922406615290230069192206564","date":"2024-10-24T10:32:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-15T10:04:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189708615570021865505150786986560524029","date":"2024-10-10T06:59:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-09T19:45:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-09T19:40:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-08T03:30:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-07T04:05:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-17T01:33:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"35837d2d-d83b-4f63-a2c0-3eeb39d05ac0","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39828836,"name":"Health sciences/Diseases/Respiratory tract diseases/Chronic obstructive pulmonary disease"},{"id":39828837,"name":"Health sciences/Medical research/Experimental models of disease"}],"tags":[],"updatedAt":"2025-04-14T16:16:44+00:00","versionOfRecord":{"articleIdentity":"rs-5100050","link":"https://doi.org/10.1038/s41598-025-95645-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-07 16:04:50","publishedOnDateReadable":"April 7th, 2025"},"versionCreatedAt":"2025-01-13 06:06:15","video":"","vorDoi":"10.1038/s41598-025-95645-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-95645-z","workflowStages":[]},"version":"v1","identity":"rs-5100050","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5100050","identity":"rs-5100050","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}