Regulation mechanism of IL-36 by neutrophil extracellular trapping in a mice of sepsis-induced ARDS

Regulation mechanism of IL-36 by neutrophil extracellular trapping in a mice of sepsis-induced ARDS | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Regulation mechanism of IL-36 by neutrophil extracellular trapping in a mice of sepsis-induced ARDS Haiyan Hu, Lifen Wang, Fu Jin, shengqin Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7688507/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Sepsis-induced acute respiratory distress syndrome (ARDS) is characterized by uncontrolled pulmonary inflammation and neutrophil-driven pathology. Neutrophil extracellular traps (NETs) have been implicated in ARDS progression, but the role of interleukin-36 (IL-36) in this process remains unclear. This study aimed to investigate the regulatory mechanism of IL-36 via NETs and its impact on NF-κB activation in a murine model of sepsis-induced ARDS. Methods A lipopolysaccharide (LPS)-induced ARDS model was established in C57BL/6 mice. Animals were divided into Control, ARDS, ARDS + IL-36, and ARDS + IL-36Ra groups. Pulmonary edema was assessed via wet/dry weight ratio, inflammatory cytokines were measured by ELISA, and histopathological changes were evaluated through H&E staining. NETs formation was analyzed using immunofluorescence and Western blot. NF-κB activation was detected via phosphorylation of p65. Results IL-36 administration exacerbated pulmonary edema, inflammatory cytokine levels (TNF-α, IL-10, MPO), and histopathological injury. Conversely, IL-36 receptor antagonist (IL-36Ra) attenuated these effects. IL-36 promoted NETs formation, as indicated by elevated citrullinated histone H3 (CitH3) and neutrophil elastase (NE) expression. NETs further enhanced IL-36-induced proinflammatory cytokine release and NF-κB activation in bronchial epithelial cells. IL-36Ra partially reversed NET-mediated NF-κB phosphorylation and inflammatory responses. Conclusions IL-36 exacerbates sepsis-induced ARDS by enhancing NETs formation and activating the NF-κB pathway, leading to amplified pulmonary inflammation and injury. Targeting IL-36 signaling may represent a therapeutic strategy to mitigate ARDS progression. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Health sciences/Pathogenesis Sepsis ARDS IL-36 NETs NF-κB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Acute respiratory distress syndrome (ARDS) is an acute-onset respiratory disorder characterized by non-cardiogenic bilateral pulmonary edema and hypoxemia, resulting from compromised pulmonary endothelial barrier integrity and excessive alveolar-capillary permeability. The pathological hallmarks include diffuse pulmonary inflammation with substantial infiltration of inflammatory cells, predominantly neutrophils and macrophages 1 , 2 ,Clinically, ARDS manifests as bilateral pulmonary infiltrates and respiratory failure 3 ,with severity classified as mild, moderate, or severe based on oxygenation parameters 4 . Epidemiological studies indicate that ARDS occurs in 10% of ICU admissions and 23% of mechanically ventilated patients, with a 28-day mortality rate of 35% that exceeds 40% in severe cases 5 .Sepsis represents the most common predisposing factor for ARDS development, with septic ARDS maintaining mortality rates between 34–45% 5,6 ,During ARDS progression, neutrophil adhesion, aggregation and activation drive inflammatory cascades through excessive neutrophil extracellular trap (NETs) formation 7 .Elevated NETs levels have been consistently documented in sepsis-induced ARDS 8 – 13 .Notably, NETs demonstrate significant correlations with alveolar epithelial/endothelial damage severity and inflammatory mediator concentrations 7 , 14 , while concurrently releasing proinflammatory cytokines such as IL-1β that exacerbate disease progression 15 – 17 ,However, the precise activation mechanisms underlying these processes remain elusive. Emerging evidence highlights IL-36/IL-36R signaling as a critical modulator of pulmonary inflammation and immune responses, with constitutive expression throughout lung tissue 18 – 20 。Bronchial epithelial cells upregulate IL-36 expression following exposure to proinflammatory cytokines and microbial molecular patterns 21 .Experimental models demonstrate that intratracheal IL-36α/γ administration induces proinflammatory cytokine/chemokine production and neutrophil recruitment 22 。The mechanism of IL-36 signaling in sepsis-induced lung inflammation remains unclear. We hypothesize that neutrophil protease-triggered IL-36 activation initiates NF-κB signaling and may regulate septic ARDS via NETs. 2 Materials and methods 2.1 Animals Male C57BL/6 mice (n = 25; aged 6–8 weeks; body weight 22–26 g) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). Animals were maintained under specific pathogen-free (SPF) conditions with controlled temperature (25 ± 1°C), relative humidity (50 ± 10%), and 12:12 h light-dark cycles, with ad libitum access to autoclaved feed and sterile water. All ARDS-related procedures strictly adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yangzhou University Institutional Animal Care and Use Committee (IACUC Approval No. 202406021). 2.2 Sepsis model Mice were randomly divided into Control, ARDS, ARDS + IL-36, ARDS + IL-36 Ra .To induce LPS-induced ARDS, All the mice were anaesthetized using a mixture of ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg). Instilled into the trachea was performed with LPS ((10mg/kg, diluted to 100ul with PBS,Beyotime,China),To facilitate LPS distribution throughout the lungs, the mice were mechanically ventilated using a 20-gauge tracheal intubation and animal ventilator (ALCBIO, China) in volumetric control mode. The mice in the PBS group were given the same amount of PBS without LPS by intratracheal instillation in the same way.The ARDS + IL-36Ra group intradermal injection of 1µg (diluted to 20 µL PBS) of IL-36 receptor antagonist(IL-1F5,NO. HY-P77007,MCE,USA) 24 hours before LPS challenge;The following parameters were applied: 7 mL/kg body weight, 120 breaths/minute, positive end-expiratory pressure (PEEP) of 2 cm H 2 O for 10 minutes. Following ventilation, the mice were monitored for the subsequent 24 hours in an animal care facility.All mice were sacrificed 24 h later. 2.3 Pulmonary Edema Quantification Lung edema was evaluated using the wet-to-dry weight ratio method. Fresh right lung tissues were weighed for wet weight and desiccated at 80°C under vacuum until constant dry weight was attained. Triplicate biological replicates were measured using an analytical balance (± 0.1 mg precision). Th lung wet/dry ratio was calculated as (W/D) × 100%, effectively eliminating interference from residual blood in tissue hydration assessment. 2.4 Histopathological Evaluation of Lung Tissues Left lungs fixed in 4% paraformaldehyde were paraffin-embedded and sectioned into 5 µm slices for H&E staining. A blinded semi-quantitative scoring system was applied by two independent pathologists to evaluate four parameters: alveolar congestion, leukocyte infiltration, septal thickening, and intra-alveolar hemorrhage. Microscopic images were acquired using a Nikon Eclipse Ni-U system with DS-Ri2 camera, enabling structural validation. 2.5 Neutrophil Isolation and NETs Analysis Human fresh anticoagulated blood was combined with saline and sedimentation solution (No. R1010, Solarbio, China) in a 1:1:1 ratio to facilitate the removal of erythrocytes. After sedimentation, the leukocyte-rich supernatant was layered onto a density gradient medium (Reagent A/C) and centrifuged (600–1000 × g, 25–30 min) to isolate neutrophils (NO. P9040,Solarbio, China). Cells were washed, resuspended in RPMI 1640 medium, and stimulated with 100 nM PMA (37°C, 5% CO₂, 4 h) to induce NETs formation. The NETs layer was collected, homogenized, and centrifuged to remove debris. DNA content was quantified using the Quant-iT™ PicoGreen® assay(NO.P11496,Thermo, USA),Fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. 2.6 NETs Viability by Trypan Blue Exclusion Isolated NETs were assessed for viability using trypan blue exclusion. A 4% trypan blue stock was prepared by dissolving 4 g powder in distilled water, grinding, adjusting to 100 mL with ddH, filtering, and storing at 4°C. The stock was diluted to 0.4% with PBS before use. Cells were trypsinized to a single-cell suspension, diluted appropriately, and mixed with 0.4% trypan blue at a 9:1 ratio. Viable (unstained) and non-viable (blue-stained) cells were counted within 3 min using a haemocytometer. 2.7 Cell Culture and Treatment NETs incubated with Cathepsin G inhibitor (5 µM) or elastase inhibitor (5 µM) for 30 min. Subsequently, IL-36α, IL-36β, or IL-36γ (50 nM) in 1 mL HBSS/0.25% BSA was added to NETs and incubated at 37°C for 30 min. The resulting IL-36-containing supernatants and isolated NETs were collected for subsequent treatments. Neutrophils cells were treated for 30 min under the following conditions: control,NETs, NETs + IL-36,NETs + IL-36Ra. 2.8 Enzyme-Linked Immunosorbent Assay (ELISA) Whole blood collected via orbital bleeding from mice was clotted (RT, 1–2 h), centrifuged (3,000 × g, 10 min), and serum stored at -80°C for subsequent analysis. Post-bleeding, bronchoalveolar lavage fluid (BALF) was obtained by saline instillation (3 × 3 mL), centrifuged (3,000 × g, 15 min, 4°C), and the supernatant analyzed for total protein (BCA assay) and cytokines. Serum and BALF levels of MPO (Mlbio ml002070), TNF-α (Mlbio ml002095), and IL-10 (Mlbio ml037873) were quantified by ELISA using analyte-specific sample dilutions. Separately, Neutrophils cells were cultured in RPMI 1640/10% FBS; PMA-stimulated neutrophils, pretreated with Cathepsin G/Elastase inhibitors (5 µM), were incubated with IL-36 isoforms (50 nM), and the resultant supernatants applied to NETs to assess TNF-α and IL-10 production by ELISA. 2.8 Immunofluorescence Staining NETs were seeded onto glass-bottomed 12-well plates (NEST) at 40–50% confluency and cultured for 24 h (37°C, 5% CO₂). Cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 20 min at 25°C, permeabilized with 1% Triton X-100 (Sigma-Aldrich Germany) for 5 min, and blocked with 5% goat serum (Gibco) for 30 min. Primary antibodies targeting citrullinated histone H3 (H3Cit, ab5103, Abcam; 1:200) and myeloperoxidase (MPO, ab90810, Abcam; 1:100) were applied overnight at 4°C. After three PBS washes, slides were incubated with species-specific secondary antibodies: Goat anti-Mouse IgG-Alexa Fluor® 647 (NO:ab150115, Abcam; 1:200) and Goat anti-Rabbit IgG-Alexa Fluor® 488 (NO:ab150077, Abcam; 1:200) for 1 h at 25°C protected from light. Nuclei were counterstained with 1 µg/mL DAPI (Sigma-Aldrich) for 5 min. Images were acquired using a Leica TCS SP8 confocal microscope with 63× oil immersion objective (LAS X software), and fluorescence intensity was quantified using Image J . 2.9 Cell Viability Assays(CCK-8) Neutrophils cells were treated with NETs, IL-36, or IL-36Ra for 30 minutes.After 24 hours, 10 µL of CCK-8 reagent(NO:C0037,Beyotime, China,)was added to each well.The plate was incubated for 2 hours Absorbance was measured at 450 nm using a microplate reader. 2.10 Western Blot NE, CitH3, p-p65 NF-κB, and p65 NF-κB expression was analyzed. Protein extraction: Lung tissue was homogenized in lysis buffer,and centrifuged (12,000 rpm, 4°C, 10 min). Bronchial epithelial cells were lysed in RIPA buffer with protease inhibitors, scraped, and centrifuged. Protein concentration was determined by BCA assay. SDS-PAGE: Samples (25 µg/lane) were denatured, loaded onto 10% gels, and electrophoresed. Proteins were transferred to PVDF membranes at 65 V for 2 h. Membranes were blocked (1 h, RT), then incubated overnight at 4°C with:NE (Affinity, NO:AF0010; 1:500),CitH3 (Abcam, NO:ab5103; 1 µg/mL),p-p65 NF-κB (Affinity, NO:AF2006; 1:500),p65 NF-κB ( Affinity,NO: AF5006; 1:500),GAPDH ( Abcam, NO:ab245355).After TBST washes, membranes were incubated with secondary antibodies:Goat anti-Mouse IgG H&L (Alexa Fluor® 647; Abcam, NO:ab150115; 1:2,000) for MPO,Goat anti-Rabbit IgG H&L (Alexa Fluor® 488; Abcam,NO: ab150077; 1:2,000) for other targets(60 min, RT, dark). Detection: ECL reagent was applied, membranes exposed to film, and imaged. Quantification: Band intensity ratios (target/GAPDH) were analyzed. 3 Results 3.1 IL-36 exacerbates pulmonary edema, inflammation, and injury in mice ARDS. The lung wet-to-dry weight ratio exhibited a progressive elevation across the experimental groups. The control group demonstrated the lowest value (2.00 ± 0.24), followed by the ARDS with IL-36Ra group (2.58 ± 0.14), then the ARDS group (3.11 ± 0.21), with the ARDS with IL-36 group showing the highest ratio (4.36 ± 0.24) (Fig. 1 A). This escalating pulmonary edema correlated with a commensurate increase in inflammatory mediators. The levels of myeloperoxidase (MPO), TNF-α, and IL-10 in both bronchoalveolar lavage fluid (BALF Fig. 1 B) and serum(Fig. 1 C) displayed an identical ascending order from control to ARDS with IL-36Ra to ARDS to ARDS with IL-36, with all pairwise comparisons being statistically significant ( p < 0.05). Consequently, IL-36 receptor blockade via IL-36Ra mitigated pathological injury, while exogenous IL-36 administration exacerbated ARDS manifestations in a dose-dependent manner, highlighting the central pathogenic role of the IL-36 pathway. 3.2 Histopathological assessment reveals IL-36 exacerbates lung injury. H&E staining demonstrated progressive histological damage across groups (Fig. 2 ). Control lungs exhibited intact alveolar architecture, absent hemorrhage, edema, or inflammatory infiltration. Both ARDS + IL-36Ra and ARDS groups displayed mild interstitial edema, minimal alveolar wall thickening, and sparse inflammatory cell infiltration. In contrast, ARDS + IL-36 mice manifested severe pathology: alveolar collapse, pronounced interstitial edema, marked alveolar wall thickening, and robust inflammatory cell (Fig. 2 ). 3.3 IL-36 drives NETs formation and neutrophilic inflammation in ARDS lungs. Western blot analysis demonstrated a progressive increase in the expression of neutrophil elastase (NE) and citrullinated histone H3 (CitH3)-both established markers of NETosis-across the experimental groups, with the lowest expression in the Control group, followed by the ARDS + IL-36Ra group, then the ARDS group, and the highest expression in the ARDS + IL-36 group. This IL-36-dependent upregulation of NETosis-associated proteins correlated with graded elevations in myeloperoxidase (MPO), pulmonary edema, and proinflammatory cytokine levels, suggesting that dysregulated neutrophil activation represents a central mechanism underlying IL-36-aggravated ARDS (Fig. 3 ) 3.4 IL-36 potentiates neutrophil NETosis, driving ARDS pathology. After isolation of human neutrophils using a commercial kit and assessment of viability by trypan blue exclusion, we confirmed that cell viability exceeded 98% (Fig. 4 A). Viable neutrophils primed with IL-36 exhibited an enhanced responsiveness to PMA stimulation. Immunofluorescence analysis demonstrated increased co-localization of MPO and CitH3, indicative of elevated neutrophil extracellular trap (NETs) formation, compared with control conditions. These results provide mechanistic evidence linking IL-36 to enhanced neutrophil-driven inflammatory responses in ARDS(Fig. 4 B,C). 3.5 NET-associated IL-36 signaling drives epithelial cytotoxicity while NETs proteases amplify IL-36-dependent inflammation. CCK-8 assays showed reduced 24-hour cell viability, with the control exhibiting the highest viability, followed by NETs, NETs + IL-36Ra, and NETs + IL-36(Fig. 5A).ELISA of supernatants from IL-36α/β/γ-stimulated cells indicated that PMA most strongly enhanced TNF-α and IL-10 release, followed by PMA with elastase, then PMA with cathepsin G, and lastly the control(Fig. 5B).Similarly, NET-associated proteases hierarchically enhanced PMA-induced cytokine production: lowest in control, higher with NETs, further elevated with NETs + IL-36Ra, and highest with NETs + IL-36(Fig. 5C.)These findings suggest NETs form a self-amplifying inflammatory circuit whereby proteases increase IL-36 expression, and IL-36 augments NET-mediated cytotoxicity. 3.6 NETs trigger sustained NF-κB activation in bronchial epithelium via IL-36 signaling. Western blot analysis of nuclear fractions demonstrated a progressive increase in p65 NF-κB phosphorylation across experimental conditions: the lowest p-p65/p65 ratio was observed in the control group, followed by NETs-treated samples, then NETs co-treated with IL-36Ra, and finally NETs with IL-36 showing the highest phosphorylation level(Fig. 6 ). This IL-36-mediated amplification of p65 phosphorylation identifies NF-κB as a central transcriptional mechanism underlying NET-induced epithelial inflammation. Notably, IL-36Ra attenuated but did not completely abolish NF-κB activation. 4 Discussion IL-36 as a member of the IL-1 cytokine family, is widely expressed in various tissues and organs such as the lungs, skin, and intestine and plays a critical role in inflammation and immune regulation 19 , 20 , 23 , 24 . Studies have shown that IL-36 is involved in the pathogenesis of multiple inflammatory diseases. In inflammatory bowel disease, IL-36 promotes disease progression by inducing the production of CXC chemokines 25 . In generalized pustular psoriasis (GPP), aberrant activation of IL-1 and IL-36 mediates the expression of neutrophil chemotactic factors, inflammatory cell infiltration, and pustule formation 26 . In chronic rhinosinusitis, IL-36 is regulated by TLR signaling, leading to impaired endothelial barrier function and exacerbation of the inflammatory response 27 . In patients with Pseudomonas aeruginosa-induced ARDS, levels of IL-36γin plasma and BALF were significantly higher than those in healthy controls 20 , confirming that IL-36 exacerbates inflammatory symptoms in ARDS. Additionally, abnormal activation of the IL-36 signaling pathway has been closely associated with various inflammatory diseases 28 . IL-36αpromotes the release of pro-inflammatory mediators and T-cell proliferation by activating the NF-κB signaling pathway in macrophages 22 . As a key upstream regulator, IL-36αenhances the activation of neutrophils, macrophages, and fibroblasts 29 , 30 . Further studies in Jurkat T cells demonstrated that IL-36 induces mitochondrial activation of MAPK and nuclear factor kappa-B (NF-κB) pathways, aggravating inflammatory responses 31 , 32 . Upon binding of IL-36α,βorγto their specific receptor-interleukin-1 receptor-related protein 2 (IL-1Rrp2)-the IL-36 signaling pathway is initiated. This process subsequently recruits the co-receptor IL-1 receptor accessory protein (IL-1RAcP) and activates the NF-κB signaling pathway, thereby driving extensive transcription and secretion of various pro-inflammatory cytokines 33 , 34 However, the precise mechanisms of action remain incompletely understood.This study demonstrates that in a sepsis-induced ARDS model, elevated IL-36 expression is closely associated with aggravated pulmonary inflammation, consistent with previous reports. Specifically, there was a significant increase in the lung wet/dry (W/D) ratio, exacerbated histopathological injury, and markedly elevated levels of inflammatory cytokines (including IL-10 and TNF-α) and MPO in serum and BALF, along with significantly increased expression of corresponding inflammatory proteins in tissues. These results suggest that in sepsis-associated ARDS, IL-36 may exert a pro-inflammatory role potentially through activation of the NF-κB signaling pathway. During the onset of ARDS, large numbers of neutrophils are recruited to lung tissue and generate neutrophil extracellular traps (NETs) 35 . NETs are complexes capable of trapping pathogens, primarily composed of nuclear chromatin, mitochondrial DNA, and neutrophil granular proteins 36 . They immobilize and eliminate invading pathogens, exert antimicrobial effects, and promote inflammation resolution, constituting an innate immune response that plays a crucial role in host defense 37 . However, excessive NET formation can damage vascular endothelial cells, promote thrombosis, cause microvascular dysfunction, and injure pulmonary cells, playing a significant role in sepsis-induced ARDS 38 , 39 . In this study, NETs were induced in neutrophils by PMA stimulation, and their activity was verified using trypan blue staining, establishing an in vitro ARDS model. Fluorescence staining revealed that IL-36 downregulated the expression of NET markers MPO and CitH3, significantly reducing NET activity. Administration of a blocking agent notably restored NET activity. Furthermore, it was observed that IL-36 (α/β/γ) increased the expression of inflammatory cytokines IL-10 and TNF-α, and elevated levels of these cytokines were also detected in the cell supernatant, further confirming that IL-36 induces NETs to secrete substantial pro-inflammatory factors. The NF-κB signaling pathway is a key transcription factor extensively involved in various biological processes including cellular regulation, inflammatory responses, and immune reactions 40 . Research indicates that the NF-κB pathway plays an important role in the formation of NETs 41 Neutrophils contain dimers composed of NF-κB1 (p50) and p65 NF-κB, and cellular stimulation can trigger nuclear translocation of NF-κB/Rel proteins and degradation of IκB-α 42 Studies have shown that disruption of NETs can promote apoptosis in gastric cancer cells by modulating the expression of Bcl-2, Bax, and NF-κB 43 . Conversely, NETs can also enhance NF-κB activation, thereby facilitating the progression and metastasis of breast tumors 44 . NETs have also been reported to participate in various pathophysiological processes such as wound healing, atherosclerosis, and acute lung injury 45 – 48 . This study found that in NET-induced ARDS, IL-36 led to increased protein expression of NF-κB, suggesting that IL-36 may regulate the NF-κB signaling pathway via NETs, activating NF-κB to promote massive secretion of inflammatory factors and resulting in pulmonary injury in ARDS. In summary, the findings of this study indicate that in sepsis-induced ARDS, IL-36 recruits large numbers of neutrophils via NETs, leading to substantial secretion of inflammatory factors and aggravated pulmonary inflammatory injury, while simultaneously activating the NF-κB signaling pathway to further promote extensive release of inflammatory mediators. However, several notable limitations must be acknowledged, including a limited sample size and the absence of independent clinical validation. Furthermore, long-term outcome data are currently unavailable. Future studies should prioritize mechanistic investigations—such as utilizing IL-36 knockout models-alongside clinical translation trials and comprehensive long-term safety assessments. Declarations Acknowledgements Not applicable. Author contributions Shenqin Li conceived and designed the study. Haiyan Hu, FuJin and Lifen Wang performed experiments. Haiyan Hu analyzed data, interpreted results, and wrote the manuscript. Shenqin Li reviewed and revised the manuscript and supervised the study. Funding This work was supported by the Medical and Health Science and Technology Project of Zhejiang Province (Grant No. 2024KY1420,2023KY468) Declarations Ethics approval and consent to participate All ARDS-related procedures strictly adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yangzhou University Institutional Animal Care and Use Committee (IACUC Approval No. 202406021). Consent for publication Not applicable. 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The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 396 , 77-80, doi:10.1038/23948 (1998). Additional Declarations No competing interests reported. Supplementary Files Originaldata.xlsx controlCitH3.tif controlDAPI.tif controlMerge.tif controlMPO.tif LungWBGAPDH.jpg NETsWBGAPDH.jpg PMACitH3.tif PMADAPI.tif PMAMerge.tif WBCitH3.jpg WBNE.jpg WBp65NFB.jpg WBpp65NFB.jpg PMAMPO.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 Dec, 2025 Reviews received at journal 05 Dec, 2025 Reviews received at journal 24 Nov, 2025 Reviewers agreed at journal 15 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers invited by journal 12 Nov, 2025 Editor assigned by journal 29 Sep, 2025 Editor invited by journal 29 Sep, 2025 Submission checks completed at journal 26 Sep, 2025 First submitted to journal 26 Sep, 2025 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. 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Scale bar: 50 μm.Sections were stained with H\u0026amp;E.\u003c/p\u003e","description":"","filename":"Fig200.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7688507/v1/d30c0f9957dbdb2ee2df2349.jpg"},{"id":96610838,"identity":"b6da3997-3057-4ed2-a8c2-1d37f8b06bf4","added_by":"auto","created_at":"2025-11-24 09:32:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":150738,"visible":true,"origin":"","legend":"\u003cp\u003eIL-36 signaling regulates CitH3 and NE expression in a sepsis mice model.Western blot analysis of CitH3 and NE in lung tissues. (* \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001;mean±SD, n=5) .\u003c/p\u003e","description":"","filename":"Fig3001.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7688507/v1/5c7251826c4b9401087eee40.jpg"},{"id":96611043,"identity":"03188125-8716-4a18-a9d2-6ebf7effbfe3","added_by":"auto","created_at":"2025-11-24 09:32:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":209261,"visible":true,"origin":"","legend":"\u003cp\u003ePMA stimulation induces neutrophil activation and MPO/CitH3 expression.\u003cbr\u003e\n \u003cstrong\u003eA\u003c/strong\u003e Viability of neutrophils assessed by trypan blue exclusion assay. Viable cells (unstained, colorless; red arrows) exclude trypan blue due to intact cell membranes, while non-viable cells (blue-stained; black arrows) uptake the dye through compromised membranes;\u003cstrong\u003eB\u003c/strong\u003e Immunofluorescence staining of MPO and CitH3 in neutrophils following PMA stimulation. Nuclei were counterstained with DAPI. Merged images show colocalization. Scale bar: 20 µm;\u003cstrong\u003eC\u003c/strong\u003e Quantification of MPO and CitH3 fluorescence intensity ratios (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001;mean±SD, n = 3).\u003c/p\u003e","description":"","filename":"Fig400.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7688507/v1/704b34007e37783ca264c0df.jpg"},{"id":96610591,"identity":"74651045-59fa-4f4d-9e22-be8fb669ac41","added_by":"auto","created_at":"2025-11-24 09:32:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":203497,"visible":true,"origin":"","legend":"\u003cp\u003eIL-36 modulates NET-mediated cytokine release;\u003cstrong\u003eA\u003c/strong\u003e Cell viability assessed by CCK-8 assay under different treatments: \u003cstrong\u003eB\u003c/strong\u003e ELISA analysis of IL-36 isoforms (IL-36α, IL-36β, IL-36γ) in cell supernatants;\u003cstrong\u003eC \u003c/strong\u003eELISA quantification of IL-10 and TNF-α in supernatants (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001;mean±SD, n = 3).\u003c/p\u003e","description":"","filename":"Fig500.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7688507/v1/c51cd63a43ed5ea2222fa6cf.jpg"},{"id":96610954,"identity":"3bb784aa-829c-4929-8c6f-044e35d3ac8a","added_by":"auto","created_at":"2025-11-24 09:32:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":139845,"visible":true,"origin":"","legend":"\u003cp\u003eIL-36 regulates NF-κB signaling in NET-treated cells.\u003cbr\u003e\n Western blot analysis of phosphorylated p65 NF-κB (p-p65 NF-κB), total p65 NF-κB \u0026nbsp;in neutrophils under the following conditions: (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e 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10:00:07","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":953244,"visible":true,"origin":"","legend":"","description":"","filename":"PMAMPO.tif","url":"https://assets-eu.researchsquare.com/files/rs-7688507/v1/8ce273b222ac472f5a334fa9.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulation mechanism of IL-36 by neutrophil extracellular trapping in a mice of sepsis-induced ARDS","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAcute respiratory distress syndrome (ARDS) is an acute-onset respiratory disorder characterized by non-cardiogenic bilateral pulmonary edema and hypoxemia, resulting from compromised pulmonary endothelial barrier integrity and excessive alveolar-capillary permeability. The pathological hallmarks include diffuse pulmonary inflammation with substantial infiltration of inflammatory cells, predominantly neutrophils and macrophages\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e,Clinically, ARDS manifests as bilateral pulmonary infiltrates and respiratory failure\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e,with severity classified as mild, moderate, or severe based on oxygenation parameters\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEpidemiological studies indicate that ARDS occurs in 10% of ICU admissions and 23% of mechanically ventilated patients, with a 28-day mortality rate of 35% that exceeds 40% in severe cases\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.Sepsis represents the most common predisposing factor for ARDS development, with septic ARDS maintaining mortality rates between 34\u0026ndash;45%\u003csup\u003e5,6\u003c/sup\u003e,During ARDS progression, neutrophil adhesion, aggregation and activation drive inflammatory cascades through excessive neutrophil extracellular trap (NETs) formation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.Elevated NETs levels have been consistently documented in sepsis-induced ARDS\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.Notably, NETs demonstrate significant correlations with alveolar epithelial/endothelial damage severity and inflammatory mediator concentrations\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, while concurrently releasing proinflammatory cytokines such as IL-1β that exacerbate disease progression\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e,However, the precise activation mechanisms underlying these processes remain elusive.\u003c/p\u003e\u003cp\u003eEmerging evidence highlights IL-36/IL-36R signaling as a critical modulator of pulmonary inflammation and immune responses, with constitutive expression throughout lung tissue\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e。Bronchial epithelial cells upregulate IL-36 expression following exposure to proinflammatory cytokines and microbial molecular patterns\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.Experimental models demonstrate that intratracheal IL-36α/γ administration induces proinflammatory cytokine/chemokine production and neutrophil recruitment\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e。The mechanism of IL-36 signaling in sepsis-induced lung inflammation remains unclear. We hypothesize that neutrophil protease-triggered IL-36 activation initiates NF-κB signaling and may regulate septic ARDS via NETs.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003eMale C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;25; aged 6\u0026ndash;8 weeks; body weight 22\u0026ndash;26 g) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). Animals were maintained under specific pathogen-free (SPF) conditions with controlled temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C), relative humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%), and 12:12 h light-dark cycles, with ad libitum access to autoclaved feed and sterile water. All ARDS-related procedures strictly adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yangzhou University Institutional Animal Care and Use Committee (IACUC Approval No. 202406021).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Sepsis model\u003c/h2\u003e\u003cp\u003eMice were randomly divided into Control, ARDS, ARDS\u0026thinsp;+\u0026thinsp;IL-36, ARDS\u0026thinsp;+\u0026thinsp;IL-36 Ra .To induce LPS-induced ARDS, All the mice were anaesthetized using a mixture of ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg). Instilled into the trachea was performed with LPS ((10mg/kg, diluted to 100ul with PBS,Beyotime,China),To facilitate LPS distribution throughout the lungs, the mice were mechanically ventilated using a 20-gauge tracheal intubation and animal ventilator (ALCBIO, China) in volumetric control mode. The mice in the PBS group were given the same amount of PBS without LPS by intratracheal instillation in the same way.The ARDS\u0026thinsp;+\u0026thinsp;IL-36Ra group intradermal injection of 1\u0026micro;g (diluted to 20 \u0026micro;L PBS) of IL-36 receptor antagonist(IL-1F5,NO. HY-P77007,MCE,USA) 24 hours before LPS challenge;The following parameters were applied: 7 mL/kg body weight, 120 breaths/minute, positive end-expiratory pressure (PEEP) of 2 cm H\u003csub\u003e2\u003c/sub\u003eO for 10 minutes. Following ventilation, the mice were monitored for the subsequent 24 hours in an animal care facility.All mice were sacrificed 24 h later.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Pulmonary Edema Quantification\u003c/h2\u003e\u003cp\u003eLung edema was evaluated using the wet-to-dry weight ratio method. Fresh right lung tissues were weighed for wet weight and desiccated at 80\u0026deg;C under vacuum until constant dry weight was attained. Triplicate biological replicates were measured using an analytical balance (\u0026plusmn;\u0026thinsp;0.1 mg precision). Th lung wet/dry ratio was calculated as (W/D) \u0026times; 100%, effectively eliminating interference from residual blood in tissue hydration assessment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Histopathological Evaluation of Lung Tissues\u003c/h2\u003e\u003cp\u003eLeft lungs fixed in 4% paraformaldehyde were paraffin-embedded and sectioned into 5 \u0026micro;m slices for H\u0026amp;E staining. A blinded semi-quantitative scoring system was applied by two independent pathologists to evaluate four parameters: alveolar congestion, leukocyte infiltration, septal thickening, and intra-alveolar hemorrhage. Microscopic images were acquired using a Nikon Eclipse Ni-U system with DS-Ri2 camera, enabling structural validation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Neutrophil Isolation and NETs Analysis\u003c/h2\u003e\u003cp\u003eHuman fresh anticoagulated blood was combined with saline and sedimentation solution (No. R1010, Solarbio, China) in a 1:1:1 ratio to facilitate the removal of erythrocytes. After sedimentation, the leukocyte-rich supernatant was layered onto a density gradient medium (Reagent A/C) and centrifuged (600\u0026ndash;1000 \u0026times; g, 25\u0026ndash;30 min) to isolate neutrophils (NO. P9040,Solarbio, China). Cells were washed, resuspended in RPMI 1640 medium, and stimulated with 100 nM PMA (37\u0026deg;C, 5% CO₂, 4 h) to induce NETs formation. The NETs layer was collected, homogenized, and centrifuged to remove debris. DNA content was quantified using the Quant-iT\u0026trade; PicoGreen\u0026reg; assay(NO.P11496,Thermo, USA),Fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 NETs Viability by Trypan Blue Exclusion\u003c/h2\u003e\u003cp\u003eIsolated NETs were assessed for viability using trypan blue exclusion. A 4% trypan blue stock was prepared by dissolving 4 g powder in distilled water, grinding, adjusting to 100 mL with ddH, filtering, and storing at 4\u0026deg;C. The stock was diluted to 0.4% with PBS before use. Cells were trypsinized to a single-cell suspension, diluted appropriately, and mixed with 0.4% trypan blue at a 9:1 ratio. Viable (unstained) and non-viable (blue-stained) cells were counted within 3 min using a haemocytometer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Cell Culture and Treatment\u003c/h2\u003e\u003cp\u003eNETs incubated with Cathepsin G inhibitor (5 \u0026micro;M) or elastase inhibitor (5 \u0026micro;M) for 30 min. Subsequently, IL-36α, IL-36β, or IL-36γ (50 nM) in 1 mL HBSS/0.25% BSA was added to NETs and incubated at 37\u0026deg;C for 30 min. The resulting IL-36-containing supernatants and isolated NETs were collected for subsequent treatments. Neutrophils cells were treated for 30 min under the following conditions: control,NETs, NETs\u0026thinsp;+\u0026thinsp;IL-36,NETs\u0026thinsp;+\u0026thinsp;IL-36Ra.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e\u003cp\u003eWhole blood collected via orbital bleeding from mice was clotted (RT, 1\u0026ndash;2 h), centrifuged (3,000 \u0026times; g, 10 min), and serum stored at -80\u0026deg;C for subsequent analysis. Post-bleeding, bronchoalveolar lavage fluid (BALF) was obtained by saline instillation (3 \u0026times; 3 mL), centrifuged (3,000 \u0026times; g, 15 min, 4\u0026deg;C), and the supernatant analyzed for total protein (BCA assay) and cytokines. Serum and BALF levels of MPO (Mlbio ml002070), TNF-α (Mlbio ml002095), and IL-10 (Mlbio ml037873) were quantified by ELISA using analyte-specific sample dilutions. Separately, Neutrophils cells were cultured in RPMI 1640/10% FBS; PMA-stimulated neutrophils, pretreated with Cathepsin G/Elastase inhibitors (5 \u0026micro;M), were incubated with IL-36 isoforms (50 nM), and the resultant supernatants applied to NETs to assess TNF-α and IL-10 production by ELISA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Immunofluorescence Staining\u003c/h2\u003e\u003cp\u003eNETs were seeded onto glass-bottomed 12-well plates (NEST) at 40\u0026ndash;50% confluency and cultured for 24 h (37\u0026deg;C, 5% CO₂). Cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 20 min at 25\u0026deg;C, permeabilized with 1% Triton X-100 (Sigma-Aldrich Germany) for 5 min, and blocked with 5% goat serum (Gibco) for 30 min. Primary antibodies targeting citrullinated histone H3 (H3Cit, ab5103, Abcam; 1:200) and myeloperoxidase (MPO, ab90810, Abcam; 1:100) were applied overnight at 4\u0026deg;C. After three PBS washes, slides were incubated with species-specific secondary antibodies: Goat anti-Mouse IgG-Alexa Fluor\u0026reg; 647 (NO:ab150115, Abcam; 1:200) and Goat anti-Rabbit IgG-Alexa Fluor\u0026reg; 488 (NO:ab150077, Abcam; 1:200) for 1 h at 25\u0026deg;C protected from light. Nuclei were counterstained with 1 \u0026micro;g/mL DAPI (Sigma-Aldrich) for 5 min. Images were acquired using a Leica TCS SP8 confocal microscope with 63\u0026times; oil immersion objective (LAS X software), and fluorescence intensity was quantified using Image J .\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Cell Viability Assays(CCK-8)\u003c/h2\u003e\u003cp\u003eNeutrophils cells were treated with NETs, IL-36, or IL-36Ra for 30 minutes.After 24 hours, 10 \u0026micro;L of CCK-8 reagent(NO:C0037,Beyotime, China,)was added to each well.The plate was incubated for 2 hours Absorbance was measured at 450 nm using a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Western Blot\u003c/h2\u003e\u003cp\u003eNE, CitH3, p-p65 NF-κB, and p65 NF-κB expression was analyzed. Protein extraction: Lung tissue was homogenized in lysis buffer,and centrifuged (12,000 rpm, 4\u0026deg;C, 10 min). Bronchial epithelial cells were lysed in RIPA buffer with protease inhibitors, scraped, and centrifuged. Protein concentration was determined by BCA assay. SDS-PAGE: Samples (25 \u0026micro;g/lane) were denatured, loaded onto 10% gels, and electrophoresed. Proteins were transferred to PVDF membranes at 65 V for 2 h. Membranes were blocked (1 h, RT), then incubated overnight at 4\u0026deg;C with:NE (Affinity, NO:AF0010; 1:500),CitH3 (Abcam, NO:ab5103; 1 \u0026micro;g/mL),p-p65 NF-κB (Affinity, NO:AF2006; 1:500),p65 NF-κB ( Affinity,NO: AF5006; 1:500),GAPDH ( Abcam, NO:ab245355).After TBST washes, membranes were incubated with secondary antibodies:Goat anti-Mouse IgG H\u0026amp;L (Alexa Fluor\u0026reg; 647; Abcam, NO:ab150115; 1:2,000) for MPO,Goat anti-Rabbit IgG H\u0026amp;L (Alexa Fluor\u0026reg; 488; Abcam,NO: ab150077; 1:2,000) for other targets(60 min, RT, dark). Detection: ECL reagent was applied, membranes exposed to film, and imaged. Quantification: Band intensity ratios (target/GAPDH) were analyzed.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 IL-36 exacerbates pulmonary edema, inflammation, and injury in mice ARDS.\u003c/h2\u003e\n \u003cp\u003eThe lung wet-to-dry weight ratio exhibited a progressive elevation across the experimental groups. The control group demonstrated the lowest value (2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24), followed by the ARDS with IL-36Ra group (2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14), then the ARDS group (3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21), with the ARDS with IL-36 group showing the highest ratio (4.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). This escalating pulmonary edema correlated with a commensurate increase in inflammatory mediators. The levels of myeloperoxidase (MPO), TNF-\u0026alpha;, and IL-10 in both bronchoalveolar lavage fluid (BALF Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) and serum(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) displayed an identical ascending order from control to ARDS with IL-36Ra to ARDS to ARDS with IL-36, with all pairwise comparisons being statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Consequently, IL-36 receptor blockade via IL-36Ra mitigated pathological injury, while exogenous IL-36 administration exacerbated ARDS manifestations in a dose-dependent manner, highlighting the central pathogenic role of the IL-36 pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Histopathological assessment reveals IL-36 exacerbates lung injury.\u003c/h2\u003e\n \u003cp\u003eH\u0026amp;E staining demonstrated progressive histological damage across groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Control lungs exhibited intact alveolar architecture, absent hemorrhage, edema, or inflammatory infiltration. Both ARDS\u0026thinsp;+\u0026thinsp;IL-36Ra and ARDS groups displayed mild interstitial edema, minimal alveolar wall thickening, and sparse inflammatory cell infiltration. In contrast, ARDS\u0026thinsp;+\u0026thinsp;IL-36 mice manifested severe pathology: alveolar collapse, pronounced interstitial edema, marked alveolar wall thickening, and robust inflammatory cell (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 IL-36 drives NETs formation and neutrophilic inflammation in ARDS lungs.\u003c/h2\u003e\n \u003cp\u003eWestern blot analysis demonstrated a progressive increase in the expression of neutrophil elastase (NE) and citrullinated histone H3 (CitH3)-both established markers of NETosis-across the experimental groups, with the lowest expression in the Control group, followed by the ARDS\u0026thinsp;+\u0026thinsp;IL-36Ra group, then the ARDS group, and the highest expression in the ARDS\u0026thinsp;+\u0026thinsp;IL-36 group. This IL-36-dependent upregulation of NETosis-associated proteins correlated with graded elevations in myeloperoxidase (MPO), pulmonary edema, and proinflammatory cytokine levels, suggesting that dysregulated neutrophil activation represents a central mechanism underlying IL-36-aggravated ARDS (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 IL-36 potentiates neutrophil NETosis, driving ARDS pathology.\u003c/h2\u003e\n \u003cp\u003eAfter isolation of human neutrophils using a commercial kit and assessment of viability by trypan blue exclusion, we confirmed that cell viability exceeded 98% (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Viable neutrophils primed with IL-36 exhibited an enhanced responsiveness to PMA stimulation. Immunofluorescence analysis demonstrated increased co-localization of MPO and CitH3, indicative of elevated neutrophil extracellular trap (NETs) formation, compared with control conditions. These results provide mechanistic evidence linking IL-36 to enhanced neutrophil-driven inflammatory responses in ARDS(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB,C).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 NET-associated IL-36 signaling drives epithelial cytotoxicity while NETs proteases amplify IL-36-dependent inflammation.\u003c/h2\u003e\n \u003cp\u003eCCK-8 assays showed reduced 24-hour cell viability, with the control exhibiting the highest viability, followed by NETs, NETs\u0026thinsp;+\u0026thinsp;IL-36Ra, and NETs\u0026thinsp;+\u0026thinsp;IL-36(Fig. 5A).ELISA of supernatants from IL-36\u0026alpha;/\u0026beta;/\u0026gamma;-stimulated cells indicated that PMA most strongly enhanced TNF-\u0026alpha; and IL-10 release, followed by PMA with elastase, then PMA with cathepsin G, and lastly the control(Fig. 5B).Similarly, NET-associated proteases hierarchically enhanced PMA-induced cytokine production: lowest in control, higher with NETs, further elevated with NETs\u0026thinsp;+\u0026thinsp;IL-36Ra, and highest with NETs\u0026thinsp;+\u0026thinsp;IL-36(Fig. 5C.)These findings suggest NETs form a self-amplifying inflammatory circuit whereby proteases increase IL-36 expression, and IL-36 augments NET-mediated cytotoxicity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 NETs trigger sustained NF-\u0026kappa;B activation in bronchial epithelium via IL-36 signaling.\u003c/h2\u003e\n \u003cp\u003eWestern blot analysis of nuclear fractions demonstrated a progressive increase in p65 NF-\u0026kappa;B phosphorylation across experimental conditions: the lowest p-p65/p65 ratio was observed in the control group, followed by NETs-treated samples, then NETs co-treated with IL-36Ra, and finally NETs with IL-36 showing the highest phosphorylation level(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). This IL-36-mediated amplification of p65 phosphorylation identifies NF-\u0026kappa;B as a central transcriptional mechanism underlying NET-induced epithelial inflammation. Notably, IL-36Ra attenuated but did not completely abolish NF-\u0026kappa;B activation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIL-36 as a member of the IL-1 cytokine family, is widely expressed in various tissues and organs such as the lungs, skin, and intestine and plays a critical role in inflammation and immune regulation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Studies have shown that IL-36 is involved in the pathogenesis of multiple inflammatory diseases. In inflammatory bowel disease, IL-36 promotes disease progression by inducing the production of CXC chemokines\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In generalized pustular psoriasis (GPP), aberrant activation of IL-1 and IL-36 mediates the expression of neutrophil chemotactic factors, inflammatory cell infiltration, and pustule formation \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In chronic rhinosinusitis, IL-36 is regulated by TLR signaling, leading to impaired endothelial barrier function and exacerbation of the inflammatory response \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In patients with Pseudomonas aeruginosa-induced ARDS, levels of IL-36γin plasma and BALF were significantly higher than those in healthy controls \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, confirming that IL-36 exacerbates inflammatory symptoms in ARDS. Additionally, abnormal activation of the IL-36 signaling pathway has been closely associated with various inflammatory diseases \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. IL-36αpromotes the release of pro-inflammatory mediators and T-cell proliferation by activating the NF-κB signaling pathway in macrophages\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. As a key upstream regulator, IL-36αenhances the activation of neutrophils, macrophages, and fibroblasts\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Further studies in Jurkat T cells demonstrated that IL-36 induces mitochondrial activation of MAPK and nuclear factor kappa-B (NF-κB) pathways, aggravating inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Upon binding of IL-36α,βorγto their specific receptor-interleukin-1 receptor-related protein 2 (IL-1Rrp2)-the IL-36 signaling pathway is initiated. This process subsequently recruits the co-receptor IL-1 receptor accessory protein (IL-1RAcP) and activates the NF-κB signaling pathway, thereby driving extensive transcription and secretion of various pro-inflammatory cytokines\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e However, the precise mechanisms of action remain incompletely understood.This study demonstrates that in a sepsis-induced ARDS model, elevated IL-36 expression is closely associated with aggravated pulmonary inflammation, consistent with previous reports. Specifically, there was a significant increase in the lung wet/dry (W/D) ratio, exacerbated histopathological injury, and markedly elevated levels of inflammatory cytokines (including IL-10 and TNF-α) and MPO in serum and BALF, along with significantly increased expression of corresponding inflammatory proteins in tissues. These results suggest that in sepsis-associated ARDS, IL-36 may exert a pro-inflammatory role potentially through activation of the NF-κB signaling pathway.\u003c/p\u003e\u003cp\u003eDuring the onset of ARDS, large numbers of neutrophils are recruited to lung tissue and generate neutrophil extracellular traps (NETs) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. NETs are complexes capable of trapping pathogens, primarily composed of nuclear chromatin, mitochondrial DNA, and neutrophil granular proteins\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. They immobilize and eliminate invading pathogens, exert antimicrobial effects, and promote inflammation resolution, constituting an innate immune response that plays a crucial role in host defense \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, excessive NET formation can damage vascular endothelial cells, promote thrombosis, cause microvascular dysfunction, and injure pulmonary cells, playing a significant role in sepsis-induced ARDS \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In this study, NETs were induced in neutrophils by PMA stimulation, and their activity was verified using trypan blue staining, establishing an in vitro ARDS model. Fluorescence staining revealed that IL-36 downregulated the expression of NET markers MPO and CitH3, significantly reducing NET activity. Administration of a blocking agent notably restored NET activity. Furthermore, it was observed that IL-36 (α/β/γ) increased the expression of inflammatory cytokines IL-10 and TNF-α, and elevated levels of these cytokines were also detected in the cell supernatant, further confirming that IL-36 induces NETs to secrete substantial pro-inflammatory factors.\u003c/p\u003e\u003cp\u003eThe NF-κB signaling pathway is a key transcription factor extensively involved in various biological processes including cellular regulation, inflammatory responses, and immune reactions\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Research indicates that the NF-κB pathway plays an important role in the formation of NETs \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Neutrophils contain dimers composed of NF-κB1 (p50) and p65 NF-κB, and cellular stimulation can trigger nuclear translocation of NF-κB/Rel proteins and degradation of IκB-α\u003csup\u003e42\u003c/sup\u003e Studies have shown that disruption of NETs can promote apoptosis in gastric cancer cells by modulating the expression of Bcl-2, Bax, and NF-κB \u003csup\u003e43\u003c/sup\u003e. Conversely, NETs can also enhance NF-κB activation, thereby facilitating the progression and metastasis of breast tumors \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. NETs have also been reported to participate in various pathophysiological processes such as wound healing, atherosclerosis, and acute lung injury \u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This study found that in NET-induced ARDS, IL-36 led to increased protein expression of NF-κB, suggesting that IL-36 may regulate the NF-κB signaling pathway via NETs, activating NF-κB to promote massive secretion of inflammatory factors and resulting in pulmonary injury in ARDS.\u003c/p\u003e\u003cp\u003eIn summary, the findings of this study indicate that in sepsis-induced ARDS, IL-36 recruits large numbers of neutrophils via NETs, leading to substantial secretion of inflammatory factors and aggravated pulmonary inflammatory injury, while simultaneously activating the NF-κB signaling pathway to further promote extensive release of inflammatory mediators. However, several notable limitations must be acknowledged, including a limited sample size and the absence of independent clinical validation. Furthermore, long-term outcome data are currently unavailable. Future studies should prioritize mechanistic investigations\u0026mdash;such as utilizing IL-36 knockout models-alongside clinical translation trials and comprehensive long-term safety assessments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShenqin Li conceived and designed the study. Haiyan Hu, FuJin and Lifen Wang performed experiments. Haiyan Hu analyzed data, interpreted results, and wrote the manuscript. Shenqin Li reviewed and revised the manuscript and supervised the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Medical and Health Science and Technology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProject of Zhejiang Province (Grant No. 2024KY1420,2023KY468)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll ARDS-related procedures strictly adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yangzhou University Institutional Animal Care and Use Committee (IACUC Approval No. 202406021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the first author on reasonable request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXiong, S.\u003cem\u003e et al.\u003c/em\u003e IL-1beta suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e130\u003c/strong\u003e, 3684-3698, doi:10.1172/JCI136908 (2020).\u003c/li\u003e\n\u003cli\u003eCorry, J.\u003cem\u003e et al.\u003c/em\u003e Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, e1010395, doi:10.1371/journal.ppat.1010395 (2022).\u003c/li\u003e\n\u003cli\u003eSaffarzadeh, M.\u003cem\u003e et al.\u003c/em\u003e Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e32366, doi:10.1371/journal.pone.0032366 (2012).\u003c/li\u003e\n\u003cli\u003eBos, L. 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J., Yamamoto, Y. \u0026amp; Gaynor, R. B. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. \u003cem\u003eNature\u003c/em\u003e\u003cstrong\u003e396\u003c/strong\u003e, 77-80, doi:10.1038/23948 (1998).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Sepsis, ARDS, IL-36, NETs, NF-κB","lastPublishedDoi":"10.21203/rs.3.rs-7688507/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7688507/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSepsis-induced acute respiratory distress syndrome (ARDS) is characterized by uncontrolled pulmonary inflammation and neutrophil-driven pathology. Neutrophil extracellular traps (NETs) have been implicated in ARDS progression, but the role of interleukin-36 (IL-36) in this process remains unclear. This study aimed to investigate the regulatory mechanism of IL-36 via NETs and its impact on NF-κB activation in a murine model of sepsis-induced ARDS.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA lipopolysaccharide (LPS)-induced ARDS model was established in C57BL/6 mice. Animals were divided into Control, ARDS, ARDS\u0026thinsp;+\u0026thinsp;IL-36, and ARDS\u0026thinsp;+\u0026thinsp;IL-36Ra groups. Pulmonary edema was assessed via wet/dry weight ratio, inflammatory cytokines were measured by ELISA, and histopathological changes were evaluated through H\u0026amp;E staining. NETs formation was analyzed using immunofluorescence and Western blot. NF-κB activation was detected via phosphorylation of p65.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIL-36 administration exacerbated pulmonary edema, inflammatory cytokine levels (TNF-α, IL-10, MPO), and histopathological injury. Conversely, IL-36 receptor antagonist (IL-36Ra) attenuated these effects. IL-36 promoted NETs formation, as indicated by elevated citrullinated histone H3 (CitH3) and neutrophil elastase (NE) expression. NETs further enhanced IL-36-induced proinflammatory cytokine release and NF-κB activation in bronchial epithelial cells. IL-36Ra partially reversed NET-mediated NF-κB phosphorylation and inflammatory responses.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eIL-36 exacerbates sepsis-induced ARDS by enhancing NETs formation and activating the NF-κB pathway, leading to amplified pulmonary inflammation and injury. Targeting IL-36 signaling may represent a therapeutic strategy to mitigate ARDS progression.\u003c/p\u003e","manuscriptTitle":"Regulation mechanism of IL-36 by neutrophil extracellular trapping in a mice of sepsis-induced ARDS","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 09:25:42","doi":"10.21203/rs.3.rs-7688507/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-10T18:52:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-05T21:09:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T16:26:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316918414605558253794384297014653073750","date":"2025-11-15T10:38:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6167981681721268421100121707053031406","date":"2025-11-13T17:41:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-13T01:02:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-30T00:47:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-29T19:40:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-26T08:50:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-26T08:46:34+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":"b4775915-61fa-4406-b9cd-00325dcf0c47","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58445987,"name":"Health sciences/Diseases"},{"id":58445988,"name":"Biological sciences/Immunology"},{"id":58445989,"name":"Health sciences/Medical research"},{"id":58445990,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-04-27T11:10:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 09:25:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7688507","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7688507","identity":"rs-7688507","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}