Hippo pathway and NLRP3-driven NETosis in macrophages: Mechanisms of viral pneumonia aggravation

Hippo pathway and NLRP3-driven NETosis in macrophages: Mechanisms of viral pneumonia aggravation | 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 Hippo pathway and NLRP3-driven NETosis in macrophages: Mechanisms of viral pneumonia aggravation Linghui Pan, Bijun Luo, Xiaoxia Wang, Jinyuan Lin, Jianlan Mo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4591287/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jul, 2025 Read the published version in Cell Death Discovery → Version 1 posted 10 You are reading this latest preprint version Abstract Background Severe viral infections can precipitate acute lung injury, causing substantial morbidity and mortality. NETosis plays a crucial role in defending against pathogens and viruses, but its excessive or dysregulated formation can cause pulmonary damage, with research into its regulation offering potential insights and treatment strategies for viral lung injuries. Methods Elevated levels of NETosis were detected in the peripheral blood of patients with viral pneumonia. To explore the correlation between NETosis and virus-induced acute lung injury, we employed a murine model, administering poly(I:C) (polyinosinic-polycytidylic acid), an artificial substitute for double-stranded RNA, intratracheally to mimic viral pneumonia. Assessment of NETosis biomarkers in afflicted patients and poly(I:C)-stimulated mice was conducted, alongside mechanistic investigations into the involvement of the Hippo signaling pathway, inflammatory factors, and chemokines in the injury process. Cytokine assays, co-culture experiments, and downstream inflammatory mediator analyses were used to ascertain the role of the Hippo pathway in macrophage to mediate NETosis. Results Enhanced expression of NETosis biomarkers was found both in patients with viral pneumonia and in poly(I:C)-stimulated mice. Hippo pathway activation in conjunction with increased levels of inflammatory actors and chemokines was observed in lung tissues of the mouse model. Elevated IL-1β was detected in cells and macrophages isolated from infected mice; this was mitigated by Hippo pathway inhibitors. IL-1β was confirmed to induce NETosis in co-culture experiments, while NLRP3, functioning downstream of the Hippo pathway, mediated its secretion. Patients with viral pneumonia exhibited increased NLRP3 and IL-1β in monocyte-macrophages relative to healthy controls. Conclusions Activated Hippo pathway in macrophages during poly(I:C) exposure upregulates NLRP3 and IL-1β expression to promote the occurrence of NETosis, thereby aggravating virus-induced lung injury. This study identifies a potential target pathway for therapeutic intervention to mitigate lung injury stemming from viral infections. Health sciences/Diseases/Infectious diseases/Viral infection Biological sciences/Molecular biology/Transcriptomics Hippo/YAP Signal Pathway Acute Lung Injury Inflammation Cellular interaction NETosis Macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Respiratory viral infections, such as influenza, SARS-CoV, and MERS-CoV, are global public health problems with substantial seasonal and pandemic morbidity and mortality [ 1 – 3 ]. Current treatments primarily focus on supportive care, antiviral medications, and in some cases, the use of corticosteroids, however, these approaches often face limitations regarding efficacy, antiviral resistance, and potential adverse effects[ 4 ]. There is a critical need to delve deeper into the underlying mechanisms of virus-host interactions. The immune microenvironment plays a pivotal role in recognizing, eliminating viruses, and infected cells during viral lung injury. Innate immune cells such as neutrophils, natural killer (NK) cells, and macrophages are rapidly recruited to the site of infection, where they exert antiviral functions[ 5 ]. The immune microenvironment within viral lung damage is critical at all stages, from initial antiviral defense to tissue damage and subsequent repair processes[ 6 ]. Understanding the dynamic changes and mechanisms of action of various immune cells within the immune microenvironment is vital for developing novel therapeutic strategies and improving patient outcomes. This comprehensive insight can lead to the identification of specific targets that can be modulated to enhance viral clearance while minimizing immune-mediated tissue injury, thus offering a balanced approach to the treatment of viral lung injuries. NETosis, the process by which neutrophils release neutrophil extracellular traps (NETs) as part of the innate immune response, plays a critical role in the body's defense against various pathogens, including viruses[ 7 , 8 ]. NETs, consisting of DNA fibers adorned with antimicrobial proteins, have been documented to play a pivotal role in trapping and neutralizing virus particles, thus impeding viral replication and dissemination[ 9 ]. While NETs serve a protective purpose by limiting viral spread within the host, excessive or dysregulated NET formation can lead to increased pulmonary inflammation and tissue damage[ 10 , 11 ]. Recent research has highlighted the induction of NETs by a variety of inflammatory mediators, emphasizing the complexity of interactions between viral infections and the host immune system[ 12 ]. Understanding the balance between beneficial and detrimental outcomes of NET formation in the context of viral infections could uncover novel insights into the pathophysiology of viral lung injury. Therefore, investigating the relationship between NETosis and viral lung injury helps us to understand the mechanisms of viral lung damage more deeply and may provide new strategies for treatment. In the present study, we identified the hub gene NLRP3 by developing a mouse model of viral lung injury with utilizing immunofluorescence, Western Blot, ELISA, and transcriptomic sequencing experiments. Our findings conclude that during poly I:C-induced lung injury, the Hippo pathway is activated in macrophages, leading to an upregulation of NLRP3 expression, which in turn promotes the production of IL-1β. Elevated IL-1β levels trigger an increase in the formation of neutrophil extracellular traps (NETs), thereby exacerbating the severity of pneumonia. These insights into the molecular pathophysiology of viral lung injury illuminate potential targets for therapeutic intervention aimed at modulating inflammation and preventing lung damage. Materials and methods Clinical sample Our study involved a total of 20 participants, which included 10 severe viral pneumonia patients and 10 healthy volunteers, all sourced from the Intensive Care Unit of the Affiliated Tumor Hospital of Guangxi Medical University. The pneumonia patients were diagnosed based on acute respiratory infection symptoms and confirmed viral infection through direct immunofluorescence antigen detection, real-time fluorescence PCR nucleic acid detection, or pathogen metagenomic next-generation sequencing. A Cycle threshold (Ct) value of ≤ 35 was considered positive for viral presence, with values between 35 and 40 considered weakly positive. Our inclusion criteria for pneumonia diagnosis required fever, cough, wheezing, accelerated breathing, and wet rales on lung auscultation, or chest imaging indicative of pneumonia. Severe pneumonia was diagnosed based on one primary criterion or three or more secondary criteria, including the need for invasive mechanical ventilation or vasoconstrictor treatment for septic shock. All participants provided informed consent, and the study protocol was approved by the Ethics Committee of the Guangxi Medical University Tumor Hospital (approval number 2023-3-7), adhering to the guidelines of the Declaration of Helsinki. Peripheral blood was collected from both groups at 8 AM, with neutrophils isolated using density gradient centrifugation and serum retained and stored at -80°C for further analysis. Mice This investigation followed the suggested guidelines from the People's Republic of China's Guide for Regulation and Administration of Laboratory Animals. The Guangxi Medical University’s Institutional Animal Care and Use Committee sanctioned the study protocols. Anesthesia, using ketamine hydrochloride and xylazine, was employed during all animal experiments to mitigate discomfort as much as possible. Wild-type C57BL/6J male mice aged from 6–8 weeks and weighing about 25 ± 5g, not previously used for any experiments, were obtained from the Animal Center of Guangxi Medical University (Nanning, China). These mice were housed in a room fitted with air-filters where they could freely access food and water. The conditions of the room were maintained at a temperature of 20–25 ºC, with 50–70% humidity levels. Cell RAW 264.7 cells were purchased from ATCC. Primary alveolar macrophages (AMs) were generated from wild-type C57BL/6 mice with or without poly(I:C) stimulation (HMW, tlrl-pic; InvivoGen, USA). Briefly, primary AMs were obtained from bronchoalveolar lavage fluid (BALF) after erythrocyte lysis. BALF was plated for 1 h, followed by thorough washing to remove unattached cells. Adherent cells were used as primary AMs [ 13 ]. RAW 264.7 cells and AMs were cultured in RMPI 1640 medium containing 10% fetal bovine serum (FBS) (10091148, Gibco, New Zealand), 20 mM HEPES, and 2 mM L-glutamine. Following this separation, AMs were further isolated using magnetic bead separation with CD14 MicroBeads (Miltenyi Biotec, Germany), targeting the CD14 + monocyte population, which is a standard practice for monocyte isolation due to its high specificity and efficiency. Approximately 95% of the harvested cells were alveolar macrophages, as confirmed by flow cytometry. Neutrophils were extracted from ethylenediaminetetraacetic acid (EDTA) (E809069, Macklin, China)-anticoagulated entire blood collected from wild-type C57BL/6 mice with or without poly(I:C) stimulation using density gradient centrifugation. The entire blood was layered upon a density gradient comprising a lower layer of Histopaque®-1119 (11191, Sigma-Aldrich, Vienna, Austria) and an upper layer of Ficoll-Paque PLUS (17-1440-03, GE Healthcare, Uppsala, Sweden), followed by centrifugation at 700 × g lasting for 30 minutes. The fraction comprising polymorphonuclear cells was located above the erythrocyte pellet and was carefully gathered, then washed with 1 × Dulbecco's phosphate-buffered saline (DPBS) (15575-020, Thermo Fisher Scientific, Vienna, Austria). Subsequently, these cells were resuspended in VersaLyse Lysing Solution (A09777, Beckman Coulter, Marseille, France) aimed at eliminating red blood cells. The purity of the neutrophil population was typically higher than 90% as evaluated via flow cytometry. Viability of the immunomagnectically isolated neutrophils was evaluated by flow cytometry using cell nucleic acid fluorescent dye, Sytox-Green. For flow cytometry, live single-cell suspensions at a concentration of 1 × 10 6 cells/ml were first blocked with anti-mouse CD16/32 Fc receptor block followed by surface labeling of anti-CD45, anti-CD11b, and anti-Ly6G antibodies at room temperature for 20 min. Cells were then washed three times, resuspended in 1 ml of DPBS, and run on an cell analyzer. In the co-culture experiment, the conditioned medium of RAW 264.7 cells in each group was co-cultured with neutrophils isolated from peripheral blood of mice in the control group at a concentration of 1 × 10 6 T cells/mL for 48 hours. Reagents administration C57BL/6 mice were intranasally challenged with 5mg/Kg high-molecular-weight poly(I:C) at a concentration of 1 mg/mL to induce ALI/ARDS [ 14 ]. This administration was performed under light anesthesia to ensure precise delivery and minimize stress to the animals. The mice were euthanized after the final treatment to collect serum, BALF, and lung tissues for further downstream examinations. In vitro , RAW 264.7 cells, primary AMs, or co-cultures of conditioned medium and neutrophils were exposed to poly(I:C) (20 µg/mL) stimulation for 48 h with or without interventions [ 15 ]. Hippo pathway inhibitor, Lats-IN-1 (MedChemExpress, USA), is a potent and ATP-competitive inhibitor of LATS1 and LATS2 kinases. The administration regimen for Lats-IN-1 involved a 10 mg/kg intraperitoneal injection daily for 3 days, initiated 24 hours before and continued simultaneously with and 24 hours after administration of Poly(I:C) [ 16 ]. Neutralizing IL-1β antibody (R&D Systems, Germany) were administered at a dose of 10 mg/kg via intraperitoneal injection daily for 2 days, timed concurrently with and 24 hours following Poly(I:C) exposure. In vitro treatments included exposure of RAW264.7 cell lines to 10 µM Lats-IN-1, and 5 µL/mL of neutralizing IL-1β antibody[ 17 ]. Plasmids, small interfering RNAs (siRNAs), and transfection All shRNAs used in this study were provided by Sangon (Shanghai, China), as listed in Supplementary Table 1. All procedures related to the experiment were carried out as per the guidelines provided by the manufacturer. The supplementary material holds the detailed methods. Measurement of pulmonary edema, permeability, and cytokines The right upper lobe with excess water was eliminated using filter paper to ascertain its weight (W). The lung tissues were subjected to a drying process at 60°C for 48 hours to attain their dry weight (D). The calculation of the W/D ratio was used as a measurement index for pulmonary edema. An evaluation of changes in lung permeability was conducted by assessing total BALF protein using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). Additionally, a hemocytometer was used to count total cell infiltration. Interleukin 1β (IL-1β), tumor necrosis factor ɑ (TNF-ɑ), dsDNA, LL-37, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in cell culture supernatant, plasma, and BALF were measured using enzyme-linked immunosorbent assay kits (CUSABIO, Wuhan, China). Histologic study The lower lobes of the right lung were preserved using 4% paraformaldehyde (30525-89-4; Sigma-Aldrich, AR, USA), and then encapsulated within the Tissue-Tek OCT compound (4583; Sakura, Tokyo, Japan). The pathological assessment of lung damage was independently evaluated by two authors on sections stained with hematoxylin and eosin, following criteria that had been reported earlier [ 18 ]. In order to analyze the accumulation of collagenous fibers in pulmonary fibrosis, the lung tissues were encased in paraffin and dyed using Masson's stain, following the guidelines provided by the manufacturer. Measurement of mRNA expression Total mRNA of the cells was extracted using TRIzol reagent (Thermo Fisher Scientific) following the guidelines listed by the manufacturer. The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) was used to prepare the cDNA, which was then quantified using the PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, A25742). The relative expression levels of mRNA were determined using the 2-△△ct cycle threshold method. The primer sequences of NLRP3 used were as follows: forward: 5′-GGAGCGGGAGCATGAACTCC-3′, reverse: 5′-GGAGCGGGAGCATGAACTCC-3′. The fold change, adjusted to GAPDH normalization, was utilized to illustrate the variances between groups. Immunoblotting The left lower lung lobes were thoroughly mixed into a uniform solution using RIPA lysis buffer (20–188, Sigma-Aldrich, AR, USA). During this process, to prevent protein degradation and dephosphorylation, both a Protease Inhibitor Tablet (product number 11836170001 from Roche, located in Basel, Switzerland) and a PhosphoSTOP Phosphatase Inhibitor Tablet (product number 4906845001, also from Roche in Basel, Switzerland) were added. This homogenization was achieved with the aid of a mechanical tissue homogenizer.The samples underwent lysis for a duration of thirty minutes at an icy temperature, followed by centrifugation at 12,000 g-force for 15 minutes. Following the measurement of protein concentrations by the bicinchoninic acid (BCA) assay, the obtained supernatants from the cell lysates were heated to 85°C for a duration of 5 minutes with a loading buffer added. Between 50 and 75 micrograms of proteins were subjected to separation through SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. After blocking a 1-hour incubation period at 22–25℃ with 5% nonfat milk, the membranes underwent an overnight incubation with primary antibodies (Supplemental Table 2) at a temperature of 4°C. This was followed by a 1-hour incubation at room temperature with secondary antibodies (Abcam, Cambridge, UK) conjugated with horseradish peroxidase. Band intensities corresponding to different proteins were quantified from digitized films through the employment of an Odyssey® CLX imaging system (LI-COR, USA). Immunostaining Air-dried for half an hour, the frozen tissue samples, cellular suspensions, or sheets of adherent cells were then stabilized with a 3.7% solution of paraformaldehyde for a quarter of an hour, followed by a chilling immersion in undiluted methanol for another fifteen minutes. The slides underwent a blocking process using a solution of phosphate-buffered saline mixed with 3% goat serum (16210064, Gibco, CA, USA), 3% bovine serum albumin (SRE0096, Sigma-Aldrich, AR, USA), 0.2% Triton X-100 (Sigma-Aldrich, Arkansas, USA), and 0.02% NaN3 (S2002, Sigma-Aldrich, Arkansas, USA). Subsequently, they were treated with both primary antibodies and appropriate secondary antibodies. Comprehensive details regarding both the primary and secondary antibodies are provided in Supplemental Table 3. The specimens underwent a treatment process using ProLong®Gold Antifade Reagent containing 4', 6-diamidino-2-phenylindole (DAPI) (8961S, CST, Massachusetts, USA). Then they were examined using multiplex confocal microscopy with a LSM980 microscope from Zeiss, located in Germany. Transcriptomics and processing of raw sequence data Transcriptomes was conducted utilizing the Visium system from 10 x Genomics. Briefly, 10 mm fresh-frozen mouse lung sections, with or without poly(I:C) stimulation, were embedded in OCT and mounted on Visium slides, and the sections underwent a permeabilization procedure for 30 minutes to facilitate the release of mRNAs. These mRNAs subsequently adhered to the spatially barcoded oligonucleotides located on the underlying spots. Following this, a reverse transcription process was executed as per the manufacturer's protocol. cDNA libraries were sequenced using the Illumina NextSeq 2000 system with a sequencing depth of over 50,000 reads for each spot, producing more than 400 million reads for each section. The software Spaceranger, at version 3.1.0 by 10 x Genomics, performed alignment of individual spots from the Visium transcriptomics slides to the reference data of the GRCh38 mouse genome, resulting in the acquisition of raw counts. The data representing the expression patterns of the selected genes were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to conduct a Gene Ontology (GO) enrichment investigation, which encompasses the analysis of biological activities, cellular constituents, and molecular functionalities. All hub genes underwent analysis using DAVID for GO enrichment and KEGG pathway investigation, with counts > 5 and p < 0.01. To assess the interactive networks connecting all targeted genes, the STRING database was employed. Statistical analysis Typically, experiments conducted in vitro were replicated three times (except where noted differently), with results shown as the average value ± standard error of the mean, based on a minimum of three separate experiments. Two groups were compared using the Student's t-test, while the one-way ANOVA with Tukey's post-hoc test was utilized for comparing more than two groups. Statistical significance was established when p -values were less than 0.05. Statistical evaluations were carried out with the GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). Results NETosis is associated with inflammation in patients with viral pneumonia To evaluate the role of NETosis in viral pneumonia, NETosis biomarkers were measured, incorporating citrullinated histone H3 (CitH3), myeloperoxidase (MPO), neutrophil elastase (NE), and LL-37 in the neutrophil from peripheral blood, as well as dsDNA and LL-37 in the serum of individuals suffering from viral pneumonia and healthy participants [ 19 – 21 ]. The of viral pneumonia patients showed increased levels of CitH3, MPO, NE, and LL37 compared to healthy individuals (Fig. 1 A–C). The levels of the CitH3 protein in patients suffering from viral pneumonia were also elevated when compared to those found in healthy individuals (Fig. 1 D-E), and serum dsDNA and LL-37 levels were both increased in these patients (Fig. 1 F). Additionally, serum inflammatory cytokine levels, such as IL-1β, TNF-α, and GM-CSF, were higher in these patients than in healthy volunteers (Fig. 1 G), suggesting that significant inflammation was observed in patients with viral pneumonia. Taken together, NETosis may be associated with a high expression of pro-inflammatory factors, which are potential risk factors for viral pneumonia. NETosis is activated in mouse acute lung injury following poly(I:C) stimulation To investigate the relationship between NETosis and viral pneumonia, C57BL/6 mice underwent intranasal exposed to poly(I:C), resulting in evident pathological injury and the accumulation of collagen fibers (Fig. 2 A). Neutrophils exhibited membrane rupture, releasing network structures containing DNA and antimicrobial proteins in mice with viral pneumonia (Fig. 2 B). Relative to the control mice, both CitH3 and MPO were exhibited at elevated levels in the pulmonary tissues of mice afflicted with poly(I:C) induced lung injury (Fig. 2 C-E). Compared with the control group, the relative expression of CitH3 protein in mice with viral pneumonia at 24h, 48h, and 72h after poly(I:C) exposure was upregulated, peaking at 48 hours post-exposure (Fig. 2 F-G). Additionally, dsDNA and LL-37 levels were higher in the serum and BALF of mice suffering from poly(I:C)-induced lung injury compared to those in the control group (Fig. 2 H). This shows that NETosis is significantly activated in mice with poly(I:C) induced lung injury, as indicated by lung injury and heightened levels of NETosis markers, peaking at 48 hours post-exposure. Hippo pathway is activated in macrophages from poly(I:C) stimulated mice Although the Hippo pathway is primarily associated with cell fate decisions, proliferation, migration, death, and organ size control [ 22 ], the Hippo pathway is critical in pulmonary inflammatory diseases[ 23 ], and its specific role in virus-induced lung injury remains unclear. Within the pulmonary tissue of mice stimulated with poly(I:C), the Hippo pathway is activated (Fig. 3 A–D), leading to increased levels of inflammatory factors and chemokines (Fig. 3 E-F). YAP, a pivotal molecule within the Hippo pathway, undergoes phosphorylation and cytoplasmic retention upon pathway activation, resulting in diminished nuclear YAP levels. Immunofluorescence was used to examine the co-localization of Yes-associated protein (YAP) with neutrophils(Fig. 3 G) or macrophages (Fig. 3 H-I), revealing that YAP co-localized with macrophages rather than neutrophils (Fig. 3 J). The YAP and Hippo pathways were assessed through the nucleocytoplasmic separation of macrophages extracted from the bronchoalveolar lavage fluid (BALF) of mice stimulated with poly(I:C). The findings verify that the stimulation of the Hippo pathway triggered by poly(I:C) stimulation takes place chiefly within macrophages (Fig. 3 K-N). Hippo pathway in macrophages is activated to promote NETosis in murine viral pneumonia following poly(I:C) stimulation Investigating how activation of the Hippo pathway in macrophages following poly(I:C) stimulation promotes NETosis in murine viral pneumonia. In RAW264.7 cells stimulated with poly(I:C), the expression of nuclear YAP was decreased, while the Hippo pathway was activated (Fig. 4 A-D). In the co-culture of conditioned medium from RAW246.7 cells and neutrophils, NETosis biomarkers in co-cultured neutrophils were upregulated under medium from poly(I:C) stimulated RAW246.7 cells. These biomarkers were abolished under medium from poly(I:C) stimulated RAW246.7 cells after the administration of Hippo pathway inhibitors(Fig. 4 E-H). This suggests that Hippo pathway activation in macrophages stimulated with poly(I:C) promotes NETosis. In vitro experiments confirm that IL-1β plays a crucial role in facilitating the interaction between macrophages and NETs Several cytokines have been found to stimulate or promote NETosis formation. For example, the activation of the Hippo pathway induces the expression of IL-1β, a critical pro-inflammatory factor implicated in the promotion of NETosis [ 24 ]. IL-1β levels were increased in RAW246.7 cells that received poly(I:C) treatment, a change abolished in RAW246.7 cells treated with poly(I:C) and Hippo pathway inhibitors (Fig. 5 A–C). In the co-culture of neutrophils and conditioned medium from RAW246.7 cells under poly(I:C) stimulation and the neutralized IL-1β antibody, the promotion of NETosis by IL-1β was attenuated, suggesting that IL-1β is the key molecule mediating macrophage-induced NETosis (Fig. 5 D-H). Validation of IL-1β as the critical mediator facilitating the interaction between macrophages and NETosis in vivo In vivo experiments revealed that IL-1β levels were increased in isolated macrophages in the BALF of mice stimulated with poly(I:C), however, administration of a Hippo pathway inhibitor simultaneously reduced IL-1β levels (Fig. 6 A–C). Mice treated with poly(I:C) and neutralizing IL-1β antibody showed an inhibition of NETosis (Fig. 6 D-H). These confirmed IL-1β as a key mediator linking macrophages to NETosis, where its elevation following poly(I:C) stimulation and reduction by Hippo pathway inhibition correlates with NETosis modulation. Transcriptome sequencing identified NLRP3 downstream of the Hippo pathway that mediates IL-1β secretion and NETosis To delve deeper into the process through which the Hippo pathway facilitates the secretion and discharge of IL-1β, lung tissues from poly(I:C) stimulated mice and PBS as controls were harvested for transcriptome sequencing. Compared to the control group, there were 1625 differentially expressed genes (DEGs), including 1027 upregulated DEGs and 598 downregulated DEGs in poly(I:C) stimulated mice (p < 0.05) (Fig. 7 A). The top five upregulated DEGs were C1rb, Ocstamp, C1qb, Irf7, and Saa3, whereas the top five downregulated DEGs included Igfbp3, Cd209a, Crispld2, 9330159F19Rik, and Sept3 (Fig. 7 B). These DEGs were enriched in the NOD receptor signaling pathway (NES = 2.0682, P.adjust = 0.0114, FDR = 0.0071) using GSEA (Fig. 7 C). Analysis of KEGG pathways also indicated that these DEGs were enriched in DNA replication, proteasomes, the Hippo signaling pathway, and taste transduction (Fig. 7 D). Sequencing data demonstrate the activation of the Hippo pathway in poly(I:C) stimulated lung tissue, further substantiating prior findings that the Hippo pathway indeed plays a role in poly(I:C) induced lung injury. To pinpoint central genes within the protein-protein interaction (PPI) network, the intersection of the top 100 genes in all 12 CytoHubba algorithms was used to obtain two hub genes, Cxcl5 and NLRP3 (Fig. 7 E). NLRP3 plays a significant role in the maturation and release of IL-1β, suggesting that the Hippo pathway may facilitate IL-1β formation and release through the upregulation of NLRP3 expression[ 25 , 26 ]. Another PPI network for hub genes was constructed using the GeneMANIA database (Fig. 7 F). GO and KEGG analyses were performed on 22 genes (including two hub genes, 20 genes related to hub genes, and 232 connections) (Fig. 7 G–H). These core genes were also found to be abundant in the NOD receptor signaling pathway. Hippo pathway regulates IL-1β secretion in macrophages via NLRP3 In the lung tissues of mice with poly(I:C) induced lung injury, there was a higher level of Cxcl5 and NLRP3 expression relative to the control group, whereas the expression of YAP1 was reduced. (Fig. 8 A). The levels of NETs in the lung tissues of mice with poly(I:C) induced lung injury were elevated compared to the control group, in contrast to the levels of neutrophil immune infiltration (Fig. 8 B). Additionally, The expression of Cxcl5 and NLRP3 showed a positive correlation with scores of NET, while the expression of YAP1 was found to have a negative relationship with NET scores (Fig. 8 C). This suggests that the Hippo pathway may be activated via the upregulation of Cxcl5 and NLRP3 to promote NETosis. NLRP3, which stands for NOD-like receptor family with a pyridine domain containing 3, plays a crucial role in inflammasomes by triggering the transformation of precursors to pro-inflammatory cytokines, such as IL-1β (pro-IL-1β), to their mature active forms. Therefore, the activation of the Hippo pathway in macrophages may be associated with IL-1β gene release via NLRP3 gene upregulation. Lentivirus vectors, with knocked down and overexpressed NLRP3, were successfully constructed (Fig. 8 D–F). The mRNA expression of NLRP3 were regulated in macrophages isolated from the BALF of mice and in RAW264.7 cells with or without poly(I:C) stimulated (Fig. 8 G). The activation of the Hippo pathway modulates NLRP3 expression and IL-1β secretion in macrophages in vitro (Fig. 8 H-J). Expression differences exist in the Hippo/NLRP3/IL-1β pathway in clinical samples To examine the function of the Hippo/NLRP3/IL-1β pathway, monocyte macrophages were procured from the circulating blood of individuals suffering from viral pneumonia and healthy volunteers. The Hippo pathway was activated in monocyte-macrophages from patients with viral pneumonia (Fig. 9 A–D), and both NLRP3 and IL-1β proteins were highly expressed in these patients compared to those in healthy volunteers (Fig. 9 E-H). Discussion NETosis refers to neutrophil cell death under the stimulation of autoinflammatory factors and antigens. This process exemplifies a series of emblematic pathological alterations, such as regulation of cellular proliferation, formation of cellular fragments, fusion of nuclear segments, disruption of the cell membrane, release of NETs, and ultimately, complete cellular disintegration [ 27 , 28 ]. NETosis plays a role not only in the immune response against bacterial infection but also in facilitating tumor metastatic processes and viral infection [ 29 – 31 ]. NETosis is beneficial for controlling infection; however, excessive NETosis activation can lead to tissue damage and inflammation, thereby exacerbating pneumonia [ 32 ]. In this study, we found that the Hippo pathway was activated in macrophages to promote NETosis via NLRP3 inflammasome formation and IL-1β release during virus-induced inflammation and lung injury. The Hippo signaling pathway is renowned for its essential role in regulating development, tissue regeneration, control of organ size, and cancer. Most studies have showed the Hippo-NLRP3 pathway is involved in regulating the release and activation of NETs, which is associated with the progression of inflammatory diseases[ 25 , 26 , 33 ]. Moreover, the Hippo pathway is triggered by MyD88-dependent Toll-like receptor signaling, with YAP enhancing the IκBα-mediated negative feedback and nuclear factor-κB expression under hepatitis B virus infection [ 34 ]. Aged mesenchymal stem cells show impaired immunosuppressive function due to the inhibition of the Hippo effector YAP and its target gene, signal transducer and activator of transcription [ 35 ]. Yang et al. recently reported that NETosis induces endothelial-to-mesenchymal transition via the Hippo-YAP pathway, revealing a relationship between NETosis and the Hippo pathway during diabetic wound healing [ 36 ]. In poly(I:C) induced lung injury, marked activation of the Hippo pathway and YAP occurred in macrophages rather than in neutrophils. The disparity in the activation of NETosis and the Hippo pathway could affect the safety of new approaches aimed at targeting receptors that recognize pathogens. NLRP3 inflammasomes are activated in neutrophils to regulate the innate immune defenses and are linked to a variety of diseases connected with Neutrophil Extracellular Traps (NETs) [ 37 , 38 ]. Under sterile conditions, the assembly of the NLRP3 inflammasome within neutrophils is facilitated by PAD4, which in turn encourages the process of NETosis [ 39 ]. The NLRP3 inflammasome is activated by cholesterol accumulation in myeloid cells, which enhances neutrophil recruitment and NETosis in atherosclerotic plaques [ 40 ]. In H9C2 cells and animals, NLRP3 inflammasome-mediated pyroptosis is reduced, and the Hippo pathway is downregulated by folic acid, effectively reducing T2DM-induced damage [ 41 ]. In turn, YAP enhances NLRP3 stability by inhibiting its interaction with E3 ligase β-TrCP1, thus preventing NLRP3's ubiquitination and subsequent proteasomal degradation at lys380 [ 26 ]. Hence, the NLRP3 inflammasome may be a bridge connecting the Hippo pathway and NETosis during the innate immune response. Our transcriptome sequencing and experiments demonstrated that NLRP3 activates the Hippo pathway to promote NETosis. Critical cytokines mediating NETosis, the Hippo pathway, and NLRP3 inflammasome activation include IL-1β, the most abundant cytokine after NLRP3 inflammasome activation and its GSDMD-mediated pyroptosis [ 42 , 43 ]. IL-1β activation leads to tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated ubiquitination of YAP at K252. This modification interferes with YAP's binding to angiomotin, facilitating its nuclear translocation. Thus, IL-1β appears to regulate the Hippo pathway through this mechanism [ 44 ]. Macrophage-derived IL-1β seems to enhance NETosis. It does this by boosting the arrival of neutrophils at plaque sites and by stimulating the activation of the NLRP3 inflammasome within neutrophils. [ 12 , 40 ]. The findings of the current research revealed that in macrophage-neutrophil co-cultures under neutralized IL-1β antibody and poly(I:C) infection, blockade of IL-1β secretion inhibited NETosis and the Hippo pathway, as well as NLRP3 inflammasome activation. However, our study has some limitations. First, it remains unclear whether other viruses also activate the Hippo pathway to promote NETosis via NLRP3 inflammasome-mediated IL-1β secretion. Secondly, the definitive mechanism underlying NETosis in viral pneumonia requires further investigation. Additionally, in vivo studies using knockout mice should be performed to explore the underlying molecular mechanisms. In conclusion, we identified a crucial function of NETosis and Hippo pathways in virus-induced lung injury. NETosis aggravates virus-induced lung injury and is increased in viral pneumonia by regulating the Hippo pathway via the NLRP3/IL-1β axis. Drug screening for Hippo and NLRP3/IL-1β pathways may suppress excessive inflammatory responses and intercept NETosis during viral infection. Collectively, Our research suggests that targeting the Hippo pathway, which is activated by NETosis through the NLRP3/IL-1β axis, could be a viable strategy for enhancing the immune system's defense against viral infections. Abbreviations ALI: Acute Lung Injury BALF: Bronchoalveolar Lavage Fluid CitH3: Citrullinated Histone H3 DEGs: Differentially Expressed Genes dsDNA: Double-Stranded DNA FDR: False Discovery Rate GSEA: Gene Set Enrichment Analysis MERS-CoV: Middle East Respiratory Syndrome Coronavirus MPO: Myeloperoxidase NETs: Neutrophil Extracellular Traps NETosis: Neutrophil Extracellular Trap Formation PAD4: Peptidyl Arginine Deiminase 4 PBS: Phosphate-Buffered Saline Poly(I:C): Polyinosinic-Polycytidylic Acid SARS-CoV: Severe Acute Respiratory Syndrome Coronavirus T2DM: Type 2 Diabetes Mellitus TNF-α: Tumor Necrosis Factor-Alpha Declarations Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding information This work was supported by the National Natural Science Foundation of China (No. 81760020, No. 82060024), "139" plan for high-level medical backbone talents of Guangxi Zhuang Autonomous Region (No. G202002015), and Guangxi Anesthesiology Clinical Medicine Research Center Construction Project (No: AD22035214). Data availability statement All relevant data were within the manuscript and its supplemental files. References Gow, N.A.R., et al., The importance of antimicrobial resistance in medical mycology . Nat Commun, 2022. 13(1): p. 5352. Gu, S., et al., Exploring Influenza A Virus-Induced Lung Injury and Immune Response Based on Humanized Lung-on-Chip. Discov Med, 2023. 35(177): p. 539–552. Wulf Hanson, S., et al., Estimated Global Proportions of Individuals With Persistent Fatigue, Cognitive, and Respiratory Symptom Clusters Following Symptomatic COVID-19 in 2020 and 2021. Jama, 2022. 328(16): p. 1604–1615. Cevik, M., et al., SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis. Lancet Microbe, 2021. 2(1): p. e13-e22. Newton, A.H., A. Cardani, and T.J. Braciale, The host immune response in respiratory virus infection: balancing virus clearance and immunopathology. Semin Immunopathol, 2016. 38(4): p. 471–82. Hanan, N., et al., The Many Faces of Innate Immunity in SARS-CoV-2 Infection. Vaccines (Basel), 2021. 9(6). Brinkmann, V., et al., Neutrophil extracellular traps kill bacteria . Science, 2004. 303(5663): p. 1532–5. Schultz, B.M., et al., Role of Extracellular Trap Release During Bacterial and Viral Infection. Front Microbiol, 2022. 13: p. 798853. Chan, L.L.Y., et al., Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses. Respir Res, 2020. 21(1): p. 160. Divolis, G., et al., Neutrophil-derived Activin-A moderates their pro-NETotic activity and attenuates collateral tissue damage caused by Influenza A virus infection. Front Immunol, 2024. 15: p. 1302489. Kim, S.J., et al., Platelet-Mediated NET Release Amplifies Coagulopathy and Drives Lung Pathology During Severe Influenza Infection. Front Immunol, 2021. 12: p. 772859. Yalcinkaya, M., et al., Cholesterol accumulation in macrophages drives NETosis in atherosclerotic plaques via IL-1β secretion. Cardiovasc Res, 2023. 119(4): p. 969–981. Cui, H., et al., Lung Myofibroblasts Promote Macrophage Profibrotic Activity through Lactate-induced Histone Lactylation. Am J Respir Cell Mol Biol, 2021. 64(1): p. 115–125. Quach, C., et al., Enhancing autophagy in CD11c(+) antigen-presenting cells as a therapeutic strategy for acute respiratory distress syndrome. Cell Rep, 2023. 42(8): p. 112990. Gao, X., et al., Interleukin-38 ameliorates poly(I:C) induced lung inflammation: therapeutic implications in respiratory viral infections. Cell Death Dis, 2021. 12(1): p. 53. Kastan, N., et al., Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues. Nat Commun, 2021. 12(1): p. 3100. Koch, A.T., et al., MyD88-Dependent Signaling Decreases the Antitumor Efficacy of Epidermal Growth Factor Receptor Inhibition in Head and Neck Cancer Cells. Cancer Res, 2015. 75(8): p. 1657–67. Jing, R., et al., Transforming growth factor-β1 attenuates inflammation and lung injury with regulating immune function in ventilator-induced lung injury mice. Int Immunopharmacol, 2023. 114: p. 109462. Duan, Z., et al., Role of LL-37 in thrombotic complications in patients with COVID-19. Cell Mol Life Sci, 2022. 79(6): p. 309. Pisareva, E., et al., Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA. Genome Med, 2022. 14(1): p. 135. Zeng, H., et al., Neutrophil Extracellular Traps may be a Potential Target for Treating Early Brain Injury in Subarachnoid Hemorrhage. Transl Stroke Res, 2022. 13(1): p. 112–131. Davis, J.R. and N. Tapon, Hippo signalling during development . Development, 2019. 146(18). Tang, W., et al., Hippo signaling pathway and respiratory diseases . Cell Death Discov, 2022. 8(1): p. 213. Li, C., et al., Hippo Signaling Controls NLR Family Pyrin Domain Containing 3 Activation and Governs Immunoregulation of Mesenchymal Stem Cells in Mouse Liver Injury. Hepatology, 2019. 70(5): p. 1714–1731. Byeon, H.E., et al., HDAC11 Regulates Palmitate-induced NLRP3 Inflammasome Activation by Inducing YAP Expression in THP-1 Cells and PBMCs. Endocrinology, 2024. 165(3). Wang, D., et al., YAP promotes the activation of NLRP3 inflammasome via blocking K27-linked polyubiquitination of NLRP3. Nat Commun, 2021. 12(1): p. 2674. Zhang, R., et al., Neutrophil autophagy and NETosis in COVID-19: perspectives . Autophagy, 2023. 19(3): p. 758–767. Sergunova, V., et al., Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study. Cells, 2023. 12(17). Bhargavan, B. and G.D. Kanmogne, SARS-CoV-2 Spike Proteins and Cell-Cell Communication Induce P-Selectin and Markers of Endothelial Injury, NETosis, and Inflammation in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for the Pathogenesis of COVID-19 Coagulopathy. Int J Mol Sci, 2023. 24(16). Hu, Y., H. Wang, and Y. Liu, NETosis: Sculpting tumor metastasis and immunotherapy. Immunol Rev, 2023. Thierry, A.R., Netosis creates a link between diabetes and Long COVID. Physiol Rev, 2023. Lin, H., et al., NETosis promotes chronic inflammation and fibrosis in systemic lupus erythematosus and COVID-19. Clin Immunol, 2023. 254: p. 109687. Wang, Y.M., et al., IL-37 improves mice myocardial infarction via inhibiting YAP-NLRP3 signaling mediated macrophage programming. Eur J Pharmacol, 2022. 934: p. 175293. Luo, X., et al., Hippo Pathway Counter-Regulates Innate Immunity in Hepatitis B Virus Infection. Front Immunol, 2021. 12: p. 684424. Yang, X., et al., Hippo Pathway Activation in Aged Mesenchymal Stem Cells Contributes to the Dysregulation of Hepatic Inflammation in Aged Mice. Adv Sci (Weinh), 2023. 10(27): p. e2300424. Yang, S., et al., Neutrophil Extracellular Traps Delay Diabetic Wound Healing by Inducing Endothelial-to-Mesenchymal Transition via the Hippo pathway. Int J Biol Sci, 2023. 19(1): p. 347–361. Byun, D.J., et al., NLRP3 Exacerbate NETosis-Associated Neuroinflammation in an LPS-Induced Inflamed Brain. Immune Netw, 2023. 23(3): p. e27. Yang, S., et al., Disulfiram accelerates diabetic foot ulcer healing by blocking NET formation via suppressing the NLRP3/Caspase-1/GSDMD pathway. Transl Res, 2023. 254: p. 115–127. Münzer, P., et al., NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions. Front Immunol, 2021. 12: p. 683803. Westerterp, M., et al., Cholesterol Efflux Pathways Suppress Inflammasome Activation, NETosis, and Atherogenesis. Circulation, 2018. 138(9): p. 898–912. Hong, L., et al., Folic Acid Alleviates High Glucose and Fat-Induced Pyroptosis via Inhibition of the Hippo Signal Pathway on H9C2 Cells. Front Mol Biosci, 2021. 8: p. 698698. Minns, M.S., et al., NLRP3 selectively drives IL-1β secretion by Pseudomonas aeruginosa infected neutrophils and regulates corneal disease severity. Nat Commun, 2023. 14(1): p. 5832. Yang, J., et al., FABP4 in macrophages facilitates obesity-associated pancreatic cancer progression via the NLRP3/IL-1β axis. Cancer Lett, 2023. 575: p. 216403. Liu, M., et al., Macrophage K63-Linked Ubiquitination of YAP Promotes Its Nuclear Localization and Exacerbates Atherosclerosis. Cell Rep, 2020. 32(5): p. 107990. Additional Declarations (Not answered) Supplementary Files SupplementalTables.xlsx WBRAWDATA.pdf Cite Share Download PDF Status: Published Journal Publication published 14 Jul, 2025 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 18 Feb, 2025 Review # 2 received at journal 03 Feb, 2025 Reviewer # 2 agreed at journal 24 Jan, 2025 Review # 1 received at journal 21 Dec, 2024 Reviewer # 1 agreed at journal 08 Dec, 2024 Reviewers invited by journal 30 Jun, 2024 Submission checks completed at journal 24 Jun, 2024 First submitted to journal 22 Jun, 2024 Unknown event 17 Jun, 2024 Editor assigned by journal 16 Jun, 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-4591287","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":320718494,"identity":"bf466e42-4ab6-4f36-9341-071a21b78523","order_by":0,"name":"Linghui Pan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3OrwvCQBTA8RsHs9xYncW/4VmUYfBf2WGwHCJY1TjLNLv/QsswPjnQMrEaFcGiQTGtuc0fbZtR8L7hbYz34Y0Qleo309AhxCGU4uuDU2xeRH+vfkOeawy+I/XSBvGwGHRgze5nQ0hilgSQaJFNbK/jIA/XPZBGYPuBJGXvAto4zCaAApC7Kz6LCdxiAjsBVHNzyPbyJuwEPCbNQrJLr/QTQvfpFauA2NP0CnJf6jXND9rMCk/d5TiH1E1RPUTukE+28ng3gkbFHLXm+yjvx54PmQzdigdL3jAbfMgwGfSat6lSqVT/2wO6eFw6qGo6QAAAAABJRU5ErkJggg==","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Linghui","middleName":"","lastName":"Pan","suffix":""},{"id":320718495,"identity":"e448152c-ebba-4c66-be1f-a33bc92967a6","order_by":1,"name":"Bijun Luo","email":"","orcid":"","institution":"Guangxi Clinical Research Center for Anesthesiology","correspondingAuthor":false,"prefix":"","firstName":"Bijun","middleName":"","lastName":"Luo","suffix":""},{"id":320718496,"identity":"67e54de1-fd9e-41aa-8515-3aa2e3835e48","order_by":2,"name":"Xiaoxia Wang","email":"","orcid":"","institution":"The Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Wang","suffix":""},{"id":320718497,"identity":"6e457732-ec1c-4a57-a177-a8e96460a6e7","order_by":3,"name":"Jinyuan Lin","email":"","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jinyuan","middleName":"","lastName":"Lin","suffix":""},{"id":320718498,"identity":"d3d299e7-b5e9-4ef0-b457-c67fe2f82bfc","order_by":4,"name":"Jianlan Mo","email":"","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jianlan","middleName":"","lastName":"Mo","suffix":""},{"id":320718499,"identity":"893d56f1-044d-4d65-a680-71ca281aa9bd","order_by":5,"name":"Jiaan Xie","email":"","orcid":"","institution":"The Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Jiaan","middleName":"","lastName":"Xie","suffix":""},{"id":320718500,"identity":"646c8ea3-e270-4745-a6f1-6f07e92e23b3","order_by":6,"name":"Yanqiong Zhou","email":"","orcid":"","institution":"The Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Yanqiong","middleName":"","lastName":"Zhou","suffix":""},{"id":320718501,"identity":"8e6a7ee5-4672-4509-b53e-a80591801ce6","order_by":7,"name":"Jifeng Feng","email":"","orcid":"","institution":"The Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Jifeng","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2024-06-17 02:16:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4591287/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4591287/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-025-02556-z","type":"published","date":"2025-07-14T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61091879,"identity":"5b14368f-b030-45c0-908b-d44957925b21","added_by":"auto","created_at":"2024-07-25 13:19:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3470903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNETosis is involved in viral pneumonia with excessive inflammatory cytokines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B)\u003c/strong\u003e Comparative analysis of NETosis biomarkers in neutrophils and serum of peripheral blood from patients with viral pneumonia versus healthy volunteers via immunofluorescence. \u003cstrong\u003e(C) \u003c/strong\u003eGraphic presentations of fluorescence mean densities of NETosis biomarkers. \u003cstrong\u003e(D)\u003c/strong\u003e Assessment of CitH3 protein expression in serum sample from patients with viral pneumonia and healthy volunteers using western blotting. \u003cstrong\u003e(E)\u003c/strong\u003e Quantitative analysis of the protein CitH3 relative to Tubulin. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of serum dsDNA and LL-37 levels conducted through Enzyme linked immunosorbent assay (ELISA). \u003cstrong\u003e(G)\u003c/strong\u003e Evaluation of serum pro-inflammatory cytokines, including TNF-α, IL-1β, and GM-CSF, via ELISA. All data are representative as means ± s.e.m of three independent experiments. L Student’s t-test for A-G; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/1bc3635cc35332a55535b609.png"},{"id":61092684,"identity":"56dcb830-852a-4010-8bb2-24a0fdf38025","added_by":"auto","created_at":"2024-07-25 13:27:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3503818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of NETosis in mouse acute \u003c/strong\u003e\u003cu\u003e\u003cstrong\u003elung injury\u003c/strong\u003e\u003c/u\u003e\u003cstrong\u003e induced by poly(I:C) stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) H\u0026amp;E and Masson staining of lung tissues from mice subjected to poly(I:C) stimulation versus control vehicle respectively. Scale bar = 50 μm. \u003cstrong\u003e(B-D)\u003c/strong\u003e Comparative analysis of NETosis biomarkers in lung tissues from mice with or without poly(I:C) stimulation via immunofluorescence. B, Scale bar = 5 μm; C-D, Scale bar = 50 μm. \u003cstrong\u003e(E) \u003c/strong\u003eGraphic presentations of fluorescence mean densities of CitH3 and MPO. \u003cstrong\u003e(F)\u003c/strong\u003e Assessment of CitH3 protein in lung tissues from mice with or without poly(I:C) stimulation using western blotting.\u003cstrong\u003e (G) \u003c/strong\u003eQuantitative analysis of the protein CitH3 relative to GAPDH. \u003cstrong\u003e(H)\u003c/strong\u003e Serum concentrations of dsDNA and LL-37 were assessed by ELISA. All data are representative as means ± s.e.m of three independent experiments. Student’s t-test for A-F; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/44d0069798662f3e2417002a.png"},{"id":61091880,"identity":"9b7b4f67-db5f-4443-bb21-00b01c60382d","added_by":"auto","created_at":"2024-07-25 13:19:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2302274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of the Hippo pathway in macrophages following poly(I:C) stimulation in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eExpression of Hippo pathway expression in lung tissues from mice treated with poly(I:C) compared to untreated controls. \u003cstrong\u003e(B) \u003c/strong\u003eThe protein expression of Hippo pathway were quantified using densitometry on ImageJ. Phospho-LATS1 and LATS1, phospho-MST and MST1, phospho-MST and MST2, phospho-YAP and YAP protein were normalised to Tubulin and the ratio is presented. \u003cstrong\u003e(C)\u003c/strong\u003e Relative expression of Yap protein in lung tissues from mice treated with or without poly(I:C) was assessed after nuclear-plasma separation. \u003cstrong\u003e(D) \u003c/strong\u003eQuantitative analysis of the protein YAP in nuclear relative to Lamin B1. \u003cstrong\u003e(E)\u003c/strong\u003e The levels of inflammatory cytokines and chemokines in BALF. \u003cstrong\u003e(F) \u003c/strong\u003eThe levels of inflammatory cytokines and chemokines in serum. (\u003cstrong\u003eG\u003c/strong\u003e) YAP and neutrophils colocalization in peripheral blood of mice infected with poly(I:C). Scale bar = 50 μm. \u003cstrong\u003e(H-I)\u003c/strong\u003e Colocalization analysis of YAP with macrophages in peripheral blood (H) and BALF (I) from poly(I:C) stimulated mice. Scale bar = 20 μm. \u003cstrong\u003e(J) \u003c/strong\u003eQuantification of the colocalization between Ly6G or F4/80 and YAP. \u003cstrong\u003e(K)\u003c/strong\u003e Expression of the Hippo pathway in macrophages from mice treated with or without poly(I:C). \u003cstrong\u003e(L) \u003c/strong\u003eQuantification of phospho-LATS1 and LATS1, phospho-MST and MST1, phospho-MST and MST2, phospho-YAP and YAP protein in macrophages from mice were normalised to Tubulin and the ratio is presented. \u003cstrong\u003e(M) \u003c/strong\u003eRelative expression of Yap protein in macrophages following nuclear-plasma separation from mice treated with or without poly(I:C). \u003cstrong\u003e(N) \u003c/strong\u003eQuantitative analysis of the protein YAP in nuclear relative to Lamin B1. All data are representative as means ± s.e.m of three independent experiments. Student’s t-test for A-I; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/78d39188e72c41da639b6c54.png"},{"id":61092686,"identity":"e8953c25-7df7-4532-a99d-ec9be9d45cb9","added_by":"auto","created_at":"2024-07-25 13:27:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1702346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of the Hippo Pathway in Macrophages Enhances NETosis under poly(I:C) stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Relative expression of Yap protein in RAW246.7 cells treated with or without poly(I:C) was assessed, following nuclear-plasma separation. \u003cstrong\u003e(B) \u003c/strong\u003eQuantitative analysis of the protein YAP in nuclear relative to Lamin B1. \u003cstrong\u003e(C)\u003c/strong\u003e Hippo pathway expression in RAW246.7 cells treated with or without poly(I:C). \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of phospho-LATS1 and LATS1, phospho-MST and MST1, phospho-MST and MST2, phospho-YAP and YAP protein in macrophages from mice were normalised to Tubulin and the ratio is presented. \u003cstrong\u003e(E)\u003c/strong\u003e Assessment of\u003cstrong\u003e \u003c/strong\u003eNETosis biomarker levels in the cocultures of neutrophils and conditioned medium for RAW246.7 cells under poly(I:C) stimulation, determined using immunofluorescence. \u003cstrong\u003e(F)\u003c/strong\u003e CitH3 protein expression in the cocultures of neutrophils and conditioned medium for RAW246.7 cells under poly(I:C) stimulation was evaluated by western blotting. \u003cstrong\u003e(G) \u003c/strong\u003eQuantitative analysis of the protein CitH3 relative to GAPDH. \u003cstrong\u003e(H)\u003c/strong\u003e dsDNA and LL-37 levels in the cocultures of neutrophils and conditioned medium for RAW246.7 cells under poly(I:C) stimulation were evaluated by ELISA. Note: IN, Lats-IN-1 is a potent and ATP-competitive inhibitor of LATS1 and LATS2 kinases. All data are representative as means ± s.e.m of three independent experiments. Student’s t-test for A-I; *, p \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/f5d0d5a6ea36ffce77b67039.png"},{"id":61091885,"identity":"77266a35-19c9-48bd-a7e0-cff52885c740","added_by":"auto","created_at":"2024-07-25 13:19:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":680452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1β mediates the macrophages-inducing NETosis under poly(I:C) stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRelative expression of IL-1β protein in RAW246.7 cells treated with poly(I:C) and Hippo pathway inhibitors was assessed. \u003cstrong\u003e(B) \u003c/strong\u003eQuantitative analysis of the protein IL-1β relative to Tubulin under different conditions. \u003cstrong\u003e(C)\u003c/strong\u003e Measurement of IL-1β level in culture supernatants from RAW246.7 cells treated with poly(I:C) and Hippo pathway inhibitors Lats-IN-1 by ELISA. \u003cstrong\u003e(D)\u003c/strong\u003e NETosis biomarker levels in the cocultures of neutrophils and conditioned medium for RAW246.7 cells under poly(I:C) stimulation and and neutralized IL-1β antibody were assessed by immunofluorescence. \u003cstrong\u003e(E) \u003c/strong\u003eGraphic presentations of fluorescence mean densities of CitH3 and MPO under different conditions. \u003cstrong\u003e(F)\u003c/strong\u003e Evaluation of CitH3 protein expression in the cocultures of neutrophils and conditioned medium for RAW246.7 cells under poly(I:C) stimulation and neutralized IL-1β antibody by western blotting. \u003cstrong\u003e(G) \u003c/strong\u003eQuantitative analysis of the protein CitH3 relative to Tubulin. \u003cstrong\u003e(H)\u003c/strong\u003e dsDNA and LL-37 levels in the cocultures of neutrophils and conditioned medium for RAW246.7 cells under poly(I:C) stimulation and neutralized IL-1β antibody were evaluated by ELISA. Note: IN, Lats-IN-1 is a potent and ATP-competitive inhibitor of LATS1 and LATS2 kinases. All data are representative as means ± s.e.m of three independent experiments. Student’s t-test for A-I; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/f3f5bbcfbf403dd464143a40.png"},{"id":61091874,"identity":"0760cb4e-2f31-4402-a7b5-7cbd4b328e91","added_by":"auto","created_at":"2024-07-25 13:19:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1570917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1β mediates the macrophages-inducing NETosis under poly(I:C) stimulation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eEvaluation of IL-1β protein expression in macrophages isolated from the BALF of mice with poly(I:C) infection and treated with Hippo pathway inhibitors. \u003cstrong\u003e(B) \u003c/strong\u003eQuantitative analysis of the protein IL-1β relative to Tubulin under different conditions. \u003cstrong\u003e(C)\u003c/strong\u003e Measurement of Serum IL-1β level from mice with poly(I:C) infection and treated with Lats-IN-1 by ELISA. \u003cstrong\u003e(D)\u003c/strong\u003e NETosis biomarker levels from Cells in the BALF of mice with poly(I:C) stimulation and neutralized IL-1β antibody were assessed by immunofluorescence. \u003cstrong\u003e(E) \u003c/strong\u003eGraphic presentations of fluorescence mean densities of CitH3 and MPO under different conditions. \u003cstrong\u003e(F)\u003c/strong\u003e CitH3 protein expression in the lung tissues from mice with poly(I:C) stimulation and neutralized IL-1β antibody was evaluated by western blotting. \u003cstrong\u003e(G) \u003c/strong\u003eQuantitative analysis of the protein CitH3 relative to Tubulin. \u003cstrong\u003e(H-I)\u003c/strong\u003e dsDNA and LL-37 levels in the peripheral blood and BALF from mice infected with poly(I:C) and treated with neutralized IL-1β antibody, evaluated by ELISA. Note: IN, Lats-IN-1 is a potent and ATP-competitive inhibitor of LATS1 and LATS2 kinases. All data are representative as means ± s.e.m of three independent experiments. Student’s t-test for A-I; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/b8a0e3f82501dbd1399eff3c.png"},{"id":61092687,"identity":"dc7f4f95-2ebb-4448-a402-b4c915ee2a39","added_by":"auto","created_at":"2024-07-25 13:27:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1416154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of lung tissues from mice treated with poly(I:C) .\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eVolcanic map describes DEGs between the samples of the virus-induced lung injury and control group. Red, blue, and gray dots represent gene expression levels associated with upregulation, downregulation, and no significant expression, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e Heat map showed the top 5 up-regulated and down-regulated DEGs. \u003cstrong\u003e(C) \u003c/strong\u003eGSEA analysis showed that NOD like receptor signaling pathway was significantly enriched in the virus-induced lung injury. \u003cstrong\u003e(D)\u003c/strong\u003e The top 20 pathways with significant differences in GSVA.\u003cstrong\u003e(E)\u003c/strong\u003e Hub genes obtained from the PPI network, including Ccxl5 and Nlrp3. \u003cstrong\u003e(F) \u003c/strong\u003eCo-expression network diagram of hub genes. (\u003cstrong\u003eG-H\u003c/strong\u003e) GO and KEGG analysis of co-expressed hub genes.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/599b60c27f2192b674164afd.png"},{"id":61093243,"identity":"9cc7d75c-e7d0-4769-8b20-dc7f897b4e6c","added_by":"auto","created_at":"2024-07-25 13:35:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":443346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHippo pathway regulates macrophage IL-1β secretion via NLRP3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eBox plot of the expression levels of Cxcl5, Nlrp3, and Yap1 between the virus-induced lung injury and control group. \u003cstrong\u003e(B)\u003c/strong\u003e Box graph of NETosis related genes using ssGSEA enrichment scores between the virus-induced lung injury and control group. \u003cstrong\u003e(C)\u003c/strong\u003e Correlation heat map. \u003cstrong\u003e(D-F) \u003c/strong\u003eThe efficiency of knockdown and overexpression of NLRP3 via lentivirus vectors were assessed using PCR and Westren blotting detection. \u003cstrong\u003e(G) \u003c/strong\u003eLevels of NLRP3 mRNA in macrophages isolated from the BALF of mice and in RAW264.7 cells with or without poly(I:C) stimulated.\u003cstrong\u003e (H-I)\u003c/strong\u003e IL-1β expression in macrophages with or without NLRP3 regulation was evaluated using Westren blotting. Hippo pathway protein expressions in macrophages with or without NLRP3 regulation was evaluated using Western blotting. \u003cstrong\u003e(J) \u003c/strong\u003eQuantitative analysis of the protein IL-1β relative to Tubulin under different conditions. All data are representative as means ± s.e.m of three independent experiments. Student’s t-test for A-I; *, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/b66e814cf2e66730b8198612.png"},{"id":61093244,"identity":"0bf8f506-3134-4519-9b1f-dd0234d0bea5","added_by":"auto","created_at":"2024-07-25 13:35:46","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":762889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHippo/NLRP3/IL-1β pathway is activated in patients with viral pneumonia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Hippo pathway expression in mononuclear macrophages isolated from peripheral blood of patients with viral pneumonia and healthy individuals. \u003cstrong\u003e(B)\u003c/strong\u003e Relative expression of YAP protein in mononuclear macrophages isolated from peripheral blood of patients with viral pneumonia and healthy individuals was assessed after nuclear-plasma separation. \u003cstrong\u003e(C)\u003c/strong\u003e NLRP3 expression in mononuclear macrophages isolated from peripheral blood of patients with viral pneumonia and healthy individuals. \u003cstrong\u003e(D)\u003c/strong\u003e Evaluation of IL-1β levels in mononuclear macrophages isolated from peripheral blood of patients with viral pneumonia and healthy individuals. All data are representative as means ± s.e.m of three independent experiments. Student’s t-test for A-I; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/4c965da2b7d6a5c7b8ded9f8.png"},{"id":61091882,"identity":"ce224318-8b11-4947-8ccf-e9133e419322","added_by":"auto","created_at":"2024-07-25 13:19:46","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":443815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed Hippo pathway and NLRP3-driven NETosis in macrophages: mechanisms of viral pneumonia aggravation (by Medpeer).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral pneumonia activates the Hippo signaling pathway within macrophages, resulting in decreased nuclear translocation of YAP. This reduction in YAP translocation leads to a weakened inhibition of NLRP3, thereby promoting the activation of the NLRP3 inflammasome complex. Consequently, there is an increase in IL-1β secretion, which further enhances the levels of NETosis.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/913ad88429017085124a1964.png"},{"id":86742706,"identity":"bbbbd068-2378-4b26-a3b0-4e7016bdb894","added_by":"auto","created_at":"2025-07-15 07:13:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18159092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/73d9bb62-719f-4657-84cf-9c82abc260cb.pdf"},{"id":61091884,"identity":"d72944ea-b159-4c63-aae1-8f81727dd04e","added_by":"auto","created_at":"2024-07-25 13:19:46","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":8208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/8d17b085e09f2011aaf70004.xlsx"},{"id":61091886,"identity":"9d1f4530-a148-4b41-8798-f0f5ffd36027","added_by":"auto","created_at":"2024-07-25 13:19:47","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":33937406,"visible":true,"origin":"","legend":"","description":"","filename":"WBRAWDATA.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4591287/v1/31be1d412634a3e8a26bd460.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Hippo pathway and NLRP3-driven NETosis in macrophages: Mechanisms of viral pneumonia aggravation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRespiratory viral infections, such as influenza, SARS-CoV, and MERS-CoV, are global public health problems with substantial seasonal and pandemic morbidity and mortality [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Current treatments primarily focus on supportive care, antiviral medications, and in some cases, the use of corticosteroids, however, these approaches often face limitations regarding efficacy, antiviral resistance, and potential adverse effects[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. There is a critical need to delve deeper into the underlying mechanisms of virus-host interactions.\u003c/p\u003e \u003cp\u003eThe immune microenvironment plays a pivotal role in recognizing, eliminating viruses, and infected cells during viral lung injury. Innate immune cells such as neutrophils, natural killer (NK) cells, and macrophages are rapidly recruited to the site of infection, where they exert antiviral functions[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The immune microenvironment within viral lung damage is critical at all stages, from initial antiviral defense to tissue damage and subsequent repair processes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Understanding the dynamic changes and mechanisms of action of various immune cells within the immune microenvironment is vital for developing novel therapeutic strategies and improving patient outcomes. This comprehensive insight can lead to the identification of specific targets that can be modulated to enhance viral clearance while minimizing immune-mediated tissue injury, thus offering a balanced approach to the treatment of viral lung injuries.\u003c/p\u003e \u003cp\u003eNETosis, the process by which neutrophils release neutrophil extracellular traps (NETs) as part of the innate immune response, plays a critical role in the body's defense against various pathogens, including viruses[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. NETs, consisting of DNA fibers adorned with antimicrobial proteins, have been documented to play a pivotal role in trapping and neutralizing virus particles, thus impeding viral replication and dissemination[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. While NETs serve a protective purpose by limiting viral spread within the host, excessive or dysregulated NET formation can lead to increased pulmonary inflammation and tissue damage[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent research has highlighted the induction of NETs by a variety of inflammatory mediators, emphasizing the complexity of interactions between viral infections and the host immune system[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Understanding the balance between beneficial and detrimental outcomes of NET formation in the context of viral infections could uncover novel insights into the pathophysiology of viral lung injury. Therefore, investigating the relationship between NETosis and viral lung injury helps us to understand the mechanisms of viral lung damage more deeply and may provide new strategies for treatment.\u003c/p\u003e \u003cp\u003eIn the present study, we identified the hub gene NLRP3 by developing a mouse model of viral lung injury with utilizing immunofluorescence, Western Blot, ELISA, and transcriptomic sequencing experiments. Our findings conclude that during poly I:C-induced lung injury, the Hippo pathway is activated in macrophages, leading to an upregulation of NLRP3 expression, which in turn promotes the production of IL-1β. Elevated IL-1β levels trigger an increase in the formation of neutrophil extracellular traps (NETs), thereby exacerbating the severity of pneumonia. These insights into the molecular pathophysiology of viral lung injury illuminate potential targets for therapeutic intervention aimed at modulating inflammation and preventing lung damage.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eClinical sample\u003c/p\u003e \u003cp\u003e Our study involved a total of 20 participants, which included 10 severe viral pneumonia patients and 10 healthy volunteers, all sourced from the Intensive Care Unit of the Affiliated Tumor Hospital of Guangxi Medical University. The pneumonia patients were diagnosed based on acute respiratory infection symptoms and confirmed viral infection through direct immunofluorescence antigen detection, real-time fluorescence PCR nucleic acid detection, or pathogen metagenomic next-generation sequencing. A Cycle threshold (Ct) value of \u0026le;\u0026thinsp;35 was considered positive for viral presence, with values between 35 and 40 considered weakly positive. Our inclusion criteria for pneumonia diagnosis required fever, cough, wheezing, accelerated breathing, and wet rales on lung auscultation, or chest imaging indicative of pneumonia. Severe pneumonia was diagnosed based on one primary criterion or three or more secondary criteria, including the need for invasive mechanical ventilation or vasoconstrictor treatment for septic shock. All participants provided informed consent, and the study protocol was approved by the Ethics Committee of the Guangxi Medical University Tumor Hospital (approval number 2023-3-7), adhering to the guidelines of the Declaration of Helsinki. Peripheral blood was collected from both groups at 8 AM, with neutrophils isolated using density gradient centrifugation and serum retained and stored at -80\u0026deg;C for further analysis.\u003c/p\u003e \u003cp\u003eMice\u003c/p\u003e \u003cp\u003e This investigation followed the suggested guidelines from the People's Republic of China's Guide for Regulation and Administration of Laboratory Animals. The Guangxi Medical University\u0026rsquo;s Institutional Animal Care and Use Committee sanctioned the study protocols. Anesthesia, using ketamine hydrochloride and xylazine, was employed during all animal experiments to mitigate discomfort as much as possible. Wild-type C57BL/6J male mice aged from 6\u0026ndash;8 weeks and weighing about 25\u0026thinsp;\u0026plusmn;\u0026thinsp;5g, not previously used for any experiments, were obtained from the Animal Center of Guangxi Medical University (Nanning, China). These mice were housed in a room fitted with air-filters where they could freely access food and water. The conditions of the room were maintained at a temperature of 20\u0026ndash;25 \u0026ordm;C, with 50\u0026ndash;70% humidity levels.\u003c/p\u003e \u003cp\u003eCell\u003c/p\u003e \u003cp\u003eRAW 264.7 cells were purchased from ATCC. Primary alveolar macrophages (AMs) were generated from wild-type C57BL/6 mice with or without poly(I:C) stimulation (HMW, tlrl-pic; InvivoGen, USA). Briefly, primary AMs were obtained from bronchoalveolar lavage fluid (BALF) after erythrocyte lysis. BALF was plated for 1 h, followed by thorough washing to remove unattached cells. Adherent cells were used as primary AMs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. RAW 264.7 cells and AMs were cultured in RMPI 1640 medium containing 10% fetal bovine serum (FBS) (10091148, Gibco, New Zealand), 20 mM HEPES, and 2 mM L-glutamine. Following this separation, AMs were further isolated using magnetic bead separation with CD14 MicroBeads (Miltenyi Biotec, Germany), targeting the CD14\u003csup\u003e+\u003c/sup\u003e monocyte population, which is a standard practice for monocyte isolation due to its high specificity and efficiency. Approximately 95% of the harvested cells were alveolar macrophages, as confirmed by flow cytometry.\u003c/p\u003e \u003cp\u003eNeutrophils were extracted from ethylenediaminetetraacetic acid (EDTA) (E809069, Macklin, China)-anticoagulated entire blood collected from wild-type C57BL/6 mice with or without poly(I:C) stimulation using density gradient centrifugation. The entire blood was layered upon a density gradient comprising a lower layer of Histopaque\u0026reg;-1119 (11191, Sigma-Aldrich, Vienna, Austria) and an upper layer of Ficoll-Paque PLUS (17-1440-03, GE Healthcare, Uppsala, Sweden), followed by centrifugation at 700 \u0026times; g lasting for 30 minutes. The fraction comprising polymorphonuclear cells was located above the erythrocyte pellet and was carefully gathered, then washed with 1 \u0026times; Dulbecco's phosphate-buffered saline (DPBS) (15575-020, Thermo Fisher Scientific, Vienna, Austria). Subsequently, these cells were resuspended in VersaLyse Lysing Solution (A09777, Beckman Coulter, Marseille, France) aimed at eliminating red blood cells. The purity of the neutrophil population was typically higher than 90% as evaluated via flow cytometry. Viability of the immunomagnectically isolated neutrophils was evaluated by flow cytometry using cell nucleic acid fluorescent dye, Sytox-Green. For flow cytometry, live single-cell suspensions at a concentration of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ml were first blocked with anti-mouse CD16/32 Fc receptor block followed by surface labeling of anti-CD45, anti-CD11b, and anti-Ly6G antibodies at room temperature for 20 min. Cells were then washed three times, resuspended in 1 ml of DPBS, and run on an cell analyzer. In the co-culture experiment, the conditioned medium of RAW 264.7 cells in each group was co-cultured with neutrophils isolated from peripheral blood of mice in the control group at a concentration of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e T cells/mL for 48 hours.\u003c/p\u003e \u003cp\u003eReagents administration\u003c/p\u003e \u003cp\u003eC57BL/6 mice were intranasally challenged with 5mg/Kg high-molecular-weight poly(I:C) at a concentration of 1 mg/mL to induce ALI/ARDS [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This administration was performed under light anesthesia to ensure precise delivery and minimize stress to the animals. The mice were euthanized after the final treatment to collect serum, BALF, and lung tissues for further downstream examinations. \u003cem\u003eIn vitro\u003c/em\u003e, RAW 264.7 cells, primary AMs, or co-cultures of conditioned medium and neutrophils were exposed to poly(I:C) (20 \u0026micro;g/mL) stimulation for 48 h with or without interventions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHippo pathway inhibitor, Lats-IN-1 (MedChemExpress, USA), is a potent and ATP-competitive inhibitor of LATS1 and LATS2 kinases. The administration regimen for Lats-IN-1 involved a 10 mg/kg intraperitoneal injection daily for 3 days, initiated 24 hours before and continued simultaneously with and 24 hours after administration of Poly(I:C) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Neutralizing IL-1β antibody (R\u0026amp;D Systems, Germany) were administered at a dose of 10 mg/kg via intraperitoneal injection daily for 2 days, timed concurrently with and 24 hours following Poly(I:C) exposure. In vitro treatments included exposure of RAW264.7 cell lines to 10 \u0026micro;M Lats-IN-1, and 5 \u0026micro;L/mL of neutralizing IL-1β antibody[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlasmids, small interfering RNAs (siRNAs), and transfection\u003c/p\u003e \u003cp\u003eAll shRNAs used in this study were provided by Sangon (Shanghai, China), as listed in Supplementary Table\u0026nbsp;1. All procedures related to the experiment were carried out as per the guidelines provided by the manufacturer. The supplementary material holds the detailed methods.\u003c/p\u003e \u003cp\u003eMeasurement of pulmonary edema, permeability, and cytokines\u003c/p\u003e \u003cp\u003eThe right upper lobe with excess water was eliminated using filter paper to ascertain its weight (W). The lung tissues were subjected to a drying process at 60\u0026deg;C for 48 hours to attain their dry weight (D). The calculation of the W/D ratio was used as a measurement index for pulmonary edema. An evaluation of changes in lung permeability was conducted by assessing total BALF protein using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). Additionally, a hemocytometer was used to count total cell infiltration. Interleukin 1β (IL-1β), tumor necrosis factor ɑ (TNF-ɑ), dsDNA, LL-37, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in cell culture supernatant, plasma, and BALF were measured using enzyme-linked immunosorbent assay kits (CUSABIO, Wuhan, China).\u003c/p\u003e \u003cp\u003eHistologic study\u003c/p\u003e \u003cp\u003eThe lower lobes of the right lung were preserved using 4% paraformaldehyde (30525-89-4; Sigma-Aldrich, AR, USA), and then encapsulated within the Tissue-Tek OCT compound (4583; Sakura, Tokyo, Japan). The pathological assessment of lung damage was independently evaluated by two authors on sections stained with hematoxylin and eosin, following criteria that had been reported earlier [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In order to analyze the accumulation of collagenous fibers in pulmonary fibrosis, the lung tissues were encased in paraffin and dyed using Masson's stain, following the guidelines provided by the manufacturer.\u003c/p\u003e \u003cp\u003eMeasurement of mRNA expression\u003c/p\u003e \u003cp\u003eTotal mRNA of the cells was extracted using TRIzol reagent (Thermo Fisher Scientific) following the guidelines listed by the manufacturer. The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) was used to prepare the cDNA, which was then quantified using the PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix (Applied Biosystems, A25742). The relative expression levels of mRNA were determined using the 2-△△ct cycle threshold method. The primer sequences of NLRP3 used were as follows: forward: 5\u0026prime;-GGAGCGGGAGCATGAACTCC-3\u0026prime;, reverse: 5\u0026prime;-GGAGCGGGAGCATGAACTCC-3\u0026prime;. The fold change, adjusted to GAPDH normalization, was utilized to illustrate the variances between groups.\u003c/p\u003e \u003cp\u003eImmunoblotting\u003c/p\u003e \u003cp\u003eThe left lower lung lobes were thoroughly mixed into a uniform solution using RIPA lysis buffer (20\u0026ndash;188, Sigma-Aldrich, AR, USA). During this process, to prevent protein degradation and dephosphorylation, both a Protease Inhibitor Tablet (product number 11836170001 from Roche, located in Basel, Switzerland) and a PhosphoSTOP Phosphatase Inhibitor Tablet (product number 4906845001, also from Roche in Basel, Switzerland) were added. This homogenization was achieved with the aid of a mechanical tissue homogenizer.The samples underwent lysis for a duration of thirty minutes at an icy temperature, followed by centrifugation at 12,000 g-force for 15 minutes. Following the measurement of protein concentrations by the bicinchoninic acid (BCA) assay, the obtained supernatants from the cell lysates were heated to 85\u0026deg;C for a duration of 5 minutes with a loading buffer added. Between 50 and 75 micrograms of proteins were subjected to separation through SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. After blocking a 1-hour incubation period at 22\u0026ndash;25℃ with 5% nonfat milk, the membranes underwent an overnight incubation with primary antibodies (Supplemental Table\u0026nbsp;2) at a temperature of 4\u0026deg;C. This was followed by a 1-hour incubation at room temperature with secondary antibodies (Abcam, Cambridge, UK) conjugated with horseradish peroxidase. Band intensities corresponding to different proteins were quantified from digitized films through the employment of an Odyssey\u0026reg; CLX imaging system (LI-COR, USA).\u003c/p\u003e \u003cp\u003eImmunostaining\u003c/p\u003e \u003cp\u003eAir-dried for half an hour, the frozen tissue samples, cellular suspensions, or sheets of adherent cells were then stabilized with a 3.7% solution of paraformaldehyde for a quarter of an hour, followed by a chilling immersion in undiluted methanol for another fifteen minutes. The slides underwent a blocking process using a solution of phosphate-buffered saline mixed with 3% goat serum (16210064, Gibco, CA, USA), 3% bovine serum albumin (SRE0096, Sigma-Aldrich, AR, USA), 0.2% Triton X-100 (Sigma-Aldrich, Arkansas, USA), and 0.02% NaN3 (S2002, Sigma-Aldrich, Arkansas, USA). Subsequently, they were treated with both primary antibodies and appropriate secondary antibodies. Comprehensive details regarding both the primary and secondary antibodies are provided in Supplemental Table\u0026nbsp;3. The specimens underwent a treatment process using ProLong\u0026reg;Gold Antifade Reagent containing 4', 6-diamidino-2-phenylindole (DAPI) (8961S, CST, Massachusetts, USA). Then they were examined using multiplex confocal microscopy with a LSM980 microscope from Zeiss, located in Germany.\u003c/p\u003e \u003cp\u003eTranscriptomics and processing of raw sequence data\u003c/p\u003e \u003cp\u003eTranscriptomes was conducted utilizing the Visium system from 10 x Genomics. Briefly, 10 mm fresh-frozen mouse lung sections, with or without poly(I:C) stimulation, were embedded in OCT and mounted on Visium slides, and the sections underwent a permeabilization procedure for 30 minutes to facilitate the release of mRNAs. These mRNAs subsequently adhered to the spatially barcoded oligonucleotides located on the underlying spots. Following this, a reverse transcription process was executed as per the manufacturer's protocol. cDNA libraries were sequenced using the Illumina NextSeq 2000 system with a sequencing depth of over 50,000 reads for each spot, producing more than 400\u0026nbsp;million reads for each section. The software Spaceranger, at version 3.1.0 by 10 x Genomics, performed alignment of individual spots from the Visium transcriptomics slides to the reference data of the GRCh38 mouse genome, resulting in the acquisition of raw counts. The data representing the expression patterns of the selected genes were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to conduct a Gene Ontology (GO) enrichment investigation, which encompasses the analysis of biological activities, cellular constituents, and molecular functionalities. All hub genes underwent analysis using DAVID for GO enrichment and KEGG pathway investigation, with counts\u0026thinsp;\u0026gt;\u0026thinsp;5 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01. To assess the interactive networks connecting all targeted genes, the STRING database was employed.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eTypically, experiments conducted \u003cem\u003ein vitro\u003c/em\u003e were replicated three times (except where noted differently), with results shown as the average value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean, based on a minimum of three separate experiments. Two groups were compared using the Student's t-test, while the one-way ANOVA with Tukey's post-hoc test was utilized for comparing more than two groups. Statistical significance was established when \u003cem\u003ep\u003c/em\u003e-values were less than 0.05. Statistical evaluations were carried out with the GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eNETosis is associated with inflammation in patients with viral pneumonia\u003c/p\u003e \u003cp\u003eTo evaluate the role of NETosis in viral pneumonia, NETosis biomarkers were measured, incorporating citrullinated histone H3 (CitH3), myeloperoxidase (MPO), neutrophil elastase (NE), and LL-37 in the neutrophil from peripheral blood, as well as dsDNA and LL-37 in the serum of individuals suffering from viral pneumonia and healthy participants [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The of viral pneumonia patients showed increased levels of CitH3, MPO, NE, and LL37 compared to healthy individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C). The levels of the CitH3 protein in patients suffering from viral pneumonia were also elevated when compared to those found in healthy individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E), and serum dsDNA and LL-37 levels were both increased in these patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Additionally, serum inflammatory cytokine levels, such as IL-1β, TNF-α, and GM-CSF, were higher in these patients than in healthy volunteers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), suggesting that significant inflammation was observed in patients with viral pneumonia. Taken together, NETosis may be associated with a high expression of pro-inflammatory factors, which are potential risk factors for viral pneumonia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNETosis is activated in mouse acute lung injury following poly(I:C) stimulation\u003c/p\u003e \u003cp\u003eTo investigate the relationship between NETosis and viral pneumonia, C57BL/6 mice underwent intranasal exposed to poly(I:C), resulting in evident pathological injury and the accumulation of collagen fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Neutrophils exhibited membrane rupture, releasing network structures containing DNA and antimicrobial proteins in mice with viral pneumonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Relative to the control mice, both CitH3 and MPO were exhibited at elevated levels in the pulmonary tissues of mice afflicted with poly(I:C) induced lung injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). Compared with the control group, the relative expression of CitH3 protein in mice with viral pneumonia at 24h, 48h, and 72h after poly(I:C) exposure was upregulated, peaking at 48 hours post-exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G). Additionally, dsDNA and LL-37 levels were higher in the serum and BALF of mice suffering from poly(I:C)-induced lung injury compared to those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). This shows that NETosis is significantly activated in mice with poly(I:C) induced lung injury, as indicated by lung injury and heightened levels of NETosis markers, peaking at 48 hours post-exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHippo pathway is activated in macrophages from poly(I:C) stimulated mice\u003c/p\u003e \u003cp\u003eAlthough the Hippo pathway is primarily associated with cell fate decisions, proliferation, migration, death, and organ size control [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the Hippo pathway is critical in pulmonary inflammatory diseases[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and its specific role in virus-induced lung injury remains unclear. Within the pulmonary tissue of mice stimulated with poly(I:C), the Hippo pathway is activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D), leading to increased levels of inflammatory factors and chemokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). YAP, a pivotal molecule within the Hippo pathway, undergoes phosphorylation and cytoplasmic retention upon pathway activation, resulting in diminished nuclear YAP levels. Immunofluorescence was used to examine the co-localization of Yes-associated protein (YAP) with neutrophils(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) or macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I), revealing that YAP co-localized with macrophages rather than neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). The YAP and Hippo pathways were assessed through the nucleocytoplasmic separation of macrophages extracted from the bronchoalveolar lavage fluid (BALF) of mice stimulated with poly(I:C). The findings verify that the stimulation of the Hippo pathway triggered by poly(I:C) stimulation takes place chiefly within macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-N).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHippo pathway in macrophages is activated to promote NETosis in murine viral pneumonia following poly(I:C) stimulation\u003c/p\u003e \u003cp\u003eInvestigating how activation of the Hippo pathway in macrophages following poly(I:C) stimulation promotes NETosis in murine viral pneumonia. In RAW264.7 cells stimulated with poly(I:C), the expression of nuclear YAP was decreased, while the Hippo pathway was activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). In the co-culture of conditioned medium from RAW246.7 cells and neutrophils, NETosis biomarkers in co-cultured neutrophils were upregulated under medium from poly(I:C) stimulated RAW246.7 cells. These biomarkers were abolished under medium from poly(I:C) stimulated RAW246.7 cells after the administration of Hippo pathway inhibitors(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). This suggests that Hippo pathway activation in macrophages stimulated with poly(I:C) promotes NETosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e experiments confirm that IL-1β plays a crucial role in facilitating the interaction between macrophages and NETs\u003c/p\u003e \u003cp\u003eSeveral cytokines have been found to stimulate or promote NETosis formation. For example, the activation of the Hippo pathway induces the expression of IL-1β, a critical pro-inflammatory factor implicated in the promotion of NETosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. IL-1β levels were increased in RAW246.7 cells that received poly(I:C) treatment, a change abolished in RAW246.7 cells treated with poly(I:C) and Hippo pathway inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). In the co-culture of neutrophils and conditioned medium from RAW246.7 cells under poly(I:C) stimulation and the neutralized IL-1β antibody, the promotion of NETosis by IL-1β was attenuated, suggesting that IL-1β is the key molecule mediating macrophage-induced NETosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eValidation of IL-1β as the critical mediator facilitating the interaction between macrophages and NETosis \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e experiments revealed that IL-1β levels were increased in isolated macrophages in the BALF of mice stimulated with poly(I:C), however, administration of a Hippo pathway inhibitor simultaneously reduced IL-1β levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C). Mice treated with poly(I:C) and neutralizing IL-1β antibody showed an inhibition of NETosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-H). These confirmed IL-1β as a key mediator linking macrophages to NETosis, where its elevation following poly(I:C) stimulation and reduction by Hippo pathway inhibition correlates with NETosis modulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTranscriptome sequencing identified NLRP3 downstream of the Hippo pathway that mediates IL-1β secretion and NETosis\u003c/p\u003e \u003cp\u003eTo delve deeper into the process through which the Hippo pathway facilitates the secretion and discharge of IL-1β, lung tissues from poly(I:C) stimulated mice and PBS as controls were harvested for transcriptome sequencing. Compared to the control group, there were 1625 differentially expressed genes (DEGs), including 1027 upregulated DEGs and 598 downregulated DEGs in poly(I:C) stimulated mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The top five upregulated DEGs were C1rb, Ocstamp, C1qb, Irf7, and Saa3, whereas the top five downregulated DEGs included Igfbp3, Cd209a, Crispld2, 9330159F19Rik, and Sept3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These DEGs were enriched in the NOD receptor signaling pathway (NES\u0026thinsp;=\u0026thinsp;2.0682, P.adjust\u0026thinsp;=\u0026thinsp;0.0114, FDR\u0026thinsp;=\u0026thinsp;0.0071) using GSEA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Analysis of KEGG pathways also indicated that these DEGs were enriched in DNA replication, proteasomes, the Hippo signaling pathway, and taste transduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Sequencing data demonstrate the activation of the Hippo pathway in poly(I:C) stimulated lung tissue, further substantiating prior findings that the Hippo pathway indeed plays a role in poly(I:C) induced lung injury. To pinpoint central genes within the protein-protein interaction (PPI) network, the intersection of the top 100 genes in all 12 CytoHubba algorithms was used to obtain two hub genes, Cxcl5 and NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). NLRP3 plays a significant role in the maturation and release of IL-1β, suggesting that the Hippo pathway may facilitate IL-1β formation and release through the upregulation of NLRP3 expression[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Another PPI network for hub genes was constructed using the GeneMANIA database (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). GO and KEGG analyses were performed on 22 genes (including two hub genes, 20 genes related to hub genes, and 232 connections) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG\u0026ndash;H). These core genes were also found to be abundant in the NOD receptor signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHippo pathway regulates IL-1β secretion in macrophages via NLRP3\u003c/p\u003e \u003cp\u003eIn the lung tissues of mice with poly(I:C) induced lung injury, there was a higher level of Cxcl5 and NLRP3 expression relative to the control group, whereas the expression of YAP1 was reduced. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The levels of NETs in the lung tissues of mice with poly(I:C) induced lung injury were elevated compared to the control group, in contrast to the levels of neutrophil immune infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Additionally, The expression of Cxcl5 and NLRP3 showed a positive correlation with scores of NET, while the expression of YAP1 was found to have a negative relationship with NET scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). This suggests that the Hippo pathway may be activated via the upregulation of Cxcl5 and NLRP3 to promote NETosis. NLRP3, which stands for NOD-like receptor family with a pyridine domain containing 3, plays a crucial role in inflammasomes by triggering the transformation of precursors to pro-inflammatory cytokines, such as IL-1β (pro-IL-1β), to their mature active forms. Therefore, the activation of the Hippo pathway in macrophages may be associated with IL-1β gene release via NLRP3 gene upregulation. Lentivirus vectors, with knocked down and overexpressed NLRP3, were successfully constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD\u0026ndash;F). The mRNA expression of NLRP3 were regulated in macrophages isolated from the BALF of mice and in RAW264.7 cells with or without poly(I:C) stimulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). The activation of the Hippo pathway modulates NLRP3 expression and IL-1β secretion in macrophages in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH-J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExpression differences exist in the Hippo/NLRP3/IL-1β pathway in clinical samples\u003c/p\u003e \u003cp\u003eTo examine the function of the Hippo/NLRP3/IL-1β pathway, monocyte macrophages were procured from the circulating blood of individuals suffering from viral pneumonia and healthy volunteers. The Hippo pathway was activated in monocyte-macrophages from patients with viral pneumonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;D), and both NLRP3 and IL-1β proteins were highly expressed in these patients compared to those in healthy volunteers (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNETosis refers to neutrophil cell death under the stimulation of autoinflammatory factors and antigens. This process exemplifies a series of emblematic pathological alterations, such as regulation of cellular proliferation, formation of cellular fragments, fusion of nuclear segments, disruption of the cell membrane, release of NETs, and ultimately, complete cellular disintegration [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. NETosis plays a role not only in the immune response against bacterial infection but also in facilitating tumor metastatic processes and viral infection [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. NETosis is beneficial for controlling infection; however, excessive NETosis activation can lead to tissue damage and inflammation, thereby exacerbating pneumonia [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, we found that the Hippo pathway was activated in macrophages to promote NETosis via NLRP3 inflammasome formation and IL-1β release during virus-induced inflammation and lung injury.\u003c/p\u003e \u003cp\u003eThe Hippo signaling pathway is renowned for its essential role in regulating development, tissue regeneration, control of organ size, and cancer. Most studies have showed the Hippo-NLRP3 pathway is involved in regulating the release and activation of NETs, which is associated with the progression of inflammatory diseases[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, the Hippo pathway is triggered by MyD88-dependent Toll-like receptor signaling, with YAP enhancing the IκBα-mediated negative feedback and nuclear factor-κB expression under hepatitis B virus infection [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Aged mesenchymal stem cells show impaired immunosuppressive function due to the inhibition of the Hippo effector YAP and its target gene, signal transducer and activator of transcription [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Yang et al. recently reported that NETosis induces endothelial-to-mesenchymal transition via the Hippo-YAP pathway, revealing a relationship between NETosis and the Hippo pathway during diabetic wound healing [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In poly(I:C) induced lung injury, marked activation of the Hippo pathway and YAP occurred in macrophages rather than in neutrophils. The disparity in the activation of NETosis and the Hippo pathway could affect the safety of new approaches aimed at targeting receptors that recognize pathogens.\u003c/p\u003e \u003cp\u003eNLRP3 inflammasomes are activated in neutrophils to regulate the innate immune defenses and are linked to a variety of diseases connected with Neutrophil Extracellular Traps (NETs) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Under sterile conditions, the assembly of the NLRP3 inflammasome within neutrophils is facilitated by PAD4, which in turn encourages the process of NETosis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The NLRP3 inflammasome is activated by cholesterol accumulation in myeloid cells, which enhances neutrophil recruitment and NETosis in atherosclerotic plaques [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In H9C2 cells and animals, NLRP3 inflammasome-mediated pyroptosis is reduced, and the Hippo pathway is downregulated by folic acid, effectively reducing T2DM-induced damage [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In turn, YAP enhances NLRP3 stability by inhibiting its interaction with E3 ligase β-TrCP1, thus preventing NLRP3's ubiquitination and subsequent proteasomal degradation at lys380 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Hence, the NLRP3 inflammasome may be a bridge connecting the Hippo pathway and NETosis during the innate immune response. Our transcriptome sequencing and experiments demonstrated that NLRP3 activates the Hippo pathway to promote NETosis.\u003c/p\u003e \u003cp\u003eCritical cytokines mediating NETosis, the Hippo pathway, and NLRP3 inflammasome activation include IL-1β, the most abundant cytokine after NLRP3 inflammasome activation and its GSDMD-mediated pyroptosis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. IL-1β activation leads to tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated ubiquitination of YAP at K252. This modification interferes with YAP's binding to angiomotin, facilitating its nuclear translocation. Thus, IL-1β appears to regulate the Hippo pathway through this mechanism [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Macrophage-derived IL-1β seems to enhance NETosis. It does this by boosting the arrival of neutrophils at plaque sites and by stimulating the activation of the NLRP3 inflammasome within neutrophils. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The findings of the current research revealed that in macrophage-neutrophil co-cultures under neutralized IL-1β antibody and poly(I:C) infection, blockade of IL-1β secretion inhibited NETosis and the Hippo pathway, as well as NLRP3 inflammasome activation.\u003c/p\u003e \u003cp\u003eHowever, our study has some limitations. First, it remains unclear whether other viruses also activate the Hippo pathway to promote NETosis via NLRP3 inflammasome-mediated IL-1β secretion. Secondly, the definitive mechanism underlying NETosis in viral pneumonia requires further investigation. Additionally, \u003cem\u003ein vivo\u003c/em\u003e studies using knockout mice should be performed to explore the underlying molecular mechanisms.\u003c/p\u003e \u003cp\u003eIn conclusion, we identified a crucial function of NETosis and Hippo pathways in virus-induced lung injury. NETosis aggravates virus-induced lung injury and is increased in viral pneumonia by regulating the Hippo pathway via the NLRP3/IL-1β axis. Drug screening for Hippo and NLRP3/IL-1β pathways may suppress excessive inflammatory responses and intercept NETosis during viral infection. Collectively, Our research suggests that targeting the Hippo pathway, which is activated by NETosis through the NLRP3/IL-1β axis, could be a viable strategy for enhancing the immune system's defense against viral infections.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALI: Acute Lung Injury\u003c/p\u003e\n\u003cp\u003eBALF: Bronchoalveolar Lavage Fluid\u003c/p\u003e\n\u003cp\u003eCitH3: Citrullinated Histone H3\u003c/p\u003e\n\u003cp\u003eDEGs: Differentially Expressed Genes\u003c/p\u003e\n\u003cp\u003edsDNA: Double-Stranded DNA\u003c/p\u003e\n\u003cp\u003eFDR: False Discovery Rate\u003c/p\u003e\n\u003cp\u003eGSEA: Gene Set Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eMERS-CoV: Middle East Respiratory Syndrome Coronavirus\u003c/p\u003e\n\u003cp\u003eMPO: Myeloperoxidase\u003c/p\u003e\n\u003cp\u003eNETs: Neutrophil Extracellular Traps\u003c/p\u003e\n\u003cp\u003eNETosis: Neutrophil Extracellular Trap Formation\u003c/p\u003e\n\u003cp\u003ePAD4: Peptidyl Arginine Deiminase 4\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003ePoly(I:C): Polyinosinic-Polycytidylic Acid\u003c/p\u003e\n\u003cp\u003eSARS-CoV: Severe Acute Respiratory Syndrome Coronavirus\u003c/p\u003e\n\u003cp\u003eT2DM: Type 2 Diabetes Mellitus\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha;: Tumor Necrosis Factor-Alpha\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding information\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 81760020, No. 82060024), \"139\" plan for high-level medical backbone talents of Guangxi Zhuang Autonomous Region (No. G202002015), and Guangxi Anesthesiology Clinical Medicine Research Center Construction Project (No: AD22035214).\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eAll relevant data were within the manuscript and its supplemental files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGow, N.A.R., et al., \u003cem\u003eThe importance of antimicrobial resistance in medical mycology\u003c/em\u003e. Nat Commun, 2022. 13(1): p. 5352.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu, S., et al., Exploring Influenza A Virus-Induced Lung Injury and Immune Response Based on Humanized Lung-on-Chip. Discov Med, 2023. 35(177): p. 539\u0026ndash;552.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWulf Hanson, S., et al., Estimated Global Proportions of Individuals With Persistent Fatigue, Cognitive, and Respiratory Symptom Clusters Following Symptomatic COVID-19 in 2020 and 2021. Jama, 2022. 328(16): p. 1604\u0026ndash;1615.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCevik, M., et al., SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis. Lancet Microbe, 2021. 2(1): p. e13-e22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewton, A.H., A. Cardani, and T.J. Braciale, The host immune response in respiratory virus infection: balancing virus clearance and immunopathology. Semin Immunopathol, 2016. 38(4): p. 471\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanan, N., et al., The Many Faces of Innate Immunity in SARS-CoV-2 Infection. Vaccines (Basel), 2021. 9(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrinkmann, V., et al., \u003cem\u003eNeutrophil extracellular traps kill bacteria\u003c/em\u003e. Science, 2004. 303(5663): p. 1532\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchultz, B.M., et al., Role of Extracellular Trap Release During Bacterial and Viral Infection. Front Microbiol, 2022. 13: p. 798853.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan, L.L.Y., et al., Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses. Respir Res, 2020. 21(1): p. 160.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDivolis, G., et al., Neutrophil-derived Activin-A moderates their pro-NETotic activity and attenuates collateral tissue damage caused by Influenza A virus infection. Front Immunol, 2024. 15: p. 1302489.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, S.J., et al., Platelet-Mediated NET Release Amplifies Coagulopathy and Drives Lung Pathology During Severe Influenza Infection. Front Immunol, 2021. 12: p. 772859.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYalcinkaya, M., et al., Cholesterol accumulation in macrophages drives NETosis in atherosclerotic plaques via IL-1β secretion. Cardiovasc Res, 2023. 119(4): p. 969\u0026ndash;981.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui, H., et al., Lung Myofibroblasts Promote Macrophage Profibrotic Activity through Lactate-induced Histone Lactylation. Am J Respir Cell Mol Biol, 2021. 64(1): p. 115\u0026ndash;125.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuach, C., et al., Enhancing autophagy in CD11c(+) antigen-presenting cells as a therapeutic strategy for acute respiratory distress syndrome. Cell Rep, 2023. 42(8): p. 112990.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, X., et al., Interleukin-38 ameliorates poly(I:C) induced lung inflammation: therapeutic implications in respiratory viral infections. Cell Death Dis, 2021. 12(1): p. 53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKastan, N., et al., Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues. Nat Commun, 2021. 12(1): p. 3100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoch, A.T., et al., MyD88-Dependent Signaling Decreases the Antitumor Efficacy of Epidermal Growth Factor Receptor Inhibition in Head and Neck Cancer Cells. Cancer Res, 2015. 75(8): p. 1657\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing, R., et al., Transforming growth factor-β1 attenuates inflammation and lung injury with regulating immune function in ventilator-induced lung injury mice. Int Immunopharmacol, 2023. 114: p. 109462.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan, Z., et al., Role of LL-37 in thrombotic complications in patients with COVID-19. Cell Mol Life Sci, 2022. 79(6): p. 309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePisareva, E., et al., Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA. Genome Med, 2022. 14(1): p. 135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, H., et al., Neutrophil Extracellular Traps may be a Potential Target for Treating Early Brain Injury in Subarachnoid Hemorrhage. Transl Stroke Res, 2022. 13(1): p. 112\u0026ndash;131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis, J.R. and N. Tapon, \u003cem\u003eHippo signalling during development\u003c/em\u003e. Development, 2019. 146(18).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, W., et al., \u003cem\u003eHippo signaling pathway and respiratory diseases\u003c/em\u003e. Cell Death Discov, 2022. 8(1): p. 213.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, C., et al., Hippo Signaling Controls NLR Family Pyrin Domain Containing 3 Activation and Governs Immunoregulation of Mesenchymal Stem Cells in Mouse Liver Injury. Hepatology, 2019. 70(5): p. 1714\u0026ndash;1731.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eByeon, H.E., et al., HDAC11 Regulates Palmitate-induced NLRP3 Inflammasome Activation by Inducing YAP Expression in THP-1 Cells and PBMCs. Endocrinology, 2024. 165(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, D., et al., YAP promotes the activation of NLRP3 inflammasome via blocking K27-linked polyubiquitination of NLRP3. Nat Commun, 2021. 12(1): p. 2674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, R., et al., \u003cem\u003eNeutrophil autophagy and NETosis in COVID-19: perspectives\u003c/em\u003e. Autophagy, 2023. 19(3): p. 758\u0026ndash;767.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSergunova, V., et al., Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study. Cells, 2023. 12(17).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhargavan, B. and G.D. Kanmogne, SARS-CoV-2 Spike Proteins and Cell-Cell Communication Induce P-Selectin and Markers of Endothelial Injury, NETosis, and Inflammation in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for the Pathogenesis of COVID-19 Coagulopathy. Int J Mol Sci, 2023. 24(16).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, Y., H. Wang, and Y. Liu, NETosis: Sculpting tumor metastasis and immunotherapy. Immunol Rev, 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThierry, A.R., Netosis creates a link between diabetes and Long COVID. Physiol Rev, 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, H., et al., NETosis promotes chronic inflammation and fibrosis in systemic lupus erythematosus and COVID-19. Clin Immunol, 2023. 254: p. 109687.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y.M., et al., IL-37 improves mice myocardial infarction via inhibiting YAP-NLRP3 signaling mediated macrophage programming. Eur J Pharmacol, 2022. 934: p. 175293.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, X., et al., Hippo Pathway Counter-Regulates Innate Immunity in Hepatitis B Virus Infection. Front Immunol, 2021. 12: p. 684424.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, X., et al., Hippo Pathway Activation in Aged Mesenchymal Stem Cells Contributes to the Dysregulation of Hepatic Inflammation in Aged Mice. Adv Sci (Weinh), 2023. 10(27): p. e2300424.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, S., et al., Neutrophil Extracellular Traps Delay Diabetic Wound Healing by Inducing Endothelial-to-Mesenchymal Transition via the Hippo pathway. Int J Biol Sci, 2023. 19(1): p. 347\u0026ndash;361.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eByun, D.J., et al., NLRP3 Exacerbate NETosis-Associated Neuroinflammation in an LPS-Induced Inflamed Brain. Immune Netw, 2023. 23(3): p. e27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, S., et al., Disulfiram accelerates diabetic foot ulcer healing by blocking NET formation via suppressing the NLRP3/Caspase-1/GSDMD pathway. Transl Res, 2023. 254: p. 115\u0026ndash;127.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;nzer, P., et al., NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions. Front Immunol, 2021. 12: p. 683803.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWesterterp, M., et al., Cholesterol Efflux Pathways Suppress Inflammasome Activation, NETosis, and Atherogenesis. Circulation, 2018. 138(9): p. 898\u0026ndash;912.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong, L., et al., Folic Acid Alleviates High Glucose and Fat-Induced Pyroptosis via Inhibition of the Hippo Signal Pathway on H9C2 Cells. Front Mol Biosci, 2021. 8: p. 698698.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinns, M.S., et al., NLRP3 selectively drives IL-1β secretion by Pseudomonas aeruginosa infected neutrophils and regulates corneal disease severity. Nat Commun, 2023. 14(1): p. 5832.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, J., et al., FABP4 in macrophages facilitates obesity-associated pancreatic cancer progression via the NLRP3/IL-1β axis. Cancer Lett, 2023. 575: p. 216403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, M., et al., Macrophage K63-Linked Ubiquitination of YAP Promotes Its Nuclear Localization and Exacerbates Atherosclerosis. Cell Rep, 2020. 32(5): p. 107990.\u003c/span\u003e\u003c/li\u003e\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":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hippo/YAP Signal Pathway, Acute Lung Injury, Inflammation, Cellular interaction, NETosis, Macrophages","lastPublishedDoi":"10.21203/rs.3.rs-4591287/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4591287/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSevere viral infections can precipitate acute lung injury, causing substantial morbidity and mortality. NETosis plays a crucial role in defending against pathogens and viruses, but its excessive or dysregulated formation can cause pulmonary damage, with research into its regulation offering potential insights and treatment strategies for viral lung injuries.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eElevated levels of NETosis were detected in the peripheral blood of patients with viral pneumonia. To explore the correlation between NETosis and virus-induced acute lung injury, we employed a murine model, administering poly(I:C) (polyinosinic-polycytidylic acid), an artificial substitute for double-stranded RNA, intratracheally to mimic viral pneumonia. Assessment of NETosis biomarkers in afflicted patients and poly(I:C)-stimulated mice was conducted, alongside mechanistic investigations into the involvement of the Hippo signaling pathway, inflammatory factors, and chemokines in the injury process. Cytokine assays, co-culture experiments, and downstream inflammatory mediator analyses were used to ascertain the role of the Hippo pathway in macrophage to mediate NETosis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eEnhanced expression of NETosis biomarkers was found both in patients with viral pneumonia and in poly(I:C)-stimulated mice. Hippo pathway activation in conjunction with increased levels of inflammatory actors and chemokines was observed in lung tissues of the mouse model. Elevated IL-1β was detected in cells and macrophages isolated from infected mice; this was mitigated by Hippo pathway inhibitors. IL-1β was confirmed to induce NETosis in co-culture experiments, while NLRP3, functioning downstream of the Hippo pathway, mediated its secretion. Patients with viral pneumonia exhibited increased NLRP3 and IL-1β in monocyte-macrophages relative to healthy controls.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eActivated Hippo pathway in macrophages during poly(I:C) exposure upregulates NLRP3 and IL-1β expression to promote the occurrence of NETosis, thereby aggravating virus-induced lung injury. This study identifies a potential target pathway for therapeutic intervention to mitigate lung injury stemming from viral infections.\u003c/p\u003e","manuscriptTitle":"Hippo pathway and NLRP3-driven NETosis in macrophages: Mechanisms of viral pneumonia aggravation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 13:19:41","doi":"10.21203/rs.3.rs-4591287/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-02-18T15:46:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-02-04T01:15:03+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-01-25T01:10:42+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-12-21T15:15:41+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-12-08T08:02:00+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-06-30T06:58:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-24T15:26:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2024-06-22T09:18:18+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-06-17T12:39:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-17T02:15:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ed017b0b-0cd2-4c10-95e3-9b83b7b33cfd","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33907181,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":33907182,"name":"Biological sciences/Molecular biology/Transcriptomics"}],"tags":[],"updatedAt":"2025-07-15T07:13:33+00:00","versionOfRecord":{"articleIdentity":"rs-4591287","link":"https://doi.org/10.1038/s41420-025-02556-z","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2025-07-14 04:00:00","publishedOnDateReadable":"July 14th, 2025"},"versionCreatedAt":"2024-07-25 13:19:41","video":"","vorDoi":"10.1038/s41420-025-02556-z","vorDoiUrl":"https://doi.org/10.1038/s41420-025-02556-z","workflowStages":[]},"version":"v1","identity":"rs-4591287","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4591287","identity":"rs-4591287","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}