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 (Supplemental Table 1). 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 or their legally authorized representatives provided written informed consent prior to enrollment in the study. For patients with severe viral pneumonia who were unable to provide consent due to their critical condition, informed consent was obtained from a legally authorized surrogate, such as a family member or guardian, following institutional ethical guidelines. The consent process was conducted by trained clinical investigators who provided verbal and written explanations of the study’s purpose, procedures, potential risks, and benefits in a clear and comprehensible manner. 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
All animal experiments were conducted using wild-type male C57BL/6 J mice, aged 6–8 weeks and weighing approximately 25 ± 5 g, obtained from the Animal Center of Guangxi Medical University (Nanning, China). Animals had not been previously used in any other experiments. They were housed in a controlled environment with filtered air, free access to food and water, a temperature of 20–25 °C, and relative humidity of 50–70%. Animals were randomly assigned to experimental groups using a computer-generated randomization sequence. Inclusion and exclusion criteria were pre-established based on animal health and viability, and no animals or samples were excluded from analysis. Anesthesia was administered using ketamine hydrochloride and xylazine to minimize discomfort. All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangxi Medical University and conducted in accordance with the Guide for Regulation and Administration of Laboratory Animals of the People’s Republic of China. The study also complied with the ARRIVE guidelines (PLoS Biol. 8(6), e1000412, 2010). Blinding was not performed during experimentation or outcome assessment.
Cell
RAW 264.7 cells were purchased from ATCC and were authenticated using short tandem repeat (STR) profiling within the past 12 months. The cells were tested and confirmed negative for mycoplasma contamination before use. 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 [28]. 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 (MiltenyiBiotec, 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 min. 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 immunomagnetically 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 × 106 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 a cell analyzer. In the co-culture experiment, neutrophils isolated from peripheral blood of control mice were cultured with conditioned medium collected from RAW 264.7 cells that had been treated under different conditions (control, poly(I:C) stimulation, or poly(I:C) plus Lats-IN-1). Neutrophils were seeded at a concentration of 1 × 106 cells/mL and co-cultured with the conditioned medium for 48 h. Following co-culture, neutrophils were collected for analysis of NETosis markers using immunofluorescence, Western blotting, and ELISA.
Reagents administration
C57BL/6 mice were intranasally challenged with 5 mg/Kg high-molecular-weight poly(I:C) at a concentration of 1 mg/mL to induce ALI/ARDS [29]. 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 [30].
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 h before and continued simultaneously with and 24 h after administration of Poly(I:C) [31]. 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 h following Poly(I:C) exposure. In vitro treatments included exposure of RAW 264.7 cell lines to 10 µM Lats-IN-1, and 5 µL/mL of neutralizing IL-1β antibody [32].
Plasmids, small interfering RNAs (siRNAs), and transfection
All shRNAs used in this study were provided by Sangon (Shanghai, China), as listed in Supplemental Table 2. 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 h 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 [33]. 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 Maste 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 30 min at an icy temperature, followed by centrifugation at 12,000g-force for 15 min. 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 min 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-h incubation period at 22–25 °C with 5% nonfat milk, the membranes underwent an overnight incubation with primary antibodies (Supplemental Table 3) at a temperature of 4 °C. This was followed by a 1-h 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 15 min. 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 4. 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 were conducted utilizing the Visium system from 10 × 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 min 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 × 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 (Supplemental Table 5). 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.
We identified differentially expressed genes (DEGs) between the normal group (Control, n = 6) and the viral lung injury group (Poly I:C, n = 6) using the R package “limma (version 3.50.0)” [33]. The screening criteria were p < 0.05 and |log2FC | > 1, and these DEGs were used for subsequent analyses. The “pheatmap” R package was employed to generate heatmaps, with clustering performed using Euclidean distance and hierarchical clustering methods.
The R package “clusterProfiler (version 4.2.2)” [34] was applied to conduct GO and KEGG enrichment analyses on the DEGs between all groups, with a significance threshold for pathway identification set at p < 0.05. Additionally, Gene Set Enrichment Analysis (GSEA) was performed using the “clusterProfiler (version 4.2.2)” R package, ranking all genes based on their log2Fold Change values and conducting 1,000 gene set permutations. The reference gene set used for GSEA was the KEGG database containing all mouse pathway gene sets [35]. Gene sets with an adjusted p-value < 0.05 were considered significantly enriched.
The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) online database, with protein pairs having an interaction score greater than 0.7 being selected [36]. The PPI network was then visualized using Cytoscape software for better presentation [37]. The top 100 genes from all 12 CytoHubba algorithms were extracted, and their intersection was taken [38]. The genes obtained from this intersection were considered to be involved in the differences between groups. The 12 CytoHubba algorithms used here include: Betweenness, Stress, Radiality, Eccentricity, node connect degree (Degree), density of maximum neighborhood component (DMNC), edge percolated component (EPC), maximal clique centrality (MCC), node connect closeness (Closeness), maximum neighborhood component (MNC), ClusteringCoefficient, and BottleNeck.
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).
