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 × 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 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 × 106 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).