Data acquisition and standardizationThe GSE171524 dataset10, which included 7 normal individuals and 20 patients, and the GSE171668 dataset11, which included 48 patients, were collected. Single-cell objects were constructed by the “Seurat” algorithm12, and qualified single-cell data were obtained with the thresholds of a gene expression number greater than 50 and a mitochondrial gene ratio less than 20%. The normalization factor was set to 10,000 to standardize the data, and 5000 highly variable genes were identified via the “vst” method. After normalization, principal component analysis was used for dimensionality reduction clustering, and the cells were grouped at a resolution of 0.6. Finally, according to the marker genes of the cell clusters, “singleR” was used to annotate cell types based on the human cell atlas. For transcriptome sequencing, the FPKM data were logarithmically normalized.Differentially expressed genes (DEGs) and enrichment analysisBased on the single-cell data, the macrophages were regrouped according to gene expression. For example, macrophages with AQP1 expression and macrophages without AQP1 expression were separated, and similar methods were applied to TLR2, TLR4 and CD86. The Wilcoxon rank sum test and the “FindMarkers” algorithm13 were used to analyze DEGs in cells with a gene expression ratio greater than 10%. According to the transcriptome sequencing results, DEGs were analyzed using “limma”14. The “clusterProfiler”15 package was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs. The activation or inhibition status of the terms was evaluated by calculating the Z score, and the “forcats” package was used to summarize the enrichment results for the different groups.Identification of overlapping genes and construction of a protein‒protein interaction networkA Venn diagram was used to visualize the overlapping DEGs. The STRING database16 was used to examine the interactions among DEGs. The MCODE algorithm17 in Cytoscape was used to investigate the important modules of the protein‒protein interaction network.Hub gene recognition and correlation analysisSix algorithms (MCC, MNC, EPC, degree, radiality, and closeness) were used to identify the top 30 DEGs. The overlapping genes identified by all six algorithms were considered hub genes. The Pearson method was used to analyze the correlation between the expression levels of the hub genes and genes related to pyroptosis.Cell culture and treatmentsThe MH-S mouse alveolar macrophage line (Procell, Wuhan, China) was partially adherent and partially suspended. After be retrieved from liquid nitrogen, MH-S cells were rapidly revived, placed in a Petri dish with complete culture medium (Procell, Wuhan, China), and incubated in a constant temperature incubator at 37 °C with 5% CO2. Once the MH-S cells reached 70% to 80% confluence, the medium was removed, and the cells were washed with PBS (HyClone, Utah, USA), digested with 0.25% trypsin (HyClone, Utah, USA), and centrifuged at 1000 r/min at room temperature for 5 min. The supernatant was discarded, and the cells were resuspended and subcultured. For western blot and RT-qPCR analyses, MH-S cells were inoculated onto a 6-well plate at a density of 2 × 106 cells/well.Construction of the M1 macrophage polarization modelMH-S cells in the logarithmic growth phase were selected, the supernatant was discarded, and the cells were washed twice with PBS, digested with trypsin and centrifuged. The centrifuged cells were suspended in complete culture medium, the cell concentration was adjusted to 5 × 105 cells/mL, and the cells were seeded in 6-well plates at 3 × 105 cells/well. After the cells had adhered to the wells for 12 h, different concentrations of LPS (1 μg/mL, 5 μg/mL or 10 μg/mL) were added, and the cells were incubated for 12 h or 24 h to establish the M1 macrophage model.AQP1 protein inhibitor and agonist screeningMH-S cells in the logarithmic growth phase were selected and seeded in 6-well plates at 3 × 105 cells/well. After 12 h, the cells were treated with LPS and AQP1 agonists or inhibitors, including different concentrations of MG132 (3 μM, 10 μM, and 30 μM), merbromin (50 μM, 100 μM, and 150 μM) and TEA (3 μM, 30 μM, and 100 μM). After 12 h, the cells were collected, and protein was extracted for verification.Evaluation of macrophage migrationOn the back of a 6-well plate, parallel horizontal lines were drawn, and approximately 3 × 106 cells were inoculated into each well. The plate was then placed in an incubator for 12 h. Subsequently, additional horizontal lines were drawn vertically on the back of the plate using a 10 µL pipette. Next, the cells were washed with PBS 2–3 times, and the medium was replaced with fresh serum-free medium. The plate was returned to the incubator for an additional 12 h. Then, the cells were observed and photographed under a microscope. ImageJ software was used to calculate the average intercellular distance between 6–8 randomly selected horizontal lines. For the transwell chamber experiment, cells were inoculated at a density of 5 × 105 cells/well. In the lower layer of the chamber, 800 μL of 1640 medium (Gibco, USA) containing 20% serum (Sigma, USA) was added, and the upper layer contained 100 μL of pretreated cells. After 12 h of incubation, the transwell chamber was removed and washed with PBS, and the cells were stained with crystal violet. The stained cells were then photographed under a microscope, and ImageJ was used to calculate the number of migrated cells.Examination of macrophage phagocytosisMH-S cells were inoculated into 12-well plates at a density of 1 × 106 cells per well. After the cells adhered to the wells, 1 μL of FITC-labeled fluorescent microspheres (Poly Sciences, Shanghai, China) was added to each well and incubated for 2 h at 37 °C. The culture supernatant was then aspirated, and 0.25% trypsin was added to digest the cells. The cells were collected, fixed with paraformaldehyde, and subjected to flow cytometry to assess the phagocytosis of fluorescent microspheres by MH-S cells. Additionally, cells subjected to the same pretreatment procedure were covered with Hoechst 33342 staining solution (Beyotime, Shanghai, China) and observed and photographed under an inverted fluorescence microscope.Examination of chemokines and cytokines by RT-qPCRTotal RNA was extracted from MH-S cells using TRIzol reagent, and RNA purity and concentration were determined using a spectrophotometer. Subsequently, reverse transcription was performed using a reverse transcription kit (Thermo Fisher Scientific, USA), and RT-qPCR was performed according to the manufacturer’s instructions (Qiagen, Germany) and the predetermined reaction time and temperature from the preliminary experiments. The procedure involved an initial activation step at 95 °C for 2 min, denaturation at 95 °C for 5 s, and annealing/stretching at 60 °C for 10 s for 40 cycles. The sequences of the primers used are listed in Table 1. Using GAPDH as the internal reference, the relative expression level of the target mRNA was analyzed using the 2(−∆∆Ct) method. The described experiment was repeated three times.Table 1 Primer sequence information.Examination of protein expression levelsMH-S cells were assessed using a BCA protein assay kit. Equal amounts of protein were separated by 10% SDS-PAGE and subsequently transferred to a PVDF membrane. The membrane was then blocked with a PBS solution containing 5% skim milk at room temperature for 2 h. Next, the membranes were incubated with primary antibodies (AQP1, NLRP3, GSDMD, N-GSDMD, Caspase-1 and iNOS) overnight at 4 °C. After three washes with TBST solution, the PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 2 h, followed by another three washes with TBST solution. The blot was developed using an enhanced chemiluminescence kit. ImageJ software was used to determine the gray values of the bands, and the ratio of the target protein to the internal reference, GAPDH, was used to calculate the relative protein expression.ELISA assayLevels of CCL4 in the macrophages were determined using commercially available ELISA kits (jianglai biology science and technology, Shanghai, China) according to the manufacture’s instructions. The absorbance of each well recorded at 450 nm using a microplate reader.Statistical analysisThe experimental values are presented as the mean ± SD. Statistical comparisons were performed using one-way analysis of variance and LSD-t test with GraphPad Prism version 9.0. Differences in values were considered statistically significant if the P values were < 0.05.
