Human clinical specimens

Thirty specimens of human gastric tissue specimens were collected from patients undergoing endoscopy and biopsy at the First Affiliated Hospital of Nanchang University in Jiangxi, China. The clinicopathological characteristics of patients were listed in Table S1. The H. pylori infection status of these clinical specimens was determined by the urea breath test (UBT) or immunohistochemistry. Based on H. pylori infection status, the specimens were divided into two subgroups: H. pylori-positive (n = 15) and H. pylori-negative (n = 15). The study protocol and informed consent waiver were approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University ((2023) CDYFYYLK (02–012).

Mice

All procedures performed on the animals were approved by the Animal Care and Ethics Committee (CDYFY-IACUC-202302QR004) of the First Affiliated Hospital of Nanchang University. The male wild-type (WT), and Tnfr1-KO mice aged 6 to 8 weeks were obtained from GemPharmatech Company (Nanjing, China). The male transgenic hypergastrinemic insulin-gastrin (INS-GAS) mice (#018149), aged 6–8 weeks old and on a FVB/N background, and Nlrp3-knockout (KO) mice (#021302) were purchased from The Jackson Laboratory and housed under a 12-h light/dark cycle with ad libitum access to food and water. The genotyping of mice was performed according to the manufacturer’s instructions. The primers used in this process are listed in Table S2. The experimental environment was maintained at a temperature of 18–26 °C and a relative humidity of 40–70%. The INS-GAS mice were infected with the mouse-adapted wild-type H. pylori strain PMSS1 (2 × 10^9 CFU/mouse) via gavage every other day for a total of five times [6, 32]. Mice were orogastrically challenged with Brucella broth (BB) as a control. Mice were fasted for 4 h before and after each gavage. Mice were euthanized 16 weeks post-infection.

Isolation of bone marrow-derived macrophages (BMDMs) and cell culture

THP-1 cells were cultured in a specialized medium (RPMI-1640 medium supplemented with 10% FBS, 0.05 mM β-mercaptoethanol, and 1% P/S (Procell, Wuhan, China)). Before experimentation, the THP-1 cells were induced to differentiate into macrophages by treatment with 200 ng/ml phorbol 12-myristate 13-acetate (PMA) (MedChemExpress, HY-18739) for 48 h.

BMDMs were isolated from the femurs and tibias of WT, Nlrp3-KO, and Tnfr1-KO mice. The BMDMs suspension was obtained by flushing the bones with PBS containing 2% FBS (Gibco, Australia) and filtered through a sterile 70 μm cell strainer. After a 10-min incubation with red blood cell lysis buffer (Absin, Shanghai, China) to lyse red blood cells, cells were cultured in complete medium (RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin/streptomycin (NCM Biotech, Suzhou, China), and 100 ng/ml recombinant murine M-CSF (PeproTech, USA)) for 7 days. All cells were maintained in a humidified atmosphere at 37 °C with 5% CO2.

Reagents and transfection

Recombinant murine M-CSF (#315–02), recombinant murine TNF-α (#315-01A), and recombinant human TNF-α (#300-01A) were purchased from PeproTech (NJ, USA). Atrosab (#HY-P990008), Nigericin (#HY-127019) and LPS (#HY-D1056) were obtained from MedChemExpress (Shanghai, China), and QNZ (#S4902) was purchased from Selleck Chemicals (HOU, USA).

Control siRNA and NLRP3 siRNA were produced by Hitrobio (Beijing, China), and then transfected into THP-1 cells using lipofectamine 3000 (Thermo Scientific, MA, USA) according to the manufacturer’s instructions. The target sequences of NLRP3 siRNA were: Sense:5′-GGAGAGACCUUUAUUGAGAATT-3′; Antisense:5′-UUCUCAUAAAGGUCUCUCCTT-3′. 48 h post-transfection, cells were treated with inhibitors of the TNF/TNFR1 signaling, 1 μM Atrosab or 400 ng/ml QNZ. Western blots and qRT-PCR analysis validated the transfection efficiency.

H. pylori strains and culture

This study employed wild-type CagA+H. pylori strains (PMSS1, NCTC11637 and 26695) [6, 33]. Isogenic PMSS1 CagA− mutant was constructed in our previous study [34]. The H. pylori isogenic 26695 VacA− mutant was kindly provided by Dr. Chunhui Lan from Daping Hospital, Third Military Medical University (Chongqing, China). Additionally, the rodent-adapted CagA+H. pylori strain pre-murine Sydney Strain 1 (PMSS1) was used for animal experiments. H. pylori were cultured under microaerophilic conditions on Brucella agar (BD Bioscience) supplemented with 5% sheep blood for in vitro passage. For in vitro co-culture with THP-1 cells, H. pylori were grown overnight in Brucella broth (BD Bioscience) supplemented with 10% FBS (Gibco, Australia). The bacteria were harvested and determined the density at 600 nm (1OD600 = 10^9 CFU/ml), then resuspended and added to THP1 or BMDM cells. Before co-culture with bacteria, each well was seeded with 1 × 10^6 cells. The bacteria were co-cultured with THP1 or BMDM cells at various multiplicities of infection (MOI = 0, 25, 50, 100 and 200) for different durations (0 h, 3 h, 6 h, 12 h and 24 h).

Assessment of H. pylori colonization and histopathology in gastric tissues of mouse models

The mouse stomach was opened along the greater curvature, and a linear strip of gastric tissue extending from the squamocolumnar junction to the proximal duodenum was selected. After fixation in formalin for 24 h, the tissue was dehydrated and then embedded in paraffin. Once cooled and solidified, the tissue was sectioned into approximately 4 μm thick gastric tissue slices. H. pylori colonization was visualized by silver staining using the Spirochete Silver Staining Kit (Warthin-Starry Method, #G1940, Solarbio Biotech, China) according to manufacturer’s instructions. All images were captured using a Nikon Eclipse microscope. Additionally, the bacterial burden was calculated by determining the number of CFU per gram of tissue. In brief, the isolated gastric tissues were weighed and homogenized, then plated in brucellar broth agar plates containing 5% sheep blood, vancomycin, trimethoprim, amphotericin and polymyxin for selective H. pylori growth. These plates were incubated under microaerophilic conditions at 37℃ for 3–5 days, followed by the calculation of bacterial burden.

The paraffin sections were stained with Hematoxylin and Eosin (H&E). A pathologist, blinded to sample identification, evaluated the degree of inflammation and the incidence of gastric injury, including atrophy and metaplasia [35]. Alcian blue coupled with periodic acid-Schiff (AB-PAS) staining was performed to identify the type of metaplasia present, following the manufacturer’s instructions (#G1285, Solarbio Biotech, China). In brief, after deparaffinization, the slides were stained with Alcian blue for 20 min, followed by oxidation and stained with Shiff reagent for 10 min. Then the slides were rinsed with running water and further stained with hematoxylin. All images were captured on a Nikon Eclipse microscope.

Immunohistochemistry

Immunohistochemical staining was conducted as previously described [6]. Briefly, the sections were sequentially soaked in xylene, 100%, 95% and 85% ethanol to remove paraffin. Antigen retrieval was then performed using sodium citrate buffer. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Non-specific binding of antibodies was avoided by incubation in 1% BSA blocking solution for 30 min. Next, the sections were incubated with the primary antibody was incubated at 4 °C overnight, followed by 3 times wash with PBS and subsequent incubation with the secondary antibody (PV-6000, Zhongshan Biotech, China) was incubated with the sections at 37 °C for 1 h. The isotype control antibody (#3900, Cell Signaling Technology, USA) was used as a negative control to determine any nonspecific background staining (Fig. S1A). Diaminobenzidine (DAB) (ZLI-9017, Zhongshan Biotech, China) was used for color development, followed by counterstaining with hematoxylin (L25050202, YULU, China). Immunohistochemical staining was assessed by two pathologists, who were blinded to the sample identities, to determine the expression profile of NLRP3. The intensity (scored 0–3) and frequency (scored 0–4) of NLRP3 expression were evaluated and graded. In statistical analyses, the expression level of NLRP3 protein was represented by an expression score, ranging from 0 to 12, calculated as the product of intensity and frequency. All images were captured using a microscope (Nikon Eclipse).

Single-cell RNA sequencing (scRNA-seq) analysis

In our previous work [36], we included 18 patients diagnosed with gastric lesions at the First Affiliated Hospital of Nanchang University. This cohort consisted of 6 cases of gastritis (GS), 6 cases of intestinal metaplasia (IM), and 6 cases of gastric cancer (GC). The GS and IM samples were collected via endoscopy, while the GC samples were obtained from surgical specimens of patients who had not received any adjuvant therapy. Patients with gastric mucosal lesions were divided into H. pylori- positive and H. pylori- negative groups based on their infection status. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University, with approval number (2023) CDYFYYLK (01–009). All tissue samples were obtained with informed consent. Tissues from different stages of gastric mucosal lesions were isolated and subsequently prepared into single-cell suspensions. Libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Cat#1000268, 10 × Genomics) according to the manufacturer’s protocol. The libraries were sequenced on the Illumina NovaSeq 6000 platform. Raw reads were processed into gene expression matrices using Cell Ranger software (10 × Genomics), followed by further quality control was performed using the Seurat package. The raw single-cell sequencing data of 18 human gastric samples from this study have been deposited in the gene expression omnibus database (GEO), under accession code GSE249874 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi).

The UMAP plots illustrate the expression of several genes, such as NLRP3, TNF, ASC, and IL1B, across various different cell types. To examine identify the activity of inflammasome pathway, we selected the crucial genes within this pathway, including HMOX1, MEFV, NFKB1, NFKB2, NLRC4, NLRP1, NLRP3, P2RX7 and PYCARD. Using the AddModuleScore function, we calculated the mean expression level of these genes in each cell. All figure visualizations were generated utilizing the SCP (Version 0.5.6) R package.

To analyze cell–cell interaction [37], we applied the CellChat R package to compare significant ligand-receptor pairs between H. pylori -positive and H. pylori -negative samples. During this procedure, we employed the netVisual_heatmap function to visualize variations in the number or strength of cell–cell interactions. The rankNet function was deployed to infer and rank the differences in information flow between signaling networks in gastric tissues, both H. pylori-infected and uninfected. The netAnalysis_contribution function was used to assess the contribution of all ligand-receptor pairs within a specific signaling network.

Western blotting

Western blotting analysis was performed according to previously describe methods [38]. Briefly, proteins were extracted using protein lysis buffer (Solarbio, Beijing, China) supplemented with phosphatase and protease inhibitors, and the protein concentrations were determined using a BCA assay kit (Thermo Scientific, MA, USA). Equal amounts of protein extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (PerkinElmer, Waltham, MA), which were subsequently blocked with a 5% non-fat milk blocking solution. The membranes were then incubated with specific primary antibody at 4 °C overnight, followed by incubation with corresponding secondary antibodies (Invitrogen, CA, USA) for 1 h at room temperature. The Western blot immunoreactive signals were visualized using the BIO-RAD ChemiDoc XRS + system with SuperSignal West Pico stable peroxide solution (Thermo Scientific, MA, USA) in a darkroom. The specific information for primary antibodies is as follows: anti-NLRP3 (#15101S), anti-Caspase-1 (#83383), anti-Cleaved Caspase-1 (#89332, #4199), anti-IL-1β (#12242), anti-Cleaved -IL-1β (#83186), anti-NF-kB p65 (#8242), anti-Phospho-NF-κB p65 (#3033) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-IL-6 (ab233706) was obtained from Abcam (Boston, MA, USA). Anti-TNF-α (A0277) and anti-TNFR1 (A1540) were from ABclonal Technology (Wuhan, China). Anti-STAT3 and anti-Phospho-STAT3 (Tyr705) were from Zen BioScience (Chengdu, China). Anti-β-actin (#HC201-01) was obtained from TransGen Biotech (Beijing, China), and anti-GAPDH (#10494–1–AP) was from Proteintech (Wuhan, China). All primary antibodies were used at a dilution of 1:1000, except for internal control antibodies such as GAPDH and β-actin, which were diluted at 1:2000 and 1:5000, respectively.

Immunofluorescence assay

For cell immunofluorescence staining, cells were washed with PBS at room temperature, followed by fixation with 4% formaldehyde in PBS for 30 min. Permeabilization of fixed cells was achieved using 0.3% Triton-100 for 15 min. After blocking with 3% bovine serum albumin (BSA) for 1 h, cells were incubated with primary antibodies or isotype antibody at 4 °C overnight, followed by incubation with secondary antibodies, Alexa Fluor Plus 488 or Alexa Fluor Plus 555 (1:500, Thermo Fisher, Weston, FL), in the dark for 1 h. Nuclei were counterstained with DAPI (P0131, Beyotime, China). All images were acquired using a confocal fluorescence microscope (Leica Stellaris 5). The primary antibodies used is as follows: anti-NLRP3 (Servicebio, GB114320-100, 1:200), anti-F4/80 (Servicebio, GB113373-100, 1:500), anti-CD68 (Santa Cruze, SC-20060, 1:50), anti-iNOS (Zen BioScience, 340668, 1:50) and anti-TNF-α (ABclonal, A0277, 1:50).

Quantitative real-time PCR analysis

Total RNA from cells or tissues was extracted using TRIzol (Invitrogen, CA, USA), followed by cDNA synthesis using the FastKing cDNA First Strand Synthesis Kit (KR116, Tiangen, China). SYBR® Premix Ex Taq (RR820B, TaKaRa, Japan) was used for RT-PCR analysis. mRNA levels were detected using the QuantStudio 5 Real-Time PCR System (Life Technologies). Gene-specific primers were obtained from the public database PrimerBank (Table S3).

ELISA assay

The ELISA kits for detecting the concentrations of IL-1β, TNF-α and IL-18 were purchased from Elabscience (Wuhan, China). The mouse H. pylori IgG antibody ELISA kit was purchased from mlbio (Shanghai, China). ELISA assay was carried out following the manufacturer’s instructions. Briefly, standards or samples was added to the antibody-coated plate and incubated for 1 h at 37℃. Then the biotinylated antibody solution, avidin-HRP solution and substrate solution were added to the microplate well. The absorbance value at 450 nm was measured after the addition of the stop solution. A standard curve was drawn according to the absorbance of standards at different concentration gradients. The concentration of cytokines in the samples was sequentially calculated using this standard curve.

Statistical analyses

All statistical analyses were performed using SPSS 21.0 software. Data are presented as means ± standard deviation (SD) from three independent experiments. Differences between the two groups were compared using Student’s t-test. One-way analysis of variance (ANOVA) was used to compare statistical differences among three or more groups. Differences were considered statistically significant at p < 0.05(*** , P < 0.001; **, P < 0.01; *, P < 0.05).