Mice

C57BL/6 (WT) mice were obtained from Jiangsu Jicui Yaokang Biotechnology Co., Ltd (Jiangsu, China). Mice were housed according to protocols approved by the Animal Ethics Committee of Guangdong Medical University.

Treatments

The cecal ligation and puncture (CLP) mouse model operative procedures were conducted following previously established methods (Marco et al., 2018). In brief, C57BL/6 female mice were anesthetized with general isoflurane (2%) anesthesia. The cecum was perforated, leading to the release of fecal material into the peritoneal cavity.

The PT3-F1α-TPO lentiviral plasmid was generously provided by Dr. Zhanghui Chen (Institute of Clinical Medicine, Zhanjiang Central Hospital, Guangdong Medical University, Zhanjiang, China). To induce TPO overexpression in mice, a hypertensive tail vein injection of 10 μl PT3-EF1α-TPO lentiviral plasmid with a titer of 1 × 10^11 virus was administered.

Preparation of mouse platelets

Blood was collected from the abdominal aortas of mice under isoflurane anesthesia using anticoagulant tubes. Platelet isolation was carried out using the Mouse Peripheral Blood Platelet Separation Kit (PLA2011M, Haoyang, Tianjin, China) following the manufacturer’s instructions. Platelet counts in whole blood were determined using a HEMAVET HV950FS multispecies hematology analyzer.

Mouse BMDM and cell treatment

BMDMs were isolated from the femurs and tibias of C57BL/6 mice, following previously established protocols (ref 19). The cells were cultured in DMEM supplemented with 10% FBS and 1% (v/v) penicillin/streptomycin.

For co-culture experiments with platelets, washed platelets at a concentration of 1 × 10^9 were added to the BMDM culture medium and incubated for 2 h, with or without pretreatment of PD-16,839 (10 μM) or Erlotinib (20 μM).

To induce pyroptosis in BMDMs in vitro, LPS (1 μg/ml) and ATP (4 mM) were added to the BMDM culture medium and incubated for 4 h.

For in vitro infection with E. coli, E. coli O157:H7 (provided by Dr. Tianwen, Guangdong Medical University, Zhanjiang, China) was cultured in Luria-Bertani (LB) medium. Prior to infection, cells were washed extensively with 1X PBS and then incubated in complete DMEM without antibiotics for 2–3 h. The concentration of the bacterial solution was determined using a standardized calibration curve of OD 600/colony-forming units (CFU).

Flow cytometry analyses

Cell surface staining and flow cytometry were performed as described previously (ref 19).

Flow cytometry acquisition was conducted using a FACS cytometer (BD Biosciences, USA), and data processing was carried out using FlowJo version 10.0 software.

Western blotting

Following the indicated treatments, cell supernatants were collected and lysed in RIPA lysis buffer (Beyotime, China) supplemented with Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail. Protein concentration was quantified using the BCA protein assay (Beyotime, China).

Quantitative real-time PCR

Total RNA was extracted from 5 × 10^5 cells following the indicated treatments using TRIzol (Invitrogen), according to the manufacturer’s instructions. cDNA was synthesized from each RNA sample using the TaKaRa reverse transcriptase kit. Quantitative PCR (qPCR) was carried out using TB Green Premix Ex Taq II (TaKaRa) on a Roche 480 instrument. Primers used are listed in Supplemental Table 1.

ELISA

Cell supernatants, mice plasma, or mice organs were collected after the indicated treatments. The concentrations of cytokines, including mouse IL-6, mouse TNF-α, mouse IL-18 and mouse IL-1β, were measured using ELISA kits (Thermo Scientific) following the manufacturer’s instructions. All ELISA assays were conducted according to the manufacturer’s protocols.

Immunofluorescence staining

Bone marrow murine neutrophils (6 × 10^5) were plated on confocal dishes. Following the indicated stimulation, cells underwent immunofluorescence staining procedures. Confocal microscopy was performed using an Olympus FV3000 confocal microscope.

Transmission electron microscopy

Mice subjected to the indicated treatments were euthanized and immediately perfused with normal saline followed by 4% paraformaldehyde solution. The lung, liver, and spleen were fixed in 2% glutaraldehyde at 4 °C overnight. Subsequently, target tissues were dissected, and selected areas were further fixed in 1% osmium tetroxide for 1 h. Following fixation, samples were dehydrated in a graded ethanol series, embedded in epoxy resin, and polymerized at 80 °C for 24 h. Ultrathin Sect. (100 nm) were cut using a Leica EM UC7 and placed on EM grids. Sections were stained with uranyl acetate and lead citrate before examination with an EM-1400 transmission electron microscope (Japan).

Colony formation experiment

E. coli was cultured at 37 °C under aerobic conditions with shaking in Luria Bertani (LB) medium overnight. The bacteria were added to macrophage cultures at a ratio of 1:2500 (v/v) when the bacterial culture reached an OD of 0.6 as determined by spectrophotometry, followed by incubation for 1 h at 37 °C. Subsequently, cell medium or cell lysates were collected for the colony formation experiment. Image capture was conducted using an EVOS XL Core Microscope (Life Technologies).

LB medium was prepared by dissolving 1 g tryptone, 0.5 g yeast extract, 1 g NaCl, and 1.4 g agar powder in 100 ml distilled water, followed by sterilization at 121 °C for 25 min. LB medium was then poured into petri dishes in an ultra-clean workbench and allowed to cool and solidify.

Equal volumes of BMDM medium or cell lysates from peritoneal lavage fluid of each group were added onto the solid LB medium and spread evenly with a sterile spreader until dry. The amount of liquid added to the plate was adjusted according to the number of colonies in the culture medium. The coated LB plates were sealed with sealant and incubated at 37 °C overnight for 24 h to observe the number of colonies.

Histological analysis

Tissues were formalin-fixed, processed, and paraffin-embedded. H&E staining was performed by Hangzhou Luoke Biotechnology Co., Ltd.

Statistical analysis

Statistical analyses were performed using Prism software (GraphPad 9), and P values were calculated using a two-tailed t-test or one-way ANOVA analysis, as appropriate. Each experiment was independently repeated at least three times, and the data are presented as the mean ± standard error (SE). Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).