Animals
Wild type (WT) male C57BL/6J mice aged 6–8 weeks (Shanghai Sippr-BK Laboratory Animal Co., Ltd., Shanghai, China) were fed under a specific pathogen-free environment in Xinhua Hospital Animal Laboratory (Shanghai, China). All animal experiments were conducted under the rules approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Approval No.: XHEC-F-2020-019).
PMNs isolation and activation
PMNs were induced in the peritoneal cavity of the mice as previously described [22]. Briefly, mice were injected intraperitoneally (i.p.) with 1 ml 9% casein solution twice overnight and killed 3 h after the second injection to harvest the peritoneal lavage fluid (PLF), which was subsequently centrifuged, and the cell pellets were washed. PMNs were isolated by discontinuous density gradient centrifugation with two commercially available solutions (Histopaque-1077 and Histopaque-1119) of differential density (Sigma (St. Louis, MO; #11191 and #10771) according to the manufacturer’s instructions. The resulting cells consisted of 90% PMNs, and viability of the isolated PMNs was 95% as assessed by flow cytometry and Trypan blue staining, respectively.
After isolation, PMNs were suspended in complete culture medium (RPMI 1640 containing 10% exosome-free FBS, supplemented with 50 mg/ml penicillin/streptomycin) at a concentration of 106 cells/ml. PMNs activation was induced upon 12-h incubation with 20 ng/mL TNF-α at 37 °C. An equal volume of phosphate buffered saline (PBS) to TNF-α was used as negative control.
Exosome isolation and characterization
Exosomes were isolated from the supernatant of PMNs treated with PBS (PBS-Exo) or TNF-α (TNF-Exo) ex vivo using Total Exosome Isolation Reagent (#4484450; Thermo Fisher Scientific, Waltham, MA, USA). The detailed isolation procedure and the methods used to determine exosomal morphology, size distribution, and surface marker expression are described in Additional file 1.
In vivo exosome administration to WT C57BL/6 mice
To explore exosome function in vivo, five WT C57BL/6 mice in each group were injected with PBS-Exo or TNF-Exo (300 μg/mouse) intraperitoneally using a 31-gauge insulin syringe, respectively. An equal volume of PBS was used as negative control. After 24 h, the obtained peritoneal lavage fluid was centrifuged, and peritoneal macrophages were detected by flow cytometry after gating with F4/80. To visualize changes in morphology and macrophage polarization, lung tissues were harvested and fixed in 4% paraformaldehyde for H&E and immunofluorescence staining. H&E staining was evaluated by a pathologist who was blinded to the experimental groups. To evaluate the lung injury, five independent random lung fields were evaluated per mouse for neutrophils in alveolar spaces, neutrophils in interstitial spaces, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening, and weighed according to the official American Thoracic Society workshop report on features and measurements of experimental ALI in animals [23]. The resulting injury score is a continuous value between 0 and 1. For immunofluorescence staining, paraffin-embedded lung tissues were sectioned, blocked with PBS containing 1% goat serum and 3% BSA, permeabilized with PBS/Triton 0.01%, and incubated with F4/80 and iNOS antibodies, and then with species-specific secondary antibodies coupled with Alexa Fluor Dyes. DNA was stained using DAPI. The sections were treated with autofluorescent quenching solution (#G1221; Servicebio, Wuhan, China) and mounted in Vectashield Mounting Media.
The in vivo miR-30d-5p inhibitors were transfected into the mouse lung through tail vein injection using the in vivo-jetPEI (Polyplus-transfection SA, New York, NY). Briefly, the miR-30d-5p inhibitor or negative control (50 μg, N/P ratio = 6, i.e., 0.12 μl of in vivo-jetPEI per μg nucleic acid) dissolved in 200 μl 5% glucose solution was injected into each mouse 1 day before exosome injection, according to the manufacturer’s protocol.
In vitro co-culture experiments
Raw264.7 macrophages or BMDMs were treated with PMN-derived exosomes (100 μg/ml) at 37 °C for 24 h. To induce pyroptosis, macrophages were primed with exosomes for 24 h before stimulation with 5 mM ATP (#HY-B2176; MedChemExpress, Monmouth Junction, NJ, USA) or 20 μM nigericin (#HY-100381; MedChemExpress) for 2 h.
For miR-30d-5p inhibition, Raw264.7 macrophages were transfected with microRNA control or miR-30d-5p inhibitor (Guangzhou Ribobio Corporation, Guangzhou, China) at a concentration of 50 nM using Lipofectamine 3000 for 24 h prior to be co-cultured with exosomes as per the manufacturer’s instructions. For miR-30d-5p overexpression, Raw264.7 macrophages were transfected with microRNA control or miR-30d-5p mimic at a concentration of 50 nM using Lipofectamine 3000 for 48 h.
Flow cytometry analysis of M1 polarization and pyroptosis
Macrophages were centrifuged and resuspended in PBS for FACS analysis. According to the manufacturer’s instructions, anti-CD11c (#117307; BioLegend, San Diego, CA, USA), anti-CD86 (#105106; BioLegend), anti-CD206 (#141706; BioLegend) and anti-F4/80 (#123116; BioLegend) antibodies were used for fluorescent staining. Isotype antibody controls were used to exclude nonspecific staining. Data were obtained using a CytoFLEX flow cytometer (Beckman, USA). Programmed cell death was analyzed with apoptosis detection kit (#559763; BD Biosciences, Franklin Lakes, NJ). Macrophages were incubated with Annexin-V and PI for 15 min at room temperature in the dark and then analyzed by flow cytometry. Cells double-stained positive for Annexin V and PI were considered as undergoing programmed death. Cell pyroptosis was detected by two-color flow cytometry. Macrophages were incubated with Alexa Fluor 488-labeled caspase-1 FLICA (#ICT098; ImmunoChemistry, Bloomington, MN, USA) at 37 °C for 1 h. After being fixed with 4% paraformaldehyde, cells were stained with TMR red-labeled In-Situ Cell Death Detection reagent (#12156792910; Roche Applied Science, Indianapolis, IN, USA), following the manufacturer’s instructions. Double-stained cells were identified as pyroptotic cells. Background and autofluorescence were determined by a control antibody with the same isotype staining.
Sequencing of exosomal miRNA and data analysis
Total RNA was extracted from PBS-Exo/TNF-Exo using the miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). The final ligation PCR products were sequenced using the BGISEQ-500 platform (BGI Group, Shenzhen, China). After acquiring the raw data, the differentially expressed miRNAs were calculated using the t test. Those with ≥ twofold upregulation and a P value < 0.05 were regarded as significantly different. A heat map was generated using the R 3.5.3 software. Pathway analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The 20 most enriched pathways related to signaling transduction are listed and were used to reveal the most associated pathways.
Quantitative real-time PCR (RT-qPCR)
Total RNA isolation was performed using TRIzol following the manufacturer’s instruction (TAKARA, Japan). mRNA was reverse transcribed using PrimeScript RT reagent Kit (#RR036; TAKARA, Tokyo, Japan), and PCR was conducted using TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (#RR420A; TAKARA) and QuantStudio™ 3 System (Applied Biosystems). Data were normalized to the expression of GAPDH. Primer sequences are shown in Additional file 1: Table S1.
For exosomal miRNA quantification, total RNA was extracted from 200 μl PMN-derived exosomes using miRNeasy Serum/Plasma kit (#217184; Qiagen, Valencia, CA, USA). RNA pellets were resuspended in 14 μl RNase-free water. Twelve microliters of RNA solution were used for reverse transcription, according to the protocol of miScript RT Kit (#218161; Qiagen). miRNA expression was quantified using a miScript SYBR Green PCR Kit (#218075; Qiagen). qPCR analysis was also performed for miR-30d-5p expression in cells or the lung tissue. Briefly, RNA was extracted by TRIzol reagent (TAKARA, Japan) and RT-qPCR was conducted using the Mir-X miRNA qRT-PCR SYBR Kit (#638316; TAKARA). Relative expression was calculated using the comparative cycle threshold (Ct) method (2−ΔΔCT) normalized to U6. The miRNA qPCR primers were purchased from Guangzhou Ribobio Corporation.
Luciferase assay
The 3′-UTR of the SOCS-1/SIRT1 sequence containing the predicted miR-30d-5p binding sites and its mutant was cloned into the plasmid vector and transfected into HEK293 cells. A renilla luciferase vector was co-transfected in all transfections described to monitor transfection efficiency. All luciferase results were reported as relative light units: the average of the photinus pyralis firefly activity observed was divided by the average of the activity recorded from the renilla luciferase vector.
Mouse model of cecal ligation and puncture (CLP)
The CLP mouse model was prepared as previously described [22]. Mice were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) via i.p. injection. After disinfection, a 1 cm midline laparotomy was made in the abdomen. The cecum was then exteriorized, and ligated below the cecal valve, and punctured with an 18-gauge needle to induce sepsis. A small drop of cecal content was extruded. The cecum was then returned to the peritoneal cavity and the abdominal incision closed with sutures. Mice were resuscitated with (5 ml/100 g) saline. Sham animals underwent the same surgical procedures without cecum ligation and puncture. The in vivo miR-30d-5p inhibitors were transfected into the mouse lung through tail vein injection using the in vivo-jetPEI one day before CLP surgery, according to the manufacturer’s protocol. At 24 h after surgery, the animals were euthanized with phenobarbital overdose (100 mg/kg body weight), followed by collection of the lung tissues as described previously.
Statistics
The normal distribution of the data was tested using the Shapiro–Wilk test. Normally distributed data are presented as means ± SEM. Comparisons between 2 groups were performed by the 2-tailed Student’s t test. Multiple group comparisons were performed by one-way ANOVA followed by Tukey's multiple comparisons test with GraphPad Prism 8 software. Comparison of survival rates between groups was performed using Log-rank test. A value of P < 0.05 was considered statistically significant.