Animals

6–8 weeks old male C57BL/6 mice (25–30 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. Healthy male Sprague Dawley rats, weighing 200–250 g were purchased from Hunan SJA Laboratory Animal Co., Ltd. The selection of both rat and mouse neutrophils for experimental studies makes our results more convincing. Mice were housed in a 12 h light /12h dark cycle, controlled room temperature (22 to 24 °C), and humidity (30 to 70%). They were fed food and water ad libitum before the experiment. The animals were grouped into the sham group and the cecal ligation and puncture (CLP) group, with 25 animals in each group. The CLP-induced sepsis model was established [17, 18]. The mice fasted and drank freely only 12 h before surgery. Mice were intraperitoneally injected with 10% chloral hydrate at an anesthetic dose of 0.3 mL/100 g. The cecum was hooked out of the right abdomen of the mouse with toothless curved forceps to expose the cecum and ligated at 3/4 of the cecum. The cecum was punctured twice parallel to the mesentery using a 0.7 mm diameter needle. The cecum was gently squeezed to allow a small amount of feces to spill out to induce infection. The sham group carried out similar manipulations but did not perform ligation and puncture. Among them, 5 animals/group were used for lung tissue permeability assessment. 5 animals/group were used for wet/dry ratio assessment. Bronchoalveolar lavage fluid (BALF) was analyzed in 5 animals/group. Flow cytometry (FCM), neutrophil extraction and Western blot were conducted in 5 animals/group. 5 animals/group were used for pathology, biochemical detection, and cryopreservation.

Furthermore, the mice were grouped into sham, CLP, CLP + BMS309403, CLP + BMS309403 + Thapsigargin (Tg), CLP + anti-Ly6G, CLP + anti-Ly6G + BMS309403, and CLP + anti-Ly6G + BMS309403 + Tg, with 15 animals in each group. The following interventions were performed on mice before modeling: BMS309403 was intraperitoneally injected, the injection dose was 5 mg/kg, the injection volume was 10 ml/kg, and the drug was administered 2 days before modeling [7]. Tg was injected intraperitoneally, the injection dose was 1 mg/kg, the injection volume was 10 ml/kg, and the drug was administered 2 days before molding [19, 20]. Anti-Ly6G antibody was given intraperitoneally at an injection dose of 125 μg/200 μl/mice 24 h after intervention with Tg or BMS309403 (or 1 day before modeling) [21]. As mentioned earlier, the sham and CLP groups were constructed. Among them, 5 animals/group were used for pathological and biochemical analysis of lung tissue. 5 animals/group were used to detect BALF. 5 animals/group were used for FCM analysis. After surgery, mice were resuscitated by subcutaneous injection of 1 mL of pre-warmed (37 °C) 0.9% normal saline. After 24 h of modeling, the experiment was completed, and mouse lung tissue, BALF, peripheral blood and cecal feces were harvested [22].

Hematoxylin–eosin (H&E) staining and lung injury score

The entire lungs were promptly preserved by fixation in 4% paraformaldehyde and then embedded in paraffin. Sections with a thickness of 3 μm were prepared and stained with hematoxylin and eosin, followed by examination under a microscope (BA210T, Motic). To assess the severity of lung injury, a semi-quantitative scoring system was employed following H&E staining. This system involved calculating scores for inflammation, edema, bleeding, and thickening of the alveolar septum in each group, with the final score reflecting a comprehensive assessment of all four injury parameters [18].

Evaluation of pulmonary edema

The lung tissue was weighed immediately after extraction to obtain the wet weight. Subsequently, the lungs were placed in a 60 °C oven for 48 h to allow for dehydration. After drying, the lungs were weighed again to determine the dry weight. The lung weight ratio (wet/dry) was calculated before and after drying.

Assessment of pulmonary vascular permeability

Pulmonary vascular permeability was assessed using the Evans blue (EB)-albumin (EBA) [23]. In brief, EB at a dosage of 20 mg/kg was injected into anesthetized mice via the caudal vein and allowed to circulate through the blood vessels for 30 min. After circulation, intravascular EB was perfused into the right ventricle by irrigation over a period of 2 min using Krebs/HEPES Buffer. Following this, mouse lungs were excised, weighed, homogenized in 1 mL of PBS, and then subjected to overnight extraction in 2 mL of formamide at 60 ℃. The concentration of EB in the supernatant of the lung homogenate was then quantified using spectrophotometry at 620 nm.

Determination of bacterial load in peripheral blood

Bacterial load was determined in blood at 24 h after CLP. Blood was collected by puncturing the heart. Blood samples were placed on ice and serially diluted with sterile PBS. Then, 100 μL of each diluted sample was placed on a trypsin soybean AGAR plate containing 5% sheep blood (BD 221261, BD Biosciences) and incubated at 37 °C for 24 h. The number of bacterial colonies was counted and expressed as colony-forming units per mL.

Biochemical testing

Mouse lung tissue and BALF were taken to measure inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, Interferon-gamma (IFN-γ), C–C motif chemokine ligand 3 (CCL3), and C-X-C motif chemokine ligand 8 (CXCL8, also known as IL-8) levels. With IL-1β (CSB-E08054m, CUSABIO), TNF-α (CSB-E04741m, CUSABIO), IL-6 (CSB-E04639m, CUSABIO), IFN-γ (KE10001, Proteintech), CCL3 (CSB-E04663m, CUSABIO), and CXCL8 (EM1592, Wuhan Fine Biotech Co., Ltd.) kits, the inflammatory factors IL-1β, TNF-α, IL-6, IFN-γ, CCL3, and CXCL8 were detected.

Isolation and purification of neutrophils

Mouse peripheral neutrophil isolation solution kit (http://www.solarbio.net/goods-12453.html, P9201, Solarbio) or rat peripheral neutrophil isolation solution kit (http://www.solarbio.net/goods-12452.html, P9200, Solarbio) were used to isolate neutrophil from mouse or rat peripheral blood according to the instruction. For isolation of neutrophil from mouse or rat peripheral blood, after 4 mL of fresh anticoagulant whole blood was taken, 4 mL of reagent A was first added into the centrifuge tube, and 2 mL of reagent C was carefully superimposed on reagent A to form a gradient interface. The blood was laid flat above the liquid level of the separation liquid, and the interface between the two liquid levels was kept clear. The samples were centrifuged at room temperature at 1000 g for 35 min. After centrifugation, two ring layers of milky white cells appeared in the centrifuge tube, the upper layer of mononuclear cells and the lower layer of neutrophil cells. Neutrophils from reagent C to reagent A and from reagent A were carefully drawn with a straw into a 15 mL clean centrifuge tube and 10 mL PBS wash cells. The cells were centrifuged at 250 g for 10 min. The supernatant was discarded, 5 mL of PBS was added to resuspend cells, and cells were centrifuged at 250 g for 10 min. Then the supernatant was discarded again, 5 mL of PBS was added to resuspend cells, and cells were centrifuged at 250 g for 10 min. The supernatant was abandoned, and the cells were resuspended.

Neutrophils were isolated from the bone marrow of mice or rats using a mouse bone marrow neutrophils isolation kit (http://www.solarbio.net/goods-12468.html, P8550, Solarbio) or a rat bone marrow neutrophils isolation kit (http://www.solarbio.net/goods-12467.html, P2610, Solarbio). For isolation of mouse or rat bone marrow neutrophils, the mouse or rat was killed by cervical dislocation, soaked in 75% ethanol solution for 5 min for disinfection, and moved to an ultra-clean bench. Pre-sterilized scissors and tweezer were used to cut the skin in a circular pattern along one thigh, and the skin was peeled towards the claw and ended at the foot. Tweezers were used to separate the leg muscles, and the femur and tibia were exposed (without damaging the bone). The ligaments between the femur and the hip were cut, and the bone below the ankle was cut. The femur and tibia were subdivided into a medium dish (sterile PBS containing 5% penicillin/streptomycin), then immersed in another PBS dish containing 5% penicillin/streptomycin, and the other leg was treated in the same way. 5 mL preheated 1640 culture solution was sucked with a 5 mL sterile syringe and filled with 1 mL syringe needle. The femur and tibia were separated from the knee joint, and the kneecap was discarded. A sterile forceps was used to clamp the femur, and ophthalmic scissors were used to remove both ends of the bone. The needle was inserted into the bone marrow cavity, and the bone marrow cavity was rinsed repeatedly with the culture solution until the bone marrow cavity turned white. The bone marrow was flushed into a new sterile 50 mL centrifuge tube. During irrigation, the bone marrow cavity was scraped by moving the needle up and down, and the tibia was treated in the same way. The precipitation was obtained by centrifugation, and 4 mL diluent was added to resuspend the cells. A new centrifuge tube was taken and 4 mL of reagent A was added first, and then 2 mL of reagent C was carefully superimposed on reagent A to form a gradient interface. The cell suspension was laid flat above the liquid surface of the separation liquid, and the interface between the two liquid surfaces was kept clear. The other methods are the same as the blood treatment.

Isolated neutrophils were cultured with 5 × 106 cells per mL in Roswell Park Memorial Institute (RPMI) 1640 with 10% fetal bovine serum and 1% penicillin/streptomycin [24]. BALF was collected within 24 h of human ARDS patients’ diagnosis, from which neutrophils were isolated using flow cytometry with anti-CD16 (Abcam, Cambridge, UK) and anti-CD45 (BD, San Jose, Califormia) double antibodies. Healthy controls were recruited from patients with an unrelated disease where necessary bronchoscopy was performed. The purity of isolated neutrophils needed to be at least 95% to be included.

RNA extraction and sequencing

Total RNA was isolated from the neutrophil samples using Trizol (Invitrogen, Carlsbad, CA, USA) and assessed for quality and quantity using a NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. Subsequently, cDNA fragments generated in the earlier step were amplified via Polymerase Chain Reaction (PCR), and the resulting products were purified using Ampure XP Beads, followed by dissolution in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double-stranded PCR products from the previous step were heat denatured and circularized by the splint oligo sequence to get the final library. The single-strand circle DNA (ssCir DNA) was formatted as the final library. This final library was amplified with phi29 to form DNA nanoballs (DNBs), with each DNB containing over 300 copies of a single molecule. The DNBs were loaded onto a patterned nanoarray, and paired-end 100-base reads were generated using the BGIseq500 platform (BGI-Shenzhen, China).

Giemsa staining

Rat neutrophils and mouse neutrophils were identified by Giemsa staining. The suspended cells were mixed with a pipette gun and smeared on slides, then rinsed with small water for 30 min. Then, Giemsa staining was added and stained for 30 s. 1–2 times the volume of Giemsa buffer was added drop by drop directly, the slides were gently shaken, and the staining solution was well mixed and the slides were stained for 3 min. The cells were treated with 75% ethanol for a duration of 2 min to achieve differentiation. The slides were baked at 60 ℃ for 30–60 min, placed in xylene for 10 min, twice, sealed with neutral gum, and observed under the microscope.

Cell culture and treatment

Rat lung epithelial cells RLE-6TN (iCell-r039, iCell) were cultured in special medium (iCell-r039-001b, iCell). Human non-small cell lung cancer cells A549 (AW-CCH011, abiowell) were cultured in F-12K medium (AW-MC004, abiowell) with 10% fetal bovine serum and 1% penicillin/streptomycin. Human promyeloid leukemia cells HL-60 (AW-CCH071, abiowell) were cultured in IMDM medium (AW-MCH071, abiowell) with 10% fetal bovine serum and 1% penicillin/streptomycin. They were all incubated at 37 °C, 5% CO2, in a saturated humidity incubator. First, rat and mouse neutrophils were treated with different concentrations of N-acetylaspartate (NaA, 127-09-3, Sigma). The groups were Control, vehicle (DMSO-treated neutrophils), 0.01 mM NaA, 0.1 mM NaA, 1 mM NaA, and 10 mM NaA. In addition, we added 25 nM polymalate (PMA, P1585, Sigma) for treatment, and the groups were: Control, PMA, PMA + vehicle, PMA + 0.01 mM NaA, PMA + 0.1 mM NaA, PMA + 1 mM NaA, and PMA + 10 mM NaA. After screening, 10 mM NaA was selected for further treatment, grouped as PMA (25 nM PMA-treated neutrophils), PMA + vehicle (25 nM PMA and DMSO-treated neutrophils), and PMA + NaA (25 nM PMA and 10 mM NaA-treated neutrophils). Moreover, we performed PMA and NaA interventions on rat and mouse neutrophils after stable transfection of si-NC or si-FFAR2. The groups were PMA + si-NC, PMA + si-FFAR2, PMA + NaA + si-NC, and PMA + NaA + si-FFAR2. After that, we treated rat neutrophils with 10 μM FABP4 inhibitor BMS309403 (HY-101903, MCE) and grouped as Control, PMA (25 nM PMA-treated rat neutrophils), PMA + vehicle (25 nM PMA and DMSO-treated rat neutrophils), and PMA + BMS309403 (10 μM BMS309403 and 25 nM PMA-treated rat neutrophils). Subsequently, the cells were treated with a 10 μM FABP4 inhibitor, BMS309403, for 30 min [8] and 100 nM ER stress inducer Thapsigargin (Tg) for 3 h before other interventions [25], and grouped as PMA + vehicle (25 nM PMA and DMSO-treated rat neutrophils), PMA + vehicle + Tg (25 nM PMA and DMSO-treated cells followed by 100 nM Tg-treated rat neutrophils), PMA + BMS309403 (10 μM BMS309403 and 25 nM PMA-treated rat neutrophils), and PMA + BMS309403 + Tg (10 μM BMS309403 and 25 nM PMA-treated cells followed by 100 nM Tg-treated rat neutrophils).

Furthermore, rat lung epithelial cells RLE-6TN were cultured and grouped as follows: RLE-6TN (cells were cultured normally), RLE-6TN + Neutrophil-PMA (normal rat neutrophil supernatant conditioned medium culture after addition of PMA intervention), and RLE-6TN + Neutrophil-(PMA + BMS309403) (supernatant conditioned medium culture of rat neutrophils after PMA and BMS309403 intervention were added) groups. In addition, after 10 μM BMS309403 advance treatment for 30 min, anti-TNF-α and anti-IL-6 intervention on neutrophils for12 h [26], supernatants of each group were collected as conditioned medium for RLE-6TN cell culture. Untreated neutrophil supernatants were used as control (Neutrophil-PMA). The groups were RLE-6TN + Neutrophil-PMA, RLE-6TN + Neutrophil-(PMA + BMS309403), RLE-6TN + Neutrophil-(anti-IL6 + PMA + BMS309403), and RLE-6TN + Neutrophil-(anti-TNF-α + PMA + BMS309403).

Additionally, HL-60 was stimulated by 1.2% DMSO for 5 days to induce differentiated HL-60 neutrophil-like cells (dHL-60) [27]. Next, the medium of PMA and BMS309403 intervention of dHL-60 cells were collected (the intervention procedure was the same as above) to treat A549 cells. A549 cells were grouped into: A549 group (A549 cells were normally cultured), A549 + Neutrophil-PMA group (The medium of PMA intervention of dHL-60 cells were collected to treat A549 cells), A549 + Neutrophil-(PMA + BMS309403) group (The medium of PMA and BMS309403 intervention of dHL-60 cells were collected to treat A549 cells).

FCM

The expression of CD45 + CD11b + Ly6G + in neutrophils was analyzed using FCM. The isolated cells were washed with PBS, precipitated once, and centrifuged at 1000 rpm for 5 min. Antibodies CD45-FITC (11-0451-82, eBioscience), CD11b-APC (17-0112-82, eBioscience), and LY-6g-PE (12-9668-82, eBioscience) were incubated for 30 min. Cells were determined using FCM (A00-1-1102, Beckman).

In addition, the content of macrophage CD45 + CD68 + F4/80 + in lung tissue was analyzed by FCM. The lung tissues were cut into 0.5 mm pieces with scissors. The clipped tissues were added to pancreatic enzyme for digestion, and digestion was terminated 1 h later. The cell suspension was pumped with a syringe to disperse the cells. The cell suspension was filtered with a 100 μm filter, and the cell suspension was collected and centrifuged at 1000 rpm for 5 min. After washing with PBS twice, CD45 antibody (25-0451-82, eBioscience™) was added and stained for 30 min without light. After washing with PBS twice, 1 mL of cell fixative was added and fixed at 4 ℃ for 30 min. Cell precipitation was obtained by centrifugation, and 1 × membrane-breaking solution was added to break the membrane for 30 min. Cell precipitates were obtained by centrifugation, cells were cleaned twice with staining buffer, CD68 antibody (MA5-23616, Invitrogen) and F4/80 antibody (11-4801-82, eBioscience™) were added, and the cells were stained for 30 min away from light. After the staining buffer was washed 3 times, 200 μL staining buffer was added to resuspension cells, and FCM was performed.

Trypan blue staining

Cell death of mouse neutrophils, rat neutrophils, and RLE-6TN was analyzed by trypan blue staining. Cells were digested and collected with trypsin and centrifuged at 1000 rpm for 5 min. Cell formation suspension was prepared with 1mL PBS to remove the supernatant. 50 μL cell suspension was mixed with 50 μL 0.08% trypan blue.

Cell apoptosis

Cell apoptosis rate was analyzed by FCM. Cells were suspended in 500 μL of Binding buffer, to which 5 μL of Annexin-APC was added. After adding and thoroughly mixing 5 μL of Propidium Iodide, the reaction mixture was incubated at room temperature, away from light for 10 min.

Immunofluorescence (IF)

The expression of MPO + FABP4 and MPO + citrullinated histone H3 (Cit-H3) in the mouse lung was evaluated through IF staining. The sections underwent a series of treatments, beginning with baking at 60 °C for 12 h, dewaxing to water, and heat-induced antigen retrieval. Subsequently, the sections were incubated in a sodium borohydride solution at room temperature for 30 min. Endogenous oxides were blocked, followed by sealing the sections with 3% hydrogen peroxide for 15 min. A solution of 10% normal serum/5% BSA was then applied for 60 min. The primary antibody MPO (ab208670, 1:50, abcam) was added and allowed to incubate overnight at 4 °C. This step was followed by incubation with an HRP secondary antibody and TYP-520 fluorescent dye. Antigen retrieval was performed again, endogenous peroxidases were blocked, and 3% hydrogen peroxide was applied for 15 min. Additionally, 10% normal serum/5% BSA was used. An appropriately diluted primary antibody FABP4 (12802-1-AP, 1:400, PTG) and Cit-H3 (ab281584, 1:2000, abcam) were added overnight at 4 ℃. Following this, HRP secondary antibodies and TYP-570 fluorescent dye were applied. Sudan Black B solution was incubated for 3 min. DAPI working solution was used for nuclear staining. Sections were sealed and observed under a fluorescence microscope (BA410T, Motic). Quantitative analysis was carried out using Image-Pro Plus software, where the images were converted to grayscale, colors were inverted, and light density was adjusted. The positive area in the images was selected, and the Integrated Optical Density (IOD) column sum value was obtained, representing the measured data.

Moreover, IF was utilized to determine FABP4 expression in mouse and rat neutrophils, and the expression of CHOP + FABP4 in rat neutrophils. The suspended cells were mixed with the pipette, coated on the slides, left to dry, rinsed with water for 30 min, and added with 0.3% Triton X-100 and permeated. Slides were blocked at 37 ℃ for 60 min. The primary antibodies FABP4 (15872-1-AP, 1:50, Proteintech) and CHOP (ab11419, 1:50, Abcam) were added at 4 ℃ overnight. 50–100 μL anti-Rabbit, Mouse and Rabbit-IgG labeled fluorescent antibodies were added and incubated. DAPI working solution was used for nuclear staining enhancement, and the slides were then sealed and observed under a fluorescence microscope (BA210T, Motic).

16S rRNA sequencing

To assess alterations in microbial diversity, fecal samples were collected from mice in sham and CLP groups. Raw data was obtained using Illumina NovaSeq PE250 for 16S amplicon sequencing. Qiime 2 analysis process was utilized, with DADA2 employed to denoise raw data. Following denoising, the sequence data was directly processed to eliminate redundancies, allowing for the extraction of feature information. Species annotation was performed for each amplicon sequence variants (ASV) sequence, and species composition in the samples was analyzed through comparison with the species database.

RNA-seq data processing and analysis

The sequencing data was filtered with SOAPnuke (v1.5.2) by removing reads with a sequencing adapter, removing reads whose low-quality base ratio (base quality less than or equal to 5) was more than 20%, and removing reads whose unknown base (‘N’ base) ratio was more than 5%. After these steps, the clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome (hg38) using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, and then the expression level of a gene was calculated by RSEM (v1.2.12). DESeq2 was employed to analyze differential expression between different groups, with genes exhibiting a false discovery rate (FDR) < 0.05 and an absolute log2 fold change (FC) > 2 between ARDS and control groups designated as differentially expressed genes.

Detection of SCFAs

Serum samples from the sham and CLP groups were collected to detect changes in SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid) on the Agilent 7890B-5977B gas chromatograph. The chromatographic conditions were set as follows: Column: DB-WAX capillary column, 30 mm × 0.250 mm × 0.25 μm, carrier gas: helium gas (99.999%), flow rate: 1 ml/min, inlet temperature: 250 °C, and auxiliary heater temperature: 260 °C. The mass spectrometry conditions were set as follows: ionization source: EI source temperature: 230 °C, quadrupole temperature: 150 °C, electron energy: 70 eV, and scanning range: 33–300.

Western blot

Western blot was performed to measure the expressions of FABP4, PGC-1α, PPARγ, p-PERK, PERK, p-eIF2α, eIF2α, and ATF4 in mouse or rat neutrophils. Total proteins were extracted from mouse or rat neutrophils using 200 μL RIPA (P0013B, Beyotime), and protein quantification was performed utilizing BCA protein assay kit (BL521A, Biosharp). 120 μL protein supernatant was taken and added with 30 μL 5 × loading buffer. The SDS-PAGE loading buffer was mixed, and the mixture was heated in boiling water bath at 100 ℃ for 5 min. 20 ± 2 μg proteins were used. The protein was wet transferred by gel electrophoresis on nitrocellulose membrane, and sealed with 5% skim milk solution at room temperature for 90 min. Proteins were incubated with FABP4 (12802-1-AP, 1:10,000, proteintech), PGC-1α (66369-1-Ig, 1:8000, proteintech), PPARγ (16643-1-AP, 1:5000, proteintech), p-PERK (29546-1-AP, 1:1000, proteintech), PERK (24390-1-AP, 1:1500. proteintech), p-eIF2α (ab32157, 1:5000, abcam), eIF2α (11170-1-AP, 1:8000, proteintech), ATF4 (60035-1-Ig, 1:2000, proteintech), FFAR2 (19952-1-AP, 1:800, proteintech), and β-actin (66009-1-Ig, 1:5000, proteintech) at 4 ℃ overnight. Proteins were then incubated with HRP-labeled secondary antibodies Goat anti-Mouse IgG (H + L) Secondary Antibody (AWS0001, 1:5000, Abiowell) and Goat anti-Rabbit IgG (H + L) Secondary Antibody (AWS0002, 1:5000, Abiowell) for 90 min. ECL color development was exposed and the protein was detected using β-actin as an internal reference.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 software. Data are presented as mean ± standard deviation (SD). Student’s t-test was used for comparison between the two groups. The significance of three or more groups was compared by one-way ANOVA, followed by Tukey’s post hoc test. Correlation between differential gut microbiota and SCFAs, and acetic acid and neutrophil content were analyzed by Pearson correlation coefficient. P < 0.05 was deemed statistically significant.