ICR male mice aged 6 weeks were fed methionine-choline-deficient and a high-fat (MCDHF) diet (Research Diet, New Brunswick, NJ, USA) as described previously23. Mice were sacrificed at day 1, 3, 7, 14, 28, or 56 after MCDHF treatment. To eliminate NETs in vivo, we treated mice with deoxyribonuclease I (DNase I). The intraperitoneal injection of DNase I (50 μg/mouse, Roche) or vehicle was performed 24 h before feeding an MCDHF diet, and then three times per week. S1PR2 knockdown mouse model was constructed by intravenous injection of chemically modified small interfering RNA (siRNA) of S1PR2 (UAA CUC CCG UGC AGU GGU UUU) purchased from Thermo Scientific (Lafayette, CO, USA). Control mice were injected with an equal volume of scramble (SCR) siRNA dissolved in phosphate-buffered saline (PBS). S1PR2-siRNAs were injected one day before MCDHF-induced liver injury, and then twice every week. Based on our published studies, six mice per group were used for the animal studies23,24,25. The animal studies were randomized and blinded according to the protocol approved by the Ethics Committee of Capital Medical University and in accordance with the approved guidelines (approval number: AEEI-2017-090).
ICR male mice aged 6 weeks received lethal irradiation (8 Gy) and immediately received transplantation by a tail vein injection of 1.5 × 107 whole bone marrow (BM) cells obtained from 3-week-old enhanced green fluorescence protein (EGFP) transgenic mice. Four weeks later, mice whose BM was rebuilt were subjected to MCDHF-induced liver injury.
Fluorescence-activated cell sorting
Nonparenchymal cells of mouse liver were isolated as described previously23. APC-Ly6G (BD Bioscience, Franklin Lakes, NJ, USA) and its isotype-matched negative control were added to the nonparenchymal cell suspension, respectively. After 15 min incubation in the dark, the cells were washed with PBS and subjected to fluorescence-activated cell sorting (FACS), which was performed on a FACSAria and analyzed with FACS Diva 4.1 (BD Biosciences).
Isolation of mouse BM neutrophils
BM cells, acquired from ICR mice aged 6 weeks as described25, were layered in a ratio of 1:3 on top of Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA), after centrifugation, and the precipitate was resuspended with PBS. The cell suspension was layered in a ratio of 1:2 on top of Histopaque 1119 (Sigma-Aldrich), after centrifugation, and neutrophils were recovered on the top of Histopaque 111926. Neutrophils were washed with PBS and then resuspended in RPMI medium 1640.
Detection of apoptosis
The Annexin V/PI apoptosis detection kit (BD Biosciences) was used to detect apoptosis according to the manufacturer’s instructions. Caspase-3 cleavage detected by western blot analysis was also used as additional indicators of apoptosis.
MPO-DNA complex detection
A capture enzyme-linked immunosorbent assay (ELISA) detecting MPO associated with DNA (MPO-DNA) was performed as described27. For the capture antibody, an MPO ELISA kit (Hycult; HK210-01) and a peroxidase-labeled anti-DNA monoclonal antibody (component 2, Cell Death ELISAPLUS; Roche) were used according to the manufacturer’s directions.
Western blot analysis
Western blot analysis was performed with 50 μg of protein extract from cells or 100 μg from liver tissue, using monoclonal antibodies: citrullinated-histone H3 (1:1000, Abcam, ab5103); ERK1/2(1:1000, Cell Signaling, 4695S) and phosphor-ERK1/2 (1:1000, Cell Signaling, 4376S), p38 (1:1000, Cell Signaling, 9212S), and phosphor-p38 (1:1000, Cell Signaling, 9211S); caspase-3 (1:1000, Cell Signaling, 9662S) and cleaved caspase-3 (1:1000, Cell Signaling, 9661S); anti-β-tubulin (1:5000, Cell Signaling, 2146S) and anti-β-actin monoclonal antibodies (1:5000, Cell Signaling, 3700S). ODYSSEY goat anti-rabbit IRDye® 800 CW antibody and goat anti-mouse IRDye® 800 CW antibody (1:10,000, LI-COR) were used as secondary antibodies. The bands were displayed using ODYSSEY and quantified by Odyssey v3.0 software. β-Tubulin or β-actin was used as reference.
Cells were fixed in 4% paraformaldehyde in PBS for 30 min and permeabilized in 0.5% Triton X-100 in PBS for 3 min; after blocking with 2% bovine serum albumin (Roche), they were incubated with the specific primary antibodies for S1PR1 (1:50, Santa Cruz, sc-25489); S1PR2 (1:50, Santa Cruz, sc-25491); S1PR3 (1:50, Santa Cruz, sc-30024); citrullinated-histone H3 (Cit-H3) (1:200, Abcam), myeloperoxidase (MPO) (1:200; Abcam, 14569), or neutrophil elastase (NE) (1:100, Abcam, ab21595), and secondary antibody conjugated with Cy3 (1:100, Jackson Immunoresearch) was a secondary antibody. The samples were observed under a confocal microscope (LSM510, Carl Zeiss MicroImaging). Fluorescence intensity and NET formation were analyzed by ImageJ 1.4 (NIH).
Total RNA was extracted from cells or liver frozen specimen using RNeasy mini kit (Qiagen, Germany) and the quantity and purity of RNA was determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA was synthesized using oligo (dT) and M-MLV reverse transcriptase (Invitrogen). Quantitative reverse transcription-PCR (RT-qPCR) was performed using SYBR Green qPCR Master Mix on the ABI 7300 TH Real-Time PCR System (Applied Biosystems). All primers were synthesized by Biotech (Beijing, China). Primers used for RT-qPCR were as follows: Rn18s: sense, 5′-GTA ACC CGT TGA ACC CCA TT-3′; antisense, 5′-CCA TCC AAT CGG TAG TAG CG-3′. Ly6g: sense, 5′-AGA AGCA AAG TCA AGA GCA ATC TCT-3′; antisense, 5′-TGA CAG CAT TAC CAG TGA TCT CAG T-3′; Sphk1: sense: 5′- TGT CAC CCA TGA ACC TGC TGT CCC TGC ACA-3′; antisense: 5′-AGA AGG CAC TGG CTC CAG AGG-3′. S1pr1: sense: 5′-ACT TTG CGA GTG AGC TG-3′; antisense: 5′-AGT GAG CCT TCA GTT ACA GC-3′. S1pr2: sense: 5′-TTC TGG AGG GTA ACA CAG TGG T-3′; antisense, 5′-ACA CCC TTT GTA TCA AGT GGC A-3′. S1pr3: sense: 5′-TGG TGT GCG GCT GTC TAG TCA A-3′; antisense, 5′-CAC AGC AAG CAG ACC TCC AGA-3′. Tnf: sense, 5′-GGC AGG TTC TGT CCC TTT CA-3′; antisense, 5′-CTG TGC TCA TGG TGT CTT TTC TG-3′. Acta2: sense: 5′-ATG CTC CCA GGG CTG TTT T-3′; antisense: 5′-TTC CAA CCA TTA CTC CCT GAT GT-3′. Col1a1: sense: 5′-AGG GCG AGT GCT GTG CTT T-3′; antisense: 5′-CCC TCG ACT CCT ACA TCT TCT GA-3′. Col3a1: sense: 5′-TGA AAC CCC AGC AAA ACA AAA-3′; antisense, 5′-TCA CTT GCA CTG GTT GAT AAG ATT AA-3′. Il6: sense, 5′-CTC TGG GAA ATC GTG GAA ATG-3′; antisense, 5′-AAG TGC ATC ATC GTT GTT CAT ACA-3′. Ilb: sense, 5′-GCA ACT GTT CCT GAA CTC AAC T-3′; antisense, 5′-ATC TTT TGG GGT CCG TCA ACT-3′.
2′,7′-Dichlorofluorescein diacetate (DCFDA) (Sigma-Aldrich) is a cell-permeable, non-fluorescent probe; it is de-esterified intracellularly and turns to highly fluorescent 2′,7′-dichlorofluorescein upon oxidation. BM neutrophils were incubated with DCFDA for 20 min, and after seeding in 96-well plates, they were treated with S1P. The plate was then transferred onto a fluorescent plate reader, EnVision 2104-0010 (PerkinElmer, MA, USA), and detected the fluorescent value.
The concentration of hepatic S1P was measured by the S1P ELISA kit (Echelon, USA) according to the manufacturer’s instructions. A standard curve was created, and the results were normalized to the protein content of the sample (pmol S1P/mg protein).
Liver damage assessment
Serum alanine aminotransferase (ALT) levels were detected by BS-200 Chemistry Analyzer (Mindray, China). Serum γ-glutamyl transpeptidase (GGT) levels were determined by a GGT ELISA kit (Cusabio, China) according to the manufacturer’s directions.
Liver tissues were fixed in 4% buffered formaldehyde. Liver tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) staining for assessment of inflammation and injury and Sirius Red staining for the extent of collagen deposition. Morphometric analysis was conducted with researcher blind to the treatment. We measured 15 randomly selected areas per sample and calculated the mean value of the percentage of inflammatory or fibrosis area accounting for total area. Morphometric analysis of H&E and Sirius Red staining was done using ImageJ 1.4 (NIH).
The results were expressed as mean ± standard error of the mean (SEM). Comparisons between two independent groups were performed using a two-sample t test. Comparisons between multiple groups were performed by one- or two-way ANOVA (analysis of variance) with post hoc Tukey’s multiple comparison tests when appropriate. Correlation coefficients were calculated by Pearson’s test. P < 0.05 was considered to be significant. All in vitro experiments were executed duplicable and repeated three times. The animal experiments were conducted with six mice in each group.