Animals
BALB/c female mice (6-8 weeks) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Mice were housed in cages with a constant-flow air exchange to support the specific pathogen-free condition, with sterile water and irradiated food from Pu Lu Teng Biotechnology Co. Ltd. (Shanghai, China) provided ad libitum. Animal care was in full compliance with the Guide for the Care and Use of Laboratory Animals and the experimental protocols were approved by the Institutional Animal Care and Use Committee of Soochow University.
Animal experiments
Bleomycin sulfate (BLM) (Abmole, M2100) was dissolved in sterile phosphate-buffered saline (PBS) at a concentration of 1 mg/mL. Mice received daily intracutaneous injections (i.c.) of 100 μL bleomycin into their shaved backs (the para-midline, lower back region) for 4 weeks, and mice injected with 100 μL PBS were used as a control group.
In the macrophage ablation experiment, mice were i.p. injected with 100 μL clodronate liposome (5 mg/mL, Yeasen) or 100 μL control liposome 3 days before the first bleomycin challenge and then administered every 4 days for 4 weeks. In the separate MerTK inhibition study, mice were daily i.p. injected with UNC2250 (5 mg/kg in 2% DMSO/30% PEG300/68% ddH2O, 1493694-70-4, Selleck) or vehicle control (2% DMSO/30% PEG300/68% ddH2O) from day 0 for 4 weeks.
In the neutrophil depletion experiment, mice were intraperitoneally injected (i.p.) with anti-Ly6G antibody (0.5 mg/kg in 200 μL PBS, BioLegend) or IgG2b isotype (0.5 mg/kg in 200 μL PBS, BioLegend) from day 3 after the first bleomycin challenge and injected every 4 days for 4 weeks. In a separate CXCR1/2 inhibition study, mice were i.p. injected with Reparixin (5 mg/kg in 2% DMSO/98% PBS, 266359-83-5, Selleck) or vehicle control (2% DMSO/98% PBS) from day 0 (the day of the first bleomycin challenge) for 4 weeks. In the experiment of NETs degradation by DNase I, mice were i.p. injected with 450 U DNase I in 200 μL PBS (D8071, Solarbio) or PBS from day 0, and injected every 2 days for 4 weeks.
Skin single cell acquisition
Mice were euthanized on day 30, the shaved back skin was removed and kept in PBS with 1% fetal bovine serum (FBS, Gibco) on ice. After scraping subcutaneous fat with forceps, the remaining skin was cut into pieces by surgical scissors as small as possible. Then the skin pieces were resuspended in Dulbecco's Modified Eagle Medium (DMEM, SH30021.01, HyClone) containing 10% FBS, 1% penicillin and streptomycin (PS, 15140163, Thermo Fisher Scientific), 0.25% type I collagenase (17100017, Gibco) and 0.01% DNase I (D8071, Solarbio) and digested for 2 h at 37°C. Next, the single cells from the digested skin tissues were filtered through a 70 μm strainer, and centrifuged at 300×g for 5 min. The precipitate was washed 1 or 2 times with 10 mL PBS. Finally, skin cells were resuspended in 1 mL PBS containing 1% FBS for subsequent experiments.
Flow cytometry
Single mouse skin cells as prepared above were first stained with Fixable Viability Stain 700 (564997, BD Pharmingen) to exclude dead cells. For cell-surface protein staining, cells were blocked with anti-mouse CD16/CD32 antibody (553141, BD Pharmingen) and then stained with the indicated antibodies for 30 min at 4°C in FACS buffer (PBS containing 1% FBS). For staining of intracellular proteins, cells were fixed and permeabilized with a Foxp3/Transcription Factor Staining Buffer Set Kit (00-5523-00, eBioscience) for 30 min at 4°C, followed by incubation with the indicated antibodies for 30 min at 4°C in Permeabilization buffer (00-5523-00, eBioscience). Cell phenotyping was performed on the Cytoflex Flow Cytometer (Beckman Coulter, U.S.A.). Cell sorting was performed on a MoFlo AstriosEQ (Beckman Coulter, U.S.A.), the purity of sorted cells was consistently above 95% for each sample. Data were analyzed using Flow Jo software (Version 10.0.7, Tree Star Inc.). Cell-surface antibodies used were as follows: PE/Cyanine7 anti-mouse CD45 (103114, BioLegend), Brilliant Violet 510 anti-mouse CD45 (563891, BD Pharmingen), PerCP/Cyanine5.5 anti-mouse CD45 (103132, BioLegend), APC/Cyanine7 rat anti-CD11b (557657, BD Pharmingen), PE/Cyanine7 anti-mouse CD24 (101822, BioLegend), PerCP/Cyanine5.5 anti-mouse CD24 (101824, BioLegend), Brilliant Violet 605 anti-mouse Ly-6G (127639, BioLegend), Brilliant Violet 510 anti-mouse Ly-6G (127633, BioLegend), FITC anti-mouse Ly-6G (127606, BioLegend), Brilliant Violet 421 anti-mouse Ly-6C(128031, BioLegend), PE anti-mouse Ly-6C(128007, BioLegend), FITC anti-mouse CD64 (139316, BioLegend), APC anti-mouse CD64 (139306, BioLegend), Brilliant Violet 605 anti-mouse I-A/I-E (107639, BioLegend), PE-Cyanine7 anti MerTK (25575182, eBioscience), Brilliant Violet 510 anti-mouse F4/80 (123135, BioLegend). Intracellular antibodies were as follows: Brilliant Violet 421 anti-mouse CD206 (MMR) (141717, BioLegend), APC anti-mouse CD206 (MMR) (141708, BioLegend), PE anti-mouse Arg-1 (12-3697-82, eBioscience), APC anti-mouse Arg-1 (17-3697-82, eBioscience) and PE anti-mouse NOS2 (12-5920-82, eBioscience).
RNA Sequencing
To obtain highly purified CD206hiMHCIIlo and CD206loMHCIIhi macrophages to perform RNA sequencing, skin single cells were first subjected to CD45+ total immune cell isolation by CD45 MicroBeads (130-052-301, Miltenyi Biotec) according to the manufacturer’s specifications. Then the isolated CD45+ cells were used for fluorescence-activated cell sorting to further isolate the macrophage subsets of CD206hiMHCIIlo and CD206loMHCIIhi. The 2 purified macrophage subsets (10,000-20,000 cells per sample) were lysed in TRIzol Reagent (15596-08, Life Technologies) and sent to LC Sciences (China) for RNA sequencing.
Hematoxylin-eosin and Sirius red staining
Skin samples from the mouse lower back were excised and fixed with 4% paraformaldehyde for 24 h, dehydrated with 70%, 75%, 85%, 95%, 100% ethyl alcohol, and then embedded into paraffin. 5 µm thick sections were stained with hematoxylin and eosin. Skin thickness was defined as the length from the top of the granular layer to the junction between the dermis and subcutaneous fat. A picrosirius red staining kit (PSR-1, ScyTek) was used to visualize dermal collagen according to the manufacturer’s instructions. Collagen content was defined as the distance from the top of the dermal layer to the junction between the dermis and subcutaneous fat.
Immunofluorescence staining
For immunofluorescence staining, slides were subjected to antigen retrieval in Tris-EDTA, pH 9.0 (R20904, Yuanye Bio-Technology), at 96°C for 30 min. Then slides were treated with 3% bovine serum albumin (BSA, CCS30014.02, MRC) for 1 h before incubation with primary antibodies overnight at 4°C. On the next day, sections were washed with PBS 3 times and incubated with the secondary antibodies for 1 h at room temperature. The nuclei were stained with Hoechst 33324 (H3570, Thermo Fisher Scientific). The primary antibodies used were as follows: anti-α-smooth muscle actin (α-SMA) (ab124964, Abcam), anti-CD64 (MA529704, Invitrogen), anti-CD64 (139302, BioLegend), anti-Ly6G (ab25377, Abcam), anti-Histone H3 (citrulline R2 + R8 + R17) (cit-H3) (ab5103, Abcam), anti-Mer (AF591, R&D) and anti-TGF-β1 (ab215715, Abcam). The secondary antibodies were Alexa Fluor 488-conjugated-goat anti-rabbit IgG (ab150077, Abcam), Alexa Fluor 594-conjugated-goat anti-rat IgG (ab150160, Abcam), Alexa Fluor 555-conjugated-donkey anti-goat IgG (A-21432, Thermo Fisher Scientific), and Alexa Fluor 647-conjugated-goat anti-rabbit IgG (ab150083, Abcam). Images were captured under a laser-scanning confocal microscope (Leica TCS SP8, Leica, Germany).
Western blotting
Skin tissues were lysed in RIPA buffer (P0013B, Beyotime) in the presence of protease inhibitors. Equal amounts of protein lysate samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. After blocking with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 for 1 h, the membranes were incubated at 4°C overnight with respective primary antibodies. Membranes were washed 3 times followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Signals were visualized by an NcmECL Ultra kit (P10300, NCM Biotech) with a FluorChem E system (Protein Simple, U.S.A.). The primary antibodies used were as follows: anti-Histone H3 (citrulline R2 + R8 + R17) (cit-H3) (ab5103, Abcam), and anti-Gas6 (67202S, CST).
Induction of NETs
Total neutrophils from mouse bone marrow were obtained by negative selection using the Mouse Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec) according to the manufacturer’s specifications. NETs were induced by overnight stimulation of neutrophils with 20 μmol/mL phorbol 12-myristate 13-acetate (PMA) (P1585, Sigma Aldrich) and 100 ng/mL LPS (L2630, Sigma Aldrich). Sytox Green dead cell stain (R37109, Thermo Fisher) was added to label the induced NETs 20 min before collection. Then the deposited NETs layer at the bottom of the culture dish was washed 4 times in PBS and collected by vigorous pipetting with 1640 medium. Cell debris was removed by centrifugation at 300g for 10 min and the supernatant containing NETs was stored at -80°C for further use.
Co-culture of macrophages and NETs
Bone marrow-derived monocytes (BMDMs) were acquired as previously described [19]. Briefly, bone marrow cells were flushed from the femurs and tibia of BALB/c mice and subjected to red blood cell lysis. Cells were passed through a 40 μm strainer and cultured in DMEM/F12 (03.2001C, EallBio) medium with 10% FBS, 1% PS and 10 ng/mL recombinant murine M-CSF (315-02, PeproTech) for 7 days. Then matured macrophages were co-cultured with or without NETs for 24 h, in the presence or absence of M1 stimuli: 100 ng/mL LPS (L2630, Sigma Aldrich) and 10 ng/mL IFN-γ (NBP2-35071, NOVUS); or M2 stimuli: 20 ng/mL IL-4 (214-14-20, PeproTech) and 20 ng/mL IL-13 (NBP2-35103, NOVUS). In the NETs degradation experiment, NETs were pre-treated with 20 U/mL DNase I for 2 h at 37°C.
Skin single-cell suspensions were obtained as described above. Then, skin cells were co-cultured with or without NETs or DNase I-treated NETs for 24 h in the presence of M2 stimuli: 20 ng/mL IL-4 (214-14-20, PeproTech) and 20 ng/mL IL-13 (NBP2-35103, NOVUS).
Detection of cytokines
The levels of IL-4 and IL-13 in skin homogenate were assayed by ELISA kits: mouse anti-IL-4 ELISA kit (abs520003, Absin) and mouse anti-IL-13 ELISA kit (abs520012, Absin). 10 mg skin tissue from the backs of the mice was removed and weighed. Then skin tissues were homogenized in 200 μL PBS with protease inhibitors. The supernatant was obtained and tested according to the manufacturer’s instructions.
Statistical analysis
Statistical analyses were performed by Prism 9 (GraphPad Software). Data are presented as the mean ± SEM in the animal experiments or the mean ± SD in the cell experiments. For comparisons between the two groups, P-values by unpaired Student's t test were reported if normality of the data could be confirmed by Shapiro-Wilk tests; otherwise, the Mann-Whitney U test was used. For comparisons among multiple groups, one-way ANOVA followed by Tukey’s multiple comparison test was used. A P-value lower than 0.05 is considered significant. *P < 0.05; **P < 0.01, ***P < 0.001; ns, not significant.