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Neutrophil extracellular trap-related mechanisms in acne vulgaris inspire a novel treatment strategy with adipose-derived stem cells – Scientific Reports

Mouse ADSCs isolation and culture

ADSCs were isolated from subcutaneous adipose tissues in the inguinal region of BALB/c mice (male, 6–8 weeks). Adipose tissues were washed with phosphate-buffered saline (PBS) three times, and the blood vessels were removed. Then, the adipose tissues were cut into small pieces and digested with 0.25% type I collagenase (Sigma, USA) at 37 ℃ for 1 h. The DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) was used to terminate the digestion. After centrifugation at 400×g for 5 min, the cells at the bottom were seeded in a culture dish with DMEM medium containing 20% FBS. The medium was replaced after 24 h to remove non-adherent cells. DMEM medium containing 10% FBS was used to culture ADSCs after passage. ADSCs from passages 3 to 7 were used for subsequent experiments. Mouse keratinocyte cell line PAM212 was cultured with RPMI Medium 1640 (Gibco, USA) containing 10% FBS (Gibco, USA).

Mouse ADSCs identification

For identification of ADSCs, ADSC suspensions were incubated with anti-CD29 (APC), anti-CD44 (PC7), anti-CD90 (FITC), anti-CD105 (APC), anti-CD31 (PE), and anti-CD34(APC) antibodies (Becton Dickinson, USA) for 30 min and were analyzed with the FACS Calibur cytometer (Becton Dickinson, USA). Mesenchymal stem cell biomarkers CD29, CD44, CD90, and CD105 were the positive markers of ADSCs. Endothelial marker CD31 and hematopoietic lineage marker CD34 were the negative markers of ADSCs. To identify the differentiation abilities of ADSCs, ADSCs were cultured with adipogenic differentiation medium, osteogenic differentiation medium, and chondrogenic differentiation medium (Cyagen Biosciences, China), according to the manufacturer’s instructions.

Bacterial strains and bacterial culture

P. acnes were purchased from BeNa Culture Collection (Henan, China). P. acnes were cultured in Columbia Blood Agar Plate (BeNa Culture Collection, China) under anaerobic conditions at 37 ℃ for 5 to 7 days using anaerobic atmosphere generation bags (Mitsubishi, Japan). For in vivo experiments, P. acnes were resuspended in PBS at 2 × 106 CFU/mL. A microplate reader (BioTek Instruments, USA) was used to measure the concentration of the bacterial suspension. The standard curve is plotted by measuring the OD value of the known concentrations at 600 nm and fitting a regression line or curve. The concentration of the bacterial sample can then be determined by comparing its OD value to the standard curve.

P. acnes skin infection mouse model

A total of 27 male BALB/c mice (male, 6 weeks) were randomly subdivided into three groups (n = 9 per group): control, P. acnes, and P. acnes + ADSCs. The sample size was calculated using G*Power software v.3.0 at alpha 0.05 and with 80% of power14. Mice in the control group were subcutaneously injected with 50 µL PBS into the middle of the left and right auricle. Mice in P. acnes and P. acnes + ADSCs group were injected with 50 μL P. acne suspension (2 × 106 CFU/mL in PBS) into the same area. After 24 h, the P. acnes + ADSCs group was subcutaneously injected with 50 μL ADSC suspension (108/mL in PBS) into the middle of the auricle. The specific concentrations of P. acnes and ADSC were determined on the basis of previous studies15,16. For comparison, the control and P. acnes groups were injected with 50 μL PBS. Mice’s ear thickness was measured with a vernier caliper, and the images of the mice’s auricle middle were photographed daily. After treatment for 48 h, the mice were euthanized with CO2 gas, and the ear tissues were obtained for Hematoxylin–Eosin (H&E) and immunofluorescence (IF) staining. The choice of P. acnes induction time and the ADSC treatment time was based on previous research and our preliminary experiments17,18. This animal experiment was approved by the ethical committee of Tongji Medical College (2022 IACUC Number: 2893) and was performed in accordance with the relevant guidelines and regulations. Besides, the animal experiment followed the recommendations in the ARRIVE guidelines.

Histological staining

The middle of the auricle were sectioned, and the interval between sections is about 5 μm. For H&E staining, the sections were fixed with 4% paraformaldehyde (Biosharp, China) and embedded in paraffin. Then, the tissue sections were stained with hematoxylin and eosin. The images were obtained with a bright microscope (SDPTOP CX40, China). For IF staining, the tissue sections and cell samples were fixed with 4% paraformaldehyde. The samples were sealed with 5% bovine serum albumin (BSA) and incubated with primary antibody at 4 °C for 12 h. After washing with PBST three times, the samples were incubated with secondary antibodies for 1 h at room temperature. Finally, DAPI nuclear dye was used to stain the nuclei. Anti-Ly6G (1:80, Proteintech, USA), Anti-MPO (1:50, Proteintech, USA), anti-citH3 (1:100, Abcam, USA), and anti-Nrf2 (1:100, Proteintech, USA) antibodies were used as the primary antibodies. Image J was used to select ROIs representing individual cells and the tissue regions of interest. Then, the average fluorescence intensity of the target protein-specific signal and the DAPI signal within the set ROIs were calculated. At least three images of each cell or tissue staining were used for statistical analysis, and the mean and standard deviation were calculated for at least three independent experiments. The relative quantification of the target protein can be calculated using the following formula. The cell fluorescence in control group was normalizated to unity.

$$text{Cell fluorescence }{text{of}}text{ protein} = frac{(text{Fluorescence intensity of target protein})}{(text{Fluorescence intensity of DAPI})} .$$

Western blot

Total proteins from ear tissue and cell samples were extracted with RIPA buffer (Beyotime, China), and were separated with 10 or 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Then, the separated proteins were electrotransferred to PVDF membranes. After blocking the membranes with 5% BSA for 1 h, the membranes were incubated with primary antibody, including anti-MPO (1:500, Proteintech, USA), anti-citH3 (1:1000, Abcam, USA), anti-Nrf2 (1:2000, Proteintech, USA), anti-GAPDH (1:10,000, Proteintech, USA), anti-IL1β (1:1000, Proteintech, USA), anti-IL6 (1:1000, Proteintech, USA), anti-NF-κB p65 (1:2000, Proteintech, USA), anti-NF-κB p65 (phospho, 1:2000, Proteintech, USA), and anti-β-Actin (1:10,000, Proteintech, USA) antibodies at 4 °C for 12 h, and then incubated with the HRP-linked secondary antibody (1:5000, Proteintech, USA). The blots were cut prior to hybridisation with antibodies. The membranes were next visualized with the enhanced chemiluminescence (ECL) assay kit (Yeasen, USA). Image Lab Software was used to detect and quantify the signals of protein bands. The original data of western blot was included in the Supplementary Information file.

The isolation of mouse bone marrow neutrophils

The mouse bone marrow neutrophils (BMNs) were isolated from the tibia and femur of BALB/c mice (male, 6 weeks). After the mice were anesthetized and euthanized by dislocation, the tibia and femur were separated and the bone marrow cavity was washed by DMEM/F12 medium containing 10% FBS. The BMNs were separated from bone marrow cell suspension with mouse BMN isolation assay kit (Solarbio, China) according to the manufacturer’s instructions. IF staining was used to examine the purity of isolated neutrophils. The purity of neutrophils was > 90%. The obtained BMN was cultured with RPMI Medium 1640 (Gibco, USA) containing 10% FBS (Gibco, USA).

In vitro NET formation assay and NET collection

The BMN was treated with PBS (100 µL), PMA (2 µM), P. acnes (106 CFU/mL in 100 µL PBS), P. acnes (106 CFU/mL in 100 µL PBS) + ADSC-CM (conditioned medium of ADSCs), P. acnes (106 CFU/mL in 100 µL PBS) + ADSC-CM + ML385 (Nrf2 inhibitor, 5 µM) and for 4 h. The IF staining (anti-MPO and anti-citH3) and SYTOX green (Solarbio, China) staining were performed to measure the formation of NETs. NETs from P. acnes, P. acnes + ADSC-CM were collected for subsequent experiments. After treatment for 4 h, the cell layer was washed softly with PBS. Then, PBS solution was added to the wash cell with vigorous agitation. The washing PBS was collected and centrifuged for 10 min at 450×g at 4 °C. NETs in the supernatant phase were collected and stored at − 80 ℃.

Assay for proliferation and migration

The proliferation ability of PAM212 cells was measured with CCK-8 assay (Yeason, China) and EdU incorporation assay kit (RiboBio, China). For the CCK-8 assay, 5 × 104 PAM212 cells were seeded into 96-well plates. After the cells were treated for 4 h, the medium was replaced and 10 μL CCK-8 reagent in 90 μL RPMI Medium 1640 was added to incubate cells for 2 h. A microplate reader (BD, USA) was used to read the absorbance at 450 nm. The cell viability was measured by OD value (450 nm).

$$mathrm{OD, value },(450text{ nm})={OD}_{test}-{OD}_{bg,}$$

where ODtest and ODbg represent the absorbance at 450 nm of the test and background respectively.

For the EdU assay, the cells in 96-well plates were incubated with culture medium containing EdU for 3 h and fixed with 4% paraformaldehyde. After being washed with PBS three times, the cells were incubated in the Apollo reaction cocktail and Hoechst staining solution in sequence. The fluorescence microscope (Olympus, Japan) was used to photograph the cells. The ratio of Edu positive cells to the total number of cells was used to measure the proliferation ability of cells

$$text{Edu positive cells }left(mathrm{%}right)=frac{text{Number of Edu positive cells} }{text{Total number of cells} }.$$

The wound healing assay was performed to assess the migration ability of PAM212 cells. When the cells in 12-well plates reached approximately 90% confluence, the cell layers were scratched with a 1 mL pipette tip and washed with PBS three times. Then, the cells were cultured in serum-free DMEM medium for 24 h. The wound image was photograph by a microscope (SOPTOP CX40, China), and the wound widths was measured by Image J software (NIH, USA). The relative percentage of wound closure was calculated with the following equation.

$$text{The relative percentage of wound closure} , left(mathrm{%}right)=frac{{W}_{0} -{W}_{24}}{{W}_{0} },$$

where W0 and W24 represent the wound area at 0 h after scratching and at 24 h after scratching.

ELISA

The secreted cytokines of PAM212 after treatment of P. acnes-induced NETs and P. acnes + ADSC-induced NETs were detected with interleukin 6 (IL-6), IL-8, chemokine ligand 1 (CXCL1), and CXCL10 ELISA kits (LiankeBio, China) according to the manufacturer’s instructions.

Statistical analysis

At least three replicates were performed in each group. All the statistical analyses were conducted with GraphPad Prism 9.0 software, and Student’s t-test was used to compare two different groups. For three or more groups, we conduct one-way ANOVA to compare the differences between different groups. If the one-way ANOVA test shows a significant difference, LSD t test was performed to determine which specific groups differ significantly from each other. P-value < 0.05 was regarded as statistically significant.

Ethics approval and consent to participate

This animal experiment was approved by the ethical committee of Tongji Medical College.Please note we have moved the section ‘Ethical approval and informed consent’ to the end of the methods, as per house style.