Mice and cell resources
Human umbilical cord samples were collected from six full-term newborns. All samples were collected with patient consent and signed informed consent. This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (approval number: SYSU-IACUC-2018-168-1).
Male NOD/ShiLtJ mice (6–8 weeks old) were purchased from Jiangsu Gempharmatech. All mice were kept under controlled temperature (24°C ± 1°C) and relative humidity (50%-60%). The light period was 12 hours, and the dark period was 12 hours. All mice were allowed free access to food and water. All surgical procedures and postoperative nursing methods were approved by the Animal Care and Use Committee of Sun Yat-sen University (approval number: SYSU-IACUC-2021-000164). On a specified date, the mice were euthanized by inhaling excessive amounts of isoflurane, according to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2013 Edition.
Isolation, culture, and pretreatment of human umbilical cord-derived MSCs
As previously described, MSCs were isolated from the umbilical cord of newborns [28]. In short, the blood vessels were carefully removed from the umbilical cord, and the remaining Wharton’s jelly was cut up and digested with 1 mg/mL collagenase II (Micropore) in Hank's balanced salt solution (HBSS, HyClone) for 30 minutes at 37°C. The isolated cells were filtered through a cell filter with a mesh diameter of 70 µm to obtain a cell suspension.
The cells were washed twice with HBSS and cultured in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (FBS, HyClone), 1% nonessential amino acids (Gibco), 2 mM GlutaMAX (Gibco) and 100 IU/mL penicillin/streptomycin (Gibco) in a humidified 5% CO2 water-jacketed incubator (Thermo Scientific). The culture medium was changed every 3 days. Cells were passaged every 2–3 days using 0.125% trypsin (Gibco) at a split ratio of 1:3. MSCs of the 4th passage were cocultured with 10 ng/ml IL-1β (PeproTech) for 48 hours.
The lentivirus carrying green fluorescent protein (GFP) was purchased from Shanghai Genechem Co., Ltd. Transfection was carried out according to the instructions. After successful transfection, flow cytometry was used to screen GFP + cells for further expansion.
Animal model
The construction of a non-obesity diabetes-experimental autoimmune prostatitis (NOD-EAP) mouse model was performed according to a previously published article [29, 30]. The EAP mouse model was established by mixing the male reproductive accessory gland (MAG) extract of male Wistar rats with the same volume of complete Freund's adjuvant (CFA, Sigma). After repeated suction and mixing, the NOD mice were injected with a mixture of MAG extract and CFA in four different parts, namely, the neck (0.025 ml), tail (0.050 ml) and bilateral shoulders (0.050 ml), on days 0 and 15. At 30 days, MSCs and IL-1β-primed MSCs were injected into EAP mice through the tail vein. On the indicated days, the spleen, blood, and prostate were collected for flow cytometry analysis, cytokine measurements, and histological analysis.
Hyperalgesia behavior testing
The test was performed 30 days after the first injection and 15 days after the treatment. According to published articles, von Frey filaments were used to test hyperalgesia in the abdomen and the plantar area of the hind paw [31, 32]. In short, five different fibers were used to test the frequency of the withdrawal response by von Frey filaments applied to the pelvis and lower abdomen with forces of 0.04, 0.16, 0.4, 1.0 and 4.0 g. There are four types of behaviors that are considered to be a positive response to filament stimulation: sharp retraction of the abdomen, immediate licking, scratching of the filament stimulation area or jumping. The frequency of response was calculated as the percentage of positive responses, and the data are reported as the average percentage of the response frequency. The 50% threshold was evaluated using an up and down method [33].
Coculture of macrophages and MSCs
The bone marrow cells surgically obtained from the femur or tibia were separated by density gradient centrifugation. The cells were grown and maintained with DMEM/F12 (HyClone) containing 10% FBS and 20% L929 conditioned medium in low adhesion plates. After 7 days of culturing, the cells were separated with TrypLETM Express (Gibco) and seeded at a density of 5×105 cells/well. Then, 1 µg/mL lipopolysaccharide (LPS, Sigma) was added to activate macrophages. Macrophages and saline/MSCs/IL-1β-primed MSCs were cocultured at a ratio of 5:1. After 72 hours of cocultivation, the cells were collected and analyzed by flow cytometry.
Coculture of CD4+ cells and MSCs
T lymphocytes were obtained from the spleen using the BioLegend Natural CD4 + T Cell Purification Kit (BioLegend) and then seeded on anti-CD3-coated plates at a density of 5×105 cells/well. CD4 + T cells and saline/MSCs/IL-1β-primed MSCs were cocultured at a ratio of 5:1. Treg cell differentiation was induced with RPMI 1640 containing 10% FBS, 3 µg/mL anti-CD28 (BioLegend), 50 ng/mL IL-2 (Novoprotein) and 5 ng/mL TGF-β (Novoprotein). After 72 hours of cocultivation, CD4 + FOXP3 + Treg cells were collected and analyzed by flow cytometry.
Ca2+ imaging in DRGs
According to the previously published literature [34, 35], dorsal root ganglia (DRGs) (T13-L2) were isolated from male mice aged 4 to 6 weeks and incubated with collagenase (Sigma). They were then incubated at 37°C for 45 minutes, digested with 0.05% trypsin-EDTA (Gibco), resuspended in complete medium (DMEM/F12), and cultured on coverslips for 3 days. A normal physiological salt wash (containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM HEPES and 10 mM glucose) was used for washing 3 times, and the pH was adjusted to 7.4. The cells were incubated with 5 µM fluo 3-AM (Molecular Probes) for 60 minutes at room temperature. The fluorescence signal was recorded by a laser scanning imaging system (IX83 research inverted microscope, Japan). Fluo 3 is excited at 488 nm and analyzed at 530 nm. After equilibrating for 3–5 minutes, the cells were treated with CCL2 and capsaicin to increase Ca2+ influx. The Ca2+ signal was captured in a 4 s interval.
Transwell migration assay
An 8-µm pore membrane filter (Millipore) was used to evaluate the targeted migration ability of MSCs. Serum-free starved MSCs and IL-1β-primed MSCs were inoculated in the upper chamber (1×105 cells per well), while the lower chamber was filled with 500 µL of DMEM/F12, which contained 50 ng/mL mouse Chemokine (C-X-Cmotif) ligand 12 (CXCL12, PeproTech). After incubating in 5% CO2 at 37°C for 12 hours, the upper chamber of the filter was removed. After fixing with 4% paraformaldehyde (PFA), the cells that migrated to the lower chambers were stained with 0.1% crystal violet and then counted by microscopy.
Flow cytometry
The obtained tissues were cut into small pieces and shaken with Liberase TL (100 U/mL; Roche) with DNase I (100 U/mL; Sigma) in a 37°C water bath for 20 minutes. Subsequently, the cell suspensions were passed through a 70-µm cell strainer to produce single cells. The collected peripheral blood samples were subjected to red blood cell lysis (BioLegend) and then washed twice with PBS. Single-cell suspensions, tissues and blood samples were incubated with the appropriate antibodies (Supplementary Table S1) for 15 minutes in the dark. The cells were washed twice, resuspended in PBS and analyzed by MoFlo Astrios EQ (Beckman Coulter) for at least three independent experiments. The data was analyzed using FlowJo V10.0 (Tree Star). Information on the antibodies is listed in Supplementary Table S1.
Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted and purified by NucleoZol reagent (Macherey-Nagel), and cDNA was obtained by a cDNA library kit (Takara). qRT-PCR was conducted on a Bio-Rad CFX96TM detection system (Roche) with SYBR PCR Master Mix (Roche). β-Actin was used for standardization. The primers used are listed in Supplementary Table S2.
Tissue preparations and histological analysis
Tissue was embedded in a tissue-Tek optimum cutting temperature (OCT) complex and frozen in a cryostat (Leica CM1900, Germany). Sections of 5-µm thickness were then placed on glass slides (Bio-Optical). Slices were prepared and sealed with 0.05% thiobarbital sodium-Tween containing 0.5% FBS for 30 minutes, incubated overnight with the primary antibody or an IgG control antibody at 4 ℃, and incubated for 60 minutes with the secondary antibody at room temperature. The slices were re-stained with 4',6-diamidino-2-phenylindole (DAPI) for 3 minutes at room temperature. Immunofluorescence (IF) signals were visualized and recorded using a laser scanning confocal microscope (LSM780, Zeiss, Germany). For hematoxylin-eosin (HE) staining, the tissue was fixed overnight in 4% PFA (Sigma), continuously dehydrated and embedded in paraffin. Then, slices with a thickness of 5 µm were placed on glass slides (Bio-Optical). The cells were stained with an HE staining kit (Servicebio). All the primary and secondary antibodies are listed in Supplementary Table S2.
Western blot analysis
Cultured cells or tissues were homogenized with RIPA lysis buffer (Millipore) containing protease inhibitors for over 30 minutes. Nuclear protein lysates were generated using a nuclear protein extract kit (Servicebio) following the manufacturer’s instructions. The homogenates were centrifuged at 15,000 ×g at 4°C for 10 minutes and then the protein concentration was detected by a BCA protein analysis kit (Sigma). Approximately 20–40 µg of total protein solution was loaded onto SDS-polyacrylamide gels and transferred onto 0.22-µm polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% bovine serum albumin for 30 minutes at room temperature and then probed with the indicated primary antibody at 4°C overnight, followed by incubation with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. A chemiluminescence kit (Millipore) was used to detect the target bands. Information about the antibodies used is provided in Supplementary Table S1.
Cytokine measurements
Tissue was homogenized with TissueLyser II (QIAGEN) and 5-mm steel balls (QIAGEN) in tissue protein extraction reagent (Thermo Fisher), and the supernatant was obtained after centrifugation. Blood samples were collected, and serum was extracted by centrifugation. According to the manufacturer's instructions, the LEGENDplex™ mouse inflammatory panel (BioLegend) was used to detect the concentration of 13 cytokines in tissue culture supernatants, serum samples and lysates extracted from RIPA. Vacuum freeze-drying equipment (Songyuan) was used to concentrate the extract to obtain the appropriate concentration. A BCA protein analysis kit (Sigma) was used to determine the concentration of total protein in the lysate extracted by RIPA, and the same amount of total protein was used for subsequent detection. The kit provides capture beads that bind to specific antibodies, making them easily distinguishable by size and fluorescence signal. The biological sample was incubated with the capture beads at room temperature for 2 hours and then a biotin-labeled detection antibody was added for flow cytometry detection. LEGENDplex 8.0 data analysis software was used to calculate the average fluorescence intensity corresponding to each cytokine. The cytokine concentration was measured by comparing the fluorescence intensity with a standard value.
Statistics
Comparisons between groups were performed by t-test or one-way analysis of variance (ANOVA), and Tukey's multiple comparison test was used. Error bars indicate the standard error of the mean. P < 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism 8.0 software.