1. Mice and EAP induction
The animal experiments were approved by the Committee for Animal Care and Use of the Animal Center of Anhui Medical University (No.LLSC20190651). Six to eight-week-old male NOD mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were maintained under specific pathogen-free conditions at the animal facility of our institution. NOD mice were induced to develop EAP model as previously reported[13, 14]; briefly, NOD mice were administered with 0.1 ml mixed emulsion prostate antigens (PAg) and complete Freund’s adjuvant (CFA; Sigma-Aldrich) by subcutaneous injection on days 0 and 14. Mice were sacrificed by an overdose of chloral hydrate (Aladdin Biotechnology) and euthanized by cervical dislocation at different times after 1st immunization. Blockade of IL-17 was achieved by intraperitoneal injection of 10 μg anti-mouse IL-17 neutralizing antibodies (R&D System, MAB421) or isotype-matched rat IgG (R&D System, 6-001-A) one day before EAP induction and then once per week. After EAP induction, parts of EAP mice were injected intraperitoneally with recombinant mouse IL-17 (rIL-17, 0.1 mg/mouse) every day for 1 week after 1st and 2nd immunization. Fucoidan (Sigma-Aldrich, F5631) or its vehicle was administered by intraperitoneal injection at a dose of 20 mg/kg body weight one day before EAP induction and then once per week thereafter.
2. Tactile allodynia assessment
The mice were tested before PAg injection (baseline, day 0) and at 14, 28, 42 days after EAP induction. Referred hyperalgesia and tactile allodynia were tested using von Frey filaments applied to the abdomen region near the prostate according to previous references. Three types of behaviors were considered to be positive responses to von Frey filament stimulation: 1) sharp retraction of the abdomen; 2) immediate licking or scratching of the area of filament stimulation; or 3) jumping. The response frequency was calculated as the percentage of positive responses and data were reported as the mean ± SEM.
3. Inflammation scoring
Prostate tissues were fixed in 10% formalin and embedded in paraffin. 5 μm sections were stained with hematoxylin and eoxin (HE) and scored blindly using the histopathological classification system for chronic prostatic inflammation. Briefly, the extent of chronic inflammation was graded from 0-3: a) 0, no inflammation; b) 1, mild but definite perivascular cuffing with mononuclear cells; c) 2, moderate perivascular cuffing with mononuclear cells; d) 3, marked perivascular cuffing, hemorrhage, and numerous mononuclear cells in the parenchyma.
4. Immunohistochemistry and immunofluorescence assays
Immunohistochemistry (IHC) and immunofluorescence (IF) assays were performed as described previously[14, 18]. Briefly, prostate tissues were fixed in 10% formalin and embedded in paraffin. Prostate sections were incubated overnight at 4℃ with anti-IL-17 (Abcam, Ab79056) primary antibodies at a dilution of 1:500. Then, an IHC kit (ZSGB-Bio, SP9000) and 3,3’-diaminobenzidine (ZSGB-Bio, ZLI-0918) were used for subsequent immunohistochemical analysis according to the manufacturer’s instructions.
Prostate sections were blocked with 5% bovine serum albumin and incubated overnight at 4℃ with rabbit anti-mouse Ly6G primary antibodies (1:200, Elabscience, E-AB-70094). The sections were then incubated with Cy3-conjugated goat anti-rabbit IgG (1:400, Beyotime, A0516) and DAPI (1:1000, Beyotime, C1005) after washed with tris-buffered saline (TBS). Fluorescence images were acquired using a Zeiss LSM510 confocal microscope.
5. RNA isolation and quantitative real-time RT-PCR
Total RNA from prostate tissues were extracted with Trizol reagent according to the manufacturer’s instructions. Total RNA was synthesized into cDNA using the Fast Quant RT kit (Tiangen, KR106-02). Quantitative real-time PCR was performed with Super Real Premix Plus (SYBR green) (Tiangen, FP205-02). The gene-specific primer sets used to amplify each gene were synthesized by Sangon Biotech Co., Ltd (Shanghai) (Supplemental Table 1). Gene expression was assessed by the comparative CT method. The results were normalized to Gapdh.
6. Analysis of chemokine and cytokine expression in prostate tissue
The concentrations of cytokines in prostate tissue homogenate samples were determined by enzyme-linked immunosorbent assay (ELISA). The levels of IL-17 (Elabscience, E-EL-M0047c), CXCL1 (Elabscience, E-EL-M0018c), and CXCL2 (Elabscience, E-EL-M0019c) in the prostate tissues were measured by ELISA kits according to the manufacturer’s instructions.
7. Analysis of prostate-infiltrating neutrophil
Freshly harvested prostate tissues were mechanically disrupted and enzymatically digested in RPMI 1640 medium containing 1mg/ml collagenase D (Sigma-Aldrich, C9891) and 0.05% DNase Ⅰ (Sigma-Aldrich, D5025) for 45 min at 37℃. After digestion, suspensions were filtered through 75-μm cell strainers and single-cell suspensions were washed with PBS. Then, cells were stained with APC-conjugated anti-Ly6G (BD Bioscience, 560599) for FACS analysis. FACS Calibur flow cytometer (BD Bioscience) was used to analyze the stained cells, and the data were analyzed by FlowJo Software X (Tree Star).
8. Analysis of MPO activity
Myeloperoxidase (MPO) is produced by activated neutrophils and is an established marker of neutrophil migration. MPO activity in prostate tissues was analyzed by an MPO activity measurement kit (Elabscience, E-BC-K074) according to the manufacturer’s instructions. MPO activity was determined by a spectrophotometer at 450 nm and expressed as U/g prostate tissues.
9. Neutrophil depletion assays
For neutrophil depletion studies, we used anti-Ly6G antibody (clone 1A8; Bio-X-Cell)[19, 20]. Briefly, EAP mice were treated with intraperitoneal injection of 0.25 mg/dose of anti-Ly6G or control rat IgG (clone GL117, Bio-X-Cell) one day before EAP induction and then once a week. Peripheral blood from mice was collected by cardiac puncture for leukocyte quantification using a HEMAVET 950 multispecies hematology cell counter.
10. Statistical analysis
Statistical analysis was performed by two-tailed Student’s t-test and two-way ANOVA with Bonferroni post hoc test. Data are representative of three independent experiments with 4 mice per group. The data are expressed as mean ± SEM. *P<0.05 was considered to indicate a significant difference in the analyses. Statistical analyses were performed using SPSS software version 21.0 and GraphPad Prism 6 software.