IL-17 exacerbates experimental autoimmune prostatitis via CXCL1/CXCL2-mediated neutrophil infiltration

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a poorly understood disease. Accumulating evidence suggests that autoimmune dysfunction is involved in the development of CP/CPPS. Interleukin-17 (IL-17) is associated with the occurrence and development of several chronic autoimmune inflammatory diseases. How-ever, the molecular mechanisms underlying the role of IL-17 in CP/CPPS are not clear. We confirmed that IL-17 was increased in the prostate tissues of experimental autoimmune prostatitis (EAP) mice. Corresponding to the increase of IL-17, neutrophil infiltration and the levels of CXCL1 and CXCL2 (CXC chemokine ligands 1 and 2) were also increased in the prostate of EAP. Treatment of EAP mice with an IL-17-neutralizing monoclonal antibody (mAb) decreased the number of infiltrated neutrophils and CXCL1 and CXCL2 levels. Depletion of neutrophils using anti-Ly6G antibodies ameliorated the inflammatory changes and hyperalgesia caused by EAP. Fucoidan, a could potent inhibitor of neutrophil migration, also ameliorate the manifestations of EAP. Our findings suggested that IL-17 promoted the production of CXCL1 and CXCL2, which triggered neutrophil chemotaxis to prostate tissues. Fucoidan might be a potential drug for the treatment of EAP via the effective inhibition of neutrophil infiltration.

various inflammatory cell types and induces proinflammatory cytokines (such as IL-1β, G-CSF, GM-CSF) and chemokines (such as CXCL1 and CXCL2) in some inflammatory diseases (Moseley et al., 2003;Y. Zhang et al., 2012). The role of IL-17 in a noninfectious autoimmune-driven mouse model of CP/CPPS experimental autoimmune prostatitis (EAP), is controversial (Motrich et al., 2016;Murphy et al., 2015). Conclusions in these studies were based on C57BL/6 mice, which were proved less susceptible to EAP induction than non-obese diabetic (NOD) mice (Rivero et al., 1998). We have demonstrated that IL-17 exerted a positive effect in EAP-NOD mice (Zhan et al., 2020). This finding suggested that the levels of IL-17 in the prostate tissues from EAP mice were higher than control mice (Zhan et al., 2020). We also found that treatment with a neutralizing antibody against IL-17 decreased the level of IL-17 in prostate tissues and ameliorated the inflammatory changes and pelvic pain in EAP (Zhan et al., 2020). However, the exact mechanisms of IL-17 involvement in the inflammatory manifestations and pelvic pain in the EAP mouse model and CP/CPPS diseases are not certain.
The current study demonstrated the mechanisms of IL-17 modulation of infiltration neutrophils into prostate tissues in an EAP model.
Meanwile, the beneficial effects of fucoidans, a sulphated polysaccharide extracted from algae, on inflammatory diseases and immune dysfunction were demonstrated previously (Aleissa et al., 2020;B. R. Chen et al., 2021). Therefore, we hypothesized that fucoidans would ameliorate EAP-induced inflammation and pain symptoms and explored its possible mechanisms. These findings improve the understanding of CP/CPPS pathogenesis and therapeutic intervention.

| Mice and EAP induction
The Committee for Animal Care and Use of the Animal Center of Anhui Medical University approved the animal experiments (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 pathogenfree conditions at the animal facility of our institution. NOD mice were induced to develop the EAP model using previously reported methods (Rivero et al., 1998;Zhan et al., 2020). Briefly, model mice were administered 0.1 ml mixed emulsion prostate antigens (PAg) and complete Freund's adjuvant (CFA; Sigma-Aldrich) via subcutaneous injection on days 0 and 14. The control group control group was injected with the same volume of normal saline. Mice were sacrificed by an overdose of chloral hydrate (Aladdin Biotechnology) and euthanized by cervical dislocation at different times after the first immunization. Blockade of IL-17 was achieved by the 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 6, 13, 20, 27, 34 and 41 days after induction (Murphy et al., 2015). After EAP induction, some EAP mice were injected intraperitoneally with recombinant mouse IL-17 (rIL-17, 0.1 mg/mouse) daily for 1 week after the first and second immunizations (Gao et al., 2010). Fucoidan (Sigma-Aldrich, F5631) or its vehicle was administered via intraperitoneal injection at a dose of 20 mg/kg body weight 1 day before EAP induction and 6, 13, 20, 27, 34 and 41 days after induction (McNamee et al., 2011). The details are shown in Supplemental Figure 1.

| Tactile allodynia assessment
The mice were tested before PAg injection (baseline, day 0) and 14, 28, and 42 days after EAP induction. Referred hyperalgesia and tactile allodynia were tested using von Frey filaments application to the abdominal region near the prostate according to previous studies (Zhan et al., 2020). Three types of behaviours were considered 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 the data were reported as means ± SEM. Tactile allodynia was measured using 50% withdrawal thresholds following stimulation with von Frey filaments, which was described as (50% threshold, g = (10[Xf + kδ])∕10,000) (Chaplan et al., 1994;Christensen et al., 2020;Quick et al., 2013).

| Inflammation scoring
Prostate tissues were fixed in 10% formalin and embedded in paraffin.
Five-micron sections were stained with haematoxylin and eoxin (HE) and scored blindly using the histopathological classification system for chronic prostatic inflammation (Breser et al., 2013). Briefly, the extent of chronic inflammation was graded from 0 to 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, haemorrhage, and numerous mononuclear cells in the parenchyma.

| RNA isolation and quantitative real-time PCR
Total RNA from prostate tissues was extracted using 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 using 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 using the comparative C T method. The results were normalized to Gapdh.

| Analysis of chemokine and cytokine expression in prostate tissue
The concentrations of cytokines in prostate tissue homogenate samples were determined using enzyme-linked immunosorbent assays (ELISAs). 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 using ELISA kits according to the manufacturer's instructions.

| Analysis of prostate-infiltrating neutrophils
Freshly harvested prostate tissues were mechanically disrupted and enzymatically digested in RPMI 1640 medium containing 1 mg/ml collagenase D (Sigma-Aldrich, C9891) and 0.05% DNase I (Sigma-Aldrich, D5025) for 45 min at 37 C. After digestion, suspensions were filtered through 75-μm cell strainers and single-cell suspensions were washed with PBS. The cells were stained with APC-conjugated anti-Ly6G (BD Bioscience, 560,599) for FACS analysis. A FACSCalibur flow cytometer (BD Bioscience) was used to analyse the stained cells, and the data were analysed by FlowJo Software X (Tree Star).

| Analysis of MPO activity
Myeloperoxidase (MPO) is produced by activated neutrophils, and it is an established marker of neutrophil migration. MPO activity in prostate tissues was analysed 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.
F I G U R E 1 EAP induction caused histological changes in prostate tissues, pelvic pain, and increased IL-17 in prostate tissues. (a,b) Representative images of HE staining (a) and inflammation scores (b) of prostate tissue sections from the control and EAP group 42 days after EAP induction. (MagnificationÂ100, scale bar = 100 μm). (c) the response frequencies to individual filament stimulation of the pelvic area on day 42. (d) IL-17 mRNA expression in prostate tissues on day 42 was measured using realtime PCR. (e) The level of IL-17 in prostate tissues on day 42 was analysed using ELISA. (f) Immunohistochemical analysis of IL-17 in prostate sections from the control and EAP groups 42 days after EAP induction. *p < 0.05, **p < 0.01. Data represent the mean ± SEM for four mice per treatment group of three independent experiments.

| Neutrophil depletion assays
For neutrophil depletion studies, we used an anti-Ly6G antibody (clone 1A8; Bio-X-Cell) (Daley et al., 2008;Sercundes et al., 2016). Briefly, EAP mice were treated with an intraperitoneal injection of 0.25 mg/dose of anti-Ly6G or control rat IgG (clone GL117, Bio-X-Cell) 1 day before EAP induction and then once weekly. Peripheral blood from mice was collected via cardiac puncture for leukocyte quantification using a HEMAVET 950 multispecies haematology cell counter.

| Statistical analysis
Statistical analysis were performed using two-tailed Student's t test and two-way ANOVA followed by Bonferroni post hoc testing. Data are representative of three independent experiments with four mice per group. The data are expressed as means ± SEM. *p < 0.05 indicated a significant difference in the analyses. Statistical analyses were performed using SPSS software version 21.0 and GraphPad Prism 6 software.

| RESULTS
3.1 | IL-17 was increased in the prostate tissues of EAP mice We first analysed the changes between control and EAP mice 42 days after the EAP first immunization. As shown in Figure 1a, pathological changes were observed in EAP mice, including a large amount of F I G U R E 2 IL-17 mRNA and protein expression paralleled EAP-induced prostate inflammation and pain symptoms. (a) IL-17 mRNA expression in prostate tissues was measured using real-time PCR at the indicated times after the first EAP immunization. (b) IL-17 levels in prostate homogenates were determined using ELISA at the indicated times after the first EAP immunization. (c,d) The mice were sacrificed at the indicated times after the first EAP immunization and formalin-fixed prostate sections were stained with HE. Representative images of HE staining (C) and inflammation scores (D) of prostate tissue sections from the control and EAP mice at different times after EAP induction. Original magnification: Â100. Scale bars = 100 μm. (E) Chronic pelvic pain was assessed by tactile allodynia using von Frey filament stimulation at different times after the first EAP immunization. *p < 0.05, **p < 0.01. Data represent the mean ± SEM for four mice per treatment group of three independent experiments.

| Neutrophil infiltration and the levels of CXCL1 and CXCL2 were increased in the prostate of EAP mice
Previous studies suggested that IL-17 promoted the recruitment of neutrophils to inflammatory sites (Catar et al., 2020;Eskan et al., 2012). We were interested in whether the high level of IL-17 in the prostate tissues of EAP could increased neutrophil infiltration. Therefore, we analysed neutrophil infiltration into the prostate tissues using various methods. As shown in Figure 3a, the percentage of Ly6G + cells in prostate tissues from EAP mice was significantly higher than control mice (p < 0.05). The IF staining results for the neutrophil marker Ly6G indicated that EAP prostate tissue sections showed significantly elevated levels of Ly6G-positive neutrophils around the prostatic glandular cavity (Figure 3b). MPO is produced by activated neutrophils, and it is an established marker of neutrophil migration. To quantitatively assess neutrophil migration, we measured MPO activity in prostate tissues from control and EAP mice. The results showed that MPO activity in prostate tissues of the EAP mice was higher than F I G U R E 3 Neutrophil infiltration and the expression levels of CXCL1, and CXCL2 were increased in the prostate tissues of EAP mice. (a) Ly6G + neutrophils in prostate tissues were analysed using flow cytometry 42 days after the first EAP induction. (b) Immunofluorescent images of neutrophil infiltration in prostate tissues 42 days after the first EAP induction. Representative images of anti-Ly6G staining in control and EAP mice are shown. (c) The MPO activity in the prostate tissues from control and EAP mice was determined by MPO colorimetric assay kit on day 42 after EAP first induction. (d) The mRNA levels of Cxcl1 and Cxcl2 in the prostate tissues from control and EAP mice were determined using qRT-PCR 42 days after the first EAP induction. The results were normalized to Gapdh. (e) The levels of CXCL1 and CXCL2 in prostate tissues were determined using ELISA 42 days after EAP induction. *p < 0.05, **p < 0.01. Data represent the mean ± SEM for four mice per treatment group of three independent experiments. control mice (Figure 3c, p < 0.01). In summary, the results shown in Neutrophils express chemokine receptors, such as CXC chemokine receptor 2 (CXCR2) and readily react to the chemokines, CXCL1 and CXCL2 (Williams et al., 2017). Neutrophil recruitment from peripheral blood is a consequence of the local production of chemokines, primarily CXCL1 and CXCL2 (Scalerandi et al., 2018), which may be elicited by IL-17 (Moseley et al., 2003;Y. Zhang et al., 2012). Therefore, we measured the mRNA and protein levels of CXCL1 and CXCL2, which are downstream of the IL-17 signalling pathway, in prostate tissues from control and EAP mice. The mRNA and protein levels of CXCL1 and CXCL2 in the prostate tissues of EAP mice were higher than control mice (Figure 3d,e, p < 0.05).
Therefore, we hypothesized that IL-17 promoted neutrophil infiltration of the prostate tissues via the high levels of CXCL1 and CXCL2 in EAP mice rather than control mice.

| IL-17 neutralization reduced CXCL1/CXCL2 production and neutrophil recruitment
To verify our hypothesis that IL-17 induced neutrophil infiltration via CXCL1 and CXCL2, we treated EAP mice with an IL-17-neutralizing mAb. As a result, IL-17 neutralization decreased IL-17 levels in prostate tissues and ameliorated the inflammatory changes and pelvic pain associated with EAP (Supplemental Figure 2, p < 0.05).
We measured neutrophil infiltration into the prostate in the EAP + IgG and EAP + anti-IL-17 groups by flow cytometry and IF staining. As shown in Figure 4a, the percentage of Ly6G + cells in prostate tissues from the EAP + anti-IL-17 group was significantly lower F I G U R E 4 Neutralization of IL-17 reduced the infiltration of neutrophils and CXCL1 and CXCL2 levels. (a) Ly6G + neutrophils in prostate tissues were analysed using flow cytometry 42 days after the first EAP immunization. (b) Representative immunofluorescent images of neutrophil infiltration (anti-Ly6G) in the prostate tissues from the EAP + IgG group and the EAP + anti-IL-17 group 42 days after the first EAP immunization. (c) The MPO activity in the prostate tissues from mice in the EAP + IgG and EAP + anti-IL-17 groups was determined using MPO colorimetric assay kit 42 days after the first EAP immunization. (d) The mRNA levels of Cxcl1 and Cxcl2 in the prostate tissues from mice in the EAP + IgG and EAP + anti-IL-17 groups were determined using qRT-PCR 42 days after the first EAP immunization. The results were normalized to Gapdh. (e) The levels of CXCL1 and CXCL2 in prostate tissues were analysed using ELISA 42 days after the first EAP induction. *p < 0.05, **p < 0.01. Data represent the mean ± SEM for four mice per treatment group of three independent experiments.

| Depletion of neutrophils ameliorated inflammatory changes and EAP-induced hyperalgesia
Recent studies demonstrated that neutrophil infiltration played a vital role in inflammatory disease (Prado et al., 2020). We hypothesized that the depletion of neutrophils would alleviate the severity of EAPinduced inflammation. To verify this hypothesis, we treated EAP mice with anti-Ly6G or IgG isotype control. As shown in Figure 5a, anti-Ly6G-treatment decreased neutrophils without affecting lymphocytes or monocytes in circulating blood. We examined the effect of neutrophil depletion treatment on the manifestation of EAP. HE staining showed that the pathological alterations were significantly alleviated in the EAP + anti-Ly6G mice compared to the EAP + IgG mice (Figure 5b,c). The response frequencies to mechanical filament stimulation of the pelvic area also exhibited a significant decrease compared to EAP + IgG mice (Figure 5d, p < 0.05). Taken together, these data suggested that neutrophils were fundamental to the development of EAP and decreasing neutrophils in prostate tissues might be a promising therapeutic strategy for EAP or CPPS. F I G U R E 5 Depletion of neutrophils using an anti-Ly6G antibody protects NOD mice from EAP. (a) Number of neutrophils, lymphocytes and monocytes in the peripheral blood from EAP + IgG and EAP + anti-Ly6G mice measured using haematology cell counter 42 days after the first EAP immunization. (b,c) representative HE staining images (b) and inflammation scores (c) of prostate tissue of EAP + IgG and EAP + anti-Ly6G mice 42 days after the first EAP immunization. (d) The response frequencies to individual filament stimulation of the pelvic area 42 days after the first EAP immunization. *p < 0.05, **p < 0.01. Data represent the mean ± SEM for four mice per treatment group of three independent experiments.

| Fucoidan ameliorated EAP-induced inflammatory changes and pain symptoms by decreasing the infiltration of neutrophils
Fucoidan has been proved to ameliorate inflammation and hyperalgesia in various inflammatory diseases (McNamee et al., 2011;Park et al., 2017;Yu et al., 2018). Its most important therapeutic effects is potent inhibition of neutrophil recruitment to inflammatory sites (McNamee et al., 2011). Fucoidan is a competitive inhibitor of selectins, which are necessary for neutrophil migration. We were curious whether fucoidan was effective for the treatment of CPPS. Therefore, we treated EAP mice with rIL-17 (to enhance neutrophil recruitment to prostate tissues) or fucoidan previously described (Gao et al., 2010;McNamee et al., 2011). The percentages of Ly6G + cells in the prostate tissues in the four groups were 5.92% ± 0.31% for EAP + vehicle, 7.45% ± 0.44% for EAP + rIL-17, 4.50% ± 0.14% for EAP + fucoidan, and 4.33% ± 0.67% for EAP + rIL-17 + fucoidan (Figure 6a,b). These results suggested that fucoidan treatment F I G U R E 6 Fucoidan treatment decreased the severity of prostate inflammation and chronic pain behaviour. (a,b) Ly6G + neutrophils in the prostate tissues from each group were analysed using flow cytometry 42 days after the first EAP immunization. (c) the MPO activity of the prostate tissues from each group analysed using the MPO colorimetric assay kit 42 days after the first EAP immunization. (d,e) representative HE staining images (d) and inflammation scores (e) of prostate tissue from each group 42 days after the first EAP immunization: 1, EAP + vehicle; 2, EAP + rIL-17; 3, EAP + fucoidan; 4, EAP + rIL-17 + fucoidan. (f) The response frequencies to individual filament stimulation of the pelvic area 42 days after the first EAP immunization. (EAP + fucoidan vs. EAP + vehicle, *p < 0.05, **p < 0.01 and EAP + rIL-17 + fucoidan vs. EAP + rIL-17, # p < 0.05, ## p < 0.01). Data represent the means ± SEM for four mice per treatment group of three independent experiments. decreased the percentage of Ly6G + neutrophil infiltration in prostate tissues (Figure 6a,b, p < 0.05). Similarly, fucoidan treatment also decreased the MPO activity in prostate tissues (Figure 6c, p < 0.01).
These results confirmed the efficiency of fucoidan-mediated neutrophil inhibition.
Finally, we investigated the effect of fucoidan treatment on the severity of prostate inflammation and chronic pain behaviour. HE staining showed showed that the histological appearance of prostate tissues from fucoidan-treated-EAP mice was alleviated, which was evidenced by a reduction in stromal mononuclear cell infiltration (Figure 6d,e, p < 0.01). Moreover, chronic pain development analysis suggested that the response frequency was ameliorated in the EAP + fucoidan group compared to the EAP + vehicle group (Figure 6f, *p < 0.05, **p < 0.01). Compared to the EAP + rIL-17 group, the inflammation changes and pain symptoms were significantly decreased in the EAP + rIL-17 + fucoidan group. (Figure 6d, f, p < 0.05). These results supported fucoidan as a potential drug for EAP treatment by inhibiting neutrophil infiltration into the prostate tissues.

| DISCUSSION
CP/CPPS is a complex syndrome with an unclear aetiology, but the involvement of immune dysfunction, especially autoimmunity, has received considerable support from a variety of human and mouse studies (Breser et al., 2017;Murphy et al., 2014;Pontari & Ruggieri, 2008). We recently demonstrated that the characteristic features of CPPS, namely, pelvic pain and infiltration of inflammatory cells into the prostate, were observed in the EAP mouse model (Zhan et al., 2020). The pelvic pain in this model was represented by referred visceral hyperalgesia of the somatic area. Our previous study demonstrated that IL-17 exerted a positive effect in an EAP mouse model (Zhan et al., 2020). In this study, we examined the molecular mechanism of IL-17, which is the main effector cytokine of Th17 cells, in the development of pelvic pain and prostate inflammation in EAP model mice. That was, IL-17 secreted by Th17 cells recruited neutrophils by increasing the expression of CXCL1/CXCL2, which promoted the occurrence of EAP.
In fact, the pathogenesis of Th17 cells in autoimmune diseases, such CP/CPPS, has attracted the attention of many scholars (Murphy et al., 2015;Zhan et al., 2020). The role of neutrophils in immune diseases was reported in many studies. As early as 1993, A.H. et al. reported the role of neutrophil cytoplasmic antibodies in autoimmune liver diseases (Mulder et al., 1993). Other scholars subsequently studied the association between neutrophils and various autoimmune diseases (Kwon et al., 2018;Lande et al., 2011). Simmons et al. showed that IL-17 promoted ELR(+) chemokine-mediated neutrophil recruitment to the brain (Simmons et al., 2014).
In this study, our group examined the pathogenesis of CPPS using Th17 cells and neutrophils together. We demonstrated that IL-17 secreted by Th17 cells upregulated the expression of CXCL1/CXCL2, which are neutrophil chemokines that recruite neutrophils by binding with CXCR1 and CXCR2 receptors. We also confirmed that fucoidan ameliorated the severity of prostate inflammation and chronic pain behaviour in EAP by inhibiting neutrophil recruitment to prostate tissues.
In conclusion, the current study revealed that IL-17 exacerbated the manifestation of EAP via neutrophil infiltration in prostate tissues.
Neutrophil recruitment was necessary for the development of EAP.