A novel murine model of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) induced by immunization with a spermine binding protein (p25) peptide

Abstract

The pathophysiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is poorly understood. Inflammatory and autoimmune mechanisms may play a role. We developed a murine model of experimental autoimmune prostatitis (EAP) that mimics the human phenotype of CP/CPPS. Eight-week-old mice were immunized subcutaneously with prostate-specific peptides in an emulsion of complete Freund's adjuvant. Mice were euthanized 10 days after immunization, and lymph node cells were isolated and assessed for recall proliferation to each peptide. P25 99-118 was the most immunogenic peptide. T-cell and B-cell immunity and serum levels of C-reactive protein and nitrate/nitrite levels were evaluated over a 9-wk period. Morphometric studies of prostate, 24-h micturition frequencies, and urine volume per void were evaluated. Tactile referred hyperalgesia was measured using von Frey filaments to the pelvic region. The unpaired Student's t-test was used to analyze differences between EAP and control groups. Prostates from p25 99-118-immunized mice demonstrated elevated gene expression levels of TNF-α, IL-17A, IFN-γ, and IL-1β, not observed in control mice. Compared with controls, p25 99-118-immunized mice had significantly higher micturition frequency and decreased urine output per void, and they demonstrated elevated pelvic pain response. p25 99-118 immunization of male SWXJ mice induced prostate-specific autoimmunity characterized by prostate-confined inflammation, increased micturition frequency, and pelvic pain. This autoimmune prostatitis model provides a useful tool for exploring the pathophysiology and new treatments.

Fig. 1.

Characterization of the immune response to the prostate-specific p25 99–118 peptide. A and B: relatively higher recall response was observed with p25 99–118, compared with STEAP 311–330, p25 109–128, and PB 64–83 peptides. Ten day-primed lymph node cells (LNC) were cultured with serial dilutions of the immunizing peptide and were pulsed with [³H]thymidine. Results are expressed as the means ± SD of four mice per group expressed as the stimulation index (A) or radioactivity in counts per minute (B). C: recall proliferative responses were observed in p25 99–118 peptide, but not in the irrelevant immunogenic inhibin-α 215–234 peptide, by splenocytes obtained 9 wk after immunization with p25 99–118 (n = 5 mice per group; values are means ± SD). D: ELISA analysis of cytokines from the 48-h supernatant of cultured 10-day primed LNCs demonstrated the response to p25 99–118 was predominantly a type-1 proinflammatory response characterized by high production of IFN-γ and IL-2 and nominal production of IL-5 and IL-10 (n = 5 per group; *P = 0.001). E: magnetic bead separation of cultured 10-day-primed LNC demonstrated that recall proliferative responses to p25 99–118 were elicited from purified (>90%) CD4+ T cells, but not from CD8+ T cells (n = 5 per group). F: ELISA analysis of serum at 1/500 dilution taken 9 wk after immunization with p25 99–118 involved both a type-1 response with high production of IgG2a and IgG3 and a type-2 response with high production of IgG1 (n = 4 per group; *P < 0.0001). All values in D–F are expressed as means ± SD.

Fig. 2.

Immunocytochemical and molecular analysis of prostate tissue from immunized and control mice. A: representative immunostaining with CD3 antibody showed a predominance of T cells in the bladder infiltrating the prostate glands of male SWXJ mice 9 wk after immunization with p25 99–118 (arrows in left panel). Control mice immunized with complete Freund's adjuvant (CFA) alone did not show T-cell infiltration (right). Scale bar: 50 μm for all tissue sections. B: qRT-PCR analysis showed significantly elevated gene expression levels of TNF-α, IL-17A, IFN-γ, and IL-1β in the prostate, but not in bladder of mice immunized 9 wk earlier with p25 99–118, compared with tissues taken from age- and sex-matched naïve mice or control mice injected with CFA alone (n = 3 per group; *P < 0.006 and **P < 0.001 compared with CFA control by unpaired t-tests with and without Welch's correction, respectively). All values are presented as means ± SD.

Fig. 3.

Production of nonspecific inflammatory factors in serum after immunization with p25 99–118. Serum levels of nonspecific inflammatory marker CRP (n = 8 per group) (A), total antioxidant enzymes (n = 8 per group) (B), and nitrate/nitrite oxidative stress markers (n = 4 per group) (C) were significantly elevated 9 wk after immunization with p25 99–118 compared with control mice immunized with CFA. *P < 0.0001 and **P = 0.039 compared with CFA controls by unpaired t-tests without and with Welch's correction, respectively. All values are presented as means ± SD.

Fig. 4.

Micturition abnormalities in male SWXJ mice immunized with p25 99–118. A: 24-h micturition frequencies were significantly higher 9 wk after immunization of male SWXJ mice with p25 99–118 compared with control mice immunized with CFA (left; P = 0.004). Inversely, mean urine output/micturition was significantly lower in p25 99–118-immunized mice compared with control mice (middle; P = 0.001). Twenty-four hour fluid ingestion (water + milk) did not differ significantly between groups (right) (n = 5 per group). All values are presented as means ± SD. B: representative frequency volume chart traces of individual mice 9 wk after immunization with p25 99–118 peptide (left) or CFA (right). Each vertical line represents a micturition event, and the height of each vertical line indicates the micturition output in grams.

Fig. 5.

Pelvic pain assessment. Referred hyperalgesia and tactile allodynia were examined with von Frey filaments applied to the pelvic region. Mice immunized with p25 99–118 developed chronic pelvic pain, as indicated by a decreased threshold of pain response to stimulus on the pelvic region, as the meaning of referred pain of the prostate organ, compared with CFA-treated mice. Values in the graph are presented as means ± SD. Estimated 50% response thresholds ± SD for p25 99–118-immunized, CFA-injected, and naïve mice were 0.086 ± 0.065, 1.21 ± 0.88, and 1.81 ± 0.94 g of force, respectively. Comparisons of 50% thresholds by unpaired t-test yielded P = 0.0030 for p25 99–118-immunized vs. CFA mice, using Welch's correction; no significant difference between CFA-treated and naïve mice was found. ANOVA of the 50% thresholds with the Tukey multiple-comparison test also revealed significant differences between p25 99–118 mice and either CFA-treated or naïve mice, but not between CFA and naïve mice.