Acyloxyacyl hydrolase modulates pelvic pain severity.

Chronic pelvic pain causes significant patient morbidity and is a challenge to clinicians. Using a murine neurogenic cystitis model that recapitulates key aspects of interstitial cystitis/bladder pain syndrome (IC), we recently showed that pseudorabies virus (PRV) induces severe pelvic allodynia in BALB/c mice relative to C57BL/6 mice. Here, we report that a quantitative trait locus (QTL) analysis of PRV-induced allodynia in F2CxB progeny identified a polymorphism on chromosome 13, rs6314295 , significantly associated with allodynia (logarithm of odds = 3.11). The nearby gene encoding acyloxyacyl hydrolase ( Aoah) was induced in the sacral spinal cord of PRV-infected mice. AOAH-deficient mice exhibited increased vesicomotor reflex in response to bladder distension, consistent with spontaneous bladder hypersensitivity, and increased pelvic allodynia in neurogenic cystitis and postbacterial chronic pain models. AOAH deficiency resulted in greater bladder pathology and tumor necrosis factor production in PRV neurogenic cystitis, markers of increased bladder mast cell activation. AOAH immunoreactivity was detectable along the bladder-brain axis, including in brain sites previously correlated with human chronic pelvic pain. Finally, AOAH-deficient mice had significantly higher levels of bladder vascular endothelial growth factor, an emerging marker of chronic pelvic pain in humans. These findings indicate that AOAH modulates pelvic pain severity, suggesting that allelic variation in Aoah influences pelvic pain in IC.

Role of PAR2 in the Development of Lower Urinary Tract Dysfunction.

PURPOSE: Lower urinary tract symptoms are a common finding in patients with chronic prostatitis/chronic pelvic pain syndrome. We previously reported that the mast cell-tryptase-PAR2 (protease activated receptor 2) axis has a critical role in the development of chronic pain in experimental autoimmune prostatitis, a mouse model of chronic prostatitis/chronic pelvic pain syndrome. Therefore, we examined whether PAR2 activation mediates lower urinary tract dysfunction. MATERIALS AND METHODS: Functional cystometry was done in male B6 mice along with immunoblotting and immunohistochemistry for the expression of COL1A1 (collagen type I α I) and α-SMA (α-smooth muscle actin). Flow cytometry analysis was performed on single cell suspensions of the prostate, bladder, lymph nodes and spleen. RESULTS: Experimental autoimmune prostatitis resulted in increased urinary voiding frequency and decreased bladder capacity 30 days after initiation. Concurrently, there was increased expression of COL1A1 and α-SMA in the prostates and bladders. In contrast, induction of experimental autoimmune prostatitis in PAR2 knockout mice did not result in altered urodynamics or increased markers of fibrosis in the prostate or the bladder. Single cell suspensions of the prostate, bladder, lymph nodes and spleen demonstrated that in the absence of PAR2 cellular inflammatory mechanisms were still initiated in experimental autoimmune prostatitis but PAR2 expression may be required to maintain chronic inflammation. Finally, antibody mediated PAR2 neutralization normalized urinary voiding frequency and bladder capacity, and attenuated chronic pelvic pain. CONCLUSIONS: PAR2 activation in the prostate may contribute to the development of lower urinary tract dysfunction through proinflammatory as well as profibrotic pathways.

Experimental autoimmune prostatitis induces microglial activation in the spinal cord.

BACKGROUND: The pathogenesis of chronic prostatitis/chronic pelvic pain syndrome is unknown and factors including the host's immune response and the nervous system have been attributed to the development of CP/CPPS. We previously demonstrated that mast cells and chemokines such as CCL2 and CCL3 play an important role in mediating prostatitis. Here, we examined the role of neuroinflammation and microglia in the CNS in the development of chronic pelvic pain. METHODS: Experimental autoimmune prostatitis (EAP) was induced using a subcutaneous injection of rat prostate antigen. Sacral spinal cord tissue (segments S14-S5) was isolated and utilized for immunofluorescence or QRT-PCR analysis. Tactile allodynia was measured at baseline and at various points during EAP using Von Frey fibers as a function for pelvic pain. EAP mice were treated with minocycline after 30 days of prostatitis to test the efficacy of microglial inhibition on pelvic pain. RESULTS: Prostatitis induced the expansion and activation of microglia and the development of inflammation in the spinal cord as determined by increased expression levels of CCL3, IL-1ß, Iba1, and ERK1/2 phosphorylation. Microglial activation in mice with prostatitis resulted in increased expression of P2X4R and elevated levels of BDNF, two molecular markers associated with chronic pain. Pharmacological inhibition of microglia alleviated pain in mice with prostatitis and resulted in decreased expression of IL-1ß, P2X4R, and BDNF. CONCLUSION: Our data show that prostatitis leads to inflammation in the spinal cord and the activation and expansion of microglia, mechanisms that may contribute to the development and maintenance of chronic pelvic pain.

Role of mast cells in male chronic pelvic pain.

PURPOSE: Chronic pelvic pain syndrome accounts for 90% of all chronic prostatitis but it has an unknown pathogenesis. We sought to understand the role of mast cells and nerve growth factor in chronic pelvic pain. MATERIALS AND METHODS: Expressed prostatic secretions in men with chronic pelvic pain syndrome and controls were tested for mast cell tryptase and nerve growth factor. Mast cell number, activation status and nerve growth factor expression were examined in the NOD/ShiLtJ experimental autoimmune prostatitis model and in mast cell deficient KitW-sh/KitW-sh mice. Tactile allodynia was quantified using von Frey filaments as a measure of pelvic pain behavior. Inhibitors of mast cell degranulation, histamine receptor antagonists and anti-nerve growth factor neutralizing antibodies were tested to decrease pelvic pain behavior. RESULTS: Men with chronic pelvic pain syndrome showed increased mast cell tryptase and nerve growth factor in expressed prostatic secretions. In the experimental autoimmune prostatitis model increased total and activated mast cells were observed in the prostate. Mast cell deficient KitW-sh/KitW-sh mice showed attenuated pelvic pain behavior but no difference in inflammatory infiltrates in the prostate from controls. Mice with experimental autoimmune prostatitis also demonstrated increased intraprostatic nerve growth factor compared to that of KitW-sh/KitW-sh mice. Treatment of experimental autoimmune prostatitis with a mast cell stabilizer combined with a histamine 1 receptor antagonist resulted in a synergistic decrease in chronic pelvic pain. In contrast, neutralization of nerve growth factor in vivo did not result in pain relief. CONCLUSIONS: Results suggest that mast cells are important mediators of chronic pelvic pain in experimental autoimmune prostatitis cases. They may be potential targets for therapeutic intervention in men with chronic prostatitis/chronic pelvic pain syndrome.

CCL2 and CCL3 are essential mediators of pelvic pain in experimental autoimmune prostatitis.

Experimental autoimmune prostatitis (EAP) is a murine model of chronic prostatitis/chronic pelvic pain syndrome (CPPS) in men, a syndrome characterized by chronic pelvic pain. We have demonstrated that chemokine ligands CCL2 and CCL3 are biomarkers that correlate with pelvic pain symptoms. We postulated that CCL2 and CCL3 play a functional role in CPPS and therefore examined their expression in EAP. Upon examination of the prostate 5 days after induction of EAP, CCL2 mRNA was elevated 2- to 3-fold, CCL8 by 15-fold, CCL12 by 12- to 13-fold, and CXCL9 by 2- to 4-fold compared with control mice. At 10 days the major chemokines were CXCL13 and CXCL2; at 20 days CCL2 (1- to 2-fold), CCL3 (2- to 3-fold) and CCL11 (2- to 3-fold); and at 30 days, CCL12 (20- to 35-fold) and smaller increases in CCL2, CCL3, and XCL1. Chemokine elevations were accompanied by increases in mast cells and B cells at 5 days, monocytes and neutrophils at day 10, CD4+ T cells at day 20, and CD4+ and CD8+ T cells at day 30. Anti-CCL2 and anti-CCL3 neutralizing antibodies administered at EAP onset attenuated pelvic pain development, but only anti-CCL2 antibodies were effective therapeutically. CCL2- and its cognate receptor CCR2-deficient mice were completely protected from development of pain symptoms but assumed susceptibility after reconstitution with wild-type bone marrow. CCL3-deficient mice showed resistance to the maintenance of pelvic pain while CCR5-deficient mice did not show any lessening of pelvic pain severity. These results suggest that the CCL2-CCR2 axis and CCL3 are important mediators of chronic pelvic pain in EAP.

Prostate secretions from men with chronic pelvic pain syndrome inhibit proinflammatory mediators.

PURPOSE: In the past numerous chemokines have been noted in the expressed prostatic secretions of patients with chronic prostatitis/chronic pelvic pain syndrome. We examined the functional effects of chemokines in expressed prostatic secretions of patients with chronic pelvic pain syndrome. MATERIALS AND METHODS: We studied the functional effects of expressed prostatic secretions on human monocytes by examining monocyte chemotaxis in response to monocyte chemoattractant protein-1, a major chemoattractant previously identified in chronic prostatitis/chronic pelvic pain syndrome cases. We determined effects on cellular signaling by quantifying intracellular calcium increase in monocytes and nuclear factor-κB activation in normal prostate epithelial cells. RESULTS: Results show that the monocyte chemoattractant protein-1 in expressed prostatic secretions is nonfunctional with an inability to mediate human monocyte chemotaxis, or mediate signaling in monocytes or prostate epithelial cells. This lack of functionality could be extended to other proinflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, when incubated with expressed prostatic secretions from patients with chronic pelvic pain syndrome. The mechanism underlying this apparent ability to modulate proinflammatory cytokines involves heat labile extracellular proteases that mediate the inhibition of immune and prostate epithelial cell function. CONCLUSIONS: These results may have implications for the design of specific diagnostic and therapeutic methods targeted toward the complete resolution of prostate inflammatory insults.

A prostate derived commensal Staphylococcus epidermidis strain prevents and ameliorates induction of chronic prostatitis by UPEC infection.

Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS) is a common syndrome with limited therapies and an unknown etiology. Previously, our laboratory has defined a potential role for pathogenic infection in disease onset. Intra-urethral infection with a uropathogenic Escherichia coli strain isolated from a CP/CPPS patient, CP1, induces prostatic inflammation and tactile allodynia in mice. We have also demonstrated that a prostate specific Staphylococcus epidermidis bacterial isolate, NPI (non-pain inducing), from a healthy subject reduces pain and inflammation in an experimental autoimmune prostatitis (EAP) murine model. Here we focus on the interplay between these human isolates in the context of prostatitis development and resolution. NOD/ShiLtJ mice were inoculated with either NP1 or CP1, or combinations of both. Infection with CP1 induced pelvic tactile allodynia after 7 days, while NPI instillation alone induced no such response. Instillation with NPI 7 days following CP1 infection resolved pelvic tactile allodynia and prophylactic instillation 7 days prior to CPI infection prevented its onset. Prophylactic NPI instillation also prevented CP1 colonization of both prostate and bladder tissues. In vitro analyses revealed that CP1 and NPI do not directly inhibit the growth or invasive potential of one another. Immunological analyses revealed that specific markers associated with CP1-induced pelvic allodynia were decreased upon NPI treatment or repressed by prophylactic colonization. This study demonstrates that a commensal bacterial isolate can inhibit the colonization, pain responses, and immunological activation to uropathogenic bacteria, emphasizing the power of a healthy prostatic microflora in controlling health and disease.

Commensal bacterial modulation of the host immune response to ameliorate pain in a murine model of chronic prostatitis.

The human commensal microflora plays an essential role in modulating the immune response to control homeostasis. Staphylococcus epidermidis, a commensal bacterium most commonly associated with the skin exerts such effects locally, modulating local immune responses during inflammation and preventing superinfection by pathogens such as Staphylococcus aureus. Although the prostate is considered by many to be sterile, multiple investigations have shown that small numbers of gram-positive bacterial species such as S. epidermidis can be isolated from the expressed prostatic secretions of both healthy and diseased men. Chronic pelvic pain syndrome is a complex syndrome with symptoms including pain and lower urinary tract dysfunction. It has an unknown etiology and limited effective treatments but is associated with modulation of prostate immune responses. Chronic pelvic pain syndrome can be modeled using murine experimental prostatitis (EAP), where CD4+ve IL17A+ve T cells have been shown to play a critical role in disease orchestration and development of pelvic tactile allodynia. Here, we report that intraurethral instillation of a specific S. epidermidis strain (designated NPI [non-pain inducing]), isolated from the expressed prostatic secretion of a healthy human male, into EAP-treated mice reduced the pelvic tactile allodynia responses and increased CD4+ve IL17A+ve T-cell numbers associated with EAP. Furthermore, a cell wall constituent of NPI, lipoteichoic acid, specifically recapitulates these effects and mediates increased expression of CTLA4-like ligands PDL1 and PDL2 on prostatic CD11b+ve antigen-presenting cells. These results identify a new potential therapeutic role for commensal S. epidermidis NPI lipoteichoic acid in the treatment of prostatitis-associated pain.

IL17 Mediates Pelvic Pain in Experimental Autoimmune Prostatitis (EAP).

Chronic pelvic pain syndrome (CPPS) is the most common form of prostatitis, accounting for 90-95% of all diagnoses. It is a complex multi-symptom syndrome with unknown etiology and limited effective treatments. Previous investigations highlight roles for inflammatory mediators in disease progression by correlating levels of cytokines and chemokines with patient reported symptom scores. It is hypothesized that alteration of adaptive immune mechanisms results in autoimmunity and subsequent development of pain. Mouse models of CPPS have been developed to delineate these immune mechanisms driving pain in humans. Using the experimental autoimmune prostatitis (EAP) in C57BL/6 mice model of CPPS we examined the role of CD4+T-cell subsets in the development and maintenance of prostate pain, by tactile allodynia behavioral testing and flow cytometry. In tandem with increased CD4+IL17A+ T-cells upon EAP induction, prophylactic treatment with an anti-IL17 antibody one-day prior to EAP induction prevented the onset of pelvic pain. Therapeutic blockade of IL17 did not reverse pain symptoms indicating that IL17 is essential for development but not maintenance of chronic pain in EAP. Furthermore we identified a cytokine, IL7, to be associated with increased symptom severity in CPPS patients and is increased in patient prostatic secretions and the prostates of EAP mice. IL7 is fundamental to development of IL17 producing cells and plays a role in maturation of auto-reactive T-cells, it is also associated with autoimmune disorders including multiple sclerosis and type-1 diabetes. More recently a growing body of research has pointed to IL17's role in development of neuropathic and chronic pain. This report presents novel data on the role of CD4+IL17+ T-cells in development and maintenance of pain in EAP and CPPS.

Tryptase-PAR2 axis in experimental autoimmune prostatitis, a model for chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) affects up to 15% of the male population and is characterized by pelvic pain. Mast cells are implicated in the murine experimental autoimmune prostatitis (EAP) model as key to chronic pelvic pain development. The mast cell mediator tryptase-ß and its cognate receptor protease-activated receptor 2 (PAR2) are involved in mediating pain in other visceral disease models. Prostatic secretions and urines from CP/CPPS patients were examined for the presence of mast cell degranulation products. Tryptase-ß and PAR2 expression were examined in murine EAP. Pelvic pain and inflammation were assessed in the presence or absence of PAR2 expression and upon PAR2 neutralization. Tryptase-ß and carboxypeptidase A3 were elevated in CP/CPPS compared to healthy volunteers. Tryptase-ß was capable of inducing pelvic pain and was increased in EAP along with its receptor PAR2. PAR2 was required for the development of chronic pelvic pain in EAP. PAR2 signaling in dorsal root ganglia led to extracellular signal-regulated kinase (ERK)1/2 phosphorylation and calcium influx. PAR2 neutralization using antibodies attenuated chronic pelvic pain in EAP. The tryptase-PAR2 axis is an important mediator of pelvic pain in EAP and may play a role in the pathogenesis of CP/CPPS.

Measurement of tactile allodynia in a murine model of bacterial prostatitis.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS). Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity. A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria. CP/CPPS is primarily characterized by the presence of tactile allodynia. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Th1-Th17 cells contribute to the development of uropathogenic Escherichia coli-induced chronic pelvic pain.

The etiology of chronic prostatitis/chronic pelvic pain syndrome in men is unknown but may involve microbes and autoimmune mechanisms. We developed an infection model of chronic pelvic pain in NOD/ShiLtJ (NOD) mice with a clinical Escherichia coli isolate (CP-1) from a patient with chronic pelvic pain. We investigated pain mechanisms in NOD mice and compared it to C57BL/6 (B6) mice, a strain resistant to CP-1-induced pain. Adoptive transfer of CD4+ T cells, but not serum, from CP-1-infected NOD mice was sufficient to induce chronic pelvic pain. CD4+ T cells in CP-1-infected NOD mice expressed IFN-γ and IL-17A but not IL-4, consistent with a Th1/Th17 immune signature. Adoptive transfer of ex-vivo expanded IFN-γ or IL-17A-expressing cells was sufficient to induce pelvic pain in naïve NOD recipients. Pelvic pain was not abolished in NOD-IFN-γ-KO mice but was associated with an enhanced IL-17A immune response to CP1 infection. These findings demonstrate a novel role for Th1 and Th17-mediated adaptive immune mechanisms in chronic pelvic pain.

O-antigen modulates infection-induced pain states.

The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic E. coli (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to E. coli lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. E. coli strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among E. coli strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.