Chronic pain is associated with memory deficits clinically and in animal models. Similarly, interstitial cystitis/bladder pain syndrome (IC/BPS) afflicts patients with severe chronic pelvic pain and urinary dysfunction as well as cognitive effects, including anxiety/depression and patient-reported memory deficits. In a genetic model of IC/BPS, mice deficient for the lipase acyloxyacyl hydrolase (AOAH) mimic key symptoms, including rodent correlates of chronic pelvic pain and anxiety/depression. Recently, we observed that pelvic pain of female AOAH-deficient mice is associated with microglial activation in the prefrontal cortex and paraventricular nucleus, and pain is dependent upon microglia. Because chronic pain is associated with memory deficits and microglia support learning and memory, we hypothesized that chronic pelvic pain is associated with memory deficits in IC/BPS models. In the hippocampus, we observed microglia in the dentate gyrus of AOAH-deficient females that exhibited a hyper-ramified morphology with increased branching, number of endpoints, and process length. In contrast, dentate gyrus microglia of AOAH-deficient males had an amoeboid morphology. Using specific memory tests, female AOAH-deficient mice exhibited deficits in both spatial and recognition memory relative to wild-type controls, whereas males had a modest phenotype. Depleting microglia pharmacologically with an antagonist of colony-stimulating factor-1 receptor resolved the deficit in recognition memory but did not improve spatial memory in females and had no significant effect in males. Overall, these data suggest a role for microglial modulation of cognitive function in pelvic pain that is sex-specific and thus identify a potential target for treating cognitive symptoms in female patients.NEW & NOTEWORTHY Interstitial cystitis/bladder pain syndrome (IC/BPS) afflicts patients with debilitating pelvic pain, voiding dysfunction, and cognitive deficits, but effective therapies are lacking. Using acyloxyacyl hydrolase-deficient mice as a genetic model of IC/BPS, we previously showed that microglia mediate pelvic pain and now show that microglia mediate sex-specific memory deficits. These findings identify microglia as potential therapeutic targets for pain and cognitive dysfunction in IC/BPS.

Chronic pelvic pain conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) remain clinical and mechanistic enigmas. Microglia are resident immune cells of the central nervous system (CNS) that respond to changes in the gut microbiome, and studies have linked microglial activation to acute and chronic pain in a variety of models, including pelvic pain. We have previously reported that mice deficient for the lipase acyloxyacyl hydrolase (AOAH) develop pelvic allodynia and exhibit symptoms, comorbidities, and gut dysbiosis mimicking IC/BPS. Here, we assessed the role of AOAH in microglial activation and pelvic pain. RNAseq analyses using the ARCHS4 database and confocal microscopy revealed that AOAH is highly expressed in wild type microglia but at low levels in astrocytes, suggesting a functional role for AOAH in microglia. Pharmacologic ablation of CNS microglia with PLX5622 resulted in decreased pelvic allodynia in AOAH-deficient mice and resurgence of pelvic pain upon drug washout. Skeletal analyses revealed that AOAH-deficient mice have an activated microglia morphology in the medial prefrontal cortex and paraventricular nucleus, brain regions associated with pain modulation. Because microglia express Toll-like receptors and respond to microbial components, we also examine the potential role of dysbiosis in microglial activation. Consistent with our hypothesis of microglia activation by leakage of gut microbes, we observed increased serum endotoxins in AOAH-deficient mice and increased activation of cultured BV2 microglial cells by stool of AOAH-deficient mice. Together, these findings demonstrate a role for AOAH in microglial modulation of pelvic pain and thus identify a novel therapeutic target for IC/BPS.

ABSTRACT Gut microbiome-host interactions play a crucial role in health and disease. Altered gut microbiome composition has been observed in patients with interstitial cystitis/bladder pain syndrome (IC/BPS), a disorder characterized by pelvic pain, voiding dysfunction, and often co-morbid with anxiety/depression. We recently showed that mice deficient for acyloxyacyl hydrolase (AOAH) mimic pelvic pain symptoms and comorbidities of IC/BPS and also exhibit gut dysbiosis. In addition, we previously identified that the conditional knockout (cKO) of two transcriptional regulators of the gene encoding corticotropin-releasing factor, Crf , that are downstream of AOAH, aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor- γ (PPAR γ ), alleviate anxiety/depressive and voiding phenotypes of AOAH-deficient mice. Here, we examined the effects of AhR and PPAR γ in CRF-expressing cells on the dysbiosis of AOAH-deficiency. AOAH-deficient mice with cKO of PPAR γ and AhR/PPAR γ exhibited reduced pelvic allodynia compared to AOAH-deficient mice, suggesting a role for PPAR γ in regulating pelvic pain. 16S rRNA sequencing of fecal stool from female AOAH-deficient mice with a cKO of AhR and/or PPAR γ in CRF-expressing cells identified altered gut microbiota distinct from AOAH-deficient stool. The cKO of AhR and PPAR γ showed improved cecum barrier function in females compared to AOAH-deficient mice, whereas males were primarily affected by PPAR γ , suggesting sex differences in gut responses. Pair-wise comparison of microbiota also suggested sex differences in response to AOAH-deficiency and conditional knockout of AhR and PPAR γ . Our findings suggest that the dysbiosis and leaky gut of AOAH deficiency is mediated by AhR and PPAR γ in CRF-expressing cells and reveal a novel mechanism and therapeutic targets for pelvic pain.

Dysbiosis of gut microbiota is associated with many pathologies, yet host factors modulating microbiota remain unclear. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating condition of chronic pelvic pain often with comorbid urinary dysfunction and anxiety/depression, and recent studies find fecal dysbiosis in patients with IC/BPS. We identified the locus encoding acyloxyacyl hydrolase, Aoah, as a modulator of pelvic pain severity in a murine IC/BPS model. AOAH-deficient mice spontaneously develop rodent correlates of pelvic pain, increased responses to induced pelvic pain models, voiding dysfunction, and anxious/depressive behaviors. Here, we report that AOAH-deficient mice exhibit dysbiosis of gastrointestinal (GI) microbiota. AOAH-deficient mice exhibit an enlarged cecum, a phenotype long associated with germ-free rodents, and a “leaky gut” phenotype. AOAH-deficient ceca showed altered gene expression consistent with inflammation, Wnt signaling, and urologic disease. 16S sequencing of stool revealed altered microbiota in AOAH-deficient mice, and GC-MS identified altered metabolomes. Cohousing AOAH-deficient mice with wild-type mice resulted in converged microbiota and altered predicted metagenomes. Cohousing also abrogated the pelvic pain phenotype of AOAH-deficient mice, which was corroborated by oral gavage of AOAH-deficient mice with stool slurry of wild-type mice. Converged microbiota also alleviated comorbid anxiety-like behavior in AOAH-deficient mice. Oral gavage of AOAH-deficient mice with anaerobes cultured from IC/BPS stool resulted in exacerbation of pelvic allodynia. Together, these data indicate that AOAH is a host determinant of normal gut microbiota, and dysbiosis associated with AOAH deficiency contributes to pelvic pain. These findings suggest that the gut microbiome is a potential therapeutic target for IC/BPS.

Urologic chronic pelvic pain syndrome patients include men chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and patients, mainly women, with interstitial cystitis/bladder pain syndrome (IC/BPS or IC). CP/CPPS is marked by severe chronic pelvic pain of unknown etiology that is differentially associated with prostatic inflammation. Microbes are known to modulate sensory responses, and microbiota are increasingly understood to drive normal biological processes and pathogenesis, including inflammation. Recent studies have linked fecal dysbiosis with chronic pelvic pain in IC/BPS, suggesting a role for microbiota in modulating UCPPS pain. Similarly, dysbiosis has been reported in CP/CPPS patients, but the relationship between with the dysbiosis of IC/BPS patients is unclear. Here, we characterized the fecal microbiota of men with CP/CPPS and women and men with IC/BPS. Similar to recent reports, we identified fecal dysbiosis in men with CP/CPPS relative to healthy controls among specific phyla and overall differences in diversity and richness. Interestingly, we also observed differences between CP/CPPS microbiota and IC/BPS microbiota that were not likely due to sex differences. These findings suggest that CP/CPPS is marked by changes in the gut microbiome, but these changes differ from IC/BPS. Taken together, UCPPS appears associated with distinct dybioses among CP/CPPS and IC/BPS, raising the possibility of distinct contributions to underlying pelvic pain mechanisms and/or etiologies.

Interstitial cystitis/bladder pain syndrome (IC) is a debilitating condition of chronic pelvic pain with unknown etiology. Recently, we used a genetic approach in a murine model of IC to identify the lipase acyloxyacyl hydrolase (AOAH) as a modulator of pelvic pain. We found that AOAH-deficient mice have elevated pelvic pain responses, and AOAH immunoreactivity was detected along the bladder-brain axis. Lipidomic analyses identified arachidonic acid (AA) and its metabolite PGE2 as significantly elevated in the sacral spinal cord of AOAH-deficient mice, suggesting AA is a substrate for AOAH. Here, we quantified the effects of AOAH on phospholipids containing AA. Spinal cord lipidomics revealed increased AA-containing phosphatidylcholine in AOAH-deficient mice and concomitantly decreased AA-phosphatidylethanolamine, consistent with decreased CoA-independent transferase activity (CoIT). Overexpression of AOAH in cell cultures similarly altered distribution of AA in phospholipid pools, promoted AA incorporation, and resulted in decreased membrane fluidity. Finally, administration of a PGE2 receptor antagonist reduced pelvic pain in AOAH-deficient mice. Together, these findings suggest that AOAH represents a potential CoA-independent AA transferase that modulates CNS pain pathways at the level of phospholipid metabolism.

INTRODUCTION AND OBJECTIVE: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating condition of chronic pelvic pain often co-morbid with voiding dysfunction and depression. We have previously identified the locus encoding acyloxyacyl hydrolase, Aoah, as a modulator of pelvic pain severity in an IC murine model. AOAH-deficient mice develop spontaneous pelvic pain and an increased response to induced pelvic pain models, in addition to voiding dysfunction and a depressive-like phenotype. Recent studies in female IC/BPS patients have also indicated fecal dysbiosis, suggesting altered gut flora in AOAH-deficient mice. Therefore, we sought to characterize the gut microbiome of AOAH-deficient mice and identify its role in modulating symptoms of IC/BPS. METHODS: For these studies we utilized male wild-type (WT) C57BL/6 and AOAH-deficient mice. To determine cecum and cecal content mass, samples were weighed and reported as the fraction of total body mass. Gut microbiome composition was analyzed using 16S rDNA sequencing and liquid chromatography mass spectrometry. Intestinal barrier integrity was measured using transepithelial/transendothelial electrical resistance (TEER). To address the role of the microbiome on symptoms of IC/BPS, AOAH-deficient mice were co-housed with WT mice or received stool slurry gavage prior to analyses of visceromotor response (VMR) to bladder distension and defensive burying. RESULTS: We observed that AOAH-deficient mice exhibited an enlarged cecum, a phenotype associated with germ-free rodents, and increased mass of cecal contents. Furthermore, AOAH-deficient mice exhibited both altered microbiota and altered metabolomes compared to WT. TEER was significantly lower in the cecum of AOAH-deficient mice, suggesting a “leaky gut” phenotype. Co-housing AOAH-deficient mice with WT mice resulted in converged microbiota and abrogated the pelvic pain phenotype of AOAH-deficient mice. Alleviation of pelvic pain and anxiety/depressive behavior was also observed by gavage of AOAH-deficient mice with stool slurry of WT mice. CONCLUSIONS: Together, these data indicate that AOAH mediates normal gut microbiota and that the dysbiosis associated with AOAH deficiency is linked to pelvic pain and depressive-like behavior. Therefore, the gut flora may be a potential therapeutic target for treating patients with IC/BPS. Source of Funding: These studies were supported by NIDDK awards U01 DK082342 and R01 DK066112-06S1

Corticotropin-releasing factor (CRF) regulates diverse physiological functions, including bladder control. We recently reported that Crf expression is under genetic control of Aoah, the locus encoding acyloxyacyl hydrolase (AOAH), suggesting that AOAH may also modulate voiding. Here, we examined the role of AOAH in bladder function. AOAH-deficient mice exhibited enlarged bladders relative to wild-type mice and had decreased voiding frequency and increased void volumes. AOAH-deficient mice had increased nonvoiding contractions and increased peak voiding pressure in awake cystometry. AOAH-deficient mice also exhibited increased bladder permeability and higher neuronal firing rates of bladder afferents in response to stretch. In wild-type mice, AOAH was expressed in bladder projecting neurons and colocalized in CRF-expressing neurons in Barrington's nucleus, an important brain area for voiding behavior, and Crf was elevated in Barrington's nucleus of AOAH-deficient mice. We had previously identified aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor-γ as transcriptional regulators of Crf, and conditional knockout of AhR or peroxisome proliferator-activated receptor-γ in Crf-expressing cells restored normal voiding in AOAH-deficient mice. Finally, an AhR antagonist improved voiding in AOAH-deficient mice. Together, these data demonstrate that AOAH regulates bladder function and that the AOAH-Crf axis is a therapeutic target for treating voiding dysfunction.

Corticotropin-releasing factor (CRF) regulates stress responses, and aberrant CRF signals are associated with depressive disorders. Crf expression is responsive to arachidonic acid (AA), where CRF is released from the hypothalamic paraventricular nucleus (PVN) to initiate the hypothalamic-pituitary-adrenal axis, culminating in glucocorticoid stress hormone release. Despite this biological and clinical significance, Crf regulation is unclear. Here, we report that acyloxyacyl hydrolase, encoded by Aoah, is expressed in the PVN, and Aoah regulates Crf through the aryl hydrocarbon receptor (AhR). We previously showed that AOAH-deficient mice mimicked interstitial cystitis/bladder pain syndrome, a condition frequently associated with comorbid anxiety and depression. With the use of novelty-suppressed feeding and sucrose preference assays to quantify rodent correlates of anxiety/depression, AOAH-deficient mice exhibited depressive behaviors. AOAH-deficient mice also had increased CNS AA, increased Crf expression in the PVN, and elevated serum corticosterone, consistent with dysfunction of the hypothalamic-pituitary-adrenal axis. The human Crf promoter has putative binding sites for AhR and peroxisome proliferator-activated receptor (PPARγ). PPARγ did not affect AA-dependent Crf expression in vitro, and conditional Pparγ knockout did not alter the AOAH-deficient depressive phenotype, despite previous studies implicating PPARγ as a therapeutic target for depression. In contrast, Crf induction was mediated by AhR binding sites in vitro and increased by AhR overexpression. Furthermore, conditional Ahr knockout rescued the depressive phenotype of AOAH-deficient mice. Finally, an AhR antagonist rescued the AOAH-deficient depressive phenotype. Together, our results demonstrate that Aoah is a novel genetic regulator of Crf mediated through AhR, and AhR is a therapeutic target for depression.

BACKGROUND:Antimicrobial resistance is an emerging problem.HYPOTHESIS/OBJECTIVE:To investigate the safety and efficacy of a live biotherapeutic product, ASB E. coli 2-12 for UTI treatment.ANIMALS:Six healthy research dogs; nine client-owned dogs with recurrent UTI.METHODS:Prospective noncontrolled clinical trial. For safety data, research dogs were sedated, a urinary catheter was inserted into the bladder; 1010 CFU/mL of ASB E. coli 2-12 was instilled. Urine was cultured on days 1, 3, and 8 post-instillation and dogs were observed for lower urinary tract signs (LUTS). For client-owned dogs, ASB E. coli 2-12 was instilled similarly and urine cultures analyzed on days 1, 7, and 14 days postinstillation.RESULTS:No LUTS were noted in any of the 6 research dogs after ASB E. coli 2-12 infusion. Pulse field gel electrophoresis (PFGE) studies confirmed the bacterial strains isolated matched that ASB E. coli 2-12 strain. Four of the nine client-owned dogs had complete or nearly complete clinical cures by day 14. Of these four dogs, 3 also had microbiologic cures at day 14; one of these dogs had subclinical bacteriuria (in addition to ASB E. coli 2-12). Three of these four dogs had ASB E. coli 2-12 isolated from their urine at day 14. With the exception of mild, temporary, self-limiting, hyporexia in two dogs on the day of biotherapeutic administration, there were no major adverse effects.CONCLUSIONS AND CLINICAL IMPORTANCE:These results suggest ASB E. coli 2-12 is safe and should be investigated in a larger controlled study evaluating clinical UTI in dogs.

Interstitial cystitis/bladder pain syndrome is a chronic bladder condition associated with pain and voiding dysfunction that is often regarded as a neurogenic cystitis. Patient symptoms are correlated with the presence of urothelial lesions. We previously characterized a murine neurogenic cystitis model that recapitulates mast cell accumulation and urothelial lesions, and these events were dependent on TNF. To further explore the role of TNF in bladder inflammation and function, we generated a transgenic mouse model with chronic TNF overexpression in urothelium under the control of the uroplakin II (UPII) promoter. Transgenic mouse lines were maintained by backcross onto wild-type C57BL/6J mice and evaluated for pelvic tactile allodynia as a measure of visceral pain, urinary function, and urothelial lesions. TNF mRNA and protein were expressed at greater levels in bladders of UPII-TNF mice than in those of wild-type mice. UPII-TNF mice showed significantly increased urinary frequency and decreased void volume. UPII-TNF mice had increased urothelial apoptosis and loss of urothelial integrity consistent with urothelial lesions. Overexpression of TNF was also associated with pelvic tactile allodynia. Consistent with these findings, UPII-TNF mice exhibited increased bladder afferent activity in response to stretch ex vivo. In summary, UPII-TNF mice display significant pelvic pain, voiding dysfunction, urothelial lesions, and sensory input. Thus UPII-TNF mice are a model for characterizing mechanisms of interstitial cystitis symptoms and evaluating therapies.

The etiology of chronic pelvic pain syndromes remains unknown. In a murine urinary tract infection (UTI) model, lipopolysaccharide of uropathogenic E. coli and its receptor TLR4 are required for post-UTI chronic pain development. However, downstream mechanisms of post-UTI chronic pelvic pain remain unclear. Because the TRPV1 and MCP-1/CCR2 pathways are implicated in chronic neuropathic pain, we explored their role in post-UTI chronic pain. Mice were infected with the E. coli strain SΦ874, known to produce chronic allodynia, and treated with the TRPV1 antagonist capsazepine. Mice treated with capsazepine at the time of SΦ874 infection failed to develop chronic allodynia, whereas capsazepine treatment of mice at two weeks following SΦ874 infection did not reduce chronic allodynia. TRPV1-deficient mice did not develop chronic allodynia either. Similar results were found using novelty-suppressed feeding (NSF) to assess depressive behavior associated with neuropathic pain. Imaging of reporter mice also revealed induction of MCP-1 and CCR2 expression in sacral dorsal root ganglia following SΦ874 infection. Treatment with a CCR2 receptor antagonist at two weeks post-infection reduced chronic allodynia. Taken together, these results suggest that TRPV1 has a role in the establishment of post-UTI chronic pain, and CCR2 has a role in maintenance of post-UTI chronic pain.

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis1 Apr 2018MP39-06 PELVIC PAIN MODULATION AND TARGETING THROUGH ARACHIDONIC ACID METABOLISM Wenbin Yang, Ryan Yaggie, Charles Rudick, Anthony Schaeffer, and David Klumpp Wenbin YangWenbin Yang More articles by this author , Ryan YaggieRyan Yaggie More articles by this author , Charles RudickCharles Rudick More articles by this author , Anthony SchaefferAnthony Schaeffer More articles by this author , and David KlumppDavid Klumpp More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1253AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Interstitial cystitis/bladder pain syndrome (IC) is a debilitating condition of chronic pelvic pain and urinary dysfunction. Using a murine neurogenic cystitis model that recapitulates key aspects of IC, we recently identified acyloxyacyl hydrolase (AOAH) as a novel modulator of pelvic pain. Here we show that AOAH modulates pain severity by mediating arachidonic acid homeostasis and metabolism and identifies novel therapeutic target for chronic pelvic pain. METHODS Metabolite analysis: Metabolites was extracted from mouse sacral spinal cord, then separated by reverse-phase liquid chromatography (LC) on a C18 column. All MS analyses were performed using QTRAP 6500 and operated in multiple reaction monitoring (MRM) mode. Eicosanoids were detected in negative electrospray ion mode and endocannabinoids detected in positive ion mode. Metabolites were quantified by measuring the area under the peak relative to internal standards. RESULTS Homology with lecithin-cholesterol acyltransferase suggested that AOAH mediates transfer of arachidonic acid between phospholipids. Spinal cord lipidomics revealed increased arachidonic acid-containing phosphatidylcholine in AOAH-deficient mice and concomitantly decreased phosphatidylethanolamine, consistent with loss of arachidonyl transferase activity. In spinal cords, AOAH deficiency was also associated with elevated arachidonic acid and PGE2, and pelvic pain was reduced in AOAH-deficient mice by a PGE2 receptor antagonist. CONCLUSIONS These findings suggest that AOAH modulates pelvic pain pathways at the level of arachidonic acid homeostasis. Furthermore, arachidonic acid metabolism offers new therapeutic targets for treating chronic pelvic pain. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e511-e512 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Wenbin Yang More articles by this author Ryan Yaggie More articles by this author Charles Rudick More articles by this author Anthony Schaeffer More articles by this author David Klumpp More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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 F2(CxB) 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.

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Neurogenic Voiding Dysfunction I1 Apr 2017MP85-20 ACYLOXYACYL HYDROLASE MODULATES PELVIC PAIN SEVERITY Wenbin Yang, Ryan Yaggie, Mingcheng Jiang, Charles Rudick, Joseph Done, Charles Heckman, Anthony Schaeffer, and David Klumpp Wenbin YangWenbin Yang More articles by this author , Ryan YaggieRyan Yaggie More articles by this author , Mingcheng JiangMingcheng Jiang More articles by this author , Charles RudickCharles Rudick More articles by this author , Joseph DoneJoseph Done More articles by this author , Charles HeckmanCharles Heckman More articles by this author , Anthony SchaefferAnthony Schaeffer More articles by this author , and David KlumppDavid Klumpp More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2682AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chronic pelvic pain causes significant morbidity to patients and is a bane 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 BALB/c mice, relative to C57BL/6 mice. Here, we use a genetic strategy to identify a novel modulator of pelvic pain expressed along the bladder-brain axis. METHODS Mouse SNP genotyping: We generated 96 F2 female mice, infected with PRV, and pelvic pain was scored in response to von Frey filament stimulus. Purified F2 mouse tail DNA was genotyped with Illumina Mouse MD arrays containing 1449 SNPs. We mapped QTL using R/qtl software. Knockout mice were evaluated for pelvic allodynia, and expression was localized by immunofluorescence. RESULTS female F1CxB mice exhibit the low-allodynia phenotype of C57BL/6 parental mice in response to PRV, indicating that the severe pelvic pain phenotype of BALB/c mice is recessive. To identify loci modulating pelvic pain, we performed a quantitative trait locus (QTL) analysis on female F2CxB progeny by quantifying PRV-induced allodynia and statistical associations between pelvic pain and recombinant genotypes. Analyses identified a polymorphism on chromosome 13, rs6314295, significantly associated with allodynia (LOD=3.11). Expression analyses revealed that the mouse gene for acyloxyacyl hydrolase (AOAH), encoded near this SNP, was induced in the sacral spinal cord of PRV-infected mice. AOAH-deficient mice exhibited pelvic hypersensitivity compared to wild-type (WT) mice and developed extreme pelvic allodynia both in neurogenic and bacterial cystitis models. AOAH deficiency results in greater bladder pathology in neurogenic cystitis consistent with increased bladder mast cell activation. AOAH expression was detected along the bladder-brain axis, and AOAH-deficient mice have elevated levels of bladder VEGF, a UCPPS biomarker. CONCLUSIONS These findings indicate that AOAH is expressed along the bladder-brain axis and modulates pelvic pain severity and UCPPS biomarker expression. Thus, allelic variation in Aoah may mediate susceptibility to UCPPS symptoms. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1155 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Wenbin Yang More articles by this author Ryan Yaggie More articles by this author Mingcheng Jiang More articles by this author Charles Rudick More articles by this author Joseph Done More articles by this author Charles Heckman More articles by this author Anthony Schaeffer More articles by this author David Klumpp More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I1 Apr 2016MP24-15 CLINICALLY DIVERSE LPS ISOLATES DIRECTLY STIMULATE DRG NEURONS: RELEVANCE FOR BLADDER PAIN Abdel Belmadani, Richard Miller, Rachel Miller, Anne-Marie Malfait, Ryan Yaggie, Anthony Schaeffer, and David Klumpp Abdel BelmadaniAbdel Belmadani More articles by this author , Richard MillerRichard Miller More articles by this author , Rachel MillerRachel Miller More articles by this author , Anne-Marie MalfaitAnne-Marie Malfait More articles by this author , Ryan YaggieRyan Yaggie More articles by this author , Anthony SchaefferAnthony Schaeffer More articles by this author , and David KlumppDavid Klumpp More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.771AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES E. coli evoke a spectrum of pain responses when instilled into the bladder, ranging from no pelvic pain to post-UTI chronic pain that persists after transient infection. Previous studies indicate that E. coli-induced pain is mediated by TLR4 independent of inflammation. Because TLR4 activation is associated with TRPV1-mediated calcium responses in DRG neurons, we evaluated the hypothesis that differential bacterial pain phenotypes correlate with LPS-induced responses of DRG neurons. METHODS LPS was purified from E. coli strains NU14, Sf874, and 83972, representing bacterial pain phenotypes of acute, chronic, and analgesic, respectively. Responses of intact murine DRGs to LPS were quantified by monitoring (Ca2+) in using PIRT-GCamP3 ratiometric Ca imaging imaging using spinning disk confocal microscopy. GCamP3 is expressed in all DRG neurons in these mice. Effects of LPS isolates on production of the proalgogenic chemokine MCP-1 by cultured DRG neurons was also measured. RESULTS All LPS isolates induced robust MCP-1 secretion in dissociated cultures of DRG neurons that was not influenced by bacterial pain phenotypes. All LPS isolates were also capable of producing robust (Ca2+) in signals in capsaicin sensitive DRG neurons in whole ganglia. Sf874 LPS increased (Ca2+) in less frequently than the other isolates investigated. CONCLUSIONS Compared to our prior observations that MCP-1 expression in intact DRG in vivo responses vary with E. coli pain phenotypes in an MCP-1 reporter mouse, these data suggest that DRG MCP-1 responses vary between dissociated and intact preparations suggesting a role for non-neuronal cells. Calcium imaging supports the idea that LPS can directly activate DRG neurons. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e275 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Abdel Belmadani More articles by this author Richard Miller More articles by this author Rachel Miller More articles by this author Anne-Marie Malfait More articles by this author Ryan Yaggie More articles by this author Anthony Schaeffer More articles by this author David Klumpp More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Interstitial cystitis/bladder pain syndrome (IC) is associated with significant morbidity, yet underlying mechanisms and diagnostic biomarkers remain unknown. Pelvic organs exhibit neural crosstalk by convergence of visceral sensory pathways, and rodent studies demonstrate distinct bacterial pain phenotypes, suggesting that the microbiome modulates pelvic pain in IC. Stool samples were obtained from female IC patients and healthy controls, and symptom severity was determined by questionnaire. Operational taxonomic units (OTUs) were identified by16S rDNA sequence analysis. Machine learning by Extended Random Forest (ERF) identified OTUs associated with symptom scores. Quantitative PCR of stool DNA with species-specific primer pairs demonstrated significantly reduced levels of E. sinensis, C. aerofaciens, F. prausnitzii, O. splanchnicus, and L. longoviformis in microbiota of IC patients. These species, deficient in IC pelvic pain (DIPP), were further evaluated by Receiver-operator characteristic (ROC) analyses, and DIPP species emerged as potential IC biomarkers. Stool metabolomic studies identified glyceraldehyde as significantly elevated in IC. Metabolomic pathway analysis identified lipid pathways, consistent with predicted metagenome functionality. Together, these findings suggest that DIPP species and metabolites may serve as candidates for novel IC biomarkers in stool. Functional changes in the IC microbiome may also serve as therapeutic targets for treating chronic pelvic pain.

Uropathogenic Escherichia coli (UPEC) accounts for 80 to 90% of urinary tract infections (UTI), and the increasing rate of antibiotic resistance among UPEC isolates reinforces the need for vaccines to prevent UTIs and recurrent infections. Previous studies have shown that UPEC isolate NU14 suppresses proinflammatory NF-κB-dependent cytokines (D. J. Klumpp, A. C. Weiser, S. Sengupta, S. G. Forrestal, R. A. Batler, and A. J. Schaeffer, Infect Immun 69:6689-6695, 2001, http://dx.doi.org/10.1128/IAI.69.11.6689-6695.2001; B. K. Billips, A. J. Schaeffer, and D. J. Klumpp, Infect Immun 76:3891-3900, 2008, http://dx.doi.org/10.1128/IAI.00069-08). However, modification of lipopolysaccharide (LPS) structure by deleting the O-antigen ligase gene (waaL) enhanced proinflammatory cytokine secretion. Vaccination with the ΔwaaL mutant diminished NU14 reservoirs and protected against subsequent infections. Therefore, we hypothesized that LPS structural determinants shape immune responses. We evaluated the contribution of LPS domains to urovirulence corresponding to the inner core (waaP, waaY, and rfaQ), outer core (rfaG), and O-antigen (waaL, wzzE, and wzyE). Deletion of waaP, waaY, and rfaG attenuated adherence to urothelial cells in vitro In a murine UTI model, the ΔrfaG mutant had the most severe defect in colonization. The mutation of rfaG, waaL, wzzE, and wzyE resulted in an inability to form reservoirs in mouse bladders. Infection with the LPS mutant panel resulted in various levels of urinary myeloperoxidase. Since the ΔwaaL mutant promoted Th1-associated adaptive responses in previous studies (B. K. Billips, R. E. Yaggie, J. P. Cashy, A. J. Schaeffer, and D. J. Klumpp, J Infect Dis 200:263-272, 2009, http://dx.doi.org/10.1086/599839), we assessed NU14 for Th2-associated cytokines. We found NU14 infection stimulated TLR4-dependent bladder interleukin-33 (IL-33) production. Inoculation with rfaG, waaL, wzzE, and wzyE mutants showed decreased IL-33 production. We quantified antigen-specific antibodies after infection and found significantly increased IgE and IgG1 in ΔwaaP mutant-infected mice. Our studies show LPS structural constituents mediate multiple aspects of the UPEC life cycle, including the ability to acutely colonize bladders, form reservoirs, and evoke innate and adaptive immune responses.