A recent study highlights a significant links between allogeneic blood transfusions (allo-BT) and primary graft dysfunction (PGD) following lung transplantation, though the underlying mechanisms remain unclear. Ischemia-reperfusion injury (IRI) is a well-established risk factor for PGD, and our prior research using a mouse orthotopic lung transplant model underscored the critical role of monocytes in IRI progression. We hypothesized that allo-BT might worsen IRI by activating monocytes and/or inducing antibody production. Using a mouse model of IRI induced by hilar clamping, we observed that administering allo-BT three days before IRI markedly aggravated lung injury. This was evidenced by increased neutrophil infiltration and impaired arterial blood gas levels. Moreover, allo-BT led to a rise in classical monocytes within the lung and elevated plasma IL-1β levels, indicating that transfusion-related injury may be driven by monocyte infiltration and the release of pro-inflammatory cytokines from activated monocytes. Interestingly, when allo-BT was administered 14 days before IRI, lung injury remained exacerbated compared to syngeneic blood transfusion. However, in this case, plasma IL-1β levels were unaffected, and non-donor-specific anti-MHC antibodies were detected. These findings suggest that non-donor-specific antibodies could contribute to early post-transplant complications, providing a complementary perspective to the traditional focus on donor-specific HLA antibodies. Supported by National Heart, Lung, and Blood Institute 5R01HL153312-04; 2R01HL145478-06; P01/FP00002771. Transplantation Immunology (TRAN)

A major limitation of immunotherapy is the development of resistance resulting from cancer-mediated inhibition of host lymphocytes. Cancer cells release CCL2 to recruit classical monocytes expressing its receptor CCR2 for the promotion of metastasis and resistance to immunosurveillance. In the circulation, some CCR2-expressing classical monocytes lose CCR2 and differentiate into intravascular nonclassical monocytes that have anticancer properties but are unable to access extravascular tumor sites. We found that in mice and humans, an ontogenetically distinct subset of naturally underrepresented CCR2-expressing nonclassical monocytes was expanded during inflammatory states such as organ transplant and COVID-19 infection. These cells could be induced during health by treatment of classical monocytes with small-molecule activators of NOD2. The presence of CCR2 enabled these inducible nonclassical monocytes to infiltrate both intra- and extravascular metastatic sites of melanoma, lung, breast, and colon cancer in murine models, and they reversed the increased susceptibility of Nod2-/- mutant mice to cancer metastasis. Within the tumor colonies, CCR2* nonclassical monocytes secreted CCL6 to recruit NK cells that mediated tumor regression, independent of T and B lymphocytes. Hence, pharmacological induction of CCR2* nonclassical monocytes might be useful for immunotherapy-resistant cancers.

Lung transplantation is a life-saving treatment for both chronic end-stage lung diseases and acute respiratory distress syndrome, including those caused by infectious agents like COVID-19. Despite its increasing utilization, outcomes post-lung transplantation are worse than other solid organ transplants. Primary graft dysfunction (PGD)—a condition affecting more than half of the recipients post-transplantation—is the chief risk factor for post-operative mortality, transplant-associated multi-organ dysfunction, and long-term graft loss due to chronic rejection. While donor-specific antibodies targeting allogenic human leukocyte antigens have been linked to transplant rejection, the role of recipient's pre-existing immunoglobulin G autoantibodies against lung-restricted self-antigens (LRA), like collagen type V and k-alpha1 tubulin, is less understood in the context of lung transplantation. Recent studies have found an increased risk of PGD development in lung transplant recipients with LRA. This review will synthesize past and ongoing research—utilizing both mouse models and human subjects—aimed at unraveling the mechanisms by which LRA heightens the risk of PGD. Furthermore, it will explore prospective approaches designed to mitigate the impact of LRA on lung transplant patients.

Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1β–mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R–dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β–dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

"Nonclassical Monocytes Promote Edema in Lung Allografts from Traumatic Brain Injury Donors." American Journal of Respiratory Cell and Molecular Biology, 64(3), pp. 391–394

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.

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 devastating condition of chronic pelvic pain. We previously linked a locus encoding acyloxyacyl hydrolase (Aoah) with pain severity in a murine model of IC. AOAH-deficient mice develop pelvic allodynia and exhibit symptoms co-morbid with IC/BPS, such as altered voiding and a depressive phenotype. Microglia are the resident immune cells of the central nervous system (CNS), and studies have linked altered microglial morphology and activation to neuropathic pain and depression. It is also well established that microglial activation can be modulated by the gut microbiota, and AOAH dysbiosis has been reported in IC/BPS patients. Therefore, we examined the microglial phenotype in AOAH-deficient mice and the potential role of microglia in modulating pelvic pain. METHODS: To analyze changes in microglial morphology, brains from wild type (WT) C57BL/6 and AOAH-deficient mice were sectioned and stained for the microglial marker P2RY12. Following immunohistochemistry, z-stacks were acquired and skeletal analyses were performed using Image J. Activation of microglia was measured through Western blotting and a proteome profiler array of mouse cytokines using brain lysates. To address the functional role of microglia in pelvic pain, microglia were eliminated from the CNS of AOAH-deficient and WT mice by oral administration of chow containing PLX5622. Mice were exposed to mechanical stimulation of the pelvic area with von Frey filaments prior to and following microglial elimination. RESULTS: Our preliminary data revealed altered microglial morphology in AOAH-deficient mice compared to WT mice. AOAH-deficient mice exhibited hyper-ramified microglia, suggesting a surveillance phenotype, in the cortex and Barrington’s nucleus, an essential region for micturition. We also observed hyper-ramified microglia in the paraventricular nucleus, a regulator of stress and depression. Microglia were less ramified in the hippocampus, consistent with an activated phenotype. Due to the morphological changes, we expect to observe differences in cytokine release between WT and AOAH-deficient mice, reflecting these differential activation states. In addition, we expect that microglia elimination will alleviate pelvic allodynia and depression in AOAH-deficient mice. CONCLUSIONS: Overall, we observed that microglial phenotype is dependent on AOAH, suggesting microglia-specific receptors or cytokines may be considered as future therapeutic targets for treating IC/BPS. Source of Funding: NIDDK awards U01 DK082342 and R01 DK066112-06S1

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.

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.

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 ...

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.