Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli.

Background Pelvic pain is a major component of the morbidity associated with urinary tract infection (UTI), yet the molecular mechanisms underlying UTI-induced pain remain unknown. UTI pain mechanisms likely contrast with the clinical condition of asymptomatic bacteriuria (ASB), where individuals have significant bacterial loads yet lack symptoms. Methods A murine UTI model was used to compare pelvic pain behavior elicited by infection with uropathogenic Escherichia coli strain NU14 and ASB strain 83972. Results NU14-infected mice developed pelvic pain, whereas mice infected with 83972 did not exhibit pain, similar to patients infected with 83972. NU14-induced pain was not dependent on mast cells, not correlated with bacterial colonization, nor correlated urinary neutrophils. UTI pain was not influenced by expression of type 1 pili, the bacterial adhesive appendage that induces urothelial apoptosis. However, purified NU14 lipopolysaccharide (LPS) induced TLR4-dependent pain, but 83972 LPS induced no pain. Indeed, 83972 LPS attenuated pain of NU14 infection, suggesting therapeutic potential. Conclusions These data suggest a novel mechanism of infection-associated pain that is dependent on TLR4 yet independent of inflammation. Clinically, these findings also provide the rational for probiotic therapies that would minimize the symptoms of infection without reliance on empiric therapies that contribute to antimicrobial resistance.

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.

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. Patients with urologic chronic pelvic pain syndromes (UCPPS) suffer chronic pelvic pain and dramatically lower quality of life, yet diagnostic markers and effective therapies remain elusive for these costly syndromes 1 . Interstitial cystitis/bladder pain syndrome (IC/BPS or IC) is a debilitating UCPPS condition of pelvic pain and voiding dysfunction that afflicts as many as 8 million U.S. women where depression is a common co-morbidity 2-5 . IC etiology remains unknown, but urothelial lesions and lamina propria mast cells are associated with patient symptoms [6][7][8] . HPA axis dysfunction has been implicated in female and male patients and cats with feline IC 9-13 , and thus may be common among UCPPS, but mechanisms that integrate pelvic pain, voiding dysfunction, HPA activity, and depression are lacking. Because of these long-standing questions, NIDDK has launched its flagship urology study, the Multi-Disciplinary Approaches to Chronic Pelvic Pain (MAPP) Research Network with the most comprehensive studies to date of UCPPS including clinical characterization and epidemiologic and mechanistic studies 14,15 . Operational taxonomic units (OTUsThe microbiome is increasingly appreciated as driving diverse physiologic processes in both health and disease 16,17 . With the notable exception of C. difficile colitis, early studies implicated pathogenic dysbioses primarily at the phylum and genus levels, but more recent studies identify individual species associated with disease. Moreover, individual species within the microbiome have recently been associated with driving disease through altered innate metabolism or altered pharmaceutical metabolism 18 . Despite these advances, it remains unclear whether microbiota contribute to pain syndromes generally and to UCPPS in particular. Consistent with the recent finding of a normal female urinary microbiome 19 , the MAPP Network is also exploring UCPPS microbiota with a focus on the urinary microbiome. MAPP Network studies have identified an association between fungi

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.