Tissue Immunity in the Bladder.

The bladder is a major component of the urinary tract, an organ system that expels metabolic waste and excess water, which necessitates proximity to the external environment and its pathogens. It also houses a commensal microbiome. Therefore, its tissue immunity must resist pathogen invasion while maintaining tolerance to commensals. Bacterial infection of the bladder is common, with half of women globally experiencing one or more episodes of cystitis in their lifetime. Despite this, our knowledge of bladder immunity, particularly in humans, is incomplete. Here we consider the current view of tissue immunity in the bladder, with a focus on defense against infection. The urothelium has robust immune functionality, and its defensive capabilities are supported by resident immune cells, including macrophages, dendritic cells, natural killer cells, and γδ T cells. We discuss each in turn and consider why adaptive immune responses are often ineffective in preventing recurrent infection, as well as areas of priority for future research.

Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments.

Central Control Circuit for Context-Dependent Micturition.

Urine release (micturition) serves an essential physiological function as well as a critical role in social communication in many animals. Here, we show a combined effect of olfaction and social hierarchy on micturition patterns in adult male mice, confirming the existence of a micturition control center that integrates pro- and anti-micturition cues. Furthermore, we demonstrate that a cluster of neurons expressing corticotropin-releasing hormone (Crh) in the pontine micturition center (PMC) is electrophysiologically distinct from their Crh-negative neighbors and sends glutamatergic projections to the spinal cord. The activity of PMC Crh-expressing neurons correlates with and is sufficient to drive bladder contraction, and when silenced impairs micturition behavior. These neurons receive convergent input from widespread higher brain areas that are capable of carrying diverse pro- and anti-micturition signals, and whose activity modulates hierarchy-dependent micturition. Taken together, our results indicate that PMC Crh-expressing neurons are likely the integration center for context-dependent micturition behavior.

A TRP Channel Senses Lysosome Neutralization by Pathogens to Trigger Their Expulsion.

Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens.

A wireless, implantable bioelectronic system for monitoring urinary bladder function following surgical recovery.

Partial cystectomy procedures for urinary bladder-related dysfunction involve long recovery periods, during which urodynamic studies (UDS) intermittently assess lower urinary tract function. However, UDS are not patient-friendly, they exhibit user-to-user variability, and they amount to snapshots in time, limiting the ability to collect continuous, longitudinal data. These procedures also pose the risk of catheter-associated urinary tract infections, which can progress to ascending pyelonephritis due to prolonged lower tract manipulation in high-risk patients. Here, we introduce a fully bladder-implantable platform that allows for continuous, real-time measurements of changes in mechanical strain associated with bladder filling and emptying via wireless telemetry, including a wireless bioresorbable strain gauge validated in a benchtop partial cystectomy model. We demonstrate that this system can reproducibly measure real-time changes in a rodent model up to 30 d postimplantation with minimal foreign body response. Studies in a nonhuman primate partial cystectomy model demonstrate concordance of pressure measurements up to 8 wk compared with traditional UDS. These results suggest that our system can be used as a suitable alternative to UDS for long-term postoperative bladder recovery monitoring.

Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs) in humans. Moreover, as one of the most common bacterial pathogens, UPEC imposes a substantial burden on healthcare systems worldwide. Epithelial cells and macrophages are two major components of the innate immune system, which play critical roles in defending the bladder against UPEC invasion. Yet, the routes of communication between these cells during UTI pathogenesis are still not fully understood. In the present study, we investigated the role of membrane-bound nanovesicles (exosomes) in the communication between bladder epithelial cells and macrophages during UPEC infection, using an array of techniques such as flow cytometry, miRNA profiling, RNA sequencing, and western blotting. Moreover, our in vitro findings were validated in a mouse model of UPEC-induced cystitis. We found that UPEC infection induced the bladder epithelial MB49 cell line to secrete large numbers of exosomes (MB49-U-Exo), which were efficiently absorbed by macrophages both in vivo and in vitro. Assimilation of MB49-U-Exo induced macrophages to produce proinflammatory cytokines, including tumor necrosis factor (TNF)α. Exposure of macrophages to MB49-U-Exo reduced their phagocytic activity (by downregulating the expression of phagocytosis-related genes) and increased their rate of apoptosis. Mechanistically, we showed that MB49-U-Exo were enriched in miR-18a-5p, which induced TNFα expression in macrophages by targeting PTEN and activating the MAPK/JNK signaling pathway. Moreover, administration of the exosome secretion inhibitor GW4869 or a TNFα-neutralizing antibody alleviated UPEC-mediated tissue damage in mice with UPEC-induced cystitis by reducing the bacterial burden of the bladder and dampening the associated inflammatory response. Collectively, these findings suggest that MB49-U-Exo regulate macrophage function in a way that exacerbates UPEC-mediated tissue impairment. Thus, targeting exosomal -release or TNFα signaling during UPEC infection may represent promising non-antibiotic strategies for treating UTIs.

PIEZO2 in sensory neurons and urothelial cells coordinates urination.

Henry Miller stated that "to relieve a full bladder is one of the great human joys". Urination is critically important in health and ailments of the lower urinary tract cause high pathological burden. Although there have been advances in understanding the central circuitry in the brain that facilitates urination1-3, there is a lack of in-depth mechanistic insight into the process. In addition to central control, micturition reflexes that govern urination are all initiated by peripheral mechanical stimuli such as bladder stretch and urethral flow4. The mechanotransduction molecules and cell types that function as the primary stretch and pressure detectors in the urinary tract mostly remain unknown. Here we identify expression of the mechanosensitive ion channel PIEZO2 in lower urinary tract tissues, where it is required for low-threshold bladder-stretch sensing and urethral micturition reflexes. We show that PIEZO2 acts as a sensor in both the bladder urothelium and innervating sensory neurons. Humans and mice lacking functional PIEZO2 have impaired bladder control, and humans lacking functional PIEZO2 report deficient bladder-filling sensation. This study identifies PIEZO2 as a key mechanosensor in urinary function. These findings set the foundation for future work to identify the interactions between urothelial cells and sensory neurons that control urination.

Creation of bladder assembloids mimicking tissue regeneration and cancer.

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer.

CD40 is a central costimulatory receptor implicated in productive antitumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway has demonstrated dose-limiting toxicities with minimal clinical activity, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe a role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of transpresented IL-15/IL-15Rα surface complexes, particularly by cross-presenting conventional type 1 DCs (Dendritic Cells), and associated enrichment of activated CD8 T cells. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, although they lack high levels of IL-15Rα at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Collectively, these data reveal an important role for IL-15 in mediating antitumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to develop combinations of these promising therapeutics for the treatment of patients with bladder cancer.

Current and Emerging Pharmacological Targets and Treatments of Urinary Incontinence and Related Disorders.

Overactive bladder syndrome with and without urinary incontinence and related conditions, signs, and disorders such as detrusor overactivity, neurogenic lower urinary tract dysfunction, underactive bladder, stress urinary incontinence, and nocturia are common in the general population and have a major impact on the quality of life of the affected patients and their partners. Based on the deliberations of the subcommittee on pharmacological treatments of the 7th International Consultation on Incontinence, we present a comprehensive review of established drug targets in the treatment of overactive bladder syndrome and the aforementioned related conditions and the approved drugs used in its treatment. Investigational drug targets and compounds are also reviewed. We conclude that, despite a range of available medical treatment options, a considerable medical need continues to exist. This is largely because the existing treatments are symptomatic and have limited efficacy and/or tolerability, which leads to poor long-term adherence. SIGNIFICANCE STATEMENT: Urinary incontinence and related disorders are prevalent in the general population. While many treatments have been approved, few patients stay on long-term treatment despite none of them being curative. This paper provides a comprehensive discussion of existing and emerging treatment options for various types of incontinence and related disorders.

Development, regeneration and tumorigenesis of the urothelium.

The urothelium of the bladder functions as a waterproof barrier between tissue and outflowing urine. Largely quiescent during homeostasis, this unique epithelium rapidly regenerates in response to bacterial or chemical injury. The specification of the proper cell types during development and injury repair is crucial for tissue function. This Review surveys the current understanding of urothelial progenitor populations in the contexts of organogenesis, regeneration and tumorigenesis. Furthermore, we discuss pathways and signaling mechanisms involved in urothelial differentiation, and consider the relevance of this knowledge to stem cell biology and tissue regeneration.

α-Hemolysin promotes uropathogenic E. coli persistence in bladder epithelial cells via abrogating bacteria-harboring lysosome acidification.

There is a growing consensus that a significant proportion of recurrent urinary tract infections are linked to the persistence of uropathogens within the urinary tract and their re-emergence upon the conclusion of antibiotic treatment. Studies in mice and human have revealed that uropathogenic Escherichia coli (UPEC) can persist in bladder epithelial cells (BECs) even after the apparent resolution of the infection. Here, we found that, following the entry of UPEC into RAB27b+ fusiform vesicles in BECs, some bacteria escaped into the cytoplasmic compartment via a mechanism involving hemolysin A (HlyA). However, these UPEC were immediately recaptured within LC3A/B+ autophagosomes that matured into LAMP1+ autolysosomes. Thereafter, HlyA+ UPEC-containing lysosomes failed to acidify, which is an essential step for bacterial elimination. This lack of acidification was related to the inability of bacteria-harboring compartments to recruit V-ATPase proton pumps, which was attributed to the defragmentation of cytosolic microtubules by HlyA. The persistence of UPEC within LAMP1+ compartments in BECs appears to be directly linked to HlyA. Thus, through intravesicular instillation of microtubule stabilizer, this host defense response can be co-opted to reduce intracellular bacterial burden following UTIs in the bladder potentially preventing recurrence.

An adeno-associated viral labeling approach to visualize the meso- and microanatomy of mechanosensory afferents and autonomic innervation of the rat urinary bladder.

The urinary bladder is supplied by a rich network of sensory and autonomic axons, commonly visualized by immunolabeling for neural markers. This approach demonstrates overall network patterning but is less suited to understanding the structure of individual motor and sensory terminals within these complex plexuses. There is a further limitation visualizing the lightly myelinated (A-delta) class of sensory axons that provides the primary mechanosensory drive for initiation of voiding. Whereas most unmyelinated sensory axons can be revealed by immunolabeling for specific neuropeptides, to date no unique neural marker has been identified to immunohistochemically label myelinated visceral afferents. We aimed to establish a non-surgical method to visualize and map myelinated afferents in the bladder in rats. We found that in rats, the adeno-associated virus (AAV), AAV-PHP.S, which shows a high tropism for the peripheral nervous system, primarily transduced myelinated dorsal root ganglion neurons, enabling us to identify the structure and regional distribution of myelinated (mechanosensory) axon endings within the muscle and lamina propria of the bladder. We further identified the projection of myelinated afferents within the pelvic nerve and lumbosacral spinal cord. A minority of noradrenergic and cholinergic neurons in pelvic ganglia were transduced, enabling visualization and regional mapping of both autonomic and sensory axon endings within the bladder. Our study identified a sparse labeling approach for investigating myelinated sensory and autonomic axon endings within the bladder and provides new insights into the nerve-bladder interface.

Role of ecto-5'-nucleotidase in bladder function.

Purinergic signaling plays an important role in regulating bladder contractility and voiding. Abnormal purinergic signaling is associated with lower urinary tract symptoms (LUTS). Ecto-5'-nucleotidase (NT5E) catalyzes dephosphorylation of extracellular AMP to adenosine, which in turn promotes adenosine-A2b receptor signaling to relax bladder smooth muscle (BSM). The functional importance of this mechanism was investigated using Nt5e knockout (Nt5eKO) mice. Increased voiding frequency of small voids revealed by voiding spot assay was corroborated by urodynamic studies showing shortened voiding intervals and decreased bladder compliance. Myography indicated reduced contractility of Nt5eKO BSM. These data support a role for NT5E in regulating bladder function through modulation of BSM contraction and relaxation. However, the abnormal bladder phenotype of Nt5eKO mice is much milder than we previously reported in A2b receptor knockout (A2bKO) mice, suggesting compensatory response(s) in Nt5eKO mouse bladder. To better understand this compensatory mechanism, we analyzed changes in purinergic and other receptors controlling BSM contraction and relaxation in the Nt5eKO bladder. We found that the relative abundance of muscarinic CHRM3 (cholinergic receptor muscarinic 3), purinergic P2X1, and A2b receptors was unchanged, whereas P2Y12 receptor was significantly downregulated, suggesting a negative feedback response to elevated ADP signaling. Further studies of additional ecto-nucleotidases indicated significant upregulation of the nonspecific urothelial alkaline phosphatase ALPL, which might mitigate the degree of voiding dysfunction by compensating for Nt5e deletion. These data suggest a mechanistic complexity of the purinergic signaling network in bladder and imply a paracrine mechanism in which urothelium-released ATP and its rapidly produced metabolites coordinately regulate BSM contraction and relaxation.

Precision Targeting: Mast Cells Wipe Out Infected Bladder Epithelia.

Exfoliating infected bladder epithelium is a powerful host defense mechanism that reduces bacterial burden. In this issue of Immunity, Choi et al. (2016) present a function of mast cells that orchestrates the infected epithelial cell exfoliation via cytolytic granules.

Ubiquitination of Innate Immune Regulator TRAF3 Orchestrates Expulsion of Intracellular Bacteria by Exocyst Complex.

Although the intracellular trafficking system is integral to most physiologic activities, its role in mediating immune responses to infection has remained elusive. Here, we report that infected bladder epithelial cells (BECs) mobilized the exocyst complex, a powerful exporter of subcellular vesicles, to rapidly expel intracellular bacteria back for clearance. Toll-like receptor (TLR) 4 signals emanating from bacteria-containing vesicles (BCVs) were found to trigger K33-linked polyubiquitination of TRAF3 at Lys168, which was then detected by RalGDS, a guanine nucleotide exchange factor (GEF) that precipitated the assembly of the exocyst complex. Although this distinct modification of TRAF3 served to connect innate immune signaling to the cellular trafficking apparatus, it crucially ensured temporal and spatial accuracy in determining which among the many subcellular vesicles was recognized and selected for expulsion in response to innate immune signaling.

Model-based analysis of the acute effects of transcutaneous magnetic spinal cord stimulation on micturition after spinal cord injury in humans.

AIM: After spinal cord injuries (SCIs), patients may develop either detrusor-sphincter dyssynergia (DSD) or urinary incontinence, depending on the level of the spinal injury. DSD and incontinence reflect the loss of coordinated neural control among the detrusor muscle, which increases bladder pressure to facilitate urination, and urethral sphincters and pelvic floor muscles, which control the bladder outlet to restrict or permit bladder emptying. Transcutaneous magnetic stimulation (TMS) applied to the spinal cord after SCI reduced DSD and incontinence. We defined, within a mathematical model, the minimum neuronal elements necessary to replicate neurogenic dysfunction of the bladder after a SCI and incorporated into this model the minimum additional neurophysiological features sufficient to replicate the improvements in bladder function associated with lumbar TMS of the spine in patients with SCI. METHODS: We created a computational model of the neural circuit of micturition based on Hodgkin-Huxley equations that replicated normal bladder function. We added interneurons and increased network complexity to reproduce dysfunctional micturition after SCI, and we increased the density and complexity of interactions of both inhibitory and excitatory lumbar spinal interneurons responsive to TMS to provide a more diverse set of spinal responses to intrinsic and extrinsic activation of spinal interneurons that remains after SCI. RESULTS: The model reproduced the re-emergence of a spinal voiding reflex after SCI. When we investigated the effect of monophasic and biphasic TMS at two frequencies applied at or below T10, the model replicated the improved coordination between detrusor and external urethral sphincter activity that has been observed clinically: low-frequency TMS (1 Hz) within the model normalized control of voiding after SCI, whereas high-frequency TMS (30 Hz) enhanced urine storage. CONCLUSION: Neuroplasticity and increased complexity of interactions among lumbar interneurons, beyond what is necessary to simulate normal bladder function, must be present in order to replicate the effects of SCI on control of micturition, and both neuronal and network modifications of lumbar interneurons are essential to understand the mechanisms whereby TMS reduced bladder dysfunction after SCI.

Mutational signature and prognosis in adenocarcinoma of the bladder.

Adenocarcinoma of the bladder is a rare urinary bladder carcinoma with limited therapy options due to lack of molecular characterization. Here, we aimed to reveal the mutational and transcriptomic landscapes of adenocarcinoma of the bladder and assess any relationship with prognosis. Between February 2015 and June 2021, a total of 23 patients with adenocarcinoma of the bladder were enrolled. These included 16 patients with primary bladder adenocarcinomas and seven patients with urachal adenocarcinoma. Whole exome sequencing (16 patients), whole genome sequencing (16 patients), bulk RNA sequencing (RNA-seq) (19 patients), and single-cell RNA-seq (5 patients) were conducted for the specimens. Correlation analysis, survival analysis, and t-tests were also performed. Prevalent T>A substitutions were observed among somatic mutations, and major trinucleotide contexts included 5'-CTC-3' and 5'-CTG-3'. This pattern was mainly contributed by COSMIC signature 22 related to chemical carcinogen exposure (probably aristolochic acid), which has not been reported in bladder adenocarcinoma. Moreover, genes with copy number changes were also enriched in the KEGG term 'chemical carcinogenesis'. Transcriptomic analysis suggested high immune cell infiltration and luminal-like features in the majority of samples. Interestingly, a small fraction of samples with an APOBEC-derived mutational signature exhibited a higher risk of disease progression compared with samples with only a chemical carcinogen-related signature, confirming the molecular and prognostic heterogeneity of bladder adenocarcinoma. This study presents mutational and transcriptomic landscapes of bladder adenocarcinoma, and indicates that a chemical carcinogen-related mutational signature may be related to a better prognosis compared with an APOBEC signature in adenocarcinoma of the bladder. © 2024 The Pathological Society of Great Britain and Ireland.