Effects of Aging on Urinary Tract Epithelial Homeostasis and Immunity

Abstract

A global increase in older individuals creates an increasing demand to understand numerous healthcare challenges related to aging. This population is subject to changes in tissue physiology and the immune response network. Older individuals are particularly susceptible to infectious diseases, with one of the most common being urinary tract infections (UTIs). Postmenopausal and older women have the highest risk of recurrent UTIs (rUTIs); however, why rUTIs become more frequent after menopause and during old age is incompletely understood. This increased susceptibility and severity among older individuals may involve functional changes to the immune system with age. Aging also has substantial effects on the epithelium and the immune system that led to impaired protection against pathogens, yet heightened and prolonged inflammation. How the immune system and its responses to infection changes within the bladder mucosa during aging has largely remained poorly understood. In this review, we highlight our understanding of bladder innate and adaptive immunity and the impact of aging and hormones and hormone therapy on bladder epithelial homeostasis and immunity. In particular, we elaborate on how the cellular and molecular immune landscape within the bladder can be altered during aging as aged mice develop bladder tertiary lymphoid tissues (bTLT), which are absent in young mice leading to profound age-associated change to the immune landscape in bladders that might drive the significant increase in UTI susceptibility. Knowledge of host factors that prevent or promote infection can lead to targeted treatment and prevention regimens. This review also identifies unique host factors to consider in the older, female host for improving rUTI treatment and prevention by dissecting the age-associated alteration of the bladder mucosal immune system.

Introduction

On a global scale, the average age of the population is increasing. It is estimated that by 2050, the population over the age of 65 will double, and this group will grow to make up more than 20% of the total population¹. Given this worldwide shift in age demographics, there is an increasing need to provide healthcare and treatments capable of meeting the needs of an aging population. This population is particularly susceptible to infectious diseases, the most common being respiratory infections, urinary tract infections (UTIs), and skin and soft tissue infections². In addition to being more susceptible to contracting these infections, older individuals also suffer greater morbidity and mortality due to increased severity when these infections occur. This is the case in the urinary tract, where aging is associated with significant changes to host physiology and immunity. Women are particularly prone to multiple conditions of the urinary tract as they age, and women over the age of 50 are highly susceptible to bladder disorders including recurrent urinary tract infections (rUTIs), urinary incontinence, and overactive bladder (OAB), among others^1,3–6. These bladder diseases all have a chronic inflammatory component as well as overlapping symptoms, known as lower urinary tract symptoms (LUTS)^7,8.

This review will address the current state of knowledge about age-associated changes to the underlying biology of the bladder that may contribute to several diseases, with a particular focus on UTIs. We also elaborate further on the innate and adaptive immune responses used by the bladder mucosa, how aging of the urothelium influences these immune responses at baseline and during an infection, and potential therapeutic strategies based on these responses.

Diseases and disorders of the bladder in women

Among the many diseases of the bladder, susceptibility may vary between men and women due to differing anatomy and physiology⁹. While men are more likely to develop bladder cancer, for instance, other bladder disorders in men are frequently mediated by the prostate rather than the bladder alone¹⁰. In women, dysfunction and disorders of the bladder are more likely to be bladder-intrinsic but may also be secondary to changes in the vagina. The most common bladder disorder among all women is bacterial infection of the bladder or kidneys, usually referred to as urinary tract infections (UTIs)¹¹. The majority of UTIs in women are limited to the bladder (cystitis) without complicating factors such as anatomic abnormality, systemic symptoms such as fever, or indwelling catheters¹¹. Other common afflictions of the bladder in women include: (1) interstitial cystitis/bladder pain syndrome (IC/BPS), (2) overactive bladder (OAB), (3) stress incontinence, and (4) incomplete bladder emptying. IC/BPS is a little-understood, sterile inflammatory disease characterized by urothelial permeability defects, inflammation, ulceration, scarring and fibrosis of the bladder, hemorrhage upon distension, and reduced bladder capacity^3,5,7,12,13. It is most commonly diagnosed in women in their 40s. OAB is characterized by uninhibited detrusor contractions with or without incontinence. Over 15 % of women report OAB and 11.0 % report OAB with urge urinary incontinence in the United States^14,15. Kuo and colleagues have recently shown that urinary TNF-α levels are significantly higher individuals with voiding dysfunction including OAB¹⁶. We have previously also demonstrated that TNF-α levels in plasma samples can reliably differentiate OAB relative to controls and can be used to distinguish OAB from the other conditions. Stress incontinence refers to when the urinary sphincter and pelvic floor muscles are too weak to prevent urine leakage during high intraabdominal pressure such as coughing, laughing, or sneezing. Women may also experience mixed incontinence condition with symptoms of OAB and stress incontinence^{4,10,14,15,17–20}. Only 3% of women under 35 years of age experience symptoms of incontinence, but nearly 25% of women over age 65 report urinary incontinence^21,22. Women generally suffer a higher rate of incontinence (urge, mixed, stress, and other) than men however, stress urinary incontinence is the most common type in women²³. Furthermore, OAB has been postulated to result from inflamm-aging, with levels of nerve growth factor (NGF) and the chemokines CCL2 and CXCL1 in the urine increasing with age and OAB severity^8,24,25. The common association of bladder disorders with both aging and chronic inflammation suggests that an underlying driver of pathology may be age-associated inflammation^24,26. Treatment for bladder diseases such as OAB may involve invasive procedures or surgeries that further increase the risk of UTIs and other infections in older women.

Urinary Tract Infections

More than 10% of women in the US experience UTIs each year, and 60% of women will be diagnosed with a UTI during their lifetime^27–29 Click or tap here to enter text.. Furthermore, the economic impact of UTIs is over $3 billion, and UTIs account for at least 15% of all antibiotic prescriptions^{11,27,30–32}. UTIs may also ascend to the kidneys, causing pyelonephritis, then the blood stream, causing bacteremia and urosepsis. Recurrence of UTIs is a common concern, as 25–50% of infections will recur within 6 to 12 months after an initial infection, termed recurrent UTIs (rUTIs)^33,34. Repeated treatment of rUTIs with antibiotics may lead to antibiotic resistance, disruption of the microbiota, and adverse effects of the drugs. Older patients are at increased risk of toxicity from certain antibiotics due to interactions with other medications and decreased renal function. Thus, there is an urgent need to identify new targets for UTI treatment and prophylaxis in women with rUTIs.

Risk factors for rUTIs vary among age demographics. The incidence of UTIs over the lifespan forms a J-shaped curve³⁵. Some risk factors can be present at any age, such as uncontrolled diabetes although the prevalence of this co-morbidity also increases with age. Infant girls are at risk for UTIs from urinary tract abnormalities and vesicoureteral reflux³⁶. After puberty, UTI risk increases and is frequently associated with sexual intercourse in adult women⁹. These risks plateau until menopause when UTIs become more frequent³⁷. Postmenopausal and older women are particularly susceptible to rUTIs; in one study in a primary care setting, 53% of women over 55 had at least one UTI recurrence within a year compared to only 36% of those under 55³⁸.

Postmenopausal vaginal atrophy and a change to the vaginal microbiota are related to reduced estrogen and increase the risk of UTIs³⁹. Estrogen also plays a protective role against UTIs and has direct protective effects on urothelial barrier function^40–45. Vaginal estrogen therapy, which has low systemic absorption, is effective in reducing UTIs in postmenopausal women and reduces inflammation in the urinary tract^46–52. Anterior vaginal wall prolapse (cystocoele) and other causes of incomplete bladder emptying promote UTIs since uropathogens can more easily grow in stagnant urine within the bladder. Older women are also more likely to have incontinence, have had urogenital surgery, or be catheterized, all of which are associated with higher rates of UTIs and rUTIs³⁷. Currently, there is a limited understanding of the extent to which age alone, or its associated co-morbidities, may have biologic effects on the bladder that mediate the increased risk of rUTIs.

Pathogenesis of UTIs in the bladder

UTIs are caused by different uropathogenic strains of bacteria. Infections from E. coli are the most common cause of UTIs, as uropathogenic E. coli (UPEC) infections alone account for approximately 80% of all UTIs³⁰. Infections are can also be caused by other gram-negative bacteria such as, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter species, Citrobacter species, and Pseudomonas aeruginosa, as well as gram-positive bacteria including group B Streptococcus (GBS), other Streptococci, Staphylococcus aureus, coagulase-negative Staphylococci, and Enterococcus species, among others^29,53. Though UPEC still accounts for the majority of UTIs in older women, they are more likely to be infected with the less common uropathogens than younger women⁵⁴. The pathogenic cycle of UPEC during an infection is well studied in vivo in a young mouse model and in vitro (Figure 1A). UPEC typically originate in the gut and go on to colonize the urethra, subsequently migrating into the bladder^55–59.

Figure 1: UPEC infection and immune response in young and aged bladders.

(A) Schematic representation of events during UPEC infection cycle in the bladder. First, UPEC attach to uroplakins present on the superficial urothelial cells and invade. Next, a chain of competing responses from UPEC and the host determines the fate of infection. (1) UPEC is expelled into the bladder lumen via RAB27B+ fusiform vesicles. (2) UPEC escape into the cytosol and establish intracellular bacterial communities. (3) UPEC enter in the autophagosome where they subvert the autophagy pathway and form quiescent intracellular reservoirs (QIRs), and seed recurrent UTIs. (4) Innate immune cells infiltrate the site of infection and kill the bacteria. (5) Superficial cells loaded with IBCs are exfoliated into the bladder lumen thereby clearing the large bacterial load.

(B) Representation of epithelial and immune changes in the aged bladder. The aged bladder elaborates high levels of pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α, and reduced levels of Estrogen relative to young bladders. The aged bladder is characterized by formation of tertiary lymphoid tissue (bTLT) with a bone fide germinal center with B cell and T cell zones and follicular dendritic cell (FDC) network. Within the bTLT, naïve B cells are recruited, undergo somatic hypermutation and respond to antigens. B cells may die by apoptosis or differentiate into memory B cells and antibody-secreting plasma cells which produce secretory IgA (sIgA). The aged urothelium harbors significantly higher numbers of QIRs and lysosomal accumulation and is increasingly permeable.

In women, UPEC can also colonize the vaginal epithelium, which may lead to an ascending infection^60,61. When UPEC ultimately reach the bladder epithelium, the bacteria bind to the surface of the urinary tract using type 1 pili and pyelonephritis-associated (P) pili⁶². Located at the tip of UPEC type 1 pili are FimH adhesins, which bind uroplakins to initiate vesicle-dependent endocytosis⁶², allowing the internalization of UPEC into superficial urothelial cells, the outermost epithelial cell layer that forms the bladder’s watertight barrier. Bacteria enter into fusiform vesicles, where they release a phospholipase that destroys membrane integrity and permits bacterial escape into the cytoplasm^63,64. After internalization, UPEC multiply within the protected intracellular niche forming biofilm-like intracellular bacterial communities (IBCs)⁶⁵. IBCs may fill nearly an entire superficial cell, and a single IBC can consist of approximately 10²-10³ bacterial cells⁶⁶. UPEC may then break out of the superficial cells, causing cell death, and go on to infect other urothelial cells where they can form quiescent intracellular reservoirs (QIRs) within autophagosomes, and can remerge to cause continued rounds of infection^63,67–69. QIRs are highly resistant to antibiotics and appear to be “hidden” from the immune system within this intracellular niche. Spontaneous reactivation of QIRs is not fully understood, but there is evidence that damage to the urothelium can trigger the release of UPEC back into the urine where the bacteria can replicate and re-infect. The urothelium can be damaged by other, transient bacteria, such as Gardnerella vaginalis, or be exfoliated by physical or chemical means to stimulate reactivation of latent infection^67,70,71. Thus, UPEC have evolved mechanisms to live long-term in the bladder and continue to cause infections without re-inoculation from the intestinal reservoir. This finding has major implications for the treatment of UPEC infections in humans. Complete treatment of UTIs may thus be dependent on how fully we understand how to inhibit the formation of these reservoirs or if we can induce their reemergence to allow the host immune response to kill the extracellular bacteria.

Innate bladder defenses

The urinary bladder has physical barriers that prevent infection in addition to several other innate mechanisms⁷² similar to those in other mucosal surfaces. Urine flow can flush many, but not all, bacteria out of the lower urinary tract. Thus, in response to UPEC infection, the host activates several pathways to offset tissue damage and expel the bacteria. This includes a surge in the secretion of pro-inflammatory cytokines such as interleukin-6 (IL-6), exfoliation of infected superficial epithelial cells, and recruitment of monocytes and neutrophils. Exfoliation of superficial cells into the urine aids in the overall reduction of the bacterial burden in the bladder by removing cells with bacteria adhered their surface or containing IBCs and other intracellular bacteria. The urothelium also has cell-autonomous defense mechanisms to limit bacterial colonization of the bladder and prevent the establishment of UTIs. One early defense strategy is the expulsion of bacteria that have invaded the cell before they can form large IBCs^69,73–75. A redox stress response is activated as part of the host response to UPEC infection and has been shown to directly drive UPEC expulsion, a major urothelial defense against UPEC⁶⁹. Joshi et al. 2021 recently demonstrated that the anti-redox pathway driven by NRF2, a transcription factor is rapidly activated to quench reactive oxygen species and thereby oxidative stress in part by directly activating the Rab GTPase, RAB27b which has been shown to play a central role in modulating UPEC entry into the urothelial cells as well as exocytosis of UPEC into the bladder lumen^{63,69,74,76,77} (Figure 1A). How this response changes with age is being explored in new work from our group and our observations suggest that the activation of the anti-redox pathway is compromised in aged bladders⁷⁸.

As an innate defense, the urinary tract also secretes antimicrobial peptides such as Tamm–Horsfall protein (also called uromodulin), which is produced in the kidneys and is the most abundant protein in the urine⁷⁹. Tamm–Horsfall protein competitively inhibits UPEC adhesion to uroplakins⁸⁰. Mice lacking Tamm–Horsfall protein show 10 to 100-fold higher bacterial counts of UPEC positive for type 1 pili or P. mirabilis in their urine^81–83.

Like the gut, a healthy urinary tract also produces secretory IgA (sIgA)^84,85. sIgA is produced by plasma cells in the lamina propria and actively secreted by epithelial cells at the mucosal surface⁸⁵. The primary function of sIgA is to limit microbial access to the epithelium and control the commensal microbiota on the mucosal surface^86,87. sIgA carries out this function via promoting agglutination-based clearance of invading pathogens⁸⁸. Interestingly, aged bladders were shown to support increased local sIgA production in urine from aged mice, and the increased levels of sIgA⁸⁹ were similar to elevated serum and intestinal IgA levels during aging^90,91. Although IgA levels appear to be increased during UTI, it is uncertain whether the increase is antigen specific, protective, or dysregulated during a UTI⁹². Studies have suggested that sIgA has a very limited role in the defense against UTI^84,93. However, new work is revealing that treatment with methanamine hippurate prevents breakthrough infections and may do so through increasing levels of urinary IgA⁹⁴. Thus, the role of sIgA in bladder mucosal immunity requires further investigation⁹⁵.

The aged urothelium has also been demonstrated to be increasingly permeable⁹⁶. Given that flexibility and impermeability are two essential features of the urothelium, alteration in these could severely impact cellular homeostasis.

Autophagy and Aging in the bladder

Urothelial superficial cells utilize a network of autophagic vesicles directed by Rab GTPase proteins for membrane recycling of uroplakins^97–100. Autophagy is a conserved cellular process that recycles macromolecules and organelles in response to starvation or stress¹⁰¹. This complex process involving many components may also be used to capture and degrade intracellular pathogens, termed xenophagy¹⁰². While autophagy may be a useful early defense against invading UPEC, these bacteria also utilize autophagosomes to establish QIRs that can cause recurrent infections^103,104. Mice that are hypomorphic for the essential autophagy gene Atg16L1 have faster UPEC clearance and form fewer QIRs than wildtype mice, indicating that UPEC normally benefit from the hosts’ initial autophagy response and sequestration in autophagosome¹⁰⁵. The early clearance of UPEC in these mice was found to be due to heightened inflammation via increased production of IL-1β by macrophages, while the reduced QIR formation was intrinsic to loss of Atg16L1 in the urothelium^104,106. While several pathogens have evolved to evade autophagy machinery, escape its compartments, or prevent lysosomal degradation, UPEC appear to use autophagosomes for long-term persistence within the bladder. UPEC are attracted to autophagosomes within urothelial cells to scavenge iron from ferritin¹⁰⁷, which requires autophagic degradation (ferritinophagy) to release free iron for use by the cell from its ferritin cage where it is stored.

However, autophagy becomes dysregulated during aging with heightened oxidative stress accumulation¹⁰⁸, affecting cellular processes like cell division, cytoskeleton architecture, membrane turnover, and vesicle trafficking. Impaired autophagy in the epithelium creates an energetic burden that promotes stem cell exhaustion and quiescence, a hallmark of premature aging¹⁰⁹ observed in muscle satellite cells¹¹⁰, hematopoietic stem cells¹¹¹, and neuronal stem cells¹⁰⁹. Given that the urothelial innate immune response heavily depends on vesicle recycling, autophagy, and lysosome activity, the change in any of these cellular processes severely impacts the urothelial ability to respond to infection. The impact of the lysosome, autophagy, and metabolism on redox stress has been recently explored by our group and others in the aged urothelium^78,112. Our studies demonstrate a significant block to autophagic flux and lysosomal degradation with low acid phosphatase activity indicative of inefficient cellular debris/waste removal. These findings align with prior studies showing accumulation of lipofuscin in aged urothelial cells¹¹³ and expanded endolysosomal compartments with accumulation of undigested material in the urothelium of aged rats⁹⁶. Concomitant decrease in cellular respiration and mitochondrial bioenergetics in aged urothelial cell cultures have recently reported¹¹². Further, a strong association with age and increase in oxidative stress^63,112 has been reported supporting that aged urothelial cells exhibit dysfunctional lysosomes and accumulate metabolic waste products and cellular debris⁹⁶. Decreased lysosomal function could lead to dysregulated autophagy and change the redox status of the urothelium. Accordingly, we note an increase in LC3I/II ratio with age and concomitant block in autophagy flux suggesting that there is age associated accumulation of autophagosomes⁶³. Since UPEC utilize autophagosomes for QIR formation, the aged urothelium may be more conducive to harboring latent UPEC within QIRs. Indeed, there increased formation of intracellular bacterial communities (IBCs) and QIRs in aged mouse bladders resulting in increased incidence of recurrent UTIs⁶³.

Overall, altered vesicular homeostasis, lysosomal function, and permeability barrier could affect the energetics of the tissue to influence its regeneration capacity and immune response to an infection. Given that these cellular functions are changed in aging tissue, a hampered urothelial response to invading pathogens is to be expected. Therefore, recurrence and persistence of urinary tract infections in older women are likely linked with aging bladder physiology. This opens several interesting questions for further investigation.

Immune responses in the bladder and rUTIs

Older individuals are more susceptible to infection and suffer disproportionate morbidity and mortality from infections than younger patients. The underlying mechanisms for this increased susceptibility and severity are not well understood but may involve functional changes to the immune system with age, known as immunosenescence. This state is characterized by several changes: (1) increased myeloid output and decreased lymphoid output from the bone marrow; (2) declining naïve repertoire of adaptive immune cells (decreased diversity of T and B cell receptors) with the oligoclonal expansion of antigen-experienced cells; (3) an increased rate of autoimmune disease; (4) poor control of latent infections and increased susceptibility to new infections, and (5) chronic low-grade inflammation of unknown origin, termed inflamm-aging^114,115. While UTIs are one of the most common infections in both older men and women, it is not well understood how immunosenescence affects the bladder.

The bladder’s susceptibility to rUTIs has long suggested that eliciting effective adaptive immune responses may not be common. Several studies have demonstrated that repeat infection with the same UPEC strain in mice results in lower bladder bacterial titers during the challenge infection^116,117. However, sterilizing immunity has not been observed, and latent UPEC reservoirs (QIRs) continue to form upon challenge infections. Furthermore, mice that have previously had severe, chronic UTIs are more susceptible to future UTIs than those that resolve their initial infection¹¹⁸, suggesting that divergent immune responses may dictate future responses to reinfection. T cell responses contribute to antigen-specific immunity in UTIs, but research has also found that bladder macrophages may be responsible for inhibiting these responses^116,117. Interestingly, the abundant γδT cells in the bladder rapidly produce IL-17 upon infection, which aids in bacterial clearance¹¹⁹. It is not known if these cells are responding in an antigen-specific manner, as this response was demonstrated in naive mice. Recent work has shown that Type 17 immunity plays an important role in bladder defense implicating ILC3s in priming rapid responses to UPEC challenge¹²⁰. This study showed presence of type 17 immune cell transcriptional factor, Rorc+ cells in the bladder. Whether the ILC3 response in increased or decreased with age is not known.

Of note, adaptive immune responses are markedly different if the infection is limited to the bladder or if it ascends to the kidneys. Kidney infection induces a robust, systemic adaptive immune response, including pathogen-specific IgG¹²¹. In humans, pyelonephritis is also marked by higher urine IgA concentrations. While antibody responses to UPEC may be antigen-specific, whether they are protective has not been demonstrated. Nevertheless, anti-UPEC vaccines are currently being investigated and several are already in use outside the U.S.^70,122–124. It is important to note that these vaccines are used as immunotherapy in patients with chronic UTIs rather than acting to prevent initial infection of the urinary tract.

Age-associated Tertiary lymphoid tissue formation in the bladder

The immune system is made up of lymphoid cells that inhabit nearly all tissues in the body and are frequently migratory between body tissues, lymphatics, and blood. The primary lymphoid organs produce new immune cells and include the bone marrow (where all hematopoietic cells are generated and B cells mature), and the thymus (which regulates T cell maturation). Secondary lymphoid organs (SLOs) include the spleen, lymph nodes, Peyer’s patches, and other, organized mucosa-associated lymphoid tissues. SLOs harbor large numbers of densely packed immune cells and function to facilitate adaptive immune responses. These organs are highly structured to facilitate a multitude of intercellular interactions between T cells, B cell, and antigen presenting cells that must take place in order to generate adaptive immune responses to pathogens¹²⁵. Tertiary lymphoid tissues (TLTs) are capable of generating adaptive immune responses, and these responses typically act at their tissue-specific location. TLTs form ectopically at sites of chronic inflammation and antigenic stimulation rather than at pre-defined locations during embryogenesis^126–129. TLTs have been found in numerous tissues such as the lung^130–133, kidneys^134–136, intestines^137–140, and meninges¹⁴¹, and in varied disease states, including chronic infection, cancer, and autoimmune diseases. Interestingly, TLTs appear to be more common in the liver, intestines, and kidneys of aged mice^{135,142–144}, indicating that an aspect of the aging process may stimulate the development of TLTs. Whether immune responses arising within TLTs are pathogenic (such as in autoimmune disease) or protective (as in some cancers and in some infections) depends on both the tissue location and type of insult. Why TLTs form during aging and what type of responses these TLTs mediate is not fully understood.

TLTs and TLT-like structures have been reported in the bladder, but little detail in the description of their structure, function, causes, and consequences had been explored. Mice given repeat UTIs have larger, more distinct T cell influx at 24 hpi^116,117. A subset of C3H/HeN mice with persistent bacteriuria, chronic cystitis, and pyelonephritis also have CD45⁺ aggregates, though specific cell types in these structures have not yet been defined^145,146. Transgenic mice with constitutive Cox-2 expression in the urothelium develop bladder cancer and lymphoid aggregates containing B and T cells¹⁴⁷. Furthermore, small lymphoid aggregates were observed in one small study of aged mice by routine and electron microscopy¹⁴⁸. TLTs have also been identified in a small sample of muscle-invasive bladder cancer¹⁴⁹. Despite these occurrences, TLTs in the bladder had been largely uncharacterized and their function entirely unexplored. A recent report from our group showed that in unperturbed, aged bladders, expanded numbers of B and T cells organize into structures termed bladder tertiary lymphoid tissue (bTLT)¹⁵⁰ (Figure 1B). These bTLT form in a sex specific manner in aged female but not aged male bladders¹⁵¹. bTLT in aged mice serve as centers for B cell recruitment, activation, and differentiation into plasma cells and express multiple B cell-associated genes (Cxcr5, Cxcl13, Cd19, fclr5)^150,151. B cell-associated pathways are enriched likely due to the redistribution of pools of B cells from the periphery to mucosal surface thus altering the mucosal landscape¹⁵². Age-dependent bTLT form in aged germ-free mice as well suggesting that microbial antigens are not required for their formation.

However aged TNFα^−/− mice have fewer and smaller bTLTs, indicating that bTLTs require age-dependent TNFα to form. TNFα is required for the maintenance or growth of bTLTs once they had formed. The effect of anti-TNFα treatment on the composition, organization, or functionality of bTLTs remains to be determined. In addition, testing whether anti-TNFα treatment at an earlier age, such as 9 or 12 months when bTLTs begin to form, could prevent the growth of bTLTs and formation of new ones over a more extended period of time. These future studies would help further elucidate the mechanisms by which age-associated TNF promotes bTLT formation in female mice. TLT development is known to be induced by LTi cells, an ILC subtype that express surface, lymphotoxin, LTα₁β₂¹⁵³ and LT signaling is potentiated by TNFα. Indeed, aged bladders also express increased lymphotoxin than bladders from young mice¹⁵⁰. Increased LT signaling through its receptor, LTβR has been described in chronic cystitis¹⁵⁴. Recent studies suggest that ILC3s are an important source of LT¹²⁰ and it would be interesting to determine whether the numbers of ILC3 are increased in aged bladders. Taken together, emerging data suggests a profound change in the bladder mucosal immune system that is fundamental to aging in this tissue.

Given our findings that aged female bladders harbor increased bacterial reservoirs and are susceptible to rUTIs concomitant with formation of bTLT would suggest that presence of these structures are ‘pathogenic’ to the aged female. Indeed, human cohort studies would support this hypothesis as bTLT occur in postmenopausal women and are significantly associated increased frequency of rUTIs and shorter time interval to next recurrent episode¹⁵⁰. bTLT in postmenopausal bladders have been shown to be associated with co-localization of E. coli species¹⁵⁵. Presence of TLTs may potentiate overexuberant or ineffective immune responses that promote inflammation rather than resolution of UTIs. Identification of these structures provides a possible explanation for the increased occurrence of sex- and age- dependent conditions in aging females and establish a foundation for therapeutic interventions to limit bTLT formation.

Hormones, Aging, and Immunity

Recurrent UTIs afflict a large proportion of postmenopausal women, suggesting that lower estrogen levels predispose older women to a significant risk of chronic and recurrent UTIs. Estrogen receptors can be found throughout the lower urogenital tract, including the vagina, bladder, urethra, and pelvic floor musculature^156,157, indicating that estrogen plays a broader role in urogenital physiology. Although the change is more dramatic in humans, mice do experience a reduction in estrogen levels during a “menopause” period around the time they lose their reproductive fitness. Interestingly, this “reproductive senescence” occurs around 9 to 12 months in mice, which coincides with the early appearance of bTLT¹⁵⁰. Since the urothelium is estrogen-responsive, processes that occur during reproductive senescence and atrophy of the ovaries and uterus may trigger changes that lead to bTLT formation. TLT structures have been described in bladders of prepubertal females. Miosevic and colleagues have shown that prepubertal females may harbor lymphoid lesions similar to those noted in postmenopausal women and these are correlated with number of UTIs in the prior year and bladder wall thickness^158,159. Together, these studies suggest that hormonal ranges may have significant implications for TLT formation and pathogenesis.

Building on this observation, in vivo data demonstrates that estrogen protects against UTIs and helps to repair urothelial permeability^40,44,160. Hass and colleagues have also shown that estrogen deficiency has a negative impact on urothelial barrier function⁴⁴. Furthermore, an ovariectomized mouse model that mimics the postmenopausal condition with or without exogenous estrogen supplementation demonstrated that mice with lower estrogen levels harbored significantly higher QIRs, implying a high risk of recurrent UTIs^40,41. The studies by Lüthje et al. and Wang et al. showed that estrogen imparts protection against UTIs by modulating mucosal bladder defenses^40,41, supporting the use of exogenous estrogen as prophylactic therapy for rUTIs^51,161–164.

Targeted use of VET has been shown to effectively reduce rUTIs and shows overall improvement of lower urinary tract symptoms (LUTS)^{46,47,165–168}. The effect of the hormone on local microbiota could be one of the factors influencing the response of the bladder. Similar to the age-associated changes that occur in the vaginal microbiome, an age-associated change in the urinary microbiome has also been observed. Both pre- and postmenopausal women show dominance of Lactobacillus in both the vagina and bladder, but Lactobacillus has a relative abundance of 77.8% in premenopausal women compared to 42.0% in postmenopausal women¹⁶⁹. VET increases the dominance of Lactobacillus in both the vagina and bladder of postmenopausal women^47,170. Microbiome analysis of urines from 34 premenopausal and 20 postmenopausal women revealed higher alpha diversity with higher abundance of the genera Gardnerella and Prevotella compared with premenopausal women who mostly presented with Lactobacillus dominated urotypes¹⁶⁹. Jung et al., recently showed that VET leads to increase in Lactobacilli species in the urobiome of postmenopausal women with rUTI⁵¹. Neugent and colleagues very recently showed strong correlations between presence of Bifidobacteria and Lactobacilli and urinary estrogen in women without a UTI history. Collectively, they suggest that rUTI and estrogen levels shape the urobiome¹⁷¹.

Additionally, estrogen modulates the thickness of the urothelial glycosaminoglycan (GAG) layer and regulates the expression of GAG-sulfation enzyme HS6ST1 over the course of UPEC infection⁴². Our work has previously shown that HS6ST1 also modulates activation of Bmp4 signaling post-infection¹⁷². These data imply that estrogen could influence BMP4-regulated regeneration and the thickness of the urothelial GAG layer post-infection. In this manner, estrogen appears to modulate cellular physiology and influence the local microbiota of the vagina and bladder.

Other studies have determined the effects of sex and hormones on UTIs in mice, finding that male mice are less likely to clear experimental UTI than female mice and that these effects are at least partially mediated by testosterone^173,174. Testosterone was recently found to impact the immune response to UTI¹⁷⁵; this finding suggests that both sex and hormones may indeed have a substantial impact on the aging immune system within the bladder. However, this hypothesis remains to be formally tested. Regarding B cells, age-associated B cells accumulate earlier and in greater quantities in female mice compared to male mice^176,177. Furthermore, these cells are found in models of autoimmune disease, which again is more common in females than males. These findings agree with the model demonstrating higher levels of inflammation response in females than males⁹, particularly in relation to aging. The interaction between age, sex, hormone levels, and reproductive senescence on the bladder is clearly complex and needs more work to be better understood.

Therapeutic Interventions

In most cases, a UTI is spontaneously resolved within days without medical intervention. However, an unresolved infection can lead to pyelonephritis and can even be fatal^178,179. The recommended antibiotic therapy for uncomplicated UTIs includes the use of fosfomycin, nitrofurantoin, amoxicillin-clavulanate, and cephalosporins¹⁸⁰. Bacterial resistance to antibiotics such as these is a growing issue of concern for the treatment of UTI. There is also no effective vaccine available that can prevent UTIs. Thus, several alternative therapeutic strategies are being pursued as treatments for UTIs and have been reviewed extensively elsewhere⁶⁹.

In addition, the U.S. FDA-approved anti-inflammatory drug dimethyl fumarate (DMF) has showed some promise, as mice treated with DMF showed decreased UPEC count in the bladder and urine⁶⁹. Another strategy is to block UPEC adhesion to superficial cells using the simple sugar D-mannose, which has reportedly lowered the risk of rUTIs when administered daily in a clinical trial^181,182. Moreover, in a retrospective cohort study of patients with rUTIs and who harboured bTLT lesions were treated with D-mannose resulting in significant decrease in UTI incidence¹⁸². Recent studies from our group suggest that D-mannose not only plays an extrinsic role but also plays important urothelial cell-intrinsic roles: we show that D-mannose treatment of aged mice promotes autophagic flux and lysosomal degradation thus improving outcomes⁷⁸.

Methenamine salts are FDA-approved in the U.S. for long-term prophylactic use in treating patients with rUTIs and have demonstrated effectiveness as antiseptics in preventing UTI in adults 60 years of age and older, regardless of kidney function status¹⁸³. Methenamine was well tolerated and helpful in reducing UTIs, antibiotic prescriptions, and hospitalization in renal transplant recipients with rUTIs¹⁸⁴ and appears to be effective as a means of improving barrier function of the urothelium⁹⁴. Importantly, there are no known resistance mechanisms to methenamine. Therapeutic approaches such as these should be considered for more widespread use and as alternatives to conventional antibiotics in the treatment of adults with UTIs, especially rUTIs.

Interventions could improve outcomes by either reducing UTIs through antibiotic and drug treatments but also via reducing TLT lesions. Indeed, Zimmern and colleagues have shown that electrofulguration by cauterization of CC/TLT led to much improved outcomes in women with antibiotic-refractory rUTIs¹⁸⁵. Milosevic and colleagues have also shown that regression of the TLT lesions improved rUTIs¹⁵⁹. Finally, recent work from our group has revealed that vaginal estrogen therapy (VET) in aged female mice promotes regression of bTLT in part by reducing levels of TNFα and CXCL13 and this is associated with reduced rUTIs⁴⁵.

Concluding remarks

Recurring UTIs will continue to be a burden on our aging population. Considering the increasing prevalence of antibiotic resistance among uropathogens, developing alternative or non-antibiotic approaches to treat and prevent UTIs is essential. Comprehensive knowledge of host factors that prevent or promote infection continues to be essential to develop new targeted treatment and prevention regimens. Since it is evident that aging affects the bladder at multiple levels, a multilayered response and treatment strategy should be pursued to effectively address rUTIs that disproportionately affect the older population. Future work to further dissect the impact of age on UTIs should also include examination of bTLT when addressing the chronic and rUTIs in older individuals. We have highlighted a new framework for studying the changing immune landscape of the bladder during old age that can be translated to clinical outcomes and potential therapies for women with rUTIs. This new concept of the interplay between aging, infection, and local immune responses within the bladder generates many new important questions for the field.

Table 1:

Cytokine/chemokines that are associated with various bladder disease conditions

Cytokines/Chemokines	Model (cell culture, mouse, rat, human/clinical, etc)	Tissue/Cell type (whole bladder, urine, urothelium, macrophages, bTLT etc)	Disease condition (UTI, OAB, cystitis, etc)	Reference(s)
mRNA levels: ↑IL-1β, IL-2, IL-4, IL-6, TNF-α/β; no change in IL-1α, IL-10 and IFN-γ	Wistar rats	Whole bladder	CYP-induced cystitis	186
Protein level: ↑IL-1β, IL-10, IL-6; No change IL-4, TNF-α	Wistar rats	Whole bladder	CYP-induced cystitis	186
↑IL-1β, CXCL1 (KC) and CCL2 (MCP-1)	C57BL/6 & CB6F1	Urine	UTI (Proteus mirabilis HI4320) (Escherichia coli CFT073)	187
↑IL-6; IL-8	Human (1–121mos.)	Urine and serum	acute UTI; Pyelonephritis (febrile UTI)	188,189
↑CXCL10, iNOS, CCL5, CXCL5, CXCL9, IL-1α, IL-1β, IL-6	C57BL/6	whole bladder & Urine	UTI (CFT073, 83972 & GBS)	190
↑IL-1β, IL-ra, IL-8, GM-CSF	Cell culture	Uroepithelial cells (T24 & 5637)	GBS cystitis	190
↑IL-8	Human	Urine	UTI	191
↑NGF, MCP-1, sCD40L, MIP-1β, IL-12p70/p40, IL-5, EGF, GRO-α, sIL-2Rα, IL-10	Human	Urine	OAB	24,25,192
↑CXCL1, CXCL8, CXCL10			UTI
↑IL-1β, IL-1α, TNF-α, IL-6	Human	Urine	Bacterial cystitis or microhematuria	193
↑MCP-1, TARC, PARC, Fas/TNFRSF6; ↓IL-5, IL-6, IL-7, GM-CSF	Human	Urine	OAB	194
↑MCP-2, MCP-3, TNF-β, G-CSF, eotaxin-3	Human	Urine	OAB and UTI	194
↑BDNF, NGF, MCP-1, GAG	Human	Urine	OAB	195
↑TNF-α, MIP-1β; ↓IL-4	Human	Plasma	OAB	8
↑CXCL1, CXCL8, IL-6	Human	Urine	Acute cystitis	196
↑IL-1β, IL-1ra, IL-6, IL-8, IL-10, TNF-α, GCSF, MCP-1, MIP-1α, MIP-1β, RANTES, VEGF, IL-4, IL-7, IL-9, IL-12 p70, IL-17A, eotaxin, GM-CSF, IP-10, FGF, IFN-γ, PDGF-BB	Human	Urine	Acute UTI	197
↑VEGF, IL-1α, IL-6	Human	Urine	HIC and NHIC	198
↑IL-6	Human	Urine	OAB	199
↑IL-6, IL-8	Human	Urine	rUTI (UPEC)	200
↑IL-6, IL-8, BDNF; ↓IL-2, NGF, VEGF	Human	Urine	rUTI with PRP injections	201
↑TNF-α	Mouse	Bladder	Age	89
↑CXCL13	Mouse	Bladder	Age	89
↓IL-8	Human	Bladder	Interstitial cystitis/BPS	202
↑IL-8	Human	Urine	Interstitial cystitis/BPS	203
↑MCP-1, RANTES, CXCL10/IP-10, Eotaxin-1	Human	Urine	Interstitial cystitis/BPS	204
↑IL-6, IL-8	Human	Urine	UTI	205

Highlights.

• Aging is associated with increased susceptibility to rUTIs

• Aged mice develop bladder tertiary lymphoid tissues (bTLT)

• bTLT are associated with significant increase in UTI frequency in mice and women.

• Urothelial permeability and urobiome species change with age and menopausal status

• Multimodal therapies can lead to bTLT regression and improved rUTI outcomes