Biological aging-related sex-disparity in bladder-associated pathologies such as bladder cancer as well as healthy bladder mucosal tissue has recently gained significant attention. Despite discrepancies in the literature and a consensus on age and sex-associated hormonal, genetic and immunological deviations, the current pre-clinical models of bladder cancer have yet to fully integrate such factors in experimental design for development of immunotherapeutic agents.

It is known that females usually mount stronger adaptive immune responses to infectious and vaccine associated immune challenges, producing higher antibody titers and more robust innate immune responses. With the similarities in mucosal immune physiology between humans and mice, and the recently reported age-associated, antigenic stimuli-independent inflammation in female murine bladders [16], we conducted a comprehensive evaluation of the sex and age-associated bladder transcriptomic and spatial immune profiling in female and male mice. Not surprisingly, an unsupervised feature selection applied to detect differences in transcriptomic profiles of bladders from all age groups led to distinct clustering of groups by age and sex. While this confirmed the unique bladder transcriptome profiles of the groups under study, many of the top upregulated genes revealed shifts in immune-associated genes with increasing age, suggesting that fundamental changes in the aged murine bladder tissue microenvironment were immune-related. These findings are in concordance with the previous report by Ligon et al. [16], where such changes were reported only in female mice.

Significant enrichment in pathways associated with B cell function was observed in the bladder transcriptome profiles, starting at 9 months of age in both sexes. Evaluation of the top 25% significantly differentially expressed genes in the oldest group revealed increased expression of a substantial number of transcripts associated with a B cell phenotype such as Cd19, Cd79a, Pax5, Mzb1, Ms4a1 and others. The age-associated increased B cell density was independently revealed by multiplex spatial immune profiling of the healthy bladder, prominently in aged female mice, that also exhibited an increased prevalence of TLSs. Following birth, such mucosa-associated lymphoid tissues (MALTs), consisting of T cells, B cells, macrophages and follicular dendritic cells, develop across various mucosal sites (e.g., respiratory tract, gut, urogenital tract). The neogenesis of TLSs occurs as a result of exposure to commensal or pathogenic microbes, IFN-1 activating vaccines that are administered via mucosal routes, or chronic local inflammation. Such induced TLSs generally provide heterologous humoral immune protection at mucosal sites [26]. As widely reported, TLSs in the bladder also form during the course of normal aging due to increased systemic levels of TNF-α [16], chronic inflammatory conditions of the urogenital tract such as those observed in interstitial cystitis-associated Hunner lesions and bladder cancer [27,28,29,30]. As per the recent TLS definitions distinguishing lymphoid aggregate, primary and secondary follicles [31], we did not observe a follicular dendritic cell network within TLSs of 18 months mouse bladders. Therefore, age-related bladder mucosal TLS formation is potentially induced by physiological increase in TNF-α with advancing age.

As observed through analysis using the Immgen MyGeneset application, the increased levels of transcripts associated with various B cell subsets are suggestive of multiple populations dispersed within the healthy bladder mucosa that may or may not be intrinsic to TLSs. Interestingly, the immunoglobulin light and heavy chain variable region genes constituted the majority of the top 50 genes distinguishing the profiles of aged female mice from the younger groups. Although such differences in these B cell-associated transcripts showed an increasing pattern of abundance with advancing age in both sexes, these observations were found to be more pronounced in bladders from 18-month-old female mice compared to all other groups.

In both humans and mice, B cells are known to exhibit age-related sex differences [12, 32]. With advancing immunologic and biologic aging, peripheral pools of B cells show significant decline with a simultaneous increase in mucosal surfaces. In the current study, the Cxcr5 gene was also significantly overexpressed in the bladders of old females with concurrent higher expression of the gene Cxcl13 that encodes the B cell-recruiting chemokine CXCL13. Higher transcripts levels of J chain also indicate a higher amount of secreted IgM and IgA antibodies in the bladder mucosa. Indeed, identification of the functional properties of these cells in the context of carcinogenesis and locally administered BCG immunotherapy in NMIBC is needed to augment the identification of newer therapeutic targets.

Another interesting and novel observation in our study was the increased expression of Pd-l1 immune checkpoint gene in bladder transcriptome profiles of aged healthy female mice, which was further also observed at the protein level within the TLSs, reflective of exhausted immune cell states. Increased expression of other immune checkpoint genes such as Lag3 and Btla, was also observed in the bladders from healthy older females compared to their male counterparts. A bulk sequencing approach does not allow the identification of specific cell types that exhibit such exhausted behavior and thus characterization of cell type specific lack of function via approaches such as single cell sequencing would allow for a comprehensive understanding of such alterations and development of precise therapeutic targeting approaches.

In a recent report, Degoricija et al., showed the higher expression of immune checkpoint molecules in tumors from young male mice at 20 weeks following exposure to BBN carcinogen [33]. Although this is one of the few reports that have characterized some local inflammatory changes accompanying carcinogens, this study was conducted in young male mice. Based on our observation of increased PD-L1 protein expression in the urothelial and endothelial cells, it can be speculated that this may result from BBN induced DNA damage and activation of cellular IFN pathways. Such increased PD-L1 expression on endothelial cells is known to cause inhibition of T cell activation [34]. Future mechanistic studies targeting this checkpoint are thus warranted to define its dynamic role in disease progression and potential immune exclusion.

Findings from our study showing higher density of plasma cells in the lamina propria of old mice, irrespective of sex, aligns with the increased recruitment of B cells to the bladder mucosa with advancing age that we observed in our findings from healthy bladder molecular profiling. It is plausible that chronic BBN exposure further amplifies the recruitment and differentiation of B cells to plasma cells. This is supported by the observation that increase in immune infiltration and lymphoid aggregate formation were observed as early as 4 weeks post-BBN initiation in the majority of mice in all age groups.

The biological and immunological aging-related similarities in humans and mice, emphasize the inclusion of aged mice in pre-clinical studies in bladder cancer. This is more important especially in studies investigating locally delivered BCG immunotherapy in NMIBC, to establish the role of resident and treatment induced or recruited immune cells that may exhibit age and sex dependent function in treatment response. Indeed, tumor associated B cells and bladder tumor-associated TLSs are gaining increased attention because of their association with response to BCG (unpublished findings) in NMIBC and positive outcomes following immune checkpoint blockade in MIBC [29, 35]. Future in-depth investigations into the interactions between these pre-existing TLSs and locally delivered immunotherapy are necessary. Finally, of the other immune cell types such as cytotoxic T cells and myeloid cells, we observed a high density of both F4/80+ and CD163+ M2-like macrophages in the bladder immune microenvironment of BBN treated mice.

The majority of studies in bladder cancer are conducted in 6- to 8-week-old mice. The influence of hormones on sex differences in time to tumor induction in the BBN model was established in a seminal study over 4 decades ago [36]. However, the impact of immunologic aging that accompanies hormonal alterations remains to be fully understood. Moreover, mice do not undergo menopause and significant decline in estrogen levels as seen in humans [37], and exhibit the phenomenon of reproductive senescence associated decline in ovarian function. It is thus challenging to model the cross-talks between hormones and immune alterations precisely. Moreover, the role of androgen and androgen receptors in mediating BBN carcinogen associated sex differences in mice is well established [38, 39].

Our study is indeed, not without limitations. Age associated increased B cell infiltrated TLSs within the bladder mucosa were first demonstrated by Ligon et al. [16], in female wild type and TNF-α knockout mice housed under specific pathogen free and germ-free conditions. It will be interesting and important to expand these findings to TNF-α deficient male mice given the significantly high levels in elderly males [40]. Future investigations could also utilize new technologies such as single cell sequencing in models that permit distinguishing hormonal and chromosomal influences. Indeed, the four-core genotype model [41, 42] permits exploration of such questions. Nonetheless, our results lay the foundation for such future studies to understand the biological mechanisms underlying sex differences in the aging urinary bladder to develop improved therapeutics that benefit both females and males.

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