Interstitial cystitis/bladder pain syndrome (IC/BPS), particularly Hunner-type IC (HIC), is characterized by epithelial barrier disruption and chronic inflammation. Our previous studies delineated the immune landscape of IC/BPS bladder tissue, revealing prominent immune cell infiltration within the urothelial compartment (Reference 1). Pseudotime trajectory analysis further demonstrated a programmed epithelial injury process in HIC, with pronounced damage to superficial umbrella cells (Reference 2). However, the upstream drivers initiating and exacerbating this epithelial injury remain unclear.
          We hypothesized that gut microbiota–mediated metabolic alterations contribute to urothelial damage through activation of innate immune signaling pathways. This multi-omics approach overcomes key limitations of previous studies, including the low resolution of 16S rRNA sequencing, the lack of metabolite specificity, and the uncertainty regarding the injury-related cell types. The study aimed to identify and functionally validate microbiota–metabolite–host signaling interactions in HIC.

A multi-omics framework was applied to cohorts of HIC patients and healthy controls, including fecal 16S rRNA sequencing and shotgun metagenomics to profile gut microbiota, blood and urine untargeted metabolomics and targeted bile acid profiling to assess systemic metabolic changes, and bulk and single-cell RNA sequencing of bladder tissue to characterize transcriptional alterations. Cross-omics integration analyses were performed to identify key microbiota–metabolite–host signaling interactions.
         Functional validation was carried out using fecal microbiota transplantation (FMT) and Enterococcus avium colonization in antibiotic-pretreated mice, as well as intravesical instillation of taurochenodeoxycholic acid (TCDCA) in rats. In vitro experiments using human urothelial cells (HUCs) evaluated barrier integrity, inflammatory responses, and TLR3 signaling, with pharmacological inhibition and pentosan polysulfate sodium (PPS) treatment for mechanistic assessment.

Gut microbial dysbiosis was observed in HIC patients, with metagenomic analysis identifying three significantly enriched bacterial species, including Enterococcus avium, Megasphaera micronuciformis, and Fusobacterium nucleatum, which demonstrated robust diagnostic performance in distinguishing HIC from controls (AUC: 0.881).
          Metabolomic profiling revealed significant alterations in metabolic pathways, with bile acid metabolism emerging as one of the most prominently dysregulated pathways. Targeted bile acid analysis further confirmed that taurochenodeoxycholic acid (TCDCA) was significantly elevated in both serum and urine of HIC patients.
         Transcriptomic analysis showed activation of innate immune and inflammatory pathways in HIC bladder tissue, particularly enrichment of Toll-like receptor signaling. Single-cell RNA sequencing further localized this activation to urothelial cells, with TLR3 markedly upregulated in epithelial populations undergoing injury.
          Integrated multi-omics analysis revealed a strong crosstalk between gut microbiota, bile acid metabolism, and host immune signaling. Notably, TCDCA levels positively correlated with E. avium abundance and were associated with TLR3 activation signatures, suggesting a microbiota–metabolite–host signaling axis.
          Functionally, fecal microbiota transplantation (FMT) from HIC patients and E. avium colonization in mice increased systemic TCDCA levels and induced voiding dysfunction, decreased pain thresholds, and histological evidence of urothelial injury. Consistently, intravesical TCDCA instillation recapitulated bladder injury phenotypes in rats.
          In vitro, TCDCA impaired urothelial barrier integrity, reduced tight junction protein expression, and induced inflammatory responses. These effects were mediated through TLR3 activation, as pharmacological inhibition of TLR3 or pentosan polysulfate sodium (PPS) treatment partially restored barrier function and attenuated inflammation.

1. Gut microbial dysbiosis in HIC patients included enrichment of three bacterial species, especially Enterococcus avium, accompanied by elevated TCDCA in serum and urine.
2. Integrated multi-omics analyses revealed a microbiota–bile acid–TLR3 signaling axis, with TLR3 prominently upregulated in injured urothelial cells.
3. Functional validation demonstrated that FMT or E. avium colonization in mice, and intravesical TCDCA instillation in rats, induced bladder injury.

Through integrated multi-omics and experimental validation, this study reveals that altered gut microbiota and bile acid metabolism activate TLR3 to induce urothelial injury in interstitial cystitis, identifying a new pathway with therapeutic potential.

Peng L, Jin X, Li BY, Zeng X, Liao BH, Jin T, Chen JW, Gao XS, Wang W, He Q, Chen G, Gong LN, Shen H, Wang KJ, Li H, Luo DY. Integrating single-cell RNA sequencing with spatial transcriptomics reveals immune landscape for interstitial cystitis. Signal Transduct Target Ther. 2022 May 20;7(1):161.
Peng L, Chen JW, Chen YZ, Zhang C, Shen SH, Liu MZ, Fan Y, Yang SQ, Zhang XZ, Wang W, Gao XS, Di XP, Ma YC, Zeng X, Shen H, Jin X, Luo DY. UPK3A+ umbrella cell damage mediated by TLR3-NR2F6 triggers programmed destruction of urothelium in Hunner-type interstitial cystitis/painful bladder syndrome. J Pathol. 2024 Jun;263(2):203-216.

Continence 19S (2026) 102635
DOI: 10.1016/j.cont.2026.102635