Bladder outlet obstruction (BOO) represents one of the most prevalent conditions in urological practice, for which timely obstruction relief remains the primary therapeutic intervention. However, approximately 20%-40% of patients exhibit persistent bladder dysfunction despite surgical decompression, largely attributable to irreversible structural and functional alterations-collectively termed bladder remodeling—induced by chronic BOO. The intricate gene regulatory networks and multidimensional molecular mechanisms underlying BOO-associated bladder remodeling remain poorly elucidated. This study aims to comprehensively characterize the cellular heterogeneity and molecular drivers, particularly focusing on macrophage subpopulations and their crosstalk with fibroblasts, during the pathological progression of BOO.

Clinical bladder tissues were obtained from BOO patients and controls. A murine BOO model was established via partial urethral ligation. Bladder samples from both human subjects and mice underwent bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) to map transcriptional dynamics. Bioinformatic analyses included differential gene expression profiling, Gene Ontology (GO) enrichment, and transcription factor prediction. Immunofluorescence staining was employed to validate immune cell infiltration. In vitro functional assays were conducted by treating mouse bladder fibroblasts with recombinant IL1F9, followed by quantification of inflammatory cytokines and extracellular matrix (ECM) components via qPCR or ELISA.

Integrated transcriptomic analysis of human and mouse BOO bladders revealed significant enrichment of upregulated differentially expressed genes (DEGs) in the GO term "Macrophage activation." Immunofluorescence confirmed elevated macrophage infiltration in BOO tissues. scRNA-seq of mouse bladders identified seven distinct macrophage subsets (C0–C6), with the C3 subpopulation substantially expanding upon BOO (11.95% vs. 0.82% in controls). The C3 subset uniquely expressed high levels of IL1F9 and exhibited a tissue-resident macrophage signature (Lyve1+Folr2+Timd4+) while lacking monocyte-derived markers (CCR2, H2-AB1, IL1b). Foxk1 was predicted as a potential transcriptional regulator of C3 macrophages. Cell-cell interaction analysis indicated robust IL1F9-IL1RL2-mediated communication between C3 macrophages and fibroblasts. In vitro stimulation of fibroblasts with IL1F9 significantly upregulated pro-inflammatory factors (CXCL1, CXCL2, CCL2, CSF1, IL6, CXCL5, CXCL16) and ECM protein fibronectin.

This study identifies a specific tissue-resident macrophage subset (C3) that expands during bladder outlet obstruction (BOO) and is potentially regulated by the transcription factor FOXK1. These C3 macrophages uniquely express high levels of the cytokine IL1F9. Analysis indicates that IL1F9 signals to fibroblasts via the IL1F9-IL1RL2 pathway. Functionally, IL1F9 stimulation activates fibroblasts, driving the expression of pro-inflammatory factors and extracellular matrix proteins. Thus, the FOXK1/IL1F9-dependent C3 macrophage-fibroblast communication axis is a key mechanism promoting inflammation and fibrosis in BOO-induced bladder remodeling, revealing a new potential therapeutic target.

This study uncovers a novel Foxk1-regulated tissue-resident macrophage subset (C3) that expands during BOO and drives bladder remodeling via IL1F9-IL1RL2 signaling to fibroblasts. The C3-fibroblast axis promotes inflammatory responses and ECM deposition, highlighting potential therapeutic targets for mitigating BOO-induced bladder dysfunction.

Continence 19S (2026) 102700
DOI: 10.1016/j.cont.2026.102700