BRD4 contributes to the cytokine-induced acquisition of an IL-13RA2-positive inflammatory fibroblast phenotype.

Abstract

Background and aims: Inflammatory bowel diseases (IBDs) are characterized by chronic intestinal inflammation, in which inflammatory fibroblasts contribute to epithelial damage and amplification of mucosal inflammation. However, the molecular mechanisms regulating their pathogenic activity remain incompletely understood. This study investigated the role of the epigenetic reader BRD4 in the differentiation and function of IL-13RA2-expressing inflammatory fibroblasts in IBD.

Methods: Single-cell RNA sequencing datasets from IBD patients and controls were analyzed to define BRD4 expression across stromal populations. Protein expression was validated by immunofluorescence and flow cytometry in intestinal tissues and primary fibroblasts. Primary fibroblasts from IBD patients were stimulated with interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-α) to induce an inflammatory phenotype, and BRD4 function was assessed using the BRD4-targeting PROTAC AT1. Cytokine and chemokine expression was evaluated by RNA sequencing, real-time polymerase chain reaction (PCR), and ELISA, while neutrophil chemotaxis assays assessed the functional effects of fibroblast-derived mediators.

Results: BRD4 was significantly upregulated in inflammatory fibroblasts from IBD patients compared with other stromal subsets and controls. Inflamed tissues showed enrichment of BRD4⁺ and IL-13RA2⁺ fibroblasts. In vitro, cytokine stimulation induced IL-13RA2 expression in the majority of fibroblasts, all of which co-expressed BRD4. BRD4 degradation significantly reduced the proportion of IL-13RA2⁺ fibroblasts. BRD4⁺ fibroblasts exhibited a pro-inflammatory transcriptional profile, including CCL2, CXCL1, CXCL2, IL-11, IL-24, IL-32, and CSF3. Degradation of BRD4 markedly decreased the expression of these mediators and reduced neutrophil migration.

Conclusions: BRD4 contributes to cytokine-induced inflammatory activation and pro-inflammatory functions of intestinal fibroblasts in IBD. Targeting BRD4 may represent a potential strategy to modulate stromal cell-driven inflammation.