Introduction
Functional dyspepsia (FD) is characterised by upper abdominal symptoms without an organic cause and has been associated with duodenal immune activation and barrier dysfunction, although findings are inconsistent.
Aims & Methods
We aimed to examine RNA‑ and protein‑level changes in duodenal biopsies, and the effects of the duodenal microenvironment on immune activation and epithelial permeability in mast cells and duodenal organoids, respectively.
Bulk RNA sequencing of duodenal biopsies of 28 healthy controls (HC) and 22 FD patients was performed on an Illumina HiSeq 4000 platform. Differential gene expression (DESeq2 in R (v4.3.0)) and gene ontology (GO biological process) enrichment analysis (ShinyGO (v0.85.1)) were conducted. Genes with FDR<0.05 were included in functional enrichment analyses. Inflammatory cytokines were quantified in total protein extracts from duodenal biopsies of 29 HC and 29 FD patients using the Olink Multiplex Inflammation I kit (Target 96).
To evaluate the effect of the microenvironment on mast cell activation and epithelial barrier function, biopsy supernatant (SN) of FD patients and HC was used (two duodenal biopsies cultured in RPMI for 24h (37°C, 5% CO2))). HMC1.1 mast cells were cultured under standard conditions and stimulated for 24h with 30% biopsy SN. Degranulation was assessed by measuring β‑hexosaminidase release. Duodenal organoids, generated from HC and FD biopsies, were expanded in Matrigel under expansion conditions (5µM CHIR in addition to (1)) and passaged mechanically before dissociation and seeding onto collagen I-coated Transwells. Transepithelial electrical resistance (TEER) was measured daily until stabilisation, after which Transwells were stimulated basolaterally with 30% biopsy SN. TEER was measured every 24h, and after 48h of stimulation epithelial permeability was also assessed using FITC-dextran (4kDa) and horseradish peroxidase passage.
Results
Only limited transcriptomic differences were observed between FD and HC samples. In total, 42 genes were differentially expressed, with 31 genes up- and 11 downregulated in FD, with no functional enrichment among the upregulated genes. However, the downregulated genes showed enrichment for pathways associated with ‘TGF‑β1 production’ (FDR: 0.008) and ‘inflammatory response’ (FDR: 0.016), indicating potential immune dysregulation involving TGF‑β1 signaling. This is further supported by reduced expression of FURIN (log₂ fold change: -0.4, FDR: 0.02), a protease required for activation of TGF‑β1. In addition, principal component analysis showed no clear separation between FD and HC groups, highlighting the modest nature of the transcriptional differences. Consistently, proteomic profiling of 96 inflammation‑related protein biomarkers revealed no differences between the two cohorts.
Stimulation of HMC1.1 did not result in any significant differences between FD and HC SN. Next, duodenal organoid-derived monolayers were stimulated with HC and FD biopsy SN for 48h. Both for monolayers derived from HC and FD patients, stimulation with neither FD nor HC SN resulted in a significant change in permeability compared to the negative control.
Conclusion
In contrast to the popular hypothesis of duodenal immune activation in FD, only minor transcriptomic and no significant proteomic alterations in inflammatory markers were present in patients with FD compared to HC. Furthermore, the duodenal microenvironment did not differentially influence mast cell activation or epithelial permeability, arguing against direct effector roles of locally secreted mediators in FD.
References
1: Vissers E et al. Inflamm Bowel Dis. 2025.