Evidence of Duodenal Epithelial Barrier Impairment and Increased Pyroptosis in Patients With Functional Dyspepsia on Confocal Laser Endomicroscopy and "Ex Vivo" Mucosa Analysis

Abstract

Introduction: Duodenal epithelial barrier impairment and immune activation may play a role in the pathogenesis of functional dyspepsia (FD). This study was aimed to evaluate the duodenal epithelium of patients with FD and healthy individuals for detectable microscopic structural abnormalities.

Methods: This is a prospective study using esophagogastroduodenoscopy enhanced with duodenal confocal laser endomicroscopy (CLE) and mucosal biopsies in patients with FD (n = 16) and healthy controls (n = 18). Blinded CLE images analysis evaluated the density of epithelial gaps (cell extrusion zones), a validated endoscopic measure of the intestinal barrier status. Analyses of the biopsied duodenal mucosa included standard histology, quantification of mucosal immune cells/cytokines, and immunohistochemistry for inflammatory epithelial cell death called pyroptosis. Transepithelial electrical resistance (TEER) was measured using Ussing chambers. Epithelial cell-to-cell adhesion proteins expression was assessed by real-time polymerase chain reaction.

Results: Patients with FD had significantly higher epithelial gap density on CLE in the distal duodenum than that of controls (P = 0.002). These mucosal abnormalities corresponded to significant changes in the duodenal biopsy samples of patients with FD, compared with controls, including impaired mucosal integrity by TEER (P = 0.009) and increased number of epithelial cells undergoing pyroptosis (P = 0.04). Reduced TEER inversely correlated with the severity of certain dyspeptic symptoms. Furthermore, patients with FD demonstrated altered duodenal expression of claudin-1 and interleukin-6. No differences in standard histology were found between the groups.

Discussion: This is the first report of duodenal CLE abnormalities in patients with FD, corroborated by biopsy findings of epithelial barrier impairment and increased cell death, implicating that duodenal barrier disruption is a pathogenesis factor in FD and introducing CLE a potential diagnostic biomarker in FD.

Figure 1.

Representative probe-based CLE images of duodenal villi: (a) a patient with functional dyspepsia with several adjacent epithelial gaps (white arrowheads indicating epithelial gaps); (b) healthy individual without epithelial gaps.

Figure 2.

Representative image of biopsy immunohistochemistry stain for activated caspase from: (a) a patient with functional dyspepsia with positive stain (green) and (b) healthy control.

Figure 3.

Epithelial gap density assessed by pCLE in the third portion of the duodenum (D3) in healthy controls and functional dyspepsia (FD) patients. pCLE, probe-based CLE.

Figure 4.

Assessment of duodenal epithelial integrity by TEER measurements in biopsied duodenal mucosa of healthy controls (n = 10) and FD patients (n = 10). TEER, transepithelial electrical resistance.

Figure 5.

Gene extpression of tight junction and adherens junction proteins evaluated by real-time reverse transcriptase PCR in the biopsied duodenal mucosa from healthy controls and patients with FD. CLDN, claudin; OCLN, occludin; ZO 1–3, zonula occludens; and JAM1, junctional adhesion molecule 1.

Figure 6.

Gene expression of tissue cytokines evaluated by reverse transcriptase PCR in the biopsied duodenal mucosa from healthy controls and patients with FD. IL, interleukin; TNF, tumor necrosis factor; INF, interferon.

Figure 7.

Quantitative assessment of duodenal epithelial cells staining positive for activated caspase-1 (immunohistochemistry) on biopsy samples from healthy controls (n = 6) and patients with FD(n = 14). *P = 0.04.