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.

Proton Pump Inhibitors Reduce Duodenal Eosinophilia, Mast Cells, and Permeability in Patients With Functional Dyspepsia

“…The presence of duodenal eosinophilia in FD, first reported in children by Friesen et al [19] and adults by Talley et al [20], was endorsed and expanded by our group in 2 patient cohorts [6,7]. Duodenal micro-inflammation with eosinophils [21] and mast cells [22] in FD was confirmed by systematic reviews and there is also evidence for impaired barrier function in FD [23,24]. We and others have hypothesized that increased antigen penetration via a defective duodenal barrier may result in inflammation [3,25].…”

The Role of Leaky Gut in Functional Dyspepsia

“…Alternatively, duodenal manometry combined with impedance allows determination of baseline impedance following the nocturnal migrating motor complex phase III, thus reducing the potential impact of luminal content ( Nakagawa et al, 2020 ). Finally, confocal laser endomicroscopy (CLE) has been used to identify breaks or extrusion zones in the intestinal epithelium through the passage of intravenously administered fluorescein during endoscopy, which are quantified as epithelial gaps or fluorescein leaks ( Nojkov et al, 2020 ). Although increasingly used, the basolateral to apical flux of fluorescein is less physiological than apical to basolateral passage of luminal compounds ( Vanuytsel et al, 2021 ).…”

“…An altered expression of AJ and desmosomal proteins can influence the expression of TJ proteins but do not directly regulate the paracellular transport. Several studies have used quantitative PCR (qPCR) for TJ (OCLN, CLDN, ZO), AJ (E-cadherin, β-catenin) and desmosomal proteins (DSG, DSC) ( Vanheel et al, 2014 ; Komori et al, 2019 ; Taki et al, 2019 ; Nojkov et al, 2020 ). Also, analysis of protein-expression of cell-to-cell adhesion molecules by western blot (WB) and semi-quantitative evaluation of immunohistochemically (IHC) stained duodenal biopsies have been done in FD ( Vanheel et al, 2014 ; Du et al, 2018 ).…”

“…These measures are, however, not standardized, with different definitions of an altered distance between adjacent enterocytes (up to 30 nm) or lack of separate assessment at the TJ, AJ and desmosomal complexes ( Tanaka et al, 2016 ). Finally, markers of inflammatory cell death or pyroptosis (caspase-1) have been used with IHC to detect potential extrusions of duodenal epithelial cells in response to environmental stimuli ( Nojkov et al, 2020 ).…”

“…In adult FD, duodenal mucosal impedance was decreased vs. controls ( Ishigami et al, 2017 ; Komori et al, 2019 ; Nakagawa et al, 2020 ). While the consistently lower but gradual increase in baseline impedance from the duodenum to jejunum in FD patients and controls was probably due to a progressive decrease in exposure to luminal content ( Nakagawa et al, 2020 ), a higher epithelial gap density was only present in the distal duodenum of FD vs. controls using CLE ( Nojkov et al, 2020 ). Although these cell extrusions were suggested to allow microbial passage in the duodenum, we found no differences in plasma LBP as a marker of intestinal bacterial translocation in FD ( Wauters et al, 2021b ).…”