IDDF2024-ABS-0314 Lipopolysaccharide leads to inflammation and impairment of esophageal epithelial barrier and causes reflux symptoms

<h3>Background</h3> Patients with gastroesophageal reflux (GER) symptoms have been shown to display altered esophageal microbiome. However, the relationship between esophageal microbiome dysbiosis and GER symptoms remains poorly understood. <h3>Methods</h3> The distal esophageal microbiome was evaluated in 12 patients with gastroesophageal reflux disease (GERD), 16 with functional esophageal disorder (FED), 10 with duodenal ulcer, and 10 healthy volunteers by 16S rRNA sequencing. Esophageal transcriptome was evaluated in 6 GERD, 4 FED and 5 healthy subjects through transcriptomic sequencing. The human esophageal epithelial cell line (HEEC) was used to explore the mechanisms by which esophageal microbiota dysbiosis induced GER symptoms. Quantitative real-time polymerase chain reaction (qPCR), Western blot and H&amp;E staining were used to analyze the expression of toll-like receptors (TLR), inflammatory cytokines and epithelial barrier proteins and the intercellular space. <h3>Results</h3> Both GERD and FED patients exhibited a significantly elevated proportion of Gram-negative bacteria in the distal esophagus (IDDF2024-ABS-0310 Figure 1. 16S rRNA sequencing. A : β diversity analysis; B, C: Microbial community cluster analysis. HV, healthy volunteer; FED, functional esophageal disorder; GERD, gastroesophageal reflux disease). At the phylum level, a higher ratio of Proteobacteria was observed, while at the genus level, Pseudomonas was predominant. Patients with GER symptoms also showed significantly higher expression of TLR2 than those in controls (IDDF2024-ABS-0310 Figure 2. Transcriptome (A), qPCR (B) and IHC (C) showed that the expression of TLR2 was significantly higher in patients with GER symtpoms. HV, healthy volunteers; FED, functional esophageal disorder; GERD, gastroesophageal reflux disease). <i>In vitro</i>, we stimulated the HEEC using lipopolysaccharide (LPS) to mimic the increased Gram-negative bacterial proportion in the esophageal mucosa of patients with GER symptoms. We found that LPS led to the upregulation of TLR2 and interleukin-6 (IL-6) and a concurrent downregulation of claudin-1 (IDDF2024-ABS-0310 Figure 3. The relative expression of TLR2 (A), IL-6 (B) and barrier proteins (C) and the intercellular space. Ctrl: control). Furthermore, dilated intercellular space (DIS) was observed following LPS exposure (IDDF2024-ABS-0310 Figure 3. The relative expression of TLR2 (D) upon LPS stimulation. Ctrl: control). The application of TLR2 inhibitors could alleviate the effects of LPS. IL-6 was also found to downregulate the expression of claudin-1, while IL-6 inhibitor could suppress the effects of LPS on claudin-1 and intercellular space (IDDF2024-ABS-0310 Figure 4. The relative expression of claudin-1 upon treatment of IL-6 (A,B) or IL-6 inhibitors (C, D), Ctrl: control; Ab: antibody). Claudin-1 knockdown could also induce the DIS (IDDF2024-ABS-0310 Figure 5. The change of intercellular space upon claudin-1 knockdown). <h3>Conclusions</h3> These findings suggest a potential role of esophageal microbiota dysbiosis in contributing to GER symptoms. An IL-6 inhibitor holds promise as a novel therapy for alleviating GER symptoms. Further co-cultivation of microbial communities and animal studies are required.