Downregulation of a choline transporter gene in equine colitis indicates a role for choline supplementation to support the large intestinal epithelial barrier.

Research Summary

• This study investigates gene expression changes associated with acute equine colitis, focusing on a gene involved in choline transport.
• It explores how choline supplementation in vitro affects epithelial barrier recovery in large intestinal cells, suggesting potential therapeutic benefits.

Introduction and Background

• Acute colitis is a severe inflammatory disease affecting the equine colon, with significant risk of illness and death.
• The colon’s epithelial barrier is crucial for maintaining intestinal health, yet the molecular mechanisms of its damage and repair during colitis are not well understood.
• Choline, an essential nutrient, plays a vital role in membrane synthesis and cell proliferation; its uptake is mediated by the protein CHT1, encoded by the gene SLC5A7.

Research Objectives

• To identify transcriptional changes in the intestinal tissue of horses with colitis, revealing pathways involved in disease pathology.
• To evaluate the impact of choline supplementation on epithelial cell growth and barrier function in cultured colonic cells.

Methods

• RNA-sequencing was performed on rectal biopsy samples from healthy horses and those with colitis (3 per group) to analyze gene expression differences.
• Downregulation of the SLC5A7 gene was confirmed by quantitative real-time PCR and immunofluorescence using additional samples from various large intestinal sites (cecum, right ventral colon [RVC], right dorsal colon [RDC], rectum) with sample sizes between 8 and 20 per group.
• Primary epithelial cells were isolated from healthy right dorsal colon biopsies and grown as monolayers in vitro.
• These cultured cells were treated with varying concentrations of choline (0-250 µM) to assess effects on cell proliferation and barrier permeability.

Key Findings

• Transcriptomic analysis identified a distinct colitis-associated gene expression pattern, with increased expression of genes related to inflammatory responses, cell cycle activity, and epithelial reorganization.
• SLC5A7 was significantly downregulated throughout the large intestine in colitis horses, with specific decreases in cecal samples (healthy horses had a mean log2 fold change of 1.8; colitis horses had -0.5), indicating reduced capacity for choline uptake.
• Immunofluorescence revealed diminished presence of the CHT1 protein at the apical surface of epithelial cells in the cecum and colon segments of colitis-affected horses, suggesting impaired choline transport at a cellular level.
• In vitro, choline supplementation enhanced epithelial cell proliferation and reduced the time required for cultured cells to reach confluency, meaning faster monolayer recovery.
• No significant effect was observed on barrier permeability after cells reached confluency, indicating choline mainly influences cell growth rather than tight junction integrity post-confluence under these conditions.

Conclusions and Implications

• Equine acute colitis is characterized by a specific intestinal gene expression profile involving inflammation and epithelial repair mechanisms.
• Downregulation of choline transporter gene SLC5A7 and its protein CHT1 suggests impaired choline uptake may contribute to epithelial barrier dysfunction in colitis.
• Choline supplementation promotes epithelial cell proliferation, potentially aiding recovery of the intestinal lining.
• These findings support the consideration of choline supplementation as a therapeutic strategy to enhance epithelial barrier restoration during equine colitis.
• Further studies are needed to evaluate clinical outcomes and mechanisms by which choline affects intestinal healing in vivo.