Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract.

The research article explores the role of pro-inflammatory cytokines, specifically those associated with T helper 17 in intestinal disorders such as inflammatory bowel disease (IBD) in horses. The study examined the expression of various reference genes in the equine intestine and determined optimal combinations for normalizing gene expression in different segments of a healthy horse’s intestine. It also reports that these combinations may need to be reevaluated when analyzing diseased tissue samples.

Research Objectives

• The primary aim of the study was to examine the role of pro-inflammatory cytokines associated with T helper 17, specifically interleukin (IL)-17 or IL-23, in equine IBD. This fills a gap because the involvement of these cytokines in equine IBD hasn’t been studied before.
• The study also aimed to identify the most suitable reference gene(s) for gene expression studies in the equine intestine. Previous studies have been limited by using a single non-validated reference gene.

Methods

• The study analyzed the expression of six potential reference genes (β2M, GAPDH, histone H2A type 1, HPRT, RPL32, SDHA, and TFRC) in the equine intestine using quantitative PCR.
• Various combinations of these genes were tested to determine which were most suitable for normalizing gene expression in different segments of the equine intestine.

Results

• It was determined that three to four reference genes were adequate for normalization of gene expression in the healthy duodenum, mid-jejunum, colon, and rectum. However, each segment required its unique combination of reference genes. None of the combinations were optimal for the caecum and ileum.
• Another combination of reference genes (GAPDH, HPRT, RPL32, and SDHA) was deemed optimal for normalizing rectal samples from both healthy and IBD-affected horses, suggesting the need for re-evaluation of reference genes when analyzing diseased tissue samples.
• Basal expression of IL-12p40, IL-17A, and IL-23p19, the cytokines associated with T helper 17 response, was detected in each segment. This opens possibilities for further studies on these cytokines’ gene expression.

Conclusion

• The study broadens our understanding of the possible role of T helper 17 associated cytokines in equine IBD and puts forward suitable reference genes for gene expression studies in the equine intestine.
• It also suggests the need for a reassessment of reference genes when analyzing samples from diseased individuals, paving the way for improved research methods.