Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract.
Abstract: There is accumulating evidence for the involvement of pro-inflammatory cytokines associated with a T helper 17 response in intestinal disorders such as inflammatory bowel disease (IBD) in humans. The involvement of interleukin (IL)-17 or IL-23 in equine IBD has not been studied and most gene expression studies in the equine intestine have been limited to the use of a single non-validated reference gene. In this study, expression of the reference gene candidates β2 microglobulin (β2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H2A type 1, hypoxanthine-guanine phosphoribosyltransferase (HPRT), 60S ribosomal protein L32 (RPL32), succinate dehydrogenase complex subunit A (SDHA) and transferrin receptor 1 protein coding (TFRC)in the equine intestine was evaluated by quantitative PCR. Three to four reference genes were adequate for normalisation of gene expression in the healthy duodenum, mid-jejunum, colon and rectum, although each segment required a unique combination of reference genes. No combination of the evaluated genes was optimal for the caecum and ileum. Another combination of reference genes (GAPDH, HPRT, RPL32 and SDHA) was optimal for normalisation of rectal samples from healthy and IBD-affected horses, indicating that reference genes should be re-evaluated if material from diseased specimens is analysed. Basal expression of IL-12p40, IL-17A and IL-23p19 was detected in each segment, which will enable gene expression studies of these cytokines by relative quantification.
Copyright © 2013 Elsevier Ltd. All rights reserved.
Publication Date: 2013-06-27 PubMed ID: 23810185DOI: 10.1016/j.tvjl.2013.05.020Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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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.
Cite This Article
APA
Hjertner B, Olofsson KM, Lindberg R, Fuxler L, Fossum C.
(2013).
Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract.
Vet J, 197(3), 817-823.
https://doi.org/10.1016/j.tvjl.2013.05.020 Publication
Researcher Affiliations
- Section of Immunology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 588, SE-751 23 Uppsala, Sweden. Electronic address: bernt.hjertner@slu.se.
MeSH Terms
- Animals
- Cytokines / genetics
- Cytokines / metabolism
- Female
- Gene Expression Regulation / physiology
- Horse Diseases / metabolism
- Horses
- Intestinal Mucosa / metabolism
- Irritable Bowel Syndrome / metabolism
- Irritable Bowel Syndrome / veterinary
- Leukocytes, Mononuclear / metabolism
- Male
- Polymerase Chain Reaction / veterinary
- RNA / genetics
- RNA / metabolism
- T-Lymphocytes, Helper-Inducer / metabolism
Citations
This article has been cited 4 times.- Collinet A, Grimm P, Jacotot E, Julliand V. Biomarkers for monitoring the equine large intestinal inflammatory response to stress-induced dysbiosis and probiotic supplementation. J Anim Sci 2022 Oct 1;100(10).
- Hellman S. Generation of equine enteroids and enteroid-derived 2D monolayers that are responsive to microbial mimics. Vet Res 2021 Aug 14;52(1):108.
- Hellman S, Hjertner B, Morein B, Fossum C. The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC. Vet Res 2018 Oct 22;49(1):108.
- Nazari F, Parham A, Maleki AF. GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells. J Anim Sci Technol 2015;57:18.
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