Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses.

The research article evaluates the expression of the molecules tlr4, md2 and cd14 in horse blood cells and in tissue samples of the horse’s intestine. It demonstrates an elevated expression of tlr4 and md2 during certain in vitro stimulations and during induced endotoxemia, whereas the expression of CD14 remains steady. The results establish a basis for future studies on clinical material.

Objective and Methodology

• The aim of this study was to examine the expression of three key molecules, toll-like receptor 4 (tlr4), myeloid differentiation factor 2 (md2), and cluster of differentiation 14 (CD14), in equine blood leukocytes and in intestinal tissues from healthy horses.
• The researchers utilized real-time quantitative polymerase chain reaction (RT-qPCR) technology to investigate the expression of these molecules. RT-qPCR is a laboratory technique used in molecular biology to amplify and simultaneously quantify a targeted DNA molecule.
• In addition, the authors evaluated the stability of three commonly used reference genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT), and succinate dehydrogenase complex subunit A (SDHA) using a tool known as qbase(PLUS).

Testing Procedures

• The study used equine peripheral blood mononuclear cells (eqPBMC), which were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynucleotide 2216 (CpG-ODN 2216).
• Leukocytes were also obtained from horses before, during, and after an infusion of endotoxin.
• Intestinal samples from healthy horses were collected from the ileum, right dorsal colon and rectum for further testing.

Results and Findings

• They found that HPRT/SDHA combination was the most suitable for ex vivo (outside the body) testing in leukocytes obtained from induced endotoxemia and in in-vitro activated eqPBMCs, while HPRT/GAPDH worked best for intestinal samples.
• It was observed that in vitro activation of the cells with CpG-ODN2216 induced a threefold increase in the relative amounts of TLR4 and MD-2 mRNA. However, these levels decreased when the cells were stimulated with PMA and ionomycin.
• Following endotoxemia, a transient increase in tlr4 and md2 transcription was noticed in equine leukocytes. Conversely, CD14 mRNA levels remained constant, regardless of in vitro stimulation or during endotoxemia.

Significance and Applications of the Study

• The study offers an understanding and a stable foundation for the real-time PCR assessment of mRNA levels for tlr4, md2, and CD14 in different segments of the equine intestine.
• The findings lend themselves to making possible future expression studies on clinical material, aiding further explorations in veterinary research and treatments.