Horses are prone to potentially lethal endotoxemia due to their surrounding fecal containing environment and their predisposition to colic. Their gastrointestinal tract and feces naturally contain gram-negative bacteria. These bacteria express lipopolysaccharide (LPS) on their cell membranes, which is recognized by Toll-like receptor 4 (TLR4). In cases where epithelial barriers are compromised or breached LPS has the potential to enter circulation and cause the inflammatory symptoms seen with endotoxemia. The objective of this study was to determine TLR4 presence and functionality on equine B cells. TLR4 expression on B lymphocytes has been studied in mouse, human and many other mammals, but has not been well characterized in the horse. Humans are highly sensitive to LPS but their B cells express non-functional TLR4. Mice in contrast are highly tolerant of LPS yet their B cells express functional TLR4. Studies in horse have perhaps been limited by the limited array of antibody markers available for use in horse. Anti-human CD21 has previously been shown to mark equine B lymphocytes. We show rat anti-mouse CD45R(B220) mAbs also accurately labels equine B lymphocytes. To investigate TLR4 expression in horses 12 Thoroughbred geldings, ages 5-10, were used for blood collection. By using the density gradient, Lympholyte, lymphocytes were separated from peripheral blood and incubated with or without LPS. B lymphocyte proliferation, TLR4 expression and mRNA changes were examined before or after culture in the presence or absence of LPS. We demonstrate TLR4 is expressed on equine B lymphocytes through the use of a mouse anti-human TLR4 antibody, clone 76B357.1, not previously used in horse. We demonstrated equine B cells fail to proliferate under LPS challenge as opposed to highly proliferative mouse B lymphocytes. However, transcriptional changes were observed in the equine cells within the TLR4 pathway upon treatment with LPS. / Master of Science / Horses are prone to potentially lethal infections due to their surrounding fecal containing environment and their predisposition to colic. Their gastrointestinal tract and environment naturally contain gram-negative bacteria such as E.coli. The gram-negative bacterial cell membrane expresses a molecule called lipopolysaccharide (LPS) which can cause inflammation by binding to an immune cell receptor called toll-like receptor 4 (TLR4). In cases where epithelial barriers are compromised or breached, LPS has the potential to enter the circulation and cause a severe inflammatory response capable of progressing to shock and death. Humans are also highly sensitive to LPS yet their B lymphocytes express non-functional TLR4. Mice, in contrast, are highly tolerant of LPS but their B lymphocytes express functional TLR4. The objective of this study was to determine TLR4 presence and functionality on equine B lymphocytes. Studies in horse have perhaps been limited by the small number of antibody markers available for use in horse. Two B lymphocyte markers not previously assessed in horse tissue were used in this experiment to enrich for B lymphocytes. A TLR4 marker, also not previously described in the horse, was used in conjunction with the two B lymphocyte markers determined TLR4 is present on equine B lymphocytes. However, equine B lymphocytes failed to proliferate in the presence of LPS similiar to human B cells. Transcriptional changes were observed in the equine cells in the TLR4 pathway upon treatment with LPS.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/80301 |
| Date | 09 November 2017 |
| Creators | Kasmark, Leah |
| Contributors | Animal and Poultry Sciences, Leeth, Caroline M., Johnson, Sally E., Witonsky, Sharon G. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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