Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 16, 2008
Publication Date: January 25, 2009
Citation: Neill, J.D., Zuerner, R.L., Ridpath, J.F. 2009. BVDV Infection Alters Toll-Like and TNF-alpha Receptor Signalling in Bovine Aortic Endothelial Cells [abstract]. 4th U.S. BVDV Symposium. Paper No. 2-5. Technical Abstract: Aim. Bovine aortic endothelial cells (BAEC) are readily available commercially and are used in many labs in a variety of experiments. However, most lots of BAEC are contaminated with BVDV. It was unknown what effect BVDV had on normal function of BAEC. Here, we examined the effect of BVDV infection on the activation of bovine aortic endothelial cells by inflammatory stimuli. Methods. To determine whether BAEC were affected functionally by the presence of the BVDV, experiments were done that examined their response to inflammatory stimuli. Near confluent non-infected, acutely infected BAEC (strain A13, 24 hours) or BAEC contaminated with BVDV (strain A13) were treated with bacterial lipopolysaccharide (LPS, 1 ug/ml), dsRNA (5 ug/ml) or TNF-alpha (25 ng/ml) for 4 hours. Total cellular RNA was purified using Trizol reagent. Real-time reverse-transcription PCR was done to examine changes in expression of P- and E-selectins and A20 (a negative regulator of NF-kappaB). All three genes are known to be upregulated following activation of NF-kappaB by these proinflammatory receptors. The real-time PCR was done using the Superscript III SYBR green qt-PCR kit (Invitrogen). All real-time PCR results were normalized to Beta2-microglobulin. Results. BAEC contaminated with BVDV showed no outward effects of the infection. Following stimulation by all three receptor agonists, non-infected BAEC showed significant increases in the expression of both selectins as well as A20 as measured by real-time PCR. Increased expression of these genes indicated activation of NF-kappaB. However, differences were observed in cells acutely and chronically infected with BVDV. In almost all cases, increases in expression of E- and P- selectins were much lower in the acutely infected BAEC as compared to the non-infected BAEC. A20 expression was similar in non-infected and acutely infected cells but showed variability in the chronically infected cells. There was variability in the expression levels of the selectins in chronically infected BAEC and was dependent on the treatment. Conclusions. BVDV infection of BAEC had a profound effect on their responses to inflammatory stimuli. BAEC are commonly used as an in vitro model in many laboratories to study endothelial cell function. As shown here, transcriptional changes resulting from BVDV infection can significantly alter cellular responses, and may have a profound impact on experimental outcome. In addition, the results describe here offers clues to the effects of BVDV infection of cattle by altering signalling through toll-like and TNF receptors.