Vet. Res. (2008) 39:11
DOI: 10.1051/vetres:2007047

Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells

Eveline M. Ibeagha-Awemu¹, Jai-Wei Lee², Aloysius E. Ibeagha¹, Douglas D. Bannerman³, Max J.Paape³ and Xin Zhao<sup>1

1</sup>  Department of Animal Science, McGill University, 21111 Lakeshore Road, Ste-Anne-De-Bellevue, Quebec, H9X 3V9 Canada
²  Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Neipu, Pingtung, 912 Taiwan
³  Bovine Functional Genomics Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705 USA

(Received 20 April 2007; accepted 11 October 2007; published online 21 December 2007)

Abstract - Bovine mammary epithelial cells contribute to the innate immune response to intramammary infections by recognizing pathogens through specialized pattern recognition receptors. Toll-like receptor 4 (TLR4) is one such receptor that binds and is activated by lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. In this study, MAC-T cells (a bovine mammary epithelial cell line) were incubated in the presence or absence of increasing concentrations of LPS for 24 h. Expression of TLR2 and TLR4 were analyzed at both mRNA and protein levels by quantitative real-time PCR (qPCR) and flow cytometry, respectively. The mRNA of both receptors were up-regulated by all concentrations of LPS used (P<0.01). Similarly, flow cytometry with specific antibodies against TLR2 and TLR4 detected increased surface expression of these proteins. Furthermore, expression of downstream TLR4 signaling molecules was examined by qPCR following varying exposure times to 1 g/mL of LPS. Results demonstrate that the required adaptor molecules and transcription factors were up-regulated in a time-dependent manner. Both the MyD88 dependent and independent pathways in TLR4 signaling were activated in MAC-T cells. Expression of TOLLIP increased in response to LPS as did the pro-apoptotic protease, CASP8. These results suggest that the bovine mammary epithelium possesses the necessary immune repertoires required to achieve a robust defense against E. coli. The current findings, coupled with previous findings that S. aureus ligands induce up-regulation of TLR4, may indicate a positive adaptation by mammary epithelial cells to effectively respond to different types of mastitis pathogens.

Key words: lipopolysaccharide / TLR4 and TLR2 / signal transduction / bovine mammary epithelial cells / mastitis

Corresponding author: xin.zhao@mcgill.ca

© INRA, EDP Sciences 2008