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United States Department of Agriculture

Agricultural Research Service

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

item Ibeagha-awemu, Eveline
item Lee, Jai-wei
item Ibeagha, A
item Bannerman, Douglas
item Paape, Max
item Zhao, Xin

Submitted to: Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/11/2007
Publication Date: 1/1/2008
Citation: Ibeagha-Awemu, E.M., Lee, J.W., Ibeagha, A.E., Bannerman, D.D., Paape, M.J., Zhao, X. 2008. Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells. Veterinary Research. 39(2):(article #11)1-12.

Interpretive Summary: Toll-like receptor 4 (TLR-4) has been identified as a receptor of bacterial lipopolysaccharide (LPS), also referred to as endotoxin. LPS is a highly inflammatory molecule released during infection with Gram-negative bacteria, the latter of which are prevalent causes of clinical mastitis. LPS activation of TLR-4 initiates cellular production of pro-inflammatory cytokines and the upregulation of adhesion molecules, which enable the host to respond to infection. However, excessive production/upregulation of these responses can be harmful to the host. The current manuscript provides evidence for a potential role of TLR-4 in contributing to mammary epithelial responses to Gram-negative infection and enhances our understanding of the immune response elicited by the mammary gland.

Technical 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 hrs. 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 ug/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.

Last Modified: 06/25/2017
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