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Title: Functional characterization of bovine TIRAP and MyD88 in mediating bacterial lipopolysaccharide-induced endothelial NF-kappaB activation and apoptosis

item Cates, Elizabeth
item Connor, Erin
item Mosser, David
item Bannerman, Douglas

Submitted to: Comparative Immunology Microbiology and Infectious Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2008
Publication Date: 8/28/2008
Citation: Cates, E.A., Connor, E.E., Mosser, D.M., Bannerman, D.D. 2008. Functional characterization of bovine TIRAP and MyD88 in mediating bacterial lipopolysaccharide-induced endothelial NF-kappaB activation and apoptosis. Comparative Immunology Microbiology and Infectious Diseases. 32(6):477-490.

Interpretive Summary: Approximately 50% of all clinical cases of mastitis are caused by Gram-negative bacteria. All of these bacteria contain a highly pro-inflammatory molecule called endotoxin or lipopolysaccharide (LPS), which is shed from the bacterial surface during bacterial replication or death. LPS has been implicated in much of the inflammation and injury associated with mastitis caused by Gram-negative bacteria. Cellular activation by LPS enables the host organism to mount an innate immune response. This immune response can be beneficial to the host by promoting clearance of the bacteria, however, an excessive inflammatory response can develop leading to the development of shock. In addition, LPS induces tissue injury and cell death (apoptosis) that can result in tissue scarring. In the setting of mastitis, this injury may contribute to decreased milk output. The present study identified functional roles for the intracellular bovine signaling molecules that promote cellular and inflammatory responses to LPS.

Technical Abstract: Mastitis is a prevalent disease in dairy cows. Gram-negative bacteria, which express the pro-inflammatory molecule lipopolysaccharide (LPS), are responsible for the majority of acute clinical cases of mastitis. Previous studies have identified differential susceptibility of human and bovine endothelial cells (EC) to the pro-inflammatory and injury-inducing effects of LPS. The Toll-like receptor (TLR)-4 signaling pathway, which is activated by LPS, has been well-studied in humans, but not in ruminants. Human myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NF-kB and apoptotic signaling pathways. To assess the role of the bovine orthologs of these proteins in bovine TLR-4 signaling, dominant-negative constructs were expressed in bovine EC, and LPS-induced NF-kB activation and apoptosis evaluated. The results from this study indicate that bovine MyD88 and TIRAP play functional roles in transducing LPS signaling from TLR-4 to downstream effector molecules involved in NF-kB activation, and that TIRAP promotes apoptotic signaling.