Bovine Streptococcus uberis mastitis results in severe damage of mammary tissue due to uncontrolled inflammation. Endothelial dysfunction is implicated in the development of uncontrolled inflammation. Oxylipids are potent lipid mediators that orchestrate pathogen-induced inflammatory responses and an abundance or imbalance of some oxylipids may contribute to pathogenesis of disease. Oxylipids are derived from polyunsaturated fatty acids (PUFA) in the cell membranes, and the enzymatic oxygenation products of linoleic acid (LA) were previously shown to modulate endothelial cell responses. Thus, the hypothesis for chapter 2 was that LA metabolites, 9- and 13-hydroxyoctadecadienoic acid (HODE), are increased during S. uberis mastitis and contribute to an inflammatory phenotype in endothelial cells. The LA-derived oxylipids were predominant and 9- and 13-HODE were significantly increased in S. uberis-infected mammary tissue. The initial oxygenation product of LA induced a pro-inflammatory phenotype in bovine mammary endothelial cells (BMEC), but 13-HODE did not change BMEC phenotype. Previous research demonstrated that 13-HPODE induced apoptosis in endothelial cells, which may disrupt the continuous, single-cell layer necessary for mediating a successful, self-limiting inflammatory response to S. uberis. However, the relative contribution of specific LA-oxygenation products to endothelial integrity was unknown. The hypothesis for chapter 3 was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Findings demonstrated that the BMEC may not be a primary source of 13-HODE in response to S. uberis. Bovine monocytes were evaluated as a potential source of synthesized 13-HODE in response to S. uberis exposure. Subsequent results suggested that exposure of cultured BMEC monolayers to 13-HPODE, but not 13-HODE, reduced endothelial barrier integrity and apoptosis and necrosis may contribute, in part, to impairment. Based on previous literature, the proposed mechanism for 13-HPODE induced apoptosis was lipid peroxidation and co-exposure with an antioxidant in the current study prevented the adverse effects of 13-HPODE. Given the potential importance of LA-derived oxylipids during S. uberis mastitis, the final objective was to change the PUFA substrate in an effort to modify predominant oxylipid pools. Other than LA, other PUFA substrates include arachidonic acid (ArA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Linoleic acid may be used for de novo synthesis of ArA and α-linolenic acid (ALA) may be used for de novo EPA and DHA synthesis. The hypothesis for chapter 4 was that supplementation of PUFA would change bovine leukocyte FA content and respective oxylipid profiles from ex vivo microbial-challenged leukocytes. Results demonstrated an ability to modify the ALA content of leukocytes following ALA supplementation but not EPA and DHA content, suggesting that dosing and timing of ALA supplementation may be important for increasing EPA and DHA. Supplementing ALA changed S. uberis-induced oxylipids derived from LA and ArA. Even though LA supplementation did not modify leukocyte PUFA content, S. uberis-induced LA and ArA-derived oxylipids were significantly decreased. Future investigations are required to determine how supplemental PUFA, without changing content, could mediate oxylipid biosynthesis. Furthermore, the ability of changing ALA content to modify ArA and LA oxylipids, but no change in ArA or LA content, is an important finding and supports the idea that the abundance of increase may not be as important as the ratio of PUFA.
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