Intramammary Lipopolysaccharide Challenge Research Articles

Liver lipid content and inflammometabolic indices in peripartal dairy cows are altered in response to prepartal energy intake and postpartal intramammary inflammatory challenge

This study evaluated the effect of feeding a control diet (CON) or a moderate energy diet (overfed, OVE) during the dry period (∼45d) and a postpartum intramammary lipopolysaccharide (LPS) challenge on blood metabolic and inflammatory indices, milk production, and hepatic gene expression. A subset of cows (n=9/diet) in CON (1.34Mcal/kg of dry matter) and OVE (1.62Mcal/kg of dry matter) received an intramammary LPS challenge (200μg; CON-LPS, OVE-LPS, respectively). Liver biopsies were harvested at −14d from calving, and postpartum at 2.5h post-LPS on d 7, 14, and 30. Prepartum, the OVE group was in more positive energy balance (EB) and had greater body condition score compared with CON. In contrast, during wk 1 postpartum and before the LPS challenge, the OVE group was in greater negative EB than CON. Prepartal diet did not affect postpartal milk production or dry matter intake. At 2h postchallenge on d 7, we observed an increase in serum nonesterified fatty acids (NEFA) and bilirubin and a decrease in hydroxybutyrate, regardless of diet. That was coupled with greater haptoglobin in OVE-LPS compared with CON-LPS. In addition, OVE-LPS cows versus CON nonchallenged, OVE nonchallenged, and CON-LPS had greater liver triacylglycerol (TAG) concentration 2.5h postchallenge on d 7. The concentration of TAG in liver of OVE-LPS remained elevated by 30d postpartum. The liver TAG concentration in OVE-LPS compared with CON-LPS cows was associated with greater serum concentration of NEFA and reactive oxygen metabolites on d 10 and 14 postpartum. Cows in OVE-LPS also had greater concentrations of ceruloplasmin, cholesterol, and vitamin E from d 10 through 21. Among 28 genes associated with fatty acid oxidation, inflammation, oxidative stress, and gluconeogenesis, only SAA3 (which encodes an acute phase protein) was greater in CON-LPS compared with OVE-LPS at 2.5h postchallenge. Expression of HP, which encodes another acute phase protein, was greater in OVE-LPS than in CON-LPS at 14 and 30d postpartum. Several inflammation-related genes (TNF, IRAK1, NFKB1, ANGPTL4) showed markedly decreased expression between 7 and 14d, after which expression remained unchanged. No differences were observed in several genes of the growth-hormone/insulin-like growth factor-1 axis, except for SOCS2, expression of which decreased markedly between 7 and 14d in OVE-LPS but not in CON-LPS. These data suggest that overfeeding a moderate-energy diet prepartum alters the response of the cow to an intramammary challenge after calving and may predispose it to sustained liver lipidosis.

Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows

The metabolic load during periods of high milk production in dairy cows causes a variety of changes of metabolite blood concentrations including dramatically decreased glucose levels. These changes supposedly impair the immune system. The goal of this study was, therefore, to evaluate adaptations of the cow's immune system in response to an intramammary lipopolysaccharide (LPS) stimulation during a 3-d modification of plasma glucose and insulin induced by different clamp infusions. Seventeen midlactating dairy cows received a hypoglycemic hyperinsulinemic clamp induced by insulin infusion (HypoG; n=5), a euglycemic hyperinsulinemic clamp induced by insulin and glucose infusion (EuG; n=6), or infusion of saline solution (NaCl; n=6) for 56h. At 48h of infusion, 2 udder quarters were challenged with 200 μg of Escherichia coli LPS. At 48h of infusion (immediately before LPS challenge), tumor necrosis factor α, lactoferrin, and serum amyloid A (SAA) mRNA abundance was increased in HypoG and Il-1β mRNA abundance was decreased in EuG. After LPS challenge, plasma glucose concentration did not decrease, although plasma insulin increased simultaneously in all groups either due to enhanced endogenous release (NaCl) or due to increased insulin infusion rate (HypoG; EuG). Plasma cortisol, rectal temperatures, and milk somatic cell count of challenged quarters increased, whereas plasma nonesterified fatty acid concentrations were similarly decreased across treatments. In mammary biopsies, increased mRNA expression of tumor necrosis factor α, IL-1β, IL-8, and IL-10, and SAA were observed in LPS-treated quarters of all groups, with a more pronounced increase in IL-1β, IL-10, and SAA expression in EuG. Nuclear factor-κB mRNA expression was upregulated in NaCl and EuG but not in HypoG in response to LPS. Lactoferrin, toll-like receptor 4, and cyclooxygenase-2 mRNA expression was increased in LPS-treated quarters of EuG only, and 5-lipoxygenase mRNA expression was decreased in LPS-treated quarters only in treatments HypoG and NaCl. In conclusion, intramammary LPS induces local and systemic inflammatory responses, as well as systemic insulin resistance. The observed treatment differences of the mammary mRNA expression of several immune parameters both before and after LPS challenge indicate a direct influence of changed glucose and insulin concentrations during the course of lactation on the immune defense against mastitis pathogens.

Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages

Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFα gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFα produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFα, which is produced by alveolar macrophages in response to LPS/TLR4 signaling and (ii) is dependent on IL8 and IL1β signaling and regulated by iNOS-derived NO.

Supplemental fish oil does not alter immune competence or the pathophysiological response to an intramammary infusion of endotoxin in peri-partum multiparous Holstein cows

The objective was to determine the effects of supplementing the diet with fish oil during the peri-partum period on the immune competence and the pathophysiological response to a lipopolysaccharide-induced mastitis challenge. Multiparous Holstein cows (n=30) were completely randomized to one of two treatments at 3 weeks pre-partum. Treatments differed only in the source of supplemental lipid and included either Energy Booster or fish oil. Treatment diets were fed from -21 d relative to expected date of parturition until 10 d post partum. Treatments were fed as a bolus prior to the a.m. feeding. The dose of lipid during the pre-partum period was 250 g/d, whereas the amount of lipid supplemented post partum was adjusted to the level of intake, approximately 0.92% of the previous day's dry matter intake. Ex-vivo analyses of immune competence were measured including the antimicrobial activity of whole blood against Escherichia coli, Salmonella typhimurium and Candida albicans as well as the production of interferon-gamma by peripheral blood mononuclear cultures. At 7 days in milk cows were infused with 100 microg of Esch. coli lipopolysaccharide into one rear quarter. Supplementing fish oil increased plasma concentrations of eicosapentaenoic and docosahexaenoic acids, but had no affect on the proportions of arachidonic acid at calving. Fish oil did not influence the production of interferon-gamma or the antimicrobial activity of whole blood against any of the microorganisms. Furthermore, fish oil had no ameliorative effect on either the local or the systemic acute phase response following an intramammary lipopolysaccharide challenge in early lactating Holstein cows. Supplementing fish oil in the diet of peri-partum cows will not protect them from deleterious effects of an excessive acute phase response.