Connecting Metabolism to Mastitis: Hyperketonemia Impaired Mammary Gland Defenses During a Streptococcus uberis Challenge in Dairy Cattle.

ß-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1ß and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Choline Regulates the Function of Bovine Immune Cells and Alters the mRNA Abundance of Enzymes and Receptors Involved in Its Metabolism in vitro.

Dietary choline can impact systemic immunity, but it remains unclear whether this is primarily via direct impacts on immune cells or secondary effects of altered metabolic function. To determine whether increased choline concentrations (3.2, 8.2, 13.2 µM) in cell culture alter the function of bovine innate and adaptive immune cells, we isolated cells from dairy cows in early and mid-lactation as models of immuno-compromised and competent cells, respectively. Phagocytic and killing capacity of isolated neutrophils were linearly diminished with increasing doses of choline. In contrast, lymphocyte proliferation was linearly enhanced with increasing doses of choline. Furthermore, increasing doses of choline increased the mRNA abundance of genes involved in the synthesis of choline products (betaine, phosphatidylcholine, and acetylcholine) as well as muscarinic and nicotinic acetylcholine receptors in a quadratic and linear fashion for neutrophils and monocytes, respectively. Phagocytic and killing capacity of neutrophils and proliferation of lymphocytes were not affected by stage of lactation or its interaction with choline or LPS. In neutrophils from early lactation cows, choline linearly increased the mRNA abundance of muscarinic and nicotinic cholinergic receptors, whereas choline-supplemented monocytes from mid-lactation cows linearly increased the mRNA abundance of several genes coding for choline metabolism enzymes. These data demonstrate that choline regulates the inflammatory response of immune cells and suggest that the mechanism may involve one or more of its metabolic products.

Continuous low-dose infusion of tumor necrosis factor alpha in adipose tissue elevates adipose tissue interleukin 10 abundance and fails to alter metabolism in lactating dairy cows.

Repeated bolus doses of tumor necrosis factor-α (TNFα) alters systemic metabolism in lactating cows, but whether chronic release of inflammatory cytokines from adipose tissue has similar effects is unclear. Late-lactation Holstein cows (n=9-10/treatment) were used to evaluate the effects of continuous adipose tissue TNFα administration on glucose and fatty acid (FA) metabolism. Cows were blocked by feed intake and milk yield and randomly assigned within block to control or TNFα treatments. Treatments (4mL of saline or 14µg/kg of TNFα in 4mL of saline) were infused continuously over 7d via 2 osmotic pumps implanted in a subcutaneous adipose depot. Plasma, milk samples, milk yield, and feed intake data were collected daily, and plasma glucose turnover rate was measured on d 7. At the end of d 7, pumps were removed and liver and contralateral tail-head adipose biopsies were collected. Results were modeled with the fixed effect of treatment and the random effect of block. Treatment with TNFα increased plasma concentrations of the acute phase protein haptoglobin, but did not alter plasma TNFα, IL-4, IL-6, or IFN-γ concentrations, feed intake, or rectal temperature. Milk yield and composition were unchanged, and treatments did not alter the proportion of short- versus long-chain FA in milk on d 7. Treatments did not alter plasma free FA concentration, liver triglyceride content, or plasma glucose turnover rate. Surprisingly, TNFα infusion tended to decrease liver TNFα and IL-1 receptor 1 mRNA abundance and significantly increased adipose tissue IL-10 protein concentration. Continuous infusion of TNFα did not induce the metabolic responses previously observed following bolus doses delivered at the same rate per day. Metabolic homeostasis may have been protected by an adaptive anti-inflammatory response to control systemic inflammation.

TNFα altered inflammatory responses, impaired health and productivity, but did not affect glucose or lipid metabolism in early-lactation dairy cows.

Inflammation may be a major contributing factor to peripartum metabolic disorders in dairy cattle. We tested whether administering an inflammatory cytokine, recombinant bovine tumor necrosis factor-α (rbTNFα), affects milk production, metabolism, and health during this period. Thirty-three Holstein cows (9 primiparous and 24 multiparous) were randomly assigned to 1 of 3 treatments at parturition. Treatments were 0 (Control), 1.5, or 3.0 µg/kg body weight rbTNFα, which were administered once daily by subcutaneous injection for the first 7 days of lactation. Statistical contrasts were used to evaluate the treatment and dose effects of rbTNFα administration. Plasma TNFα concentrations at 16 h post-administration tended to be increased (P<0.10) by rbTNFα administration, but no dose effect (P>0.10) was detected; rbTNFα treatments increased (P<0.01) concentrations of plasma haptoglobin. Most plasma eicosanoids were not affected (P>0.10) by rbTNFα administration, but 6 out of 16 measured eicosanoids changed (P<0.05) over the first week of lactation, reflecting elevated inflammatory mediators in the days immediately following parturition. Dry matter and water intake, milk yield, and milk fat and protein yields were all decreased (P<0.05) by rbTNFα treatments by 15 to 18%. Concentrations of plasma glucose, insulin, ß-hydroxybutyrate, non-esterified fatty acids, triglyceride, 3-methylhistidine, and liver triglyceride were unaffected (P>0.10) by rbTNFα treatment. Glucose turnover rate was unaffected (P=0.18) by rbTNFα administration. The higher dose of rbTNFα tended to increase the risk of cows developing one or more health disorders (P=0.08). Taken together, these results indicate that administration of rbTNFα daily for the first 7 days of lactation altered inflammatory responses, impaired milk production and health, but did not significantly affect liver triglyceride accumulation or nutrient metabolism in dairy cows.

Anti-inflammatory salicylate treatment alters the metabolic adaptations to lactation in dairy cattle.

Adapting to the lactating state requires metabolic adjustments in multiple tissues, especially in the dairy cow, which must meet glucose demands that can exceed 5 kg/day in the face of negligible gastrointestinal glucose absorption. These challenges are met through the process of homeorhesis, the alteration of metabolic setpoints to adapt to a shift in physiological state. To investigate the role of inflammation-associated pathways in these homeorhetic adaptations, we treated cows with the nonsteroidal anti-inflammatory drug sodium salicylate (SS) for the first 7 days of lactation. Administration of SS decreased liver TNF-α mRNA and marginally decreased plasma TNF-α concentration, but plasma eicosanoids and liver NF-κB activity were unaltered during treatment. Despite the mild impact on these inflammatory markers, SS clearly altered metabolic function. Plasma glucose concentration was decreased by SS, but this was not explained by a shift in hepatic gluconeogenic gene expression or by altered milk lactose secretion. Insulin concentrations decreased in SS-treated cows on day 7 compared with controls, which was consistent with the decline in plasma glucose concentration. The revised quantitative insulin sensitivity check index (RQUICKI) was then used to assess whether altered insulin sensitivity may have influenced glucose utilization rate with SS. The RQUICKI estimate of insulin sensitivity was significantly elevated by SS on day 7, coincident with the decline in plasma glucose concentration. Salicylate prevented postpartum insulin resistance, likely causing excessive glucose utilization in peripheral tissues and hypoglycemia. These results represent the first evidence that inflammation-associated pathways are involved in homeorhetic adaptations to lactation.

Toll-like receptor 4 signaling is required for induction of gluconeogenic gene expression by palmitate in human hepatic carcinoma cells.

Saturated free fatty acids (FFA) can activate inflammatory cascades including the toll-like receptor 4 (TLR4) pathway. TLR4 is expressed by hepatocytes and may help link FFA to altered hepatic gluconeogenesis in type 2 diabetes mellitus. This study examined the role of TLR4 in mediating palmitate effects on the expression of phosphoenolpyruvate carboxykinase (PCK1) and the catalytic subunit of glucose-6-phosphatase (G6PC), rate-determining gluconeogenic enzymes. Human hepatocellular carcinoma cells (HepG2 and HuH7) were incubated in media including 2% bovine serum albumin and 250 to 1000 µM palmitate for 24 h. Signaling mediated by TLR4 was blocked by a TLR4 decoy peptide or small interfering RNA knockdown of TLR4. Palmitate induced dose-dependent increases in PCK1 and G6PC mRNA abundance, which were prevented by the TLR4 decoy peptide. Palmitate doubled PCK1 promoter activity, and TLR4 knockdown ablated this response. Lipopolysaccharide and monophosphoryl lipid A also up-regulated G6PC and PCK1 transcript abundance in a TLR4-dependent manner. Addition of oleate attenuated palmitate-induced increases in G6PC and PCK1 mRNA abundance. Palmitate increased nuclear factor κ-light-chain-enhancer of activated B cells reporter gene activity, which was unaffected by TLR4 blockade, but increased mRNA abundance of hepatocyte-specific cyclic AMP response element binding protein, a transcriptional regulator of PCK1, in a TLR4-dependent manner. Finally, TLR4 activation by palmitate increased subsequent cellular uptake of palmitate, and inhibiting ceramide synthesis ablated palmitate effects on PCK1 mRNA abundance and promoter activity. These results suggest that TLR4 signaling could play a critical role in linking elevated saturated FFA to increased transcription of gluconeogenic genes.

Daily injection of tumor necrosis factor-{alpha} increases hepatic triglycerides and alters transcript abundance of metabolic genes in lactating dairy cattle.

To determine whether inflammation can induce bovine fatty liver, we administered recombinant bovine tumor necrosis factor-alpha (rbTNF) to late-lactation Holstein cows. Cows (n = 5/treatment) were blocked by feed intake and parity and randomly assigned within block to control (CON; saline), rbTNF at 2 microg/(kg.d), or pair-fed control (saline, intake matched) treatments. Treatments were administered once daily by subcutaneous injection for 7 d. Plasma samples were collected daily for analysis of glucose and FFA and a liver biopsy was collected on d 7 for triglyceride (TG) and quantitative RT-PCR analyses. Data were analyzed using treatment contrasts to assess effects of tumor necrosis factor-alpha (TNFalpha) and decreased feed intake. By d 7, feed intake of both rbTNF and pair-fed cows was approximately 15% less than CON (P < 0.01). Administration of rbTNF resulted in greater hepatic TNFalpha mRNA and protein abundance and 103% higher liver TG content (P < 0.05) without affecting the plasma FFA concentration. Hepatic carnitine palmitoyltransferase 1 transcript abundance tended to be lower (P = 0.09) and transcript abundance of fatty acid translocase and 1-acyl-glycerol-3-phosphate acyltransferase was higher (both P < 0.05) after rbTNF treatment, consistent with increased FFA uptake and storage as TG. Transcript abundance of glucose-6-phosphatase (P < 0.05) and phosphoenolpyruvate carboxykinase 1 (P = 0.09), genes important for gluconeogenesis, was lower for rbTNF-treated cows. These findings indicate that TNFalpha promotes liver TG accumulation and suggest that inflammatory pathways may also be responsible for decreased glucose production in cows with fatty liver.