Effect of Holstein genotype on innate immune and metabolic responses of heifers to lipopolysaccharide (LPS) administration.

Heifers (n = 4/genotype) from unselected (stable genotype since 1964, UH) and contemporary (CH) Holsteins that differed in milk yield (6,200 and 11,100 kg milk/305 d) were used to assess the impact of selection on innate immune and acute-phase response to an endotoxin (lipopolysaccharide; LPS). Jugular catheters were implanted 24 h before LPS administration. Blood samples were collected at -1, -0.5, 0, 1, 2, 3, 4, 6, 8, and 24 h relative to iv administration of 0.5 µg LPS/kg BW. Rectal body temperature (BT) was determined at these sampling times and at 5 and 7 h. Dermal biopsies were collected after the 24 h blood sample and processed to isolate fibroblasts. Plasma was analyzed for tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), serum amyloid A (SAA), xanthine oxidase (XO), and nitrate + nitrite (NOx), cortisol, glucose, and IGF-1 content. Isolated fibroblasts were exposed to IL-1ß or LPS and IL-6 and IL-8 content of culture media determined. Exposure to LPS increased BTs and plasma concentrations of TNF-α, IL-6 SAA, XO, cortisol, and glucose (P < 0.05) in both genotypes. Plasma concentrations of TNF-α, XO, NOx, and glucose did not differ (P > 0.25) between the genotypes, but IL-6 and SAA concentrations were reduced (P < 0.05) in CH relative to UH heifers while cortisol and IGF-1 concentrations tended (P < 0.08) to be reduced in CH heifers. After 36 h exposure to LPS, concentrations of IL-6 were greater (P < 0.05) in culture media from incubations of CH than UH fibroblasts but concentrations of IL-8 did not differ between genotypes. There was a trend (P = 0.08) for IL-8 concentrations to be reduced in media from CH fibroblasts exposed to IL-1ß for 24 h but IL-6 concentrations did not differ between genotypes. Results indicate 50 yr of selection has reduced the robustness of the innate immune and acute-phase response to LPS in the contemporary Holstein heifer.

Variation in fibroblast expression of toll-like receptor 4 and lipopolysaccharide-induced cytokine production between animals predicts control of bacterial growth but not severity of Escherichia coli mastitis.

Mastitis caused by environmental pathogens such as Escherichia coli is highly problematic to the dairy industry because it incurs substantial cost and tends to be difficult to manage. An effective innate immune response by the host is key to controlling infection, but it should also limit collateral damage to the mammary gland. Between-animal differences in mastitis severity have been attributed to variability in the innate response. In the current study, we used primary dermal fibroblast as a model to rank animals based on composite expression of the toll-like receptor 4 gene (TLR4) and lipopolysaccharide (LPS)-induced IL-8 and IL-6 protein production. Animals ranked as high and low responders (HR and LR, respectively) were then infected with the P4 strain of E. coli to determine how difference in rank would affect response to mastitis. All animals developed an acute response to the infection with varying degrees in severity; however, HR animals had an elevated somatic cell count and fever response at 12 h post-infection and greater production of milk IL-8 at 24 h post-infection. The HR animals were also significantly more capable of limiting bacterial growth. No differences in post-infection milk production or concentrations of milk BSA were measured. The current study indicates that HR animals have an early upregulation in their innate response that is beneficial for bacterial clearance; however, they are equally susceptible to tissue damage caused by an exuberant response to the infection. The dermal fibroblast may be used in conjunction with other cell types to determine how the innate response is regulated to mitigate unnecessary injury to the mammary gland while still effectively clearing the pathogen.

The effects of feeding mixed tocopherol oil on whole-blood respiratory burst and neutrophil immunometabolic-related gene expression in lactating dairy cows.

The 4 major tocopherol isoforms differ in their biochemical reactivity and cellular effects due to basic chemical structural differences. Alpha-tocopherol has been well studied regarding effects on bovine polymorphonuclear leukocyte (PMN) function and its involvement in respiratory burst. However, no studies to date have identified the effects of supplementing a mixed tocopherol oil (Tmix) particularly enriched in non-α tocopherol isoforms (i.e., γ- and δ-isoforms) on fundamental immunometabolic changes in dairy cows. Therefore, the objectives of this study were to determine whether short-term feeding of vegetable oil-derived Tmix alters specific biomarkers of metabolism, whole-blood leukocyte populations, respiratory burst, immunometabolic-related gene expression of PMN, or gene expression of isolated PMN when challenged with lipopolysaccharides (LPS). Clinically healthy multiparous lactating Holstein cows (n = 12; 179 ± 17 d in milk, 40.65 ± 3.68 kg of milk yield) were fed Tmix (620 g/d) for 7 consecutive days. Jugular blood (EDTA anticoagulant) was collected from all cows on d 0 before treatment initiation and again on d 7 after Tmix feeding. Total stimulated respiratory burst activity (RBA) and leukocyte populations were assessed in whole blood, and tocopherol isoform concentrations, metabolites, and hormones were measured in plasma. For gene expression analysis, isolated PMN from cows before and after Tmix feeding were incubated with LPS at a final concentration of either 0.0 or 1.5 µg/mL. Feeding of Tmix for 7 d increased the concentrations of α- and γ-tocopherol. The Tmix did not alter plasma insulin but decreased cholesterol. The Tmix did not alter whole-blood RBA or the leukocyte populations. The LPS challenge increased the expression of proinflammatory genes TNFA and IL6. However, Tmix treatment did not alter the patterns of LPS-affected expression of genes (e.g., TNFA, ITGB2, PPARA, and RXRA) associated with the immune or metabolic response. In conclusion, short-term feeding of Tmix may have no negative effect on animal health as Tmix increased α- and γ-tocopherol concentrations in blood and did not impair whole-blood RBA or alter leukocyte populations. The data provide further support that the α- and γ-tocopherol isoforms do not interfere with normal immune or metabolic function.

Neonatal lipopolysaccharide exposure does not diminish the innate immune response to a subsequent lipopolysaccharide challenge in Holstein bull calves.

The innate immune response following experimental mastitis is quite variable between individual dairy cattle. An inflammatory response that minimizes collateral damage to the mammary gland while still effectively resolving the infection following pathogen exposure is beneficial to dairy producers. The ability of a lipopolysaccharide (LPS) exposure in early life to generate a low-responding phenotype and thus reduce the inflammatory response to a later-life LPS challenge was investigated in neonatal bull calves. Ten Holstein bull calves were randomly assigned to either an early life LPS (ELL) group (n=5) or an early life saline (ELS) group (n=5). At 7d of age, calves received either LPS or saline, and at 32d of age, all calves were challenged with an intravenous dose of LPS to determine the effect of the early life treatment (LPS or saline) on the immune response generated toward a subsequent LPS challenge. Dermal fibroblast and monocyte-derived macrophage cultures from each calf were established at age 20 and 27d, respectively, to model sustained effects from the early life LPS exposure on gene expression and protein production of components within the LPS response pathway. The ELL calves had greater levels of plasma IL-6 and tumor necrosis factor-α than the ELS calves following the early life LPS or saline treatments. However, levels of these 2 immune markers were similar between ELL and ELS calves when both groups were subsequently challenged with LPS. A comparison of the in vitro LPS responses of the ELL and ELS calves revealed similar patterns of protein production and gene expression following an LPS challenge of both dermal fibroblast and monocyte-derived macrophage cultures established from the treatment groups. Whereas an early life exposure to LPS did not result in a dampened inflammatory response toward a later LPS challenge in these neonatal bull calves, the potential that exposure to inflammation or stress in early life or in utero can create an offspring with a low-responding phenotype as an adult is intriguing and has been documented in rodents. Further work is needed to determine if an inflammatory exposure in utero in a dairy animal would result in a low-responding innate immune phenotype.

Short communication: Amino acid supplementation and stage of lactation alter apparent utilization of nutrients by blood neutrophils from lactating dairy cows in vitro.

Glutamine is the preferred AA used by polymorphonuclear leukocytes (PMN) during the inflammatory response. However, the effect of other AA on bovine PMN response during inflammation and how this is altered by stage of lactation has not been fully elucidated. The objective of this study was to determine the effect of additional AA supplementation (pool of AA excluding Gln) on AA profiles, gene expression, and inflammatory function of PMN from dairy cows in early and mid lactation in vitro. We used 18 Holstein cows for this study. Polymorphonuclear leukocytes were isolated. Working solutions of AA (0 or 4 mM) and LPS (0 or 50µg/mL) were added to cell populations suspended in RPMI and incubated for 2h at 37°C. We used a subset of samples for gene and protein expression. Concentrations of AA in medium were determined using gas chromatography-mass spectrometry with norleucine as an internal standard. Apparent AA and glucose utilization were calculated by subtracting the concentration after from that of before incubation. Data were analyzed as a randomized block design. Challenge with LPS increased the expression of proinflammatory genes and AA supplementation decreased both the expression of some proinflammatory genes and the media concentrations of tumor necrosis factor-α. Neither stage of lactation, LPS challenge, nor AA supplementation altered the chemotactic or phagocytic abilities of PMN in vitro. Polymorphonuclear leukocytes supplemented with AA had greater concentrations and apparent utilization of most of the supplemented AA, whereas the unsupplemented group had greater apparent utilization of glucose. Alanine was not provided in the media but was present in spent media, and Ile, Gly, and Pro were greater in spent media than in media before incubation indicating synthesis of these AA. Regarding expression of genes involved in nutrient metabolism, the expression of G6PD, coding for the enzyme glucose 6-phosphate dehydrogenase, was increased and that of PDHA1, coding for the enzyme pyruvate dehydrogenase α 1, tended to increase with AA supplementation. Due to the lower concentration of tumor necrosis factor-α in media coupled with a downregulation of several proinflammatory genes, we concluded that AA, rather than Gln, alter the inflammatory response of bovine blood PMN. Independent from Gln, blood PMN from cows in early lactation may use certain AA as their primary carbon source for energy than cows in later lactation. Evaluating cows during the early postpartum period will provide additional information on the effect of stage of lactation and nutrient supplementation on PMN function.

Glucose supplementation has minimal effects on blood neutrophil function and gene expression in vitro.

During early lactation, glucose availability is low and the effect of glucose supply on bovine polymorphonuclear leukocyte (PMNL) function is poorly understood. The objective of this study was to determine the effect of glucose supplementation on the function and transcriptomic inflammatory response of PMNL from cows in early and mid-lactation in vitro. Twenty Holstein cows in early (n=10; days in milk=17±3.1) and mid-lactation (n=10; days in milk=168±14.8) were used for this study. Jugular blood was analyzed for serum concentrations of nonesterified fatty acids, ß-hydroxybutyrate, and glucose. Polymorphonuclear leukocytes were isolated and diluted using RPMI (basal glucose concentration was 7.2 mM) to different concentrations of PMNL/mL for phagocytosis, chemotaxis, gene expression, and medium analyses. Working solutions of glucose (0 or 4 mM of d-glucose) and lipopolysaccharide (0 or 50µg/mL) were added and tubes were incubated for 120 min at 37°C. Media were analyzed for concentrations of glucose and tumor necrosis factor-α (TNF-α). Data were analyzed in a randomized block (stage of lactation) design. Challenge with lipopolysaccharide increased the expression of the genes encoding for nuclear factor kappa B (NFKB1), IL-10 (IL10), IL1B, IL6, IL8, TNF-α (TNFA), glucose transporter 3 (SLC2A3), and the concentration of TNF-α in medium (147.3 vs. 72.5 pg/mL for lipopolysaccharide and control, respectively). Main effect of stage of lactation was minimal where the expression of IL10 increased for cows in early compared with cows in mid-lactation. After lipopolysaccharide challenge, cows in early lactation experienced more marked increases in the expression of IL6, TNFA, and IL8 when compared with cows in mid-lactation. Glucose supplementation had minimal effects on gene expression where glucose supplementation increased the expression of lysozyme (LYZ). Glucose supplementation increased PMNL phagocytosis but did not alter chemotaxis, morphology, or concentration of TNF-α in the medium. Under the conditions of the experiment, stage of lactation had minimal effects on PMNL response to glucose supply where only the expression of NFKB1 and the production of TNF-α were greater for cows in mid-lactation when compared with early lactation. Metabolic profiles for cows in early lactation did not parallel those for cows during the early postpartum period and may partly explain results for this study. Future studies investigating the effect of glucose supply on bovine PMNL function in vivo and how this may be altered by stage of lactation are warranted.

COMPARATIVE GUT PHYSIOLOGY SYMPOSIUM: Comparative physiology of glucagon-like peptide-2: Implications and applications for production and health of ruminants.

Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide derived from proteolytic cleavage of proglucagon by prohormone convertase 1/3 in enteroendocrine L cells. Studies conducted in humans, in rodent models, and in vitro indicate that GLP-2 is secreted in response to the presence of molecules in the intestinal lumen, including fatty acids, carbohydrates, amino acids, and bile acids, which are detected by luminal chemosensors. The physiological actions of GLP-2 are mediated by its G protein-coupled receptor expressed primarily in the intestinal tract on enteric neurons, enteroendocrine cells, and myofibroblasts. The biological activity of GLP-2 is further regulated by dipeptidyl peptidase IV, which rapidly cleaves the N-terminus of GLP-2 that is responsible for GLP-2 receptor activation. Within the gut, GLP-2 increases nutrient absorption, crypt cell proliferation, and mesenteric blood flow and decreases gut permeability and motility, epithelial cell apoptosis, and inflammation. Outside the gut, GLP-2 reduces bone resorption, can suppress appetite, and is cytoprotective in the lung. Thus, GLP-2 has been studied intensively as a therapeutic to improve intestinal function of humans during parenteral nutrition and following small bowel resection and, more recently, as a treatment for osteoporosis and obesity-related disorders and to reduce cellular damage associated with inflammation of the gut and lungs. Recent studies demonstrate that many biological actions and properties of GLP-2 in ruminants are similar to those in nonruminants, including the potential to reduce intestinal nitro-oxidative stress in calves caused by parasitic diseases such as coccidiosis. Because of its beneficial impacts on nutrient absorption, gut healing, and normal gut development, GLP-2 therapy offers significant opportunities to improve calf health and production efficiency. However, GLP-2 therapies require an extended time course to achieve desired physiological responses, as well as daily administration because of the hormone's short half-life. Thus, practical means of administration and alternative strategies to enhance basal GLP-2 secretion (e.g., through specific feed additives), which are more likely to achieve consumer acceptance, are needed. Opportunities to address these challenges are discussed.

Environmental heat stress modulates thyroid status and its response to repeated endotoxin challenge in steers.

The objective of this study was to evaluate in cattle, the effects of acute exposure to a heat stress (HS) environment on the status of the pituitary (thyrotropin, TSH)-thyroid (thyroxine, T4)-peripheral tissue T4 deiodination (type 1 5'-deiodinase [D1]; triiodothyronine [T3]; reverse-triiodothyronine [rT3]) axis, and the further response of this pituitary-thyroid-peripheral tissue axis (PTTA) to perturbation caused by the induction of the proinflammatory innate immune state provoked by the administration of gram-negative bacteria endotoxin (lipopolysaccharide [LPS]). Ten steers (318 ± 49 kg body weight) housed in controlled environment chambers were subjected to either a thermoneutral (TN: constant 19°C) or HS temperature conditions (cyclical daily temperatures: 32.2°C-40.0°C) for a total period of 9 d. To minimize the effects of altered plane of nutrition due to HS, steers in TN were pair-fed to animals in HS conditions. Steers received 2 LPS challenges 3 d apart (LPS1 and LPS2; 0.2 µg/kg body weight, intravenously, Escherichia coli 055:B5) with the first challenge administered on day 4 relative to the start of the environmental conditioning. Jugular blood samples were collected at 0, 1, 2, 4, 7, and 24 h relative to the start of each LPS challenge. Plasma TSH, T4, T3, and rT3 were measured by radioimmunoassay. Liver D1 activity was measured in biopsy samples collected before the LPS1 (0 h) and 24 h after LPS2. Before the start of LPS1, HS decreased (P < 0.01 vs TN) plasma TSH (40%), T4 (45.4%), and T3 (25.9%), but did not affect rT3 concentrations. In TN steers, the LPS1 challenge decreased (P < 0.01 vs 0 h) plasma concentrations of TSH between 1 and 7 h and T4 and T3 at 7 and 24 h. In HS steers, plasma TSH concentrations were decreased at 2 h only (P < 0.05), whereas plasma T3 was decreased at 7 and 24 h (P < 0.01). Whereas plasma T4 concentrations were already depressed in HS steers at 0 h, LPS1 did not further affect the levels. Plasma rT3 concentrations were increased in all steers at 4, 7, and 24 h after LPS1 (P < 0.01). The patterns of concentration change of T4, T3, and rT3 during LPS2 mirrored those observed in LPS1; the responses in plasma TSH were of smaller magnitude than those incurred after LPS1. The LPS challenges reduced (P < 0.01) hepatic activity of D1 in all animals but no differences were observed between steers subjected to TN or HS environment. The data are consistent with the concept that acute exposure of cattle to a HS environment results in the depression of the pituitary and thyroid components of the PTTA, whereas a normal capacity to generate T3 from T4 in the liver is preserved. The data also suggest that LPS challenge further suppresses all components of the PTTA including liver T3 generation, and these PTTA perturbations are more pronounced in steers that encounter a HS exposure.

Short communication: Glucagon-like peptide-2 and coccidiosis alter tight junction gene expression in the gastrointestinal tract of dairy calves.

Tight junction (TJ) proteins are integral factors involved in gut barrier function, and therapy with glucagon-like peptide-2 (GLP-2) enhances gut integrity. Our aim was to assess effects of GLP-2 treatment on mRNA expression of 8 TJ complex proteins in the intestine of dairy calves not infected or infected with Eimeria bovis at 11±3d of age. Mucosal epithelium from jejunum, ileum, and cecum was collected at slaughter from Holstein bull calves assigned to 4 groups: noninfected, buffer-treated (n=5); noninfected, GLP-2 treated (n=4); E. bovis-infected, buffer-treated (n=5); and E. bovis-infected, GLP-2-treated (n=4). Infected calves were orally dosed with 100,000 to 200,000 sporulated E. bovis oocysts on d 0; GLP-2-treated calves received 50 µg of GLP-2/kg of body weight subcutaneously twice daily for 10d beginning on d 18; and buffer-treated calves received an equal injection volume of 0.01 M Na bicarbonate buffer. All calves were killed on d 28. The mRNA expression of coxsackie and adenovirus receptor (CXADR), claudins 1, 2, and 4 (CLDN1, CLDN2, and CLDN4), F11 receptor (F11R), junction adhesion molecule 2 (JAM2), occludin (OCLN), and tight junction protein ZO-1 (TJP1) was determined by real-time quantitative PCR. In jejunum and ileum, an interaction of E. bovis infection and GLP-2 treatment on gene expression was noted. In jejunum of noninfected calves, GLP-2 increased CXADR, CLDN2, OCLN, and TJP1 mRNA expression but had no effect on mRNA expression in infected calves. Treatment with GLP-2 also increased tight junction protein ZO-1 protein expression in jejunum of noninfected calves as determined by immunohistochemistry. In ileum, E. bovis decreased expression of JAM2, OCLN, and TJP1 in buffer-treated calves, and GLP-2 increased TJP1 expression in infected calves. In cecum, E. bovis infection reduced expression of CXADR, CLDN4, F11R, and OCLN, and GLP-2 therapy increased expression of CLDN4, F11R, OCLN, and TJP1. Results are consistent with studies in nonruminants showing decreased expression of TJ complex proteins in the intestinal tract during pathogen-induced diarrhea and increased TJ protein expression in intestinal tissues in response to GLP-2 treatment. In conclusion, E. bovis reduces gene expression of TJ proteins primarily in cecum of calves 28d postinfection, and GLP-2 increases expression of selected TJ genes in intestinal tissues. Use of GLP-2 to improve gut barrier function in ruminants during pathogen-induced diarrhea warrants additional study.

The effect of citrus-derived oil on bovine blood neutrophil function and gene expression in vitro.

Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing. Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity or duration of mastitis. To our knowledge, effect of cold-pressed terpeneless Valencia orange oil (TCO) on bovine PMNL function has not been elucidated. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood PMNL chemotaxis and phagocytosis capabilities and the expression of genes involved in inflammatory response in vitro. Polymorphonuclear leukocytes were isolated from jugular blood of 12 Holstein cows in mid-lactation and were incubated with 0.0 or 0.01% TCO for 120min at 37°C and 5% CO2, and phagocytosis (2×10(6) PMNL) and chemotaxis (6×10(6) PMNL) assays were then performed in vitro. For gene expression, RNA was extracted from incubated PMNL (6×10(6) PMNL), and gene expression was analyzed using quantitative PCR. The supernatant was stored at -80°C for analysis of tumor necrosis factor-α. Data were analyzed using a general linear mixed model with cow and treatment (i.e., control or TCO) in the model statement. In vitro supplementation of 0.01% of TCO increased the chemotactic ability to IL-8 by 47%; however, migration of PMNL to complement 5a was not altered. Treatment did not affect the production of tumor necrosis factor-α by PMNL. Expression of proinflammatory genes (i.e., SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins was not altered by incubation of PMNL with TCO. However, downregulation of TLR2 [fold change (FC=treatment/control)=-2.14], NFKBIA (FC=1.82), IL1B (FC=-2.16), TNFA (FC=-9.43), and SOD2 (FC=-1.57) was observed for PMNL incubated with TCO when compared with controls. Interestingly, expression of IL10, a well-known antiinflammatory cytokine, was also downregulated (FC=-3.78), whereas expression of IL8 (FC=1.93), a gene coding for the cytokine IL-8 known for its chemotactic function, tended to be upregulated in PMNL incubated with TCO. Incubation of PMNL with TCO enhanced PMNL chemotaxis in vitro. The expression of genes involved in the inflammatory response was primarily downregulated. Results showed that 0.01% TCO did not impair the function of PMNL in vitro. Future studies investigating the use of TCO as an alternative therapy for treatment of mastitis, including dose and duration, for cows during lactation are warranted.

Dynamics of Progesterone, TNF- α , and a Metabolite of PGF2 α in Blood Plasma of Beef Cows following Embryo Transfer.

Lactating beef cows previously synchronized for estrus (d 0) were assigned to four treatments to assess their effectiveness in increasing blood progesterone (P4) and its effects on tumor necrosis factor-α (TNF-α) and prostaglandin F2α (PGF2α) after the transfer of embryos. At the time of transfer (d 7), cows received no treatment (control; n = 16), a controlled internal drug releasing device (CIDR; n = 16), human chorionic gonadotropin (hCG; n = 15), or gonadotropin releasing hormone (GnRH; n = 15). Blood samples were taken on d 7, 14, and 21 for analysis of P4 and tumor necrosis factor-α (TNF-α). Blood was collected (every 15 min for 2 h) in half the animals in each treatment group on d 14 and the remaining half on d 21 for analysis of prostaglandin F2α metabolite (PGFM). Retention rates were 56.2, 62.5, 46.7, and 13.3% for cows in the control, CIDR, hCG, and GnRH groups, respectively. Progesterone was greater (P ≤ 0.05) in cows receiving hCG compared to others on d 14. Progesterone in all treatment groups increased from d 7 to d 14 and declined (P ≤ 0.05) from d 14 to d 21. Contrary to pregnant cows, P4 and TNF-α declined from d 7 to d 21 in nonpregnant cows (P ≤ 0.05). Although PGFM increased by d 21, there was no difference between pregnant and nonpregnant cows.

Identification and characterization of a nuclear factor-κ B-p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture.

Hepatic responses to proinflammatory signals are controlled by the activation of several transcription factors, including, nuclear factor-κ B (NF-κB). In this study, hepatocytes prepared from suckling pigs and maintained in serum-free monolayer culture were used to define a novel proinflammatory cytokine-specific NF-κB subunit modification. The immunoreactive p65 protein was detected by Western blot analysis at the appropriate molecular weight in the cytosol of control cultures and those incubated with tumor necrosis factor-α (TNF). However, in nuclei, the p65 antisera cross-reacted with a protein of approximately 38 kDa (termed p38) after TNF addition, which was not observed in the cytosol of control or cytokine-treated cells. Specifically, incubation with TNF also resulted in phosphorylation (P < 0.05) of the inhibitor complex protein (IκB), whereas incubation with other cytokines, IL-6, IL-17a, or oncostatin M was not associated with either phosphorylation of IκB or nuclear translocation of p65. Intracellular endothelial nitric oxide synthase was deceased (P < 0.05) and plasminogen activator inhibitor-1 secretion was increased (P < 0.05) after TNF incubation. The TNF-induced p38 protein was purified from hepatocyte nuclei by immunoprecipitation, concentrated by electrophoresis, and subsequently analyzed by mass spectrometry. Ten unique NF-κB p65 peptides were identified after digestion with trypsin and chymotrypsin; however, all were mapped to the N-terminus and within the first 310 amino acid residues of the intact p65 protein. Although low molecular weight immunoreactive p65 molecules were previously observed in various human and rodent systems, this is the first report to positively identify the p38 fragment within hepatocyte nuclei or after specific cytokine (TNF) induction.

The use of dermal fibroblasts as a predictive tool of the toll-like receptor 4 response pathway and its development in Holstein heifers.

The innate immune system comprises the host's first line of defense against invading pathogens, and variation in the magnitude of this response between animals has been shown to affect susceptibility to mastitis. The toll-like receptor (TLR) family of proteins initiates the response to invading bacteria, specifically with TLR4 recognizing lipopolysaccharide (LPS) of gram-negative microbes. The underlying genetic variation in the TLR4 pathway leading to differential response is not well understood; therefore, the objective of this work was to determine the efficacy in which the response to LPS by dermal fibroblasts could be used to predict the actual systemic response of that animal to an intravenous endotoxin challenge. To accomplish this, dermal fibroblasts were isolated from 15 Holstein heifers at 5, 11, and 16 mo of age and exposed to either LPS or IL-1ß; then, the production of IL-8 in medium was quantified by ELISA. Animals were ranked based upon the magnitude of the fibroblast IL-8 response, and 8 heifers were selected [4 low responders (LR) and 4 high responders (HR)] for challenge with an intravenous bolus dose (0.5 µg/kg of body weight) of LPS. Overall, between-animal variation in fibroblast IL-8 production following LPS or IL-1ß was high, indicating appreciable differences in the TLR4 pathway of the animals. Ranking of the fibroblast responses was consistent across the 3 sampling times for each animal; however, the absolute response increased, and the age at which the fibroblasts were obtained was consistent with the potential for age-related changes in cell function to affect immune function processes. Following systemic LPS challenge, HR heifers had higher plasma concentrations of tumor necrosis factor-α and IL-8 than LR heifers. However, LR heifers had a stronger febrile response than HR heifers. The use of dermal fibroblasts under laboratory conditions appears to represent a practical model for predicting the innate immune response in vivo and could act as an important tool in mapping genetic differences of the TLR4 pathway.

Modeling the effects of estradiol and progesterone on the acute phase proinflammatory axis: variability in tumor necrosis factor-α, nitric oxide, and xanthine oxidase responses to endotoxin challenge in steers.

The severity of host response in some diseases differs between sexes, and this dimorphism has been attributed to the immunomodulating effects of reproductive steroid hormones. In females, susceptibility to disease stress has been associated with reproductive status and attributed to prevailing progesterone (P4) or estrogen concentrations during different estrous cycle phases. Our objective was to clarify and define the effect of P4 or 17ß-estradiol (E2) on the acute proinflammatory component of the innate immune system by administering these hormones to steers and evaluating initial and tolerance-associated concentration patterns of circulating proinflammatory immune response mediators after two consecutive lipopolysaccharide (LPS) challenges (LPS1 and LPS2, 6 d apart; 2.5 µg/kg BW, intravenously, Escherichia coli 055:B5). Plasma concentrations of the proinflammatory initiation cytokine tumor necrosis factor-α (TNF-α), nitrate+nitrite [NO(x), estimate of nitric oxide (NO) production], haptoglobin (HG; acute phase protein) and plasma xanthine oxidase activity (mediator of superoxide production) were measured. Crossbred steers (392 ± 7 kg) were fed a forage-concentrate diet (15% CP) to appetite and assigned to control (C; n = 7), P4 (n = 8), or E2 (n = 5) treatment. Jugular blood samples were obtained at 0, 1, 2, 3, 4, 7, and 24 h relative to each of the two LPS injections. For each proinflammatory biomarker, the area under the time by concentration curve (AUC) was used to evaluate and compare responses to the LPS challenge. Treatment with E2 disrupted LPS tolerance as observed in augmented plasma TNF-α (P < 0.01) and NO(x) (P < 0.01) responses to LPS2. Compared with C, P4 treatment decreased plasma NO(x) AUC after LPS2 (P < 0.05) and tended to reduce TNF-α AUC after LPS1 (P = 0.08). Plasma xanthine oxidase activity AUC was increased (P < 0.01) over C by E2 treatment after both LPS1 and LPS2. HG response to LPS1 within 24 h was not affected by any treatment. However, 6 d after LPS1 plasma HG concentration remained higher (P < 0.01) in steers treated with E2 than with C or P4. Results indicate that in cattle, P4 and E2, respectively, attenuate or amplify the response to LPS challenge at several points critical to the regulation of the progression of the proinflammatory cascade.

Tumor necrosis factor-alpha and acute-phase proteins in early pregnant ewes after challenge with peptidoglycan-polysaccharide.

Bacterial infection shortly after mating interferes with establishment of pregnancy. Injection of peptidoglycan-polysaccharide (PG-PS), a component of gram-positive bacteria, into sheep on day 5 after mating reduces pregnancy rate. Experiments were designed to evaluate the acute-phase response (APR) in ewes to injection of PG-PS on day 5 after mating (day 0). Catheters were inserted into the jugular and posterior vena cava on day 4. On day 5, ewes were challenged with saline or 30 microg/kg body weight (BW) PG-PS (Exp 1) or 60 microg/kg BW PG-PS (Exp 2). Blood samples were collected every 15 min for 6 h (Exp 1) and every 15 min for 2 h, hourly for 12 h, and at 24, 36, and 48 h (Exp 2). Body temperature and clinical signs of infection were monitored in Exp 2. Plasma was assayed for concentrations of a pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha); 2 APR proteins, serum amyloid A (SAA) and haptoglobin (Hp); and progesterone (P(4)). Ewes injected with 60 microg/kg BW PG-PS exhibited fever, vaginal discharge, loss of appetite, and lethargy. After challenge with either 30 microg/kg or 60 microg/kg BW PG-PS, TNF-alpha increased in the posterior vena cava. Concentrations of SAA and Hp in the jugular increased after challenge with 60 microg/kg BW PG-PS. Only half (5/10) of the ewes treated with 60 microg/kg BW PG-PS had ultrasonically visible embryos, and none of them had functional corpora lutea (CL) (<1 ng/mL of P(4)) on day 21. On the other hand, 8/9 (88.9%) control ewes had visible embryos and all had functional CL on day 21. Using logistic regression, pregnancy on day 21 was predicted to depend on concentrations of TNF-alpha and Hp on day 5 and concentration of P(4) on day 14. In summary, injection of PG-PS on day 5 after mating resulted in fever; increased concentrations of TNF-alpha, Hp, and SAA on the day of and the day after the PG-PS challenge; and decreased concentrations of P(4) on days 14 and 21. These factors were related to failure to establish pregnancy.

Variability in tumor necrosis factor-alpha, nitric oxide, and xanthine oxidase responses to endotoxin challenge in heifers: effect of estrous cycle stage.

The severity of host response to some disease agents differs between sexes and this dimorphism has been attributed to the immunomodulating effects of steroid hormones. Our objective was to determine in heifers whether the phase of estrous cycle affected immune response mediators after endotoxin challenge (LPS, 2.5microg/kg BW, i.v.). Sixteen beef heifers (426+/-9kg) were reproductively synchronized with the two-injection protocol of dinoprost tromethamine (Lutalyse, Pfizer) to establish diestrus and estrus stages of the estrous cycle. Heifers were challenged with LPS on day 3 (E, estrus; n=8) or day 10 (D, diestrus, n=8) after the last i.m. injection of Lutalyse. In all heifers, plasma concentrations of tumor necrosis factor-alpha (TNF-alpha) peaked 2h after LPS treatment (P<0.01) and returned to basal level by 7h. However, the integrated TNF-alpha response (area under the time x concentration curve, AUC) was greater in E than in D (P<0.05). Plasma concentrations of nitrate+nitrite (NO(x), an estimate of NO production) increased (P<0.01) in all heifers at 7 and 24h after LPS; plasma NO(x) AUC after LPS was greater in E than D (P<0.01). Plasma xanthine oxidase activity (XO, a mediator of superoxide production) responses were also greater in E than D (P<0.05). A companion LPS challenge study in steers validated that the protocol for and use of Lutalyse did not affect any of the immune parameters studied in heifers in response to LPS. Results indicate that the underlying physiological attributes of the estrus and diestrus phases of the estrous cycle constitute a major source of variability in the magnitude of proinflammatory response to bacterial toxins like LPS.

Critical control points in the impact of the proinflammatory immune response on growth and metabolism.

Intrinsic in the equation for successful animal production is the efficiency of nutrient use for assimilation into useful animal-derived products. However, when young growing animals encounter various stressors that activate the proinflammatory response (PR), the biochemical effects of the resulting cascade of PR mediators [cytokines, prostaglandin and prosta-cyclin derivatives, nitric oxide (NO), superoxide anion (O2(.-)), etc.] override the regulatory signals normally ascribed to anabolic tissue accretion and growth. The efficiency of energy and nutrient use will proportionally decrease for growth rate due to the redirection of nutrient use to support immune defense processes. These proinflammatory events can develop in association with infectious disease but also are apparent in and a part of the natural response to birth, parturition, and weaning. If growth patterns are tracked during the PR, growth deficits are often apparent. Some growth deficits are relatively transient in duration, whereas others are quite long lasting, persisting although traditional clinical markers of PR are no longer evident. Recent evidence indicates that the PR cascades initiated by cytokines like tumor necrosis factor-alpha play a major role in these growth deficits. Perturbations in mitochondrial energetics and NO and O2(.-) interactions further affect metabolic balance. Free radicals and reactive nitrogen intermediates interact with select molecular targets in proteins (i.e., enzymes, histone proteins, and signal transduction proteins), causing the nitration and nitrosylation of select amino acids. If these posttranslational modifications occur in proteins associated with control points critical in metabolic stability, the resulting altered protein structure blocks its functionality. Attenuation of these overt posttranslational protein modifications at their site of production offers a strategy to minimize their detrimental impact while preserving needed cytokine, NO, and O2(.-) functions.

Limit-feeding a high-energy diet to meet energy requirements in the dry period alters plasma metabolite concentrations but does not affect intake or milk production in early lactation.

Limit-feeding dry cows a high-energy diet may enable adequate energy intake to be sustained as parturition approaches, thus reducing the extent of negative energy balance after parturition. Our objective was to evaluate the effect of dry period feeding strategy on plasma concentrations of hormones and metabolites that reflect energy status. Multiparous Holstein cows (n = 18) were dried off 45 d before expected parturition, paired by expected calving date, parity, and previous lactation milk yield, and randomly assigned to 1 of 2 dry-period diets formulated to meet nutrient requirements at ad libitum or limited intakes. All cows were fed the same diet for ad libitum intake after parturition. Prepartum dry matter intake (DMI) for limit-fed cows was 9.4 kg/d vs. 13.7 kg/d for cows fed ad libitum. During the dry period, limit-fed cows consumed enough feed to meet calculated energy requirements, and ad libitum-fed cows were in positive calculated net energy for lactation (NE(L)) balance (0.02 vs. 6.37 Mcal/d, respectively). After parturition, milk yield, milk protein concentration, DMI, body condition score, and body weight were not affected by the prepartum treatments. Cows limit fed during the dry period had a less-negative calculated energy balance during wk 1 postpartum. Milk fat concentration and yield were greater for the ad libitum treatment during wk 1 but were lower in wk 2 and 3 postpartum. Plasma insulin and glucose concentrations decreased after calving. Plasma insulin concentration was greater in ad libitum-fed cows on d -2 relative to calving, but did not differ by dietary treatment at other times. Plasma glucose concentrations were lower before and after parturition for cows limit-fed during the dry period. Plasma nonesterified fatty acid concentrations peaked after parturition on d 1 and 4 for the limit-fed and ad libitum treatments, respectively, and were greater for limit-fed cows on d -18, -9, -5, and -2. Plasma tumor necrosis factor-alpha concentrations did not differ by treatment in either the pre- or postpartum period, but tended to decrease after parturition. Apart from a reduction in body energy loss in the first week after calving, limit feeding a higher NE(L) diet during the dry period had little effect on intake and milk production during the first month of lactation.

Short communication: suppressor of cytokine signaling-2 mRNA increases after parturition in the liver of dairy cows.

After parturition, the somatotropic axis of the dairy cow is uncoupled, partly because of reduced concentration of liver-specific GH receptor (GHR) 1A. Estradiol-17 beta(E(2)) concentrations increase at parturition and E(2) upregulates suppressors of cytokine signaling-2 (SOCS-2) mRNA expression, potentially inhibiting GH signaling. Therefore, we hypothesized that SOCS-2 mRNA is upregulated after parturition. Multiparous Holstein cows (n = 18) were dried off 45 d before expected parturition and fed diets to meet nutrient requirements at ad libitum or limited dry matter intake during the dry period. All cows were fed the same diet ad libitum from calving until 4 wk after parturition. Blood samples were collected weekly and more frequently near parturition. Liver biopsies obtained at - 21, - 7, 2, and 28 d relative to parturition were assessed for SOCS-2 and GHR 1A mRNA by quantitative real-time reverse-transcription PCR. The relative amount of SOCS-2 mRNA increased after parturition with both treatments and was greater on d 2 for cows limit-fed during the dry period compared with cows fed at ad libitum dry matter intake. Plasma E(2) concentrations increased on d - 13, - 5 and 1 relative to parturition and the increases were greater in limit-fed cows. Plasma GH concentration was greater for limit-fed cows and increased after parturition in all cows. The amount of GHR 1A mRNA did not differ between diets but decreased on d 2. In addition to reduced GHR 1A, increased SOCS-2 mRNA after parturition, perhaps because of increased E(2), may further uncouple GH signaling in the liver of the transition dairy cow.

Modeling growth factor activity during proinflammatory stress: methodological considerations in assessing cytokine modulation of IGF binding proteins released by cultured bovine kidney epithelial cells.

The present research was conducted to model potential mechanisms through which IGFBPs might be affected by a key proinflammatory response initiating cytokine tumor necrosis factor (TNF-)-alpha. Madin-Darby bovine kidney epithelial (MDBK) cells, known to release IGFBPs in response to several stimuli, were grown under several conditions and challenged with forskolin (F) or recombinant TNF-alpha for 24h. Forskolin increased IGFBP-3 gene expression and media content of BP-3 protein. TNF-alpha increased basal and augmented F-mediated IGFBP-3 gene expression. However, TNF-alpha effects on the measurable media content of IGFBPs were influenced by culture conditions; in the absence of added protease inhibitors (PIs) or sufficient media albumin concentration (high BSA, 1mg/ml), the effect of TNF-alpha was to decrease (P<0.02) measurable IGFBPs. In the presence of PI and high BSA, media IGFBP-3 levels were shown to be increased by TNF-alpha consistent with the gene expression data. Changes in media IGFBP-3 protease activity were examined further to explain the observed effects of TNF-alpha on production and destruction of IGFBPs in media. When recombinant human IGFBP-3 (500 ng/ml) was added to PI-free, low BSA 100 microg/ml) media from TNF-treated MDBK cells, less than 10% of the BP-3 was recognizable by Western blot in 30 min; conversely, inclusion of High BSA and PI in media resulted in attenuation of the protease effect on the IGFBPs. The data suggest that the MDBK model of cellular response to proinflammatory stimulus is affected by culture conditions and that TNF-alpha affects media content of IGFBPs through effects on IGFBP gene expression coupled with degradation of IGFBPs via enhanced proteolytic enzyme release.