Mild heat stress-induced adaptive immune response in blood mononuclear cells and leukocytes from mesenteric lymph nodes of primiparous lactating Holstein cows.

Heat stress negatively affects the metabolism and physiology of the bovine gut. However, it is not known whether heat stress induces an inflammatory response in mesenteric lymph nodes (MLN), the primary origin of gut immune cells, and thus contributes to inflammatory processes in the circulation. Therefore, our objective was to elucidate the effects of chronic heat stress on the systemic activation of acute-phase response in blood, proinflammatory cytokine production in peripheral blood mononuclear cells (PBMC), and the activation of the toll-like receptor signaling (TLR) 2/4 pathway in MLN leucocytes and their chemokines and chemokine receptor profiles in Holstein cows. Primiparous Holstein cows (n = 30; 169 ± 9 d in milk) were exposed to a temperature-humidity index (THI) of 60 [16°C, 63% relative humidity (RH)] for 6 d. Thereafter, cows were evenly assigned to 3 groups: heat-stressed (HS; 28°C, 50% RH, THI = 76), control (CON; 16°C, 69% RH, THI = 60), or pair-feeding (PF; 16°C, 69% RH, THI = 60) for 7 d. On d 6, PBMC were isolated and on d 7 MLN. Plasma haptoglobin, TNFα, and IFNγ concentrations increased more in HS than CON cows. Concomitantly, TNFA mRNA abundance was higher in PBMC and MLN leucocytes of HS than PF cows, whereas IFNG mRNA abundance tended to be higher in MLN leucocytes of HS than PF cows, but not for chemokines (CCL20, CCL25) or chemokine receptors (ITGB7, CCR6, CCR7, CCR9). Furthermore, the TLR2 protein expression tended to be more abundant in MLN leucocytes of HS than PF cows. These results suggest that heat stress induced an adaptive immune response in blood, PBMC, and MLN leukocytes involving the acute-phase protein haptoglobin, proinflammatory cytokine production, and TLR2 signaling in MLN leucocytes. However, chemokines regulating the leucocyte trafficking between MLN and gut seem not to be involved in the adaptive immune response to heat stress.

The Effects of Oral Quercetin Supplementation on Splanchnic Glucose Metabolism in 1-Week-Old Calves Depend on Diet after Birth.

BACKGROUND: Inadequate colostrum supply results in insufficient intake of macronutrients and bioactive factors, thereby impairing gastrointestinal development and the maturation of glucose metabolism in neonatal calves. The flavonoid quercetin has been shown to have health-promoting properties, including effects in diabetic animals. However, quercetin interacts with intestinal glucose absorption and might therefore exert negative effects in neonates. OBJECTIVE: We evaluated the interaction between neonatal diet and quercetin feeding on splanchnic glucose metabolism in neonatal calves. METHODS: Calves (n = 28) were assigned to 4 groups and fed either colostrum or a milk-based formula on days 1 and 2 and supplemented daily with 148 µmol quercetin aglycone/kg body weight [colostrum with quercetin (CQ+)/formula with quercetin (FQ+)] or without this substance [colostrum without quercetin (CQ-)/formula with quercetin (FQ-)] from days 2-8. From day 3 onward, all calves received milk replacer. A xylose absorption test was performed on day 3, and on day 7, blood samples were collected to study glucose first-pass uptake after [(13)C6]-glucose feeding and intravenous [6,6-(2)H2]-glucose bolus injection. Plasma concentrations of metabolites and hormones were measured by taking additional blood samples. A biopsy specimen of the liver was harvested on day 8 to measure the mRNA expression of gluconeogenic enzymes. RESULTS: Higher postprandial plasma concentrations of glucose, lactate, urea, adrenaline, noradrenaline, insulin, and glucagon on day 7 in colostrum-fed calves indicate that metabolic processes were stimulated. Postabsorptive xylose and glucose plasma concentrations each increased by an additional 26%, and splanchnic glucose turnover decreased by 35% in colostrum-fed calves, suggesting improved glucose absorption and lower splanchnic glucose utilization in colostrum-fed calves. Quercetin supplementation resulted in higher noradrenaline concentrations and enhanced peak absorption and oxidation of [(13)C6]-glucose by 10%. Liver mitochondrial phosphoenolpyruvate carboxykinase mRNA abundance was reduced by 34% in colostrum-deprived calves. CONCLUSIONS: Feeding colostrum during the first 2 d of life is crucial for maturation of splanchnic glucose metabolism in calves. Supplementing quercetin improves gastrointestinal absorption capacity, particularly in colostrum-deprived calves.

Effects of a 6-wk intraduodenal supplementation with quercetin on energy metabolism and indicators of liver damage in periparturient dairy cows.

Periparturient dairy cows experience metabolic challenges that result in a negative energy balance (EB) and a range of postpartum health problems. To compensate for the negative EB, cows mobilize fatty acids from adipose tissues, which can lead to fatty liver disease, a periparturient metabolic disorder. Flavonoids, such as quercetin (Q), are polyphenolic substances found in all higher plants and have hepatoprotective potential and the ability to prevent or reduce lipid accumulation in the liver. In ruminants, few studies on the metabolic effects of Q are available, and thus this study was conducted to determine whether Q has beneficial effects on EB, lipid metabolism, and hepatoprotective effects in periparturient dairy cows. Quercetin was supplemented intraduodenally to circumvent Q degradation in the rumen. Cows (n=10) with duodenal fistulas were monitored for 7wk. Beginning 3wk before expected calving, 5 cows were treated with 100mg of quercetin dihydrate per kilogram of body weight daily in a 0.9% sodium chloride solution for a total period of 6wk, whereas the control cows received only the sodium chloride solution. The plasma flavonoid levels were higher in the Q-treated cows than in the control cows. A tendency for higher postpartum (pp) than antepartum (ap) plasma flavonoid levels was observed in the Q-treated cows than in the controls, which was potentially caused by a reduced capacity to metabolize Q. However, the metabolic status of the Q-treated cows did not differ from that of the control cows. The pp increases in plasma aspartate aminotransferase and glutamate dehydrogenase activities were less in the Q-treated cows than in the control cows. The Q had no effect on energy expenditures, but from ap to pp the cows had a slight decline in respiratory quotients. Irrespective of the treatment group, the oxidation of fat peaked after calving, suggesting that the increase occurred because of an increased supply of fatty acids from lipomobilization. In conclusion, supplementation with Q resulted in lower pp plasma aminotransferase and glutamate dehydrogenase, which indicated reduced liver damage. However, the direct effects of Q on the liver and the implications for animal performance remain to be investigated.

Activation of indoleamine 2,3-dioxygenase by LPS in a porcine model.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway, and its increased activation is associated with immunologic disorders. Because the specific role of IDO activation is not yet completely clear, the aim of the present study was to establish a pig model of IDO activation for further research. The activation of IDO in pigs was induced experimentally by LPS stimulation in vivo and ex vivo. IDO activation was characterized by measuring Trp, Trp metabolites and IDO protein expression in blood, liver, lung, muscle and different brain areas. The results show that the in vivo LPS administration induced increased plasma concentrations of TNF-α and IL-10, a depletion of Trp and an increase of Kyn, indicating an elevated enzymatic activity of IDO. This was supported by an LPS-induced IDO protein expression in blood, liver and lung. The ex vivo LPS stimulation also resulted in increased TNF-α concentrations and an IDO activation, characterized by an increase of Trp metabolites and IDO protein expression. In conclusion, our data emphasize that the LPS stimulation is a suitable model for IDO activation in the domestic pig, which provides a basis for further research on immunoregulatory IDO functions.

Tumour necrosis factor receptor deficiency alters anxiety-like behavioural and neuroendocrine stress responses of mice.

Tumour necrosis factor (TNF)-alpha is known to be involved in anxiety and the regulation of the hypothalamic-pituitary-adrenal axis. To examine the role of its receptors in neuroendocrine immunomodulation, we studied behaviour, corticosterone production and T-cell activation in mice with a C57BL/6J background and deficient for one or both TNF receptors (TNFR1-/-, TNFR2-/-, and TNFR1+2-/-) compared to wildtype C57BL/6J mice with and without psychological stress. Stress was induced by social disruption (SDR), and anxiety-like behaviour was examined using the elevated plus maze (EPM). Anxiety of unstressed TNFR1+2-/- mice was increased compared to C57BL/6J mice as shown by reduced ratios of entries into open arms relatively to total entries. SDR-stressed TNFR1+2-/- mice showed reduced ratios of entries into open arms relatively to total entries, reduced ratios of distances walked in open relatively to distances walked in both arms and reduced time in open arms compared to C57BL/6J mice. Locomotor activity of unstressed and SDR-stressed TNFR1-/- and TNFR2-/- mice was reduced. Serum corticosterone concentrations of control mice do not differ between mouse strains. However, TNFR1+2-/- mice had significantly higher corticosterone concentrations than C57BL/6J mice after SDR. EPM testing significantly increased corticosterone concentrations in all strains. Mitogen-induced activation-marker expression was reduced in TNFR1-/- T-helper cells under control and stress conditions, while activation marker expression of TNFR2-/- and TNFR1+2-/- cells was only slightly affected by stress compared to C57BL/6J T cells. Our study suggests that both TNF receptors contribute to anxiety-like behaviour and corticosterone responses, whereas TNFR1 has a larger impact on T-cell activation.

High and low protein∶ carbohydrate dietary ratios during gestation alter maternal-fetal cortisol regulation in pigs.

Imbalanced maternal nutrition during gestation can cause alterations of the hypothalamic-pituitary-adrenal (HPA) system in offspring. The present study investigated the effects of maternal low- and high-protein diets during gestation in pigs on the maternal-fetal HPA regulation and expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-HSD1 and 11ß-HSD2) and c-fos mRNAs in the placenta and fetal brain. Twenty-seven German Landrace sows were fed diets with high (HP, 30%), low (LP, 6.5%) or adequate (AP, 12.1%) protein levels made isoenergetic by varying the carbohydrate levels. On gestational day 94, fetuses were recovered under general anesthesia for the collection of blood, brain and placenta samples. The LP diet in sows increased salivary cortisol levels during gestation compared to the HP and AP sows and caused an increase of placental GR and c-fos mRNA expression. However, the diurnal rhythm of plasma cortisol was disturbed in both LP and HP sows. Total plasma cortisol concentrations in the umbilical cord vessels were elevated in fetuses from HP sows, whereas corticosteroid-binding globulin levels were decreased in LP fetuses. In the hypothalamus, LP fetuses displayed an enhanced mRNA expression of 11ß-HSD1 and a reduced expression of c-fos. Additionally, the 11ß-HSD2 mRNA expression was decreased in both LP and HP fetuses. The present results suggest that both low and high protein∶carbohydrate dietary ratios during gestation may alter the expression of genes encoding key determinants of glucocorticoid hormone action in the fetus with potential long-lasting consequences for stress adaptation and health.

Alterations in anxiety-like behavior following knockout of the uncoupling protein 2 (ucp2) gene in mice.

AIMS: Uncoupling protein 2 (UCP2) is a mitochondrial protein that reduces oxidative stress and has a protective function in chronic inflammatory diseases such as multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. UCP2 is strongly expressed in areas implicated in the central regulation of stress and anxiety. Therefore, we compared the neuroendocrine regulation of stress responses, immunity and behavior in UCP2-deficient and wildtype C57BL/6J mice under psychological stress. MAIN METHODS: Stress was induced by social disruption (SDR) and anxiety-like behavior was examined using the elevated plus-maze (EPM). Serum corticosterone was determined by radioimmunoassay and brain neurotransmitter concentrations were analyzed by HPLC of whole brain homogenates. T cell activation and tumor necrosis factor (TNF)-α production of mitogen-activated splenocytes were determined in vitro by flow cytometry staining of CD25, CD69 and CD71 on CD4 cells, and ELISA, respectively. The influence of corticosterone on UCP2 expression of splenocytes was analyzed by Western blot. KEY FINDINGS: At baseline, UCP2-deficient mice were significantly more anxious in the EPM than wildtype mice, and this phenotype was exacerbated after SDR stress. The corticosterone response after SDR+EPM was reduced in UCP2-deficient mice compared to wildtype mice. Corticosterone in turn downregulates UCP2 expression in splenocyte cultures of wildtype mice at physiological concentrations. Dopaminergic and serotonergic turnovers were increased in UCP2-deficient mice after SDR+EPM. While T-helper cell activation-marker expression was reduced, TNF-α production was increased in UCP2-deficient mice after SDR+EPM. SIGNIFICANCE: Our study shows that UCP2 is involved in anxiety-like behavior and modulates neuroendocrine and immune responses after stress.

Psychological stress-induced, IDO1-dependent tryptophan catabolism: implications on immunosuppression in mice and humans.

It is increasingly recognized that psychological stress influences inflammatory responses and mood. Here, we investigated whether psychological stress (combined acoustic and restraint stress) activates the tryptophan (Trp) catabolizing enzyme indoleamine 2,3-dioxygenase 1(IDO1) and thereby alters the immune homeostasis and behavior in mice. We measured IDO1 mRNA expression and plasma levels of Trp catabolites after a single 2-h stress session and in repeatedly stressed (4.5-days stress, 2-h twice a day) naïve BALB/c mice. A role of cytokines in acute stress-induced IDO1 activation was studied after IFNgamma and TNFalpha blockade and in IDO1(-/-) mice. RU486 and 1-Methyl-L-tryptophan (1-MT) were used to study role of glucocorticoids and IDO1 on Trp depletion in altering the immune and behavioral response in repeatedly stressed animals. Clinical relevance was addressed by analyzing IDO1 activity in patients expecting abdominal surgery. Acute stress increased the IDO1 mRNA expression in brain, lung, spleen and Peyer's patches (max. 14.1+/-4.9-fold in brain 6-h after stress) and resulted in a transient depletion of Trp (-25.2+/-6.6%) and serotonin (-27.3+/-4.6%) from the plasma measured 6-h after stress while kynurenine levels increased 6-h later (11.2+/-9.3%). IDO1 mRNA up-regulation was blocked by anti-TNFalpha and anti-IFNgamma treatment. Continuous IDO1 blockade by 1-MT but not RU486 treatment normalized the anti-bacterial defense and attenuated increased IL-10 inducibility in splenocytes after repeated stress as it reduced the loss of body weight and behavioral alterations. Moreover, kynurenic acid which remained increased in 1-MT treated repeatedly stressed mice was identified to reduce the TNFalpha inducibility of splenocytes in vitro and in vivo. Thus, psychological stress stimulates cytokine-driven IDO1 activation and Trp depletion which seems to have a central role for developing stress-induced immunosuppression and behavioral alteration. Since patients showed Trp catabolism already prior to surgery, IDO is also a possible target enzyme for humans modulating immune homeostasis and mood.

Alteration of reproductive hormone levels in pregnant sows induced by repeated ACTH application and its possible influence on pre- and post-natal hormone secretion of piglets.

Prenatal stress has been seen as a reason for reproductive failures in pig offspring mostly originated or mediated by changed maternal functions. Experiments were conducted in pregnant gilts (n=32) to characterize effects of elevated maternal glucocorticoids on the secretion of reproductive hormones (LH, progesterone) during the 1st (EXP 1), 2nd (EXP 2) and 3rd (EXP 3) trimester of pregnancy (TP). Transiently elevated cortisol release was repeatedly achieved by application of 100 IU adenocorticotropic hormone (ACTH) (Synacthen Depot) six times every second day beginning either on day 28 (EXP 1), day 49 (EXP 2) or day 75 of pregnancy (EXP 3). Glucocorticoid concentrations were examined in umbilical blood vessels of fetuses which mothers were subjected to ACTH at 2nd and 3rd TP (EXP 4). Furthermore, the pituitary function of newborn piglets of EXP 2 was checked by a LH-RH challenge test. In sows, LH concentrations were at low basal level (0.1-0.2 ng/ml) but with pulsatory release pattern during each TP. The number of LH pulses/6 h (LSM +/- SE) of saline treated Controls increased with ongoing pregnancy and decreased to the 3rd TP (1.3 +/- 0.2 in EXP 1 vs. 2.0 +/- 0.1 in EXP 2 vs. 1.4 +/- 0.1 in EXP 3, p<0.05). After ACTH treatment the number of LH pulses left unchanged in Experiments 1 and 2 (1.3 +/- 0.2 and 1.5 +/- 0.1) and decreased in EXP 3 (0.8 +/- 0.2, p<0.05). Differences (p<0.05) were obtained comparing the LH pulse number of ACTH and saline treated sows at the 2nd and 3rd TP. Moreover, areas under the curve (AUC) of each LH pulse and of LH over baseline were significantly reduced by treatment. Levels of progesterone increased (p<0.05) for 150 to 170 min after each ACTH application both in EXP 1 and EXP 2, but not in EXP 3. The mean progesterone concentration was different between trimesters, and ACTH and Controls (1st TP: 30.0 +/- 0.9 and 24.4 +/- 0.7 ng/ml; 2nd TP: 35.5 +/- 0.9 and 29.1 +/- 1.0 ng/ml; 3rd TP: 13.6 +/- 0.2 and 13.1 +/- 0.1 ng/ml; p<0.05). In fetuses (n=87) recovered 3 h after ACTH or saline (EXP 4), the plasma cortisol concentrations were significantly increased in umbilical vein (93.7 +/- 5.5 vs. 47.0 +/- 5.3 nmol/l) and artery (95.7 +/- 5.4 vs. 66.4 +/- 5.4 nmol/l), and in periphery (46.8 +/- 5.3 vs. 27.1 +/- 5.3 nmol/l) compared to controls. Plasma ACTH concentrations, however, did not differ in fetuses of both treatment groups. Postnatal LH-RH challenge tests (1st and 28th day post partum) induced LH surges in female piglets (n=67) both of ACTH and saline treated sows, but did not differ between groups (1st day: 7.2 +/- 0.8 vs. 8.1 +/- 0.7 ng/ml; 28th day: 10.5 +/- 1.7 vs. 13.6 +/- 2.2 ng/ml). However, basal LH of piglets whose mothers were submitted to ACTH during 2nd TP was lower on 1st day (1.7 +/- 0.2 vs. 2.3 +/- 0.2 ng/ml, p<0.05) but not on 28th day (1.0 +/- 0.2 vs. 1.1 +/- 0.2 ng/ml). However in both groups, the basal LH was always higher on 1st as on 28th day (p<0.05). Thus, chronic intermittent ACTH administration is able to influence the release pattern of maternal reproductive hormones. However, these findings demonstrate that these effects are dependent on the stage of pregnancy. Furthermore, it was shown that maternal cortisol can cross the placenta during gestation and thus may affect maternal-fetal interactions and, as a result, reproductive function of offspring.

Stress-related gene expression in brain and adrenal gland of porcine fetuses and neonates.

This study was conducted to examine stress-induced effects on gene expression of specific markers for HPA axis and neuronal activity in fetuses and neonatal pigs. Brain, pituitary gland, and adrenal gland were obtained to determine the mRNA levels for corticotropin-releasing hormone (CRH), CRH receptor 1 (CRHR1), pro-opiomelanocortin (POMC), ACTH receptor (MC2R), c-jun and c-fos. The suitability of these molecular markers was determined in neonatal pigs which were maternally deprived for two hours. It was found that maternal deprivation caused significantly higher transcript levels of c-fos and CRH in brain accompanied by a down-regulation of CRHR1 mRNA and an up-regulation of c-jun in the pituitary gland. To determine the effect of elevated maternal cortisol levels on gene expression of these molecular markers in fetuses, pregnant sows were treated with 100 IU ACTH (Synacthen Depot) s.c. every two days between Day 49 and Day 75 of gestation (normal gestation length 114 days). Animals were killed 48 hours after the last ACTH administration and fetuses of each sow were isolated. The ACTH treatment of sows significantly increased mRNA expression of c-fos but not of CRH in the fetal brain, and significantly decreased MC2R mRNA expression in the adrenal gland. However, HPA axis seems not to be fully developed in Day 77-fetuses because fetal pituitary CRHR1 and POMC mRNA expression was low in most of the fetuses. Although the expression of endocrine regulatory factors was partially incomplete in fetuses at the beginning of the third-trimester, ACTH dependent activation of c-fos mRNA in brain indicates a stress-related increase of neuronal activity. Based on these results it is assumed that prenatal stress in pigs may also have effects on the activity of the HPA axis in the offspring.