Dysregulation of immune responses results in the development of chronic inflammatory conditions. The current frontline therapy, glucocorticoids, are effective immunosuppressive drugs but come with a trade-off of cumulative, debilitating side effects with sustained use. Clearly, alternative drug options with improved safety profiles are urgently needed. Macrophage Migration Inhibitory Factor (MIF) is a pleotropic pro-inflammatory cytokine and integral component of immune and inflammatory responses. MIF counter-regulates the immunosuppressive effects of glucocorticoids and promotes NLRP3 inflammasome activation.1, 2 Elevated MIF is a feature of multiple diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematous. Given the association of increased MIF in serum with multiple disease models, it is considered MIF may be a plausible, specific druggable target in treatment of chronic inflammatory and autoimmune diseases, particularly as a target for glucocorticoid-sparing therapy to reduce the dose or duration of glucocorticoid treatment. The organosulfur isothiocyanate phytochemical sulforaphane (SFN) is extracted from cruciferous vegetables, including broccoli and Brussel sprouts following hydrolysis of its inactive precursor, glucoraphanin. SFN has antioxidant and cancer chemoprotective properties, and promotes NRF2 antioxidant signalling to upregulate the expression of numerous antioxidant enzymes. SFN has been shown to covalently modify MIF with high reactivity and is a potent inhibitor of MIF tautomerase activity. However, to date, no such study has evaluated the role of SFN as a novel inhibitor of MIF-mediated inflammatory pathway activation. Using cell-based assays, we have sought to investigate the role of SFN as an inhibitor of multiple inflammatory pathways which have previously implicated MIF as a possible regulator. Our initial work has examined SFN as an inhibitor of NF-κB activity, inflammasome activation, and evaluated if MIF is required for this effect. RAW264.7 murine macrophage cells stably expressing NF-κB-luciferase reporter construct were pre-treated with SFN (2.5 µM) before the induction of inflammation, via LPS (100ng/mL). For NLRP3 inflammasome activation, cells were subsequently treated with the NLRP3-specific inflammasome activator, nigericin (10 µM). TNF, IFN-β and IL-1β cytokine expression was measured by ELISA and NF-κB activity by luciferase reporter assay. We found SFN is a potent inhibitor of NF-κB activity and inhibits release of the pro-inflammatory cytokine IL-1β through inhibition of NLRP3 inflammasome activation. Finally, co-incubation of SFN with the glucocorticoid dexamethasone significantly suppressed TNF and IFN-β expression, demonstrating steroid sparing activity of SFN in vitro. Thus, SFN may be a suitable treatment for disruption of inflammatory pathways and suggest some of these effects may be mediated through direct interactions with MIF.

The ‘coping hypothesis’ of stereotypic behaviour — that stereotypies are performed as a means of helping the animal to cope with its environment by reducing stress — was tested using two adult female margays (Leopardus wiedii), an endangered neotropical small cat species. Within-individual and between-individual comparisons of the duration of stereotypic pacing and glucocorticoid concentration, measured non-invasively in faeces, indicated that stereotypic pacing did not help the two margays to cope with their captive environment by reducing their physiological stress level. However, hiding appeared to serve as a coping function in the two margays, not by immediately reducing the faecal glucocorticoid concentration, but rather as a long-term effect.

This study evaluated the short-term hormonal and behavioural responses to capture and radio-collar fitting in free-ranging pampas deer (Ozotoceros bezoarticus). Twenty adult deer (eleven females and nine males) were captured in the South Pantanal wetland (Brazil) and equipped with VHF radio-collars (marked deer). Untreated adult deer of the same sex were randomly chosen as the control group (nine females and nine males). On the day following capture, an observer followed all deer for faecal collection and behaviour evaluation. Faecal samples were immediately refrigerated and frozen at-20°C within a maximum of 12 h. Faecal glucocorticoid metabolites (FGM) were measured using an 11-oxoaetiocholanolone enzyme immunoassay. A qualitative behaviour assessment and the consequences of capture were evaluated using pre-defined terminologies and scores. Flight distance was recorded using a range finder. FGM increased from 19-22 h after capture onwards and peak concentrations were five times (median) higher as the respective baseline values. FGM values of marked deer were significantly higher at 22-25 and 25-28 h compared with controls. Marked male but not female deer had significantly higher FGM values at 22-25 and 25-28 h compared with their baseline values. Marked deer were significantly more fearful, less sociable and defensive than controls. The absences of significant increases of FGM in the captured female deer may indicate that females are less prone to capture stress. The significantly more fearful, and less sociable and defensive patterns observed in marked deer may be relevant during capture of lactating females or in areas with high predator pressure.

The welfare and productivity of South American camelids may be affected by stressful events. The purpose of this study was to validate a non-invasive method for stress monitoring using faecal samples and to apply it to evaluate a stressful event, such as confinement. For physiological validation, nine alpacas (Vicugna pacos) and six llamas (Lama glama) were subjected to pharmacological stimulation of their adrenal cortex. Serial faecal samples were collected during 48 h before and after stimulation. During confinement, faecal samples from six llamas were collected twice per day during six consecutive days. Faeces belonging to 18 vicuñas (Vicugna vicugna) were collected before and one day after their capture for confinement (Chacu). Faecal cortisol metabolites (FCM) were extracted from each sample and quantified by an 11-oxoaetiocholanolone enzyme immunoassay. Thirty-three and 28 h (median) after ACTH stimulation, FCM concentrations peaked with a ten- and eight-fold increase (median) above baseline in alpacas and llamas, respectively. There were no significant differences in FCM concentrations between sexes. In llamas, FCM concentrations peaked (4.7 times higher than baseline) after five days of confinement in females and after three days (2.7 times) in males. In vicuñas, three times higher FCM levels were observed the day after the start of confinement (in comparison to the starting values). Based on our findings, this non-invasive method is well suited to measure adrenocortical activity in alpacas, llamas and vicuñas. Thus, this method could help to improve management, handling and welfare in wild and domesticated South American camelids.

Animal welfare is important for the humane treatment of animals under our care. Zoos and rescue centres manage various charismatic animals, such as big cats, with limited resources. It is therefore essential for caretakers to understand the needs of an individual big cat to ensure its welfare. However, these needs may differ due to a big cat's personality, which may be identified by its coping style in a stressful situation. In addition, stress is one of the major factors affecting animal welfare. There is limited evidence showing strong associations between personality and stress physiology in big cats. This review focuses on the integration of personality and stress physiology of captive big cats, to highlight possible improvements in their husbandry. Our review identifies key factors that may influence big cat responses to stressors. These influencing factors include: i) social interactions; ii) environment; iii) life history and evolutionary traits; iv) genetics; and v) health. The first two factors are relatively well covered in the literature; however, the final three are potentially very promising avenues for future research to better understand how we can improve big cat welfare.

Prenatal glucocorticoid overexposure has been shown to programme adult cardiovascular function in a range of species, but much less is known about the long-term effects of neonatal glucocorticoid overexposure. In horses, prenatal maturation of the hypothalamus–pituitary–adrenal axis and the normal prepartum surge in fetal cortisol occur late in gestation compared to other precocious species. Cortisol levels continue to rise in the hours after birth of full-term foals and increase further in the subsequent days in premature, dysmature and maladapted foals. Thus, this study examined the adult cardiovascular consequences of neonatal cortisol overexposure induced by adrenocorticotropic hormone administration to full-term male and female pony foals. After catheterisation at 2–3 years of age, basal arterial blood pressures (BP) and heart rate were measured together with the responses to phenylephrine (PE) and sodium nitroprusside (SNP). These data were used to assess cardiac baroreflex sensitivity. Neonatal cortisol overexposure reduced both the pressor and bradycardic responses to PE in the young adult males, but not females. It also enhanced the initial hypotensive response to SNP, slowed recovery of BP after infusion and reduced the gain of the cardiac baroreflex in the females, but not males. Basal diastolic pressure and cardiac baroreflex sensitivity also differed with sex, irrespective of neonatal treatment. The results show that there is a window of susceptibility for glucocorticoid programming during the immediate neonatal period that alters cardiovascular function in young adult horses in a sex-linked manner.

Several preclinical studies have demonstrated neuronal effects of glucocorticoids on the hippocampus (HC), a limbic structure with anterior–posterior anatomical and functional segmentation. We propose a volumetric magnetic resonance imaging analysis of hippocampus head (HH), body (HB) and tail (HT) using Cushing's disease (CD) as model, to investigate whether there is a differential sensitivity to glucocorticoid neuronal damage in these segments. We found a significant difference in the HH bilaterally after 12 months from trans-sphenoidal surgical selective resection of the adrenocorticotropic hormone (ACTH)-secreting pituitary micro-adenomas. This pre–post surgery difference could contribute to better understand the pathopysiology of CD as an in vivo model for stress-related hypercortisolemic neuropsychiatric disorders.

To prove the hypothesis of a common defect in the central nervous system and immune system in patients with major depression, we examined the inhibition of phytohemagglutinin (PHA)-induced lymphocyte proliferation by in vitro and in vivo administration of glucocorticoids in 20 patients with major depression. The patients were divided into two groups according to the results of the dexamethasone suppression test (DST). Six patients showed cortisol non-suppression. They had significantly higher baseline plasma cortisol levels in comparison with cortisol suppressors. The lymphocytes from cortisol non-suppressors were significantly stronger inhibited by in vitro administration of glucocorticoids compared to the lymphocytes from cortisol suppressors. However, there were no significant differences of lymphocyte inhibition by in vivo administration of glucocorticoids between both patient groups. From our results we conclude that cortisol non-suppressors reveal impaired down-regulation of glucocorticoid receptors of lymphocytes. Furthermore, the effect of glucocorticoids on lymphocyte reactivity may reflect the effects on brain structures in patients with major depression.

Exposure to glucocorticoid levels higher than appropriate for current developmental stages induces offspring metabolic dysfunction. Overfed/obese (OB) ewes and their fetuses display elevated blood cortisol, while fetal Adrenocorticotropic hormone (ACTH) remains unchanged. We hypothesized that OB pregnancies would show increased placental 11β hydroxysteroid dehydrogenase 2 (11β-HSD2) that converts maternal cortisol to fetal cortisone as it crosses the placenta and increased 11β-HSD system components responsible for peripheral tissue cortisol production, providing a mechanism for ACTH-independent increase in circulating fetal cortisol. Control ewes ate 100% National Research Council recommendations (CON) and OB ewes ate 150% CON diet from 60 days before conception until necropsy at day 135 gestation. At necropsy, maternal jugular and umbilical venous blood, fetal liver, perirenal fat, and cotyledonary tissues were harvested. Maternal plasma cortisol and fetal cortisol and cortisone were measured. Fetal liver, perirenal fat, cotyledonary 11β-HSD1, hexose-6-phosphate dehydrogenase (H6PD), and 11β-HSD2 protein abundance were determined by Western blot. Maternal plasma cortisol, fetal plasma cortisol, and cortisone were higher in OB vs. CON (p < 0.01). 11β-HSD2 protein was greater (p < 0.05) in OB cotyledonary tissue than CON. 11β-HSD1 abundance increased (p < 0.05) in OB vs. CON fetal liver and perirenal fat. Fetal H6PD, an 11β-HSD1 cofactor, also increased (p < 0.05) in OB vs. CON perirenal fat and tended to be elevated in OB liver (p < 0.10). Our data provide evidence for increased 11β-HSD system components responsible for peripheral tissue cortisol production in fetal liver and adipose tissue, thereby providing a mechanism for an ACTH-independent increase in circulating fetal cortisol in OB fetuses.

Both parasitism and social contact are common sources of stress that many gregarious species encounter in nature. Upon encountering such stressors, individuals secrete glucocorticoids and although short-term elevation of glucocorticoids is adaptive, long-term increases are correlated with higher mortality and deleterious reproductive effects. Here, we used an experimental host-parasite system, social rodents Acomys cahirinus and their characteristic fleas Parapulex chephrenis, in a fully-crossed design to test the effects of social contact and parasitism on stress during pregnancy. By analysing faecal glucocorticoid metabolites, we found that social hierarchy did not have a significant effect on glucocorticoid concentration. Rather, solitary females had significantly higher glucocorticoid levels than females housed in pairs. We found a significant interaction between the stressors of parasitism and social contact with solitary, uninfested females having the highest faecal glucocorticoid metabolite levels suggesting that both social contact and infestation mitigate allostatic load in pregnant rodents. Therefore, the increased risk of infestation that accompanies group-living could be outweighed by positive aspects of social contact within A. cahirinus colonies in nature.

Vitamin C (VC) is a vital micronutrient for humans and some other mammals and also has antioxidant activity. Stress-induced elevation of glucocorticoid production is well known to cause immunosuppression. The present study evaluated the effect of high VC intake on glucocorticoid-induced immune changes in mice. Senescence marker protein 30 knockout mice with genetic VC deficiency were fed a diet containing the recommended VC content (20 mg/kg per d; 0·02 %VC group) or a high VC content (200 mg/kg per d; 0·2 %VC group) for 2 months, then dexamethasone was given by intraperitoneal injection. After administration of dexamethasone, the plasma ascorbic acid concentration decreased significantly in the 0·02 %VC group and was unchanged in wild-type C57BL/6 mice on a VC-deficient diet (wild-type group), while it was significantly higher in the 0·2 %VC group compared with the other two groups. In the 0·02 %VC and wild-type groups, dexamethasone caused a significant decrease in the cluster of differentiation (CD)4+ and CD8+ T cells among splenocytes as well as a significant decrease in IL-2, IL-12p40 and interferon-γ protein production by splenocytes and a significant decrease in T-cell proliferation among splenocytes. In the 0·2 %VC group, these dexamethasone-induced immunosuppression improved when compared with the other two groups. In addition, reduction in the intracellular levels of ascorbic acid, superoxide dismutase and glutathione in splenocytes by dexamethasone as well as elevation in thiobarbituric acid-reactive substances were significantly suppressed in the 0·2 %VC group. These findings suggest that high dietary VC intake reduces glucocorticoid-induced T-cell dysfunction by maintaining intracellular antioxidant activity.

Recent findings highlight that there are prenatal risks for affective disorders that are mediated by glucocorticoid mechanisms, and may be specific to females. There is also evidence of sex differences in prenatal programming mechanisms and developmental psychopathology, whereby effects are in opposite directions in males and females. As birth weight is a risk for affective disorders, we sought to investigate whether maternal prenatal cortisol may have sex-specific effects on fetal growth. Participants were 241 mothers selected from the Wirral Child Health and Development Study (WCHADS) cohort (n=1233) using a psychosocial risk stratifier, so that responses could be weighted back to the general population. Mothers provided saliva samples, which were assayed for cortisol, at home over 2 days at 32 weeks gestation (on waking, 30-min post-waking and during the evening). Measures of infant birth weight (corrected for gestational age) were taken from hospital records. General population estimates of associations between variables were obtained using inverse probability weights. Maternal log of the area under the curve cortisol predicted infant birth weight in a sex-dependent manner (interaction term P=0.029). There was a positive and statistically significant association between prenatal cortisol in males, and a negative association in females that was not statistically significant. A sex interaction in the same direction was evident when using the waking (P=0.015), and 30-min post-waking (P=0.013) cortisol, but not the evening measure. There was no interaction between prenatal cortisol and sex to predict gestational age. Our findings add to an emerging literature that suggests that there may be sex-specific mechanisms that underpin fetal programming.

The reduction of reproductive performance associated with stress is a known phenomenon in domestic birds. This review demonstrates the involvement of glucocorticoids, a stress hormone, in the decision-making process regarding energy ingestion and distribution in laying hens. During the energetic challenge induced by a stressful environment, corticosterone stimulates energy intake and a preference for a high-fat diet by up-regulating neuropeptide Y (NPY) expression via the AMP-activated protein kinase (AMPK) pathway. The elevated corticosterone levels in response to stressors may be associated with suppressed reproduction in laying hens via a possible perturbation of the hypothalamic-pituitary-gonadal (HPG) axis. Corticosterone suppresses follicular development and is energy dependent by decreasing the availability of the circulating yolk precursor and the prevention of yolk deposition in follicles. Energy status is also involved in rejuvenation in moult hens.