Glucocorticoid production in lymphoid organs: Acute effects of lipopolysaccharide in neonatal and adult mice

Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of GCs, steroidogenic enzymes expression, and components of the HPA axis (e.g., CRH) expression. We administered LPS (50µg/kg i.p.) or vehicle control to male and female C57BL/6J neonatal (post-natal day (PND) 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hr later. We measured progesterone, 11-deoxycorticosterone (DOC), corticosterone, and 11-dehydrocorticosterone (DHC) via liquid chromatography tandem mass spectrometry (LC-MS/MS). We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via qPCR. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominately in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.

Isoflurane stress induces glucocorticoid production in mouse lymphoid organs

Glucocorticoids (GCs) are secreted by the adrenal glands and locally produced by lymphoid organs. Adrenal GC secretion at baseline and in response to stressors is greatly reduced during the stress hyporesponsive period (SHRP) in neonatal mice (postnatal day (PND) 2-12). It is unknown whether lymphoid GC production increases in response to stressors during the SHRP. Here, we administered an acute stressor (isoflurane anesthesia) to mice before, during, and after the SHRP and measured systemic and local GCs via mass spectrometry. We administered isoflurane, vehicle control (oxygen), or neither (baseline) at PND 1, 5, 9, or 13 and measured progesterone and 6 GCs in blood, bone marrow, thymus, and spleen. At PND1, blood and lymphoid GC levels were high and did not respond to stress. At PND5, blood GC levels were very low and increased slightly after stress, while lymphoid GC levels were also low but, increased greatly after stress. At PND9, blood and lymphoid GC levels were similar at baseline and increased similarly after stress. At PND13, blood GC levels were higher than lymphoid GC levels at baseline, and blood GC levels showed a greater response to stress. These data demonstrate the remarkable plasticity of GC physiology during the postnatal period, show that local steroid levels do not reflect systemic steroid levels, provide insight into the SHRP, and identify a potential mechanism by which early-life stressors can alter immunity in adulthood.

Cellular mechanisms of impaired mineralocorticoid and glucocorticoid production by endocrine disruptor DDT

Developmental exposure to persistent endocrine disruptors is of a great concern. Affection of adrenal hormones production by low-dose exposure to dichlorodiphenyltrichloroethane (DDT) has been revealed recently. Objective of the research – investigation of ultrastructural mechanisms of impaired mineralocorticoid and glucocorticoid production after prenatal and postnatal exposure to endocrine disruptor DDT. Male Wistar rats exposed to low doses of DDT during prenatal and postnatal development were studied. Aldosterone and corticosterone levels in serum were measured by enzyme-linked immunosorbent assay, histological examination and electron microscopy of the adrenals were performed. Pubertal rats, developmentally exposed to low doses of DDT, demonstrated lowered aldosterone and elevated corticosterone serum levels. After puberty the rats showed tendency to normalization of hormones’ production. Morphological examination of the adrenals revealed less developed zona glomerulosa in pubertal period and its relative hyperplasia after puberty. Microcirculatory disorders and focal cell death were observed in outer zona fasciculata. Electron microscopy of glomerulosa cells found signs of suppressed secretory activity in pubertal period and no significant reduction of mitochondria size in adult rats. Total number of mitochondria in 1 µm2 of cytoplasm and percent of mitochondria with swollen matrix were diminished compared to the control. Cells of inner zona fasciculata demonstrated increased functional activity. Age-dependent changes in fine structure of fasciculata cells were similar to the control, but were more pronounced. In this way, cellular mechanism of impaired mineralocorticoid production in rats prenatally and postnatally exposed to low doses of endocrine disruptor DDT, are insufficient mitochondrion function and impaired reorganization of mitochondrial apparatus, which occurs during pubertal period. Mechanisms of elevated glucocorticoid secretion are attributed to enhanced function of mitochondria in fasciculata cells.

PSII-37 Program Chair Poster Pick: The effect of glucocorticoids on circulating plasma concentrations of ghrelin in sheep

Activation of the hypothalamic-pituitary-adrenal (HPA) axis to release adrenocorticotropic hormone (ACTH) culminates in glucocorticoid production in sheep. Glucocorticoids alter metabolic function, which suggests concentrations of metabolically important hormones, ghrelin and growth hormone (GH), might be affected by components of the HPA axis. To clarify the relationship between the HPA axis and these hormones in ruminants, 10 Katahdin ewe lambs (1 year old; 43.3 +/- 1.3 kg) received an intravenous injection of ACTH (0.2 µg/kg BW) or saline. In a second experiment, the same lambs received an intravenous injection of the synthetic glucocorticoid, dexamethasone (DEX; 2 mg/kg BW) or saline. Blood samples were collected in 15 min intervals from 1 h before injection to 2 h after. Plasma concentrations of cortisol, ghrelin, and GH were analyzed by radioimmunoassay analysis. Data were tested for effect of treatment (saline or ACTH and saline or DEX), time, and treatment by time interaction using procedures for repeated measures with JMP software (version 10; SAS Inst. Inc.). Cortisol concentrations increased in response to both ACTH and dexamethasone administration (P < 0.0001). Neither ghrelin nor GH concentrations were affected by ACTH or DEX administration (P ≥ 0.4372). These data confirm ACTH influences cortisol secretion but suggest neither ACTH nor glucocorticoids impact plasma concentrations of ghrelin and GH in sheep.

OR19-04 Glucocorticoid Production in the Nervous and Immune Systems: Evidence for a Local HPA Axis Homolog

Glucocorticoid Production in the Nervous and Immune Systems: Evidence for a Local HPA Axis Homolog The hypothalamic-pituitary-adrenal (HPA) axis is a critical stress response system in vertebrates. The hypothalamus secretes corticotropin-releasing hormone (CRH), which binds its receptor (CRH-R1) in the anterior pituitary. The anterior pituitary then secretes adrenocorticotropic hormone (ACTH), which binds its receptor (MC2R) in the adrenal glands and stimulates secretion of glucocorticoids into the bloodstream. Glucocorticoids are critical modulators of neural and immune system development. During early development (postnatal day (PND) 2 to 12), mice show decreased adrenal glucocorticoid secretion at baseline and in response to stressors, termed the stress hyporesponsive period (SHRP) (1). Traditionally, glucocorticoids have been thought to be synthesized only in the adrenal glands. However, recent evidence demonstrates that glucocorticoids are also produced in extra-adrenal tissues, such as the brain and lymphoid organs (2). This may be of particular importance during the SHRP, as local production allows glucocorticoid modulation of specific tissues and cells, without general effects throughout the organism. Importantly, the factors that regulate local glucocorticoid production remain unknown. To study the regulation of local glucocorticoid production, we examined whether mediators of the HPA axis are locally expressed at baseline and in response to an immune stressor. We assessed systemic and local glucocorticoid levels in neonatal (PND5) C57BL/6J mice 4hr after an immune challenge with lipopolysaccharide (50µg/kg i.p.) or vehicle control. We examined blood, microdissected brain regions (prefrontal cortex, hippocampus, hypothalamus), and lymphoid organs (thymus, spleen, bone marrow). A panel of 7 steroids was measured via liquid chromatography tandem mass spectrometry (LC-MS/MS). Gene expression of Crh, Crh-R1, Pomc, and Mc2r was quantified via qPCR. Preliminary data indicate that corticosterone was 2-fold higher in tissues than in blood after an immune stressor. The thymus expressed all genes of interest, supporting the existence of a local HPA axis “homolog” in the thymus. Brain, spleen and bone marrow expressed a subset of the genes of interest. These exciting data demonstrate that all the mediators of the HPA axis are locally expressed within the thymus, likely to regulate thymocyte development and reactivity. Greater understanding of local glucocorticoid production will provide crucial insight into neural and immune development and function. Reference: (1) Sapolsky et al., Brain Res Rev. 1986 11(1):65–76. (2) Taves et al., Endocrinology. 2015 156(2):511–522.

Hair glucocorticoids are not a historical marker of stress – exploring the time-scale of corticosterone incorporation into hairs in a rat model

Hair glucocorticoids are increasingly popular biomarkers, used across numerous research fields as a measure of stress. Although they are suggested to be a proxy of the average HPA axis activity spanning a period of weeks or months into the past, this theory has never been tested.In the present study, adrenalectomized rats with no endogenous (adrenal) glucocorticoid production were used to study how circulating glucocorticoid levels would be reflected in the glucocorticoid levels found in hair samples. By dosing the animals daily with high levels of corticosterone for seven days, while sampling hairs before, during, and after treatments, a timeline for glucocorticoid uptake into hairs was constructed. This kinetic profile was compared to two hypothetical models, and the theory that hair glucocorticoids are a record of historical stress had to be rejected.Corticosterone concentrations in hairs were found to increase within three hours of the first injection, the highest concentrations were found on the seventh day of treatments, and the decrease in concentrations post-treatment suggests rapid elimination. We speculate that hair glucocorticoid levels can only be used to characterize a stress-response for a few days following a postulated stressor.An updated model, where glucocorticoids diffuse into, along, and out of hairs needs to be adopted to reconcile the experimentally obtained data. The inescapable consequence of this updated model is that hair glucocorticoids become a marker of – and can only be used to study – recent, or ongoing, stress, as opposed to historical events, weeks or months in the past.

Effect of Vasopressin on the Hypothalamic-Pituitary-Adrenal Axis in ADPKD Patients during V2 Receptor Antagonism

<b><i>Background:</i></b> Patients with autosomal dominant polycystic kidney disease (ADPKD) are treated with a vasopressin V2 receptor antagonist (V2RA) to slow disease progression. This drug increases vasopressin considerably in these patients with already elevated baseline levels. Vasopressin is known to stimulate the hypothalamic-pituitary-adrenal (HPA) axis through V1 and V3 receptor activation. It is unknown whether this increase in vasopressin during V2RA treatment affects glucocorticoid production. <b><i>Methods:</i></b> Twenty-seven ADPKD patients were studied on and off treatment with a V2RA and compared to age- and sex-matched healthy controls and IgA nephropathy patients, the latter also matched for kidney function. Vasopressin was measured by its surrogate copeptin. Twenty-four-hour urinary excretions of cortisol, cortisone, tetrahydrocortisone, tetrahydrocortisol, allotetrahydrocortisol, and the total glucocorticoid pool were measured. <b><i>Results:</i></b> At baseline, ADPKD patients demonstrated a higher copeptin concentration in comparison with healthy controls, while urinary excretion of cortisol and cortisone was lower (medians of 0.23 vs. 0.34 μmol/24 h, <i>p</i> = 0.007, and 0.29 vs. 0.53 μmol/24 h, <i>p</i> &#x3c; 0.001, respectively). There were no differences in cortisol and cortisone excretion compared to IgA nephropathy patients. Cortisol, cortisone, and total glucocorticoid excretions correlated with kidney function (<i>R</i> = 0.37, 0.58, and 0.19, respectively; all <i>p</i> &#x3c; 0.05). Despite that V2RA treatment resulted in a 3-fold increase in copeptin, only cortisone excretion increased (median of 0.44 vs. baseline 0.29 μmol/24 h, <i>p</i> &#x3c; 0.001), whereas no changes in cortisol or total glucocorticoid excretion were observed. <b><i>Conclusions:</i></b> Increased concentration of vasopressin in ADPKD patients at baseline and during V2RA treatment does not result in activation of the HPA axis. The impaired glucocorticoid production in these patients is related to their degree of kidney function impairment.