Glucocorticoid Sensitivity

Overview
The neuroendocrine and immune systems play a major role in adaptation to stress. Glucocorticoids secreted as end hormones of the hypothalamus-pituitary-adrenal (HPA) axis belong to the most frequently used biological outcomes in stress research. This is based on their dynamic response to acute situations, and changes of their circadian secretion in some conditions of chronic psychological stress, and on their effects on target tissues throughout the body, which are involved in regulating body homeostasis at rest and in conditions of stress. Due to the complexity of the signal transduction cascade from cortisol to actual biological effects on a given target cell, cortisol effects on target cells can vary within and between individuals. Assessing the ability of the target tissue to respond to the cortisol signal, i.e. cortisol, also referred to as Glucocorticoid Sensitivity (GC sensitivity), as well as Corticosteroid Sensitivity or relative Glucocorticoid Resistance allows to determine the actual net effect on the affected system of interest. This adds important information about health effects, and can, therefore, be considered an important measure to improve understanding of the CNS-to-periphery pathway specifically, or more broadly, stress pathways to disease

Background
Glucocorticoid effects on target tissues vary depending on the target tissue of interest, more specifically, the presence of receptors (GRs or MRs), the intracellular status of receptors with regard to chaperone proteins, the availability of transcription factors and DNA status (e.g. epigenetic factors). While one approach to understanding GC effects on target tissues is to interrogate the transcriptome for transcripts activated or deactivated by GCs (see for example 1,2 and https://www.stressmeasurement.org/gene-expression), GC sensitivity assays usually apply a simpler black-box approach by determining the effects of typically different doses of GCs on a specific target system function. The most frequently studied system is the inflammatory system, in which a mitogen-activated inflammatory cascade is subjected to different concentrations of GCs in vitro, and inflammatory cytokine production is measured after incubation periods of usually 6 to 24 hours. The most frequently used assay is a whole blood assay, in which diluted whole blood is incubated with lipopolysaccharide (LPS) and one to six different concentrations of dexamethasone (DEX) or hydrocortisone (HC), including saline as control (e.g. 3). Separated peripheral blood mononuclear cells (PBMCs) can also be used (e.g. 4, but the method is less frequently employed due to the more time consuming and expensive preparation.

Using the standard technique of incubating whole blood with LPS and DEX or HC, it was for example found that GC sensitivity changes in response to acute laboratory stress and that changes are related to age, sex hormone status, and BMI, for example, 3,5–8. The main application, however, for GC sensitivity assays testing for GC regulation of inflammation has been on chronic stress and related psychiatric conditions such as depression and posttraumatic stress disorder. The chronic stress of caregiving for example was found to be associated with lower GC sensitivity cross-sectionally 9, and decreases in GC sensitivity over time were also found in longitudinal studies 10,11. Furthermore, declines in GC sensitivity were related to decreased expression of anti-inflammatory proteins as well as increased plasma inflammation 10,11. Lower GC sensitivity was also found following major life events, and predictive of higher vulnerability for rhinovirus infections, as well as higher plasma inflammation 12. GC sensitivity is further related to adverse psychological factors such as vital exhaustion 13. While GC sensitivity is typically found to be decreased in depression 14, it typically is found increased compared to controls in posttraumatic stress disorder (PTSD) (15,16, also summarized in 17). Altered GC sensitivity might also be a risk factor for the development of psychiatric symptoms after trauma. It was found that GC sensitivity assessed in soldiers before deployment was predictive of post-deployment symptoms, including PTSD and fatigue 18.
Alterations in GC sensitivity are also linked with and might be predictive of physical disease development. In addition to the findings of Cohen et al. reported above (2012), showing increased susceptibility to experimental rhinovirus infection 12, van den Akker et al. (2008) reported that variants of the GR gene linked with lower sensitivity were predictive of higher systemic inflammatory markers, higher carotid intima-media thickness, as well higher cardiovascular disease risk 19. Given the role of systemic inflammation in the development of a host of diseases 20, this latter finding also points to reduced GC sensitivity playing a role also in Alzheimer’s disease, diabetes, obesity, and cancer.

There are several possible variations of this assay. For example, other mitogens can be used to activate specific components, for example, phytohemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA), to specifically activate components of cellular immunity. In line with findings on inflammatory GC sensitivity, do Prado et al. (2017) for example reported increased GC sensitivity of PHA stimulated lymphocytes in adolescents with childhood maltreatment 21. GC sensitivity has also been operationalized as leukocyte distributional sensitivity (e.g. 22,23, by statistically analyzing blood counts with regard to GC’s associations with neutrophil/lymphocyte ratio or neutrophil/monocyte ratio. Using this alternative marker, loneliness was found to be related to relative GC resistance, in line with findings using the in vitro whole blood approach. Distributional GC sensitivity, therefore, is an alternative for data sets in which leukocyte counts are available, but laboratory analyses are not an option.

GC sensitivity can also be determined for non-immunological target tissues, as compared by Ebrecht et al. 24. One example is GC induced skin bleaching, which reflects vasoconstrictive GC effects and has been proposed as a marker for vascular GC sensitivity in general (see for example 25). Finally, the systemic application of GCs to patients or research participants can be considered tests of GC sensitivity in a broader sense. Examples would be dexamethasone suppression test (DST), with systemic hormonal or gene-expression readouts 26, or probes of specific cognitive functions 17. It is important to highlight that different methods to assess GC sensitivity (for example, systemically vs. in vitro) bring slightly different information, as recently shown by a meta-analysis on this topic 27. Interestingly, in this meta-analysis the in vitro methods presented here were strong predictors of systemic inflammation in depression, thus confirming the clinical relevance of this approach. However, ideally, multiple measurements of GG sensitivity should be collected at the same time, especially combining GC sensitivity with the measurement of glucocorticoid receptor expression (mRNA) in the blood.

Collection and measurement
Measurement of in vitro GC sensitivity requires access to a laboratory with biosafety cabinet / sterile flow hood and CO2 incubator in addition to standard laboratory equipment. If using whole blood, collection using heparin is recommended, as other anticoagulants (such as DHEA) are toxic. Heparinized whole blood is typically diluted with saline (for example at the ratio of 10:1) before being co-incubated with mitogen and GCs. It is recommended to standardize time between collection and culture to ideally 0 to 30 minutes. New batches of LPS and other mitogens should be tested with regard to their stimulative capacity before the start of experiments, because of batch-to-batch variation in inflammatory stimulative capacity. The GCs used most frequently are dexamethasone (DEX) or hydrocortisone (HC). DEX is typically used because of its higher affinity, which will displace endogenous cortisol present in the blood and render the assay robust against circulating cortisol. HC in contrast has the advantage to be more comparable in its effects, because it is similar to the naturally circulating cortisol. Typical DEX concentrations range from 10-6 M, which in healthy participants typically induces almost complete suppression of cytokine production to 10-8 or 10-9 M, which show very weak to no suppression. Diluted whole blood is then co-incubated in sterile cell culture plates for 6 to 18 hours (standardized within each study) at 5% CO2 and 37C, followed by centrifugation and harvest of plasma supernatant. Supernatant can be stored at -80C and then subjected to assay for pro- or anti-inflammatory cytokines such as interleukin(IL)-6, IL-1beta, tumor necrosis factor(TNF)-alpha, or IL-10. Concentrations tend to be higher than the detection range of regular cytokine ELISAs, requiring dilution before assaying. Alternative assay methods such as multiplexing are possible as well, and can sometimes eliminate the requirement of pre-diluting supernatants, due to higher dynamic range. To compute GC sensitivity, a non-linear regression is fitted for each individual dose-response curve, and the half-maximal inhibitory concentration (IC50) is computed as the most frequently used index. Alternative approaches are to use individual data points in multilevel modeling approaches, to calculate percentage inhibition at each concentration of DEX or HC, or to compute the area under curve (AUC) or other markers of the curve’s slope.

Specific protocols and advice can be obtained by contacting Nicolas Rohleder (nicolas.rohleder@fau.de).

Author(s) and Reviewer(s): Prepared by Nicolas Rohleder, PhD. Reviewed by Gregory Miller, PhD and Carmine Pariante. Please direct suggestions and feedback to Dr. Rohleder (nicolas.rohleder@fau.de).

References:

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11. Walsh, C. P. et al. Development of glucocorticoid resistance over one year among mothers of children newly diagnosed with cancer. Brain Behav. Immun. (2017) doi:10.1016/j.bbi.2017.12.011 .