Effects of Coupled Dose and Rhythm Manipulation of Plasma Cortisol Levels On Leukocyte Transcriptional Response to Endotoxin in Humans

Healthy function of the immune system is intrinsically tied to the biological rhythms running in harmony. There is a bi-directional information flow between the immune and neuroendocrine systems such that; while a wide range of immune parameters go through daily fluctuations under the control of autonomic and endocrine rhythms; immune effectors, such as cytokines, can, in turn, influence the phase of these rhythms (1). In the case of acute stress, such as trauma or infection, inflammatory response is mounted by the coordinated action of immune and neuroendocrine systems. Hypothalamus-pituitary-adrenal (HPA) axis is a crucial mediator of this action because of its control on circadian and ultradian glucocorticoid (GC) secretion patterns (2). These fluctuating patterns are hypothesized to provide the basis for a “continuous dynamic equilibration”, fundamental to establishment of homeostasis and allostasis (3). Cortisol is the major among circulating human GCs which, through feedforward and feedback regulatory loops, maintains this dynamic state essential for proper response to stressors. Although GCs are mostly famous for their immunosuppressive and anti-inflammatory actions that have been clinically utilized for the treatment of autoimmune and inflammatory disorders for decades; due to their non-linear effects on transcription and highly complex interactions with other inflammatory mediators; their downstream reactions are highly diverse. Effect of GCs on the immune response can be so disparate, such as to permit, suppress or stimulate the response, depending on their secretion frequency, concentration and timing with respect to a stressor (4-7).

Since GCs are both major endogenous hormones regulating stress response as well as clinically used pharmacological agents; building a solid understanding on their effects on immune cells is of primary importance. With this vision in mind, this in vivo study in healthy human subjects seeks to assess the responses of whole blood leukocytes to coupled dose and rhythm manipulation of cortisol preceding an immune challenge induced by endotoxin (LPS) administration. Healthy human model of LPS administration has been used as a reproducible experimental platform providing mechanistic insights into how cells and organs respond to systemic inflammation. The model transiently alters many physiologic and metabolic processes in a qualitatively similar manner to those observed after acute injury and systemic inflammation (8); therefore served as a suitable platform for investigating the changes in immune response due to hormonal manipulations. Transcriptional responses of leukocytes as well as the change in total leukocyte count in blood and plasma cortisol levels were monitored over 24 hours both before and after LPS administration. Differential gene expression and clustering analyses were done separately for these two periods to allow focusing both on the response to cortisol infusion alone and on the effects of this treatment to subsequent LPS exposure.

Through continuous infusion; plasma cortisol concentration was increased to and kept constant at a level associated with major physical stress and almost twice as much as the normal daily peak. In response to this treatment, total leukocyte count in blood was increased both during cortisol infusion period and within 2 hours after LPS exposure, after which it started to decrease. Transcriptional programming of leukocytes was also significantly altered where a priming response at the end of 24hr infusion period was evident. Enhanced expression of a number of pattern recognition and cytokine receptors, receptor regulatory and signal transduction elements; as well as tuned down protein translation and mitochondrial function portrayed a phenotype sensitized against potential infectious threats. Promoter analysis within cortisol responsive genes revealed that only less than half of this transcriptional response might have been as a direct consequence of glucocorticoid receptor (GR)-DNA binding, therefore transcriptional regulation mechanisms other than DNA binding, such as tethering, may play a significant role. Furthermore, absence of any correlations between the direction of the response and different modes of transcriptional regulation underscored the complexity of mechanisms that govern the transcriptional programming through GR.

Despite these significant changes in transcriptional programming in leukocytes prior to LPS administration, response to LPS followed very similar patterns in both cortisol and saline pre-treated groups except one significantly different cluster including probesets associated with major players known to regulate the inflammatory response such as cytokines and chemokines, anti-inflammatory signal transduction elements and receptors mediating recruitment of leukocytes to sites of inflammation. Observed much more intense response in the cortisol group in this cluster indicated that although continuous cortisol infusion did not directly affect the transcription of major inflammatory players, it indirectly affected their regulation, possibly through modulating their up-stream signaling cascades.

In sum, altered natural fluctuation coupled with increased concentration of plasma cortisol levels induced a primed phenotype in the circulating leukocytes associated with sensitization of surveillance function and tuning down of energy-intensive processes which may be linked to increasing their efficiency in responding to subsequent infectious threats.

References

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