Final Report Summary - GCS-CNS-IS (Pro-inflammatory and anti-inflammatory effects of glucocorticosteroids in the Central Nervous System.) Glucocorticoids (GCs), the adrenal steroid hormones released during stress, can have an array of adverse effects in the nervous system, including disruption of hippocampal-dependent plasticity and cognition. Of relevance to this proposal, GCs can also compromise the ability of hippocampal and cortical neurons to survive an array of necrotic neurological insults. Amid this picture of deleterious effects, there is the accepted paradigm in which GCs are seemingly protective in the context of necrotic insults, namely concerning the well-known anti-inflammatory effects of the hormone. There has been a growing appreciation for the extent to which inflammation can worsen the neurotoxicity of necrotic insults, and the anti-inflammatory capacity of GCs has long been exploited pharmacologically to protect against circumstances of post-insult edema. Most importantly, and as the centerpiece of this project, there is the growing recognition that there are numerous circumstances such as social stress where GCs are pro-inflammatory, rather than anti-inflammatory. Here, as a new and innovative area of research, the group of the outgoing host institution (Dept. Biological Sciences, Stanford University) and the return host institution (Dept. Psychobiology, Basque University) have explored this project collaboratively, by way of carrying out the proposed experiments at Stanford University. The purpose of this project is to better understand when stress released GCs are classically anti-inflammatory, and when unexpected pro-inflammatory effects occur in the challenged nervous system, and to explore the mechanisms underlying such unexpected effects. This represents an innovative and fascinating question, i.e. understanding how a signaling molecule such as GCs can have diametrically opposite and simultaneous effects. At the same time, understanding this phenomenon is of great potential clinical relevance, given the common use (and often counterproductive effects) of GCs in clinical neurology. In order to achieve these goals three specific aims were proposed for the outgoing phase of the project: Specific Aim I:To better define the circumstances where GCs have pro-inflammatory effects, Specific Aim II:Explore gene expression patterns under conditions where GCs are pro-inflammatory, and Specific Aim III: Examine the cellular specificity of GC-induced pro-inflammatory gene expression profiles. We designed experiments through which we determined that the most interesting proinflammatory effects following immune challenge were observed at a time point of 24 hours after termination of stress, a time point when glucocorticoids in blood were back to baseline. We used this time point to examine gene expression patterns for inflammatory markers (IL-1β, IL-6, TNFα, IL-10, IL4 and IL-1ra) in proposed regions such as hippocampus, hypothalamus, cortex and amygdala. Interestingly a majority of the effects of stress that we observed were pro-inflammatory (IL-1b, IL-6 and TNFa) in regions such as the hippocampus and hypothalamus, however in the amygdala; stress induced a reduction in the pro- and anti-inflammatory effects of lypopolysacharides (LPS). Motivated by the potential impact of these regional differences, we sampled additional regions (striatum, prefrontal cortex, frontal cortex, brain stem, cerebellum, pituitary and liver) and increased the extent of the markers for the inflammatory response and GC receptors (BDNF, GR, MR, GILZ, CD200, CX3CL1, CX3CR1 and IL-17A, G-CSF, GM-CSF, Leptin, EGF, IFN-alpha, MCP-1, GRO/KC, MIP-2, RANTES, Eotaxin, IL-1#, MIP-1alpha, IL-13, IL-12p70, IL-5, IL-18, IP-10, VEGF, LIX and TNF-alpha). We found that stress and inflammation effects follow a different pattern in the all of the different brain structures . We observed that independent of whether stress is acting in a pro-inflammatory or anti-inflammatory manner it has the greatest effects in the parts of the brain traditionally most sensitive to glucocorticoids: hippocampus, amygdala and frontal cortex. One of the most interesting findings concerns IL-1b. Stress appears to be acting in a pro-inflammatory manner potentiating the effect of LPS on IL-1b in the hippocampus, hypothalamus, frontal cortex, prefrontal cortex and pituitary. Stress does not have this proinflammatory effect in the amygdala, brainstem, cerebellum and striatum. Of these, amygdala is the most relevant given how strong of a target tissue it is for glucocorticoid actions. Now, by using cluster analysis we continue analyzing those data to find specific patterns between the most interesting structures, amygdala and hippocampus. Finally, based on the data explained above, we examined the cellular specificity of proinflammatory effects of stress in the hippocampus and amygdala using the 24 hours post stress time point. We collected and sectioned brains for immunohistochemistry and stained for the neurochemical markers for gabaergic neurons (GAD 67), astrocytes (GFAP), microglia (Iba-1), neurons (NeuN) and a marker of neuronal activation (C-fos). While we have finished imaging these markers using confocal microscopy, data analyses are an ongoing process right now. However, preliminary data examining C-fos mRNA from the same experimental design suggest that we are going to see something interesting with the immunohistochemistry as amygdala showed lower C-fos expression in the stressed animals inoculated with LPS while no effects were observed in hippocampal C-fos mRNA expression. The analysis of the images is an ongoing project, as those analyses are necessary for the culmination of the manuscript. These studies have generated data characterizing stress induced GC effects, and test a large number of predictions. This project has an impact on the classic concept of GCs as anti-inflammatory, which must be modified in a number of ways: a) In the CNS, GCs are not uniformly anti-inflammatory, and are even pro-inflammatory, an effect that is regional dependent. b) In the CNS, prior exposure to GCs can result in a “priming” of the immune response to a subsequent inflammatory challenge. c) Pro-inflammatory GC effects in the brain are not homogeneous throughout the brain. Of all the different responses within 11 distinct structures, opposite effects of GCs between amygdala and hippocampus are the most relevant given how strong of a target tissue they are for glucocorticoid actions. d) Stress induced GC in the brain are pro-inflammatory and depend upon the brain region in question, and the timing of stress exposure with respect to the inflammatory challenge. The results obtained in this project help to redefine the research approaches of the endocrinology and neuroscience studies. Ultimately the most important future direction of this work would be to examine its clinical relevance, work that I would anticipate to be highly collaborative. Finally, these findings might as well have important implications for the reallocation of the economical resources applied in health treatments.This project has clearly strengthen the scientific, biological and clinical bases of less favored region of the European Community and encourage international competitiveness in an area that, despite its importance, it is not already sufficiently cultivated in Europe. The techniques used in this project are completely innovative for the return host institution, which has actualize not only methodology, but also scientific concepts, and has increased the quality of our research.
