Andrea Engler

The time of prenatal immune challenge determines the specificity of inflammation-mediated brain and behavioral pathology.

Disturbance to early brain development is implicated in several neuropsychiatric disorders including autism, schizophrenia, and mental retardation. Epidemiological studies have indicated that the risk of developing these disorders is enhanced by prenatal maternal infection, presumably as a result of neurodevelopmental defects triggered by cytokine-related inflammatory events. Here, we demonstrate that the effects of maternal immune challenge between middle and late gestation periods in mice are dissociable in terms of fetal brain cytokine responses to maternal inflammation and the pathological consequences in brain and behavior. Specifically, the relative expression of pro- and anti-inflammatory cytokines in the fetal brains in response to maternal immune challenge may be an important determinant among other developmental factors for the precise pathological profile emerging in later life. Thus, the middle and late gestation periods correspond to two windows with differing vulnerability to adult behavioral dysfunction, brain neuropathology in early adolescence, and of the acute cytokine responses in the fetal brain.

Stress in puberty unmasks latent neuropathological consequences of prenatal immune activation in mice.

Double Whammy Psychopathologies that cannot be explained by simple genetic or environmental circumstances may sometimes result from complex interplay between multiple inputs. Giovanoli et al. (p. 1095) analyzed the interactions between prenatal and postnatal stressors in mice to see what synergies give rise to psychopathologies in the adult mice. The results suggest that susceptibilities arise when mice are exposed to prenatal infection and also exposed to stressors around puberty. Stressors delivered later in adolescence did not seem to produce the same susceptibility. Although the mechanisms that impose the delay between stressors and psychopathology remain obscure, the timing and sequence of the triggers hint at possible cellular causes. Unfortunate synergies between stressors at vulnerable stages may underlie psychopathologies in mice. Prenatal infection and exposure to traumatizing experiences during peripuberty have each been associated with increased risk for neuropsychiatric disorders. Evidence is lacking for the cumulative impact of such prenatal and postnatal environmental challenges on brain functions and vulnerability to psychiatric disease. Here, we show in a translational mouse model that combined exposure to prenatal immune challenge and peripubertal stress induces synergistic pathological effects on adult behavioral functions and neurochemistry. We further demonstrate that the prenatal insult markedly increases the vulnerability of the pubescent offspring to brain immune changes in response to stress. Our findings reveal interactions between two adverse environmental factors that have individually been associated with neuropsychiatric disease and support theories that mental illnesses with delayed onsets involve multiple environmental hits.

Catechol-O-methyltransferase Val158Met polymorphism is associated with somatosensory amplification and nocebo responses.

A large number of unwanted adverse events and symptoms reported by patients in clinical trials are not caused by the drug provided, since most of adverse events also occur in corresponding placebo groups. These nocebo effects also play a major role in drug discontinuation in clinical practice, negatively affecting treatment efficacy as well as patient adherence and compliance. Experimental and clinical data document a large interindividual variability in nocebo responses, however, data on psychological, biological or genetic predictors of nocebo responses are lacking. Thus, with an established paradigm of behaviorally conditioned immunosuppressive effects we analyzed possible genetic predictors for nocebo responses. We focused on the genetic polymorphisms in the catechol-O-methyltransferase (COMT) gene (Val158Met) and analyzed drug specific and general side effects before and after immunosuppressive medication and subsequent placebo intake in 62 healthy male subjects. Significantly more drug-specific as well as general side effects were reported from homozygous carriers of the Val158 variant during medication as well as placebo treatment compared to the other genotype groups. Val158/Val158 carriers also had significantly higher scores in the somatosensory amplification scale (SSAS) and the BMQ (beliefs about medicine questionnaire). Together these data demonstrate potential genetic and psychological variables predicting nocebo responses after drug and placebo intake, which might be utilized to minimize nocebo effects in clinical trials and medical practice.

Neurobehavioural activation during peripheral immunosuppression.

Like other physiological responses, immune functions are the subject of behavioural conditioning. Conditioned immunosuppression can be induced by contingently pairing a novel taste with an injection of the immunosuppressant cyclosporine A (CsA) in an associative learning paradigm. This learned immunosuppression is centrally mediated by the insular cortex and the amygdala. However, the afferent mechanisms by which the brain detects CsA are not understood. In this study we analysed whether CsA is sensed via the chemosensitive vagus nerve or whether CsA directly acts on the brain. Our experiments revealed that a single peripheral administration of CsA increases neuronal activity in the insular cortex and the amygdala as evident from increased electric activity, c-Fos expression and amygdaloid noradrenaline release. However, this increased neuronal activity was not affected by prior vagal deafferentation but rather seems to partially be induced by direct action of CsA on cortico-amygdaloid structures and the chemosensitive brainstem regions area postrema and nucleus of the solitary tract. Together, these data indicate that CsA as an unconditioned stimulus may directly act on the brain by a still unknown transduction mechanism.

Chemical destruction of brain noradrenergic neurons affects splenic cytokine production

The neurotransmitter noradrenaline (NA) plays a pivotal role in immune regulation. Here we used the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to investigate the impact of central NA depletion on cytokine production by splenic monocytes/macrophages and T cells. Intraperitoneal administration of DSP-4 in adult rats induced a substantial reduction of noradrenergic neurons in the locus coeruleus and the A5 cell group. The degeneration of brainstem noradrenergic neurons was accompanied by a significant decrease in the production of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha by lipopolysaccharide-stimulated splenocytes. In addition, upon T cell receptor stimulation with anti-CD3, isolated splenocytes of DSP-4 treated animals produced significantly less interferon (IFN)-gamma but not IL-2 and IL-4. The proportion of monocytes/macrophages and T cells in the spleen remained unaffected by the neurotoxin treatment, however, the percentage of natural killer cells decreased significantly. The findings suggest that a certain level of central noradrenergic tone is required for normal functioning of peripheral immune cells.

Mitochondrial DNA: An Endogenous Trigger for Immune Paralysis.

Background:Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. Methods:Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. Results:Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. Conclusions:The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.

Extinction of conditioned taste aversion is related to the aversion strength and associated with c-fos expression in the insular cortex

Taste aversion learning is a type of conditioning where animals learn to associate a novel taste (conditioned stimulus; CS) with a stimulus inducing symptoms of poisoning or illness (unconditioned stimulus; US). As a consequence animals later avoid this taste, a reaction known as conditioned taste aversion (CTA). An established CTA extinguishes over time when the CS is repeatedly presented in the absence of the US. However, inter-individual differences in CTA extinction do exist. Using a model of behavioral conditioning with saccharin as CS and the immunosuppressant cyclosporine A as US, the present study aimed at further elucidating the factors underlying individual differences in extinction learning by investigating whether extinction of an established CTA is related to the strength of the initially acquired CS-US association. In addition, we analyzed the expression of the neuronal activation marker c-fos in brain structures relevant for acquisition and retrieval of the CTA, such as the insular cortex and the amygdala. We here show that animals, displaying a strong CS-US association during acquisition, maintained a strong CTA during unreinforced CS re-exposures, in contrast to animals with moderate CS-US association. Moreover, the latter animals showed increased c-fos mRNA expression in the insular cortex. Our data indicate that CTA extinction apparently depends on the strength of the initially learned CS-US association. In addition, these findings provide further evidence that the memory for the initial excitatory conditioning and its subsequent extinction is probably stored in those structures that participate in the processing of the CS and the US.

Reduced Peripheral Expression of the Glucocorticoid Receptor α Isoform in Individuals with Posttraumatic Stress Disorder: A Cumulative Effect of Trauma Burden

Background Posttraumatic stress disorder (PTSD) is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis and changes in glucocorticoid (GC) responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR) of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants. Methods GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42) as well as in ethnically matched reference subjects (non-PTSD, n = 35). Results Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups. Conclusions Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences.

Establishment of an easy and straight forward heparinase protocol to analyse circulating and myocardial tissue micro-RNA during coronary artery-bypass-graft surgery

Coronary artery-bypass-graft (CABG) surgery is associated with myocardial damage and increased blood concentrations of circulating microRNAs (miRNA). However, whether and to what extent these miRNAs relate to cardiac tissue miRNA expression have not yet been explored. Since plasma miRNA quantification in samples from cardiopulmonary bypass (CPB) patients is severely hampered by heparin, we established and validated successfully a protocol to reliably measure miRNA in 49 heparinized patients undergoing CABG so as to investigate the relationship between circulating and right atrial miRNAs. Plasma and right atrial expression of miR-1, miR-133a, miR-423-5p, and miR-499 were measured before and after CPB, as well as miRNAs in plasma 24 h thereafter. All plasma miRNAs increased significantly with surgery while cardiac tissue expression of only miR-133a (1.4-fold; p = 0.003) and miR-423-5p (1.3 fold; p = 0.025) increased as well. Right atrial and plasma miR-133a expression correlated positively before CPB (r = 0.288, p = 0.045) but miR-499 expression inversely (r = −0.484, p = 0.0004). There was a strong association between plasma miR-133a and miR-499 concentrations and postoperative troponin I concentrations, the marker for myocardial damage. Increased myocardial miR-133a and miR-423-5p expression together with unchanged miR-1 and miR-499 expression might suggest active release of these miRNAs rather than their origin from damaged cells.

The GRK2 Promoter Is Regulated by Early-Growth Response Transcription Factor EGR-1

The G‐protein‐coupled receptor kinase 2 (GRK2) plays a major role in cardiovascular diseases, and its expression is increased in heart failure. However, only little is known about factors being involved in up‐regulation of GRK2 expression through transcriptional regulation of its promoter. Since the transcription factor early‐growth response 1 (EGR‐1) is also up‐regulated in patients with heart failure, we tested the hypothesis that EGR‐1 regulates GRK2 transcription. Stimulation of immortalized rat cardiomyocytes (H9c2) with phorbol 12‐myristate 13‐acetate (PMA) resulted in up‐regulation of Egr‐1 and subsequently of Grk2 mRNA expression, with maximum Grk2 expression (p = 0.008) 5 hr after PMA stimulation and being abolished by actinomycin D, indicating a transcriptional mechanism. To identify naturally occurring variants affecting promoter transcriptional activity, we identified a novel G(‐43)A polymorphism (rs182084609), which surrounded a putative EGR‐1‐binding site. While the minor A allele frequency was rare (0.02), this variant was used to explore regulation by EGR‐1 and promoter construct with altered alleles at nt‐43 were subjected of reporter assays in human embryonic kidney cells (Hek293). Here, EGR‐1 over‐expression resulted in a more than twofold increase in GRK2 promoter activity but only in the presence of the G‐allele (p = 0.04). In electrophoretic mobility shift assays, EGR‐1 over‐expression resulted in a specific binding of transcription factors only to the G oligonucleotide. Finally, EGR‐1 over‐expression resulted in increased GRK2 mRNA expression (p = 0.03). We identified EGR‐1 as a regulator of GRK2 transcription. Suppression of GRK2 expression by inhibition of EGR‐1 binding to GRK2 might be a promising approach to mitigate adrenergic desensitization.