Inflammation shapes neural processing of interoceptive fear predictors during extinction learning in healthy humans.

Inflammation could impact on the formation and persistence of interoceptive fear and hypervigilance, with relevance to psychiatric disorders and chronic pain. To systematically analyze effects of inflammation on fear learning and extinction, we performed two complementary randomized, double-blind, placebo-controlled functional magnetic resonance imaging (fMRI) studies combining experimental endotoxemia as a translational model of acute systemic inflammation with a two-day multiple-threat fear conditioning paradigm involving interoceptive and exteroceptive unconditioned stimuli (US). Healthy volunteers (N = 95) were randomized to receive intravenous injections of either endotoxin (lipopolysaccharide, LPS; 0.4 ng/kg) or placebo prior to fear acquisition (study 1) or extinction training (study2). Treatment effects on behavioral and neural responses to conditioned stimuli (CS) predicting interoceptive or exteroceptive threat were assessed during fear learning and extinction phases, along with US valence ratings. Despite robust inflammatory and emotional responses triggered by LPS, no direct effects of inflammation on US ratings or on the formation or extinction of conditioned fear, as assessed with CS valence ratings, were observed. However, in the group treated with LPS prior to acquisition (i.e., study 1), we found enhanced neural responses to the interoceptive but not the exteroceptive CS in key regions of the central fear circuitry during extinction learning. After extinction, this group further showed enhanced negative valence ratings selectively for the interoceptive US during unexpected US re-exposure when compared to the placebo group. Together, inflammation during fear acquisition may promote the establishment of a more robust neural signature of the interoceptive fear memory trace, which may contribute to altered interoceptive pain perception. The fear extinction circuitry engaged during interoceptive fear memory processing may be particularly vulnerable to inflammation, with transdiagnostic implications for gut-brain mechanisms underlying disturbed interoception in psychiatric conditions and chronic visceral pain.

Circulating Pro-inflammatory Cytokines Do Not Explain Interindividual Variability in Visceral Sensitivity in Healthy Individuals.

A role of the immune system in the pathophysiology of pain and hyperalgesia has received growing attention, especially in the context of visceral pain and the gut-brain axis. While acute experimental inflammation can induce visceral hyperalgesia as part of sickness behavior in healthy individuals, it remains unclear if normal plasma levels of circulating pro-inflammatory cytokines contribute to interindividual variability in visceral sensitivity. We herein compiled data from a tightly screened and well-characterized sample of healthy volunteers (N = 98) allowing us to assess associations between visceral sensitivity and gastrointestinal symptoms, and plasma concentrations of three selected pro-inflammatory cytokines (i.e., TNF-α, IL-6, and IL-8), along with cortisol and stress-related psychological variables. For analyses, we compared subgroups created to have distinct pro-inflammatory cytokine profiles, modelling healthy individuals at putative risk or resilience, respectively, for symptoms of the gut-brain axis, and compared them with respect to rectal sensory and pain thresholds and subclinical GI symptoms. Secondly, we computed multiple regression analyses to test if circulating pro-inflammatory markers predict visceral sensitivity in the whole sample. Despite pronounced subgroup differences in pro-inflammatory cytokine and cortisol concentrations, we observed no differences in measures of visceroception. In regression analyses, cytokines did not emerge as predictors. The pain threshold was predicted by emotional state and trait variables, especially state anxiety, together explaining 10.9% of the variance. These negative results do not support the hypothesis that systemic cytokine levels contribute to normal interindividual variability in visceroception in healthy individuals. Trajectories to visceral hyperalgesia as key marker in disorders of gut-brain interactions likely involve complex interactions of biological and psychological factors in keeping with a psychosocial model. Normal variations in systemic cytokines do not appear to constitute a vulnerability factor in otherwise healthy individuals, calling for prospective studies in at risk populations.

When gut feelings teach the brain to fear pain: Context-dependent activation of the central fear network in a novel interoceptive conditioning paradigm.

The relevance of contextual factors in shaping neural mechanisms underlying visceral pain-related fear learning remains elusive. However, benign interoceptive sensations, which shape patients' clinical reality, may context-dependently become conditioned predictors of impending visceral pain. In a novel context-dependent interoceptive conditioning paradigm, we elucidated the putative role of the central fear network in the acquisition and extinction of pain-related fear induced by interoceptive cues and pain-predictive contexts. In this fMRI study involving rectal distensions as a clinically-relevant model of visceroception, N = 27 healthy men and women underwent differential conditioning. During acquisition training, visceral sensations of low intensity as conditioned stimuli (CS) predicted visceral pain as unconditioned stimulus (US) in one context (Con+), or safety from pain in another context (Con-). During extinction training, interoceptive CS remained unpaired in both contexts, which were operationalized as images of different rooms presented in the MRI scanner. Successful contextual conditioning was supported by increased negative valence of Con+ compared to Con- after acquisition training, which resolved after extinction training. Although interoceptive CS were perceived as comparatively pleasant, they induced significantly greater neural activation of the amygdala, ventromedial PFC, and hippocampus when presented in Con+, while contexts alone did not elicit differential responses. During extinction training, a shift from CS to context differentiation was observed, with enhanced responses in the amygdala, ventromedial, and ventrolateral PFC to Con+ relative to Con-, whereas no CS-induced differential activation emerged. Context-dependent interoceptive conditioning can turn benign interoceptive cues into predictors of visceral pain that recruit key regions of the fear network. This first evidence expands knowledge about learning and memory mechanisms underlying interoceptive hypervigilance and maladaptive avoidance behavior, with implications for disorders of the gut-brain axis.

Associative learning and extinction of conditioned threat predictors across sensory modalities.

The formation and persistence of negative pain-related expectations by classical conditioning remain incompletely understood. We elucidated behavioural and neural correlates involved in the acquisition and extinction of negative expectations towards different threats across sensory modalities. In two complementary functional magnetic resonance imaging studies in healthy humans, differential conditioning paradigms combined interoceptive visceral pain with somatic pain (study 1) and aversive tone (study 2) as exteroceptive threats. Conditioned responses to interoceptive threat predictors were enhanced in both studies, consistently involving the insula and cingulate cortex. Interoceptive threats had a greater impact on extinction efficacy, resulting in disruption of ongoing extinction (study 1), and selective resurgence of interoceptive CS-US associations after complete extinction (study 2). In the face of multiple threats, we preferentially learn, store, and remember interoceptive danger signals. As key mediators of nocebo effects, conditioned responses may be particularly relevant to clinical conditions involving disturbed interoception and chronic visceral pain.

Acute Versus Chronic Administration of Calcineurin-Inhibitors Differentially Affect T-Cell Function.

BACKGROUND: Calcineurin-inhibitors (CNI) are used in renal transplant patients (RTX) to prevent rejection. CNI mainly suppress T-cell mediated immunity but very little is known about the impact of long-term treatment with CNI on T-cell function. OBJECTIVE: We investigated the immunological effects of long-term CNI intake in RTX patients in comparison to short-term CNI administration in healthy controls (HC). METHODS: Blood was drawn from 30 RTX patients with long-term CNI treatment. In addition, blood was sampled from HC with short-term CNI treatment (four dosages) before the first and 2 hours after the last CsA intake. T-cells were analyzed for cytokine production, proliferation, and CD25 expression. RESULTS: Short-term CNI reduced T-cell derived IL-2 and IFNγ as well as T-cell proliferation in HC. IFNγ was not suppressed in patients with long-term CNI treatment. IL-2 production, CD25 expression, and T-cell proliferation were enhanced in long-term CNI patients. CONCLUSION: Suppression of IFNγ/IL-2 and T-cell proliferation is weaker during long-term CNI treatment in patients compared to short-term treatment in healthy subjects. Enhanced CD25 expression may lower the threshold for T-cell activation during long-term CNI treatment.

Are Adverse Events Induced by the Acute Administration of Calcineurin Inhibitor Cyclosporine A Behaviorally Conditioned in Healthy Male Volunteers?

PURPOSE: The learned immunosuppressive placebo response has been demonstrated in experimental animals, healthy humans, and patients, and is suggested as a therapy for improving immunopharmacologic treatment. It remains unclear, however, whether potential adverse events induced by the drug are also behaviorally conditioned. Employing an established taste-immune learning paradigm in healthy humans using the calcineurin inhibitor and immunosuppressive drug cyclosporine A (CsA) as an unconditioned stimulus, we investigated whether and to what extent perceived adverse events induced by acute CsA administration are behaviorally conditioned. METHODS: A total of 68 healthy male subjects were exposed to the established taste-immune learning paradigm, receiving either placebo or CsA (10 mg/kg) as an unconditioned stimulus, and a novel-tasting drink as a conditioned stimulus. FINDINGS: Subjects repeatedly receiving CsA during acquisition reported significantly more adverse events than did placebo-receiving subjects. However, during reexposure to the conditioned stimulus, the reported adverse events did not differ from those in the placebo control condition. IMPLICATIONS: These data indicate that acute adverse events are not behaviorally conditioned during the learned immunosuppressive response. Our results further strengthen the great potential clinical relevance of employing the learned immunosuppressive placebo response as a therapy to support immunopharmacologic regimens, ultimately aiming to reduce the medical dosages required, thereby minimizing adverse drug events while maximizing the therapeutic benefit in patients. German Clinical Trial Register (www.drks.de) identifier: DRKS00007693.

Learned immunosuppressive placebo responses in renal transplant patients.

Patients after organ transplantation or with chronic, inflammatory autoimmune diseases require lifelong treatment with immunosuppressive drugs, which have toxic adverse effects. Recent insight into the neurobiology of placebo responses shows that associative conditioning procedures can be employed as placebo-induced dose reduction strategies in an immunopharmacological regimen. However, it is unclear whether learned immune responses can be produced in patient populations already receiving an immunosuppressive regimen. Thus, 30 renal transplant patients underwent a taste-immune conditioning paradigm, in which immunosuppressive drugs (unconditioned stimulus) were paired with a gustatory stimulus [conditioned stimulus (CS)] during the learning phase. During evocation phase, after patients were reexposed to the CS, T cell proliferative capacity was significantly reduced in comparison with the baseline kinetics of T cell functions under routine drug intake (ƞp2 = 0.34). These data demonstrate, proof-of-concept, that learned immunosuppressive placebo responses can be used as a supportive, placebo-based, dose-reduction strategy to improve treatment efficacy in an ongoing immunopharmacological regimen.

Plasma cortisol response cannot be classically conditioned in a taste-endocrine paradigm in humans.

RATIONALE: Peripheral immune responses can be modified by associative learning procedures. Less is known, however, whether and to what extent neuroendocrine parameters can be classically conditioned. OBJECTIVES: In this randomized double-blind study, we modified an established paradigm to behaviorally condition endocrine responses in humans. METHODS: Thirty-one healthy male participants received a distinctively flavored green drink as the conditioned stimulus (CS) and intravenous injections of corticotropin-releasing hormone (CRH) (CRH group, N = 17) or NaCl (placebo group, N = 14) as the unconditioned stimulus (US) during two subsequent acquisition trials. Plasma levels of cortisol and noradrenaline, heart rate, and psychological parameters were analyzed before and 15, 30, 60, 120, and 180 min after injection. The two acquisition trials were followed by two evocation trials, during which participants underwent the same procedure but now receiving NaCl injections. RESULTS: CRH administration induced pronounced increases in cortisol and noradrenaline plasma concentrations, heart rate, and anxiety levels. However, re-exposure to the CS during evocations trials did not provoke conditioned increases in neuroendocrine parameters. Median split of the CRH group based on the cortisol baseline level into "cort-high" and "cort-low" subgroups showed that the "cort-high" subgroup displayed a significantly increased cortisol production on evocation days compared to the "cort-low" subgroup and the placebo group. CONCLUSION: This taste-endocrine paradigm employing CRH injection as the US in healthy male volunteers failed to induce a behaviorally conditioned cortisol release as a learned endocrine response. Future studies should clarify a possible role of higher baseline cortisol levels in perhaps facilitating a conditioned cortisol response.

Salivary alpha-amylase and noradrenaline responses to corticotropin-releasing hormone administration in humans.

Salivary alpha-amylase (sAA) is a digestive enzyme mainly responsible for the hydrolysis of starch and glycogen in the oral cavity. Since the secretion of sAA is largely under the control of the sympathetic nervous system, sAA activity is also considered to be a non-invasive marker of sympathetic activation. However, the direct association between sAA activity and other sympathetic parameters remains questionable. Therefore, we employed the corticotropin-releasing hormone (CRH) stimulation test to pharmacologically activate the sympathetic nervous system and to analyze plasma noradrenaline response together with sAA activity. Thirty-one healthy male volunteers (mean age of 25.2±3.1years) were randomized into two groups and received injections with either CRH (100µg, N=17) or placebo (0.9% NaCl, N=14). Blood samples were taken at baseline and 15, 30, 60, 120min after injection. Results showed that CRH administration increased plasma noradrenaline and cortisol concentrations, sAA activity, heart rate, as well as self-reported side effects (i.e. flushing in the facial area, heart rate changes, giddiness, malaise and restlessness) and stress perception, while plasma adrenaline levels remained unaffected. In the CRH group, the total increase of sAA activity significantly correlated with noradrenaline release, indicating that sAA activity reflects pharmacologically induced sympathetic activation.

Psychosocial Stress Increases Salivary Alpha-Amylase Activity Independently from Plasma Noradrenaline Levels.

Salivary alpha-amylase activity (sAA) and plasma noradrenaline (NA) concentrations are often considered to be surrogate markers of sympathetic activation in response to stress. However, despite accumulating evidence for a close association between sAA and noradrenaline and other indicators of sympathetic activity, reliability and generality of this relation remains unclear. We employed the Trier Social Stress Test (TSST) in order to directly compare the responses in sAA and NA to psychological stress in healthy volunteers (n = 23). The TSST significantly increased sAA and NA plasma levels with no significant differences in females and males. However, when subjects were divided according to their NA responses into low versus high responders, both groups did not significantly differ in their sAA before, during or after stress exposure. These data suggest that in response to acute psychological stress both plasma NA levels and sAA reflect sympathetic activity, however seemed to increase independently from each other.