Placebo Effects on the Immune Response in Humans: The Role of Learning and Expectation

<div><p>Placebo responses are primarily mediated via two neuropsychological mechanisms: patients’ expectation towards the benefit of a treatment and associative learning processes. Immune functions, like other physiological responses, can be modulated through behavioral conditioning. However, it is unknown whether learned immune responses are affected by the number of re-expositions to the conditioned stimulus (CS) during evocation. Moreover, it is unclear whether immune functions can also be modulated through mere verbally induced expectation. In the experiments reported here, we investigated in healthy male volunteers with an established model of learned immunosuppression whether a single re-exposition to the CS is able to induce a behaviorally conditioned immunosuppression. This conditioned immunosuppression is reflected through a significantly decreased interleukin (IL)-2 production by anti-CD3 stimulated peripheral blood mononuclear cells. Our data revealed that in contrast to four CS re-expositions (control group n = 15; experimental group n = 17), a single CS re-exposition was not sufficient to significantly suppress IL-2 production (control group n = 9, experimental group n = 10). Furthermore, we could demonstrate that mere expectation of taking an immunosuppressant did not cause an immunosuppressive response (n = 8–9 per expectation condition). Together, these findings extend our knowledge about the kinetics and mechanisms of placebo-induced immunosuppression and provide therewith information for designing conditioning protocols, which might be employed as a supportive therapy in clinical settings.</p> </div

Behavioral conditioning induced cytokine response.

<p>Behaviorally conditioned suppression of IL-2 release was observed after four CS re-expositions (control group n = 15, experimental group n = 17) (Fig. 2A). In contrast, a single CS re-exposition did not induce a significant inhibition in IL-2 production (control group n = 9, experimental group n = 10) (Fig. 2B). Data are expressed as percental changes from baseline. Bars represent mean ± SEM; ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Experimental design.

<p>(A) During the acquisition phase in conditioning <i>experiment A</i>, subjects of the experimental group received four times cyclosporin A (CsA) as an US together with a green-colored, novel tasting drink, the CS. During evocation, subjects were re-exposed to the drink four times but received identically looking placebo capsules instead of CsA. The control group was treated in an identical way but received placebo capsules throughout the study. Blood was drawn on the first day (baseline), on day 3 to determine the CsA-effect, on day 8 to analyze possible residual drug effects and on day 10 in order to determine the conditioned effect on IL-2 production <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049477#pone.0049477-Wirth1" target="_blank">[19]</a>. (B) During the acquisition phase in conditioning <i>experiment B</i> subjects were identically treated as in <i>experiment A</i>. However, during evocation, subjects were re-exposed to the drink and the placebo capsules only once. Blood was drawn on the first day (baseline), on day 3 to determine the CsA-effect, on day 8 to analyze possible residual drug effects and on day 10 in order to determine the conditioned effect on IL-2 production. (C) In <i>experiment C</i>, subjects were told to have a probability of either 25%, 50%, 75% or 100% of receiving CsA to manipulate subjects’ expectation of receiving an active drug. Capsules were given at four time points on 3 consecutive days. Blood was drawn on the first day for baseline measurement and on day 3 to determine the potential effect of expectation on IL-2 production of anti-CD3 stimulated PBMC.</p

Expectation induced cytokine response.

<p>(A) Expectation did not induce a significant reduction in IL-2 production in any of the four expectation groups differing in the probability of receiving CsA. IL-2 (pg/ml) concentration in supernatants of anti-CD3 stimulated PBMC was analyzed before and after placebo pill intake (25% n = 9, 50% n = 8, 75% n = 8, 100% n = 8). Data are shown as mean ± SEM. (B) Expectation did not reduce the percentage of IL-2 producing CD4⁺T cells. IL-2 producing CD4⁺T cells were analyzed as percent of total CD4⁺T cells before and after placebo pill intake. Data are shown as mean ± SEM.</p

Catechol-O-Methyltransferase Val158Met Polymorphism Is Associated with Somatosensory Amplification and Nocebo Responses

<div><p>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 (<i>COMT</i>) 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.</p></div

Reported CsA-specific side effects after <i>Medication</i> (A) and “<i>Placebo</i>” intake, respectively (B).

<p>Significantly higher CsA-specific side effects, after four medication intakes, were reported by homozygous Val158/Val158 carriers (<b>A</b>). This difference was even more pronounced after fourteen “<i>Placebo</i>” intakes (<b>B</b>). Bars represent mean ±SEM. In case of significant F tests, these were followed by Bonferroni post hoc tests; *<i>p</i><0.05, **<i>p</i><0.001.</p

On experimental days 1 (6 pm), 2 (8 am and 6 pm) and 3 (8 am) during medication intake, all subjects in each of the 3 treatment groups received four oral doses of 2.5 mg/kg body weight of the immunosuppressive drug CsA (Sandimmun optoral, Novartis) in capsule form.

<p>In addition, subjects in groups 1 (n = 24) and 2 (n = 26) received the CS (drink) with each capsule (CsA) intake whereas subjects in group 3 (n = 12) were not exposed to the CS <b>(A)</b>. After five days wash out time, subjects received either identical looking capsules containing a placebo (lactose powder) or a subtherapeutic dose of CsA (0.25 mg/kg) fourteen times, twice a day (8 am and 6 pm respectively) with (groups 1 and 2) or without (group 3) the CS <b>(B)</b>. In order to analyze possible general treatment side effects (nocebo effects), participants were asked before the start of the study (<i>Pre I</i>), after <i>Medication</i> intake (<i>Post I</i>) as well as before (<i>Pre II</i>) and after “<i>Placebo</i>” (<i>Post II</i>) intake to fill out the GASE. Blood was drawn on the first day for baseline measurement (<i>Pre I</i>), on day 3 (<i>Post I</i>) to analyze the pharmacological effect of CsA as well as on day 8 (<i>Pre II</i>) and 15 (<i>Post II</i>) to determine possible residual effects of the drug as well as effects on physiological parameters after treatment with “<i>Placebo</i>” (sub-therapeutical doses of CsA).</p

CsA serum levels and IL-2 protein concentrations of the three treatment groups during <i>Medication</i> and “<i>Placebo</i>” intake.

<p>CsA treatment during <i>Medication</i> significantly increased CsA serum levels and significantly suppressed IL-2 protein concentrations after anti-CD3 stimulation in all groups. During the “<i>Placebo</i>” condition, treatment with subtherapeutical doses of CsA slightly increased CsA levels (groups 2 and 3), however did not effect IL-2 concentration in these groups. (ANOVA, time effect; *<i>p</i><0.001) (n.d. =  not detectable). Data are shown as mean ±SEM.</p><p>CsA serum levels and IL-2 protein concentrations of the three treatment groups during <i>Medication</i> and “<i>Placebo</i>” intake.</p

CsA-specific and general side effects during the <i>Medication</i> and “<i>Placebo</i>” condition.

<p>Treatment groups did not significantly differ in reported CsA-specific and general side effects analyzed with the GASE, neither before study participation (GASE Baseline), nor during the <i>Medication</i> and the “<i>Placebo</i>” condition (all p>0.05). Data are shown as mean ±SEM.</p><p>CsA-specific and general side effects during the <i>Medication</i> and “<i>Placebo</i>” condition.</p

Sociodemographic and psychological characteristics of the three experimental groups.

<p>Age, body mass index, physical activity, trait anxiety (STAI), SSAS, BMQ_ general harm, BMQ_general overuse, BMQ_general benefit, BMQ_sensitive soma, BMQ_specific necessity and BMQ_specific were compared between all three treatment groups using univariate ANOVA. Groups did not significantly differ in any of the variables listed (all p>0.05). Data are shown as mean ±SEM.</p><p>Sociodemographic and psychological characteristics of the three experimental groups.</p