LETTERS 940 VOLUME 22 | NUMBER 8 | AUGUST 2016 NATURE MEDICINE Positive expectations contribute to the clinical benefits of the placebo effect 1,2 . Such positive expectations are mediated by the brain’s reward system 3,4 ; however, it remains unknown whether and how reward system activation affects the body’s physiology and, specifically, immunity. Here we show that activation of the ventral tegmental area (VTA), a key component of the reward system, strengthens immunological host defense. We used ‘designer receptors exclusively activated by designer drugs’ (DREADDs) to directly activate dopaminergic neurons in the mouse VTA and characterized the subsequent immune response after exposure to bacteria (Escherichia coli), using time-of-flight mass cytometry (CyTOF) and functional assays. We found an increase in innate and adaptive immune responses that were manifested by enhanced antibacterial activity of monocytes and macrophages, reduced in vivo bacterial load and a heightened T cell response in the mouse model of delayed-type hypersensitivity. By chemically ablating the sympathetic nervous system (SNS), we showed that the reward system’s effects on immunity are, at least partly, mediated by the SNS. Thus, our findings establish a causal relationship between the activity of the VTA and the immune response to bacterial infection. Despite its elusive nature, the potency of the placebo effect is recog- nized in modern medicine. For 50 years, clinical trials have included a placebo group to control for nonspecific effects, such as statistical errors, spontaneous recovery and patient expectations. Such expecta- tion of clinical improvement has a key role in the placebo effect 5,6 and has been associated with activation of the brain’s reward system 3,4 . It is, however, unknown whether and how activity of the brain’s reward system can affect physical health, specifically immunity, and, if it does, what peripheral components mediate this interaction. The immune system responds to psychological and cognitive factors 7,8 , including positive emotions 9–14 , in which the reward system has a crucial role 15,16 . Thus, we designed our study to examine whether reward system activation, which is evident in the placebo response and in positive expectation, can impact immunity. To determine the effects of the reward system on immunity, we focused on dopaminergic neurons in the VTA, a central mediator of reward-related stimuli and positive expectations 17 . To manipulate neuronal activity, we used DREADDs, which are G-protein-coupled receptors (GPCRs) that have been mutated to be activated only by an otherwise inert ligand (clozapine-N-oxide; CNO) 18 . These DREADDs activate endogenous downstream signal transduction pathways to augment neuronal activity 18 . To express DREADDs and a fluorescent reporter marker in VTA dopaminergic neurons, we stereotactically injected a viral vector that expresses a Cre-dependent DREADD construct (AAV8-hSyn1-DIO-hM3D(Gq)- mCherry) under the control of the human synapsin I (Syn1) promoter in mice that express Cre under the tyrosine hydroxylase (Th) pro- moter (which we refer to as TH-Cre mice). As a control, we infected another group of mice with a sham virus that expresses only the fluo- rescent reporter (Fig. 1a). Throughout the study, mice injected with the sham virus were used as the control group. Immunohistochemical analysis revealed that TH + cells in the VTA expressed the fluorescent reporter (Fig. 1b and Supplementary Fig. 1). Among these TH + cells, the efficiency of viral infection was 58% ± 4% (Fig. 1c). We verified DREADD-induced neuronal activation by analyzing the expression of c-Fos, an immediate-early activation marker, following intraperi- toneal (i.p.) injection of CNO. c-Fos expression was evident in 42% ± 0.5% of DREADD-expressing cells, as compared to 3% ± 1% in con- trol mice infected with the sham virus (Fig. 1d,e). To determine the behavioral effects of DREADD-induced VTA activation, we evalu- ated mouse behavior using the ‘conditioned place preference’ (CPP) paradigm. This assay quantifies an animal’s preference for a location in which it had a subjectively positive experience 19 . Despite the low temporal resolution of DREADDs, mice in which VTA activation was induced (hereafter referred to as VTA-activated mice) showed a 31.5% ± 12.6% increased preference for the chamber in which they were injected with CNO (Fig. 1f–h). In addition, VTA-activated mice participated in more social interactions with cage mates (44.4% ± 12.9% increase; Supplementary Fig. 2), consistent with a recent report that found VTA activation increases social behavior 20 . Thus, we concluded that DREADDs were specifically expressed in VTA TH + neurons and that they activated the reward system in the experimental mice. Activation of the reward system boosts innate and adaptive immunity Tamar L Ben-Shaanan 1,2 , Hilla Azulay-Debby 1,2 , Tania Dubovik 1 , Elina Starosvetsky 1 , Ben Korin 1,2 , Maya Schiller 1,2 , Nathaniel L Green 1,2 , Yasmin Admon 1 , Fahed Hakim 1,3 , Shai S Shen-Orr 1,4,5 & Asya Rolls 1,2,5 1 Department of Immunology, Rappaport Faculty of Medicine, Technion–Israel Institute of Technology, Haifa, Israel. 2 Center of Science and Engineering of Neuronal Systems, Rappaport Faculty of Medicine, Technion–Israel Institute of Technology, Haifa, Israel. 3 Pediatric Pulmonary Unit, Rambam Health Care Campus, Haifa, Israel. 4 Faculty of Biology, Technion–Israel Institute of Technology, Haifa, Israel. 5 These authors jointly directed this work. Correspondence should be addressed to A.R. (rolls@technion.ac.il) or S.S.S.-O. (shenorr@technion.ac.il). Received 15 January; accepted 1 June; published online 4 July 2016; doi:10.1038/nm.4133 npg © 2016 Nature America, Inc. All rights reserved.