Abstract The orphan receptor sigma-1 (sigmar-1) is a transmembrane chaperone protein expressed in both the central nervous system and in immune cells. It has been shown to regulate neuronal differentiation and cell survival, and mediates anti-inflammatory responses and immunosuppression in murine in vivo models. Since the details of these findings have not been elucidated so far, we studied the effects of the endogenous sigmar-1 ligands N,N-dimethyltryptamine (NN-DMT), its derivative 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and the synthetic high affinity sigmar-1 agonist PRE-084 hydrochloride on human primary monocyte-derived dendritic cell (moDCs) activation provoked by LPS, polyI:C or pathogen-derived stimuli to induce inflammatory responses. Co-treatment of moDC with these activators and sigma-1 receptor ligands inhibited the production of pro-inflammatory cytokines IL-1β, IL-6, TNFα and the chemokine IL-8, while increased the secretion of the anti-inflammatory cytokine IL-10. The T-cell activating capacity of moDCs was also inhibited, and dimethyltryptamines used in combination with E. coli or influenza virus as stimulators decreased the differentiation of moDC-induced Th1 and Th17 inflammatory effector T-cells in a sigmar-1 specific manner as confirmed by gene silencing. Here we demonstrate for the first time the immunomodulatory potential of NN-DMT and 5-MeO-DMT on human moDC functions via sigmar-1 that could be harnessed for the pharmacological treatment of autoimmune diseases and chronic inflammatory conditions of the CNS or peripheral tissues. Our findings also point out a new biological role for dimethyltryptamines, which may act as systemic endogenous regulators of inflammation and immune homeostasis through the sigma-1 receptor.

Citation: Szabo A, Kovacs A, Frecska E, Rajnavolgyi E (2014) Psychedelic N,N-Dimethyltryptamine and 5-Methoxy-N,N-Dimethyltryptamine Modulate Innate and Adaptive Inflammatory Responses through the Sigma-1 Receptor of Human Monocyte-Derived Dendritic Cells. PLoS ONE 9(8): e106533. https://doi.org/10.1371/journal.pone.0106533 Editor: Thomas Langmann, University of Cologne, Germany Received: May 30, 2014; Accepted: August 8, 2014; Published: August 29, 2014 Copyright: © 2014 Szabo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 „National Excellence Program” to AS, and OTKA-NK101538 to ER. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The term sigma receptor dates back historically to the sigma/opioid receptor described by Martin et al. [1] and reported to mediate the psychotropic effects of N-allylnormetazocine (NANM). It was originally thought to be an opioid receptor due to its modulation by NANM that could be antagonized by naloxone, a universal opioid antagonist [2]. Later, Su and colleagues clarified the pharmacological features of the ligand-binding site and the name was changed to ‘sigma receptor’ differentiating it from the sigma/opioid receptor [3], [4]. According to its tissue expression profile and ligand selectivity the receptor was subsequently classified to the sigma-1 and sigma-2 receptor subtypes (sigmar-1/2) [5]. In the last two decades several clinical studies demonstrated the importance of sigmar-1 in many diseases ranging from cancer, pain and addiction to different psychiatric and neurological disorders among them Major depression, Alzheimer’s disease, schizophrenia, and stroke [2]. Early studies showed that sigmar-1 is expressed not only in distinct regions of the CNS but also in immune cells [4], [6]. It was shown to regulate cell differentiation and survival by acting as a chaperone at the mitochondria-associated endoplasmic reticulum membrane [7], [8]. Murine studies also demonstrated that the specific activation of sigmar-1 resulted in immunosuppression [9], and in vivo decreased lymphocyte activation and proliferation [10]. Sigma-1 receptor ligands possess potent immunoregulatory properties via increasing the secretion level of anti-inflammatory IL-10 [11], and suppressing IFNγ and GM-CSF expression [10]. These important studies showed that sigmar-1 may cause significant alterations in immune functions. The endogenous ligands for sigmar-1 involve neurosteroids, dehydroepiandrosterone (DHEA), and naturally occuring indole alkaloids/tryptamines, such as N,N-dimethyltryptamine (NN-DMT) and its closely related analogue 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT). Hallucinogen indole alkaloids are widespread in nature and abundant in plants, which are used in preparation of sacramental psychoactive decoctions such as yage and ayahuasca [12]. NN-DMT and 5-MeO-DMT have also been detected in animal tissues; furthermore, NN-DMT is considered as an endogenous trace amine neurotransmitter that regulates brain physiology [13]–[15]. It has recently been shown that NN-DMT is a natural ligand for sigmar-1 [16], and its administration was reported to influence the number of circulating lymphocytes in humans, but the exact mechanism has not been uncovered yet [17]. In the light of these findings it is tempting to speculate that NN-DMT and 5-MeO-DMT may have impact on inflammatory responses through sigmar-1 [12]. Dendritic cells (DCs) are key players of innate immunity in higher vertebrates and their most prominent functions involve the continous sampling of the neighbouring enviroment. Harboring a selected spectrum of pathogen-sensing pattern recognition receptors (PRRs), such as intracellular Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), they can be activated by specific self and non self ligands [18]. They also exhibit the unique and crucial ability to translate PRR-mediated signals to adaptive immunity, thereby orchestrating natural and acquired immune responses [19]. Specialized subsets of DCs are working together as a network of vigilant gatekeepers in literally all tissues of the body [20]. In vitro differentiated human monocyte-derived DCs (moDCs) are considered as gold standards of DC biology and are used in various clinical and experimental settings [21]. Since human monocytes have recently been shown to migrate to the brain and are able to modulate the neuroinflammatory profile of the CNS [22], moDCs may represent a cell type, which, besides microglia, could also contribute to the immunoregulation of the neural tissue. In this study we aimed to investigate the effects of NN-DMT and 5-MeO-DMT-mediated activation of sigmar-1 on human primary moDC functions under inflammatory conditions as compared to resting state. To our best knowledge this is the first study reporting that dimethyltryptamines are potent anti-inflammatory agents, which have the capacity to modulate the functions of moDCs in a sigmar-1-dependent manner. Our results envision that dimethyltryptamines targeted to the sigmar-1 receptor could emerge as promising candidates for future pharmacological therapies in chronic inflammatory and autoimmune conditions of the CNS or peripheral tissues. We also propose a new biological role for NN-DMT, which, through the sigmar-1 of myeloid immune cells, may act as an endogenous regulator of inflammation and immune homeostasis.

Materials and Methods Cell isolation and culturing Leukocyte-enriched buffy coats were obtained from healthy blood donors drawn at the Regional Blood Center of the Hungarian National Blood Transfusion Service (Debrecen, Hungary) in accordance with the written approval of the Director of the National Blood Transfusion Service and the Regional and Institutional Ethics Committee of the University of Debrecen, Faculty of Medicine (Debrecen, Hungary). Written informed consent was obtained from the donors prior blood donation, and their data were processed and stored according to the directives of the European Union. Peripheral blood mononuclear cells (PBMCs) were separated by a standard density gradient centrifugation with Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden). Monocytes were purified from PBMCs by positive selection using immunomagnetic cell separation with anti-CD14 microbeads according to the manufacturer’s instruction (Miltenyi Biotech, Bergisch Gladbach, Germany). After separation on a VarioMACS magnet, 96–99% of the cells were CD14+ monocytes as measured by flow cytometry. Monocytes were cultured in 12-well tissue culture plates at a density of 2×106 cells/ml in serum-free AIMV medium (Invitrogen, Carlsbad, CA) supplemented with 80 ng/ml GM-CSF (Gentaur Molecular Products, Brussels, Belgium) and 100 ng/ml IL-4 (Peprotech EC, London, U.K.). On day 2, the same amounts of GM-CSF and IL-4 were added to the cell cultures without changing their media. Autologous naive T-cells were separated from mononuclear cells of the same donor using human naive CD4+ T Cell Isolation Kits (Miltenyi Biotech). Activation of dendritic cells Bacterial lipopolysaccharide (LPS) (Sigma, Schnelldorf, Germany), and high molecular weight polyinosinic: polycytidylic acid (polyI:C) (InvivoGen, San Diego, CA) were used at working concentrations of 500 ng/ml (LPS) and 20 µg/ml (polyI:C), respectively. N,N-dimethyltryptamine (R&D Systems, Abingdon, UK), 5-methoxy-N,N-dimethyltryptamine (Sigma), and 2-(4-morpholinoethyl-1)-phenylcyclohexane-1-carboxylate hydrochloride (PRE084) (Tocris Bioscience, Bristol, UK) were used in a working concentration of 100 µM. Pre-treatments were performed 1 h prior to DC activation. Controlled substances (Schedule I drugs) were used with the approval and monitoring of the Hungarian Institute for Forensic Sciences and the Hungarian National Police Department. Purified and inactivated A/Brisbane/59/7 (H1N1) influenza virus (kindly provided by the National Center for Epidemiology, Hungary) of 6×106 PFU/mL was used for in vitro treatment of 1×106 per mL DC in serum-free AIMV medium for 24 h. Activated cells were washed two times in sterile medium and then co-cultured with autologous naive T cells (ELISPOT). For activation of cells with bacteria, 5×104 heat-killed Escherichia coli 058 (Institute of Microbiology and Virology of National Academy of Science, Hungary) was added to 105 moDCs in 300 µl medium on a 96 well plate and incubated at 37°C for 24 h prior to ELISPOT assay. MoDCs were washed two times in sterile AIMV before co-culturing, as above. To prepare cell lysates for Western blotting, or collect supernatants for ELISA cells were activated for 24 h. Cell lysates were made after 8 h for Q-PCR measurements (if not stated otherwise). RNA isolation, cDNA synthesis and QPCR Real-time quantitative polymerase chain reaction (QPCR) was performed as described previously [23]. Briefly, total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA). 1.5–2 µg of total RNA were reverse transcribed using SuperScript II RNase H reverse transcriptase (Invitrogen) and Oligo (dT) 15 primers (Promega, Madison, WI). Gene-specific TaqMan assays (Applied Biosystems, Foster City, CA) were used to perform QPCR in a final volume of 12 µl in triplicates using AmpliTaq Gold DNA polymerase and ABI StepOnePlus real-time PCR instrument (Applied Biosystems). Amplification of 36B4 was used as a normalizing control. Cycle threshold values (Ct) were determined by using the StepOne 2.1 software. Constant threshold values were set for each gene throughout the study. Details of TaqMan assays are shown in Table S1. Cytokine measurements Culture supernatants were harvested 24 hours after activation and the concentrations of IL-1β, IL-6, TNFα, IL-8, and IL-10 cytokines were measured using OptEIA kits (BD Biosciences, San Jose, CA) following the manufacturer’s recommendations. The precision of the kits were the following: Intra-Assay variation: CV<10%; Inter-Assay variation: CV<12% (CV% = SD/meanX100). In each cases non-diluted supernatant samples were used for the assays, except IL-6 (3X dilution) and IL-8 (6X dilution) measurements. Western blotting Cells were lysed in Laemmli buffer and the protein extracts were tested by Ab specific for OPRS1/Sigmar-1 (Abcam, Cambridge, UK), and β-actin (Sigma) diluted at 1∶500 and 1∶000, respectively. Anti-rabbit Ab conjugated to horseradish peroxidase (GE Healthcare, Little Chalfont Buckinghamshire, UK) was used as the secondary Ab at a dilution of 1∶5000. The SuperSignal enhanced chemiluminescence system was used for probing target proteins (Thermo Scientific, Rockford, IL). After the membranes had been probed for OPRS1/Sigmar-1, they were stripped and re-probed for β-actin. ELISPOT assay Activated, pathogen-loaded DCs (2×105 cells/well) were co-cultured with naïve autologous CD4+ T cells (106 cells/well) in serum-free AIMV medium for 4 days at 37°C in humidified atmosphere containing 5% CO 2 . Phytohaemagglutinin (PHA) and Concanavalin A (ConA) activated T cells were used as positive controls, non-treated DC+T cell co-cultures and T cells without DC served as negative controls. Detection of activated, cytokine secreting CD4+ T cells producing IFNγ or IL17 was performed by the avidin-horseradish peroxidase system (NatuTec). Plates were analyzed on ImmunoScan plate reader (CTL Ltd., Shaker Heights, OH). RNA interference Gene-specific siRNA knockdown was performed by Silencer Select siRNA (Applied Biosystems) transfection using Gene Pulser Xcell instrument (Bio-Rad, Hercules, CA). Pulse conditions were square-wave pulse, 500 V, 0.5 ms. Immediately after electroporation, cells were transferred to fresh AIMV medium supplemented with, penicillin, streptomycin, and L-glutamine in addition to GM-CSF and IL-4. Silencing of sigmar1 gene expression was performed by using a mix of three of the available SIGMAR1 siRNAs. Silencer negative control nontargeting siRNA (Applied Biosystems) was used as a negative control. The efficacy of siRNA treatments was tested two days post-transfection by Western blotting. Statistical analysis Data are presented as mean ± SEM. A t-test was used for comparison of two groups. One-way ANOVA, followed by Bonferroni post hoc test, was used for multiple comparisons. Differences were considered to be statistically significant at p values <0.05 (*).

Discussion Hallucinogenic trypamines are members of the indole alkaloid family, the largest and most common class of alkaloids in the Animal and Plant Kingdoms. NN-DMT and bufotenine, the metabolic product of 5-MeO-DMT in mammals, have been detected in animal and human blood, urine, cerebrospinal fluid, brain, intestine and many other tissues suggesting that these compounds may have important biological roles other than their psychotropic and neuromodulatory properties [12], [35]–[37]. The orphan receptor sigmar-1 has been shown to regulate many physiological processes inculding cell survival and proliferation [7], [8]. The expression of sigma receptors is not limited to the brain as high level expression was detected in mammalian liver, kidney, gut and other tissues as well [5], [38]. Sigmar-1 has also been detected in immune cells mediating strong immunosuppressive and anti-inflammatory effects [9]–[11]. It has recently been reported that NN-DMT is an endogenous ligand for sigmar-1, and its agonistic activity may be expanded to analogues, such as the methoxy derivative 5-MeO-DMT [16]. However, very little is known about the physiological functions of dimethyltryptamines in human and the emphasis of contemporary research is mostly related to understanding its psychedelic properties and to our best knowledge, the biological effects of DMT via sigmar-1 has not been investigated yet. In this study we adressed the question whether sigmar-1 is expressed in human primary myeloid cells, and if so, what is its functional role in human physiology. According to our results, sigmar-1 is expressed in human monocytes and its expression is increasing during the differentiation process to macrophages and dendritic cells (Figure 1). Although sigmar-1 expression has been documented in murine macrophages [24], [25], this is the first report on characterizing sigmar-1 expression in resting and activated human myeloid immune cells. To test the hypothesis that dimethyltryptamines may have impact on immune cell functions through sigmar-1, we tested the effects of DMT treatment on the cytokine profile of activated moDCs. In these experiments we used the TLR3/RLR ligand polyI:C and the TLR4 agonist LPS as strong inducers of innate immune responses [18]. Our results revealed that NN-DMT and 5-MeO-DMT pre-treatment potently inhibited pro-inflammatory cytokine and chemokine (IL-1β, TNFα, IL-6, IL8) expression in human moDCs stimulated by specific PRR ligands, while had opposing effect on the mRNA and protein expression of the anti-inflammatory cytokine IL-10 (Figures 2 and 3). Furthermore, NN-DMT and 5-MeO-DMT interfered with the activation and polarization of naive T-lymphocytes toward Th1 and Th17 effector T cells when co-cultured with E. coli or influenza-virus loaded human moDCs (Figure 4). These results are in good agreement with reports showing that sigmar-1 activation results in elevated IL-10, decreased IFNγ and GM-CSF levels, and inhibition of lymphocyte proliferation in mice [9]–[11]. The results also demonstrated for the fist time that NN-DMT and 5-MeO-DMT have the capability to inhibit the polarization of human moDC-primed CD4+ T helper cells towards inflammatory Th1 and Th17 effector lymphocytes in infectious/inflammatory settings. This is of particular importance, since Th1 and Th17 cells and the cytokines they secrete are key players in the etiology and symptomatology of many chronic inflammatory and autoimmune diseases of the CNS and other tissues [29], [30], [39]. Moreover, the mobilization of innate immune mechanisms is also well established in many psychiatric and neurological disorders [39]. In neuropsychiatric research it is an increasingly accepted hypothesis that a number of diseases affecting large populations, such as Alzheimer’s, Parkinson’s disease, Major depression are caused by chronic inflammation of the central nervous system. High-resolution whole genome-wide association studies found significant correlations between gene polymorphisms of innate immune receptors and the frequency of late onset Alzheimer’s disease (AD) [40], [41]. It has also been demonstrated in mice that the ligand specific activation of the mother’s TLRs and RLRs by LPS and polyI:C results in decreased neurogenesis, cognitive deficits, and a marked increase in the appearance and deposition of Aβ aggregates in the brain of the offspring [42], [43]. Since blood-derived monocytes were shown to be able to translocate to the CNS [22], our results could expand the role of moDCs to a more global context by suggesting their regulatory role under autoimmune or infectious inflammatory conditions in the brain. In order to verify the contribution of sigmar-1 to the observed immunomodulatory effects we used the approach of gene-specific silencing. The results clearly demonstrated that downregulation of sigmar-1 abrogated the immunomodulatory effects of both NN-DMT and 5-MeO-DMT on cytokine secretion by innate immune cells (Figures 5) and also inhibited the moDC-mediated polarization of Th1 and Th17 effector cells (Figure 6). Remarkably, knock-down of sigmar-1 upon DMT pre-treatments could not restore cytokine levels and the T cell priming capacity of moDCs completely (Figures 5 and 6). These findings suggest that additional mechanisms through which DMT could modulate moDC functions may exist. NN-DMT and 5-MeO-DMT also bind to the 5-HT 2 (particularly 5-HT 2A ) and 5-HT 1A serotonin receptors with high affinity [44], [45]. This agonism was suggested to take part in psychological effects of dimethyltryptamines but may also contribute to immunological functions, since the neurotransmitter serotonin also exerts anti-inflammatory and immunoregulatory effects in DCs [46], [47]. As the experiments with human cells were performed in serum-free medium, the „serotonin background” effect can be excluded [48]. Thus, it is very likely that the observed phenomenon is the result of DMT-mediated serotonin receptor activation. To further verify the role of sigmar-1 in the modulation of moDC functions, we used the highly selective, high-affinity sigmar-1 agonist PRE-084 hydrochloride as a substitute of NN-DMT/5-MeO-DMT in the activation protocol. Similarly to NN-DMT and 5-MeO-DMT, PRE-084 treatment strongly interfered with TNF-α and IL-10 secretion by LPS or polyI:C stimulated moDCs (Figure S2). However, sigmar-1 knock-down could completely restore cytokine levels in PRR-activated and PRE-084-treated cells showing that sigmar-1 plays an essential role in the immunomodulation of moDCs (Figure S2). We conclude that the function of dimethyltryptamines may extend the central nervous system activity and may play a more universal role in immune regulation. Here we demonstrate for the first time that NN-DMT and 5-MeO-DMT have potent immunomodulatory effects on the functional activities of human dendritic cells operating through the sigma-1 receptor. We also show that DMT-mediated sigmar-1 activation can interfere with both innate and adaptive immune responses. On the one hand, it strongly decreases the levels of pro-inflammatory cytokines and chemokines such as IL-1β, IL-6, TNFα and IL8, while upregulates the production of the anti-inflammatory cytokine IL-10. On the other hand, NN-DMT and 5-MeO-DMT pre-treatment of pathogen-activated moDCs abolishes their capacity to initiate adaptive immune responses mediated by inflammatory Th1 and Th17 cells. These findings greatly expand the biological role of dimethyltryptamines, which may act not only as neuromodulators or psychedelics, but also as important regulators of both innate and adaptive immunity. Thus, the DMT-sigmar-1 axis emerges as a promising candidate for novel pharmacotherapies of chronic inflammatory and autoimmune diseases.

Supporting Information Figure S1. Time-dependence of the effects of sigmar-1 stimulation on moDC cytokine expression profiles. A-B: Expression of TNFα and IL-10 genes in 8 h 500 ng/ml LPS-stimulated moDCs. Tryptamines were added to the cells either at the time of LPS treatment (co-administration; co-adm) or 1 h prior to activation with LPS (1 h pre-treatment). Red bars represent LPS-only treated positive controls. White bars demonstrate co-treatments with 100 µM NN-DMT and 500 ng/ml LPS, while black bars show co-treatments with same concentrations of 5-MeO-DMT and LPS. Results are demonstrated as Mean ± SEM of three independent donors. C: Concentration-dependence of tryptamines in hindering TNFα production of 24 h 500 ng/ml LPS (red line) or 20 µg/ml polyI:C (blue line)-treated moDCs. Non-activated controls are shown in green. Data of a representative experiment out of two are shown. (*) represents p values<0.05. https://doi.org/10.1371/journal.pone.0106533.s001 (RAR) Figure S2. Results of sigmar-1 gene silencing on the PRE-084 hydrochloride-modulated cytokine profile of moDCs activated by LPS or polyI:C. Non-treated, 24 h ctrl siRNA-only, and 24 h targeting siRNA-only treated cells were used as negative controls (black bars). Red and blue bars represent 24 h 500 ng/ml LPS (red) or 20 µg/ml polyI:C (blue)-treated cells, while white bars show ctrl siRNA and 100 µM PRE-084 hydrochloride-treated cells 1 h prior to activation with LPS (A–B) or polyI:C (C–D) for one day. Green bars demonstrate 1 h PRE-084 hydrochloride pre-treated and then 24 h LPS (A–B) or polyI:C (C–D) activated sigmar-1 knockdown cells. Results are shown as Mean ± SEM of three independent donors. (*) represents p values<0.05. https://doi.org/10.1371/journal.pone.0106533.s002 (RAR) Table S1. QPCR assay information. https://doi.org/10.1371/journal.pone.0106533.s003 (RAR)

Acknowledgments We thank Erzsébet Nagy, Ágota Veres, and Anikó Kiss for their excellent technical assistance.

Author Contributions Conceived and designed the experiments: AS AK EF ER. Performed the experiments: AS AK EF. Analyzed the data: AS AK EF ER. Contributed reagents/materials/analysis tools: AK EF ER. Contributed to the writing of the manuscript: AS AK EF ER.