This in vitro (neuronal cell culture) study investigated the anti-inflammatory effects of NN-DMT and 5-MeO-DMT (100 μM), and demonstrate that its immunomodulatory effects on the functional activities of human dendritic cells operate through the sigma-1 receptor.
Abstract
“Introduction: 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.
Methods: 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.
Results: 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.”
Authors: Attila Szabo, Attila Kovacs, Ede Frecska & Eva Rajnavolgyi
Summary
The orphan receptor sigmar-1 regulates neuronal differentiation and cell survival, and mediates anti-inflammatory responses and immunosuppression in murine in vivo models. We show that the endogenous ligands NN-DMT and 5-MeO-DMT have immunomodulatory potential on human primary monocyte-derived dendritic cells via sigmar-1.
Psychedelics modulate innate and adaptive inflammatory responses through the Sigma-1 Receptor of human monocyte-derived dendritic cells.
Introduction
The sigma receptor was originally thought to be an opioid receptor, but later it was classified into sigma-1 and sigma-2 receptor subtypes. These subtypes are involved in many diseases, including cancer, pain and addiction, and psychiatric and neurological disorders.
Early studies showed that sigmar-1 regulates cell differentiation and survival by acting as a chaperone at the mitochondria-associated endoplasmic reticulum membrane, and may cause significant alterations in immune functions.
Endogenous ligands for sigmar-1 include neurosteroids, dehydroepiandrosterone (DHEA), and naturally occurring indole alkaloids/tryptamines, such as NN-DMT and 5-MeO-DMT. NN-DMT and 5-MeO-DMT may have impact on inflammatory responses through sigmar-1.
Dendritic cells (DCs) are key players of innate immunity in higher vertebrates and harbor a selected spectrum of pathogen-sensing pattern recognition receptors (PRRs). They are also able to translate PRR-mediated signals to adaptive immunity, thereby orchestrating natural and acquired immune responses.
In this study, we investigated the effects of NN-DMT and 5-MeO-DMT on human primary moDC functions under inflammatory conditions as compared to resting state. We propose a new biological role for NN-DMT.
Cell isolation and culturing
Leukocyte-enriched buffy coats were obtained from healthy blood donors, and monocytes were purified from PBMCs using immunomagnetic cell separation with anti-CD14 microbeads. The monocytes were cultured in serum-free AIMV medium supplemented with 80 ng/ml GM-CSF and 100 ng/ml IL-4.
Activation of dendritic cells
Bacterial lipopolysaccharide and high molecular weight polyinosinic: polycytidylic acid were used as pretreatments, and controlled substances 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 was used to activate DC in vitro and then co-cultured with autologous naive T cells.
Heat-killed Escherichia coli 058 was added to 105 moDCs and incubated at 37uC for 24 h prior to ELISPOT assay.
RNA isolation, cDNA synthesis and QPCR
Real-time quantitative polymerase chain reaction was performed to determine the expression of genes in the nephron. Gene-specific TaqMan assays were used to perform QPCR.
Cytokine measurements
Culture supernatants were harvested 24 hours after activation and cytokine concentrations were measured using OptEIA kits (BD Biosciences, San Jose, CA).
Western blotting
Cells were lysed in Laemmli buffer and protein extracts were tested for OPRS1/Sigmar-1 and b-actin. The SuperSignal enhanced chemiluminescence system was used for probing target proteins.
ELISPOT assay
Activated, pathogen-loaded DCs were cocultured with nave autologous CD4+ T cells for 4 days at 37uC in humidified atmosphere containing 5% CO2. The cytokine production of the T cells was detected by an avidin-horseradish peroxidase system.
RNA interference
Gene-specific siRNA knockdown was performed by using Silencer Select siRNA (Applied Biosystems) transfection using Gene Pulser Xcell instrument (Bio-Rad, Hercules, CA). The efficacy of siRNA treatments was tested two days post-transfection by Western blotting.
Statistical analysis
Sigmar-1 is an intracellular receptor located on the mitochondria-associated endoplasmic reticulum membrane. It has already been detected in various neuronal cell types and brain macrophages of rodents, but little is known about its expression in other immune cells. Despite strong inflammatory responses via TLRs and RLRs, high-dose bacterial lipopolysaccharide (LPS) or viral nucleic-acid analogue polyinosinic: polycytidylic acid (polyI:C) did not alter the expression level of sigmar-1 in moDCs.
inflammatory but boosts anti-inflammatory responses in LPS or polyI:C-stimulated moDCs
Based on the detectable levels of sigmar-1 in moDCs, we tested the effects of NN-DMT and 5-MeO-DMT treatments on the cytokine profile of activated moDCs. We found that 100 mM DMT was optimal for modulating TNFa levels in activated moDCs.
Further experiments showed that pre-treatment of LPS-activated moDCs with either NN-DMT or 5-MeO-DMT strongly reduced the expression levels of pro-inflammatory cytokines and chemokines, while the expression and production of the anti-inflammatory cytokine IL-10 was significantly higher in DMT+LPS treated cells as compared to LPS-activated control. NN-DMT and 5-MeO-DMT have a strong inhibitory capacity on inflammatory responses in moDCs, suggesting that they can be used as pharmacological modulators.
moDCs inhibits their capacity to prime autologous naive T helper 1 (Th1) and T helper 17 (Th17) cells
We found that short-term DMT pre-treatment of LPS or polyI:C activated moDCs can suppress innate pro-inflammatory responses but concomitantly promote IL-10 secretion. We tested whether this could result in an unique T cell response by co-culturing naive autologous CD4+ T cells with pathogen-activated moDCs.
MoDCs were stimulated with heat-killed E. coli and influenza virus, and the number of activated IFNc (Th1) and IL-17 (Th17) secreting effector T cells was measured by ELISPOT assays. NN-DMT and 5-MeO-DMT inhibited the processing and/or presentation of bacterial and viral peptide antigens by moDCs.
NN-DMT and 5-MeO-DMT modulate the cytokine production and T cell-priming capacity of moDCs in a sigma-1 receptor dependent manner
After demonstrating that NN-DMT and 5-MeO-DMT inhibit inflammatory cytokine production and CD4+ helper T cell priming capacity, we aimed to test whether sigmar-1 plays a role in these processes. We performed sigmar1 gene knock-down experiments to clarify the immunomodulatory role of sigmar-1 in moDC functional activities and to check its contribution to the observed immunomodulatory effects. We found that sigmar-1 knock-down resulted in decreased modulatory capacity of NN-DMT and 5-MeO-DMT on priming Th1 and Th17 T-lymphocyte differentiation.
Dimethyltryptamines can modulate the cytokine responses of human primary macrophages. PRE-084 hydrochloride can be used as a substitute of NN-DMT and 5-MeO-DMT, and can completely abrogate TNF-a production and increase IL-10 secretion.
Discussion
Hallucinogenic trypamines are members of the indole alkaloid family and have been detected in animal and human blood, urine, cerebrospinal fluid, brain, intestine and other tissues.
The orphan receptor sigmar-1 has been shown to regulate many physiological processes, including cell survival and proliferation. It has also been detected in immune cells mediating strong immunosuppressive and anti-inflammatory effects, but its functional role in human physiology has not been investigated yet. We used TLR3/RLR ligand polyI:C and TLR4 agonist LPS to stimulate human moDCs and found that dimethyltryptamines inhibited pro-inflammatory cytokine and chemokine expression while increasing the expression of the anti-inflammatory cytokine IL-10.
Human moDCs stimulated with E. coli or influenza-virus loaded cells secreted IL-10, decreased IFNc and GM-CSF, and inhibited lymphocyte proliferation. NN-DMT and 5-MeO-DMT inhibited the polarization of CD4+ T helper cells towards inflammatory Th1 and Th17 effector lymphocytes. In neuropsychiatric research, it is increasingly accepted that chronic inflammation of the central nervous system is a major cause of Alzheimer’s, Parkinson’s disease, and Major depression. Bloodderived monocytes may play a key role in controlling inflammation in the brain.
We used gene-specific silencing to verify the contribution of sigmar-1 to the observed immunomodulatory effects of dimethyltryptamines. The results clearly demonstrated that downregulation of sigmar-1 abrogated the immunomodulatory effects of dimethyltryptamines on cytokine secretion by innate immune cells and also inhibited the moDC-mediated polarization of Th1 and Th17 effector cells.
The sigmar-1 agonist PRE-084 hydrochloride strongly interfered with TNFa and IL-10 secretion by LPS or polyI:C stimulated moDCs. However, sigmar-1 knock-down could completely restore cytokine levels.
We conclude that dimethyltryptamines may extend the central nervous system activity and may play a more universal role in immune regulation. They can interfere with both innate and adaptive immune responses.
Supporting Information
Figure S1 shows the time-dependence of the effects of sigmar-1 stimulation on moDC cytokine expression profiles. Tryptamines inhibited TNFa production in both 24 h 500 ng/ml LPS-treated moDCs and 1 h pre-treatment LPS-stimulated moDCs.
PRE-084 hydrochloride modulated cytokine profile of moDCs activated by LPS or polyI:C. Cells were treated with PRE-084 hydrochloride 1 h prior to activation with LPS or polyI:C for one day.