Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
Molecules 29 (21): 5084 (2024)
Wiens, Kennedy R.
Brooks, Noah A. H.
Riar, Ishvin
Greuel, Bridget K.
Klegeris, Andis
Lindhout, Ivan
Neuroinflammation that is caused by microglia, the main immune cells of the brain, contributes to neurodegenerative diseases. Psychedelics, including psilocybin and lysergic acid diethylamide (LSD), possess certain anti-inflammatory properties and, therefore, should be considered as drug candidates for treating neuroinflammatory pathologies. When ingested, psilocybin is rapidly dephosphorylated to yield psilocin, which crosses the blood–brain barrier and exerts psychotropic activity by interacting with the 5-hydroxytryptamine 2A receptors (5-HT₂ARs) on neurons. Since microglia express all three 5-HT₂AR isoforms, we hypothesized that, by interacting with these receptors, psilocin beneficially modulates select neuroimmune functions of microglia. We used microglia-like cell lines to demonstrate that psilocin, at non-toxic concentrations, did not affect the secretion of tumor necrosis factor (TNF) by immune-stimulated microglial cells, but significantly inhibited their phagocytic activity, the release of reactive oxygen species (ROS), and nitric oxide (NO) production. The inhibitory activity of psilocin on the latter two functions was similar to that of two selective 5-HT₂R agonists, namely, 25I-NBOH and Ro60-0175. The role of this subfamily of receptors was further demonstrated by the application of 5-HT₂R antagonists cyproheptadine and risperidone. Psilocin should be considered a novel drug candidate that might be effective in treating neuroimmune disorders, such as neurodegenerative diseases, where reactive microglia are significant contributors.
Citation: Wiens, K.R.; Brooks, N.A.H.;Riar, I.; Greuel, B.K.; Lindhout, I.A.;Klegeris, A. Psilocin, the PsychoactiveMetabolite of Psilocybin, ModulatesSelect Neuroimmune Functions ofMicroglial Cells in a 5-HT2Receptor-Dependent Manner.Molecules 2024, 29, 5084. https://doi.org/10.3390/molecules29215084Academic Editor: JoséMarco-ContellesReceived: 29 August 2024Revised: 16 October 2024Accepted: 17 October 2024Published: 28 October 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).moleculesArticlePsilocin, the Psychoactive Metabolite of Psilocybin, ModulatesSelect Neuroimmune Functions of Microglial Cells in a 5-HT2Receptor-Dependent MannerKennedy R. Wiens, Noah A. H. Brooks, Ishvin Riar, Bridget K. Greuel , Ivan A. Lindhout and Andis Klegeris *Laboratory of Cellular and Molecular Pharmacology, Department of Biology, University of British ColumbiaOkanagan Campus, Kelowna, BC V1V 1V7, Canada* Correspondence: andis.klegeris@ubc.caAbstract: Neuroinflammation that is caused by microglia, the main immune cells of the brain,contributes to neurodegenerative diseases. Psychedelics, including psilocybin and lysergic acid di-ethylamide (LSD), possess certain anti-inflammatory properties and, therefore, should be consideredas drug candidates for treating neuroinflammatory pathologies. When ingested, psilocybin is rapidlydephosphorylated to yield psilocin, which crosses the blood–brain barrier and exerts psychotropicactivity by interacting with the 5-hydroxytryptamine 2A receptors (5-HT2ARs) on neurons. Sincemicroglia express all three 5-HT2R isoforms, we hypothesized that, by interacting with these receptors,psilocin beneficially modulates select neuroimmune functions of microglia. We used microglia-likecell lines to demonstrate that psilocin, at non-toxic concentrations, did not affect the secretion oftumor necrosis factor (TNF) by immune-stimulated microglial cells, but significantly inhibited theirphagocytic activity, the release of reactive oxygen species (ROS), and nitric oxide (NO) production.The inhibitory activity of psilocin on the latter two functions was similar to that of two selective5-HT2R agonists, namely, 25I-NBOH and Ro60-0175. The role of this subfamily of receptors wasfurther demonstrated by the application of 5-HT2R antagonists cyproheptadine and risperidone.Psilocin should be considered a novel drug candidate that might be effective in treating neuroimmunedisorders, such as neurodegenerative diseases, where reactive microglia are significant contributors.Keywords: affective disorders; depression; 5-hydroxytryptamine; neurodegenerative diseases;neuroprotection; phagocytic activity; reactive oxygen species; reactive nitrogen species; serotonin1. IntroductionWhile the aggregation of amyloid β and the hyperphosphorylation of tau play well-established roles in the pathophysiology of Alzheimer’s disease (AD), the involvementof persistent neuroinflammation in the development and progression of this and otherneurodegenerative disorders is increasingly accepted [1]. Neuronal death and neuroin-flammatory responses in AD are accompanied by the chronic overactivation of glial cells,particularly microglia [2,3]. When confronted with abnormal protein aggregates, microgliafacilitate the innate neuroimmune response. In part, this involves the clearance of harmfulparticles by phagocytosis and the release of cytokines, such as the pro-inflammatory tumornecrosis factor (TNF), and cytotoxins, including reactive oxygen species (ROS) and nitricoxide (NO) [4]. While these functions are essential for mounting an effective acute immuneresponse, neurodegeneration, once triggered, leads to the chronic adverse reactivity ofmicroglia. This causes the sustained secretion of pro-inflammatory mediators and excessphagoptosis, contributing to neuronal death [5,6]; therefore, anti-inflammatory therapiesaimed at ameliorating reactive microglia represent feasible options in the search for effectivetreatments for these disorders [7,8].Anti-inflammatory drugs are a class of medication aimed at suppressing or mod-ulating inflammatory responses. The long-term use of conventional non-steroidal anti-Molecules 2024, 29, 5084. https://doi.org/10.3390/molecules29215084 https://www.mdpi.com/journal/moleculesMolecules 2024, 29, 5084 2 of 18inflammatory drugs (NSAIDs) has previously been associated with a decreased risk ofAD in epidemiological studies [9]; however, conflicting results have been observed inclinical trials [10]. Additionally, several other commonly used inflammation-reducing ther-apies, such as corticosteroids, have proven to lack protective effects in AD treatment [11];therefore, novel molecular targets and drug candidates that can be used to effectivelysuppress neuroinflammation in a broad range of neurodegenerative diseases need to beidentified. Anti-inflammatory drugs often exert their effects by reducing the activity ofenzymes and transcription factors, such as cyclooxygenase (COX)-2, nicotinamide ade-nine dinucleotide phosphate (NADPH)-dependent oxidase (NOX), inducible NO synthase(iNOS), and nuclear factor (NF)-κB. Such inhibitory activities diminish the production ofinflammation-inducing and -propagating prostaglandins, ROS, NO, and cytokines [12–15].Psychedelics, including psilocybin, mescaline, and lysergic acid diethylamide (LSD), haverecently been shown to possess anti-inflammatory properties [16]. Since this heterogenousgroup of compounds remains relatively unexplored in the context of neuroinflammationand neurodegenerative diseases, they may possess untapped potential as drug candidatesfor the treatment of these disorders [17].Depression is often associated with alterations in 5-hydroxytryptamine (5-HT, sero-tonin) neurotransmission. Most first-line antidepressants modulate specific aspects of 5-HTsignaling, such as its reuptake, but there are treatment-resistant cases that do not respondto such therapies [18]. Notably, increasing evidence indicates that neuroinflammationalso contributes to the pathogenesis of depression [19]. Treatment-resistant depressioncases in particular are associated with increased TNF concentrations [20]. In addition,higher-than-normal plasma concentrations of both ROS and NO have been detected inindividuals experiencing depressive symptoms [21,22]. Recently, antidepressant drugs,such as selective serotonin reuptake inhibitors (SSRIs), have been shown to reduce neuroin-flammation [23]. Therefore, drugs with antidepressant activity that are also confirmed tocross the blood–brain barrier should be considered for use to suppress neuroinflammationin neurodegenerative diseases [24,25].Psilocin (4-hydroxy-N,N-dimethyltryptamine) is the bioactive metabolite of the natu-rally occurring psychedelic alkaloid psilocybin, which was first isolated from mushroomsof the Psilocybe genus [26]. Since their discovery, methods of synthetic production for bothpsilocin and psilocybin have been developed. When ingested, psilocybin undergoes rapiddephosphorylation by alkaline phosphatase in the small intestine, kidneys, and plasmato yield psilocin, which freely crosses the blood–brain barrier and exerts psychotropicactivity [27]. The distinctive psychedelic effects induced by psilocin result from alteredserotonin signaling, primarily due to the activation of the 5-HT2A receptor (5-HT2AR) iso-form on frontal cortex pyramidal neurons [28,29]. Likely due to this stimulatory activity ofits main metabolite, psilocybin has emerged as a promising novel antidepressant, as shownin multiple human clinical trials, significantly alleviating symptoms in various forms ofdepression, including in treatment-resistant cases [30–32]. Recently, it has been proposedthat the clinically meaningful antidepressant activity of psilocin could be due in part to itsanti-inflammatory effects, which are also mediated by 5-HTRs expressed by neurons andglial cells [16,33]. Therefore, we hypothesized that the anti-neuroinflammatory activity ofpsilocin is mediated by its activation of 5-HT2R isoforms expressed by microglia, therebydownregulating their phagocytic activity and reducing their secretion of cytotoxins.To test this hypothesis, in addition to psilocin, we employed two established ligands of5-HT2Rs, namely, 25I-NBOH and Ro60-0175. All three compounds exhibit unique bindingpatterns with regard to 5-HTRs [34,35]. Psilocin displays a high level of affinity for the5-HT2AR but is also capable of binding to the 5-HT2BR, 5-HT2CR, and 5-HT1AR isoforms,albeit with a lower level of affinity [36,37]. 25I-NBOH demonstrates a high level of affinityfor the 5-HT2AR and, in contrast to psilocin, is highly selective for this receptor isoformin relation to other members of the 5-HT2R subfamily [38]. Conversely, Ro60-0175 has ahigher level of affinity towards the 5-HT2CR isoform than 5-HT2A [39]. In this study, wefound that psilocin significantly inhibited phagocytic activity and the production of ROSMolecules 2024, 29, 5084 3 of 18and NO by immune-activated microglial cells. We also demonstrated that the inhibitoryactivity of psilocin on ROS and NO production by microglial cells was similar to that ofthe two established 5-HT2R agonists, 25I-NBOH and Ro60-0175. Additionally, the 5-HT2Rantagonists cyproheptadine and risperidone were employed to provide further evidencethat the identified modulatory effects of psilocin were 5-HT2R-dependent.2. Results2.1. Effects of Psilocin on Phagocytosis of Latex Beads and Secretion of Tumor Necrosis Factor byBV-2 Murine MicrogliaThe phagocytic activity of primary murine microglia has been shown to decrease in re-sponse to treatment with the 5-HT2R agonists 5-HT and 2,5-dimethoxy-4-iodoamphetamine(DOI) [40]. In a more recent investigation, Kozlowska et al. [41] showed that psilocindownregulates the phagocytosis of viable neurons by primary murine CD11b+ microglia.Additionally, psilocin has been demonstrated to inhibit TNF secretion by RAW 264.7 murinemacrophages [42] and exposure to psilocybin reduces TNF levels in mouse brains andhuman sera [33,43]; however, the opposite effect has been reported on serum TNF levelsin rats [44]. Therefore, we first investigated whether psilocin inhibited the phagocyticactivity and TNF secretion of immune-stimulated BV-2 murine microglia. The bacterialendotoxin lipopolysaccharide (LPS) and the combination of LPS plus interferon (IFN)-γwere employed as known inducers of phagocytic activity and cytokine secretion in multiplecell types, including BV-2 cells [45,46]. Psilocin was used at up to the maximum non-toxicconcentration (10 µM), which was determined in preliminary experiments. The phagocyticactivity of BV-2 cells was nearly doubled after their incubation with LPS for 24 h (Figure 1A).On its own, psilocin did not affect this function but completely inhibited the stimulatoryeffect of LPS. This effect was not caused by the cytotoxicity of psilocin since, at the 0.01 to10 µM range, it did not inhibit TNF secretion by BV-2 cells (Figure 1B). The lack of toxiceffects of psilocin towards immune-stimulated BV-2 cells at this concentration range wasalso confirmed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay (Figure A1).Molecules 2024, 29, x FOR PEER REVIEW 3 of 20 albeit with a lower level of affinity [36,37]. 25I-NBOH demonstrates a high level of affinity for the 5-HT2AR and, in contrast to psilocin, is highly selective for this receptor isoform in relation to other members of the 5-HT2R subfamily [38]. Conversely, Ro60-0175 has a higher level of affinity towards the 5-HT2CR isoform than 5-HT2A [39]. In this study, we found that psilocin significantly inhibited phagocytic ctivity and the production of ROS and NO by immune-activated microglial cells. We also demonstrated that the inhibitory activity of psilocin on ROS and NO production by microglial cells was similar to that of the two established 5-HT2R agonists, 25I-NBOH and Ro60-0175. Additionally, the 5-HT2R antagonists cyproheptadine and risperidone were employed to provide further evidence that the identified modulatory effects of psilocin were 5-HT2R-dependent. 2. Results 2.1. Effects of Psilocin on Phagocytosis of Latex Beads and Secretion of Tumor Necrosis Factor by BV-2 Murine Microglia The phagocytic activity of primary murine microglia has been shown to decrease in response to treatment with the 5-HT2R agonists 5-HT and 2,5-dimethoxy-4-iodoampheta-mine (DOI) [40]. In a more recent investigation, Kozlowska et al. [41] showed that psilocin downregulates the phagocytosis of viable neurons by primary murine CD11b+ microglia. Additionally, psilocin has been demonstrated to inhibit TNF secretion by RAW 264.7 mu-rine macrophages [42] and exposure to psilocybin re ces TNF levels in mouse brains and human sera [33,43]; howeve , posite effect has been reported on serum TNF levels in rats [44]. Theref e first investigated whether psilocin inh bited the phagocytic c-tivity F secretion of im une-stimulated BV-2 murine microglia. The b cterial en-dotoxin lipopolysaccharide (LPS) and the combination of LPS plus interferon (IFN)-γ were employed as known inducers of phagocytic activity and cytokine secretion in mul-tiple cell types, including BV-2 cells [45,46]. Psilocin was used at up to the maximum non-toxic concentration (10 µM), which was determined in preliminary experiments. The phagocytic activity of BV-2 cells was nearly doubled after their incubation with LPS for 24 h (Figure 1A). On its own, psilocin did not affect this function but completely inhibited the stimulatory effect of LPS. This effect was not caused by the cytotoxicity of psilocin since, at the 0.01 to 10 µM range, it did not inhibit TNF secretion by BV-2 cells (Figure 1B). The lack of toxic effects of psilocin towards immune-stimulated BV-2 cells at this concen-trati n range was also confir ed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl te-trazoliu bromide (MTT) assay (Figure A1). Figure 1. The effect of psilocin on the phagocytosis of latex beads and the secretion of TNF by BV-2 murine microglia. Varying concentrations of psilocin, shown on the abscissa, were administered 20 Figure 1. The effect of psilocin hagocyto is of latex beads and the secretion of TNF by BV-2murine microglia. Varying concentrations of psilocin, shown on the abscissa, were administered20 min prior to the addition of LPS (A) and LPS plus IFN-γ (B). After a 24 h incubation period, cellswere exposed to fluorescent latex beads for 1 h and corrected total cell fluorescence values, takenfrom 189 to 192 randomly selected cells per experimental condition, were measured in a blindedmanner (A). In a separate experiment, TNF concentration in cell-free supernatants was quantifiedafter 24 h of exposure to stimuli. The dashed line represents the detection limit of the enzyme-linkedimmunosorbent assay (ELISA) (B). Data from 3 to 4 experiments, completed on different days, areshown as means ± SEM. The p and F values displayed in the figures were calculated using one-way(A) or randomized block one-way (B) ANOVA. * p < 0.05 and ** p < 0.01, as determined according toTukey’s post hoc test.Molecules 2024, 29, 5084 4 of 182.2. Effects of Psilocin, 25I-NBOH, and Ro60-0175 on Reactive Oxygen Species Generation byHL-60 Human Microglia-Like CellsNkadimeng et al. [47] report that the use of extracts from psilocybin-containing mush-rooms decreases intracellular ROS levels in cardiomyocytes. To the best of our knowledge,the effects of psilocin or psilocybin on the generation of ROS by primary microglia or mi-croglial model cells have not been determined. To investigate the effects of psilocin on ROSproduction in microglia-like cells, we used differentiated human HL-60 myelomonocytes,which respond similarly to microglia with regard to the respiratory burst when they areprimed with LPS and subsequently stimulated with the bacterial peptide N-formyl-Met-Leu-Phe (fMLP) [48–50]. While psilocin has a high affinity for 5-HT2Rs, it also binds toother 5-HTR isoforms. Therefore, in addition to psilocin, we studied the effects of twoother 5-HT2R ligands on the respiratory burst response: the specific 5-HT2AR agonists25I-NBOH and Ro60-0175. The latter is more selective towards 5-HT2CR than 5-HT2AR.We hypothesized that psilocin would display a level of biological activity comparable tothat of these established specific agonists when applied to microglial 5-HT2Rs. All threedrugs were used at or below the maximum non-toxic concentrations determined in thepreliminary experiments.In comparison to the unprimed cells, LPS priming upregulated ROS production byHL-60 cells in response to fMLP stimulation (Figure 2A–C). Psilocin (1 and 10 µM), 25I-NBOH (3 µM), and Ro60-0175 (10 µM) significantly decreased the production of ROSby LPS-primed and fMLP-stimulated HL-60 cells. 25I-NBOH was the most effectivedrug, inhibiting approximately 75% of the maximal CHL signal (Figure 2B), followed byRo60-0175 (Figure 2C) and psilocin (Figure 2A), which caused 58% and 52% reductions,respectively. At the concentrations tested, psilocin, 25I-NBOH, and Ro60-0175 did notsignificantly decrease the viability of HL-60 human cells (Figure 2D–F). This confirmedthat the observed inhibitory effects of these drugs on ROS production were not due to theinduction of HL-60 cell death.Molecules 2024, 29, x FOR PEER REVIEW 5 of 20 Figure 2. The effects of psilocin, 25I-NBOH, and Ro60-0175 on ROS generation by HL-60 human microglia-like cells and their viability. Varying concentrations of psilocin, 25I-NBOH, or Ro60-0175, shown on the abscissa, were added to differentiated HL-60 cells 20 min prior to their priming with LPS for 24 h. The respiratory burst response was induced by fMLP, and CHL was measured to quantify ROS production (A–C). The viability of HL-60 cells in separate wells was measured by the MTT assay (D–F). The dashed line represents the mean baseline CHL signal from unprimed but fMLP-stimulated cells. Data from 6 to 7 experiments completed on different days are shown as means ± SEM. p and F values displayed in the figures were calculated using randomized block one-way ANOVA. * p < 0.05 and ** p < 0.01 according to Dunnett’s post hoc test. 2.3. Effects of Psilocin, 25I-NBOH, and Ro60-0175 on Nitric Oxide Production by BV-2 Murine Microglia Cells Due to the established relationship between oxidative and nitrosative stress, we also investigated the effects of psilocin, 25I-NBOH, and Ro60-0175 at non-toxic concentrations on NO production by immune-stimulated BV-2 murine microglia cells. Unstimulated BV-2 microglia do not produce detectable levels of NO; therefore, the combination of LPS plus IFN-γ was used to induce NO secretion by BV-2 cells [45]. Figure 3A–C illustrates that BV-2 microglia, stimulated for 24 h with LPS plus IFN-γ, secreted detectable levels of NO. Psilocin, 25I-NBOH, and Ro60-0175 administration in a concentration-dependent manner decreased NO production by stimulated BV-2 cells. The most effective drug was 25I-NBOH, inhibiting maximal NO production by approximately 36% (Figure 3B). At the con-centrations studied, psilocin, 25I-NBOH, or Ro60-0175 did not significantly affect the via-bility of BV-2 cells (Figure 3D–F); therefore, the observed inhibitory effects of these drugs on NO production were not caused by decreased BV-2 cell viability. Figure 2. The effects of psilocin, 25I-NBOH, and Ro60-0175 on ROS generation by HL-60 humanmicroglia-like cells and their viability. Varying concentrations of psilocin, 25I-NBOH, or Ro60-0175,shown on the abscissa, were added to differentiated HL-60 cells 20 min prior to their priming withLPS for 24 h. The respiratory burst response was induced by fMLP, and CHL was measured toquantify ROS pro (A–C). The v ability of HL-60 cells in separate wells was measured bythe M T assay (D–F). The dashed line represents the mea baseline CHL signal from unprimedbut fMLP-stimulated cells. Data from 6 to 7 experiments completed on different days are shown asmeans ± SEM. p and F values displayed in the figures were calculated using randomized blockone-way ANOVA. * p < 0.05 and ** p < 0.01 according to Dunnett’s post hoc test.Molecules 2024, 29, 5084 5 of 182.3. Effects of Psilocin, 25I-NBOH, and Ro60-0175 on Nitric Oxide Production by BV-2 MurineMicroglia CellsDue to the established relationship between oxidative and nitrosative stress, we alsoinvestigated the effects of psilocin, 25I-NBOH, and Ro60-0175 at non-toxic concentrationson NO production by immune-stimulated BV-2 murine microglia cells. UnstimulatedBV-2 microglia do not produce detectable levels of NO; therefore, the combination of LPSplus IFN-γ was used to induce NO secretion by BV-2 cells [45]. Figure 3A–C illustratesthat BV-2 microglia, stimulated for 24 h with LPS plus IFN-γ, secreted detectable levelsof NO. Psilocin, 25I-NBOH, and Ro60-0175 administration in a concentration-dependentmanner decreased NO production by stimulated BV-2 cells. The most effective drug was25I-NBOH, inhibiting maximal NO production by approximately 36% (Figure 3B). At theconcentrations studied, psilocin, 25I-NBOH, or Ro60-0175 did not significantly affect theviability of BV-2 cells (Figure 3D–F); therefore, the observed inhibitory effects of these drugson NO production were not caused by decreased BV-2 cell viability.Molecules 2024, 29, x FOR PEER REVIEW 6 of 20 Figure 3. The effects of psilocin, 25I-NBOH, and Ro60-0175 on NO production and the viability of BV-2 murine microglia. Varying concentrations of psilocin, 25I-NBOH, or Ro60-0175, shown on the abscissa, were added to BV-2 cells 20 min prior to stimulation with LPS plus IFN-γ. Following a 24 h incubation period, nitrite in cell-free supernatants was quantified by the Griess assay (A–C) and cell viability was measured by the MTT assay (D–F). The dashed line represents the detection limit of the Griess assay (A–C). Data from eight independent experiments completed on different days are shown as means ± SEM. The p and F values displayed in the figures were calculated using ran-domized block one-way ANOVA. * p < 0.05, determined according to Dunnett’s post hoc test. 2.4. Effects of Psilocin on Nitric Oxide Production by BV-2 Murine Microglia Exposed to Diverse Stimuli To further elucidate the inhibitory effects of psilocin on NO production by BV-2 mi-croglia, four different stimuli relating to inflammatory signaling were utilized. LPS, IFN-γ, zymosan A, and polyinosinic:polycytidylic acid (poly (I:C)) were selected based on their demonstrated ability to induce NO secretion by BV-2 cells as well as the diversity of sig-naling pathways activated and immune responses initiated by these stimuli [45]. Figure 4 shows that exposure to the selected stimuli or two combinations, namely, LPS plus IFN-γ and zymosan A plus poly (I:C), upregulated NO production by BV-2 cells. The inhibitory activities of psilocin towards the generation of NO by BV-2 murine microglia were similar (11–22% reduction) with all stimuli and with all the combinations used in this study. At the concentrations studied, psilocin did not significantly affect the viability of BV-2 cells under any of these stimulatory conditions (Figure A1). Therefore, the observed inhibitory effect of psilocin on NO production was not caused by decreased BV-2 cell viability. Figure 3. The effects of psilocin, 25I-NBOH, and Ro60-0175 on NO production and the viability ofBV-2 urine icroglia. Varying concentrations of psilocin, 25I- BO , or Ro60-0175, sho n on thei , t -2 cells 20 min prior to stimulation with LPS plus IFN-γ. Following a 24 hincubation period, nitr e in cell-free supernatants was quantified by the Griess a ay (A–C) and cellviability was measured by the MTT assay (D–F). The dashed line represents the detection limit of theGriess assay (A–C). Data from eight independent experiments completed on different days are shownas means ± SEM. The p and F values displayed in the figures were calculated using randomizedblock one-way ANOVA. * p < 0.05, determined according to Dunnett’s post hoc test.. . ff f il i i i i i i i lii erse ti lif rt r elucidate the inhibitory effects of psilocin on NO production by BV-2microglia, four different stimuli relating to inflammatory signaling were utilized. LPS,IFN-γ, zymosan A, and polyinosinic:polycytidylic acid (poly (I:C)) were selected basedon their demonstrated ability to induce NO secretion by BV-2 cells as well as the diversityof signaling pathways activated and immune responses initiated by these stimuli [45].Figure 4 shows that exposure to the selected stimuli or two combinations, namely, LPSplus IFN-γ and zymosan A plus poly (I:C), upregulated NO production by BV-2 cells. Theinhibitory activities of psilocin towards the generation of NO by BV-2 murine microgliawere similar (11–22% reduction) with all stimuli and with all the combinations used inMolecules 2024, 29, 5084 6 of 18this study. At the concentrations studied, psilocin did not significantly affect the viabilityof BV-2 cells under any of these stimulatory conditions (Figure A1). Therefore, the ob-served inhibitory effect of psilocin on NO production was not caused by decreased BV-2cell viability.Molecules 2024, 29, x FOR PEER REVIEW 7 of 20 Figure 4. The effect of psilocin on NO production, induced by diverse stimuli, by BV-2 murine mi-croglia. Varying concentrations of psilocin were administered 20 min prior to the addition of four different stimuli and their combinations, as displayed in the figure panels (A–F). Following a 24 h incubation period, the level of nitrite in cell-free supernatants was quantified by the Griess assay. The dashed line represents the detection limit of the Griess assay. The corresponding cell viability data are presented in Figure A1. Data from five independent experiments completed on different days are shown as means ± SEM. p and F values displayed in the figures were calculated using randomized block one-way ANOVA. * p < 0.05, determined according to Dunnett’s post hoc test. 2.5. Effects of 5-HT2 Receptor Antagonists on the Inhibitory Activity of Psilocin To confirm that the observed cellular effects of psilocin were mediated by 5-HT2R isoforms, we employed two antagonists of this 5-HTR subtype, cyproheptadine and risperidone, at non-toxic concentrations determined in preliminary experiments. Similar to Figure 3A, Figure 5 shows that psilocin (10 µM) inhibited LPS-induced NO secretion by BV-2 microglia. In this experiment, cells treated with psilocin and LPS exhibited ap-proximately 80% of the maximum level of NO production compared to cells stimulated with LPS in the absence of psilocin or antagonists. At the two concentrations used (2 and 10 µM), both cyproheptadine (Figure 5A) and risperidone (Figure 5B) significantly sup-pressed the inhibitory effect of psilocin on NO generation by LPS-stimulated BV-2 cells. At these concentrations, risperidone was not toxic (Figure 5D) but cyproheptadine low-ered the viability of BV-2 microglia when subsequently treated with both psilocin and LPS by up to 7% (Figure 5C). However, this small cytotoxic effect of cyproheptadine cannot account for the observed increase in NO production, as reduced cell viability typically results in decreased NO levels. Figure 4. The effect of psilocin on NO production, induced by diverse stimuli, by BV-2 murinemicroglia. Varying concentrations of psilocin were administered 20 min prior to the addition offour different stimuli and their combinations, as displayed in the figure panels (A–F). Following a24 h incubation period, the level of nitrite in cell-free supern tants was quantified by the Grie s assay.li .ata are rese te i Fi re 1. ata fr fi e i e e e t ex eri e ts c lete iffere tdays are shown as means ± SEM. p and F values displayed in the figures were calculated usingrandomized block one-way ANOVA. * p < 0.05, determined according to Dunnett’s post hoc test.ff 2 eceptor ntagonists on the I hibitory ctivity of silocico fir t at the observe cell l r effects of psiloci ere ediate by 5- T2s, we employed two ant gonists of this 5-HTR subtype, c roheptadine and risperi-don , at on- oxic c ncentrations determined preliminary experiments. Similar toFigure 3A, Figure 5 shows that psilocin (10 µM) inhibited LPS-induced NO secr tion byBV-2 microglia. In this experiment, cells treated with psilocin and LPS exhibited approxi-mately 80% of the maxi um level of NO production compared to cells stimulated withLPS in the absence of psilocin or antagonists. At the two concentrations used (2 a10 µ ), both cyproheptadine (Figure 5 ) and risperidone (Figure 5B) significantly sup-pressed the inhibitory effect of psilocin on NO generation by LPS-stimulated BV-2 cells. Atthese concentrations, risperidone was not toxic (Figure 5D) but cyproheptadine loweredthe viability of BV-2 microglia when subsequently treated with both psilocin and LPS by upto 7% (Figure 5C). However, this small cytotoxic effect of cyproheptadine cannot accountfor the observed increase in NO production, as reduced cell viability typically results indecreased NO levels.Molecules 2024, 29, 5084 7 of 18Molecules 2024, 29, x FOR PEER REVIEW 8 of 20 Figure 5. Effects of 5-HT2R antagonists cyproheptadine and risperidone on the inhibitory activity of psilocin on NO production by BV-2 murine microglia. Psilocin (10 µM) was administered 20 min after cyproheptadine or risperidone and 20 min prior to stimulation with LPS. Following a 24 h incubation period, the presence of nitrite in cell-free supernatant was quantified by the Griess assay (A,B) and cell viability was assessed by the MTT assay (C,D). The dashed line represents the detec-tion limit of the Griess assay, while the dotted line represents NO production by LPS-stimulated BV-2 cells in the absence of psilocin or 5-HT2R antagonists (A,B). Data from five experiments com-pleted on different days are shown as means ± SEM. p and F values displayed in the figures were calculated using randomized block one-way ANOVA. * p < 0.05 and ** p < 0.01, determined accord-ing to Dunnett’s post hoc test. 3. Materials and Methods 3.1. Reagents The following reagents were obtained from Sigma Aldrich (Oakville, ON, Canada): MTT (M2128), bisbenzimide Hoechst 33258 trihydrochloride (B1155), carboxylate-modi-fied polystyrene fluorescent yellow-green latex beads (L4655), LPS (from Escherichia coli O55:B5; L6529), luminol sodium salt (A4685), N-(1-naphthyl)-ethylenediamine dihydro-chloride (222488), fMLP (F3506), poly (I:C) (P0913), psilocin (P-098), and zymosan A (from Saccharomyces cerevisiae; Z4250). The 5-HT2R agonists (S)-6-chloro-5-fluoro-1H-indole-2-propanamine (Ro60-0175; 29520) and 2-((2-(4-iodo-2,5-dimethoxyphenyl)ethylamino)me-thyl)phenol (25I-NBOH; 14909) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The 5-HT2R antagonists cyproheptadine hydrochloride sesquihydrate (C3218) and risperidone (R0087) were obtained from the Tokyo Chemical Institute (TCI; Tokyo, Japan). Recombinant murine IFN-γ (315-05) and ELISA development kits, used to detect murine TNF (900-T54) levels, were purchased from PeproTech (Embrun, ON, Canada). All cell culture media and other reagents were obtained from ThermoFisher Scientific (Ottawa, ON, Canada). 3.2. Cell Culture Figure 5. Effects of 5-HT2R antagonists cyproheptadine and risperidone on the inhibitory activity ofpsilocin on NO production by BV-2 murine microglia. Psilocin (10 µM) was administered 20 min aftercyproheptadine or risperidone and 20 min prior to stimulation with LPS. Following a 24 h incubationperiod, the presence of nitrite in cell-free supernatant was quantified by the Griess assay (A,B) andcell viability was assessed by the MTT assay (C,D). The dashed line represents the detection limit ofthe Griess assay, while the dotted line represents NO production by LPS-stimulated BV-2 cells in theabsence of psilocin or 5-HT2R ntagonists (A,B). Data from five experiments completed on differentdays are shown as means ± SEM. p and F values displayed in the figures wer calcul te usingrandomized block one-way ANOVA. * p < 0.05 and ** p < 0.01, determined according to Dunnett’spost hoc test.3. Materials and Methods3.1. ReagentsThe following reagents were obtained from Sigma Aldrich (Oakville, ON, Canada):MTT (M2128), bisbenzimide Hoechst 33258 trihydrochloride (B1155), carboxylate-modifiedpolystyrene fluorescent yellow-green latex beads (L4655), LPS (from Escherichia coli O55:B5;L6529), luminol sodium salt (A4685), N-(1-naphthyl)-ethylenediamine dihydrochloride(222488), fMLP (F3506), poly (I:C) (P0913), psilocin (P-098), and zymosan A (from Saccha-romyces cerevisiae; Z4250). The 5-HT2R agonists (S)-6-chloro-5-fluoro-1H-indole-2-propanamine(Ro60-0175; 29520) and 2-((2-(4-iodo-2,5-dimethoxyphenyl)ethylamino)methyl)phenol (25I-NBOH; 14909) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The 5-HT2Rantagonists cyproheptadine hydrochloride sesquihydrate (C3218) and risperidone (R0087)were obtained from the Tokyo Chemical Institute (TCI; Tokyo, Japan). Recombinant murineIFN-γ (315-05) and ELISA development kits, used to detect murine TNF (900-T54) levels,were purchased from PeproTech (Embrun, ON, Canada). All cell culture media and otherreagents were obtained from ThermoFisher Scientific (Ottawa, ON, Canada).3.2. Cell CultureThe HL-60 human myelomonocytic cell line was purchased from the American TypeCulture Collection (ATCC, Manassas, VA, USA) and the BV-2 murine microglia cell lineMolecules 2024, 29, 5084 8 of 18was a generous donation from Dr. G. Garden (University of Washington, Seattle, WA, USA).All cells were maintained in Dulbecco’s modified Eagle medium/Ham’s F-12 nutrientmixture (DMEM/F-12), supplemented with 10% heat-inactivated bovine calf serum (CBS),penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (500 ng/mL). HL-60cells were used following differentiation with dimethyl sulfoxide (DMSO), as outlined inSection 2.5, while BV-2 cells were used undifferentiated. The media used in cell cultureexperiments contained 2%, 5%, or 10% heat-inactivated CBS, as well as the aforementionedantibiotics and antimycotic agents. Cells were cultured at 37 ◦C in a humid atmospherecomprising 5% CO2 and 95% O2.3.3. Measurement of Phagocytic ActivityBV-2 murine microglia cells were utilized as microglial models to display phagocyticactivity and were induced to secrete inflammatory cytokines and cytotoxins. Immunestimulation also induces the expression of iNOS in BV-2 microglia, leading to the productionof NO [45,48,51,52]. Fluorescent latex beads were used to assess the phagocytic activity ofBV-2 microglia as previously described [48]. The experimental conditions of this assay andall other assays performed in this study are summarized in Table 1. Cells were seeded infour-chambered glass-bottom Petri dishes at a density of 0.05 million per mL in 500 µL ofDMEM/F-12 containing 5% CBS. They were then allowed to adhere for 24 h. Psilocin orits vehicle solution (20% v/v acetonitrile in deionized water) was administered on its ownor 20 min prior to the addition of LPS or its vehicle solution (phosphate-buffered saline,known as PBS). After a 24 h incubation period, we added 2 µL of fluorescent latex beads(1 µm diameter; 0.1 million particles/µL) to the wells for 1 h. Following the removal ofexcess beads, the cells were fixed with 500 µL of cold 70% ethanol for 5 min and washedwith warm PBS. After the cell nuclei were stained by adding 5 µL of bisbenzimide solution(2 µg/mL), cells were imaged at 40X magnification with a Zeiss AxioObserver.Z1 invertedwidefield fluorescence microscope using ZEN 2.0 software. Measurements of correctedtotal cell fluorescence intensities were performed by an investigator, who was blinded tothe experimental conditions, using the NIH ImageJ (version 1.53, FIJI build).Table 1. Experimental conditions used to measure the activation parameters of microglia-like cells.ParameterStudied and theCell Type UsedAssayDrugs Added20 min BeforeStimulants(Solvent Usedin Controls)Concentrationsof DrugsStimulants(Solvent Used inControls)Concentrationsof StimulantsDuration ofExposure toStimulantsPhagocyticactivity of BV-2murine microgliaEngulfment offluorescentlatex beadsPsilocin (20%v/v acetonitrile) 10 µM LPS (PBS) 400 ng/mL 24 hTNF secretion byBV-2 murinemicrogliaELISAPsilocin (20%v/v acetonitrile)0.1–10 µMLPS (PBS) 150 ng/mL24 hLPS + IFN (PBS) 150 ng/mL +30 ng/mLProduction of NOby BV-2 murinemicrogliaGriess assay Psilocin (20%v/v acetonitrile) 0.01–10 µM LPS (PBS) 150 ng/mL 24 hIFN (PBS) 30 ng/mL25I-NBOH(DMSO) 0.1–3 µM LPS + IFN (PBS)150 ng/mL +30 ng/mLZymosan A (PBS) 10 µg/mLRo60-0175(DMSO) 0.1–10 µMPoly (I:C)(deionized H2O)10 µg/mLZymosan (PBS) +Poly (I:C)(deionized H2O)10 µg/mL +10 µg/mLMolecules 2024, 29, 5084 9 of 18Table 1. Cont.ParameterStudied and theCell Type UsedAssayDrugs Added20 min BeforeStimulants(Solvent Usedin Controls)Concentrationsof DrugsStimulants(Solvent Used inControls)Concentrationsof StimulantsDuration ofExposure toStimulantsGeneration ofROS by DMSO-differentiatedhuman HL-60cellsLuminol-dependentchemilumines-cencePsilocin (20%v/v acetonitrile) 0.01–10 µMPriming: LPS(PBS)Stimulation:fMLP(PBS)500 ng/mL1 µM24 h25 min25I-NBOH(DMSO) 0.1–3 µMRo60-0175(DMSO) 0.1–10 µMViability of all celltypes MTT assayPsilocin (20%v/v acetonitrile)25I-NBOH(DMSO)Ro60-0175(DMSO)Correspond tothe functionalassays listedaboveCorrespond tothe functionalassays listedaboveCorrespond tothe functionalassays listedabove24 h3.4. Measurement of Nitric Oxide and Tumor Necrosis FactorBV-2 murine microglia were seeded in 24-well plates at a volume of 1 mL and a densityof 0.2 million cells per mL in DMEM/F-12 containing 5% CBS. After a 24 h incubationperiod, the following drugs (or their vehicle solutions) were added as described in theresults section: psilocin (dissolved in 20% v/v acetonitrile), the 5-HT2R agonists 25I-NBOH(DMSO) and Ro60-0175 (DMSO), and the 5-HT2R antagonists cyproheptadine (DMSO)and risperidone (DMSO). Following a 20 min incubation period, LPS, IFN-γ, LPS plusIFN-γ, zymosan A, poly (I:C), zymosan A plus poly (I:C), or their vehicle solutions (PBS ordeionized water for poly (I:C) only) were added to the wells. Following a 24 h incubationperiod, the concentration of nitrite, a stable metabolite of NO, in cell-free supernatantswas measured by adding the Griess reagent (1% sulfanilamide, 2.5% orthophosphoric acid,and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in deionized water) in a 1:1ratio with the well volume. A calibration curve was created using standard nitrite concen-trations in a cell culture medium. Optical density at 570 nm was measured immediatelyusing a microplate reader. The collected cell-free supernatants were also used to measureconcentrations of secreted TNF using ELISAs according to the instructions provided by themanufacturer (PeproTech).3.5. Measurement of Reactive Oxygen SpeciesDMSO-differentiated HL-60 human myelomonocytic cells were used to model themicroglial respiratory burst since they upregulate the expression of the NOX subunits,required for ROS production during this cellular response [53]. The chemiluminescence(CHL) assay was performed as described previously, albeit with minor modifications [48,54].HL-60 cells were seeded in 6 cm cell culture plates in DMEM/F-12 containing 10% CBSand 1.3% DMSO at a volume of 20 mL and a density of 0.2 million cells per ml. Aftera six-day incubation period, chosen to allow for differentiation, cells were resuspendedin fresh medium at a density of one million cells per ml. One mL aliquots of the culturewere then added to 24-well plates and exposed to psilocin, 25I-NBOH, Ro60-0175, ortheir vehicle solutions. LPS was used as a priming agent and applied 20 min after theaddition of the drugs. After a 24 h incubation period, the contents of each well wereindividually resuspended in phenol red-free DMEM/F-12 containing 2% CBS, and thenMolecules 2024, 29, 5084 10 of 18seeded into a 96-well plate at a volume of 85 µL and a concentration of 0.5 million cells perml. Using the injectors of a FLUOstar Omega microplate reader (BMG Labtech; Ortenberg,Germany), 10 µL of luminol sodium salt (10 mg/mL in PBS) and 5 µL of fMLP (20 µM inPBS) solutions were sequentially added into each well. This step was followed by CHLintensity measurement for a 25 min period. The ROS production induced by fMLP wasconsidered to be luminol-dependent CHL [55].3.6. Measurement of Cell ViabilityViable cells converted MTT into insoluble purple formazan crystals [56]. The cellcultures used in this study were exposed to MTT (0.5 mg/mL) for 1 h. This was followedby the addition of a solubilizing solution (20% w/v sodium lauryl sulfate and 50% v/vN,N-dimethylformamide in water) in a 1:1 ratio with well volume. The crystals weredissolved by using an orbital plate shaker for 3 h. Optical density was measured at570 nm using a microplate reader. The data were normalized using values obtained fromcells incubated in fresh growth medium only.3.7. Statistical AnalysesStatistical analyses and graphing were completed using Prism GraphPad software(version 10.1.0, GraphPad Software Inc., La Jolla, CA, USA). Randomized block design one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison post hoctest, was used in most cases. One-way ANOVA was applied to phagocytosis experimentaldata. This was followed by Tukey’s multiple comparisons test. Data were collected fromindependent experiments, performed on separate days, and are shown as means± standarderror of the mean (SEM). Significance was established at p < 0.05.4. DiscussionWhile the use of psilocybin-containing mushrooms for their consciousness-alteringproperties spans centuries and many cultures, the non-hallucinogenic effects of this sub-stance, as well as its main metabolite psilocin, have only recently been identified and are notyet well understood [57]. 5-HT2R-mediated antidepressant and anti-neuroinflammatoryactivities are two of the significant non-psychotropic properties reported for both psilocybinand psilocin with relevance in the context of affective disorders and neurodegenerativediseases [16,58]. Since microglia play a central role in neuroimmune responses associatedwith these pathologies, we tested the hypothesis that psilocin modulates select neuroim-mune functions of microglia in a manner that could be beneficial for the treatment of thedisorders mentioned above. Additionally, we hypothesized that 5-HT2Rs are responsiblefor the observed beneficial effects of psilocin. To test these hypotheses, we used BV-2murine microglia and HL-60 human myelomonocytic cells as microglia models, sinceseveral functional responses to immune stimulation in both these types of cell mimic thoseof their primary counterparts. Importantly, both BV-2 [59] and HL-60 cells [60,61] havealready been reported to express all three 5-HT2R isoforms (A, B, and C).First, by demonstrating the inhibitory effect of psilocin on phagocytosis of latexbeads by BV-2 murine microglia (see Figure 6), we confirmed the validity of a previousstudy [40] and preliminary data by Kozlowska et al. [41] showing the downregulation ofthe phagocytic activity of murine microglia by 5-HT2R agonists and psilocin, respectively.Notably, psilocin did not affect the phagocytic activity of unstimulated BV-2 microglia andonly reduced the LPS-induced uptake of latex particles. Such a selective activity towardsimmune-stimulated cells could be used to modulate the phagocytic activity of microgliaunder pathological conditions. Many researchers agree that the phagocytosis of cell frag-ments, myelin debris, and abnormal protein aggregates is a beneficial activity of microgliathat should not be interfered with or even facilitated under specific pathological conditions;however, the suppression of the exacerbated phagocytic activity of reactive microglia,leading to cellular damage and overactive synaptic pruning, could be advantageous in thetreatment of other conditions such as AD, neurotrauma, and ischemic stroke [62–66].Molecules 2024, 29, 5084 11 of 18Molecules 2024, 29, x FOR PEER REVIEW 14 of 20 Figure 6. Intracellular signaling pathways activated by the binding of psilocin to microglial 5-HT2R. Herein, we demonstrate that the psychotropic alkaloid psilocin inhibits three differ-ent neuroimmune functions of microglia model cells without affecting their viability or having an effect on TNF secretion. The roles of 5-HT2Rs, as mediators of the cellular effects of psilocin, are indicated by the observations that (1) two other agonists of this receptor subfamily display effects that are comparable to those of psilocin in two of the cellular assays employed, and (2) the inhibitory effect of psilocin on BV-2 microglia NO release is blocked by two different antagonists of 5-HT2Rs. Our study has significant limitations since only in vitro models of neuroinflammatory activation of microglia were employed, which involved using immortal cell lines as microglial model cells. There are several im-portant experimental steps that are required for the validation of psilocin as a candidate anti-neuroinflammatory drug that could potentially prevent or slow down neurodegen-erative diseases that are characterized by the adverse activation of microglia. They include additional in vitro studies using primary human microglia or human induced pluripotent stem cell (iPSC)-derived microglia. Human iPSC-derived brain organoids, which contain all major brain cell types as well as humanized animal models of neurodegenerative dis-eases, could be used subsequently to study the anti-neuroinflammatory and neuroprotec-tive effects of psilocin before it advances to human clinical trials. Nevertheless, to the best of our knowledge, this is the first study demonstrating the 5-HT2R-mediated inhibition of select neuroimmune functions of microglia by psilocin. While the inhibitory effect on the phagocytic activity of microglia could have adverse outcomes under certain neuropatho-logical conditions, the lowering of excess ROS and NO production by microglia likely Figure 6. Intracellular signaling pathways activated by the binding of psilocin to microglial 5-HT2R.The observed lack of the effect of psilocin in the 0.1 to 10 µM range on TNF secretionby BV-2 microglia supports the mounting evidence that the modulation of microglial TNFexpression by this alkaloid is cell type-depend nt. F r exampl , no effect on TNF secretionby immune-stimulated huma THP-1 micr glia-like cell and human T-cells was reportedafter th ir exposu to 0.3–30 µM psilocin, which is similar t the concentr tion range usedin our experime ts [67]. However, an inhibitory effect of ps locin on TNF production wasbserved when usi g LPS-stimulated RAW 264.7 mur ne macrophages [42]. Even thoughelevated TNF is impl cated in the development of neuro nflammation, particularly in thecontext of neu odegenerative diseases and depression [19], psilocin m y not be effective atsuppressing the production of this cytokine by microglia.Ou study revealed the pr viously unknown inhibitory effect f psilocin on the respi-ratory burst activity of human microglia-like cells, resulting in the reduced production ofROS. This activity was a c aracteristic of not only psilocin but also two specific 5-HT2Ragonists, which implicated this subfamily of 5-HTRs in the protective effect exhibited bypsilocin. ROS play multifaceted roles in the CNS, and at high concentrations, such as thoseassociated with increased microglial respiratory burst activity, they are linked to oxidativedamage and the cytotoxicity observed in a multitude of neurological disorders [68].Similar to their effects on ROS production, psilocin and the two 5-HT2R agonists hadsmall yet significant inhibitory effects on NO release by BV-2 microglia. Our observationsimplicating the 5-HT2R subfamily in these suppressive actions of psilocin align well withother studies reporting that the activation of 5-HT2AR [69] and 5-HT2CR [70] isoforms isMolecules 2024, 29, 5084 12 of 18associated with lowered iNOS activity, leading to decreased NO levels. To the best of ourknowledge, reduced NO production by microglia in the presence of psilocin has not beenreported before, but it is noteworthy that extracts from Psilocybe natalensis, a psilocybin-containing mushroom, have been shown to reduce NO release from LPS-stimulated RAW264.7 murine macrophages. Nitric oxide modulates multiple CNS signaling pathways underphysiological conditions. However, when present at high concentrations, it is also capableof reacting with ROS to form peroxynitrite, a significant contributor to oxidative damage invarious neuropathologies, including neurodegenerative diseases and neurotrauma [71,72].Thus, the observed inhibitory effects of psilocin on ROS and NO production by microglialcells could explain its reported protective activity in certain human neuropathologies andmakes it a suitable candidate for further preclinical studies aimed at identifying drugswhich are effective at suppressing microglia-driven neuroinflammatory diseases.Most previous studies investigating the effects of psilocin or psilocybin on microglialfunctions used LPS to induce a reactive state in this cell type. The Toll-like receptor (TLR)4-dependent signaling pathways and functional outcomes triggered by LPS exposure are wellcharacterized in microglia and their documented involvement in various neuropatholo-gies makes this endotoxin an appropriate immune stimulant for the in vitro and in vivomodeling of neuroinflammatory responses [73]. Since our experiments demonstrated thesuppressive activity of psilocin on three different LPS-induced inflammatory functions ofmicroglial cells, we next aimed to investigate whether psilocin was effective under otherstimulatory conditions known to engage alternative signaling pathways in microglia [45].We discovered that psilocin had a similar inhibitory effect on NO production triggeredby LPS, IFN-γ, zymosan A, poly (I:C), and two different combinations of these stimu-lants. LPS is typically used to simulate bacterial infection [74], zymosan A mimics fungalpathogens and interacts with microglial TLR2 [75], poly (I:C) models viral particles andbinds to TLR3 [76], and IFN-γ is a potent endogenous pro-inflammatory cytokine thatactivates IFN-γ receptors (IFNGR) and is known to be upregulated in several humanneuropathologies [77,78]. The activation of these different receptors induces distinct down-stream mechanisms [45]. The fact that psilocin inhibited NO release induced by thesediverse stimuli could indicate that, by interacting with 5-HT2Rs, it modulated multiplesignaling pathways regulating the inflammatory response of microglia. The ability tomodify microglial responses to diverse pro-inflammatory stimuli makes psilocin a gooddrug candidate for the treatment of neuroinflammatory conditions that are triggered bydiverse causative agents and conditions.Finally, we obtained direct evidence that the inhibitory effect of psilocin on one ofthe pro-inflammatory responses of microglia, namely NO production, was mediated by5-HT2Rs. We were not interested in the contributions of the individual isoforms of 5-HT2Rs;therefore, we selected two structurally distinct antagonists, cyproheptadine and risperidone,which are known to bind with different affinities to all three (A, B, and C) isoforms ofthis receptor subfamily [79–81]. Both these drugs effectively blocked the inhibitory effectof psilocin on BV-2 microglia, which indicated that this alkaloid exerted its activity bybinding and activating at least one of the 5-HT2R isoforms. Detailed molecular studies willbe needed to pinpoint the exact molecular targets when applying psilocin to microglia,but 5-HT2AR and 5-HT2CR isoforms are the likely candidate receptors due to their moreprominent expression across various brain regions compared to 5-HT2BR [82] Notably, theA and C isoforms of 5-HT2R expressed by neurons are directly linked to the psychotropiceffects of psilocybin and psilocin [28,36]; therefore, designing and testing novel derivativesof psilocin that possess anti-neuroinflammatory properties but do display hallucinogenicactivity is an avenue of research that can be pursued in the future [83].Downstream signaling pathways responsible for the suppressive actions of psilocinobserved in this study also require further investigation (see Figure 6). It is known that5-HT2AR is a G protein-coupled receptor (GPCR) and that psychedelics, after binding to thisreceptor, activate both Gq and β-arrestin-2 transducers [83]. Coupling through β-arrestin-2, in turn, has been shown to negatively regulate NF-κB activity [84]. It has also beenMolecules 2024, 29, 5084 13 of 18reported that the expression of NOX, a primary producer of ROS in macrophages, dependson NF-κB activity [85] and that the expression and activity of iNOS in macrophages arehighly upregulated by NF-κB [86]; therefore, this transcription factor may mediate theinhibitory effects of psilocin on ROS and NO production by microglia. Similarly, due to thepreviously reported dependence of the phagocytic activity of murine macrophages on NF-κB activity [87], it could also be responsible for the reduced phagocytosis by BV-2 microglia,which was observed in this study. In this regard, the previously reported inhibitory effectof 10–15 µM psilocybin on NF-κB activity in LPS-stimulated human THP-1 microglia-likecells is notable [88]. In addition to NF-κB, protein kinase C (PKC) and signal transducerand activator of transcription (STAT) have been linked to the inhibitory effect of 5-HT2Ragonists on iNOS expression [69,89,90], but the contribution of these pathways needs to beconfirmed regarding psilocin-induced inhibitory effects in microglia.Herein, we demonstrate that the psychotropic alkaloid psilocin inhibits three differentneuroimmune functions of microglia model cells without affecting their viability or having aneffect on TNF secretion. The roles of 5-HT2Rs, as mediators of the cellular effects of psilocin,are indicated by the observations that (1) two other agonists of this receptor subfamilydisplay effects that are comparable to those of psilocin in two of the cellular assays employed,and (2) the inhibitory effect of psilocin on BV-2 microglia NO release is blocked by twodifferent antagonists of 5-HT2Rs. Our study has significant limitations since only in vitromodels of neuroinflammatory activation of microglia were employed, which involved usingimmortal cell lines as microglial model cells. There are several important experimentalsteps that are required for the validation of psilocin as a candidate anti-neuroinflammatorydrug that could potentially prevent or slow down neurodegenerative diseases that arecharacterized by the adverse activation of microglia. They include additional in vitro studiesusing primary human microglia or human induced pluripotent stem cell (iPSC)-derivedmicroglia. Human iPSC-derived brain organoids, which contain all major brain cell types aswell as humanized animal models of neurodegenerative diseases, could be used subsequentlyto study the anti-neuroinflammatory and neuroprotective effects of psilocin before it advancesto human clinical trials. Nevertheless, to the best of our knowledge, this is the first studydemonstrating the 5-HT2R-mediated inhibition of select neuroimmune functions of microgliaby psilocin. While the inhibitory effect on the phagocytic activity of microglia could haveadverse outcomes under certain neuropathological conditions, the lowering of excess ROSand NO production by microglia likely induces protective effects that could be beneficial inthe treatment of neuroimmune disorders such as stroke, neurotrauma, and neurodegenerativediseases [72]. We conclude that psilocin holds merit not only as a novel antidepressant, butalso as a potential therapeutic agent that can be used to mitigate neuroinflammation invarious neuropathologies where reactive microglial play a prominent role [91]. This justifiesfurther preclinical research to characterize the precise molecular mechanism of action ofpsilocin and also studies that aim to create non-psychotropic derivatives of this alkaloid thatstill possess its anti-neuroinflammatory activity.Author Contributions: K.R.W.: conceptualization, methodology, formal analysis, data curation,writing—original draft, and writing—review and editing. N.A.H.B.: methodology, formal analysis,data curation, writing—original draft, and writing—review and editing. I.R.: methodology, formalanalysis, data curation, and writing—review and editing. B.K.G.: methodology, formal analysis, datacuration, and writing—review and editing. I.A.L.: methodology, formal analysis, data curation, andwriting—review and editing. A.K.: funding acquisition, project administration, conceptualization,methodology, writing—original draft, and writing—review and editing. All authors have read andagreed to the published version of the manuscript.Funding: This work was supported by grants from the Jack Brown and Family Alzheimer’s DiseaseResearch Foundation, the Natural Sciences and Engineering Research Council of Canada, and theUniversity of British Columbia Okanagan Campus.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Molecules 2024, 29, 5084 14 of 18Data Availability Statement: The data presented in this study are available on request fromthe authors.Acknowledgments: The authors would like to thank Z. Mohammad and E. Creed for their assistancewith the experiments and formatting of the figures as well as all members of the Laboratory ofCellular and Molecular Pharmacology for the helpful discussions and comments on the manuscript.Figure 6 in this manuscript was created with BioRender.com.Conflicts of Interest: The authors declare no conflicts of interest.Abbreviations5-HT, 5-hydroxytryptamine, serotonin; 5-HTR, 5-hydroxytryptamine receptor; AD,Alzheimer’s disease; ANOVA, analysis of variance; CBS, calf bovine serum; CHL, chemilu-minescence; CNS, central nervous system; COX, cyclooxygenase; DMEM/F-12, Dulbecco’smodified Eagle medium/Ham’s F-12 nutrient mixture; DMSO, dimethyl sulfoxide; ELISA,enzyme-linked immunosorbent assay; fMLP, N-formyl-Met-Leu-Phe; IFN, interferon; iNOS,inducible nitric oxide synthase; iPSC, induced pluripotent stem cell; LPS, lipopolysaccha-ride; LSD, lysergic acid diethylamide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide; NADPH, nicotinamide adenine dinucleotide phosphate; NF-κB, nuclearfactor-κB; NO, nitric oxide; NOX, nicotinamide adenine dinucleotide phosphate-dependentoxidase; NSAID, non-steroidal anti-inflammatory drug; PBS, phosphate-buffered saline;poly (I:C), polyinosinic:polycytidylic acid; PKC, protein kinase C; ROS, reactive oxygenspecies; SEM, standard error of the mean; SSRI, selective serotonin reuptake inhibitor;STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; TLR,Toll-like receptor.Appendix AMolecules 2024, 29, x FOR PEER REVIEW 16 of 20 Appendix A Figure A1. 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affective disorders
depression
5-hydroxytryptamine
neurodegenerative diseases
neuroprotection
phagocytic activity
reactive oxygen species
reactive nitrogen species
serotonin
Psilocin, the Psychoactive Metabolite of Psilocybin, Modulates Select Neuroimmune Functions of Microglial Cells in a 5-HT₂ Receptor-Dependent Manner
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