PsilocybinDMT

The DMT and Psilocin Treatment Changes CD11b+ Activated Microglia Immunological Phenotype

This study on neuronal-glial cells (CD11b+ microglia, from mice) found that the direct application of psilocin (a metabolite of psilocybin) and DMT, led to increased capacity for the cells to fulfill their immune responses. Specifically, it reduced levels of TLR4, p65, CD80 proteins (markers of the immune response), and upregulated TREM2 (neuroprotective receptor).

Authors

Figiel, M.
Klimczak, A.
Kozłowska, U.

Published

March 8, 2021

Biorxiv

individual Study

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Abstract

Psychedelics are new, promising candidate molecules for clinical use in psychiatric disorders such as Treatment-Resistant Depression (TRD) and Post Traumatic Stress Disorder (PTSD). They were recently also proposed as molecules supporting neural tissue repair by anti-inflammatory properties. Here we reported that two classic psychedelics, DMT and psilocin, can influence microglial functions by reducing the level of TLR4, p65, CD80 proteins, which are markers of the immune response, and upregulat TREM2 neuroprotective receptor. Psilocin also secured neuronal survival in the neuron-microglia co-culture model by attenuating the phagocytic function of microglia. We conclude that DMT and psilocin regulate the immunomodulatory potential of microglia. Of note, psychedelics were previously reported as a relatively safe treatment approach. The demonstrated regulation of inflammatory molecules and microglia phagocytosis suggests that psychedelics or their analogs are candidates in the therapy of neurological disorders where microglia and inflammation significantly contribute to pathogenic disease mechanisms.

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Research Summary of 'The DMT and Psilocin Treatment Changes CD11b+ Activated Microglia Immunological Phenotype'

Introduction

Psychedelic compounds such as DMT and psilocin have been proposed not only for psychiatric indications (for example treatment-resistant depression and PTSD) but also as agents that may support neural repair through immunomodulatory and pro‑regenerative effects. Previous animal and in vitro work has suggested psychedelics can influence neurogenesis, neural plasticity and inflammation, but the cellular and molecular mechanisms—particularly how these drugs affect microglial phenotype and microglia–neuron interactions—remain incompletely characterised. Kozłowska and colleagues set out to test whether two classic tryptamine psychedelics, DMT and psilocin, alter the immunological phenotype and function of primary mouse CD11b+ microglia. The study measured expression of innate and adaptive immune markers (TLR4, NF-κB p65, CD80), the neuroprotective receptor TREM2, morphological features, and microglial phagocytosis of healthy neurons in mono- and co-culture models to approximate effects relevant to neuroinflammatory conditions and tissue repair.

Methods

Primary microglia were isolated from 3–6 month old DBA/1 mice. Brains were perfused, homogenised and microglia-enriched fractions obtained with Percoll centrifugation; cells were expanded in DMEM/F12 with supplements including TGF‑β and IL‑34 until a monolayer was reached. The heterogeneous cultures were characterised by immunofluorescence and flow cytometry; the extracted population contained a majority CD11b+ microglia (reported mean ~65%). For activation, cultures were serum-/growth-factor deprived for 24 h (vehicle, VEH) and then either left in VEH or stimulated with 150 ng/mL LPS for 24 h. After that, cultures were treated for 12 h with 100 µM DMT or 100 µM psilocin, or left untreated, prior to downstream assays. Neural cells were generated from C57BL/6 E10 embryos as neurospheres, dissociated and differentiated into neurons using Neurobasal/DMEM-F12 with BDNF and GDNF. For co-cultures, microglia (LPS‑pretreated or VEH) were labelled with PKH67 and added to 5‑day differentiated neuronal cultures at 5×10^4 cells per well. Six experimental co-culture conditions were observed continuously for 24 h: VEH, VEH+DMT, VEH+psilocin, LPS, LPS+DMT, LPS+psilocin. Outcomes included cell morphology measured from phase‑contrast images (skeletonisation and FracLac metrics: branch length, fractal dimension, lacunarity, span ratio, circularity), immunofluorescent measurement of protein expression (TLR4, NF‑κB p65, TREM2, CD11b) analysed by corrected total cell fluorescence (CTCF) in ImageJ, and flow cytometry quantification of surface markers including CD80 (with standard controls and DAPI dead‑cell gating). Phagocytosis of healthy neurons was assessed in the PKH‑labelled co‑cultures with time‑lapse microscopy. Experiments were performed at least three times, typically in duplicates; some figure legends report biological replicates n = 6 and event counts per condition. Statistical comparisons used unpaired t‑tests with Welch’s correction (GraphPad Prism 5).

Results

Cell composition and baseline morphology: The isolated heterogeneous cultures contained on average ~64–65% CD11b+ cells with remaining astrocytic cells evident by GFAP staining. In VEH conditions (no LPS), microglia in untreated cultures were often rod/bipolar or small and round; after 12 h treatment with DMT or psilocin more ramified cells with long branches were frequently observed. Morphology metrics after LPS and psychedelic treatment: LPS alone tended to make microglia rounder. Compared with LPS control, 12 h DMT or psilocin treatment produced changes consistent with an altered activation state: both treatments showed lower span ratio (DMT p < 0.0063; psilocin p < 0.0098), higher circularity (DMT p < 0.0003; psilocin p < 0.001), and lower fractal dimension (DMT p < 0.0083). These metrics indicate a shift in cellular shape induced by the psychedelics in the context of stimulation. TLR4 expression: In VEH (unstimulated) conditions, application of DMT or psilocin did not significantly change TLR4 fluorescence intensity. After LPS stimulation, TLR4 fluorescence intensity on CD11b+ cells was significantly lower in the DMT‑ and psilocin‑treated groups compared with LPS control (reported ***p < 0.0001). NF‑κB (p65) expression: Immunofluorescence showed that NF‑κB (p65) signal in CD11b+ microglia decreased after DMT and psilocin treatment in both VEH and LPS conditions (reported **p < 0.002 and ***p < 0.0001 for various comparisons), indicating reduced activation of this pro‑inflammatory transcription factor. CD80 co‑stimulatory molecule: Flow cytometry of CD11b+ cells indicated that both DMT and psilocin reduced CD80 expression in VEH and LPS conditions. The extracted text reports statistical significance for psilocin in the VEH group and for both DMT and psilocin in LPS‑treated cells, but the reported p‑values in the extracted text use '>' signs (for example *p > 0.0172), which is inconsistent with conventional reporting; the direction (reduction) and presence of significance are stated by the authors but the inequality symbols in the extraction are unclear. TREM2 expression: Psilocin, but not DMT, significantly increased TREM2 fluorescence intensity in VEH conditions (reported **p < 0.0023). LPS treatment alone increased TREM2 compared with VEH (authors report a 72% increase). In the LPS group, psilocin maintained TREM2 at levels similar to VEH, whereas DMT significantly decreased TREM2 fluorescence (reported ***p < 0.0001). The authors also report percentage differences: in VEH psilocin increased TREM2 by ~23% more than DMT; in LPS, psilocin increased TREM2 by ~42% whereas DMT reduced it by ~20%. Phagocytosis in co‑culture: Time‑lapse co‑culture observations indicated that 100 µM psilocin reduced microglial phagocytosis of healthy neurons compared with DMT in both VEH and LPS conditions. The authors interpret this as psilocin exhibiting stronger neuroprotective behaviour by attenuating engulfment of healthy neuronal elements. Supplementary videos are cited as supporting evidence. Quantitative comparisons reported by the authors: In VEH, psilocin reduced CD80 and NF‑κB p65 fluorescence intensity ~11–12% more than DMT; in LPS conditions psilocin reduced TLR4 and CD80 ~7–10% more than DMT; conversely, DMT reduced NF‑κB p65 ~28% more than psilocin in the LPS group. These percentage figures are presented in the Discussion as summary comparisons.

Discussion

Kozłowska and colleagues interpret their findings as evidence that DMT and psilocin modulate the immunological phenotype of primary CD11b+ microglia in vitro, with psilocin generally producing stronger immunomodulatory and putatively neuroprotective effects. Both compounds reduced markers associated with pro‑inflammatory activation—TLR4, NF‑κB p65 and CD80—particularly after LPS stimulation, while psilocin uniquely increased or maintained TREM2 expression in several conditions. The authors suggest that downregulation of TLR4 and NF‑κB and lowering of CD80 could reduce microglial pro‑inflammatory signalling and antigen‑presentation capacity, which might be beneficial in contexts where excessive immune activation impairs neural repair or complicates cell graft survival. They situate these in vitro results within broader therapeutic interest in selective anti‑inflammatory strategies for neurological disease, noting limitations of broad immunosuppressive drugs and the challenge of blood–brain barrier penetration for some biologics. The authors highlight TREM2 as a potentially important mediator: TREM2–DAP12 signalling can antagonise TLR responses and support phagocytic and reparative microglial functions, so psilocin’s capacity to upregulate TREM2 while reducing pro‑inflammatory markers may contribute to neuroprotective outcomes. Limitations and uncertainties acknowledged by the authors include incomplete understanding of the mechanistic basis for the differential effects of DMT and psilocin (for example, differing receptor affinities) and the still‑uncertain precise role of TREM2 in phagocytosis. The study is in vitro, using primary murine cells and acute pharmacological exposures; the authors therefore imply that further work is required to determine whether these molecular and cellular changes translate into beneficial effects in vivo and in disease models. They call for further investigation of psychedelics' selective immunomodulatory actions and their potential utility in conditions where microglial inflammation contributes to pathology.

Conclusion

The authors conclude that both DMT and psilocin alter the phenotype of mouse primary CD11b+ microglia in vitro and change microglia–neuron interactions in co‑culture. Psilocin exhibited a stronger overall immunomodulatory profile than DMT in these experiments: it downregulated pro‑inflammatory markers (TLR4, NF‑κB p65, CD80), upregulated or preserved TREM2 expression, and attenuated microglial phagocytosis of healthy neurons. The authors propose that these properties make psychedelics, and psilocin in particular, candidates for further study as agents that might support neural tissue cleansing and regeneration in disorders with an inflammatory component.

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INTRODUCTION

Psychedelic therapy is undoubtedly a recent breakthrough in psychiatry for treatmentresistant depression, post-traumatic stress disorder. Recent studies in animal models suggest that psychedelics may also display a broader spectrum of applications in neuronal damage and neuropsychiatric conditions such as Traumatic Brain Injury (TBI), Alzheimer's Disease (AD), and others. Their therapeutic effects are related to immunomodulatory potential, ability to induce neurogenesis, and neural plasticity; however exact mechanisms are yet to be identified. The reported anti-inflammatory potential of psychedelics is appealing since the state of acute brain inflammation is the direct cause of neural cell death and limitation of the regeneration process. Such conditions are frequently observed in neurodegenerative diseases or Traumatic Brain Injury,. Anti-inflammatory drugs are recently proposed as a therapeutic strategy since they can reduce microglia's harmful activity. However, anti-inflammatory pharmacologic approaches used in the clinical treatment so far may cause serious adverse effects related to broad immune system suppression. Microglia is a particular type of brain-specific macrophages, which display a broad spectrum of actions to support neuronal network rearrangement, neural tissue regeneration, and protection from pathogens. In the healthy brain, the microglial cells work in favor of tissue rearrangement and regeneration, induction of NPC neurogenesis, and control of synaptic pruning directly or indirectly by recruiting astrocytes. The phagocytosis activity of microglia serves for cleansing the local environment from pathogens, damaged or infected cells, cellular debris, and toxic protein aggregates. Like macrophages, the microglia infiltrates the local environment detecting the infection or tissue damage signals through Pattern Recognition Receptors (PRRs), and can present the antigen to T-cells. During infection, microglia responds to bacterial LPS via Toll-like Receptor 4 (TLR4), starting the innate immune response mediated by NF-κβ (p65) activation. The NFκβ regulates pro-inflammatory protein secretion by microglia, which may attract other immune cells to cross the Blood-Brain Barrier. The activated microglia upregulates CD40, CD80, or CD86 co-stimulatory molecules expression, which provides the second signal for antigen presentation via MHC to T-cells. If the process of microglia immune-regulation excessively amplifies, it may lead to the attraction of unwanted immune cells, the expansion of microglial phagocytosis to functional neurons, and other pro-inflammatory actions which leads to acute tissue damage. Another important regulator of several microglia functions is the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), which plays an essential role in proper microglial phagocytosis. TREM2 reacts to anionic molecules, like Danger associated molecular patterns (DAMPs), such as toxic protein aggregates and Pathogen associated molecular patterns (PAMPs), including bacterial lipopolysaccharide (LPS). Activation of TREM2 initiates anti-inflammatory pathways in microglia; therefore, dysregulation of its expression may lead to neurodegenerative diseases, where it may be a promising therapeutic target. In the current study, we aimed to investigate whether the application of classic psychedelics, such as DMT and psilocin, could be beneficial for neural tissue homeostasis and the promotion of pro-regenerative and anti-inflammatory features. We tested DMT and psilocin's influence on mouse primary CD11b+ microglia by measuring the protein expression of factors crucial for innate and adaptive immune response regulation (TLR-4, NF-κβ, CD80). We also measured the changes in TREM2 protein expression, which is recently studied in the context of neuroprotection and proper microglial phagocytosis. Finally, we checked if DMT and psilocin affect microglial phagocytosis of healthy neurons after LPS stimulation. Our investigation examines classic psychedelics' influence on the model of activated microglia also together in neurons. Therefore, we mimic the therapeutic influence of psychedelics in neurological disorders where inflammation and microglia significantly contribute to pathogenic disease mechanisms.

MICROGLIA CULTURE:

DBA1 mice age between 3 to 6 months were euthanized according to local ethical law regulations. Shortly after death (up to 1 min), the bodies underwent full-body perfusion with ice-cold PBS. After the brain extraction, mixed glial cell culture from each mouse was isolated by homogenizing the brain in a glass homogenizer (Sigma) and then purified by mixing 17 mL PBS brain lysate with 4,5 mL Percoll Plus (Sigma, Saint Louis, Missouri, USA). After 15 min 500g centrifugation in +4 C, the pellet was resuspended in DMEM/F12 (Thermo Fisher, Waltham, Massachusetts, USA), 10% Fetal Bovine Serum (Gibco, Origin: Brazil, Campinas, Brazil), 1% penicillin/streptomycin, 1% L-glutamine (Sigma-Aldrich, Steinheim, Germany), 1.5 µg/mL, sheep wool cholesterol, 2 ng/mL TGF-β, 15 ng/mL IL-34 and 20 ng/mL bFGF (Sigma, Saint Louis, Missouri, USA). The culture media was changed every 4 days until cell cultures reached full confluence. The ratio of astrocytes to microglia was then assessed with immunofluorescence (IF) and flow cytometry (FCM) techniques. In order to activate the microglia with LPS, after reaching the monolayer, the culture at P0 was washed 2 times with PBS and incubated 24h in growth factor-free DMEM/F12 media, supplemented with 1% penicillin/streptomycin, 1% L-glutamine, and 1.5 µg/mL sheep wool cholesterol (VEH). After 24h incubation in VEH media, new VEH media containing 150 ng/mL LPS (Sigma, Saint Louis, Missouri, USA) was added for 24h (Fig.). For detaching, the cells were washed twice with PBS and then digested with pre-warmed TrypLE (Thermo Fisher, Waltham, Massachusetts, USA) for 10 min in +37 C. After incubation, the media was neutralized 1:1 with culture media, and the cells that remained attached were taken off by gentle but dynamic pipetting. The cells were then centrifuged 470 g for 5 min in +4 C.

NEUROSPHERES CULTURE:

The neurospheres were isolated from C57BL/6 mouse E10 embryos according to the modified protocol described in Ebert 2013. In brief, embryonic brains were dissociated by trypsin (Merck, Darmstadt, Germany), washed 2 times in HBSS (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 1x penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), and pre-plated on uncoated plates for 2 hours. The neurospheres were then cultured on non-adherent 6-well plates in neurosphere medium containing: 7:3 mixture of DMEM with Hams F12 Nutrient mix supplemented with 2% of B27 supplement, 100 ng/mL EGF, 100 ng/mL bFGF (Thermo Fisher, Waltham, Massachusetts, USA), 5 μg/mL heparin (Sigma, Saint Louis, Missouri, USA) and 1% penicillin/streptomycin, 1% L-glutamine (Sigma, Saint Louis, Missouri, USA). The maintenance medium was changed every 2-3 days, and passages were performed every 4-5 days using mechanical disassociation.

NSC DIFFERENTIATION:

Neurospheres were dissociated to single-cell and were seeded on poly-Lornithine/laminin (Sigma, Saint Louis, Missouri, USA) coated plates for 24h in neurosphere medium. After the cells fully attached to the surface, the medium was changed to neuronal differentiation medium: Neurobasal and DMEM/F12 1:1, 1% of B27 supplement, 30 ng/mL GDNF, 50 ng/mL BDNF (Thermo Fisher, Waltham, Massachusetts, USA), 1% penicillin/streptomycin, 1% L-glutamine (Fig.).

IMMUNOFLUORESCENT STAINING:

The cells attached to the 48-well plate were fixed for 10 min with 10% buffered formalin Plus (Sigma, Saint Louis, Missouri, USA) and then permeabilized for 15 min with 0.01% Tween-20 (Serva, Heidelberg, Germany). The cells were then incubated in 1% BSA (Abcam Cambridge, Great Britain) and 10% Goat Serum (Thermo Fisher, Waltham, Massachusetts, USA) for 1h RT. Then the primary antibody solution was added and incubated (GFAP, Thermo Fisher, 1:200 4h, RT), (TREM2, Bioss, Woburn, MA, USA 1:200, overnight, +4 C), (Nf-κβ, Invitrogen, Carlsbad, CA, USA 1:200, overnight, +4 C), (TLR4, Bioss, 1:200, overnight, +4 C), (CD11b, Bio-Rad, Hercules, CA, USA, 1:100, overnight, +4 C). After incubation, the primary antibody was rinsed off with PBS, and the secondary antibody (Alexa Fluor 488nm or 594nm, Thermo Fisher, Waltham, Massachusetts, USA) was applied in a concentration of 1:400 (488nm) or 1:200 (594nm) for 1h in RT. After rinsing off three times with PBS the secondary antibody solution, the cell nuclei were visualized with DAPI Sigma, Saint Louis, Missouri, USA staining.

IMMUNOFLUORESCENCE ANALYSIS:

The stained cells were analyzed with Zeiss Axio Observer 2 microscope, using Alexa Fluor 488 nm settings for CD11b staining: 60% blue laser strength, 1000 ms acquisition, Alexa Fluor 594 nm settings for TLR4, TREM2, and Nf-κβ stainings: 100% orange laser strength, 5000 ms acquisition. The optimal acquisition time and laser strength were assessed as compared to negative control staining. The image analysis was adjusted in ZEN 2.0 software, setting 1050 units lower -3500 units upper curve threshold for TLR4 and TREM2 staining, and 1050 units lower -4000 units upper curve threshold for Nf-κβ staining. The Corrected Total Cell Fluorescence (CTCF) was measured using ImageJ Software and calculated using equation CTCF = D n -(A n x B n ), where D-integrated density, A-area of the selected cell, B-mean background, n-event number (Fig.).

FLOW CYTOMETRY:

The cells (dethatched by TrypLE digestion as previously described) were incubated for 10 min in 1% BSA (Abcam Cambridge, Great Britain) containing PBS to block unspecific reactions. Then the cells were centrifuged 470 g, 5 min, +4 C, and resuspended in PBS. The cells were divided into two fractions -one fraction dedicated for compatible isotype control stainings (Thermo Fisher, Waltham, Massachusetts, USA) and the second fraction for CD11b (Bio-Rad, Hercules, CA, USA) staining (1 h on ice) and secondary antibody (Alexa Fluor 488 nm, 1:400 for 30 min on ice). After rinsing in ice-cold PBS and +4 C 470 g centrifugation, the cells were stained for 30 min with 2 µL of Thermo Fisher antibodies (Armenian Hamster IgG, CD80, PE), (Rat IgG2, CD11c, APC), (Armenian Hamster IgG CD163, APC). Each test was performed with density 1x 10 5 cells in 150 µL. The reaction was ended by adding 2 mL of PBS into the probes, and the cells were 10 min centrifuged in +4 C with 470 g speed. The measurements were made with BD Fortessa Cytometer in DIVA software after proper compensation. Before measurements, DAPI was added into cell suspension as a control to gate out the dead cells.

PKH CELL STAINING:

The cells collected from the culture plate were stained with PKH (Sigma, Saint Louis, Missouri, USA) by 5 min incubation in a mixture containing 2 µL PKH and 500 µL diluent C on the ice. Subsequently, the dye was neutralized by adding 10 mL of PBS containing 1% BSA and centrifuged 470 g in +4 C for 5 min. The cells were again washed with 5 mL 1% BSA in PBS.

NEURAL-GLIAL CO-CULTURES:

The neural cells were differentiated in 24-well plates for 5 days. The microglia cells (LPS-stimulated and non-stimulated) from monolayers were detached from culture flasks, PKH67 stained, and added to neural cultures (5 x 10 4 per well). The heterogeneous populations of cells isolated from DBA mice brains were 24h pre-treated with LPS or nontreated (VEH). After 24h, cells were taken off the culture flask, stained with PKH67, and added into wells with neural cells to form co-cultures, and observed for 24h in six experimental variants: 1) 100 µM of DMT in VEH media, 2) 100 µM of DMT + 150 ng/mL LPS, 3) 100 µM of psilocin in VEH media, 4) 100 µM of psilocin + 150 ng/mL LPS, 5) control (VEH), 6) control with 150 ng/mL of LPS. In the LPS treated groups, cells were pretreated with 150 ng/mL LPS for 24h before initiation of the experiment. The continuous observation was done for 24h using a Zeiss Axio Observer microscope in 10 min intervals.

ANALYSIS OF CELL MORPHOLOGY:

The microglia cells morphology was measured from contrast-phase pictures using ImageJ software. The branch length was measured using the skeletonization method, and fractal dimension, lacunarity, span ratio, and circularity were measured using FracLac plug-in, both described in the protocol by.

STATISTICAL ANALYSIS:

The experiments were made at least three times in duplicates. Statistical analysis was done using Graph Pad Prism 5. All p-values were calculated by using a t-test for unpaired samples with Welch's correction. The graphs are presented as mean values with a standard error of the mean (SEM).

DMT AND PSILOCIN MODIFY THE MORPHOLOGY OF MICROGLIA

The primary microglial cells were isolated from DBA mice brains as a heterogeneous population. The heterogenous population was evaluated by estimating the percent of CD11b+ microglia vs CD11b negative cells. After reaching the monolayer in 3 weeks, an average of 65.3% of cells expressed marker CD11b+ as analyzed by FCM (Fig.). The staining for GFAP also revealed the presence of numerous astrocytes in the population (Fig.). After reaching the monolayer, to limit external stimuli, microglia cultures were pretreated for 24h in serum-free defined media in which all grow factors (except cholesterol) were omitted (VEH media). The microglia cultures were either preincubated for 24h in media supplemented with 150 ng/mL LPS or cultured in fresh VEH media. The cultures with LPS and without LPS were subsequently treated for 12h with 100 µM DMT or 100 µM psilocin or left untreated (Fig.). There were subtle changes in microglia morphology in the VEH group (Fig.). Cells cultured in VEH media stayed mostly in the rod/bipolar or small, round shape; however, in the group treated with psilocin and DMT, long, ramified cells with long branches were often observed (Fig 3B). After 24h incubation with LPS, the microglia were rather round and acquire a ramnified morphology. However, after 12h of DMT or psilocin treatment, many cells acquired ameboid morphology (Fig.). DMT or psilocin-treated cells in comparison to control (just LPS-treated group) were characterized with lower span ratio (**p<0.0063 DMT) ,( **p<0.0098 psilocin), higher circularity (*** p<0.0003 DMT), (**p<0.001-psilocin) and lower fractal dimension (** p < 0.0083 DMT) (Fig.). The TLR4 protein level is downregulated in the psychedelic-treated group after LPSstimulation To investigate how DMT and psilocin affect microglia immunological phenotype, we studied the expression of markers involved in inflammatory pathways: TLR4, Nf-κβ, and CD80; and TREM2, which is suggested to be involved in neuroprotection and regulation of microglial phagocytosis. We measured TLR4, TREM2, and Nf-κβ (p65) expression using the CTCF analysis of CD11b+ microglia. TLR4 receptor binds LPS which activates pro-inflammatory cascade, leading to upregulation of co-stimulatory molecules expression in antigen-presenting cells (APC) such as microglia. We observed that the expression of TLR4 did not significantly change in the VEH group after the application of both psychedelics. However, after LPS stimulation, the TLR4 expression on CD11b+ cells measured by fluorescence intensity was significantly lower in groups stimulated with DMT and psilocin (***p < 0.0001) as compared to the control group (Fig., Fig.).

DMT AND PSILOCIN DOWNREGULATE NF-ΚΒ P65 PROTEIN LEVEL IN BOTH CONTROL AND LPS CONDITIONS

The signaling cascade from LPS -stimulated TLR4 receptor under normal conditions results in activation of Nf-κβ (p65), a transcription factor that activates the expression of the pro-inflammatory genes. The microglial Nf-κβ (p65) fluorescence intensity decreased after DMT and psilocin stimulation both in VEH, and LPS-treated groups (** p < 0.002, ***p < 0.0001). In the picture (Fig.), which presents DMT-treated cells in the VEH group, the cells tagged with yellow arrows are the CD11b+ microglia cells (for the doublestained picture, see Fig.).

DMT AND PSILOCIN DOWNREGULATE CD80 CO-STIMULATORY MOLECULE

CD80 co-stimulatory molecule is necessary for antigen presentation via MHC T-cells, which is crucial for adaptive immune response development. Blocking the co-stimulatory molecule protein domains with specific factors for their inactivation is one of the immunosuppressive strategies called co-stimulatory blockade. The FCM analysis of CD80 marker in CD11b+ (H2 gated) cells revealed that DMT and psilocin application to the culture media decreases CD80 co-stimulatory molecule expression on both VEH and LPS-treated microglia. In the untreated group, both DMT and psilocin decreased CD80 protein level; however, the statistical significance (*p > 0.0172) was observed only in the group where the psilocin was applied. In the LPS-treated microglia, the decrease of CD80 protein level in DMT and psilocin stimulated groups was greater than in the group where no psychedelics were applied. Statistical significance was calculated for both DMT (*p > 0.0115) and psilocin (**p > 0.0069) (Fig.).

PSILOCIN BUT NOT DMT UPREGULATES TREM2 PROTEIN LEVEL IN THE VEH GROUP

TREM2 is a receptor present on myeloid cells, recently studied in the context of proper regulation of microglial phagocytosis and its neuroprotective properties. The stimulation with psilocin, but not DMT significantly (**p < 0.0023), increased the expression of TREM2 in the VEH group. Interestingly, in the LPS-treated group, treatment with DMT significantly (*** p< 0.0001) decreased TREM2 fluorescence intensity. In contrast, psilocin administration did not cause this effect, leaving TREM2 expression on a level similar to the VEH control group (Fig., Fig.).

PSILOCIN BUT NOT DMT ATTENUATES HEALTHY NEURONS MICROGLIAL PHAGOCYTOSIS

The microglia is essential in preserving tissue homeostasis, but sometimes proinflammatory and pro-regenerative microglia functions become imbalanced. Hyperactive microglia may react to compounds on the surface of stressed but healthy neurons and proceed with phagocytosis, seriously deteriorating the process of neural healing or even triggering neural damage. The study in co-cultures revealed that the application of 100 µM psilocin into culture media resulted in reduced phagocytosis of healthy neurons by microglia compared to DMT, both in VEH and LPS groups. This observation suggests that psilocin may display more robust neuroprotective properties than DMT by reducing healthy neuron phagocytosis by microglia (Fig., see supplementary videos V1, V2, V3. V4).

DISCUSSION:

The inflammatory environment which limits brain regeneration can be caused by hyperactive microglial cells as often seen in neurodegenerative disorders and brain damage, such as ALS, Alzheimer Disease, and Parkinson Disease, Multiple Sclerosisand TBI. Moreover, stem cell grafts are often proposed as cell therapy for neurodegeneration and brain damage; however, host vs. graft immune response can be a severe limitation. Current anti-inflammatory strategies that attenuate hyperactive immune response and microglia are recently proposed as a therapy for neurologic disorders. Unfortunately, therapeutics with anti-inflammatory properties such as antibiotic minocycline failed in the experimental treatment of AD patients and were not well tolerated at a higher dose (400 mg). The limitation of minocycline and other antiinflammatory drugs, whether steroid or non-steroid, is their broad-band influence on immune response and their severe adverse effects after long-term use. Many other novel therapeutics based on humanized antibodies targeting specific anti-inflammatory molecules are not suitable to crosse blood-brain barrier to treat brain inflammationThe anti-inflammatory therapeutic strategy should be more selective and involve cytokine-suppressive anti-inflammatory drugs (CSAIDs), that would target pro-inflammatory cytokine production pathways in astrocytes and microglia. In particular, the activity of neuroprotective receptors AT1, Sigma-1, and TREM2, as well as induction on Nrf2 pathways in order to secrete neurogenic factors and anti-oxidative enzymes, would be beneficial. Psychedelics are recently reported as agents which display neurogenic, neuroplastic, and anti-inflammatory properties, and they are characterized as physiologically relatively safe agents. Therefore, psychedelics might be an attractive strategy to consider and should be further investigated. The aim of the present study was to investigate in vitro if and how psychedelics can modulate the microglia immune response, which may influence the process of neural tissue cleaning and regeneration. Here we presented results supporting the hypothesis that psychedelic tryptamines (DMT and psilocin) have an immunomodulatory effect on CD11b+ microglia cells, limiting neural cell phagocytosis (psilocin). Special attention was paid to the expression of hallmark immunoregulatory proteins such as TLR4, Nf-κβ (p65), and CD80 and to recently wider studied TREM2 receptor, which might be a target in the treatment strategy for the neurodegenerative disorders. We observed changes in the protein level of the major markers of the microglial immune response: CD80, TLR4, TREM2, and Nf-κβ on the CD11b+ microglia cells after 100 µM DMT or 100 µM psilocin treatment. Both DMT and psilocin displayed immunomodulatory effects; however, those observed in the psilocin-treated group were stronger on average. In the VEH group, psilocin caused a reduction of CD80 and Nf-κβ (p65) fluorescence intensity by 11-12% more than DMT. In the LPS group, psilocin reduced TLR4 and CD80 fluorescence intensity by 7-10% more than DMT. The difference was observed in the LPS group, where DMT reduced Nf-κβ (p65) fluorescence intensity by 28% stronger than psilocin. The TLR4 receptor is the component of the innate immunity that reacts to PAMPS such as LPS and DAMPs, (ex. S-100, heat shock proteins, histones, and some other cellular debris). TLR4 stimulation activates the NF-κβ, a key factor involved in the inflammatory response acting via the canonical pathway. It leads to upregulation of co-stimulatory molecules, such as CD80, on the surface of antigen-presenting cells (APC), which is essential to introduce the adaptive immune response into play. Upregulation of co-stimulatory molecule protein level resulting from TLR4 -NFκβ pathway induction is known as a hallmark response to LPS stimulation in APC cells. The visible decrease in CD80 protein level on microglial cells, both: LPS-stimulated and in the VEH group after psilocin treatment, suggests that it may limit APC ability to present the antigen to T-cells, which might be helpful when considering cellular grafts as a therapeutic strategy. The adaptive immune response is essential for protection against new pathogens. However, its outstanding sensitivity and rapid activation of host immune responses provide a severe limitation when using cell therapy based on brain grafts or transplants. Therefore, the observed decrease in the CD80 protein expression after psilocin and DMT treatment, together with their reported activity towards induction of neurogenesis and neuroplasticity, is promising. TREM2 is the transmembrane amine receptor of the immunoglobulin family that regulates inflammatory response and phagocytosis. It is present on tissue-specific macrophages, including microglia and osteoclasts.. The results presented in our study showed that in the VEH group, psilocin increases TREM2 fluorescence intensity on primary microglia by 23% stronger than DMT. LPS treatment increased TREM2 expression on microglia cells by 72% as compared to the VEH group, as LPS is one of the TREM2 ligands. In the LPS group, psilocin increased TREM2 protein expression by 42%, whereas DMT reduced it by 20%. The ability of psilocin to promote upregulation of TREM2 protein level while decreasing pro-inflammatory proteins, even in the situation of LPS-treatment, suggests its potent anti-inflammatory properties. The particular activity of psilocin might be involved in its reported therapeutic effects. In vivo, TREM2 supports microglia-mediated rearrangements of synaptic connectivity in a process called synaptic pruningand phagocytosis of apoptotic neurons. Abnormalities in TREM2 regulation might be one of the causes of the progression of diseases such as Alzheimer's, Parkinson's, epilepsy, and schizophrenia. Moreover, the TREM2 expression is involved in phagocytosis, but its exact role in the process is still poorly understood. However, it is already known that TREM2 activation induces DAP12 phosphorylation, leading to cytoskeleton reorganization crucial for phagocytosis.. Interestingly, the abnormalities in TREM2 expression result in dysfunctional phagocytosis of Amyloid β and apoptotic neurons by microgliaand impairing engulfed particle metabolism. It is not completely clear why psilocin but not DMT increased TREM2 protein level in microglia after LPS-stimulation. Both psychedelics share similarities in molecular structure but display significantly varying binding affinities towards receptors, resulting in different activation of anti-inflammatory pathways depending on conditions. In the physiological conditions, miroglia-expressing TREM2 is abundantly localized throughout the brain. During brain damage, the TREM2 pathway may protect the brain and stimulate the transition of microglia towards anti-inflammatory phenotype. It was reported in bone-marrow-derived macrophages that TREM2-DAP12 signaling antagonizes TLRs receptors, causing a downregulation in TLRs proteins level and pro-inflammatory cytokine production, whereas silencing of TREM2 attenuated LPS-induced TLR4 signaling and its pro-inflammatory response. The downregulation of TLR4 was reported to act protectively for tissues in the model of cerebral ischemia/reperfusion through reduction of inflammatory protein levels. Therefore, the psychedelicinduced downregulation of TLR4 might be beneficial in brain regeneration.

CONCLUSION:

The study provides evidence supporting the hypothesis that the DMT and psilocin influence mouse microglia phenotype in vitro, and microglia-neural interactions in cocultures. The psilocin displays more substantial immunomodulatory potential on CD11b+ microglia than DMT, leading to downregulation of pro-inflammatory factors (TLR4, p65, and CD80) and upregulation of TREM2. Psilocin but not DMT also attenuate healthy neurons' phagocytosis by microglia. Summarizing, DMT and psilocin attenuate microglial proinflammatory response and can be considered therapeutic molecules to support neural tissue cleansing and regeneration in multiple conditions with an inflammatory pathogenic component.

FIGURE LEGEND

Fig.: Characteristics of the cells isolated from 3 -6 months old DBA/1 mice brain: A) After 7 days, the cells formed numerous, dense colonies. B) The GFAP and CD11b staining revealed a mixed population of astrocytes and microglia cells. C) FCM analysis revealed that 64.3% of the population in mean express CD11b marker (SEM = 1.96), error bars: SEM; total number of samples n = 12 per experimental group (grey -unstained control, green -CD11b. Scale bar -100 µm. revealed that in the VEH group, the incubation with DMT and psilocin did not change the TLR4 expression. However, after LPS treatment, the application of DMT and psilocin significantly (***p < 0.0001) decreased TLR4 fluorescence intensity, which could be comparable with the intensity measured in the VEH group. For double staining pictures, please see supplementary data. . Unpaired Student's t-test; error bars: SEM; the total number of biological replicates in VEH group: n = 6, the total number of events: n = 418, events per variant: n= 131 -153. Total number of biological replicates in LPS group: n = 6 , total number of events: n = 437, events per variant: n= 143 -150 Scale bar -50 µm.

FIG: 5: DMT AND PSILOCIN DOWNREGULATED THE EXPRESSION OF NF-ΚΒ IN CD11B+ MICROGLIA CELLS UNSTIMULATED AND AFTER LPS STIMULATION:

The IF staining revealed that CD11b microglia loses the expression of NF-κβ in VEH and in LPS-stimulated groups when treated both with DMT (** p < 0.002 VEH group, *** p < 0.000 LPS-stimulated group) and psilocin (*** p < 0.001 VEH and LPS-stimulated groups). For double staining pictures, please see supplementary data. Unpaired Student's t-test; error bars: SEM; total number of samples in VEH group: n = 6, total number of events: n = 410, events per variant: n= 133 -143. Total number of biological replicates in LPS group: n = 6 , total number of events: n = 421, events per variant: n= 131 -159. Scale bar -50 µm The IF staining revealed that the incubation with psilocin but not DMT increased the TREM2 expression (**p < 0.0023) in the VEH group. After LPS treatment, the application of DMT significantly decreased TREM2 expression (***p < 0.0001), whereas its expression was maintained in the psilocin group. For double staining pictures, please see supplementary data. Unpaired Student's t-test; error bars: SEM; total number of biological replicates in VEH group: n = 6, total number of events: n = 444, events per variant: n= 138 -158. Total number of biological replicates in LPS group: n = 6 , total number of events: n = 435, events per variant: n= 142 -151. Scale bar -50 µm The cells were characterized with an ability to differentiate in 7 days in DMEM/F12 supplemented with 1% B27, 1% Pen/Strep, 1% L-glutamine, 50 ng/mL BDNF, and 30 ng/mL GDNF into cells displaying neural morphology (A). Positive immunofluorescent staining with Synapsin and β-III-tubulin confirmed successful neural differentiation (B). In the differentiated neuronal culture, there was no GFAP-positive fraction; however, the population of single microglial cells could be identified with IBA-1+ staining(arrows). Control staining revealed no unspecific reactions (E), scale bar = 50µm.