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).

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.“

Authors: Urszula Kozłowska, Aleksandra Klimczak, Kalina Wiatr & Maciej Figiel

Notes

The study found that psilocin led to a stronger response on the immune (cell-)system than DMT.

“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.”

Summary

Psychedelics are new, promising candidate molecules for clinical use in psychiatric disorders and neural tissue repair by anti-inflammatory properties. DMT and psilocin regulate the immunomodulatory potential of microglia and may be useful in the therapy of neurological disorders.

Psychedelic therapy has been used for treatment-resistant depression, post-traumatic stress disorder, and other neuropsychiatric conditions. Its therapeutic effects are related to immunomodulatory potential, ability to induce neurogenesis, and neural plasticity.

Psychedelics may reduce brain inflammation, which is a direct cause of neural cell death and limitation of the regeneration process. However, anti-inflammatory drugs may cause serious adverse effects.

Microglia are brain-specific macrophages that play an important role in tissue rearrangement, neural tissue regeneration, and protection from pathogens. The activated microglia secrete pro-inflammatory proteins that attract other immune cells to the brain, which may lead to acute tissue damage.

Microglia are important regulators of several cellular functions, including phagocytosis. The Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) reacts to anionic molecules, such as toxic protein aggregates and pathogen associated molecular patterns.

In the current study, we investigated whether classic psychedelics could be beneficial for neural tissue homeostasis and the promotion of pro-regenerative and anti-inflammatory features. We tested whether DMT and psilocin affected microglial phagocytosis of healthy neurons after LPS stimulation.

DBA1 mice were euthanized and their brains were extracted. Microglia were then cultured in ice-cold PBS and purified using Percoll Plus.

Neurospheres were isolated from C57BL/6 mouse E10 embryos and cultured in a medium containing DMEM with Hams F12 Nutrient mix supplemented with 2% of B27 supplement, 100 ng/mL EGF, 100 ng/mL bFGF, 5 g/mL heparin, 1% penicillin/streptomycin, 1% L-glutamine.

NSCs were dissociated to single-cell and seeded on poly-L-ornithine/laminin coated plates for 24h in neurosphere medium. The medium was changed to neuronal differentiation medium.

Immunofluorescence analysis was performed using Zeiss Axio Observer 2 microscope, with Alexa Fluor 488 nm settings for CD11b staining and Alexa Fluor 594 nm settings for TLR4, TREM2, and Nf- stainings. The Corrected Total Cell Fluorescence (CTCF) was calculated using ImageJ Software.

The neural cells were differentiated in 24-well plates for 5 days, and the microglia cells were detached from monolayers, PKH67 stained, and added to neural cultures (5 x 104 per well). The co-cultures were observed for 24h in six experimental variants.

The experiments were performed at least three times in duplicates, and the results are presented as mean values with SEM.

The primary microglial cells were isolated from DBA mice brains and 65.3% expressed marker CD11b+. GFAP staining revealed the presence of numerous astrocytes in the population.

After reaching the monolayer, microglia cultures were pretreated for 24h in serum-free defined media in which all grow factors (except cholesterol) were omitted (VEH media). The cultures were either preincubated for 24h in media supplemented with 150 ng/mL LPS or cultured in fresh VEH media.

We measured the expression of markers involved in inflammatory pathways in CD11b+ microglia and found that the expression of TLR4, TREM2, and Nf- (p65) was significantly lower in groups stimulated with DMT and psilocin as compared to the control group.

LPS-stimulated TLR4 receptor activates Nf- (p65), a transcription factor that activates the expression of the pro-inflammatory genes in microglia. The Nf- (p65) fluorescence intensity decreased after DMT and psilocin stimulation.

A co-stimulatory molecule (CD80) is necessary for antigen presentation via MHC T-cells, which is crucial for adaptive immune response development. DMT and psilocin decrease CD80 co-stimulatory molecule expression on both VEH and LPS-treated microglia.

Psilocin, but not DMT, increased the expression of TREM2 in VEH cells, while LPS treated cells showed a decrease in TREM2 expression.

Psilocin but not DMT attenuates healthy neurons microglial phagocytosis in co-cultures, suggesting that psilocin may display more robust neuroprotective properties than DMT by reducing healthy neuron phagocytosis by microglia.

Hyperactive microglial cells can cause brain inflammation, which limits brain regeneration. Current anti-inflammatory strategies are not effective for treating neurodegenerative disorders and brain damage, such as minocycline, and humanized antibodies targeting specific anti-inflammatory molecules are not suitable to cross the blood-brain barrier. Psychedelics might be an attractive strategy to consider for the treatment of chronic inflammation, as they display neurogenic, neuroplastic, and anti-inflammatory properties. In vitro studies with psychedelics have shown that they can modulate the immune response of microglia cells, limiting neural cell phagocytosis.

The expression of hallmark immunoregulatory proteins such as TLR4, Nf- (p65), and CD80 was observed on CD11b+ microglia cells after 100 m DMT or 100 m psilocin treatment. Psilocin caused a reduction of CD80 and Nf- (p65) fluorescence intensity by 11-12% more than DMT.

The TLR4 receptor is involved in the innate immunity response to PAMPs, such as LPS, and DAMPs, such as S-100, heat shock proteins, histones, and some other cellular debris. TLR4 stimulation activates the NF-, which leads to the upregulation of co-stimulatory molecules on APC cells. The adaptive immune response is essential for protection against new pathogens. The results presented in this study showed that psilocin increases TREM2 fluorescence intensity on tissue-specific macrophages.

Psilocin increased TREM2 expression on microglia cells by 42% compared to VEH group, and decreased pro-inflammatory proteins by 20%. This suggests its potent anti-inflammatory properties.

TREM2 is involved in synaptic pruning, phagocytosis of apoptotic neurons, and the regulation of inflammatory pathways. It is not completely clear why psilocin but not DMT increased TREM2 protein level in microglia after LPS-stimulation.

In physiological conditions, TREM2 is abundantly localized throughout the brain and may protect the brain and stimulate the transition of microglia towards anti-inflammatory phenotype. Psychedelic-induced downregulation of TLR4 might be beneficial in brain regeneration.

The study showed that DMT and psilocin influence mouse microglia phenotype in vitro, and microglia-neural interactions in cocultures. DMT and psilocin can be considered therapeutic molecules to support neural tissue cleansing and regeneration in multiple conditions.

We thank friends from the Institute of Immunology and Experimental Therapy Polish ska for help and advice in lab and handymen for adjusting the store room.

CSAIDs are cytokine-suppressive anti-inflammatory drugs, DAMPS are danger associated molecular patterns, and TLR4 is a toll-like receptor 4.

UK conceived, designed, supervised all experiments, analyzed the data and wrote the manuscript. AK provided scientific guidance, KW obtained NPC cells, and MF provided scientific guidance and funding.

Microglia are lifelong modulators of neural circuits, and are involved in the development of neurodegenerative diseases. TREM2 is a protein that regulates microglial phagocytosis and is required for microglial instruction of astrocytic synaptic engulfment in neurodevelopment. Kigerl, K. A., de Rivero Vaccari, J. P., Dietrich, W. D., Popovich, P. G., and Keane, R. W. (2014) reviewed the role of pattern recognition receptors in central nervous system repair and NF- B signaling in inflammation.

Matejuk, A., Ransohoff, R. M., Nayak, D., Roth, T. L., McGavern, D. B., et al. (2020) reviewed the crosstalk between astrocytes and microglia and the application of TREM2 antagonists. Nichols, D. E. (2016) reviewed psychedelics and their potential for treating disorders of consciousness. A new method for the rapid and long term growth of human neural precursor cells was described in 1998. Tan, Y.-L., Yuan, Y., and Tian, L. (2020). Microglial regional heterogeneity and its role in the brain.

The microglia were cultured for 2-3 weeks until monolayer, and then the media were changed for 24h, then 12h, then 100 mM DMT or Psilocin was added, and the tests were performed immediately after all the incubations.

The LPS, DMT, and psilocin treatment altered the morphology of CD11b+ microglia cells in the VEH and LPS groups. The DMT and psilocin treatment resulted in reassuming ameboid morphology in the LPS group as compared to just LPS-treated cells, which was ramified.

After LPS treatment, the DMT and psilocin 12h stimulation decreased TLR4 fluorescence intensity on CD11b+ microglia cells, which could be comparable with the intensity measured in the VEH group.

DMT and psilocin downregulated the expression of NF- in CD11b+ microglia cells unstimulated and after LPS stimulation.

The DMT and psilocin 12h stimulation changed the TREM2 fluorescence intensity on CD11b+ microglia cells. The psilocin group maintained the TREM2 expression after LPS treatment, whereas the DMT group decreased the expression.

Fig. 8 shows that co-cultures of neural cells and adherent cells isolated from DBA mice brains resulted in greater neural phagocytosis in the DMT group as compared with the psilocin group.

The brain isolated E10 embryonic bodies differentiated into cells displaying neural morphology in 7 days, and the population of single microglial cells could be identified with IBA-1+ staining.

DMT and psilocin downregulate Nf-p65 protein level in control and LPS-stimulated cells, whereas psilocin upregulates TREM2 protein level in VEH and LPS-stimulated cells.