Amanita muscaria extract (AME-1) was provided by Psyched Wellness Inc. (Toronto, Ontario) and can be purchased directly from the manufacturer. AME-1 is a commercial product sold for human consumption.

HMC3 cells were purchased from ATCC (Manasass, VA, United States). The cells were cultured in minimum essential medium (MEM) supplemented with 10% FBS (Hyclone, Logan, UT, US), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Waltham, MA, United States) and maintained in a humidified atmosphere of 5% CO₂ in air at 37°C. The cells were trypsinized in 0.25% trypsin-EDTA (Gibco) and maintained at 2 × 10⁴ cells/cm² once they reached maximum confluency.

Stock concentrations of LPS (1 mg/mL, Sigma, St. Louis, MO, US), poly(I:C) (10 mg/mL, Sigma), recombinant human TNF (100 µg/mL, ThermoFisher, Waltham, MA, United States), Trehalose (250 mM, Sigma) and AME-1 (diluted 1:2) were prepared in sterile phosphate buffered saline (PBS, Gibco) pH 7.4 without calcium or magnesium. Substance P (Bachem, Bubendorf, CH) was prepared in 0.1 M acetic acid to a concentration of 1 mg/mL. Working stocks of treatments were diluted in culture medium prior to addition to cells.

HMC3 were seeded at 0.4 × 10⁶ cells per well in a 6-well plate and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C overnight to adhere. For single treatments the cells were then treated with AME-1 (50–5,000 µg/mL), 1 µg/mL LPS (Sigma), 0.5 µg/mL poly(I:C) (Sigma), 0.25 µg/mL recombinant human TNF (ThermoFisher), 0.5 µg/mL substance P (Bachem) or 0.15–15 mM trehalose (Sigma) for 24 h at 37°C, 5% CO₂ and cell free supernatants were collected. For co-treatments, the cells were treated with AME-1 (50–5,000 µg/mL), or trehalose (0.15–15 mM) for 3 h at 37°C, 5% CO₂. Following incubation, the media was aspirated, replenished with fresh media and the cells were stimulated with poly(I:C) (0.5 µg/mL; Sigma), LPS (1 µg/mL; Sigma), substance P (0.5 µg/mL; Bachem), recombinant human TNF (0.25 µg/mL; ThermoFisher) or untreated for 24 h. Cell free supernatants were isolated. For ELISA analysis for human IL-8 and IL-6 commercial ELISA kits (R&D Systems, Minneapolis, MIN, US) were used and plates were read using a VarioSkan Lux plate reader (ThermoFisher). Electrochemiluminescent multiplex analysis was performed using a custom-made multiplex plate with capture antibodies specific for IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-22, IFN-γ, CCL2 and TNF (Meso Scale Diagnostics LLC., Rockville, MD, United States) and the plate was read using a MESO QuickPlex SQ 120MM plate reader (Meso Scale Diagnositcs LLC.).

HMC3 were seeded at 0.1 × 10⁵ cells/mL in a 96-well plate and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C overnight to adhere. The cells were then treated with AME-1 (50–5,000 µg/mL), poly(I:C) (0.5 µg/mL) (Sigma), LPS (1 µg/mL; Sigma), substance P (0.5 µg/mL) (Bachem), recombinant human TNF (0.25 µg/mL; ThermoFisher), trehalose (0.15–15 mM (Sigma), or untreated for 24 h. Metabolic activity was analysed using the Cell Proliferation Kit II (XTT Assay, Roche, Basel, Switzerland) according to the manufacturer’s instructions and the results are presented as percent metabolic activity relative to untreated cells.

HMC3 were seeded at 0.4 × 10⁶ cells/mL in a 6-well plate and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C overnight to adhere. The cells were then treated with AME-1 (50–5,000 µg/mL) for 24 h at 37°C in 5% CO2. Following incubation, the cells were trypsinized and mixed 1:1 with 0.4% Typan Blue (Gibco) and counted on a haemocytometer. Percent viable cells was calculated relative to untreated.

HMC3 were seeded at a density of 0.5 × 10⁶ cells/mL in a 6-well dish and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C for 2 h to adhere. Cells were treated with AME-1 (50–5,000 μg/mL), LPS (1 mg/mL), poly(I:C) (10 μg/mL), or trehalose (0.15–15 μM) for 24 h. The cells were removed with 0.25% trypsin-EDTA (Gibco), washed with PBS, blocked for 10 min with 3% bovine serum albumin (BSA) in PBS and subsequently labelled with mouse anti-human CD125-phycoerytherin (PE) (555902, BD Biosciences), mouse anti-human, CXCR4-PE (12–9999, Invitrogen), rat anti-mouse CD86-allophycocyanin (APC) (558703, BD Biosciences, Franklin Lakes, NJ, United States), mouse anti-human TLR4-FITC (sc-13593, Santa Cruz Biotechnology Inc., Dallax, TX, United States), or mouse anti-human CD45-fluorescein isothiocyanate (FITC) (A85816, antibodies-online Inc., Limerick, PA, United States) in 0.1% BSA in PBS for 1 h at 4°C. Cells were labelled with isotypes (IgG1-PE (568275, BD Biosciences), IgG2a-PE (12–4724–82, Invitrogen), IgG2a-APC (553932, BD Biosciences), or IgG2a-FITC (sc-2856, Santa Cruz Biotechnology Inc.) for comparison. Flow cytometry was performed using the CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, United States) and data analysis was performed using FlowJo software version 10.6.1 (FlowJo LLC, Ashland OR, United States).

HMC3 were seeded at a density of 1 × 10⁶ cells/mL and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C overnight to adhere. The cells were then treated with AME-1(50–5,000 µg/mL) for 3 h at 37°C in 5% CO2 and RNA was isolated using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). Complementary DNA was synthesized using 1 µg of total RNA and M-MLV reverse transcriptase (ThermoFisher). qPCR was performed using PrimeTime qPCR primer probes (IDT, Brighton, United Kingdom) for TLR3 and a StepOnePlus qPCR instrument (Applied Biosystems) Results are presented as ΔΔCT relative to untreated expression levels.

HMC3 were seeded at a density of 0.5 × 10⁶ cells/mL in a 6-well dish and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C overnight to adhere. The cells were then treated with LPS (1 µg/mL, Sigma), poly(I:C) (0.5 µg/mL, Sigma), trehalose (0.15–15 μM, Sigma) or AME-1 (50–5,000 ug/mL) for 24 h. For cells treated with trehalose and poly(I:C), the cells were treated with trehalose (0.15–15 μM) for 3 h, the media was then removed and replaced with media containing 0.5 ug/mL poly(I:C) and the cells were incubated a further 24 h. The cells were then lysed in Laemmli buffer (50 mM Tris pH 6.8, 1% SDS, 9% glycerol, 1.8% β-mercaptoethanol, and 0.002% bromophenol blue) and boiled at 100°C for 15 min. Cell lysates were loaded on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was subsequently blocked for 1 h at room temperature with 5% skim milk in TBS-T followed by incubation for1hr with 0.4 µg/mL of mouse anti-human TLR3 (sc-32232, Santa Cruz Biotechnology, Dallas, TX, United States), 0.5 µg/mL of mouse anti-human IRF3 (550428, BD biosciences), 2.5 µg/mL of rabbit anti-human MDA5 (33H12L34, Invitrogen), 1 µg/mL rabbit anti-human RIG-1 (PA510297, Invitrogen), or 1 µg/mL of mouse anti-β-actin loading control (PA1-183, Invitrogen) in 3% BSA-TBS-T, and finally incubated for 1 h with goat anti-mouse IRDye 680 (Li-Cor, Lincoln, NE, United States) or goat anti-rabbit IRDye 800 (Li-Cor) diluted 1:15,000 in 3% BSA-TBS-T. The membrane was imaged using a Li-Cor Odyssey CLx (Li-Cor). Densitometry data was generated using Image Studio software version 5.2 (Li-Cor) and presented as a ratio of the indicated band signal of treated samples to untreated samples and all samples are corrected to the signal of their respective β-actin band.

AME-1 was diluted 100 times with D₂O containing 0.15 mM TMSP (3-(trimethylsilyl)propionate-2,2,3,3-d4, Cambridge Isotope Laboratories, Inc., Tewksbury, MA, United States) for NMR measurement. NMR experiments were performed on a Varian Direct Drive VNMRS 600 spectrometer, operating at a magnetic field strength of 14.1 T (599.49 MHz proton frequency) and equipped with an autoX dual broadband probe. One dimensional (1D) ¹H NMR spectra were measured for all samples using 1D ¹H with water suppression sequence (NOESY 1D) at 298 K. TMSP was used as an internal reference standard for spectra calibration. Metabolites were identified and quantified using Chenomx NMR Suite 8.6 Professional (ChenomX Inc., Edmonton, Canada).

LC separation was performed using an Agilent Zorbax NH₂ column (4.6 × 250 mm, 5 um) on an Agilent 1260 HPLC system. The mobile phase was composed of 20: 80 water: acetonitrile (EMD Millipore Corporation) with 2 mM ammonium acetate (HPLC grade, EM Science) at a flow rate of 1 mL/min. The volume of injection was 10 µL. Analyte detection was achieved using an Agilent Single-Quad MSD (G6135B) with an electrospray ionization (ESI) source. Identification and quantification were achieved by comparison of peak retention time and area of reference standards.

Experiments were conducted in triplicate and values represent mean of n = 3± standard error of the mean (SEM) or standard deviation (SD) as indicated. p values for statistical significance were determined by two-way ANOVA with Tukey post hoc analysis for co-treatments and, one-way ANOVA with Dunnett’s multiple comparison or Tukey post hoc analysis for single treatments, p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). GraphPad Prism software version 7.01 was used to generate figures and perform statistical analysis (GraphPad Software, San Diego, CA, United States).

Since HMC3 are considered an imprecise model of primary human microglia (He et al., 2021; dello Russo et al., 2018), we characterized some of their common features. Light microscopic analysis indicated that some of the cells had the expected globular morphology while most of the cells were elongated with thick processes (Figure 1A). We measured HMC3 expression of CD125, CXCR4, CD86, TLR4, and CD45, by flow cytometry. Non-stimulated HMC3 showed little to no expression of any of these receptors (Figures 1B–F). These data support previous data that show that HMC3 express few microglial surface receptors in the absence of stimuli (Li et al., 2009; Rai et al., 2020). Our data contribute to the characterization of HMC3 as a model of microglial research by further indicating that, in the absence of stimulation, HMC3 express few microglial receptors.

To better understand the response of HMC3 to common neuroinflammatory stimuli, we activated HMC3 with LPS, TNF, poly(I:C) and substance P and measured their release of IL-8 and IL-6 (Figures 2A, B). HMC3 response to poly(I:C) and substance P had not been previously described. HMC3 constitutively released significant amounts of both IL-6 and IL-8, and when they were activated with LPS, TNF and poly(I:C) they produced approximately 30% more of both mediators compared to untreated cells. Substance P did not significantly increase IL-8 release and significantly reduced the basal amount of IL-6 released from HMC3.

Since the metabolic rate of microglia is often increased after activation, we measured their metabolic rate using a standard formazan reduction assay by which XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium) is reduced by mitochondrial produced NADH to a soluble orange-yellow formazan salt. Activation of HMC3 with LPS, TNF, poly(I:C) or substance P did not alter the metabolic rate of HMC3 cells (Figure 2C).

Since LPS and poly(I:C) differentially modulate primary microglia activation (He et al., 2021), we tested their effect on HMC3 receptor expression (Figure 3). We focused on poly(I:C) since poly(I:C) effects on HMC3 had not been previously described. We chose LPS as a positive control since LPS effects on HMC3 have been extensively described in the literature. Our data indicates that 24 h exposure to LPS or poly(I:C) do not alter the expression of microglial surface receptors CD125, CXCR4, CD86, TLR4, and CD45.

We next determined the effect of A. muscaria extract (AME-1) on HMC3 production of cytokines using two different approaches: ELISA and electrochemiluminescent multiplex analysis. ELISA analysis indicates that at higher concentrations, AME-1 significantly increased basal IL-8 production but did not significantly increase IL-6 production, even at extremely high concentrations of 5,000 µg/mL (Figures 4A, B). AME-1 also did not affect HMC3 metabolic activity (Figure 4C), although the very highest concentration of 5,000 µg/mL decreased their metabolic activity by approximately 60% compared to the untreated control (UT). AME-1 did not affect cell viability after 24 h at even the highest concentration of 5,000 µg/mL (Figure 4D) which indicates that although the metabolic rate of cells treated with 5,000 μg/mL was significantly reduced, the cells were still viable and had not undergone necrosis.

Electrochemiluminescent multiplex analysis showed that AME-1 significantly induced the production of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-22, IFN-γ, CCL2 and TNF (Figure 5). HMC3 did not significantly release IL-12p70 or IFN-γ upon LPS stimulation (Figures 5F, H). Poly(I:C) and substance P did not significantly induce the release of any of the mediators that were measured.

We next determined the effect of AME-1 on receptor expression by HMC3 (Figures 6A–J). AME-1 increased the expression of CD125 CXCR4, TLR4, CD86 and CD45 when compared to untreated cells. MFI analysis of three independent experiments confirmed that AME-1 significantly and reproducibly increased expression of CD125, CXCR4, CD86, TLR4 and CD45. AME-1 increased expression of CD125, CXCR4, TLR4 and CD45 in a concentration-dependent manner, with the highest AME-1 concentration (5,000 μg/mL) showing the highest increase in receptor expression. AME-1 upregulation of CD86 was only significant at the 500 µg/mL concentration. Although AME-1 appeared to also increase expression of TLR4, this was not statistically significant in this study.

Next, the combined effect of AME-1 and other stimulants on HMC3 was evaluated. HMC3 were treated with AME-1 and then stimulated with LPS, poly(I:C), or TNF and their production of IL-8 and IL-6 was measured. AME-1 had no effect on LPS-induced production of either IL-8 or IL-6 (Figures 7A, B). However, AME-1 potentiated poly(I:C)-induced production of IL-8 (Figure 7C). AME-1 did not enhance poly(I:C)-induced IL-6 production (Figure 7D). AME-1 had no effect on TNF-induced production of IL-8 or IL-6 (Figures 7E, F).

Since poly(I:C) is a potent ligand of TLR3, we next measured the effect of AME-1 on TLR3 expression by HMC3. AME-1 had a significant effect on the expression of TLR3 mRNA (Figure 8A) by HMC3 cells, although this effect was modest (approximately 2-fold increase with 500 μg/mL of AME-1). The highest concentration of AME-1 of 5,000 μg/mL did not modify expression of TLR3 mRNA, suggesting that this effect reaches saturation at these extremely high concentrations.

Western blot analysis indicated that AME-1 had no significant effect on TLR3 protein expression or expression of the interferon regulatory transcription factor (IRF3), even at the highest concentration of 5,000 μg/mL (Figures 8B, E, F). RIG-I and MDA5 are other receptors that can bind and sense dsRNA (Brisse and Ly, 2019) and thus we evaluated their expression following AME-1 treatment. AME-1 appeared to upregulate MDA5 expression (Figure 8C) and RIG-I (Figure 8D), although this effect was not statistically significant over three experiments (Figures 8G, H).

To get a better understanding of the components of AME-1, a metabolomic profile of the extract was achieved by combining NMR spectroscopy with metabolites identification and quantification software, i.e., ChenomX NMR suite. Some typical metabolites were identified (Table 1) and several signals could not be assigned to a particular metabolite due to the limitations of the database. The strongest signals in the aliphatic region of ¹H NMR spectrum were due to trehalose which was the most abundant metabolite found in the extract (Figure 9A). 4-Aminobutyrate (GABA) was also observed with a relatively low content level in the proton NMR spectrum of AME-1. In the aromatic region, uridine, cytosine, ADP, tyrosine, phenylalnine, guanosine, and nicotinate were detected. The identity of trehalose was further confirmed by the NMR spiking experiments (Figure 9B). The concentration of trehalose in the 100X dilute solution is 1.38 mM as revealed by the ¹H NMR analysis.

Although the trehalose in AME-1 has been identified and quantified, we wanted to confirm using HPLC-MS (Figures 9C–E). Therefore, we used a straightforward HPLC-MS based assay for the detection and quantification of trehalose in AME-1, demonstrating that trehalose could be separated from other metabolites and detected using a single quadrupole mass spectrometer with electrospray ionization (ESI-MS). In the positive scan mode, a mass range of 50—1,000 was chosen to cover the m/z value of 360 corresponding to the [M + NH₄ ⁺] parent ion. As can be seen, the retention time for trehalose is 30.1 min (Figure 9C), and the trehalose signal was separated from other metabolite signals in the AME-1 extract (Figure 9D).

Having determined that trehalose can be easily separated and detected using this HPLC-MS method, a calibration curve for trehalose was constructed (Figure 9E). In these experiments, the mass spectrometer was operated in single ion monitoring (SIM) mode in which an m/z of 360 due to [M + NH₄ ⁺] parent ion was used. The relative response of SIM was used to plot the standard curve. The standard curves are best fit by a single polynomial. The polynomial fits produce R ² value of greater than 0.998 indicating low accuracy errors.

To determine the concentration of trehalose in the AME-1 by HPLC-MS, 100x diluted AME-1 in water was used for this experiment. The peak for trehalose in SIM mode was detected at the expected retention time for 100x diluted AME-1 sample. Based on the standard curve, the trehalose concentration in the 100x diluted AME-1 was calculated to be 0.91 ± 0.06 mM (n = 4). These data demonstrate, for the first time, that trehalose contained within the AME-1 extract can be successfully detected and quantified using HPLC-MS.

It is noteworthy that the trehalose concentration in 100X dilute AME-1 was determined by NMR spectroscopy and HPLC-MS. HPLC-MS shows a relatively low trehalose concentration compared to NMR analysis. This fact may be due to the complex composition of AME-1, and the NMR signals were overlapped in the spectrum. The strong water signal in AME-1 could cause the baseline of the NMR spectrum to become distorted, and the relative signal intensities due to metabolites may have therefore been over-estimated. Therefore, the trehalose concentration in the original AME-1 without dilution would be 0.091 M based on the HPLC-MS.

Since AME-1 contained high levels of trehalose, the effect of trehalose on HMC3 metabolic activity, IL-8 production and receptor expression was determined (Figure 10). Like AME-1, trehalose had no effect on HMC3 metabolic activity (Figure 10A), and potentiated poly(I:C)-activated production of IL-8 (Figure 10B). Trehalose potentiated poly(I:C)-activated production of IL-8 even at the lowest concentration of 0.15 μg/mL and increasing trehalose concentrations did not increase this effect. Trehalose also significantly potentiated poly(I:C) production of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-22, IFN-γ, CCL2, and TNF (Figures 11A–J). Trehalose treatment alone and trehalose followed by poly(I:C) stimulation did not significantly increase MDA5 expression (Figures 10C, D), although there was an increased trend in expression. Similarly, trehalose alone and combined treatment with poly(I:C) did not significantly affect RIG-I expression (Figures 10E, F).

Since AME-1 significantly increased receptor expression by HMC3 we investigated the effects of trehalose. Trehalose treatment did not alter surface expression of CD125, CXCR4, CD86, TLR4, or CD45 (Figures 12A–J) by HMC3 cells.