Six- to eight-week-old female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed within the Animal Resource Facilities (ARF) at the University of South Carolina School of Medicine. Mice were cohoused for a period of 2 weeks prior to experimentation. On day 0, mice were randomly divided into either vehicle or treatment groups prior to the induction of EAE. Lck-Cre AHR^flox/flox mice on C57Bl/6 background were bred in house using AHR^tm3.1Bra/J (Jackson stock number 006203) and dLCK-hcre³⁷⁷⁹/J (Jackson stock number 012837) mice purchased from Jackson Laboratories. The resulting Lck-Cre AHR^flox/flox are a cell-specific knockout that lack expression of a functional AHR in all T Cells. All donor Lck-Cre AHR^flox/flox were age matched with WT donor mice prior to immunization for all passive EAE experiments. All animal work was conducted in accordance with protocols that follow the National Institute of Health guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of South Carolina.

The following reagents were used during the course of the experiments and were purchased as following: red blood cell (RBC) lysis buffer was purchased from Millipore Sigma (Burlington, MA); Percoll from GE Healthcare Life Sciences (Pittsburgh, PA); Myelin oligodendrocyte glycoprotein peptide subunit 35–55 (MOG35-55) from PolyPeptide Laboratories (San Diego, CA); Pertussis Toxin (PTX) from List Biological Laboratories (Campbell, CA); Heat killed Mycobacterium tuberculosis (H37Ra) from Difco (Detroit, MI); β-mercaptoethanol, Freund’s Advjuvant, propionic acid, n-butyric acid, isovaleric acid, 2-ethylbutyric acid, sulfuric acid and Tween-80 from Sigma-Aldrich (St. Louis, MO); absolute ethanol, acetic acid, lithium carbonate and tryptamine were purchased from Fisher Scientific (Hamptom, NH); RPMI 1640, fetal bovine serum (FBS), L- glutamine, phosphate buffered saline (PBS), luxol fast blue (LFB), cresyl violet, eosin, hematoxylin, and HEPES were purchased from VWR (West Chester, PA; flourophore conjugated antibodies and ELISA kits from Biolegend (San Diego, CA); Illumina MiSeq reagents from Illumina Inc. (San Diego, CA); QIAamp Stool Mini Kits from Qiagen (Germantown, MD); 10% formalin from Azer Scientific (Morgantown, PA).

Chronic progressive EAE was induced in C57BL/6 mice according to previously published protocols (Constantinescu et al., 2011; Glatigny and Bettelli, 2018). Briefly, mice were immunized on day 0 with subcutaneous injections containing 150 µg of myelin oligodendrocyte glycoprotein subunit 35–55 (MOG35-55) and 600 mg heat killed Mycobacterium tuberculosis (H37Ra) suspended within an emulsion of PBS and Freund’s complete adjuvant On day 0 and 2, mice received a single intraperitoneal injection containing 200 and 400 ng of PTX, respectively. Beginning on day 1, mice received a 50 µL intraperitoneal injection containing either a vehicle (sterile corn oil (CO) with 2% DMSO v/v) or a treatment suspension (12.5 mg/kg tryptamine in sterile CO with 2% DMSO v/v) every 48 h. This dose of 12.5 mg/kg was chosen in accordance with previous studies that have demonstrated it to be a safe dose that will not induce short term behavioral changes in mice (Mousseau, 1993). This dose was also deemed to be safe and clinically relevant when converted to a human equivalent dose of 1 mg/kg (Nair and Jacob, 2016; Wüst et al., 2017).

The weight and paralysis symptoms were recorded within EAE mice daily. The scoring of paralysis symptoms was done according to the following key: 0 = no symptoms, 1 = inability to curl the distal end of tail, 2 = complete tail atony/impaired movement, 3 = partial hind limb paralysis, 4 = complete hind limb paralysis, 5 = tetraplegia/moribund state.

Single cell suspensions were prepared using neural tissue dissociation kits purchased from Miltenyi according to manufacturer instructions (Pennartz et al., 2009). After dissociation of CNS tissue, myelin was removed from the single cell suspensions using two washes in 30% percoll. Remaining cells were then subjected to RBC lysis buffer, washed and suspended in RPMI supplemented with FBS and penicillin/streptomycin (cRPMI) for downstream use. Immune cells were isolated from the spleens and lymph nodes by use of mechanical dissociation followed by RBC lysis and filtration using 70 µm filters. After washing, the remaining mononuclear fractions were suspended in cRPMI for downstream use.

Media was collected from primary cultures of mononuclear cells isolated from the CNS and spleen plated at 10 × 10⁶ cells/ml under unstimulated conditions from EAE mice treated in vivo and MOG35-55 (20 µg/ml) stimulated conditions from MOG35-55 immunized mice treated in vitro for ELISA quantification of IL-10 and IL-17. ELISAs were performed according to Biolegend protocols. Briefly, high affinity protein-binding plates were coated by incubating a 100 µL suspension of capture antibody overnight at 4°C. The plates were then washed three times using wash solution of (PBS + 0.05% Tween80). The plates were then incubated with blocking solution for 1 h at room temperature (RT). After incubation, the plate was washed three times using wash solution and incubated for 2 h at RT with 100 µL of standards prepared using serial dilutions according to manufacturer instructions and 100 µL of supernatant collected from overnight cell cultures. Plates were again washed three times with wash solution before incubating with 100 µL of a biotinylated detection antibody solution diluted according to the manufacture’s instruction in blocking solution at RT for 1 h. After incubation with the detection antibody, plates were washed three times with solution and incubated with horseradish peroxidase (HRP) conjugated avidin antibody for 30 min at RT. After incubation with HRP-avidin, the ELISA plates were washed five times with wash solution and incubated with TMB substrate for fast color development. After color development, the reaction was stopped simultaneously in all wells using 1N hydrosulfuric acid. The concentration of captured protein content was calculated by comparing relative absorbance of variable samples at a wavelength of 450 nm to the standard curve calculated from standards of known concentration. Plates were analyzed with a PerkinElmer Victor² plate reader.

Suspensions of single mononuclear cells isolated from splenic and CNS tissue using previously described methods were phenotyped using a BD FACSCelesta flow cytometer. Single cell suspensions were tagged with fluorescently labeled monoclonal antibodies purchases from Biolegend (APC conjugated anti-RORγT, BV786 conjugated anti-CD4, FITC conjugated anti-CD3, BV421 conjugated anti-Foxp3, PE conjugated and BV605 conjugated anti-CD45). Analysis of .fcs files was conducted using FlowJo software.

C57BL/6 mice were immunized with subcutaneous injections of 150 µg of MOG35-55 and 600 mg H37Ra suspended within an emulsion of sterile PBS and Freund’s adjuvant. After 7 days, the mice were euthanized using isoflurane. Spleens were excised from the mice and cells were isolated via mechanical dissociation, RBC lysis and 70 µm filtration. Live cells were plated at 10 × 10⁶ cells/ml in complete cRPMI activated with 20 µg/ml MOG35-55. Cells were activated in the presence or absence of 100 µM tryptamine in the medium and cultured for 48 h. Statistical analysis was performed using paired t-tests to compare changes across donor samples in the presence or absence of tryptamine.

Passive polyclonal EAE was induced as previously described (McPherson et al., 2014). Briefly, C57BL/6 (wild-type) and Lck-Cre AHR^flox/flox mice were immunized with subcutaneous injections of 150 µg of MOG35-55 and 600 mg H37Ra suspended within an emulsion of sterile PBS and Freund’s complete adjuvant. After 7 days, the mice were humanely euthanized. Cells were isolated from the spleens and inguinal lymph nodes of these donor mice as previously described. Live cells were incubated at a concentration of 4 × 10⁶ cells/ml in cRPMI medium supplemented with MOG35-55 (10 µg/ml), rIL-2 (10 U/ml), rIL-12 (25 ng/ml) and rIL-18 (25 ng/ml) in the presence or absence of tryptamine (100 µM). After 48 h, the cells were washed and plated in cRPMI medium supplemented with MOG35–55 (10 µg/ml), rIL-2 (20 U/ml), rIL-12 (25 ng/ml) and rIL-18 (25 ng/ml) in the presence or absence of tryptamine (100 µM). After 24 h, the cells were washed, counted and brought to a final concentration of 4 × 10⁶ live cells/100 µL within sterile PBS. 4 × 10⁶ live cells were given via retro-orbital injection to recipient C57BL/6 (wild-type) mice. After 2 h, mice received an intraperitoneal injection containing 200 ng of PTX suspended within sterile PBS. After 48 h, the mice received a second intraperitoneal injection containing 400 ng of PTX suspended in sterile PBS. Paralysis symptoms were recorded daily as previously described.

Euthanized mice were perfused with 10 ml of heparinized PBS followed by 10 ml of 10% formalin. After isolation, whole brains were held in 70% EtOH and embedded in paraffin blocks. 7 µm sections were cut using a microtome and placed onto microscope slides using a water bath. Slides were deparaffinized with washes of xylene and ethanol prior to staining with Luxol Fast Blue and Hematoxylin and Eosin (H&E) according to previously published protocols (Feldman and Wolfe, 2014; Carriel et al., 2017).

Phylogenetic profiling of the cecal microbiota was conducted by use of 16s rDNA sequencing of DNA isolated from cecal contents of vehicle and tryptamine treated mice. Briefly, DNA was isolated from cecal contents using the QIAamp Stool Mini Kit according to the manufacturer instructions. Sequencing was performed using the Illumina MiSeq platform. Downstream analysis of .fastq files was conducted using the Nephele platform provided via the National Institute of Allergy and Infectious Diseases (NIAID) (Weber et al., 2018). Further downstream analysis of Nephele operational taxonomic unit (OTU) outputs was conducted using linear discrimination of effect size (LEfSe) analysis (Segata et al., 2011).

SCFA content was quantified from cecal content using methods previously described (Zhao et al., 2006; Busbee et al., 2020). Briefly, 100 mg of cecal content was homogenized in 400 µL deionized H20. After homogenization, samples were acidified by adding 1:4 of the total volume of 25% metaphosphoric acid. After 30 min of incubation, samples were centrifuged (12,000 g for 15 min), with the supernatant being collected and filtered using Ultra-free MC Columns (ThermoFisher). Ethyl butyric acid was added to each sample at a final concentration of 0.0375 mM for use as an internal standard to accurately calculate experimental concentrations. Samples then received 400 µL of methyl tert-butyl ether (MTBE) and were vortexed vigorously. Samples were then spun at 400 g for 5 min. Lastly, 100 µL of the upper organic layer was transferred to a fresh glass tube. Samples were then analyzed with an HP 5890 gas chromatograph configured with flame-ionized detection (GC-FID).

Statistical analysis was conducted using GraphPad Prism software version 8.4.3. Unpaired t tests were used to compare experimental groups separated by a single variable. Paired t tests were used to compare changes in experimental groups that shared a single subject prior to subjection to a variable. Common subjects are annotated by a solid line connecting individual values across groups. To test the efficacy of tryptamine in EAE, we used four experiments containing five mice per group equating to a total of 20 mice. The number of animals used in other experiments have been shown in Figure legends. All graphs show individual values with the mean ± standard error of the mean (SEM). Samples were considered statistically significant if p < 0.05. Degree of significance was demonstrated using the following key: *p < 0.05, **p < 0.01, ***p < 0.001.

In order to investigate the immunosuppressive potential of tryptamine, we administered tryptamine (12.5 mg/kg) via i.p injection every 48 h in a murine model of chronic progressive EAE. We analyzed mice daily to observe paralysis symptoms and body weight until any individual mice displayed a severity of symptoms that reflected a moribund state and required euthanasia. At this point, observed at day 13, all mice were humanely euthanized for uniform sample collection. Tryptamine treated mice demonstrated a significant reduction in paralysis symptoms beginning from day 8 that persisted for the remaining duration of the study (Figure 1A). The sum of paralysis scores per mouse further demonstrates a significant reduction in observed paralysis symptoms experienced between tryptamine vs. vehicle treatment groups (Figure 1B). The average weight of mice was also measured throughout the time course of chronic progressive EAE and the data expressed as percent of starting body weight, clearly showed that while the vehicle-treated group started losing weight, especially on days 11–13, the tryptamine-treated mice showed a significant retention of weight when compared to the vehicle controls (Figure 1C). These results together demonstrated that tryptamine treatment of EAE mice ameliorates the clinical symptoms of paralysis and weight loss associated with EAE.

In order to understand if the attenuation of clinical symptoms by tryptamine results from decreased neuroinflammation, we next investigated the T cell responses in the CNS of these mice. A representative flow plot demonstrated the reduction in CD4⁺ T cells found within the CNS of tryptamine treated mice (Figure 2A). When CNS infiltrating CD4⁺ T cells were analyzed using flow cytometry among the CD45⁺ population (Supplementary Figure S1), we noted that while the percentage of CD4⁺ T cells did not significantly decrease in comparison to the vehicle group (Figure 2B), there was significant reduction in the total number of infiltrating CD4⁺ T cells present in the CNS (Figure 2C). Analysis of anti-inflammatory (IL-10) and pro-inflammatory (IL-17) cytokines secreted by cultured mononuclear cells isolated from the CNS revealed that tryptamine treated mice displayed no significant change in IL-10 secretion (Figure 2D) while significantly suppressing IL-17 secretion in comparison to the vehicle group (Figure 2E). Because the MOG antigen is injected peripherally, we also tested the spleens of the tryptamine for Tregs vs. Th-17 cells to test if tryptamine also altered the immune response in the periphery. CD4⁺ T Cells were identified from the spleens as being CD3⁺CD4⁺CD8⁻ prior to looking at transcription factor expression (Supplementary Figure S2). This increase in the Treg population of tryptamine treated mice is represented with overlaying histograms demonstrating Foxp3 expression among CD4⁺ T cells between the groups (Figure 2F). We found that tryptamine treated mice displayed a higher proportion (Figure 2G) as well as total number (Figure 2H) of anti-inflammatory Tregs in comparison to the spleens of vehicle treated mice. After discovering an increase in the anti-inflammatory Treg subset, we then studied the pro-inflammatory counterpart of RORγT+ T cells. The spleens of tryptamine treated mice demonstrated a reduction in the percentage of RORγT+ T cells as shown by a representative histogram (Figure 2I). Tryptamine treatment significantly reduced the proportion of the pro-inflammatory RORγT T cell subset within the spleens in comparison to the vehicle group (Figure 2J) while displaying a trend in the total number of Th17 lymphocytes, however this trend was not statistically significant (Figure 2K). Collectively these results showed that treatment with tryptamine suppresses inflammation in both the periphery and in the CNS.

In order to better demonstrate that tryptamine ameliorates EAE by regulating CD4⁺ T cell reactivity in an AHR-dependent manner we tested the immunosuppressive capacity of tryptamine on MOG35-55 reactive T cells donated from wild WT and Lck-Cre AHR^flox/flox mice prior to the induction of CD4⁺ T cell mediated passive polyclonal EAE. Briefly, C57BL/6 wild-type and Lck-Cre AHR^flox/flox mice were immunized with MOG35-55 antigen and immune cells from the spleens and lymph nodes were further activated with a combination of MOG35-55 and pro-inflammatory cytokines IL-2, IL-12 and IL-18 in vitro, in the presence or absence of tryptamine (100 µM). Following a 72 h incubation the cell suspensions were then transferred into naïve wild-type mice via retroorbital injection. The development of EAE symptoms were studied on a daily basis as described in Materials and Methods.

The mice which received transfer of cells from wildtype+Vehicle, Lck-Cre AHR^flox/flox +Vehicle and Lck-Cre AHR^flox/flox +Tryptamine demonstrated significant paralysis symptoms, while those that received transfer of cells from wildtype + tryptamine group developed only mild paralysis symptoms (Figure 3A). These results are further corroborated by the cumulative scores of paralysis scores per mouse (Figure 3B). Flow cytometry analysis of the spleen (Figure 3C) and CNS (Figure 3D) revealed that mice which received a transfer of tryptamine treated encephalitogenic T cells from wildtype mice displayed increased Treg abundance when compared to similar cells that were treated with vehicle. Measurement of IL-10 in in vivo sensitized cells with MOG that were activated in vitro with MOG in the presence of tryptamine showed an increase in IL-10 secretion when compared to vehicle controls, but it was not statistically significant (Figure 3E). Histological analysis of brain in passive EAE experiments showed a retention of myelin indicated by a deeper intensity of Luxol fast blue staining in the areas surrounding the dark blue stained neuronal cell bodies and a reduction of immune cell infiltration denoted by a lack of dark colored hematoxylin stained infiltrates in the mice that received cells treated with tryptamine in vitro when compared to mice that received similar cells treated with vehicle (Figure 3F).

Because culture of MOG-sensitized T cells in vitro with tryptamine and adoptive transfer led to decreased EAE when compared to vehicle-treated cells, we next investigated the mechanisms involved. Representative flow plots demonstrated the percentage of CD4⁺ and PCNA+ cells among all lymphocytes collected from immunized mice stimulated in vitro with MOG35-55 in the presence or absence of tryptamine demonstrated a reduction in proliferative activity (Figure 3G). Tryptamine treatment in vitro reduced the percentage of proliferating CD4⁺ T cells as demonstrated by PCNA+ cells (Figure 3H). Also, tryptamine treatment in vitro significantly reduced the percentage of CD4⁺ RORγT as shown in a representative flow cytometric analysis (Figure 3I) and the percentage of such cells from multiple samples (Figure 3J).

Previous literature has demonstrated that AHR ligands exert their anti-inflammatory properties by a combination of direct interactions on the immune system and indirect interactions mediated by the GI microbiota (Lefever et al., 2016; Brawner et al., 2019; Busbee et al., 2020). For this purpose, we studied the effects of tryptamine treatment in vivo on the composition of the cecal microbiota in EAE mice. The chao1 index (Figure 4A) and Shannon index (Figure 4B) of 16s sequencing reads indicated there were slight increases in the alpha diversity of the cecal microbiome composition in tryptamine treated mice. PCoA plotting demonstrated that the cecal microbiota of treated mice was distinctly clustered from the vehicle group (Figure 4C). LEfSe analysis was conducted on the 16s sequencing outputs to distinguish differentially regulated bacterial phylogenies (Figure 4D). Three LEfSe identified phylogenies in Dehalobacterium (Figure 4E), Bacteroides (Figure 4F) and Peptostreptococcaceae (Figure 4G) to be significantly altered in tryptamine-treated groups when compared to the vehicle controls. When Short Chain Fatty Acids (SCFAs) were quantified using mass spec the data revealed a significant increase in n-Butyric Acid following tryptamine treatment when compared to vehicle controls (Figure 4H). These results suggested that tryptamine treatment alters the microbiota in the gut and promotes the induction of n-Butyric Acid.