Animals

Female C57BL/6 mice were obtained from the Experimental Animal Center of Guangdong (Guangzhou, China) and were kept in a pathogen-free facility at the Guangzhou Medical University. All experiments were approved by the Bioethics Committee of Guangzhou Medical University.

EAE induction and SCFAs treatments

EAE was induced in 6–8-week-old female C57BL/6 mice [27] by subcutaneous immunization with 200 µg myelin oligodendrocyte glycoprotein (MOG35–55, GL Biochem Ltd, China) peptide emulsified in complete Freund’s adjuvant (CFA, Sigma-Aldrich, USA, F5881) including 500 μg Mycobacterium tuberculosis H37RA (BD Biosciences, USA, 231141) on day 0 and 7. Immediately thereafter and on day 2, the mice received an intraperitoneal injection of 300 ng pertussis toxin (PTX, KKL MED, USA, KM10754) in 100 μL PBS. Clinical signs of EAE were scored daily and blindly by two researchers individually ranging from 0 to 5 as follows: grade 5, death; grade 4.5, near death, moribund; grade 4, complete paralysis of two limbs; grade 3, complete paralysis of a single limb; grade 2.5, partial limb paralysis and ataxia; grade 2, dysfunctional gait with limp tail and ataxia; and grade 1, dysfunctional gait with tail tonicity or limp tail; 0, no signs of disease [16]. On day 0, the drinking water of mice was supplemented with SCFAs mix (sodium acetate 67.5 mM, 241245; sodium propionate 40 mM, P5436; and sodium butyrate 25.9 mM, 303410, Sigma–Aldrich, USA) and changed every 3 days as described [28]. A control group received only sodium chloride. Throughout the duration of the experiment, mice were continuously provided with SCFAs in their drinking water.

16S rRNA gene sequencing and bioinformatic analysis

Freshly extruded stools were collected immediately after the EAE induction for 16S rRNA sequencing analysis at BGI Co. (Shenzhen, China) following the methodology described in our previously published study [16]. Briefly, DNA was extracted using QuickGene DNA tissue kit from Kurabo Company (Neyagawa, Japan) and next used for PCR amplification and sequencing of the V3 and V4 region of bacterial 16S rRNA genes using Illumina MiSeq technology. Taxonomic annotation was performed using the GreenGene database. Partial least squares discrimination analysis (PLS-DA) was performed based on operational taxonomic units (OTU) abundance information using QIIME (Quantitative Insights into Microbial Ecology, version1.8.0). The differential microbial flora biomarkers among groups were performed using linear discriminant analysis (LDA) effect size (LEfSe) analysis.

SCFAs quantification based on LC–MS

The freshly extruded stools and serum were collected on the 22nd day post-induction (p.i.) to initiate EAE. For fecal samples, 20 mg of the samples were accurately weighed and placed in EP tube. After adding 0.5% v/v phosphoric acid solution and a small steel ball, the mixture was ground, vortexed, and ultrasonicated. After centrifugation of the mixture at 12,000 rpm for 10 min at 4 °C, the supernatant was collected. 500 μL MTBE (containing internal standard) solution was added to the centrifugal tube.

For serum samples, the samples were thawed and vortexed for 1 min prior to analysis. Then, 50 μL of the sample was added to an eppendorf tube, followed by the addition of a 0.5% v/v phosphoric acid solution. The mixture was vortexed for 3 min. 150 μL MTBE (containing internal standard) solution was added.

Both the fecal and serum mixtures were vortexed and ultrasonicated separately. After ultrasonication, each mixture was centrifuged at 12,000 r/min for 10 min at 4 °C. The resulting supernatants obtained from the centrifugation of the fecal and serum samples were collected for SCFAs detection using the Agilent 7890B-7000D GC–MS/MS platform [29, 30].

Large scale medical targeted metabolomics based on UHPLC-MS

The brain samples were collected on the 22nd day p.i. to initiate EAE. The metabolites were extracted from brain samples followed by incubation and centrifugation at 16,000 g and 4 °C for 20 min. The resulting supernatant was transferred to a sampling vial for subsequent analysis using UHPLC-MS. Quality control (QC) samples were prepared and analyzed using the same procedure as the experimental samples in each batch. The dried extracts were dissolved in 50% acetonitrile and stored at −80 °C until analysis.

UHPLC/MS was employed for metabolite detection in both electrospray negative-ionization and positive-ionization modes, utilizing a Shimadzu Nexera X2 LC-30AD system. The ACQUITY UPLC HSS T3 column (1.7 μm, 2.1 mm × 100 mm, Wasters) was used in this experiment. A gradient elution method was employed to separate the compounds. During the acquisition, QC samples were injected periodically to monitor the reproducibility of the analysis. Widely targeted metabolites were quantified using multiple-reaction monitoring mode set up as described in previous report [31, 32]. All transitions from large-scale metabolites were detected with optimized decluttering potential and collision energy. MultiQuant 3.0.2 software was used to extract the original MRM data of large-scale metabolites and obtain the peak area of each metabolite for quantification from different samples. The discriminating metabolites were obtained using a statistically significant threshold of variable influence on projection values obtained from the OPLS-DA model.

RNA sequencing and bioinformatic analysis

For RNA from brain tissue, cDNA was generated using random oligonucleotides, Super Script II, DNA Polymerase I and RNase H. The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.

Quantitative real-time PCR

RNA from brain tissue was extracted using miRNeasy kits (Qiagen, Germany, #217,004). The extracted RNA was then reverse transcribed using the Evo M-MLV RT Master Mix kit (Accurate Biology, China, #AG11706). Subsequently, quantitative PCR (qPCR) was performed on the generated cDNA samples using the SYBR Green Pro Taq HS kit (Accurate Biology, China, #AG11701) and the real-time polymerase chain reaction system (Roche LightCycler 480). The primer sequences used for qPCR are listed in Table 1. β-actin was chosen as the internal control, and the comparative cycle threshold (ΔΔCt) method was employed to determine gene expression levels.

Table 1 Primers used for RT-PCRImmunofluorescence

Brains were collected on the 22nd day p.i. to initiate EAE and were dissected and embedded into optimal cutting temperature compound (OCT, Tissue-Tek) and snap-frozen for cryo-sectioning. Slides were incubated in 1% Triton X-100 for 30 min and then incubated in 10% goat serum at room temperature for 30 min to block nonspecific antibody binding. For GFAP/AhR/AQP4 staining, the slices were incubated with primary antibodies against GFAP (1:1000, Cell Signaling Technology, USA, #3670), AhR (1:200, Boster Biological Technology, China, A00225-4) or AQP4 (1:1000, Proteintech, USA, CL488-16473) at 4 °C overnight after washing with PBS. Slices were then incubated with Alexa 488-conjugated (Cell Signaling Technology, USA, #4412S), Alexa 555-conjugated (Cell Signaling Technology, USA, #4409) secondary antibodies at a concentration of 1:1000 at room temperature for 2 h in the dark. The slides were mounted in Slowfade gold antifade mountant with DAPI (Thermo Fisher Scientific, USA, S36942). Slides were taken on an Leica DM6B microscope or Carl Zeiss 880 confocal microscope.

Western blots analysis

Brain tissues isolated from mice were lysed with RIPA lysis buffer with 1% protease inhibitor cocktail, and supernatants were collected as total protein. Protein concentration was determined with the BCA Protein Assay Reagent. Lysates (100 μg) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA in TBS, membranes were incubated with the following primary antibodies: anti-AhR (1:500, Boster Biological Technology, China, A00225-4), anti-Claudin 5 (1:1000, Abcam, UK, ab131259), anti-Occludin (1:1000, Abcam, UK, ab167161), anti-ZO-1 (1:1000, Abcam, UK, ab276131), anti-AQP4 (1:1000, Proteintech, USA, 16473-1-AP), anti-GAPDH (1:5000, Affinity, USA, #AF7021), and anti-β-tubulin (1:5000, Affinity, USA, #AF7011). After incubation with a HRP-conjugated anti-rabbit secondary antibody (1:2000, Abcam, UK, ab97051), the proteins were visualized with ECL reagents (Millipore, USA, #WBKLS0100) for the immunoreactive bands visualization. The intensity of each target protein band was quantified by densitometry analysis using ImageJ software.

Histopathology

Brains and lumbar spinal cords tissues were collected on the 22nd day p.i. to initiate EAE and were fixed by cardiac perfusion with 4% (w/v) PFA, and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE) and luxol fast blue (LFB). ImageJ Software analysis (NIH, Bethesda, MD, USA) was used to evaluate inflammatory cell infiltration and demyelination.

Flow cytometry

Brains and spinal cords tissues from different treated mice were collected on the 22nd day p.i. to initiate EAE for the flow cytometry analysis. Tissues were subjected to 0.5 mg/mL collagenase solution digestion at 37 °C for 30 min, then pressed through a 40 μm cell strainer. CNS-infiltrating mononuclear cells were separated from myelin debris by centrifugation in 30%/70% Percoll solution [16]. Collected cells were stained with CD4-BV510 (BD Biosciences, USA, 563106), IFN-γ-PE-Cy7 (BD Biosciences, USA, 557649) and IL-17A-BV421 (BD Biosciences, USA, 563354). Samples were measured by a fluorescence-activated cell sorter flow cytometer (BD Biosciences), and then analyzed by FlowJo (Tree Star, Ashland, OR).

Transmission electron microscopy

Brains were isolated and fixed in electron microscope fixative (Servicebio) overnight at 4 °C for transmission electron microscopy (TEM) [16]. Specimens were then rinsed with phosphoric acid, dehydrated with acetone, immersed, embedded in epoxy resin, and cut into 60 nm ultrathin sections using an ultramicrotome (Leica, EM UC7). Grids were observed using a transmission electron microscope (FEI, Czech Republic).

Evans blue perfusion

Mice were injected with 0.4% evans blue dye at a dose of 200 mg/kg into the tail vein and circulated for 30 min [16]. Then mice were fixed by cardiac perfusion with PBS and 4% (w/v) PFA. Brains were obtained and tissue cryo-sections were analyzed by fluorescence microscopy.

Virus preparation

AAV8-GFAP-shAhR were purchased from (Sunbio Medical Biotechnology Company, China) at the titer of 1.0*1013 vg/mL. Sequences of shAhR was 5′-CCGGAGAGCTCTT TCCGGATAATAACTCGAGTTATTATCCGGAAAGAGCTCTTTTTTTG-3′.

Intracisterna magna and intracerebral injection

Mice were anesthetized by intraperitoneal injection of a mixed solution of ketamine (100 mg/kg) and xylazine (10 mg/kg) in saline. For intracisterna magna injection, the skin of the neck was shaved and cleaned with 70% ethanol. A 50 μL Hamilton syringe was used to deliver a 10 μL bolus injection of viral suspension diluted at 1012 vg/mL or Alexa Fluor 555-conjugated ovalbumin (OVA, Thermo Fisher Scientific, USA, O34782) into the cisterna magna at a rate of 2.5 μL/min. After injecting, the syringe was left in place for at least 2 min to prevent backflow of CSF. For intracerebral injection, the mice were maintained in a stereotaxic frame. A hole was drilled in the localization: Bregma AP, −3.0 mm, ML, ±1.3 mm, DV, −4.7 mm [33]. 1 μL bolus injection of Alexa Fluor 555-conjugated OVA was infused using a 10 μL glass Hamilton syringe at a rate of 0.2 μL/min. The mice were allowed to recover from anesthesia on warmed pad (about 37 °C). Mice were sacrificed at 30 min after OVA intracisterna magna injection or 2 h after OVA intracerebral injection.

Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed by Graphpad Prism v9. Statistical significance was determined: using Student’s t test (comparison of two groups), using Mann–Whitney tests (comparison of two groups, non-parametric data), one-way ANOVA or two-way ANOVA (comparison of three or more groups). When ANOVA showed significant differences, comparisons between means were tested by Bonferroni’s multiple comparisons test or Tukey’s multiple comparisons test. Values of p < 0.05 were considered statistically significant.