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Gut Microbes
Gut Microbes
Gut Microbes
1949-0976
1949-0984
Taylor & Francis

39287045
10.1080/19490976.2024.2402547
2402547
Version of Record
Research Article
Research Paper
Prebiotic inulin controls Th17 cells mediated central nervous system autoimmunity through modulating the gut microbiota and short chain fatty acids
N. LI ET AL.
GUT MICROBES
Li Ning a b c *
Han Xinyan a *
Ruan Ming b c *
Huang Fei a
Yang Liu a
Xu Tianhao b c
Wang Huijun a
Wu Hui a
Shi Songshan a
Wang Yongjun b c
Wu Xiaojun a
Wang Shunchun a
a The MOE Key Laboratory for Standardization of Chinese Medicines and the MOE Innovation Centre for Basic Medicine Research on Qi-Blood TCM Theories, Shanghai Key Laboratory of Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine , Shanghai, China
b Longhua Hospital, Shanghai University of Traditional Chinese Medicine , Shanghai, China
c Spine Institute, Shanghai University of Traditional Chinese Medicine , Shanghai, China
CONTACT Yongjun Wang wangyongjun@shutcm.edu.cn Spine Institute, Shanghai University of Traditional Chinese Medicine, 725 Wan-Ping South Road, Shanghai 200032, China
Xiaojun Wu xiaojunwu320@126.com
Shunchun Wang shunchunwang@126.com Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, P. R. China
* These authors contributed equally.

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© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
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The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination occurring in the central nervous system (CNS). Inulin is a common prebiotic that can improve metabolic disorders by modulating the gut microbiota. However, its capacity to affect CNS autoimmunity is poorly recognized. Experimental autoimmune encephalomyelitis (EAE) is a classical mouse model of MS. Herein, we found that oral administration of inulin ameliorated the severity EAE in mice, accompanied by reductions in inflammatory cell infiltration and demyelination in the CNS. These reductions were associated with decreased proportion and numbers of Th17 cells in brain and spleen. Consistent with the findings, the serum concentrations of IL-17, IL-6, and TNF-α were reduced in inulin treated EAE mice. Moreover, the proliferation of auto-reactive lymphocytes, against MOG35-55 antigen, was attenuated ex vivo. Mechanistically, inulin treatment altered the composition of gut microbiota. It increased Lactobacillus and Dubosiella whereas decreased g_Prevotellaceae_NK3B31_group at the genus level, alongside with elevated concentration of butyric acid in fecal content and serum. In vitro, butyrate, but not inulin, could inhibit the activation of MOG35-55 stimulated lymphocytes. Furthermore, fecal microbiota transplantation assay confirmed that fecal contents of inulin-treated normal mice had an ameliorative effect on EAE mice. In contrast, antibiotic cocktail (ABX) treatment diminished the therapeutic effect of inulin in EAE mice as well as the reduction of Th17 cells, while supplementation with Lactobacillus reuteri restored the amelioration effect. These results confirmed that the attenuation of inulin on Th17 cells and inflammatory demyelination in EAE mice was dependent on its modulation on gut microbiota and metabolites. Our findings provide a potential therapeutic regimen for prebiotic inulin supplementation in patients with multiple sclerosis.

KEYWORDS

Inulin
multiple sclerosis
gut microbiota
short chain fatty acids
inflammatory demyelination
Th17 cells
the Key New Drug Creation and Manufacturing Program in China 2019ZX09735001-004 National Natural Science Foundation of China 10.13039/501100001809 82003010 the ‘Innovation Team’ development projects IRT1270 the Three-Year Action to Accelerate the Development of Traditional Chinese Medicine Plan ZY(2018-2020)-CCCX-3003 the Shanghai Natural Science Fund 21ZR1462900 This work was supported by the Key New Drug Creation and Manufacturing Program in China [2019ZX09735001-004 to SW], the National Natural Science Foundation of China [No. 82003010 to NL, No. 82074043 to XW], the ‘Innovation Team’ development projects [IRT1270 to YW], and the Three-Year Action to Accelerate the Development of Traditional Chinese Medicine Plan [ZY(2018-2020)-CCCX-3003 to YW], and the Shanghai Natural Science Fund [21ZR1462900 to SW].
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pmc1. Introduction

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination affecting the brain and spinal cord. Demyelination deflects neuronal conduction, resulting in hypokinesia, sensory impairment, cognitive dysfunction, and ultimately paralysis.1 Epidemiological studies have shown that there are approximately 3 million MS patients worldwide, with higher prevalence in Europe and the United States and lower prevalence in Asian countries.2 Clinical treatment of MS is mainly aimed at delaying the exacerbation period and improving symptoms using disease-modifying medications, including interferon beta, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, and alemtuzumab, etc.3 Although, the use of these drugs is beneficial in the control of MS, they usually have certain side effects, all of these drugs are potentially hepatotoxic;4 besides, fingolimod may reduce heart rate and increase susceptibility to infection;5 and dimethyl fumarate can cause a decrease in the number of white blood cells and gastrointestinal (GI) disorders.6 Therefore, the current therapeutic drugs cannot fulfill the treatment demands of MS patients, and thus, development of high safety and efficacy MS management strategies is valuable.

MS is a disease caused by abnormalities of the immune system, T lymphocytes especially CD4+ T helper (Th) cells play an important role in mediating autoimmune reaction of MS.7 Th cells are differentiated from naive CD4+ T cells under different cytokine stimulations. Among them, the IFN-γ secreting Th1 cells and IL-17 secreting Th17 cells are proven to be critical for the autoimmune response against myelin proteins. Meanwhile, they also contribute to the disruption of the blood–brain barrier and inflammatory response in central nerve system (CNS).8 In contrast, regulatory T cells (Tregs) inhibit the aberrant activation of immune responses and protect against MS.9

The probandwise concordance rates are only about 25.3% in monozygotic twins and as low as about 5.4% in dizygotic twins, indicating genetic factors cannot fully elucidate the activation of auto-reactive immune responses and the development of MS.10 Therefore, the critical roles of environmental factors, especially gut microbiota, in the initiation of autoimmune responses and the progression of demyelinating lesions are receiving increasing attention. Recently, several articles have reported significant differences in the composition of gut microbiota between MS patients and healthy individuals. For instance, the increased number of Methanobrevibacter and Akkermansia, and decreased number of Butyricimonas may be associated with the activation of CD4+ T cells.11 Experimental autoimmune encephalomyelitis (EAE) mice are a classic animal model of MS. It has been reported that germ-free RR (relapsed) mice are completely unaffected by EAE during their lifetime,12 suggesting a functional role of gut microbiota in the development of EAE. Interestingly, transplantation of gut microbiota from MS patients into germ-free RR mice exacerbates the severity of EAE, confirming that intestinal commensal flora is essential in initiating the immune process and contributes to the inflammatory demyelination of MS and EAE.13 Particularly, gut-colonized segmented filamentous bacteria (SFB) promote pro-inflammatory T-cell responses (Th17 cells) and exacerbate EAE in germ-free mice.14 Clearing the gut microbiota with antibiotics or altering it with probiotics alleviates EAE symptoms by inducing protective Foxp3+ Treg cells,15,16 suggesting modulation of the gut microbiota may provide a novel management strategy for EAE and MS.

Inulin is a naturally occurring polysaccharide composed of more than ten fructose units with β-2,1 fructosyl-fructose linkages. It is well recognized as a prebiotic or dietary fiber that specifically modulates the composition of the gut microbiota and short-chain fatty acids (SCFAs) production.17,18 Inulin supplementation has been reported to be beneficial in the treatment of type 2 diabetes,19 obesity,20 tumor,21 and rheumatoid arthritis.22 However, studies regarding the efficacy of inulin on inflammatory demyelinating lesions of the CNS are limited. The aim of this study was to investigate the beneficial effects of inulin on EAE and disclose the mechanisms by which inulin regulates gut microbiota and metabolites, thereby inhibits autoimmune responses. It also aimed to provide an experiment basis for the use of prebiotics in MS patients to improve their clinical symptoms.

2. Materials and methods

2.1. Reagents

RPMI 1640 medium, PBS, and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA). Myelin oligodendrocyte glycoprotein (MOG35–55) was purchased from Shanghai Qiangyao Biotechnology Company (Shanghai, China). Pertussis toxin (PTX) was purchased from Merck Millipore Company (Darmstadt, Germany). Acetate, propionate, butyrate, acetic acid, propionic acid, butyric acid, valeric acid, Concanavalin – A (ConA), lipopolysaccharides (LPS) and collagenase D were obtained from Sigma-Aldrich (St. Louis, MO, USA). Freund’s incomplete adjuvant was purchased from Chondrex Company (Redmond, WA, USA). Perm/Wash buffer, Cell stimulation cocktail (00-4970-03), Fixation buffer, Fixable Viability Dye eFluor™ 780, Fluorescent-labeled anti-CD4, anti-IL-17A, anti-CD3, anti-IFN-γ, anti-CD25, anti-CD11c, anti-CD86, anti-MHC-II, recombinant TNF-α, IL-1β, IFN-γ, IL-6, IL-17A, Foxp3, Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE), Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent and enzyme-linked immunosorbent assay (ELISA) kits were obtained from eBioscience (San Diego, CA, USA). DNase I was purchased from Roche (Mannheim, Germany). Percoll was obtained from Cytvia (Chicago, IL, USA). Annexin V was purchased from BD Bioscience (New York, NY, USA). Mouse CD4 Microbeads, LS columns were obtained from Miltenyi (Bergisch Gladbach, Germany). Recombinant mouse IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were obtained from Peprotech (Rocky Hill, NJ, USA). Lactobacillus reuteri (L. reuteri) strains (ATCC23272) were purchased from Ning Bo Testobio Co., Ltd (Ningbo, China).

2.2. Animals

Female C57BL/6 mice at 6 weeks old were purchased from Shanghai Silaca Company and maintained in the Experiment Animal Centre of the Shanghai University of Traditional Chinese Medicine. Rodent laboratory feed and tap water were provided ad libitum, and the living conditions were maintained at a humidity of 50 ± 10% and a 12/12 h light/dark cycle with temperature of 24 ± 1°C. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee at the Shanghai University of Traditional Chinese Medicine (Approval number: SZY20191002, PZSHUTCM2308260012).

2.3. Preparation of inulin

Inulin was prepared as described previously.22 In brief, the herbs of Artemisia japonica were defatted by 95% ethanol and extracted with 10 volumes of boiling water. Then, the water extractions were concentrated and mixed with three volumes of 95% ethanol to precipitate the crude polysaccharides, which were then fractionated on a DEAE-cellulose column and eluted with distilled water. The water eluates were concentrated and further purified on a superdex-75 column using a 0.2 M NaCl solution. The eluate was dialyzed, concentrated, and lyophilized to obtain lyophilized powder of inulin. The purity and homogeneity of inulin were determined by the High-Performance Gel Permeation Chromatography (HPGPC) method using series-connected KS-804 and KS-802 columns (Shodex Co., Tokyo, Japan).

2.4. EAE induction and treatment

Induction of EAE was performed on C57BL/6 mice acclimatized for 1 week. Briefly, mice were subcutaneously injected with 300 μg of MOG35–55 emulsified in complete Freund’s adjuvant containing 0.5 mg of heat-killed Mycobacterium tuberculosis H37Ra. This was followed by intraperitoneal injection of 300 ng pertussis toxin (PTX) on day 0 and day 2. To assess the efficacy of pro-phylactic treatment, mice were intragastrically administered with inulin (200 mg/kg) or vehicle (distilled water) daily from day 0 to day 24. To assess the therapeutic efficacy of inulin against ongoing EAE, mice were divided into two groups on day 16 post-immunization and were orally administered either vehicle or inulin from day 16 to day 24 post-immunization. The dose of inulin was selected based on our preliminary experiment, where a dose of 200 mg/kg showed a superior protective effect compared to 400 and 100 mg/kg. According to previously study, the clinical signs of EAE were assessed and scored using a 5-point scale: 0, no clinical signs; 0.5, partially limp tail; 1, paralyzed tail; 2, loss of coordinated movement and hind limb paresis; 2.5, one hind limb paralyzed; 3, both hind limbs paralyzed; 3.5, hind limbs paralyzed and weakness in forelimbs; 4, forelimbs paralyzed; and 5, moribund or death.23

2.5. Histology assessment

On day 24 post immunization, the spinal cords were collected from mice perfused with PBS and fixed in 4% paraformaldehyde for 24 h. For the pro-phylactic treatment experiment, six mice were randomly selected from each group to collect spinal cords. After fixation, the tissues were embedded in paraffin and cut into 5 μm sections. Paraffin sections were subsequently stained with hematoxylin and eosin (H&E) to assess inflammatory cell infiltration and Luxol Fast Blue (LFB) to assess demyelination of the CNS. Histopathological examination was performed under blinded conditions. Inflammation was evaluated according to the following criteria: 0 points, no inflammatory cells; 1 point, few scattered inflammatory cells; 2 points, perivascular inflammatory infiltrative tissue; and 3 points, extensive perivascular cuffs with extension into the adjacent parenchyma, or parenchymal infiltration without obvious cuffs. Spinal cord demyelination was scored as previously described24: 1, traces of subsurface demyelination; 2, marked subsurface and perivascular demyelination; 3, perivascular or subsurface fused demyelination; 4, extensive perivascular and subcutaneous demyelination involving half of the spinal cord with cellular infiltration into the parenchyma of the CNS; and 5, extensive perivascular and subcutaneous demyelination involving the entire portion of the spinal cord with cellular infiltrating into the parenchyma of the CNS.

2.6. Quantitative PCR

Spinal cord tissues were collected on day 24 post immunization. Total RNA was extracted from the spinal cord tissues using the Trizol method. Total RNA was reverse transcribed into cDNA using PrimerScript RT reagent kit with genomic DNA eraser (Takala Bio Inc, Japan) following the manufacturer’s instructions. Real-time quantitative PCR analysis was performed using SYBR green master mix on ABI ViiA7 instrument (Applied Biosystems, USA). The mRNA expression levels of IL-6, IL-17 and TNF-α were normalized to the level of β-Actin. The primer sequences used were as follows: GAPDH forward, 5′-ATGTGTCCGTCGTGGATCTGA-3′, GAPDH reverse, 5′-ATGCCTGCTTCACCACCTTCT-3′; TNF-α forward, 5′-AACCTCCTCTCTGCCGTCAAG-3′, TNF-α reverse, 5′-CCTCCCAGGTATATGGGCTCAT-3′; IL-17 forward, 5′-TGTCTCTGATGCTGTTGCT-3′, IL-17 reverse, 5′-GTTGACCTTCACATTCTGG-3′; IL-6 forward, 5′-TAGTCCTTCCTACCCCAATTTCC-3′, IL-6 reverse, 5′- TTGGTCCTTAGCCACTCCTTC-3′.

2.7. Lymphocyte proliferation assay

Lymphocyte proliferation assay was performed with slight modifications as previously described.25 Firstly, spleen tissues were isolated from C57BL/6 mice and homogenized in PBS solution. Splenocytes were filtered through a 70 μm cell strainer. Red blood cells were removed from splenocytes by lysing in a solution of Tris – HCl NH4Cl (pH 7.2). Then cells were washed with PBS solution and resuspended in RPMI-1640 medium containing 10% FBS and 1% penicillin & streptomycin to a concentration of 2 × 106 cells/mL. The cells were cultured in a 96-well plate and stimulated with mitogen ConA at 2.5 μg/mL, in the presence of inulin (400, 200, and 100 μg/mL), acetate, propionate, or butyrate (2, 1, 0.5, 0.25 mM, respectively). After incubation at 37°C in a 5% CO2 incubator for 48 h, the lymphocyte proliferation was detected using CCK-8 reagent. The absorbance was measured at 450 nm using a microplate absorbance reader (BIO-RAD, USA).

2.8. [3H]-thymidine incorporation assay

[3H]-thymidine incorporation assay was performed to evaluate the proliferation of MOG35-55 autoreactive lymphocytes proliferation ex vivo. Splenocytes of mice were prepared as described above and resuspended in complete RPMI-1640 medium to a concentration of 2 × 106 cells/mL. The cells were seeded into a 96-well plate and stimulated with 20 μg/mL MOG35-55 in the presence of inulin (400, 200, and 100 μg/mL) or butyrate (2, 1, 0.5 mM) for 48 h. During the final 10 h, cells were incubated with one microcurie of [3H]-thymidine, followed by collection on glass fiber filters. The incorporated [3H]-thymidine was measured using a MicroBeta Tri-lux liquid scintillation counter (Perkin-Elmer, USA).

2.9. Flow cytometry analysis

To detect Th17 cells and Th1 cells, splenocytes were isolated from mice in each group on day 10 post immunization as described above. CNS mononuclear cells were isolated on day 24 as following: brains from mice in each group were collected, homogenized, and digested by a digest solution containing collagenase D (2.5 mg/mL) and DNAase1 (500 μg/mL) for 25 min. Then, filtered through a 70 μm nylon cell strainers and centrifuged, cell pellets were purified using a 40/70% (vol/vol) discontinuous Percoll gradient to obtain mononuclear cells. The splenocytes or CNS mononuclear cells were treated with a cell stimulation cocktail containing PMA, ionomycin, and Golgiplug (eBioscience, USA) for 4 h. The stimulated cells were collected and incubated with anti-CD4 antibody and Fixable Viability Dye eFluor™ 780 for 30 min. The cells were then washed twice with PBS containing 1% FBS. After fixation in IC Fixation Buffer for 30 min, intracellular cytokines were stained with anti-IL-17A and anti-IFN-γ antibodies prepared in permeabilization buffer for 30 min.

To detect Treg cells, splenocytes were collected and incubated with anti-CD4 antibody, anti-CD25 antibody and Fixable Viability Dye eFluor™ 780 for 30 min. The cells were then washed twice with PBS containing 1% FBS. After fixation in Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent at room temperature for 30 min, intracellular cytokines were stained with anti-FOXP3 antibody prepared in permeabilization buffer for 30 min.

To analyze the proportion and function markers of dendritic cells, splenocytes were stained with Fixable Viability Dye eFluor™ 780, CD11c, CD86, and MHC-II for 30 min, then washed and resuspended with PBS containing 1% FBS.

The stained cells were washed twice with permeabilization buffer and resuspended in PBS for flow cytometry analysis, which was performed on a Attune NxT analysis cytometer (ThermoFisher), and the results were analyzed using FlowJo software. Dead cells were excluded by Fixable Viability Dye eFluor™ 780 staining, and live CD4+ T cells were gated to analyze the subsets of Th17, Th1 and Tregs.

2.10. T cell-DCs coculture

The coculture experiment was performed as described previously with slight modifications.26 In brief, 10 × 106 bone marrow cells/well were cultured in 6-well plates, and GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) were added to induce dendritic cells (DCs). On day 6, the trypsin-digested adherent cells were collected and seeded into 96-well plate, stimulated with 1 μg/mL LPS for 24 h to induce the maturation of DCs. Meanwhile, CD4+ T cells were sorted by magnetic separation on LS columns after labeling with CD4 (L3T4) microbeads according to the manufacturer’s instructions, and 0.5 μM CFSE was used for labeling at 37°C for 15 min. The obtained CFSE-labeled CD4+ T cells were added into the 96-well plate containing the pre-matured DCs, and co-cultured in the presence of 20 μg/mL MOG35-55 for 72 h. The cocultured cells were collected to detect the incorporation of CFSE in CD4+ T cells by flow cytometry.

2.11. Fecal bacteria DNA sequencing and bioinformatic analysis

Feces were collected under sterile conditions and promptly stored at −80°C. Bacterial genomic DNA was extracted from feces using QIAamp DNA stool mini kit (QIAGEN, Germany). For the initial microbiota study, DNA samples were amplified targeting the V1-V3 region of the bacterial 16S rRNA gene using forward primer 5′-AGAGTTTGATCCTGG-3′ and reverse primer 5′-TTACCGCGGCTGCT-3′. The amplified DNA samples were sequenced using a Roche 454 GSFLX+ sequencer. Bioinformatics analysis was performed with QIIME software (Quantitative Insights into Microbial Ecology). Chimeric sequences were detected and removed using the Pintail algorithm 49, and the sequences were de-noised and error-corrected with Acacia50. Operational taxonomic units (OTU) were picked at a 97% sequence identity using the uclust algorithm in QIIME. Taxonomies were assigned in QIIME using BLAST against the Greengenes database 51.27

2.12. Measurement of SCFAs

The content of SCFAs in mouse feces and serum was determined by gas chromatography with an Agilent 6890 N instrument. The standard solutions of acetic acid, propionic acid, butyric acid, and valeric acid at different concentrations were used to create standard curves for quantitative analysis. Precisely weighed 200 mg of dried feces or 0.1 mL serum was mixed with 1 mL distilled water. After centrifugation, the supernatant was collected and mixed with 200 μL of 50% sulfuric acid. The mixture was extracted by the addition of 1 mL of ether. After hand shaking for 30 times, the mixture was centrifuged and the ether layer was collected for injection. The chromatographic column used was HP-INNOWAX Polyethylene Glycol (30 m × 0.25 mm × 0.25 μm) (Agilent, The Netherlands), and high-purity nitrogen, at a flow rate of 1.0 mL/min, was used as carrier gas. The injection volume was 2 μL, and the splitting ratio was set to 3:1. The initial temperature of the column was maintained at 130°C for 16 min, then ramped up to 140°C at a rate of 5℃/min, and finally ramped up to 250°C at a rate of 30℃/min and maintained for 3 min.

2.13. Measurement of apoptosis of CD4+ T cells

Splenocytes of EAE mice were collected and resuspended in complete RPMI-1640 medium to a concentration of 2 × 106 cells/mL. The cells were seeded into a 96-well plate and stimulated with 20 μg/mL MOG35-55 in the presence of butyrate (2, 1, 0.5, 0.25 mM) for 48 h. Then, cells were collected and stained with anti-CD4 antibody and Annexin-V for 30 min at 4°C. After washed twice, the stained cells were analyzed using a Attune NxT analysis cytometer (ThermoFisher), and the results were analyzed using FlowJo software.

2.14. Fecal microbiota transplantation (FMT) assay

FMT was performed as reported previously with slight modifications.28 In brief, 6-week-old female donor mice (n = 6 per group) were oral administrated with inulin (200 mg/kg) or vehicle for 2 weeks. Then, stools were collected from the cecum and colon of donor mice. Stools from donor mice of each group were pooled together and 100 mg was resuspended in 1 mL of sterile saline. The solution was vigorously mixed and centrifugated at 800 g for 3 min. The supernatant was collected and stored at −80°C. EAE was induced in recipient mice, and the prepared supernatants from the stool of donor mice were oral administrated to the EAE mice once daily.

2.15. Antibiotic cocktails (ABX) treatment

Six-week-old female C57BL/6 mice (n = 6 per group) were administrated with a cocktail of antibiotics to deplete their gut microbiota. The antibiotic treatment consisted of a mixture of metronidazole, vancomycin, neomycin and ampicillin which were given in the drinking water for one week (antibiotics per 1 L of water: metronidazole 1 g, vancomycin 500 mg, neomycin 1 g, ampicillin 1 g). Additionally, an antibiotic mixture of 200 µL was administered through oral gavage every alternate day for another week. Then, mice were randomly divided into 5 groups: EAE+Vehicle group, EAE+ABX group, EAE+ABX+Inulin group, EAE+ABX+L. reuteri group, and EAE+ABX+ Inulin+L. reuteri group. Inulin or L. reuteri (109 CFU) administration was started from day 0 to day 24 post immunization.

2.16. Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 software. Comparisons between two groups were performed using the paired and unpaired Student’s t-test or Mann–Whitney test. Differences in α-diversity of microbiota between different groups were tested by one-way analysis of variance (ANOVA) followed by non-parametric Kruskal–Wallis test. The measurement data between different groups were compared using the one-way or two-way ANOVA with Bonferroni’s posthoc test. Data are representative of two/three independent experiments and are expressed as the mean ± SD. p values less than 0.05 was considered statistically significant.

3. Results and discussions

3.1. Inulin treatment alleviated the clinical symptoms and pathological changes of EAE mice

EAE was induced in female C57BL/6 mice through immunization with MOG35-55 emulsified in CFA. In terms of treatment regimen, oral administration of inulin (200 mg/kg) from the peak of disease (day 16 post-immunization) significantly reduced disease severity at day 24 post-immunization (p < 0.05, Figure 1a). In the pro-phylactic treatment regimen, inulin administration starting from day 0 postimmunization was even more effective in decreasing the clinical score of EAE mice (Figure 1b). Surprisingly, the incidence of EAE also decreased after inulin treatment, and by the time of sacrifice, all mice in the EAE+Vehicle group showed clinical signs (100% incidence), while 4 of the 12 mice in the EAE+Inulin group had no clinical signs (Figure 1c). Inflammatory demyelination is one of the most critical pathological manifestations of MS and EAE. The spinal cords of EAE mice treated with vehicle showed significant inflammatory cell infiltration and demyelination. In accordance with the reduced clinical severity, mice in the EAE+Inulin group had less inflammatory cell infiltration and demyelination in the spinal cord (Figure 1d,e). The pathological scores of inulin treated mice were significantly lower than vehicle treated mice (p = 0.022 for the infiltration score and p = 0.034 for the demyelination score, respectively) as shown in Figure 1f,g. Collectively, these results indicated inulin could effectively alleviate the severity and reduce the incidence of EAE in mice. Figure 1. Inulin treatment attenuated the pathologic changes of EAE mice.

EAE mice were oral administrated daily with vehicle or inulin (200mg/kg), and normal mice treated with vehicle as the Control group. (a) Clinical scores of mice starting treatment on day 16 post immunization, n = 8/group. (b) Clinical scores and (c) disease incidence of mice starting treatment from day 0 post immunization, n = 12/group. Data are representative of more than three independent experiments and presented as mean ± SD. On day 24, spinal cords from normal mice or EAE mice administrated with vehicle or inulin from day 0 were analyzed for (d) inflammation using H&E staining, scale bar = 200 or 50μm and (e) for degree of demyelination using LFB staining, scale bar = 200 or 50μm. Pathology scores of (f) inflammation and (g) demyelination. Data are representative of two independent experiments and presented as mean ± SD (n = 6). *p < 0.05; **p < 0.01 compared with EAE+Vehicle group.

3.2. Inulin decreased the pathogenic Th17 cell infiltration and inflammatory cytokine production in CNS of EAE mice

Th17 cells are a unique subtype of CD4+ T cells which is characterized by the expression of transcription factor ROR-γt and the production of IL-17A.29 It has been reported that MOG-specific Th17 cells not only cause CNS inflammation, by secreting IL-17A, but also directly interact with neuronal cells, leading to the induction of extensive axonal damage.30,31 In addition, Th1 cells are also enriched in EAE and contribute to CNS inflammation.32 Therefore, we analyzed the Th1 and Th17 cells in the CNS by flow cytometry with the gating strategy shown in Figure S1a. The percentages of Th17 and Th1 cells were highly elevated in EAE mice and the inulin treatment significantly decreased the proportion and numbers of Th17 cells (Figure 2a–c). Meanwhile, the frequency of Th1 cells was not changed but the infiltrated numbers of Th1 cells were decreased (Figure 2d–f). These results suggested that inulin is preferred to inhibit the infiltration and activation of Th17 cells in the CNS. Inflammatory cytokines such as IL-17A, IL-6 and TNF-α are considered to contribute to neuroinflammation and axonal damage.33 As shown in Figure 2g–i, the mRNA expressions of IL-17A, IL-6 and TNF-α in the spinal cords of inulin-treated mice were significantly decreased compared to untreated EAE mice (p < 0.05 or p < 0.01). These results indicated that the protective effect of inulin on the demyelination process was associated with the inhibition of Th17 cells and the production of inflammatory cytokines. Figure 2. Inulin treatment downregulated the proportion of Th17 cells and mRNA expression of inflammatory cytokines in CNS.

C57BL/6 mice were immunized with MOG33–35 emulsified in CFA and treated with vehicle or inulin from day 0 to day 24. (a) Representative graph of Th17 cells in brain tissues of mice from Control, EAE+Vehicle and EAE+Inulin groups at day 24 post immunization (n = 6/group). (b, c) Quantitative analysis of the percentages and numbers of Th17 cells (IL-17A+ CD4+). (d) Representative graph of Th1 cells in brain tissues of mice from Control, EAE+Vehicle and EAE+Inulin groups at day 24 post immunization (n = 6/group). (e, f) Quantitative analysis of the percentages and numbers of Th1 cells (ifn-γ+ CD4+). (g, h, i) Relative mRNA expression of IL-17A, IL-6, and tnf-α in spinal cord tissues of mice from the Control, EAE+Vehicle and EAE+Inulin groups at day 24 post immunization. Data are representative of three independent experiments and presented as mean ± SD; ns, no significance, *p < 0.05; **p < 0.005 compared to the EAE+Vehicle group.

3.3. Autoreactive T-cell proliferation and Th17 cell activation were suppressed in inulin-treated EAE mice

It is generally accepted that inflammatory demyelination occurred in the CNS is induced by myelin-specific T lymphocytes, which are activated in the periphery.7 To evaluate the inhibitory effect of inulin on systemic immune response in EAE mice, we firstly determined the serum levels of inflammatory cytokines IL17A, IL-6, IFN-γ, and TNF-α. The results demonstrated that the serum concentrations of these inflammatory cytokines except for IFN-γ were all downregulated in mice from the inulin treated group compared to mice from the EAE+Vehicle group (Figure 3a–d), suggesting inulin treatment might inhibit the systemic autoreactive immune response. Then, we isolated lymphocytes from mice in different groups, and cultured them with MOG35–55 to induce autoreactive proliferation. As shown in Figure 3e, there was no significant difference in lymphocyte proliferation between the EAE+Inulin and EAE+Vehicle groups in the absence of MOG35–55. However, after MOG35–55 stimulation, lymphocyte proliferation was significantly elevated in the EAE+Vehicle group. Although, increased lymphocyte proliferation had been observed in the inulin treated group, the level was significantly lower than that in the EAE+Vehicle group (Figure 3f). These results confirmed that the T lymphocytes mediated autoreactive responses were decreased in mice from the EAE+Inulin group. As decreased frequency of pathogenic Th17 cells was observed in the CNS of mice from the EAE+Inulin group, we further analyzed the changes of Th17 and Th1 cells in mice spleens. Consistently, inulin treatment resulted in a reduction of the frequency and numbers of Th17 cells in spleen of EAE mice (Figure 3g–i), did not affect the proportion but reduced the number of Th1 cells (Figure 3j–l), which is associated with the reduced number of total CD4+ T cells compared to vehicle treated EAE mice (Figure S1b-d). In addition, we detected the changes of Tregs, however, both the frequency and total numbers of Tregs were decreased, and the serum level of IL-10 secreted by Tregs was reduced in inulin treated EAE mice (Figure S1e-h). Dendritic cells are specialized antigen-presenting cells that express MHC-II and the co-stimulatory molecules to activate T cells. We found no change in the proportion of CD11c+ dendritic cells in spleen (Figure S 1i), but the mean fluorescence intensity (MFI) of MHC- II was decreased in inulin-treated EAE mice, while the MFI of CD86 was comparable between different groups (Figure S 1j,k), suggesting downregulated MHC-II expression of DCs may correlated with the diminished autoreactive T cell response in inulin treated EAE mice. Furthermore, CD4+ T cells from each group were sorted (with a purity higher than 95% determined by flow cytometry, data not shown), labeled with CFSE and cocultured with pre-matured dendritic cells in the presence of MOG35-55. The results indicated the proliferation ability of CD4+ T cells in EAE mice treated with inulin is lower than that of EAE mice treated with vehicle (Figure 3m,n). Taken together, these results indicated the peripheral autoreactive lymphocyte response and Th17 cell activation, in EAE mice, were diminished by inulin treatment. Figure 3. Inulin treatment inhibited systemic immune responses in EAE mice.

C57BL/6 mice were immunized with MOG33–35 emulsified in CFA and treated with vehicle or inulin. Mice were sacrificed, and serum were collected on day 24 post immunization, the concentrations of IL-17A (a), ifn-γ (B) IL-6 (c) and tnf-α (d) were determined by ELISA. (e) Splenocytes from mice in different groups were cultured for 48 h in vitro, and the proliferation was measured using the [3H]-thymidine incorporation assay. (f) Splenocytes from mice in different groups were stimulated with MOG35–55 (20μg/mL) for 48 h in vitro, and the proliferation was measured using the [3H]-thymidine incorporation assay. Data are representative of two independent experiments and presented as mean ± SD. Th1 and Th17 cells in spleen of mice from Control, EAE+Vehicle and EAE+Inulin groups at day 24 post immunization (n = 6/group). (G) Representative graph of Th17 cells (IL-17A+ CD4+), (h, i) Quantitative analysis of the percentages and numbers of Th17 cells. (j) Representative graph of Th1 cells (ifn-γ+ CD4+), (k, l) Quantitative analysis of the percentages and numbers of Th1 cells. (m) CD4+ T cells were sorted and incubated with 0.5 μM CFSE at 37°C for 15 min, after which they were co-cultured with DC cells in the presence of MOG at 20 μg/mL for 72 h. After coculture, the proliferation rate of CD4+ T cells was detected by flow cytometry. The representative graph of CFSE in gated CD4+ T cells.(n) Quantitative analysis of the proliferation percentages of CD4+ T cells. *p < 0.05 compared to the EAE group. Data are representative of two independent experiments and presented as mean ± SD (n = 3). ns, no significance. **p < 0.01 vs the EAE+Vehicle group.

3.4. Inulin treatment had a profound effect on the composition of gut microbiota

Gut microbiota has been proven to be correlated with the disease progression of EAE.34 We hypothesized that inulin, as a common prebiotic, might modulate the gut microbiota to exert its therapeutic effects on EAE mice. We performed 16S rRNA sequencing to compare the composition of gut microbiota among the Control, EAE+Vehicle and EAE+Inulin groups. Venn diagram analysis was performed to access the species richness across groups. It was observed the number of operational taxonomy units (OTUs) in the EAE+Inulin group was slightly decreased compared to that in the EAE+Vehicle group (Figure 4a). Next, we analyzed the community composition at various taxonomic levels. Compared to untreated EAE mice, inulin treatment resulted in increased richness of Firmicutes phylum and decreased relative abundance of Bacteroidota phylum (Figure 4b). Additionally, there was a significant increase in the abundance of Lactobacillus genus in the EAE+Inulin group (Figure 4c), and this increase was also observed in normal mice treated with inulin for 2 weeks (data not shown). Furthermore, based on the Chao1 index, Sobs index and Shannon index, there was no difference of α-diversity between the normal mice and the EAE mice. However, the α-diversity of EAE mice treated with inulin was significantly decreased compared to mice treated with vehicle (Figure 4d). This phenomenon may be related to the increased abundance of Lactobacillus by inulin treatment, which has been reported to produce soluble factors that limit the growth of other bacteria.35 To further compare the structural differences of microbiota composition, principal component analysis (PCA) was performed. Significant clustering variations were observed among the three groups (Figure 5a), indicating gut dysbiosis was appeared in EAE mice, and inulin treatment intended to restore the composition of gut bacteria. Then, the linear discriminant analysis effect size (LEfSe) assessment was used to identify high-dimensional biomarkers of gut microbiota in different groups. As shown in Figure 5b, at genus level, mice in the Control group exhibited a more diverse bacteria composition, including g__norank_f__Muribaculaceae, g_Alloprevotella, g_Blautia, g_Lachnospiraceae_NK4A136_group, and g_Tuzzerella. The featured bacteria in the mice of EAE+Vehicle group were g_Prevotellaceae_NK3B31_group, g_Parvibacter, g_Lachnospiraceae_UCG-006, g_Family_XIII_AD3011_group, and g_NK4A214_group. Meanwhile, g_Lactobacillus, g_Dubosiella, g_Rikenella, g_Bifidobacterium and g_Gordonibacter were enriched in the mice from the EAE+Inulin group (Figure 4b). Notably, Lactobacillus, Bifidobacterium and Dubosiella are well recognized probiotics,34 the relative abundance of Lactobacillus, Dubosiella and Prevotellaceae_NK3B31_group were shown in Figure 5c. Furthermore, the hypervariable regions of 16S gene allow distinction of different species in Lactobacillaceae.36 By aligning the sequences of Lactobacillus with the NCBI database (>99% similarity and 100% coverage), it was determined that the proportion of L. reuteri and L. gasseri at species level was higher in the EAE+Inulin group compared to the EAE+Vehicle group (Figure 5d). Figure 4. Inulin treatment modulated the composition of gut microbiota in EAE mice.

Stool samples were collected from mice in the Control, EAE and Inulin groups on day 24 post immunization (n = 6/group). (a) Venn diagrams illustrated the differences of OTU among groups. (b) Histogram of species distribution at the phylum level. (c) Histogram of species distribution at the genus level. (d) Alpha diversity analysis (Chao1 index, species richness, Shannon index) of microbial species in mice from the Control, EAE+Vehicle and EAE+Inulin groups. Data are representative of two or three independent experiments and presented as mean ± SD, and statistical significance was determined by non-parametric Kruskal–Wallis test. *p < 0.05; **p < 0.01 compared to the EAE+Vehicle group.

Figure 5. Inulin treatment altered gut microbiome composition and concentrations of SCFAs in EAE mice.

(a) Principal component analysis (PCA), at the genus level, for mice from the indicated groups. (b) The lineages with LDA values of 4.0 or higher, as determined by LEfSe, at the genus level. (c) The relative abundance of Lactobacillus, Dubosiella and Prevotellaceae_NK3B31_group. (d) The relative abundance of L. reuteri, L. gasseri and L. intestinalis. (e) The fecal levels of acetic acid, propionic acid, butyric acid. (f) The serum levels of acetic acid, propionic acid, butyric acid. Data are representative of two or three independent experiments and presented as mean ± SD, n = 6. ns, no significance, **p < 0.01, ***p < 0.001 compared to the EAE+Vehicle group.

3.5. Inulin elevated the concentrations of short chain fatty acids

Inulin cannot be digested by host digestive enzymes but can be fermented by gut microbiota to generate SCFAs, such as acetic acid, propionic acid and butyric acid.37 To assess the impact of inulin on SCFAs concentrations, we analyzed the concentrations of these SCFAs in feces and serum of mice using GC chromatography. The results revealed inulin treatment had little impact on acetic acid level, but significantly promoted the production of propionic acid and butyric acid in fecal content (Figure 5e). In addition, only serum concentration of butyric acid was elevated in the EAE+Inulin group compared to the EAE+Vehicle group, but there was no significant change in the serum concentration of other SCFAs (Figure 5f). These findings were consistent with the changes of gut microbiota post inulin treatment, which indicated that the administration of inulin altered the composition of gut microbiota and the concentrations of SCFAs in EAE mice.

3.6. SCFAs but not inulin inhibited autoreactive T lymphocyte activation

Inulin has been reported to have immunostimulatory activity on macrophages and T cells,38,39 and in this case, we hypothesized that the diminished autoimmune response in inulin-treated EAE mice was associated with changes in the gut microbiota and SCFAs. To clarify whether inulin itself or SCFAs exhibited immunosuppressive effects, we performed lymphocyte proliferation assay in vitro. We found inulin did not inhibit ConA-induced T-lymphocyte proliferation (Figure S2a), whereas propionate and butyrate could, dose-dependently, inhibit ConA-induced T-lymphocyte proliferation (Figure S2b). We further compared the inhibitory effects of inulin and butyrate on the proliferation of MOG35-55-induced autoreactive lymphocytes derived from the spleen of EAE mice. Once again, inulin treatment had no effect on the proliferation, while butyrate (0.5 mM and 1 mM) treatment significantly reduced the proliferation of autoreactive lymphocytes, (Figure 6c). In particularly, butyrate has been reported to inhibit CD4+ T cells by inducing CD4+ T cell apoptosis at high concentrations40 and affecting CD4+ T cell proliferation through histone deacetylation at low concentrations.41 We examined the effect of butyrate on CD4+ T cell apoptosis, and similar to the above findings, 2 mM butyrate induced CD4+ T cell apoptosis, 1 mM and 0.5 mM had no significant effect on apoptosis, while 0.25 mM inhibited apoptosis (Figure S2c). The inhibition of 0.5 mM and 1 mM butyrate on lymphocyte proliferation may not correlated with the apoptotic effect. These results suggested that inulin inhibited the autoimmune response, in EAE mice, mainly through modulating the gut bacteria composition and butyrate concentration. Figure 6. Gut microbiota and SCFAs contributed to the inhibition effects of inulin on autoimmune response and severity of EAE mice.

(a) Lymphocytes were isolated from the spleen of mice in the EAE+Vehicle group and treated with or without 20μg/mL MOG35-55 in the presence of inulin or butyrate. Proliferation was measured using [3H]-thymidine incorporation assay n = 4. Data are representative of two independent experiments and presented as mean ± SD. *p < 0.05, ***p < 0.001, ns (no significance) vs. MOG group. (b) Mice (n = 6/group) were immunized with MOG33–35 emulsified in CFA to induce EAE. Meanwhile, they were orally administrated with fecal contents collected from normal mice treated with vehicle (EAE+FMT Vehicle group) or inulin (EAE+FMT inulin group). Clinical scores were recorded until day 24 post immunization. **p < 0.01 compared to the EAE+FMT Vehicle group. (c) Representative graph of H&E staining and LFB staining, scale bar = 200μm. (d) Pathological scores of inflammation and demyelination are presented as mean ± SD (n = 6). *p < 0.05; **p < 0.01 compared to the EAE+FMT Vehicle group. (e) Representative graph of Th17 cells (IL-17A+ CD4+) in brain tissues of mice from the EAE+FMT Vehicle and EAE+FMT inulin group at day 24 post immunization (n = 6/group). (f) The percentages and numbers of Th17 cells. (g) Representative graph of Th1 cells (ifn-γ+ CD4+) in brain tissues of mice from the EAE+FMT Vehicle and EAE+FMT Inulin group at day 24 post immunization (n = 6/group).(h) The percentages and numbers of Th1 cells. Data are representative of three independent experiments and presented as mean ± SD. *p < 0.05; **p < 0.01; ns (no significance) compared to the EAE+FMT Vehicle group.

3.7. Fecal microbiota transplant (FMT) from inulin treated mice attenuated the pathological changes of EAE

Based on our results that short-chain fatty acids, but not inulin, inhibited autoreactive lymphocyte responses, we speculated that the fecal microbiota of inulin-treated mice might be effective in reducing the severity of EAE in mice. We collected fecal matter from mice treated with solvent control or inulin and transferred them by oral gavage to recipient C57BL/6 mice, which were subsequently immunized with MOG35–55 to induce EAE. We found that FMT from inulin-treated mice reduced the severity of EAE in recipient mice compared to FMT from vehicle-treated mice (Figure 6b), and the disease incidence was also reduced (67% in EAE+FMT Inulin group vs 100% in EAE+FMT Vehicle group, data not shown). In addition, the inflammation and demyelination in spinal cord and the pathological scores of H&E and LFB staining were decreased in the EAE+FMT Inulin group (Figure 6c,d). Importantly, the frequency and numbers of Th17 cells in the CNS were reduced in the EAE+FMT Inulin group, and the numbers of Th1 cells were also decreased but the proportion was not changed (Figure 6e–h). These results indicated a similar effect of FMT from inulin on EAE mice compared to inulin treatment, which confirmed that the modulation of gut microbiota and SCFAs contributed to the beneficial effects of inulin on EAE mice.

3.8. Antibiotic treatment abolished the beneficial effects of inulin on EAE mice

Antibiotics cocktail treatment (ABX) to clear the gut microbiota could facilitate the determination of a causal relationship between the changes in the gut microbiota and the therapeutic effects of drugs.42 Since we observed an increased abundance of L. reuteri in the EAE+Inulin group, which is one of the Lactobacillus species widely distributed in the gastrointestinal tract of mammals, and a synbiotic formulation of L. reuteri and inulin alleviates ASD-like behaviors in a mouse model.43 Therefore, we performed ABX experiment to evaluate the impact of ABX and L. reuteri supplementation on the amelioration effect of inulin in EAE mice. The results showed that the alleviative effect of inulin on EAE mice largely disappeared after antibiotic clearance, and the pathologic changes were comparable between mice treated with inulin or vehicle. Interestingly, administration with L. reuteri could reduce the clinical scores, disease incidence and pathological changes of ABX treated EAE mice, and the simultaneous treatment with L. reuteri and inulin was even more effective, reducing the disease incidence to 16.6% (Figure 7a–f). In addition, Th17 cells in the CNS were not changed in EAE+ABX+Inulin group, but significantly decreased in EAE+ABX+L. reuteri group and EAE+ABX+ Inulin+L. reuteri group compared to EAE+ABX group (Figure 7e–h). Collectively, these results further confirmed the alteration of gut microbiota and SCFAs contributes to the therapeutic effects of inulin on EAE mice. Figure 7. Antibiotics treatment abolished the alleviative effects of inulin on EAE mice.

Mice (n = 6/group) were treated with antibiotics cocktail for 2 weeks to deplete the gut microbiota, thereafter, ABX treated mice were immunized with MOG33–35 emulsified in CFA to induce EAE, and divided into 5 groups: EAE+Vehicle group, EAE+ABX group, EAE+ABX+Inulin group, EAE+ABX+L. reuteri group, and EAE+ABX+ Inulin+L. reuteri group. Vehicle, inulin or L. reuteri (109 CFU) administration was started from day 0 to day 24 post immunization. (a) Clinical scores. (b) Disease incidence. (c) H&E staining, scale bar = 200μm. (d) LFB staining, scale bar=200μm. (e) Pathological scores of inflammation. (f) Pathological scores of demyelination. (g) Representative graph of Th17 cells (IL-17A+ CD4+) in brain tissues of mice from different groups (n = 6/group). (h) The percentages and numbers of Th17 cells. Data are representative of two independent experiments and presented as mean ± SD. ns, no significance, *p < 0.05; **p < 0.01 compared to the ABX+Vehicle group.

4. Discussion and conclusion

The benefits of inulin as a widely used prebiotic for metabolic diseases have been well investigated, and we have previously reported that inulin improves symptoms in collagen induced arthritis mice.22 In this study, we found that inulin administration started at either day 16 or day 0 post-immunization could attenuate inflammatory demyelination in EAE mice, associated with decreased infiltration of Th17 cells in the CNS and reduced proportion of Th17 cells in the spleen. Ex vivo study indicated the autoreactive immune response of lymphocytes to MOG35-55 was inhibited by inulin treatment, and this inhibitory effect was attributed to the alteration of gut microbiota and increased concentrations of SCFAs which confirmed by FMT and ABX experiments. Importantly, these studies suggested that inulin-induced changes in intestinal microecology could suppress the systemic autoimmune response and improve inflammatory demyelinating lesions in the CNS.

T-cell mediated immune responses occurring in the periphery, especially Th17 cell activation, play a vital role in the inflammatory demyelinating lesions of CNS.8 Th17 cells can be detected in the spinal cord of MS patients and their secretion of IL-17 is thought to be associated with the disruption of blood–brain barrier, the activation of microglia, and the macrophages induced inflammation in the CNS.44 Clinically, a proof-of-concept study provides evidence that a monoclonal antibody targeting IL-17A (secukinumab) may reduce MRI lesion activity in MS. Meanwhile, the treatment efficacy of fingolimod (FTY-720) or dimethyl fumarate on MS patients are believed to be related to the reduction of Th17 response.45 In our study, oral administration of inulin reduced the proportion and numbers of Th17 cells in spleen and brain tissues of EAE mice. Th1 cells are also known as pro-inflammatory cells that can exacerbate neuroinflammation by secreting IFN-γ.46 Natural compounds such as Eriocalyxin B and Kirenol ameliorate EAE by suppressing both Th1 and Th17 cells.47,48 Interestingly, inulin administration does not reduce the proportion of Th1 cells but decrease the numbers of Th1 cells in the brain and spleen, indicating the differentiation of Th17 cells but not Th1 cells was inhibited. This is in accordance with the finding that inulin treatment boosts Th1 immunity and IFN-γ production during the murine whipworm Trichuris muris infection.49 These results suggest that the improvement of inflammatory demyelination symptoms, by inulin administration, might be primarily owing to its systemic inhibition of Th17 cells.

The gut microbiota composition is altered in EAE mice, and this alteration is associated with Th17 cell responses.14 Thus, intervention of gut microbiota composition is expected to inhibit the activation of autoimmune response and improve the symptoms of MS and EAE. For instance, intermittent fasting confers protective effects on EAE by altering the composition and metabolic pathways of gut microbiota.50 In addition, the supplementation of probiotics also ameliorates neuroinflammation in EAE mice by modulating the balance of CD4+ T cell subsets.51,52 However, the availability, convenience, and cost might limit the clinical application of intermittent fasting or probiotics for MS patients. On the other hand, inulin, as an intervention for modulating gut microbiota, has the advantage in terms of tolerability, low cost and easy to manufacture. Our study found differences of gut microbiota composition between EAE mice and normal mice. The administration of inulin changed the gut microbiota composition of EAE mice, which was featured by increased abundance of Lactobacillus, Dubosiella, Bifidobacterium and decreased abundance of g_Prevotellaceae_NK3B31_group at genus level. Lactobacillus is commonly used as a probiotic due to its beneficial effects on a variety of diseases including EAE. Oral administration of Lactobacillis containing myelin antigens, induces tolerance in EAE models,53 and the enrichment of the intestinal microbiota with Lactobacillis is associated with resistance to EAE.54 Similarly, the improvement of EAE by intermittent fasting is associated with increased abundance of L. johnsonii and L. reuteri, which exhibit immunomodulatory functions.50 Importantly, L. reuteri can enhances the fermentation of inulin and promote butyrate production in vitro batch fermentation systems.55 And supplementation of L. reuteri can increase the cecal butyrate levels in house dust mite (HDM)-treated mice.56 Moreover, inulin administration also increased the abundance of Dubosilla, which also had positive correlation with the levels of SCFAs.57 As reported previously, butyrate can inhibit the differentiation of Th17 cells, and thus, reduce colitis in mice.58 In addition, butyrate can also inhibit the antigen presentation ability of DCs.59 We found that inulin significantly increased butyrate concentration in EAE mice while decreasing the activation of Th17 cells and DCs. Indeed, in vitro assay demonstrated that butyrate inhibited ConA or MOG35-55 induced lymphocyte proliferation. In conclusion, the downregulated autoimmune reaction, in EAE mice treated with inulin, probably was associated with increased probiotics and the content of butyrate.

Fecal bacteria transplantation is a reliable method for establishing a causal relationship between gut microbiota and disease progression. Hsin-Chih Lai et al., have reported that the aqueous extract from fecal samples of mice treated with Ganoderma lucidum (WEGL) has weight loss effect on obese mice, confirming the anti-obesity effect of WEGL is achieved by modulating gut microbiota.28 Similarly, the fecal contents from inulin treated normal mice did have an ameliorating effect on EAE mice. The critical role of gut microbiota is further confirmed by ABX experiment, in which inulin could not inhibit the Th17 cells and alleviate the disease severity in antibiotics treated EAE mice. In contrast, L. reuteri added back in ABX-treated mice restored the protection effect of inulin in EAE mice, suggesting that ameliorative effect of inulin is reliant on the presence of Lactobacillus. In summary, the current results from EAE mice indicated that intervention of gut microbiota and metabolic pathways, by inulin, contributed to the inhibition of systemic autoimmune response and protection against inflammatory demyelination occurred in the CNS.

There are several limitations in this study. Firstly, we observed reductions in IL-17A levels and the frequency of Th17 cells in EAE mice treated with inulin, while γδ T cells and group 3 innate lymphoid (ILC3) cells are also a source of IL-17A, it is not clear whether these cells are altered after inulin administration.60,61 Secondly, although significant elevation of L. reuteri have been observed in EAE mice treated with inulin, it remains unclear how it utilizes inulin and affects the growth of other bacteria. In the future, we will explore the in-depth mechanisms by which L. reuteri regulates the production of short-chain fatty acids in EAE mice treated with inulin. Thirdly, L. reuteri derived from the microbiota of genetically distinct wild-derived PWD/PhJ (PWD) mice has been reported to increase host susceptibility to CNS autoimmunity,62 whereas another study demonstrated that L. reuteri (DSM 17,938) reduced the severity of EAE in mice,63 which is consistent with our findings. These conflicting results suggest that different sources of L. reuteri may exert distinct roles in the development of EAE, and further studies are needed to elucidate the mechanisms by which L. reuteri exerts opposite effects on the CNS autoimmunity. Nevertheless, our study provides valuable evidence that inulin ameliorates the CNS inflammation through the modulation of gut ecosystem and Th17 cells in EAE mice.

In conclusion, the findings from our mouse model of MS indicated that administration of inulin exhibited promising amelioration effects on inflammatory demyelination in CNS. These findings provide evidence that inulin or L. reuteri may be beneficial to MS patients.

Supplementary Material

Supplemental Material

Acknowledgments

The authors would like to thank Jieqiong Chen for providing language help.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors contributions

L.N., H.XY. and R.M. performed the experiments, analyzed data, prepared figures and wrote the manuscript; H.F., Y.L. and X.TH participated in experiments and analyzed data; W.H., W.H. and S.SS. revised the manuscript and performed formal analysis; W.YJ., W.XJ. and W.SC. contributed to the study concept, design, and revised the manuscript.

Data availability statement

All data included in this study are available upon request by contact with the corresponding author.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2024.2402547
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