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Link to original content: https://pubmed.ncbi.nlm.nih.gov/21965673
N-acetylglucosamine inhibits T-helper 1 (Th1)/T-helper 17 (Th17) cell responses and treats experimental autoimmune encephalomyelitis - PubMed Skip to main page content
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. 2011 Nov 18;286(46):40133-41.
doi: 10.1074/jbc.M111.277814. Epub 2011 Sep 29.

N-acetylglucosamine inhibits T-helper 1 (Th1)/T-helper 17 (Th17) cell responses and treats experimental autoimmune encephalomyelitis

Ani Grigorian  1 , Lindsey Araujo, Nandita N Naidu, Dylan J Place, Biswa Choudhury, Michael Demetriou
Affiliations

N-acetylglucosamine inhibits T-helper 1 (Th1)/T-helper 17 (Th17) cell responses and treats experimental autoimmune encephalomyelitis

Ani Grigorian et al. J Biol Chem. .

Abstract

Current treatments and emerging oral therapies for multiple sclerosis (MS) are limited by effectiveness, cost, and/or toxicity. Genetic and environmental factors that alter the branching of Asn (N)-linked glycans result in T cell hyperactivity, promote spontaneous inflammatory demyelination and neurodegeneration in mice, and converge to regulate the risk of MS. The sugar N-acetylglucosamine (GlcNAc) enhances N-glycan branching and inhibits T cell activity and adoptive transfer experimental autoimmune encephalomyelitis (EAE). Here, we report that oral GlcNAc inhibits T-helper 1 (Th1) and T-helper 17 (Th17) responses and attenuates the clinical severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE when administered after disease onset. Oral GlcNAc increased expression of branched N-glycans in T cells in vivo as shown by high pH anion exchange chromatography, MALDI-TOF mass spectroscopy and FACS analysis with the plant lectin l-phytohemagglutinin. Initiating oral GlcNAc treatment on the second day of clinical disease inhibited MOG-induced EAE as well as secretion of interferon-γ, tumor necrosis factor-α, interleukin-17, and interleukin-22. In the more severe 2D2 T cell receptor transgenic EAE model, oral GlcNAc initiated after disease onset also inhibits clinical disease, except for those with rapid lethal progression. These data suggest that oral GlcNAc may provide an inexpensive and nontoxic oral therapeutic agent for MS that directly targets an underlying molecular mechanism causal of disease.

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Figures

FIGURE 1.
FIGURE 1.
Regulation of GlcNAc-branched N-glycan biosynthesis by the hexosamine and N-glycan pathways. UDP-GlcNAc is required by the N-acetylglucosaminyltransferases Mgat1, 2, 4, and 5 and iGnT. Cytosolic UDP-GlcNAc enters the Golgi via antiporter exchange with Golgi UMP, a reaction product of the N-acetylglucosaminyltransferases. Galectins bind N-acetyllactosamine, with avidity increasing in proportion to the number of N-acetyllactosamine units (i.e. branching). β1,6-GlcNAc branching by Mgat5 promotes poly-N-acetyllactosamine production, further enhancing avidity for galectins; although poly-N-acetyllactosamine extension is possible on all branches. GalT3, galactosyltransferase 3; NAGK, N-acetylglucosamine kinase; GFAT, glutamine fructose-6-phosphate amidotransferase.
FIGURE 2.
FIGURE 2.
MALDI-TOF mass spectrum and HPAEC profile of N-glycans. N-Glycans were isolated from purified CD3+ T cells from age- and sex-matched wild-type control mice or mice treated orally with GlcNAc for 7 days and analyzed by MALDI-TOF mass spectrometry (A–D) or HPAEC (E and F). Oral GlcNAc was administered by supplementing the drinking water at 0.25 mg/ml, with intake confirmed by measuring the amount of drinking water consumed. 6 mice were used per group, and the harvested CD3+ T cells were pooled prior to analyses. The assignment of likely structures in the MALDI-TOF spectrum is based on mass and those in HPAEC on N-glycan standards from RNase B, IgG, and fetuin and correlations with the major structures observed in the MALDI-TOF spectrum. Definitive structure assignment of the latter requires HPAEC-mass spectrometry in-line as well as linkage analysis. Abbreviations for individual sugars are defined in Fig. 1.
FIGURE 3.
FIGURE 3.
Oral GlcNAc treatment inhibits CD25 expression and proinflammatory cytokine production. A, splenocytes were isolated from mice and cultured in vitro with the indicated concentrations of GlcNAc under resting or activated (1000 ng/ml anti-CD3) conditions for 3 days, stained with l-PHA-FITC in triplicate, and analyzed by FACS. B–D, mice were immunized with MOG 35-55 peptide emulsified in Complete Freund's adjuvant and treated orally with GlcNAc at the same time by supplementing the drinking water at 0.25 mg/ml. After 14 days, splenocytes harvested from mice treated with GlcNAc had reduced CD25+ T cells upon restimulation with MOG 35-55 peptide in culture (B and C) and promoted development of CD4+CD25+FoxP3+ regulatory T cells (D). The reduction in CD25+ T cell blasts at the highest antigen concentrations is likely secondary to the observed increase in cell death, consistent with antigen-induced cell death at high antigen stimulation levels. E, oral GlcNAc treatment in vivo also inhibited secretion of Th1 cytokines (IFN-γ and TNF-α) and Th17 cytokines (IL-17 and IL-22) upon restimulation with MOG 35-55 peptide in vitro. 3 mice were used per group, and the harvested CD3+ T cells were pooled within each group prior to analyses. p values in C were determined by t test. *, p < 0.01. Error bars represent the means ± S.E. of duplicate or greater values unless otherwise stated.
FIGURE 4.
FIGURE 4.
Oral GlcNAc treatment attenuates the clinical course of EAE. A, EAE was induced in C57BL/6 mice by immunization with MOG 35-55 peptide emulsified in Complete Freund's adjuvant and pertussis toxin. Mice were treated orally with GlcNAc by supplementing the drinking water at 0.25 mg/ml starting on the second day after disease onset and continued for the duration of the study (n = 9 per control group, n = 7 per GlcNAc group). Day 1 indicates the first day of disease onset. Mice were examined daily for clinical signs of EAE over the next 30 days with the observer blinded to treatment conditions and scored daily as follows: 0, no disease; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb weakness or paralysis and hindlimb paralysis; 5, moribund or dead. Mean clinical scores per group daily were compared by the Mann-Whitney U test. B, GlcNAc treatment increased N-glycan branching in T cells as seen by l-PHA staining in representative mice taken at the peak of disease. The results are representative of at least three mice compared from each group. C, a significant reduction in CD25+ T cells was observed in representative GlcNAc-treated mice taken at the peak of disease compared with control mice. The results are representative of at least three mice compared from each group. D, in vivo treatment with GlcNAc inhibited production of proinflammatory cytokines IFN-γ, TNF-α, IL-17, and IL-22 upon restimulation with MOG 35-55 peptide in vitro. p values in D were determined by t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent the means ± S.E. of duplicate or greater values unless otherwise stated.
FIGURE 5.
FIGURE 5.
Oral GlcNAc treatment after disease onset attenuates the clinical course of EAE in moderate-severe disease, but not disease with rapid lethal progression. A and B, EAE was induced in 2D2 TCR transgenic mice by immunization with MOG 35-55 peptide emulsified in Complete Freund's adjuvant and pertussis toxin. Mice were treated orally with GlcNAc by supplementing the drinking water at 0.25 mg/ml starting on the second day after disease onset and continued for the duration of the study (n = 10 per control group, n = 8 per GlcNAc group). Day 1 indicates the first day of disease onset. Mice were examined daily for clinical signs of EAE over the next 40 days with the observer blinded to treatment conditions and scored daily as follows: 0, no disease; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb weakness or paralysis and hindlimb paralysis; 5, moribund or dead. Mice that scored ≤4 were included in the moderate-severe disease group (A), and mice with scores of 5 (i.e. died within the first 40 days after disease onset) (B) were included in the lethal disease group. Mean clinical scores per group daily (A and B) were compared by the Mann-Whitney U test. C, 2 representative mice from each group of the 2D2 TCR transgenic EAE experiment were used to assess expression of branched N-glycans. Splenocytes were harvested from the immunized mice and stained with anti-CD4 and l-PHA lectin in triplicate and analyzed by FACS. p values in C were determined by t test. Error bars represent the means ± S.E. of triplicate or greater values unless otherwise stated.
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