D-mannose induces regulatory T cells and suppresses immunopathology

Abstract

D-mannose, a C-2 epimer of glucose, exists naturally in many plants and fruits, and is found in human blood at concentrations less than one-fiftieth of that of glucose. However, although the roles of glucose in T cell metabolism, diabetes and obesity are well characterized, the function of D-mannose in T cell immune responses remains unknown. Here we show that supraphysiological levels of D-mannose safely achievable by drinking-water supplementation suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation, and increased the proportion of Foxp3⁺ regulatory T cells (T_reg cells) in mice. In vitro, D-mannose stimulated T_reg cell differentiation in human and mouse cells by promoting TGF-β activation, which in turn was mediated by upregulation of integrin α_vβ₈ and reactive oxygen species generated by increased fatty acid oxidation. This previously unrecognized immunoregulatory function of D-mannose may have clinical applications for immunopathology.

Figure 1

d-mannose induces T_reg cell differentiation in vitro and in vivo. (a–d) We cultured CD4⁺CD25⁻ (naive) T cells from spleen and peripheral lymph nodes of C57BL/6 mice with anti-CD3 and anti-CD28 for up to 3 d in 10% FBS glucose-free DMEM complete medium (CTRL) or with added 25 mM mannose (Man) or other sugars (Fru, fructose; Glu, glucose; Gal, galactose). We measured proliferation by labeling with CFSE dye. (a) Representative FACS plots gated on unproliferated cells among CD4⁺ T cells cultured for 24, 48 or 72 h. Values in the upper left corners are mean ± s.d. Bars indicate the gate of unproliferated cells. (b,c) Representative FACS plots (b) and the frequency of CD25⁺Foxp3⁺ T_reg cells among CD4⁺ T cells (c, top) after 3 d in culture. (c, bottom) Absolute numbers of CD25⁺Foxp3⁺ T_reg cells. In the FACS plots, numbers adjacent to outlines indicate the percentage of cells in the gate. (d) Foxp3 mRNA expression at 24 h. (e) The ratio of CD4⁺CD25⁺GFP⁺(Foxp3⁺) T_reg cells among CD4⁺CD25⁻GFP⁻(Foxp3⁻) T cells from spleens and peripheral lymph nodes of transgenic mice expressing Foxp3–GFP cultured with anti-CD3 and anti-CD28 in 25 mM mannose-supplemented DMEM or control medium for 3 d. (f) The frequency of CD25⁺Foxp3⁺ T_reg cells among naive T cells from spleens and lymph nodes of C57BL/6 mice after 3 d of culture with soluble anti-CD3 plus APCs. (g) KJ1–26⁺CD4⁺CD25⁻ naive T cells from spleens and lymph nodes of DO11.10 TCR-transgenic Rag2^−/− mice were cultured with OVAp_323–339 plus APCs. The plot shows the frequency of KJ1–26⁺CD25⁺Foxp3⁺ T_reg cells after 4 d of culture. (h) KJ1–26⁺CD4⁺CD25⁻ naive T cells from spleens and lymph nodes of DO11.10 TCR-transgenic Rag2^−/− mice were adoptively transferred into BALB/cJ mice. The plot shows the frequency of KJ1–26⁺CD4⁺Foxp3⁺ T_reg cells among adoptively transferred cells in the indicated organs after 5 d of ovalbumin gavage (n = 3). PP, Peyer’s patches; MLN, mesenteric lymph nodes; LPL, lamina propria. Data are presented as mean ± s.d. In box plots (c–g), center lines indicate the median, box limits represent the upper and lower quartiles, and whiskers extend to the minimum and maximum values. Data were analyzed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test (c,h) or unpaired two-tailed Student’s t-test (d–g). *P < 0.05, **P < 0.01. Data are pooled from three (e,g) or five (c,d,f) experiments or are representative of three (a,h) or five (b) independent experiments.

Figure 2

d-mannose induces T_reg cell differentiation via activation of TGF-β. (a,b) CD4⁺CD25⁻ (naive) T cells from spleens and peripheral lymph nodes of C57BL/6 mice were cultured with anti-CD3 and anti-CD28, as well as latent TGF-β1, anti-TGF-β or SB431542 (an inhibitor of TGF-β receptor). “Med” indicates TCR stimulation only. (a) Foxp3 mRNA expression at 24 h. (b) The frequency of CD25⁺Foxp3⁺ T_reg cells among CD4⁺ T cells after 3 d of culture. (c,d) Naive CD4⁺ T cells deficient in TβRI (c) or TβRII (d) from spleens and lymph nodes of Tgfbr1^f/fCD4-Cre or tamoxifen-treated Tgfbr2^f/fER-Cre (Tamoxifen) mice were cultured with anti-CD3 and anti-CD28, with or without latent TGF-β1. Tamoxifen was dissolved with sunflower oil (Oil). The plots show the frequency of CD25⁺Foxp3⁺ T_reg cells after 3 d of culture. (e) KJ1–26⁺CD4⁺CD25⁻ naive T cells from spleens and lymph nodes of DO11.10 TCR-transgenic Rag2^−/− mice were adoptively transferred into BALB/cJ mice. The plot shows the frequency of KJ1–26⁺CD4⁺Foxp3⁺ T_reg cells among adoptively transferred cells in the indicated organs after 5 d of ovalbumin gavage (n = 5). Data are presented as mean ± s.d. (f) Naive CD4⁺ T cells purified from human peripheral blood mononuclear cells were cultured with anti-CD3, anti-CD28 and IL-2, with or without anti-TGF-β and SB431542. The plot shows the frequency of CD25^hiFoxp3⁺ T_reg cells after 4 d of culture. (g) Naive CD4⁺ T cells from spleens and lymph nodes of C57BL/6 mice were cultured with anti-CD3 and anti-CD28, with or without latent TGF-β1 or anti-TGF-β plus SB431542. The plot shows the frequency of CD25⁺Foxp3⁺ T_reg cells after 3 d of culture. (h) Naive CD4⁺ T cells from spleens and lymph nodes of C57BL/6 mice were cultured with anti-CD3 and anti-CD28 plus different doses of latent TGF-β1. The plot shows the frequency of CD25⁺Foxp3⁺ T_reg cells after 3 d of culture (mean ± s.d.). In box plots (a–d,f,g), center lines indicate the median, limits represent the upper and lower quartiles, and whiskers extend to the minimum and maximum values. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (a–g) or by unpaired two-tailed Student’s t-test (h). **P < 0.01. Data are pooled from five (a–d,f,g) or three (h) experiments or are representative of two (e) independent experiments.

Figure 3

Integrin α_vβ₈ and ROS are required for d-mannose-mediated TGF-β1 activation and T_reg cell generation. (a,b) CD4⁺CD25⁻ (naive) T cells from spleens and peripheral lymph nodes of C57BL/6 mice were cultured with anti-CD3 and anti-CD28. The plots show relative Itgb8 (a) and Itgav (b) mRNA expression at 24 h. (c) CD4⁺CD25⁻ (naive) T cells from spleens and peripheral lymph nodes of control or Itgb8^f/fCD4-Cre mice were cultured with or without latent TGF-β1 or NAC. The plot shows the frequency of CD25⁺Foxp3⁺ T_reg cells after 3 d of culture. (d) CD4⁺CD25⁻ (naive) T cells from spleens and peripheral lymph nodes of C57BL/6 mice were cultured with anti-CD3 and anti-CD28. The plot shows ROS expression after 24 h of culture. In box plots (a–c), center lines indicate the median, limits represent the upper and lower quartiles, and whiskers extend to the minimum and maximum values. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (c) or by unpaired two-tailed Student’s t-test (a,b). *P < 0.05, **P < 0.01. Data are pooled from three to five (a–c) experiments or are representative of three (d) independent experiments.

Figure 4

d-mannose suppresses type 1 diabetes in NOD mice. (a–c) Female NOD mice were supplemented with d-mannose in drinking water from 7.5 weeks of age and were subsequently checked for the development of type 1 diabetes. (a) The frequency of mice without type 1 diabetes in the indicated groups over time (n = 20). (b) Representative histology sections of pancreas from the mice in a. White arrows indicate pancreatic islands. (c) The frequency of islets with grade X insulitis in the indicated groups. The stages refer to diabetes progression (also applies to j). (d–g) Female NOD mice were treated with d-mannose in drinking water daily from 7.5 weeks of age, and were euthanized when the mice were 14 weeks old. The plots in d–f show the frequencies of CD25⁺Foxp3⁺ T_reg cells (d), IFN-γ⁺CD4⁺ T cells (e) and IFN-γ⁺CD8⁺ T cells (f) in the spleens and DLNs of the mice. (g) Splenocytes from the NOD mice were cultured with GAD65 peptide (1 μg/ml). The plot shows the concentration of IFN-γ in the culture medium after 3 d, as determined by ELISA. (h–k) NOD mice with blood glucose levels between 140 and 160 mg/dL were supplemented with d-mannose in drinking water and then monitored for the progression of diabetes. (h) The levels of blood glucose in individual mice from the indicated groups over time (n = 7–8). (i) Representative histology sections of pancreas from the mice in h. White arrows indicate pancreatic islands. (j) The frequency of islets with grade X insulitis in the indicated groups. (k) The frequencies of CD25⁺Foxp3⁺ T_reg cells, CD4⁺IFN-γ⁺ T cells, and CD8⁺IFN-γ⁺ T cells in the pancreases of NOD mice. Summary data are presented as mean ± s.e.m. (d–f,g,k). *P < 0.05, **P < 0.01, Mantel–Cox log-rank test (a) or unpaired two-tailed Student’s t-test (d–h,k). Data are pooled from two (h,j) or four (a,c) experiments or are representative of two (g,i,k) or four (b,d–f) independent experiments.

Figure 5

T_reg cells and TGF-β are involved in d-mannose-mediated suppression of autoimmune diabetes in NOD mice. (a–f) Female NOD mice were supplemented with d-mannose in drinking water from 7.5 weeks of age, injected with anti-CD25 or isotype-control antibody twice at 13–14 weeks of age and then checked for the development of type 1 diabetes. (a) The frequency of diabetes-free mice in the indicated groups over time (n = 10). (b) Representative histology sections of pancreas from the mice in a. White arrows indicate pancreatic islands. (c) The frequency of islets with grade X insulitis in the indicated groups. Stages refer to diabetes progression (also applies to h). (d–f) The frequency of CD25⁺Foxp3⁺ T_reg cells (d), the ratio of CD25⁺Foxp3⁺ T_reg cells to IFN-γ⁺CD4⁺ T cells (e), and the ratio of CD25⁺Foxp3⁺ T_reg cells to IFN-γ⁺CD8⁺ T cells (f) in the spleens and DLNs of female NOD mice. Data in d–f are presented as mean ± s.e.m. (g,h) Female NOD mice were supplemented with mannose in drinking water from 7.5 weeks of age, injected with anti-TGF-β or isotype-control antibody once a week for 6 weeks and then checked for the development of type 1 diabetes. (g) The frequency of diabetes-free mice in the indicated groups over time (n = 10). (h) The frequency of islets with grade X insulitis in the indicated groups. *P < 0.05, **P < 0.01, ***P < 0.001, Mantel–Cox log-rank test (a,g) or one-way ANOVA with Tukey’s post hoc test (d–f). Data are pooled from two (a,c,g,h) experiments or are representative of two (b,d–f) independent experiments.

Figure 6

d-mannose induces antigen-specific T_reg cells and suppresses ovalbumin-induced airway inflammation in BALB/cJ mice. (a–f) Disease suppression by d-mannose treatment in BALB/cJ mice with ovalbumin-induced airway inflammation. (a) Representative lung histology sections with periodic acid Schiff staining. Insets show magnified views (10× magnification) of the outlined regions. (b) Collated inflammation scores. (c) Absolute numbers of polymorphonuclear neutrophils (PMN), basophils (Bas), eosinophils (Eos), macrophages (Mac) and lymphocytes (Lymph) in bronchoalveolar lavage fluid of BALB/cJ mice after intranasal challenge with ovalbumin. (d–f) Frequencies of IL-4⁺ T cells (d), IL-13⁺ T cells (e) and CD25⁺Foxp3⁺ T_reg cells (f) among KJ1–26⁺CD4⁺ T cells in BALB/cJ mice after induction of airway inflammation. (g–l) Disease amelioration by d-mannose treatment in BALB/cJ mice with ovalbumin-induced airway inflammation. (g) Representative lung histology sections with periodic acid Schiff staining. Insets show magnified views (10× magnification) of the outlined regions. (h) Collated inflammation scores. (i) Absolute numbers of polymorphonuclear neutrophils, basophils, eosinophils, macrophages and lymphocytes (abbreviated as in c) in bronchoalveolar lavage fluid of BALB/cJ mice after intranasal challenge with ovalbumin. (j–l) Frequencies of IL-4⁺ T cells (j), IL-13⁺ T cells (k) and CD25⁺Foxp3⁺ T_reg cells (l) among KJ1–26⁺CD4⁺ T cells in BALB/cJ mice. Summary data (b–f,h–l) are presented as mean ± s.e.m.; data points represent individual mice (n = 4 mice per group). *P < 0.05, **P < 0.01, unpaired two-tailed Student’s t-test. All data are representative of two independent experiments.