Promotion of regulatory T cell induction by immunomodulatory herbal medicine licorice and its two constituents

Ao Guo, Dongming He, Hong-Bo Xu, Chang-An Geng, Jian Zhao, Ao Guo, Dongming He, Hong-Bo Xu, Chang-An Geng, Jian Zhao

Abstract

Regulatory T cells (Treg) play a critical role to control immune responses and to prevent autoimmunity, thus selective increase of Treg cells in vivo has broad therapeutic implications for autoimmune and inflammatory diseases. Licorice is a well-known herbal medicine used worldwide for over thousands of years, and accumulating evidence has shown its immunomodulatory potential. However, it is not clear whether licorice could regulate the induction and function of Treg cells. Here we found licorice extract could promote Treg cell induction, and then we used a rational approach to isolate its functional fractions and constituents. The results showed that two constituents, isoliquiritigenin and naringenin, promoted Treg cell induction both in vitro and in vivo. The effective fractions and two constituents of licorice also enhanced immune suppression of Treg cells, and they further reduced severity of DSS-induced colitis in mice. This study suggested that promotion of regulatory T cell induction could be an underlying mechanism of the historically and widely used herbal medicine licorice, providing its two effective molecules against autoimmune and inflammatory diseases.

Figures

Figure 1. Licorice extract and its fractions…
Figure 1. Licorice extract and its fractions promote Treg cell induction in vitro.
(a) Naive CD4+ T cells were stimulated with immobilized anti-CD3, soluble anti-CD28 monoclonal antibodies and TGFβ (1 μg/ml) under the indicated concentrations of licorice extract and analyzed by FACS. (b) Naive CD4+ T cell were stimulated as in (a) under the indicated licorice fractions and analyzed by FACS. (c) Analysis of Foxp3 protein expression of a per-cell basis in Treg cells generated in the presence of licorice extract or Gly1 fraction treatment. Data are shown as mean fluorescence intensity (MFI). (d,e) CD4+CD25+ Treg cells untreated or treated with Gly1 were incubated with CFSE labelled CD4+CD25− conventional T cells in an in vitro suppression assay. The suppression was assayed by FACS analysis for dilution of CFSE in gated conventional T cells. Results are expressed as means ± SEM and are representative of more than three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by one-way ANOVA followed by Bonferroni’s test.
Figure 2. Licorice extract and its active…
Figure 2. Licorice extract and its active fraction Gly1 promote Treg cells in vivo.
(a) C57BL/6 mice were orally administrated with licorice or water, Foxp3+CD4+ Treg cells in peripheral blood were monitored every three days (n = 5 mice per group). (b) Analysis of Foxp3 protein expression of a per-cell basis in (a). Data are shown as mean fluorescence intensity (MFI). (c–f) C57BL/6 mice were orally administrated with licorice (c) or Gly1 fraction (d), Foxp3+CD4+ Treg cells in colonic lamina propria and spleen were analyzed after two weeks (n = 5 mice per group). (e) Quantification of the result in (c) (n = 5 mice per group). (f) Quantification of the result in (d). (g,h) Mice were treated with Gly1 fraction and induced colitis using dextran sulfate sodium, and the body weight (g) and colon shortness (h) were analyzed (n = 6 mice per group). Results are expressed as means ± SEM and are representative of three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by Man-Whitney U test (e,f), one-way ANOVA followed by Bonferroni’s test (b), or two-way ANOVA (a,g).
Figure 3. Isoliquiritigenin and naringenin are two…
Figure 3. Isoliquiritigenin and naringenin are two active constituents of licorice to promote Treg induction and function.
(a) Structures of four major constituents from Gly1 fraction. (b) Naive CD4+ T cells were stimulated with Treg-inducing conditions in the absence or presence of isoliquiritigenin, naringenin, licoricidin, liquiritigenin. Foxp3+CD4+ Treg cells were analyzed by FACS. (c) CD4+CD25+ Treg cells untreated or treated with isoliquiritigenin were incubated with CFSE labelled CD4+CD25− conventional T cells. The suppression was assayed by FACS analysis for dilution of CFSE in gated Tconv cells. (d) Quantification of the result in (c). (e–g) C57BL/6 mice were orally administrated with isoliquiritigenin or naringenin for two weeks, colonic lamina propria Treg cells (e,f) and peripheral blood Treg cells (g) were monitored (n = 5 mice per group). Results are expressed as means ± SEM and are representative of more than three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by One-way ANOVA followed by Bonferroni’s test (b,f,g), or two-way ANOVA (d).
Figure 4. Isoliquiritigenin and naringenin attenuate DSS…
Figure 4. Isoliquiritigenin and naringenin attenuate DSS induced IBD.
C57BL/6 mice were given 2.5% (w/v) DSS in drinking water for 6 days. Isoliquiritigenin and naringenin were orally administrated every day, starting 7 days prior to the DSS treatment (n = 5 mice for each group). (a,b) Body weight of isoliquiritigenin (a) or naringenin (b) treated mice with the DSS-induced colitis. (c,d) Colon length of mice treated with isoliquiritigenin or naringenin and control mice. (e) FACS profile of colonic lamina propria Treg, Th17 and Th1 cells isolated from mice treated with isoliquiritigenin, naringenin or water. Results are expressed as means ± SEM and are representative of more than three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by one-way ANOVA followed by Bonferroni’s test (d,e), or two-way ANOVA (a,b).
Figure 5. Isoliquiritigenin reduces Akt-mTOR signaling pathway…
Figure 5. Isoliquiritigenin reduces Akt-mTOR signaling pathway activity.
(a) Immunoblotting of phosphorylation P70 S6 kinase and total P70 S6 kinase in CD4+ T cells under Treg-inducing conditions and treated with isoliquiritigenin or naringenin for indicated times. (b) Quantitative PCR analyses of gene expressions in CD4+ T cells under Treg-inducing conditions treated with isoliquiritigenin or naringenin. (c) Immunoblot of phosphorylation Akt and Erk in CD4+ T cells under Treg-inducing conditions and treated with isoliquiritigenin or naringenin for indicated times. (d) Immunoblotting of phosphorylation Akt and Erk in CD4+ T cells under Treg-inducing conditions and treated with licorice or Gly1 fraction for indicated times. (e) Quantitative PCR analyses of Cyp1a expression in CD4+ T cells treated with licorice, Gly1 fraction, isoliquiritigenin or naringenin. The immunoblot in (a,c,d) were run under the same experimental conditions. Cropped blots were shown in (a,c,d) and the full-length blots were presented in Supplementary Figure 10. Results are expressed as means ± SEM and are representative of more than three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by one-way ANOVA followed by Bonferroni’s test (b,e).

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