Tannic acid, a higher galloylated pentagalloylglucose, suppresses antigen-specific IgE production by inhibiting ɛ germline transcription induced by STAT6 activation

Abstract

Interleukin (IL)-4 is a critical stimulator that induces ɛ germline transcripts (ɛGT) for switch recombination to initiate immunoglobulin (Ig) E and is important in allergic disease pathogenesis. We found pentagalloylglucose (PGG) inhibited IL-4-induced ɛGT expression. PGG exerted its inhibitory function by suppressing IL-4-induced activation of IL-4Rα, JAK3 and STAT6. Furthermore, tannic acid, a higher galloylated PGG, attenuated ovalbumin-induced IgE production in vivo by inhibiting IL-4-induced ɛGT expression and the IL-4 signaling pathway. In conclusion, our results suggest that tannic acid may attenuate allergic diseases by suppressing IgE production by inhibiting IL-4-induced signaling.

Keywords: IFN-γ, interferon-gamma; IL, interleukin; IgE; IgE, immunoglobulin E; JAK, Janus kinase; OVA, ovalbumin; PGG, 1,2,3,4,6-penta-O-galloyl-β-d-glucose; Pentagalloylglucose; STAT, signal transducer and activator of transcription; Signal transducers and activators of transcription6; TGF-β, transforming growth factor-beta; Tannic acid; ɛ Germline transcript; ɛGT, ɛ germline transcript.

Graphical abstract

Fig. 1

PGG inhibits IL-4-induced ɛGT expression. (A) The structure of galloyl group, PGG, digallic acid and tannic acid. (B) DND39 cells were treated with PGG (1, 10, 25 μM) or TGF-β (2 ng/ml, positive control) in the presence of IL-4 (250 U/ml) for 48 h, and then assessed for the expression of ɛGT by RT-PCR followed by Southern hybridization. Each experiment was repeated three times in triplicate.

Fig. 2

PGG suppresses IL-4-induced STAT6 activation by inhibiting the IL-4 signal pathway. DND39 cells were stimulated with IL-4 (250 U/ml) in the presence of 25 μM of PGG for 10 min or 30 min. Lysates were then immunoprecipitated with anti-JAK3, anti-IL-4Rα, or anti-STAT6 antibodies and analyzed by western blotting using anti-phosphotyrosine antibody (4G10) for phosphorylated JAK3 and IL-4Rα or anti-phosphotyrosine antibody (PY20) for phosphorylated STAT6. Each experiment was repeated three times in triplicate.

Fig. 3

PGG specifically inhibits IL-4 and IL-13 signaling, but not IFN-γ signaling. (A) DND39 cells were treated with IFN-γ (400 ng/ml) and PGG for 30 min. Lysates were then immunoprecipitated with anti-STAT1 antibody and analyzed by western blotting using an anti-pTyr-STAT1 antibody. (B) NIH-3T3 cells were treated with IL-13 (10 ng/ml) and PGG (50 μM) for 30 min. Lysates were then immunoprecipitated with anti-STAT6 antibody and analyzed by western blotting using an anti-phosphotyrosine antibody (PY20). Each experiment was repeated three times in triplicate.

Fig. 4

Tannic acid inhibits IL-4-induced ɛGT expression by suppressing the IL-4 signaling pathway. (A) DND39 cells were treated with tannic acid (1, 10 μg/ml) or TGF-β (2 ng/ml) in the presence of IL-4 (250 U/ml) for 48 h, and then assessed for the expression of ɛGT by RT-PCR followed by Southern hybridization. (B) Cells were treated with tannic acid (1, 10 μg/ml) in the presence of IL-4 (250 U/ml) for 30 min. (C) Cells were stimulated with IL-4 (250 U/ml) in the presence of tannic acid (1, 10 μg/ml) for 10 min. Lysates were then immunoprecipitated with an anti-STAT6, anti-JAK3 and anti-IL-4Rα antibodies and analyzed by western blotting using anti-phosphotyrosine antibody (4G10) for phosphorylated JAK3 and IL-4Rα or anti-phosphotyrosine antibody (PY20) for phosphorylated STAT6. Each experiment was replicated three times with triplicate.

Fig. 5

Tannic acid inhibits antigen-specific IgE production in ovalbumin-treated mice. Female, 6-week-old C57BL/6J mice were divided into two groups of 5 mice each. Mice were administered water or water containing 4 mg/ml tannic acid ad libitum for 17 days. Mice were intraperitoneally injected with 50 μg/ml OVA with aluminum hydroxide on day 3 to induce specific IgE responses. At 1 month after administration, the levels of total (A) and OVA-specific (B) IgE, IgM and IgG were measured in serum by ELISA. Statistical analysis was performed using unpaired t-test. Mean values were significantly different from the control group: ^*p < 0.05 (n = 5). n.s.: not significant.