Glyteer, Soybean Tar, Impairs IL-4/Stat6 Signaling in Murine Bone Marrow-Derived Dendritic Cells: The Basis of Its Therapeutic Effect on Atopic Dermatitis

Abstract

Atopic dermatitis (AD) is a common inflammatory skin disease. Recent studies have revealed the involvement of T helper (Th)2 cytokines including Interleukin 4 (IL-4) in the pathogenesis of AD. Since epidermal Langerhans cells (LCs) and dermal myeloid dendritic cells (DCs) produce CCL17 and CCL22 that chemoattract Th2 cells, interfering with CCL17 and CCL22 production from LCs and dermal myeloid DCs may be beneficial in the treatment of AD. To investigate this, we stimulated murine bone marrow-derived DCs (BMDCs) with IL-4. IL-4 stimulation produced Ccl17 and Ccl22, which was attenuated by soybean tar Glyteer, a known aryl hydrocarbon receptor (Ahr) activator. Notably, Glyteer treatment blocked the nuclear translocation of Stat6 induced by IL-4 stimulation, suggesting that this treatment impairs the IL-4/Stat6 signaling pathway in BMDCs. Unexpectedly, Glyteer treatment did not potently upregulate the expression of Cyp1a1, a specific Ahr-responsive gene, suggesting that its inhibitory machinery for Ccl17 and Ccl22 expression is likely to operate in an Ahr-independent manner. These findings indicate that Glyteer may exhibit therapeutic potential for AD by downregulating the CCL17 and CCL22 production from DCs in a Th2-deviated microenvironment.

Keywords: Ccl17; Ccl22; aryl hydrocarbon receptor; atopic dermatitis; dendritic cell.

Figure 1

IL-4 stimulation induced Ccl17 and Ccl22 production in bone marrow-derived dendritic cells (BMDCs). (a–d) Data are expressed as mean ± standard error of the mean (S.E.M.); n = 3 for each group; * p < 0.05. Expression of Ccl17 (a) and Ccl22 (e) in BMDCs stimulated with IL-4 (0.1, 1 and 10 ng/mL) for 24 h and production of Ccl17 (b) and Ccl22 (f) in the culture supernatant were measured. Expression of Ccl17 (c) and Ccl22 (g) in BMDCs stimulated with IL-4 (10 ng/mL) for 1, 3, 6 and 24 h and production of Ccl17 (d) and Ccl22 (h) in the culture supernatant were measured.

Figure 2

Glyteer treatment inhibited IL-4-induced Ccl17 and Ccl22 production in BMDCs. (a–f) Data are expressed as mean ± S.E.M.; n = 3 for each group; * p < 0.05. IL-4-simulated BMDCs were treated with Glyteer or 6-formylindolo[3.2-b]carbazole (FICZ) at the indicated dose for 24 h. Expression of Ccl17 (a) and Ccl22 (c) in BMDCs was analyzed by Quantitative Reverse Transcription (qRT)-PCR. Production of Ccl17 (b) and Ccl22 (d) in culture supernatant was measured by ELISA. Expression of Cyp1a1 in BMDCs treated with Glyteer (e) or FICZ (f) was analyzed by qRT-PCR.

Figure 3

Glyteer treatment inhibited IL-4-induced Ccl17 and Ccl22 production in an aryl hydrocarbon receptor (Ahr)-independent manner in BMDCs. (a–d) Data are expressed as mean ± S.E.M.; n = 3 for each group; * p < 0.05. IL-4-stimulated BMDCs were treated with Glyteer in the absence or presence of CH223191 for 24 h. Glyteer and CH223191 were administered at the same timing. Expression of Ccl17 (a) and Ccl22 (c) in BMDCs was analyzed by qRT-PCR. Production of Ccl17 (b) and Ccl22 (d) in culture supernatant was measured by ELISA.

Figure 4

Glyteer treatment inhibited nuclear translocation of Stat6 induced by IL-4 stimulation without affecting the phosphorylation of Stat6 in BMDCs. (a) BMDCs treated with Glyteer for 24 h were stimulated with IL-4 (10 ng/mL) for 10, 20 and 30 min and then total protein or nuclear protein of the BMDCs was extracted; (b) BMDCs treated with Glyteer for 24 h were stimulated with IL-4 (10 ng/mL) for 1, 2 and 3 h and then total protein or nuclear protein of the BMDCs was extracted. Expression of phosphorylated Stat6 (pStat6) and Stat6 was analyzed by western blotting using anti-pStat6 antibodies. Histone H3 served as an internal loading control. The data are representative of experiments repeated three times with similar results.