Oral Administration of 4-Hydroxy-3-Methoxycinnamaldehyde Attenuates Atopic Dermatitis by Inhibiting T Cell and Keratinocyte Activation

Abstract

Atopic dermatitis (AD) is a skin condition caused by an imbalance of distinct subsets of T helper cells. Previously, we showed that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) inhibits T cell activation but does not induce apoptosis. Here, we examined the mechanism underlying the inhibitory effect of 4H3MC on AD both in vivo and in vitro. We sought to test the pharmacological effects of 4H3MC using a mouse model of 2, 4-'2,4-dinitrocholorobenzene' (DNCB)- and mite-induced AD. Also, we determined whether 4H3MC affects T cell differentiation and proliferation. Oral administration of 4H3MC attenuated the symptoms of DNCB- and mite-induced AD, including increased ear thickness, serum IgE levels, immune cell infiltration into inflammatory lesions, and pathogenic cytokine expression in ear tissues. In vitro, 4H3MC blocked T cell differentiation into Th1 and Th2 subtypes, as reflected by suppression of T-bet and GATA3, which are key transcription factors involved in T cell differentiation. In addition, 4H3MC downregulated T cell proliferation during Th1 and Th2 differentiation and keratinocyte activation. Collectively, these findings suggest that 4H3MC ameliorates AD symptoms by modulating the functions of effector T cells and keratinocytes.

Fig 1. Oral delivery of 4H3MC ameliorates the symptoms of atopic dermatitis in mice.

(A) A schematic diagram showing the induction and treatment of atopic dermatitis (AD). (B) Representative pictures of mouse ears on Day 28 (n = 3–6/group). Con, control mice; 4H3MC, control mice receiving 4H3MC; AD, AD mice; AD+4H3MC, AD mice receiving 4H3MC. (C) Ear thickness during the course of AD. (D) Levels of serum IgE and mite-specific IgE in mice were measured by ELISA. Blood samples were collected by cardiac puncture at Day 28 post-induction. Data are expressed as the mean ± SEM. *P < 0.05, versus the AD control group.

Fig 2. 4H3MC reduces tissue inflammation and infiltration of immune cells in AD mice.

Microphotographs of left ear sections isolated at Day 28 post-AD induction and stained with (A) hematoxylin and eosin (H&E), (C) toluidine blue, and (F) an anti-CD4 antibody. Original magnification, ×200. White bar, 10 μm (F). (B) Epidermal and dermal thickness was measured in H&E-stained microphotographs. (D) Infiltrating mast cells were counted after toluidine blue staining. (E) mRNA levels of mast cell markers (CD117, FCER1G and CCR2) in the ear tissues at Day 28 were analyzed by real-time PCR. (G) Infiltrating CD4⁺ T cells were counted after staining with anti-CD4 antibodies. Data are expressed as the mean ± SEM (n = 3–6/group). *P < 0.05, versus the AD control group.

Fig 3. 4H3MC inhibits the production of pro-inflammatory cytokines in ear tissues of AD mice.

Expression of cytokines in left ear tissues from each group was measured by real-time RT-PCR. mRNA was isolated at Day 28 post-AD induction. Data are expressed as the mean ± SEM (n = 3–6/group). *P < 0.05, versus the AD control group.

Fig 4. 4H3MC induces morphological changes in immunological organs and down-regulates the production of pro-inflammatory cytokines in dLNs and spleens (but not in non-dLNs).

(A) Weight and size of dLNs, non-dLNs, and spleens from each mouse were measured at Day 28 post-AD induction. (B) The number of total cells and CD4⁺ T cells in the dLNs was counted. (C) CD4, CD19 and CD11c mRNA in the dLNs was analyzed. (D) and (E) Expression of genes encoding pro-inflammatory cytokines in CD4⁺ T cells from (D) dLNs and (E) non-dLNs. On Day 28, dLNs and non-dLNs were removed and CD4⁺ T cells isolated by MACS. After isolation of total RNA, expression of cytokine genes was measured by real-time RT-PCR. Data are expressed as the mean ± SEM (n = 3–6/group). *P < 0.05, versus the AD control group.

Fig 5. 4H3MC inhibits T cells both before and after differentiation.

(A–D) CD4⁺ T cells were isolated from normal C57BL/6 mice and differentiated in vitro, as described in Materials and Methods, in the absence or presence of 4H3MC. On Day 5 of culture (A) Th1-polarized cells or (C) Th2-polarized cells were collected and total RNA isolated. Expression of (A) T-bet and (C) GATA3 mRNA was measured by real-time RT-PCR. (B) and (D) On Day 5 of culture (B) Th1-polarized cells and (D) Th2-polarized cells were treated with PMA/A23187 for 4 h. Two hours before cell harvest, brefeldin A was added to the cultures. Production of IFN-γ (B) or IL-4 (D) was measured by FACS analysis. (E) and (F) CD4⁺ T cells were isolated from normal mice and differentiated. On Day 5 of culture, differentiated Th1 (E) cells or Th2 (F) cells (1 × 10⁶/sample) were pre-incubated with 4H3MC (10 μM) for 30 min and stimulated with anti-CD3/CD28 antibodies (left) or PMA/A23187 (right) for 24 h. The levels of IFN-γ (E) or IL-4 (F) were then measured by ELISA. Data are expressed as the mean ± SD from three independent experiments. *P < 0.05, versus the cells not treated with 4H3MC.

Fig 6. 4H3MC attenuates T cell proliferation during T cell differentiation.

(A) CD4⁺ T cells from LNs and spleens were differentiated into Th1- or Th2-polarized cells, as described in Materials and Methods, in the absence or presence of 4H3MC (10 μM) for 5 days. On Day 5, cells were harvested and total cell numbers measured. (B) CD4⁺ T cells from LNs and spleens were labeled with CFSE (10 μM) for 30 min and differentiated in the absence or presence of 4H3MC. After 72 h of culture, cells were harvested and the CFSE intensity measured by flow cytometry. 0 h indicates naïve CD4⁺ T cells. The numbers above 72 h population indicates the number of cell division. Data are representative of three independent experiments. Data are expressed as the mean ± SD from three independent experiments. *P < 0.05, versus the cell not treated with 4H3MC.

Fig 7. 4H3MC reduces both the production of pro-inflammatory cytokines and PKC activity in keratinocytes.

(A) HaCaT (1 × 10⁶) cells were pretreated with 4H3MC (0–20 μM) for 30 min and activated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for 3 hr and then harvested mRNA from the cells. The expression of mRNA for pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and TSLP) was measured by conventional RT-PCR. (B) HaCaT (1 × 10⁶) cells were pretreated with 4H3MC (10 μM) for 30 min and activated with TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for the indicated times (0–6 h) and then harvested mRNA from the cells. Expression of mRNA for pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and TSLP) was measured by conventional RT-PCR. Data are representative of three independent experiments. (C) HaCaT cells (1 × 10⁶) were lysed and the cytosolic fraction was incubated with various concentrations of 4H3MC (0, 2, 10, and 20 μM) for 30 min. The cell lysate was then treated with PMA (100 nM) for 3 min and 2.5 μg protein from the cytosolic fraction was used to measure PKC activity. Dara are expressed as the mean ± SD from three independent experiments. *P < 0.05, versus the mock-treated cells (A), or versus treatment with 4H3MC (B) and (C).