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. 2020 Oct;41(10):1337-1347.
doi: 10.1038/s41401-020-0435-1. Epub 2020 Jun 18.

Free fatty acid receptor 4 (FFA4) activation ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis by increasing regulatory T cells in mice

So-Eun Son  1 , Soo-Jin Park  1 , Jung-Min Koh  2 , Dong-Soon Im  3   4
Affiliations

Free fatty acid receptor 4 (FFA4) activation ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis by increasing regulatory T cells in mice

So-Eun Son et al. Acta Pharmacol Sin. 2020 Oct.

Abstract

High dose intake of docosahexaenoic acid showed beneficial effects on atopic dermatitis in patients and was found to increase regulatory T cells in mice, but its molecular target has not been identified. Free fatty acid receptor 4 (FFA4, also known as GPR120) is a receptor sensing polyunsaturated long-chain fatty acids including docosahexaenoic acid. In the present study, we examined whether FFA4 acted as a therapeutic target of docosahexaenoic acid for treating atopic dermatitis. Experimental atopic dermatitis was induced in mice by 2,4-dinitrochlorobenzene (DNCB) sensitization on day 0, followed by repeated DNCB challenges from D7 to D48. The mice were treated with a selective agonist compound A (30 mg· kg-1· d-1, ip) from D19 to D48, and sacrificed on D49. We found that DNCB-induced atopic dermatitis-like skin lesions, i.e. hypertrophy and mast cell infiltration in skin tissues, as well as markedly elevated serum IgE levels. Administration of compound A significantly suppressed the atopic responses in ears and lymph nodes, such as hypertrophy and mast cell infiltration in the ears, enlarged sizes of lymph nodes, and elevated serum IgE and levels of cytokines IL-4, IL-13, IL-17, and IFN-γ in ear tissue. The therapeutic effects of compound A were abolished by FFA4 knockout. Similarly, increased CD4+Foxp3+ regulatory T-cell population in lymph nodes was observed in wide-type mice treated with compound A, but not seen in FFA4-deficient mice. In conclusion, we demonstrate that activation of FFA4 ameliorates atopic dermatitis by increasing CD4+Foxp3+ regulatory T cells, suggesting FFA4 as a therapeutic target for atopic dermatitis.

Keywords: 2,4-dinitrochlorobenzene; FFA4 agonist; Omega-3; Treg cells; atopic dermatitis; free fatty acid receptor 4; polyunsaturated fatty acids; skin.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Experimental protocol for the induction of AD and the effect of compound A on DNCB-induced AD responses in the ears of FFA4 WT mice.
a Experimental protocol. A murine model of DNCB‐induced AD was established through DNCB sensitization on day 0 as well as through repeated DNCB challenges on days 7–49 to induce AD. FFA4 WT mice were treated with either PBS or DNCB, and compound A (an FFA4 agonist) was administered by i.p. injection 30 min before DNCB challenge. b Macroscopic view of the ears. c H&E staining of ear tissue sections. d Toluidine blue staining of ear samples. e The number of mast cells (dark violet‐stained cells) was counted using a light microscope. f Blood was collected on day 49 of the experiment. Serum levels of IgE were measured by ELISA. The results are presented as the means ± SEM (n = 5). ***P < 0.001 vs. the vehicle-treated group, ###P < 0.001, ##P < 0.01 vs. the DNCB-treated group.
Fig. 2
Fig. 2. Effect of compound A on the expression levels of the cytokines of Th2, Th1, and Th17 cells in the ear tissues of DNCB-induced AD FFA4 WT mice.
Analysis of the mRNA expression of Th2 (IL-4 and IL-13), Th1 (IFN-γ), and Th17 (IL-17A) cytokines in ear tissues from the DNCB-induced and compound A-treated FFA4 WT groups. a–d The relative mRNA levels of cytokines were quantified by determining the ratios of their levels to GAPDH transcript levels. e Representative RT-PCR images are shown. The results are presented as the means ± SEM (n = 5). ***P < 0.001 vs. the vehicle-treated group, ###P < 0.001, ##P < 0.01, #P < 0.05 vs. the DNCB-treated group.
Fig. 3
Fig. 3. Effect of compound A on DNCB-induced AD in the lymph nodes of FFA4 WT mice.
a The cervical lymph nodes were photographed to record morphological changes. b Weights of lymph nodes that were measured. c–f RT-PCR analyses of Th2 cytokines (IL-4 and IL-13), Th17 cytokines (IL-17A), and Th1 cytokines (INF-γ) were performed using mRNA isolated from cervical lymph node tissues. mRNA levels were also expressed as the ratios versus the levels of GAPDH mRNA. The results are presented as the means ± SEM (n = 5). ***P < 0.001, *P < 0.05 vs. the vehicle-treated group, ###P < 0.001, ##P < 0.01 vs. the DNCB-treated group.
Fig. 4
Fig. 4. Effect of compound A on the generation of CD4+Foxp3+ T cells in the lymph nodes of FFA4 WT mice.
a Lymph nodes of FFA4 WT mice with DNCB-induced AD were labeled with rat anti-Foxp3 (green), and counterstaining was conducted using DAPI (blue). b FACS analysis of CD4+Foxp3+ T cells. c Representative flow cytometry dot plots. The results are presented as the means ± SEM (n = 5). #P < 0.05 vs. the DNCB-treated group.
Fig. 5
Fig. 5. Effect of compound A on AD in the ears of FFA4 KO mice.
A murine model of DNCB‐induced AD was generated in FFA4 KO mice by DNCB sensitization on day 0 as well as through repeated DNCB challenges on days 7–49 to induce AD. FFA4 KO mice were treated with either PBS or DNCB, and compound A (an FFA4 agonist) was administered by i.p. injection 30 min before DNCB challenge. a Macroscopic view of the ears. b H&E staining of ear tissue sections. c Toluidine blue staining of ear samples. d The number of mast cells (dark violet‐stained cells) was counted using a light microscope. e Blood was collected on day 49 of the experiment. The serum levels of IgE were measured by ELISA. The results are presented as the means ± SEM (n = 5). ***P < 0.001 vs. the vehicle-treated group.
Fig. 6
Fig. 6. Effect of compound A on the expression levels of Th2, Th1, and Th17 cytokines in the ear tissues of DNCB-induced AD FFA4 KO mice.
Analysis of the mRNA expression of Th2 (IL-4 and IL-13), Th1 (IFN-γ), and Th17 (IL-17A) cytokines in ear tissues from the DNCB-induced and compound A-treated FFA4 KO mice groups. a–d Relative mRNA levels of cytokines were quantified by determining the ratios of their levels to GAPDH transcript levels. e Representative RT-PCR images are shown. The results are presented as the means ± SEM (n = 5). ***P < 0.001, **P < 0.01 vs. the vehicle-treated group.
Fig. 7
Fig. 7. Effect of compound A on DNCB-induced AD in the lymph nodes of FFA4 KO mice.
a The cervical lymph nodes were photographed to record morphological changes. b The weights of the lymph nodes were measured. c–f RT-PCR analyses of Th2 cytokines (IL-4 and IL-13), Th17 cytokines (IL-17A), and Th1 cytokines (INF-γ) were performed using mRNA isolated from cervical lymph node tissues. mRNA levels were also expressed as ratios versus the level of GAPDH mRNA. The results are presented as the means ± SEM (n = 5). ***P < 0.001, *P < 0.05 vs. the vehicle-treated group.
Fig. 8
Fig. 8. Effect of compound A on the generation of CD4+Foxp3+ T cells in the lymph nodes of FFA4 KO mice.
a The lymph nodes of FFA4 WT mice with DNCB-induced AD were labeled with rat anti-Foxp3 (green), and counterstaining was conducted using DAPI (blue). b FACS analysis of CD4+Foxp3+ T cells. The results are presented as the means ± SEM (n = 5). c Representative flow cytometry dot plots.
Fig. 9
Fig. 9. Compound A-induced activation of FFA4 in macrophages leads to M2 polarizarion.
M2 polarized macrophages induces generation of CD4+FoxP3+ Treg cells in lymph nodes. Treg cells suppress inflammatory cytokine production, resulting in reduced lymph node size, suppressed serum IgE levels, and suppressed hypertrophy of epidermis.
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