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Research ArticleDermatologyInflammation
Open Access | 
10.1172/JCI156501
1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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Peng, G.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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1Atopy (Allergy) Research Center and
2Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
3Juntendo Itch Research Center, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
4Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
5Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
6Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
Address correspondence to: François Niyonsaba, Atopy (Allergy) Research Center and Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81.3.5802.1591; Email: francois@juntendo.ac.jp.
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Published July 14, 2022 - More info
Published in Volume 132, Issue 17 on September 1, 2022
AbstractHuman β-defensin-3 (hBD-3) exhibits antimicrobial and immunomodulatory activities; however, its contribution to autophagy regulation remains unclear, and the role of autophagy in the regulation of the epidermal barrier in atopic dermatitis (AD) is poorly understood. Here, keratinocyte autophagy was restrained in the skin lesions of patients with AD and murine models of AD. Interestingly, hBD-3 alleviated the IL-4– and IL-13–mediated impairment of the tight junction (TJ) barrier through keratinocyte autophagy activation, which involved aryl hydrocarbon receptor (AhR) signaling. While autophagy deficiency impaired the epidermal barrier and exacerbated inflammation, hBD-3 attenuated skin inflammation and enhanced the TJ barrier in AD. Importantly, hBD-3–mediated improvement of the TJ barrier was abolished in autophagy-deficient AD mice and in AhR-suppressed AD mice, suggesting a role for hBD-3–mediated autophagy in the regulation of the epidermal barrier and inflammation in AD. Thus, autophagy contributes to the pathogenesis of AD, and hBD-3 could be used for therapeutic purposes.
Graphical Abstract
Introduction
Atopic dermatitis (AD) is the most common inflammatory skin disorder, and it has a complex etiology that is dependent on interactions between the skin barrier and the environment (1). Emerging evidence indicates that the skin barrier has important roles in immune surveillance and homeostasis (2). In particular, inherited defects in epidermal barrier proteins facilitate the interaction between external antigens and skin-resident immune cells, resulting in local inflammation (1, 3). Inflammation may in turn cause skin barrier damage, which further exacerbates inflammation and allergic sensitization to environmental allergens (4). These observations suggest that it is important to maintain skin barrier function for both the effective management of AD and the prevention of the subsequent development of allergic diseases.
Autophagy is an essential process through which the cell breaks down unwanted components to maintain homeostasis, and it is typically triggered by nutrient starvation (5). Upregulation of microtubule-associated protein light chain 3-II (LC3-II) and downregulation of sequestosome-1/p62 (p62) result in autophagy activation (5). p62 acts as a selective autophagy adaptor and enhances inflammation in skin conditions, including AD and psoriasis, which are characterized by defects in the epidermal barrier and keratinocyte differentiation, through signaling of nuclear factor κ light chain enhancer of activated B cells (NF-κB) and mechanistic target of rapamycin (mTOR) (6). mTOR functions as an upstream regulator of autophagy. Activation of mTOR suppresses autophagy, while its inhibition initiates autophagy (7, 8), and it is associated with a defective epidermal barrier (9). Interestingly, fasting and dietary restriction, which activate autophagy, have been shown to improve the symptoms of allergic dermatitis (10), implying that there are system-wide benefits of autophagy activation. In addition, the application of rapamycin, an autophagy inducer, ameliorates AD-like skin lesions in NC/Nga mice (11). Notably, an association between AD and autophagy-related (ATG) genes, such as ATG16L2, ATG4s, and unc-51–like autophagy activating kinase (ULK1), has also been proposed (12). Furthermore, given that autophagosome-lysosome fusion supports epidermal differentiation (13) and that functional lysosome-related proteins such as cathepsins D and L are downregulated in AD skin lesions (14), it appears that autophagy plays a crucial role in AD.
Recent studies have demonstrated that autophagy contributes to keratinocyte differentiation, host defense, and immune responses in the epidermal barrier (15). The multilayered structure of the epidermis is continuously renewed by basal-layer keratinocytes that differentiate to form a physical barrier that is mainly composed of the stratum corneum (SC) barrier and tight junction (TJ) barrier. Autophagy contributes to the mature differentiation and development of the epidermis via the regulation of recycling endosomes (16), and ablation of autophagy suppresses the expression of differentiation markers in keratinocytes (17) and the epidermis (18). In addition, Staphylococcus aureus, which frequently colonizes AD skin, can persist within keratinocytes by exploiting autophagy (19). Activation of autophagy attenuates Toll-like receptor 3–mediated (TLR3-mediated) inflammatory responses in epidermal keratinocytes (20), while higher TLR3 expression is correlated with the occurrence of severe lesions of AD (21). These findings indicate that degradative autophagy is involved in the physiological mechanisms of the epidermal barrier.
In addition to antimicrobial activities, human β-defensin-3 (hBD-3) participates in pleiotropic immunomodulatory processes, including keratinocyte cytokine/chemokine production, cell proliferation, migration and differentiation, and the regulation of skin barrier function (22). hBD-3 is also involved in the pathogenesis of various skin diseases, including AD, in which abnormal expression of autophagy-related proteins was recently reported (12, 22, 23); however, the precise role of hBD-3 in autophagy regulation remains unclear, and the contribution of autophagy to epidermal barrier function in AD is also poorly understood.
Although nutrient starvation–induced autophagy enhances TJ barrier function in intestinal epithelial cells (24), little information is currently available on the association of autophagy with the skin barrier in AD. Here, we reveal an immunoregulatory mechanism of autophagy in AD and highlight the therapeutic role of the skin-derived antimicrobial peptide hBD-3 in AD, whose effects are mediated through the regulation of autophagy.
ResultsAutophagy-related proteins are functionally inactive in AD skin lesions. LC3, which comprises cytosolic LC3-I and lipidated LC3-II, and p62 are widely used autophagy markers in mammalian cells (5). Following activation of autophagy, LC3-I is converted to LC3-II, leading to higher expression of LC3-II. At the same time, p62 is degraded, resulting in lower expression. To determine the autophagic status in AD, the expression patterns of LC3 and p62 were analyzed in skin biopsies from patients with AD and compared with those from normal healthy volunteers. In the epidermis of healthy volunteers, LC3 was displayed in all epidermal layers, with the strongest expression in the upper layer, which is consistent with a previous report (25), while this expression was remarkably reduced in the skin lesions of patients with AD (Figure 1A, top panels). In contrast, while p62 was absent in the epidermal layers of healthy skin, it accumulated in the parakeratotic regions of the AD epidermis (Figure 1A, bottom panels).
Figure 1Autophagy-related proteins are dysregulated in AD skin lesions. (A) Immunofluorescence staining of LC3 and p62 in the epidermis of patients with AD and normal participants. Representative immunofluorescence images of skin (left) and quantification of the staining intensity in the epidermis (right). The white dashed line indicates the basement membrane between the epidermis and dermis. Scale bars: 50 or 100 μm; n = 5 per group. (B) Immunofluorescence staining of LC3 and p62 in the epidermis of DNCB-treated AD mice and normal mice. Representative immunofluorescence images of skin (left) and quantification of the staining intensity in the epidermis (right). The white dashed line indicates the basement membrane between the epidermis and dermis. Scale bars: 20 μm; n = 3–6 per group. (C) Expression of p62 and LC3 in the back skins of DNCB-induced AD mice and normal mice; n = 6 per group. Representative immunoblots of the indicated proteins from mouse skin lysates (left) and quantification of the band intensities of LC3 and p62 (right). GAPDH was used as a loading control. (D) Representative electron microscopic images of keratinocytes in lesional skin from DNCB-induced AD mice and keratinocytes in normal mouse skin (left) and quantification of autophagic vacuoles (right). The yellow arrowheads indicate autophagic vacuoles. Scale bars: 5 μm. Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, §§P < 0.01. Statistical significance was determined by 2-tailed Student’s t test. All of the data are representative of 3 independent experiments.
Furthermore, microarray data of skin lesions from 84 patients with AD and normal skin from 199 healthy volunteers were obtained from the ArrayExpress database (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI156501DS1). A comparison of the differences in the ATG genes between the 2 groups revealed that the fold changes in the gene expression of LC3B and ULK1, as well as mitophagy-related genes such as PINK1 and PARK2, were significantly downregulated in patients with AD (Supplemental Figure 1, B and C). This observation implies that autophagy dysregulation in skin might be involved in AD pathogenesis.
To confirm whether autophagy is dysregulated in AD, a 2,4-dinitrochlorobenzene–induced (DNCB-induced) AD-like mouse model (26) was established. Similar to the results seen in patients with AD, LC3 was downregulated, while p62 was increased in the epidermis of AD mice compared with normal mice (Figure 1B). We further established 2 other AD murine models, an AD-like mouse model induced by the vitamin D3 analog MC903 (27) and a Dermatophagoides farinae extract–induced NC/Nga AD model (28). Consistently, decreases in LC3 and increases in p62 in skin samples from both AD models were observed (Supplemental Figure 1, D and E), further confirming that autophagy is inactivated in AD skin.
The difference in the levels of LC3-II between samples in the absence and presence of lysosome inhibitors represents the level of autophagic flux (5). To measure autophagic flux in mouse skin, we injected the lysosome inhibitor chloroquine (CQ) subcutaneously 4 hours before skin tissue collection. Immunoblot analysis showed that LC3-II levels were markedly decreased in the skin tissues from AD mice compared with those from normal mice in the absence of CQ treatment. Likewise, CQ treatment increased the levels of LC3-II and p62 in skin tissues from normal mice, while there was no remarkable difference among AD mice, indicating partial blockade of autophagic flux in the skin tissues of AD mice (Figure 1C). Moreover, ultrastructural transmission electron microscopy image analysis revealed that keratinocytes of DNCB-induced AD skin lesions exhibited fewer autophagic vesicles than those in normal mouse skin (Figure 1D), suggesting that autophagy activation in keratinocytes might play a crucial role in AD pathogenesis.
Th2-derived cytokines are involved in autophagy inactivation in AD keratinocytes. T helper type 2 (Th2) cytokines, such as interleukin-4 (IL-4) and IL-13, have long been associated with the pathogenesis of AD (1), and keratinocytes treated with a mixture of IL-4 and IL-13 constitute an in vitro AD-like keratinocyte model (29). The effects of IL-4 and IL-13 on human keratinocyte autophagy were evaluated in the presence or absence of the lysosomal enzyme inhibitors E64d and pepstatin A (E&P), which prevent lysosomal acidification and autophagosome-lysosome fusion and are used to exclude the possibility of simple blockade of lysosomal degradation rather than autophagy activation (30). As shown in Figure 2, treatment of keratinocytes with IL-4 or IL-13 alone and the combination of both IL-4 and IL-13 significantly increased p62 levels in the absence of E&P, while there was no difference in p62 levels in the presence of E&P. These cytokines markedly reduced the LC3-II amounts in the presence of E&P, indicating that IL-4 and IL-13 partially block autophagic flux in keratinocytes (Figure 2, A–C), which is consistent with the in vivo results described in Figure 1C.
Figure 2Th2-derived cytokines are involved in the inactivation of autophagy in AD keratinocytes. (A–C) Keratinocytes were stimulated for 12 hours with 100 ng/mL IL-4 or IL-13 alone or in combination in the presence (+) or absence (–) of 10 μg/mL E&P; n = 3 per group. Representative p62 and LC3 immunoblots (left) and quantification of band intensities (right). GAPDH was used as a loading control. (D) Keratinocytes were stimulated for 12 hours with or without IL-4 or IL-13 alone or in combination in the presence of 10 μM rapamycin (Rap); n = 5 per group. Representative immunofluorescence images (left) and quantification of LC3 puncta in keratinocytes (right). Scale bars: 10 μm. Mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001, #P < 0.05, ###P < 0.001, ####P < 0.0001. Statistical significance was determined by 2-tailed Student’s t test or 1-way ANOVA with Tukey’s multiple-comparison test. All of the data are representative of 3 independent experiments.
Moreover, while the autophagy inducer rapamycin increased the appearance of LC3-positive puncta, an indicator of autophagy occurrence (5), the administration of IL-4 or IL-13 alone as well as their combination significantly diminished the number of LC3-positive puncta in rapamycin-treated keratinocytes (Figure 2D). Interestingly, treatment of keratinocytes with other Th2 cytokines, such as IL-33 and thymic stromal lymphopoietin (TSLP), affected neither p62 accumulation nor LC3-II levels in the presence of E&P (Supplemental Figure 2, A and B) and did not affect the number of LC3-positive puncta in keratinocytes (Supplemental Figure 2F), implying that not all Th2 cytokines involved in AD pathogenesis play a role in the autophagy process in keratinocytes.
Given that the activation of Th1- and Th17-mediated responses has been reported in chronic AD skin lesions (1), we investigated the effects of Th1 and Th17 cytokines on keratinocyte autophagy. Treatment with the Th1 cytokine interferon-γ (IFN-γ) and the Th17 cytokine IL-17 decreased the accumulation of p62 and increased the LC3-II amounts in keratinocytes, while IL-23 did not show any significant effect (Supplemental Figure 2, C–E). Interestingly, both IFN-γ and IL-17 significantly increased LC3-II levels in the presence of E&P compared with the absence of E&P (Supplemental Figure 2, C and D), suggesting that these cytokines may induce activation rather than inhibition of autophagic flux in keratinocytes. Taken together, the Th2 cytokines IL-4 and IL-13 may inhibit autophagic flux in keratinocytes.
Keratinocyte-specific autophagy deficiency exacerbates AD. Although autophagy is involved in the homeostasis of intestinal barrier function (24), the role of autophagy in epidermal barrier function remains unclear. To examine the role of autophagy in the maintenance of the barrier function in keratinocytes, we established autophagy-deficient keratinocytes by transfecting mutant Atg3C264S, which has a mutation at the active-site cysteine that leads to autophagy inactivation, using an adenovirus system as described in a previous study (31). Note that Atg3 is an E2-like enzyme required for Atg8 conjugation, which is indispensable for the proper development of autophagic isolation membranes (31). Following Atg3C264S transfection into keratinocytes, Atg3 expression was not affected, while LC3-II was decreased and p62 was increased in the transfected cells, confirming the deficiency of autophagy in these cells and the equal transfection levels between the Atg3- and Atg3C264S-transduced cells (Supplemental Figure 3A). In autophagy-deficient keratinocytes, the mRNA expression of TJ-related proteins, including claudin-1 and zonula occludens-1 (ZO-1; tight junction protein 1 [TJP1]), and the expression of SC barrier proteins, such as filaggrin and loricrin, were significantly decreased (Supplemental Figure 3B). Likewise, claudin-1 and ZO-1 were noticeably reduced at the protein level, and a similar tendency was observed for filaggrin and loricrin expression, although it was not significant (Supplemental Figure 3C). We further confirmed that the intercellular distribution of claudin-1 and ZO-1 was attenuated in autophagy-deficient keratinocytes (Supplemental Figure 3D). Both claudin-1 and ZO-1 are important TJ barrier components, as claudin-1–null mice die within 1 day of birth owing to dehydration (32), and ZO-1 drives TJ barrier formation (33).
To identify the role of autophagy in the regulation of the skin barrier in AD, we crossed Atg7-floxed mice (referred to as Atg7fl/fl mice hereafter) (34) with K14-Cre transgenic mice to generate mice with selective ablation of Atg7 in keratinocytes (referred to as K14Cre Atg7fl/fl mice hereafter). We first confirmed the keratinocyte-specific deletion of autophagy in K14Cre Atg7fl/fl mice, as evidenced by the absence of Atg7 (Supplemental Figure 3E) and LC3 (Supplemental Figure 3F, left) and the accumulation of p62 (Supplemental Figure 3F, right). Interestingly, from day 10 to day 40 postnatally, the weight gain of K14Cre Atg7fl/fl mice was significantly less than that of K14Cre mice, and transepidermal water loss (TEWL) was higher in K14Cre Atg7fl/fl mice (Figure 3A). Furthermore, skin sections at both newborn age (day 0) and young adult age (day 42) showed downregulation of claudin-1, ZO-1, filaggrin, loricrin, and involucrin in K14Cre Atg7fl/fl mice (Figure 3B). Moreover, we established a DNCB-induced AD-like murine model using both K14Cre mice and K14Cre Atg7fl/fl mice and observed that K14Cre Atg7fl/fl AD mice displayed more severe inflammatory symptom
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