Osthole attenuates mouse atopic dermatitis by inhibiting thymic stromal lymphopoietin production from keratinocytes

Atopic dermatitis is one of the most common skin diseases. Dysregulation of immune system and chronic inflammation were believed to be associated with atopic dermatitis. Osthole was reported to play important roles in antitumor and anti‐inflammation. However, whether osthole has effects on atopic dermatitis remains unclear. In this present study, we explored the biological role of osthole in atopic dermatitis and the molecular mechanism. Atopic dermatitis was induced by 2,4‐dinitrochlorobenzene. Pathological damage of ear was detected by H&E staining. IgE level in serum or thymic stromal lymphopoietin (TSLP) level in supernatant was detected by ELISA. Interleukin (IL)‐4 expression and IL‐13 expression in CD4⁺ T cells were detected using flow cytometry. The expression levels of mRNA or protein levels were detected by RT‐PCR or Western blot. Osthole attenuated atopic dermatitis development in mouse model. Osthole inhibits Th2 cell response, but have on influence on Th1 or Th17 cell response in the skin. In mouse model, osthole treatment significantly inhibited atopic dermatitis via directly inhibiting TLSP expression levels in keratinocytes. Osthole treatment alleviates atopic dermatitis through directly down‐regulating TSLP production from keratinocytes. Osthole may serve as a potential choice for atopic dermatitis treatment in clinic.     

The Th2 cytokine,interleukin‐4, abrogates the cohesion of normal stratum corneum in mice: implications for pathogenesis of atopic dermatitis

There is mounting evidence that Th2 cytokines adversely affect skin barrier functions and contribute to the pathogenesis of atopic dermatitis (AD). AD is also characterized by abnormal cohesion in the stratum corneum (SC). However, the contribution of Th2 cytokines to this abnormality remains unknown. This study examined the effects of IL‐4, a prototypic Th2 cytokine, on the cohesion of the SC. Structural and physiological assessments revealed that repeated intradermal injections of IL‐4 compromised the cohesion of the SC of normal hairless mice. Two potential mechanisms were explored to account for the altered cohesion. First, IL‐4 decreased the amount of corneodesmosomes and down‐regulated the expression of desmoglein 1, but not of corneodesmosin (CDSN) or loricrin expression, in murine skin and in cultured human keratinocytes (KC). IL‐4 did not affect the skin surface pH, and in situ zymography revealed no net change in total serine protease activity in the IL‐4‐treated SC. Yet, IL‐4 enhanced expression of kallikrein (KLK)7, while simultaneously down‐regulating KLK5 and KLK14. Finally, IL‐4 did not alter the expression of the lympho‐epithelial Kazal‐type inhibitor (LEKTI) in KC. This study suggests that IL‐4 abrogates the cohesion of SC primarily by reducing epidermal differentiation.     

Toll‐like receptor 4 attenuates a murine model of atopic dermatitis through inhibition of langerin‐positive DCs migration

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that is often associated with skin barrier dysfunction leading to a higher frequency of bacterial and viral skin infections. Toll‐like receptor (TLR) 4 on resident skin cells was involved in sensing pathogens and eliciting pathogen‐specific innate and adaptive immune responses. Previous studies have demonstrated that TLR4 was linked to AD severity in context of pathogen infection. However, the immune regulatory role of TLR4 in AD remains to be defined. We here investigated the immune regulatory function of TLR4 in AD induced by repeated epicutaneous application of a hapten, 2,4‐dinitrochlorobenzene (DNCB). Our results showed that TLR4‐deficient (TLR4^?/?) mice exhibited more severe AD symptoms than WT mice after DNCB challenge. The DNCB‐treated TLR4^?/? mice also displayed higher expression levels of inflammatory cytokines and stronger Th2 response than WT counterparts. Moreover, the skin expression of thymic stromal lymphopoietin (TSLP), an important potential contributor to allergic inflammation, was significantly elevated in TLR4^?/? mice compared with that in WT mice upon DNCB administration. Furthermore, we demonstrated that the migration of langerin‐positive dendritic cells (DCs) into draining lymph nodes was enhanced in TLR4^?/? mice following DNCB challenge, which is partially dependent on the production of pro‐inflammatory cytokine TNF‐α. Together, these results determined that TLR4 affected the hapten‐induced skin inflammation in the absence of exogenous pathogen infection, suggesting that TLR4 not only regulates infection but also may serve as a modulator of the immune response during AD development.     

The skin microbiome of caspase‐14‐deficient mice shows mild dysbiosis

Caspase‐14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase‐14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2‐pyrrolidone‐5‐carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase‐14^?/? mice show enhanced antibacterial response compared to wild‐type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild‐type and caspase‐14^?/? mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase‐14 ablation leads to an increase in bacterial richness and diversity during steady‐state conditions. Although both wild‐type and caspase‐14^?/? skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase‐14^?/? mice. Altogether, our data demonstrated that caspase‐14 deficiency causes the imbalance of the skin‐resident bacterial communities.     

Therapeutic potential of topically administered γ‐AlOOH on 2,4‐dinitrochlorobenzene‐induced atopic dermatitis‐like lesions in Balb/c mice

Boehmite (γ‐AlOOH) has a wide range of applications in a variety of industrial and biological fields. However, little is known about its potential roles in skin diseases. The current study investigated its effect on atopic dermatitis (AD). Following characterization, cytotoxicity, pro‐inflammatory response and oxidative stress associated with boehmite were assessed, using TNF‐α‐induced keratinocytes and mast cells. In addition, therapeutic effects of boehmite, topically administered to Balb/c mice induced by 2,4‐dinitrochlorobenzene (DNCB), were evaluated. Expression of cytokines (TLSP, IL‐25 and IL‐33) and the generation of ROS from keratinocytes induced by TNF‐α were significantly inhibited by boehmite without affecting cell viability. MAPKs (ERK, JNK and p38) required for cytokine expression were suppressed by boehmite treatment. Up‐regulation of cytokines (TSLP, IL‐4, IL‐5, IL‐13, RANTES) in human mast cells treated with phorbol 12‐myristate 13‐acetate and calcium ionophore was also suppressed by boehmite. Boehmite improved the AD severity score, epidermal hyperplasia and transepidermal water loss in DNCB‐induced AD‐like lesions. Moreover, Th2‐mediated cytokine expression, mast cell hyperplasia and destruction of the skin barrier were improved by boehmite treatment. Overall, we demonstrated that boehmite may potentially protect against AD.     

Antioxidant soybean tar Glyteer rescues T‐helper‐mediated downregulation of filaggrin expression via aryl hydrocarbon receptor

Soybean tar Glyteer (Gly) has been widely used for the treatment of various inflammatory skin diseases in Japan since 1924 as an alternative to coal tar remedy. Recently, coal tar has been shown to induce barrier repair in atopic dermatitis via aryl hydrocarbon receptor (AhR). In this study, we demonstrated that Gly activated AhR by inducing its cytoplasmic to nuclear translocation in keratinocytes. The AhR ligation by Gly was biologically active, with significant and dose‐dependent upregulation of CYP1A1 expression, which is a specific marker for AhR activation. Gly upregulated the expression of filaggrin in an AhR‐dependent manner because its enhancing effect was completely abrogated in AhR‐knockdown keratinocytes. T‐helper (Th)2 cytokines inhibited the expression of filaggrin; however, Gly completely restored the Th2‐mediated inhibition of filaggrin expression. Furthermore, Gly coordinately upregulated a series of epidermal differentiation complex genes, including involucrin, loricrin and hornerin. In addition, Gly exhibited potent antioxidant activity through the activation of nuclear factor‐erythroid 2‐related factor‐2 (Nrf2) and downstream antioxidant enzymes such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), which actually inhibited the generation of reactive oxygen species in keratinocytes treated with tumor necrosis factor‐α or benzo[α]pyrene. In conclusion, antioxidant Gly rescues the downregulated expression of filaggrin (and plausibly other barrier proteins) in a Th2‐skewed milieu via AhR activation, which may partly explain its empirical anti‐inflammatory therapeutic effects.     

角质形成细胞与特应性皮炎相关研究进展

角质形成细胞（keratinocytes，KCs）的异常导致角蛋白的表达出现异常，从而导致表皮屏障功能失调。在特应性皮炎(Atopic Dermatitis，AD)皮损中，KC大量表达胸腺淋巴基质生成素、肿瘤坏死因子α以及一些白细胞介素如IL-1α、IL-1β和IL-18等介导皮肤的炎症反应。在AD中KC还可表达模式识别受体，通过先天免疫系统，产生和维持炎症反应。另外，AD皮损中KC损伤导致抗菌肽的表达缺乏可能有助于增加AD患者皮肤对感染病毒、细菌和真菌的易感性。本文对角质形成细胞与特应性皮炎相关研究进展进行综述。     

Thymic stromal lymphopoietin regulates eosinophil migration via phosphorylation of l‐plastin in atopic dermatitis

Infiltration of eosinophils in atopic dermatitis (AD), which contains inflammatory molecules and cytokines, recruits more inflammatory cells and causes further skin damage. Thymic stromal lymphopoietin (TSLP) is an epithelial cytokine that induces the proinflammatory Th2 immune response and plays an important role in allergic disease. In this study, we aimed to identify a novel protein that regulates TSLP in eosinophils to further understand the role of eosinophils in atopic dermatitis. Using a proteomics approach, we identified the TSLP‐inducible protein l ‐plastin and confirmed upregulation of l ‐plastin and p‐l ‐plastin in TSLP‐treated human eosinophilic leukaemic (EoL‐1) cells and in eosinophils from AD patients. Migration assays showed that migration of eosinophils increased when cells were treated with TSLP and when cells were treated with TSLP and an additional cytokine such as interleukin (IL)‐3, IL‐4, IL‐5 or IL‐13, when compared to migration of untreated eosinophils. We also confirmed a positive correlation between phosphorylation of l ‐plastin and an increase in migration of TSLP and cytokine‐treated eosinophils. In addition, phosphorylation of l ‐plastin was sensitive to PKCβII inhibition. Our results suggest that TSLP‐induced phosphorylation of l ‐plastin affects eosinophil migration, which may be mediated by the protein kinase C signalling pathway in atopic dermatitis, thus suggesting p‐l ‐plastin as a potential drug target for eosinophil‐targeted allergy therapy.     

Does therapeutic intervention in atopic dermatitis normalize epidermal Notch deficiency?

This viewpoint presents a unifying concept for the treatment of atopic dermatitis (AD) that is based on the improvement of deficient Notch signalling, which appears to represent the fundamental epithelial defect of AD resulting in epidermal and immunological barrier dysfunction. One study of AD patients demonstrated a marked epidermal deficiency of Notch receptors and several mouse models with genetically suppressed Notch signalling exhibit dry skin, signs of scratching, skin barrier abnormalities, increased transepidermal water loss and Th2 cell‐mediated immunological changes closely resembling human AD. Notch signalling is critically involved in the differentiation of regulatory T cells, in the feedback inhibition of activated innate immunity, in the repression of activating protein‐1 (AP‐1), the regulation of late epidermal differentiation associated with filaggrin‐ and stratum corneum barrier lipid processing, in aquaporin 3‐ and claudin‐1 expression and in keratinocyte‐mediated release of thymic stromal lymphopoietin (TSLP), which promotes Th2‐driven immune responses with TSLP‐ and IL‐31‐mediated stimulation of cutaneous sensory neurons involved in the induction of itch. Translational evidence will be provided that all major therapeutic regimens employed for the treatment of AD such as glucocorticoids, calcineurin inhibitors and UV radiation may converge in the upregulation of impaired Notch signalling, the proposed pathogenic defect of AD.     

DAMP molecules S100A9 and S100A8 activated by IL‐17A and house‐dust mites are increased in atopic dermatitis

S100A9 and S100A8 are called damage‐associated molecular pattern (DAMP) molecules because of their pro‐inflammatory properties. Few studies have evaluated S100A9 and S100A8 function as DAMP molecules in atopic dermatitis (AD). We investigated how house‐dust mites affect S100A9 and S100A8 expression in Th2 cytokine‐ and Th17 cytokine‐treated keratinocytes, and how secretion of these molecules affects keratinocyte‐derived cytokines. Finally, we evaluated expression of these DAMP molecules in AD patients. S100A9 expression and S100A8 expression were strongly induced in IL‐17A‐ and Dermatophagoides (D.) farinae‐treated keratinocytes, respectively. Furthermore, co‐treatment with D. farinae and IL‐17A strongly increased expression of S100A9 and S100A8 compared with D. farinae‐Th2 cytokine co‐treatment. The IL‐33 mRNA level increased in a dose‐dependent manner in S100A9‐treated keratinocytes, but TSLP expression did not change. S100A8/A9 levels were also higher in the lesional skin and serum of AD patients, and correlated with disease severity. Taken together, S100A9 and S100A8 may be involved in inducing DAMP‐mediated inflammation in AD triggered by IL‐17A and house‐dust mites.     

Expressions of peroxisome proliferator‐activated receptors (PPARs) are directly influenced by permeability barrier abrogation and inflammatory cytokines and depressed PPARα modulates expressions of chemokines and epidermal differentiation‐related molecules in keratinocytes

Previous studies have demonstrated that the activation of peroxisome proliferator‐activated receptors (PPARs) not only has positive effects on permeability barrier homoeostasis but also has anti‐inflammatory effects by an as yet unknown mechanism. Reduced expression of PPARα in lesion of human atopic dermatitis (AD) and in epidermis of murine AD‐like dermatitis has been demonstrated. This study revealed that expression of PPARα alone among PPARs (α, β/δ and γ) was suppressed by both permeability barrier abrogation and additional existence of Th2 cytokine in cultured normal human keratinocytes. In addition, expressions of transglutaminase 1 and loricrin and those of thymus and activation‐related chemokine and regulated on activation normal T‐cell expressed in cultured human keratinocytes were reduced and enhanced, respectively, by transfection with siRNA for PPARα. In conclusion, depressed PPARα in keratinocytes might be involved in a relationship between permeability barrier abrogation and allergic inflammation and could be a therapeutic target which accounts for both the aspects in AD.     

Heparinoid suppresses Der p‐induced IL‐1β production by inhibiting ERK and p38 MAPK pathways in keratinocytes

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro‐inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen‐induced interleukin (IL)‐1β release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase‐1 release, suggesting that heparinoid did not affect HDM allergen‐induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro‐IL‐1β, but also suppressed IL‐1β messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal‐regulated kinase and p38 pathways, which are required for IL‐1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL‐1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte‐mediated skin inflammation.     

Interleukin‐1 from epithelial cells fosters T cell‐dependent skin inflammation

Background Chronic inflammatory skin diseases such as atopic dermatitis and psoriasis are characterized by the infiltration of lymphocytes into the epidermal compartment. Several studies point to an active role of skin epithelial cells in the pathophysiology of such diseases. Objectives In this study we addressed the regulatory function of primary human keratinocytes in the interaction with autologous T cells and monocytes. Methods We used a human coculture model with keratinocytes grown from epidermal stem cells of the outer root sheath of human hair follicles and autologous T cells. Results In our coculture system we observed a high production of interferon (IFN)‐γ, but not Th2 cytokines, in the presence of superantigen or antigen‐pulsed autologous monocytes. Critical parameters for this effect were: (i) T‐cell receptor activation, (ii) an intercellular adhesion molecule‐1/lymphocyte function‐associated antigen (LFA)‐1‐dependent interaction between keratinocytes and T cells, and (iii) secretion of interleukin (IL)‐1β. Remarkably, in the presence of activated T cells, epithelial cells seemed to be a more significant source of IL‐1β than monocytes. Application of the LFA‐1 blocker efalizumab or IL‐1 receptor antagonist anakinra enabled us to suppress completely the production of IFN‐γ by T cells in the coculture. Conclusions IL‐1 secretion and the physical contact between keratinocytes and activated, infiltrating T cells may be central for the development of chronic inflammatory skin conditions.     

Suppression of IL-17A-induced CCL20 production by cytokine inducible SH2-containing protein 1 in epidermal keratinocytes

BackgroundLesions of atopic dermatitis have fewer Th17 cells than those of psoriasis, resulting in frequent skin infections. Expression of CCL20, a chemokine that is important for recruiting Th17 cells, is suppressed in the lesions of atopic dermatitis. We previously reported that IL-4 induces the expression of cytokine-inducible SH2-containing protein 1 (CIS1), a member of the CIS/SOCS family, in epidermal keratinocytes.ObjectiveTo investigate whether CIS1 influences CCL20 production in epidermal keratinocytes.MethodsExpression of CIS1 was examined in atopic dermatitis skin and in cultured keratinocytes. The effects of overexpression of CIS1 on CCL20 production by IL-17A, and on signaling pathways inhibited by CIS1, were assessed in vitro.ResultsExpression of CIS1 was enhanced in the basal layer of the lesional epidermis of skin with atopic dermatitis. When CIS1 was expressed in keratinocytes using adenoviral vectors, IL-17A-induced CCL20 expression, but not HBD2 or S100A7 expression, was significantly suppressed. TNF-α/IL-1-induced CCL20 production was not altered by CIS1. Overexpression of CIS1 attenuated IL-17A-induced ERK phosphorylation. ERK phosphorylation was mediated by the Act1 and Src family kinase pathways. CIS1 overexpression suppressed Src phosphorylation. Among the Src family kinases, the Yes kinase may have an important role because knockdown of Yes in epidermal keratinocytes resulted in suppression of ERK phosphorylation and CCL20 mRNA expression by IL-17A.ConclusionCIS1 induced by Th2 cytokines has the ability to change the response of epidermal keratinocytes to IL-17A by suppression of Src family kinases.     

Immune escape phenomenon in molluscum contagiosum and the induction of apoptosis

Molluscum contagiosum (MC) may persist for many weeks, evading host immunity. We studied the mechanism of immune escape phenomenon in MC, and the possible inducer of apoptosis. Using tissue samples of MC, we examined the numbers of epidermal Langerhans cells (LC), the expression levels of macrophage inflammatory protein‐3α (MIP‐3α) and thymic stromal lymphopoietin (TSLP), and the apoptotic signals. After molluscum contagiosum virus (MCV) genotyping, we studied the expression of MCV‐encoded MC148 mRNA and MC159 mRNA which correspond to viral antagonist for CCR8 and viral Fas‐linked interleukin (IL)‐1β converting enzyme (FLICE)‐like inhibitor protein (vFLIP), respectively. The nutlin‐3‐induced apoptosis in MC was observed ex vivo. The numbers of CD1a⁺ or Langerin⁺ epidermal LC and the expression levels of MIP‐3α were markedly decreased in MC. The expression of TSLP was enhanced in the lesional epidermis of atopic dermatitis and human papillomavirus‐induced warts, whereas the expression was observed locally in MC. All 14 MC samples examined harbored MCV type 1. The MC148 mRNA was detected in all 14 samples and the MC159 mRNA was detected in 13 samples. Apoptotic cells were absent or at a background level in the living layers of MC, but their numbers were increased in the molluscum bodies by overnight incubation with 5 μmol/L nutlin‐3 in culture medium. In conclusion, molluscum bodies are protected from host immune responses and apoptotic signals by being surrounded by LC‐depleted epidermal walls and viral immunosuppressive molecules, but could be eradicated by reagents inducing p53‐dependent apoptosis.     

E‐cadherin mediates ultraviolet radiation‐ and calcium‐induced melanin transfer in human skin cells

Skin pigmentation is directed by epidermal melanin units, characterized by long‐lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X‐mediated control of MC filopodia. Both MC and KC express Ca²⁺‐dependent E‐cadherins. These homophilic adhesion contacts induce transient increases in intra‐KC Ca²⁺, while ultraviolet radiation (UVR) raises intra‐MC Ca²⁺ via calcium‐selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca²⁺ triggers melanin transfer remains unclear. Here we evaluated the role of E‐cadherin in UVR‐mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia‐associated E‐cadherin, Cdc42, VASP and β‐catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E‐cadherin revealed that this cadherin is essential for UVR‐induced MC filopodia formation and melanin transfer. Moreover, Ca²⁺ induced a dose‐dependent increase in filopodia formation and melanin transfer, as well as increased β‐catenin, Cdc42, Myosin X and E‐cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E‐cadherin, which are upregulated by intracellular (UVR‐stimulated) and extracellular Ca²⁺ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca²⁺ signalling influences human pigmentation.     

The atopic skin‐like microenvironment modulates the T‐cell‐polarising cytokine production of myeloid dendritic cells,as determined by laser scanning cytometry

Because it is not known exactly when or where myeloid dendritic cells (mDCs) acquire their atopic dermatitis (AD)‐specific T‐cell‐polarising ability in patients with this condition, we used laser scanning cytometry (LSC) to determine whether isolated peripheral blood mDCs from AD patients differed from cells from controls in their cytokine expression profiles de novo and after stimulation with Staphylococcus enterotoxin B (SEB) and thymic stromal lymphopoietin (TSLP), which represents an AD‐like microenvironment. Unstimulated mDCs from AD patients showed pluripotent T‐cell‐polarising capacity, and the surrounding skin microenvironment was essential for the distinctive, disease‐specific activity of mDCs (Th2‐Th22 bias). We also emphasise that LSC is an attractive technique to study the effect of new DC‐targeted therapeutic modalities in AD.