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Ameliorating effect of dipotassium glycyrrhizinate on an IL-4- and IL-13-induced atopic dermatitis-like skin-equivalent model
Abstract
Atopic dermatitis (AD) is a chronic inflammatory skin disease that is not fully understood. Defects in skin barrier function and dysregulation of the Th2 immune response are thought to be pivotal in AD pathogenesis. In this study, we used keratinocytes and AD-like skin equivalent models using Th2 cytokines IL-4 and IL-13. The keratinocytes and AD-like skin model were used to investigate the effect of dipotassium glycyrrhizinate (KG), which is widely used as an anti-inflammatory agent for AD treatment. KG decreased AD-related gene expression in keratinocytes stimulated with Th2 cytokines. KG alleviated AD-like phenotypes and gene expression patterns and inhibited release of AD-related cytokines in the AD-like skin equivalent models. These findings indicate KG has potential effectiveness in AD treatment and AD-like skin equivalent models may be useful for understanding AD pathogenesis.
... This will enable researchers to develop more effective therapies for these skin diseases. Most skin disease models use a basic form of HSE generated using manual deposition of an epidermal compartment composed of keratinocytes and cell media over a dermal compartment composed of fibroblasts and collagen type I [12][13][14][15]. While these models do provide more insight into the pathogenesis of skin disease than a typical 2D in vitro model would, they fail to fully capture the intricacies of both healthy and diseased skin. ...
... An AD-like 3D HSE was also used to investigate the effect of dipotassium glycyrrhizinate (KG) on AD [15]. The HSE was constructed using a mixture of NHFs and collagen I with NHKs seeded on top. ...
Models of skin diseases, such as psoriasis and scleroderma, must accurately recapitulate the complex microenvironment of human skin to provide an efficacious platform for investigation of skin diseases. Skin disease research has been shifting from less complex and less relevant 2D (two-dimensional) models to significantly more relevant 3D (three-dimensional) models. Three-dimensional modeling systems are better able to recapitulate the complex cell–cell and cell–matrix interactions that occur in vivo within skin. Three-dimensional human skin equivalents (HSEs) have emerged as an advantageous tool for the study of skin disease in vitro. These 3D HSEs can be highly complex, containing both epidermal and dermal compartments with integrated adnexal structures. The addition of adnexal structures to 3D HSEs has allowed researchers to gain more insight into the complex pathology of various hereditary and acquired skin diseases. One method of constructing 3D HSEs, 3D bioprinting, has emerged as a versatile and useful tool for generating highly complex HSEs. The development of commercially available 3D bioprinters has allowed researchers to create highly reproducible 3D HSEs with precise integration of multiple adnexal structures. While the field of bioengineered models for study of skin disease has made tremendous progress in the last decade, there are still significant efforts necessary to create truly biomimetic skin disease models. In future studies utilizing 3D HSEs, emphasis must be placed on integrating all adnexal structures relevant to the skin disease under investigation. Thorough investigation of the intricate pathology of skin diseases and the development of effective treatments requires use of highly efficacious models of skin diseases.
... A similar phenomenon was observed by Lee et al., who treated atopic dermatitis skin models with a licorice-derived compound. As revealed by IHC staining, the FLG protein expression was nearly completely restored after treatment with the highest compound concentration, while the mRNA levels were increased but still strikingly lower than the healthy control 51 . Hence, crossing a certain threshold of mRNA expression seems to be abundant for restoration of the FLG protein production. ...
Psoriasis is a prevalent, inflammatory skin disease without cure. Further research is required to unravel dysregulated processes and develop new therapeutic interventions. The lack of suitable in vivo and in vitro preclinical models is an impediment in the psoriasis research. Recently, the development of 3D skin models has progressed including replicas with disease-like features. To investigate the use of in vitro models as preclinical test tools, the study focused on treatment responses of 3D skin replicas. Cytokine-priming of skin organoids induced psoriatic features like inflammation, antimicrobial peptides (AMP), hyperproliferation and impaired differentiation. Topical application of dexamethasone (DEX) or celastrol (CEL), a natural anti-inflammatory compound reduced the secretion of pro-inflammatory cytokines. DEX and CEL decreased the gene expression of inflammatory mediators. DEX barely affected the psoriatic AMP transcription but CEL downregulated psoriasis-driven AMP genes. Subcutaneous application of adalimumab (ADM) or bimekizumab (BMM) showed anti-psoriatic effects via protein induction of the differentiation marker keratin-10. Dual blockage of TNF-α and IL-17A repressed the inflammatory psoriasis phenotype. BMM inhibited the psoriatic expression of AMP genes and induced KRT10 and cell-cell contact genes. The present in vitro model provides a 3D environment with in vivo-like cutaneous responses and represents a promising tool for preclinical investigations.
... [1][2][3] As the principal anti-viral and anti-inflammatory bioactive ingredient, glycyrrhizic acid (GA) has been widely used in the clinical treatment of atopic dermatitis-like skin disease and rapidly skin wound healing. 4 F I G U R E 1 Glycyrrhizic acid (GA)-based medical dressing effectively improved photodynamic therapy (PDT)-caused skin roughness damage in port-wine stains patients. The data are presented as the means ± the standard error of mean (SEM), n = 29. ...
... The main biologically active components of G. glabra are dipotassium glycyrrhizinate, glycyrrhizin, also known as glycyrrhizic acid, and its aglycone, glycyrrhetinic acid [206]. Dipotassium glycyrrhizinate has similar properties to those of corticosteroids, namely, anti-inflammatory, antiallergic, and antibiotic activities, without the side effects of allergic reactions on the skin [208]. This property is due to dipotassium glycyrrhizinate's ability to efficiently inhibit the activity of phospholipase A2 enzyme, which is necessary for several inflammatory processes [209][210][211]. ...
... Full-thickness (FT) skin wrinkle mimics were reconstructed mainly based on our previous studies with slight modifications. 23 ...
Background
Wrinkles represent a characteristic symptom of skin aging. In recent years, various studies have focused on their prevention and/or cure. However, clinical tests are still the only method available to directly detect and evaluate the anti‐wrinkle efficacy of various substances. Moreover, no in vitro strategy for such anti‐aging skin analysis has been reported. Therefore, in this study, we aimed to develop a novel technology to overcome these limitations.
Materials and methods
Full‐thickness (FT) skin wrinkle mimics with various widths and depths were fabricated using a collagen stamping method. These were analyzed and compared using 2D and 3D Swept Source‐Optical Coherence Tomography (SS‐OCT) imaging technologies.
Results
SS‐OCT demonstrated superficial and cross‐sectional images of the wrinkle mimics, and the size of the wrinkles was validated using image analysis. Retinoic acid treatment significantly decreased both the depth and width of wrinkles formed in the FT skin wrinkle mimics.
Conclusions
Using 3D tissue engineering and SS‐OCT imaging technologies, we developed a novel in vitro technique that can directly detect skin wrinkles. This significantly efficient method could lead to an alternative strategy for animal experiments and preclinical anti‐aging research on the skin.
... Biyolojik olarak aktif ana bileşenleri, glisirizik asit olarak da bilinen dipotasyum glisirizinat, glisirizin ve bunun aglikonu, glisiretinik asittir (El-Saber Batiha et al., 2020). Dipotasyum glisirizinat, ciltte alerjik reaksiyonların yan etkileri olmaksızın, kortikosteroidlerinkine benzer özelliklere, yani antienflamatuvar, antialerjik ve antibiyotik aktivitelere sahiptir (Lee et al., 2019). Bu özellik, dipotasyum glisirizinatın, birkaç enflamatuvar süreç için gerekli olan fosfolipaz A2 enziminin aktivitesini etkili bir şekilde inhibe etme yeteneğinden kaynaklanmaktadır (Vitali et al., 2013). ...
Cancer is still a primary public health problem all over the world today. Around 19.3 million new cases of cancer were diagnosed worldwide in 2020. Chemotherapy, which cause serious
side effects, which is pharmacological method often used to treat cancer. Oral mucositis is one of the most significant side effects of chemotherapy; It is a solemn symptom characterized by the presence of ulcers in the oral mucosa that causes pain, bleeding, and difficulty in swallowing liquid and solid foods or speaking. The incidence of oral mucositis in cancer patients ranges from 40% to 100%. The type of tumor and treatment methods, the
patient's age, nutritional status, and oral hygiene is associated with the development of oral mucositis. Ulcerations that require the use of local analgesics in the oral mucosa are quite painful, cause eating difficulties and malnutrition, increase the risk of developing sepsis, the need for enteral/parenteral nutrition, and the need for systemic analgesics. This adversely affects the quality of life of cancer patients, delays chemotherapy, and consequently increases repeated hospitalizations and health expenditures. However, natural products such as Aloe
vera, Glycyrrhiza glabra, Camellia sinensis, and Calendula officinalis, which are nonpharmacological methods with lower costs, and honey bees have antimicrobial, antiviral, antiinflammatory, analgesic, and wound healing properties. For this reason, it is considered that the properties of these nonpharmacological products can also be used in the management of oral mucositis. In this review, the use of pharmacological and non-pharmacological methods
in the management of oral mucositis in cancer patients will be discussed in light of the literature.
Keywords: Pharmacological methods, Non-pharmacological methods, Oral Mucositis.
... Dipotassium glycyrrhizinate (95) is the dipotassium salt of glycyrrhizic acid and is a widely used anti-inflammatory agent. Dipotassium glycyrrhizinate is chemically stable, water-soluble, and is used as an ingredient in cosmetics, with no reported side effects, even with continuous use [167][168][169]. ...
The global cosmetics market reached US800 billion by 2023, at around a 7% annual growth rate. The cosmetics industry is emerging as one of the fastest-growing industries of the past decade. Data shows that the Chinese cosmetics market was US450 billion. The influence of social media and the internet has raised awareness of the risks associated with the usage of many chemicals in cosmetics and the health benefits of natural products derived from plants and other natural resources. As a result, the cosmetic industry is now paying more attention to natural products. The present review focus on the possible applications of natural products from various biological sources in skin care cosmetics, including topical care products, fragrances, moisturizers, UV protective, and anti-wrinkle products. In addition, the mechanisms of targets for evaluation of active ingredients in cosmetics and the possible benefits of these bioactive compounds in rejuvenation and health, and their potential role in cosmetics are also discussed.
... The specific mechanism needs to be studied further. DG has been shown in previous studies to reduce inflammation, but no study has reported its whitening effect [26]. The results showed that DG inhibited melanin production and tyrosinase activity in B16-F10 cells and showed no significant inhibition in the zebrafish model for the first time ( Figures 2G, 4G, 5G, 6G,8G, 9G). ...
Abstract The mushroom tyrosinase assay, B16-F10 mouse melanoma cell model, and zebrafish model are frequently used for high-throughput screening and are widely used for developing anti-hyperpigmentation compounds, although these systems cannot be compared. We used each of these three systems to evaluate the seven anti-hyperpigmentation compounds. We investigated 1. tyrosinase activity using a mushroom tyrosinase assay, 2. viability, tyrosinase activity, and melanin content in B16-F10 cells, and 3. embryonic toxicity, tyrosinase activity, and melanin content in zebrafish. α-Arbutin, raspberry ketone (RK), raspberry ketone glucoside (RKG), glabridin (GLA), and 3-o-ethyl-ascorbic (EA), inhibited the activity of mushroom tyrosinase; dipotassium glycyrrhizinate (DG) did not inhibit mushroom tyrosinase activity, and glycyrrhetic acid (GA) promoted tyrosinase activity. Tyrosinase activity was inhibited by α-arbutin, GLA, GA and DG in B16-F10 cells; RK, RKG and EA did not inhibit tyrosinase activity. α-Arbutin, RK, RKG, EA, and GA, inhibited tyrosinase activity in zebrafish; GLA and DG did not inhibit tyrosinase activity. α-arbutin, RK, RKG, EA, GLA, and DG reduced melanin synthesis in B16-F10 cells in a dose-dependent manner without significant toxicity; GA did not inhibit melanin synthesis. α-arbutin, RK, RKG and GA significantly reduced melanin content on the zebrafish body surface. Mushroom tyrosinase analysis was the most practical assay among the three systems but had poor reliability. The B16-F10 mouse melanoma cell system was the most sensitive but had the worst stability. The zebrafish system had better reproducibility than other systems; however, most compounds were difficult to screen in this system.
... The main biologically active components of G. glabra are dipotassium glycyrrhizinate, glycyrrhizin, also known as glycyrrhizic acid, and its aglycone, glycyrrhetinic acid [206]. Dipotassium glycyrrhizinate has similar properties to those of corticosteroids, namely, anti-inflammatory, antiallergic, and antibiotic activities, without the side effects of allergic reactions on the skin [208]. This property is due to dipotassium glycyrrhizinate's ability to efficiently inhibit the activity of phospholipase A2 enzyme, which is necessary for several inflammatory processes [209][210][211]. ...
Cancer, a major world public health problem, is associated with chemotherapy treatments whose administration leads to secondary concerns, such as oral mucositis (OM). The OM disorder is characterized by the presence of ulcers in the oral mucosa that cause pain, bleeding, and difficulty in ingesting fluids and solids, or speaking. Bioactive compounds from natural sources have arisen as an effective approach for OM. This review aims to summarize the new potential application of different natural products in the prevention and treatment of OM in comparison to conventional ones, also providing a deep insight into the most recent clinical studies. Natural products, such as Aloe vera, Glycyrrhiza glabra, Camellia sinensis, Calendula officinalis, or honeybee crops, constitute examples of sources of bioactive compounds with pharmacological interest due to their well-reported activities (e.g., antimicrobial, antiviral, anti-inflammatory, analgesic, or wound healing). These activities are associated with the bioactive compounds present in their matrix (such as flavonoids), which are associated with in vivo biological activities and minimal or absent toxicity. Finally, encapsulation has arisen as a future opportunity to preserve the chemical stability and the drug bioa vailability of bioactive compounds and, most importantly, to improve the buccal retention period and the therapeutic effects.
... Chitosan (CS) has been tried for drug delivery in wound dressings, and has proven to be biocompatible, biodegradable, non-toxic, and antibacterial [ 33 , 34 ]. Dipotassium glycyrrhizinate (DG), the water-soluble salt of glycyrrhizin acid (GA) isolated from licorice root and widely used in pharmaceuticals and cosmetics, has demonstrated anti-allergic, anti-tumor, antibacterial, anti-virus, and anti-inflammatory effects [35][36][37][38] . Therefore, we adopted hydroxypropyl chitosan (HPCS) reversibly cross-linked with PNIPAM to build large-crack self-healing and thermosentivie hydrogels. ...
Severe skin injuries are hard to repair and susceptible to bacterial infection. Development of a versatile antimicrobial anti-inflammatory hydrogel dressing that eliminates concern over antibiotic resistance is urgently needed but remains an elusive goal. Our research, described herein, the design and fabrication of a new family of supramolecular hydrogels based on hydroxypropyl chitosan (HPCS) and poly(N-isopropylacrylamide) (PNIPAM) may prove to be that goal. Employing the reversible cross-linking by β-cyclodextrin (β-CD) and adamantyl (AD) pre-assembly, the hydrogels can be formed in a facile one-pot method. Additionally, the structure and performance of the hydrogels can be controlled by a simple adjustment of the AD content. The obtained hydrogels exhibit an abundance of desired properties; they are injectable, thermosensitive, highly ductile, self-healable (will self-heal recurring damage to the hydrogel bandage of up to several millimeters wide), biocompatible, and have antimicrobial activity against Staphylococcus aureus when infused with dipotassium glycyrrhizinate (DG). Using a mouse full-thickness skin defect model, in vivo wound healing evaluations revealed that the DG-loaded hydrogels (HP-3/DG10) applied to the wound resulted in rapid wound closure. The hydrogels promoted efficient tissue remolding, collagen deposition, decreased inflammation and performed better than the control groups of commercial TegadermTM film and 3M dressing. Given their multifunctionality and in vivo efficacy, the DG-loaded HP hydrogels hold great potential as a wound dressing for full-thickness skin repair.
Statement of Significance
: Injectable hydrogels are receiving increasing attention as an ideal wound dressing. To the best of our knowledge, however, injectable and wide-crack self-healing hydrogel dressings have been hardly studied. A versatile antimicrobial hydrogel without drug resistance or cytotoxicity is also highly required. Therefore, in the present study, we constructed injectable thermosensitive and wide-crack self-healing hydrogels with antibacterial and anti-inflammatory properties. These hydrogels were developed through novel strategies of the wide-crack self-healing design and the loading of the bioactive antibacterial and anti-inflammatory agent dipotassium glycyrrhizinate. The simple preparation method and multifunctionality of the studied hydrogel composites may provide important insights for the development of future biomaterials for wound dressings and other biomedical applications.
... 78 These cytokine-induced FTSE models can be utilized to evaluate the possible effects of small molecules in alleviating AD inflammation. For example, Lee, et al. 79 used dipotassium glycyrrhizinate in IL-4/IL-13-induced AD skin equivalents and showed that dipotassium glycyrrhizinate successfully suppressed cytokine release and ameliorated epidermal phenotype and gene expression patterns. ...
The skin is the first line of defense of our body, and it is composed of the epidermis and dermis with diverse immune cells. Various in vitro models have been investigated to recapitulate the immunological functions of the skin and to model inflammatory skin diseases. The simplest model is a two-dimensional (2D) co-culture system, which helps understand the direct and indirect cell-to-cell interactions between immune and structural cells; however, it has limitations when observing three-dimensional (3D) interactions or reproducing skin barriers. Conversely, 3D skin constructs can mimic the human skin characteristics in terms of epidermal and dermal structures, barrier functions, cell migration, and cell-to-cell interaction in the 3D space. Recently, as the importance of neuro-immune-cutaneous interactions in the inflammatory response is emerging, 3D skin constructs containing both immune cells and neurons are being developed. A microfluidic culture device called "skin-on-a-chip," which simulates the structures and functions of the human skin with perfusion, was also developed to mimic immune cell migration through the vascular system. This review summarizes the in vitro skin models with immune components, focusing on two highly prevalent chronic inflammatory skin diseases: atopic dermatitis and psoriasis. The development of these models will be valuable in studying the pathophysiology of skin diseases and evaluating the efficacy and toxicity of new drugs.
... A full-thickness human 3-D-skin model was reconstructed through slight modification of our previously described protocols [37,38]. Briefly, dermal mixture was prepared by mixing Type-I collagen (3 mg/mL, Nitta gelatin, Osaka, Japan), reconstruction buffer (Nitta gelatin), DE concentrated culture solution (Nitta gelatin), fibrinogen (10 mg/mL, Sigma-Aldrich. ...
Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.
... KG decreased AD-related gene expression in Th2 cytokine-stimulated keratinocytes. KG alleviated AD phenotypes and gene expression patterns and inhibited AD-related cytokine release in disease models [94]. ...
This review aims to be an update of Bioengineered Artificial Skin Substitutes (BASS) applications. At the first moment, they were created as an attempt to replace native skin grafts transplantation. Nowadays, these in vitro models have been increasing and widening their application areas, becoming important tools for research. This study is focus on the ability to design in vitro BASS which have been demonstrated to be appropriate to develop new products in the cosmetic and pharmacology industry. Allowing to go deeper into the skin disease research, and to analyze the effects provoked by environmental stressful agents. The importance of BASS to replace animal experimentation is also highlighted. Furthermore, the BASS validation parameters approved by the OECD (Organisation for Economic Cooperation and Development) are also analyzed. This report presents an overview of the skin models applicable to skin research along with their design methods. Finally, the potential and limitations of the currently available BASS to supply the demands for disease modeling and pharmaceutical screening are discussed.
... ere are also experimental evidences supporting that these herbs have anti-inflammatory and antiallergic effects, which may help with AD treatment. Angelicae Gigantis Radix and the main component (glycyrrhizin) and derivatives (dipotassium glycyrrhizinate) of Glycyrrhizae Radix et Rhizoma showed excellent anti-inflammatory effects in the in vitro and in vivo AD models [37][38][39]. Moreover, these two herbs have shown to reduce inflammation and improve AD symptoms such as itching in the AD model even in topical use [40,41]. ...
Objectives:
Herbal medicine (HM) is attracting attention for treating atopic dermatitis (AD). This overview was conducted to summarize and critically evaluate the current systematic reviews (SRs) on HM for the treatment of AD.
Methods:
Through comprehensive searches, all relevant SRs on HM for AD published until May 2020 were included. The quality of included SRs was assessed using the AMSTAR-2 tool. Moreover, original randomized controlled trials (RCTs) included in the SRs were resynthesized to investigate the efficacy and safety of oral HM for AD. The quality of evidence for the main findings was evaluated using the GRADE approach.
Results:
Nine SRs were included in this overview. HM showed significantly better efficacy in terms of total effective rate (TER), itching and sleep symptom scores, quality of life, and the dose of topical treatment used compared with placebo. HM as a monotherapy and/or an adjunctive therapy to conventional medication (CM) showed significantly better results on the efficacy, symptom relief, and some laboratory parameters related to the inflammatory response. The methodological quality was generally low. When 58 original RCTs were reanalyzed, HM showed significantly lower SCORing Atopic Dermatitis (SCORAD) score and higher TER than the placebo or CM. In terms of the safety profile, HM was not significantly different from the placebo and was better than CM. The quality of evidence ranged from "moderate" to "very low."
Conclusion:
The results suggested that HM as a monotherapy or an adjunctive therapy is promising for the treatment of AD. However, due to low methodological quality and low quality of evidence, further rigorous, well-designed, high-quality SRs, and RCTs are needed to make clinical recommendations on HM use.
... Epidermal skin models were established through slight modification of our previously described protocol [16]. Briefly, keratinocytes were seeded in inserts of the Snapwell TM device (3407, Corning, Kennebunk, ME, USA) and cultured in keratinocyte medium. ...
Objective
Skin aging is inevitably exposed through its typical features such as wrinkles and sagging. Therefore, skin anti‐aging is a major issue in cosmetic research to prevent and improve aging symptoms using effective ingredients. Chondroitin sulfate (CS), a type of glycosaminoglycan, is an important structural component of the extracellular matrix (ECM) and is involved in various biological processes, such as cell proliferation, differentiation, and migration. Here, we aimed to investigate the effects of CS on skin regeneration and examine its efficacy as a potential safe and effective skin anti‐aging ingredient.
Methods
We investigated the effects of CS on cell proliferation in normal human keratinocytes and fibroblasts. Then, cell migration, ECM synthesis, and related signaling pathways were examined in fibroblasts through gene and protein expression analysis. Finally, the effect on skin wound healing and regeneration was validated using a full‐thickness skin wound model and an aged skin model.
Results
CS treatment increased the proliferation of keratinocytes and fibroblasts. It also stimulated the migration and synthesis of ECM components of fibroblasts. Further analysis revealed that CS induced the expression of type I procollagen by activating the extracellular signal‐regulated kinase pathway. Using a full‐thickness skin wound model and an aged skin model, we confirmed that CS treatment promoted skin wound healing and regeneration.
Conclusion
Together, our results indicated that CS has the potential to facilitate skin regeneration, implying that CS could be clinically applied to improve skin aging.
... The variation in the production of interferon-b appeared depending on the experiments was noteworthy, and we did not test other proinflammatory cytokines such as IL-1b and IL-8 (Mori et al. 2018). Higher concentrations of GK2 presumably show stronger inhibitory effects, given that much higher concentrations of GK2 (up to 400 lM) were reportedly avirulent and showed higher anti-inflammatory effects (Lee et al. 2019). ...
HaCaT cells have been widely used as undifferentiated epidermal keratinocytes, since these non-tumorigenic cells can be readily maintained in conventional medium and partly retain epidermal differentiation potential upon stimulation with high concentration of calcium. In contrast to primary epidermal keratinocytes, however, these cells never form tight junction (TJ), a specific structure in highly differentiated keratinocytes, solely by the differentiation stimulation. Here, we show that HaCaT cells secrete a considerable amount of high mobility group box-1 protein (HMGB1), one of major inflammatory mediator, which appeared to be responsible, at least in part, for such aberrant differentiation response. So far, inhibition of c-Jun N-terminal kinase (JNK) in high calcium medium has been supposed to be the only way to induce TJ formations in HaCaT cells; however, SP600125, a potent inhibitor of JNK showed cytostatic effects and clearly attenuated epidermal differentiation and stratification. In contrast, dipotassium glycyrrhizate (GK2), a soluble analogue of HMGB1-blocker Glycyrrhizin, down-regulated interferon-β, a typical inflammatory cytokine induced by secreted HMGB1, and accelerated differentiation responses to the calcium treatment in these cells. In addition, GK2-treatmenrt resulted in the formation of double cell layers in cultured HaCaT cells, where the stratified upper cells transiently accumulated TJ proteins at the cell-cell contact sites. These results highlight the importance of attenuation of secreted HMGB1-signals in cultured HaCaT cells for studies of functional keratinocytes.
Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL‐4 and IL‐13 induce AD‐like features in keratinocytes in vitro . However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single‐cell RNA sequencing data (scRNA‐seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL‐4 and IL‐13 induced the closest in vivo ‐like AD phenotype which was observed in the scRNA‐seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM‐associated genes such as POSTN . While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo . Taken together, our comprehensive study shows that the simple model using IL‐4 or IL‐13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro , which may aid the discovery of novel treatment options.
Atopic dermatitis (AD) is one of the most prevalent inflammatory skin diseases that is characterized by eczematous rashes, intense itching, dry skin, and sensitive skin. Although AD significantly impacts the quality of life and the number of patients keeps increasing, its pathological mechanism is still unknown because of its complexity. The importance of developing new in vitro three-dimensional (3D) models has been underlined in order to understand the mechanisms for the development of therapeutics since the limitations of 2D models or animal models have been repeatedly reported. Thus, the new in vitro AD models should not only be created in 3D structure, but also reflect the pathological characteristics of AD, which are known to be associated with Th2-mediated inflammatory responses, epidermal barrier disruption, increased dermal T-cell infiltration, filaggrin down-regulation, or microbial imbalance. In this review, we introduce various types of in vitro skin models including 3D culture methods, skin-on-a-chips, and skin organoids, as well as their applications to AD modeling for drug screening and mechanistic studies.
Epidermal changes are histological hallmarks of secondary lymphedema, but it is unknown if keratinocytes contribute to its pathophysiology. Using clinical lymphedema specimens and mouse models, we show that keratinocytes play a primary role in lymphedema development by producing T-helper 2 (Th2) -inducing cytokines. Specifically, we find that keratinocyte proliferation and expression of protease-activated receptor 2 (PAR2) are early responses following lymphatic injury and regulate the expression of Th2-inducing cytokines, migration of Langerhans cells, and skin infiltration of Th2-differentiated T cells. Furthermore, inhibition of PAR2 activation with a small molecule inhibitor or the proliferation inhibitor teriflunomide (TF) prevents activation of keratinocytes stimulated with lymphedema fluid. Finally, topical TF is highly effective for decreasing swelling, fibrosis, and inflammation in a preclinical mouse model. Our findings suggest that lymphedema is a chronic inflammatory skin disease, and topically targeting keratinocyte activation may be a clinically effective therapy for this condition.
The environment is being continuously deteriorated by air pollution and global warming, increased pressure of work or family on life, and changes in lifestyle; these factors have contributed to an increase in the proportion of both females and males with sensitive skin. To this end, the cosmetics market has focused on developing hypoallergenic or skin-soothing and make-up products. Here, we summarize the definition and causes of sensitive skin, list the mechanisms underlying the development of this condition, and review the natural plant products and purified active components that have potential for use in anti-allergic cosmetics. A review of studies that evaluated the anti-allergic properties of natural products was conducted. There has been a progressive increase in the number of natural products identified as potential anti-allergic raw materials in cosmetics. Finally, we suggest strategies for developing anti-allergic cosmetics in the future.
Objective:
The purpose of this study was to develop pluronic F127/D-a-tocopheryl polyethylene glycol 1000 succinate mixed micelles-based hydrogel (MMs-gel) for topical delivery of GL to improve its skin permeability and atopic dermatitis (AD) treatment.
Significance:
GL loaded MMs-gel (GL-MMs-gel) could be potentially used as a promising nanocarrier for the treatment of AD.
Methods:
GL-MMs were prepared by thin film hydration method and then loaded into carbopol gel. The formulation of GL-MMs-gel was optimized by full factorial design and systematically characterized for drug content, pH, spreadability, in vitro drug release and percutaneous permeation, etc. The therapeutic effect of GL-MMs-gel was also investigated in AD-like skin lesion model in BALB/c mice and compared with GL solution-based gel (GL-sol-gel).
Results:
Spherical GL-MMs with particle size of ∼30 nm were successfully incorporated into carbopol gel to form GL-MMs-gel with drug content of (98.80 ± 1.30) %, pH of 6.0 ± 0.08, and spreadability of (7.1 ± 0.2) cm. In vitro drug release profile of GL-MMs-gel exhibited a sustained-release behavior. The permeation flux for GL-MMs-gel (5.15 ± 0.33 µg/cm2/h) was significantly higher than that of GL-sol-gel (3.08 ± 0.34 µg/cm2/h) and GL-MMs-gel increased the accumulative amounts of GL in rats' skin 8.41 times than GL-sol-gel. The GL-MMs-gel was more effective than GL-sol-gel in suppressions of various AD symptoms including skin lesions, edema, high IgE levels, epidermal hyperplasia, and mast cell infiltration.
Conclusion:
All results revealed that MMs-gel could be a promising carrier for topical delivery of GL for the treatment of AD.
Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of actin filaments, thereby modulating tight junction (TJ) integrity, but the detailed molecular mechanisms of this remain unclear. In this study, we first found that GL binds to the first PDZ domain of zonula occludens-1 (ZO-1(PDZ1)) through NMR experiments. The structure of the GL-ZO-1(PDZ1) complex was then constructed using HADDOCK with the transferred nuclear Overhauser effect-based inter-hydrogen distance constraints as well as restrictions on the interfacial residues identified from 1H-15N HSQC spectral changes. We identified the relevant interactions between the glucuronate-2 moiety of GL and the carboxylate binding loop of the ligand binding site of ZO-1(PDZ1). We further examined the interaction of ZO-1(PDZ1) with glycyrrhetinic acid and with GA-3-monoglucuronide and observed a much lower affinity for each than for that with GL, with good agreement with the model. The other contacts found in the model were examined by using an amino acid substitution mutant of ZO-1(PDZ1). Finally, we reproduced the experiments reported by Sakai et al. in which high-dose GL prolonged the TJ-opening mediated with sodium deoxycholate as indicated by reduced transepithelial electrical resistance.
Safe and efficient drugs to combat the current COVID-19 pandemic are urgently needed. In this context, we have analyzed the anti-coronavirus potential of the natural product glycyrrhizic acid (GLR), a drug used to treat liver diseases (including viral hepatitis) and specific cutaneous inflammation (such as atopic dermatitis) in some countries. The properties of GLR and its primary active metabolite glycyrrhetinic acid are presented and discussed. GLR has shown activities against different viruses, including SARS-associated Human and animal coronaviruses. GLR is a non-hemolytic saponin and a potent immuno-active anti-inflammatory agent which displays both cytoplasmic and membrane effects. At the membrane level, GLR induces cholesterol-dependent disorganization of lipid rafts which are important for the entry of coronavirus into cells. At the intracellular and circulating levels, GLR can trap the high mobility group box 1 protein and thus blocks the alarmin functions of HMGB1. We used molecular docking to characterize further and discuss both the cholesterol- and HMG box-binding functions of GLR. The membrane and cytoplasmic effects of GLR, coupled with its long-established medical use as a relatively safe drug, make GLR a good candidate to be tested against the SARS-CoV-2 coronavirus, alone and in combination with other drugs. The rational supporting combinations with (hydroxy)chloroquine and tenofovir (two drugs active against SARS-CoV-2) is also discussed. Based on this analysis, we conclude that GLR should be further considered and rapidly evaluated for the treatment of patients with COVID-19.
The development of an efficient ocular drug delivery system is helpful in improving the ocular diffusion of topically delivered drugs as well as enhancing drugs therapeutic efficacy. The objective of this study was to explore the potential of self-assembled nanomicelles based on glycyrrhizin in ocular topical applications. In brief, a dipotassium glycyrrhizinate (DG)-based nanomicelle ophthalmic solution encapsulating thymol (DG-THY) was developed using a simple thin-film dispersion method. The optimal formulation featured a DG/thymol (THY) weight ratio of 9:1 and an encapsulation efficiency of 98.25 ± 1.16%; the nanomicelles were ultra-small spheres with an average particle size of 3.30 ± 0.39 nm, a polydispersity index of 0.22 ± 0.02, and an electrically negative surface (-[10.03 ± 1.31] mV) for the optimized DG-THY. This DG-THY ophthalmic solution was observed to be stable upon good storage at both 4 °C and 25 °C for 12 weeks. The DG-THY was observed to remarkably improve in vitro antioxidant activity, in vitro release, and the membrane permeation of THY. The DG-THY ophthalmic solution proved to be very well-tolerated in a rabbit model. The DG-THY ophthalmic solution also demonstrated distinct improvements in the ex vivo and in vivo intraocular permeations of THY. The DG-THY ophthalmic solution also exhibited decreased minimal inhibitory concentrations and minimum bactericidal concentrations of THY. Treatment with the DG-THY ophthalmic solution significantly relieved ocular infection symptoms in rabbit eyes by lowering the number of colony-forming units recovered from the corneas. Therefore, these results demonstrate that DG-THY may be a promising new ophthalmic formulation for the treatment of ocular diseases, especially in terms of oxidative stress-, bacteria-, and inflammation-related eye diseases.
The human skin is involved in protecting the inner body from constant exposure to outer environmental stimuli. There is an evident need to screen for toxicity and the efficacy of drugs and cosmetics applied to the skin. To date, animal studies are still the standard method for substance testing, although they are currently controversially discussed Therefore, the multi-organ chip is an attractive alternative to replace animal testing. The two-organ chip is designed to hold 96-well cell culture inserts (CCIs). Small-sized skin equivalents are needed for this. In this study, full-thickness skin equivalents (ftSEs) were generated successfully inside 96-well CCIs. These skin equivalents developed with in vivo-like histological architecture, with normal differentiation marker expressions and proliferation rates. The 96-well CCI-based ftSEs were successfully integrated into the two-organ chip. The permeation of fluorescein sodium salt through the ftSEs was monitored during the culture. The results show a decreasing value for the permeation over time, which seems a promising method to track the development of the ftSEs. Additionally, the permeation was implemented in a computational fluid dynamics simulation, as a tool to predict results in long-term experiments. The advantage of these ftSEs is the reduced need for cells and substances, which makes them more suitable for high throughput assays.
The skin barrier is an important shield regulating the outside-in as well as inside-out penetration of water, nutrients, ions and environmental stimuli. We can distinguish four different barrier compartments, the physical, chemical, immunological and microbial skin barrier. Well-functioning of those is needed to protect our body from the environment. To better understand the function and the contribution of barrier dysfunction in skin diseases, 3D skin or epidermal models are a valuable tool for in vitro studies. In this review we summarize the development and application of different skin models in skin barrier research. During the last years enormous effort was made on optimizing these models to better mimic the in vivo composition of the skin, by fine-tuning cell culture media, culture conditions and including additional cells and tissue components. Thereby in vitro barrier formation and function has been improved significantly. Moreover, in this review we point towards changes and chances for in vitro 3D skin models to be used for skin barrier research in the nearby future. This article is protected by copyright. All rights reserved.
Atopic dermatitis (AD) is a chronic inflammatory skin disease of increasing prevalence, especially in industrialized countries. Roughly 25% of the children and 1-3% of adults are affected. Although significant progress has been made in the understanding of the pathogenesis of AD, many aspects remain poorly understood. Moreover, there is a pressing need for improved therapeutic options. Studies to elucidate the pathophysiological pathways of AD and to identify novel therapeutic targets over the last few decades have been conducted almost exclusively in animal models. However, in vitro approaches such as 3D skin disease models have recently emerged due to an increasing awareness of distinct inter-species related differences that hamper the effective translation of results from animal models to humans. In addition, there is growing political and social pressure to develop alternatives to animal models according to the 3Rs principle (reduction, refinement and replacement of animal models). In this review, we briefly summarize commonly used animal models of AD and discuss the advancements and limitations of human-based in vitro models in AD research. Moreover, we address aspects where further improvements are urgently required.
Atopic dermatitis (AD) is a chronic inflammatory skin disease causing a strong impact on quality of life. Its pathophysiology is the result of complex interactions involving immunological, genetic and environmental factors. Although there are several published in vitro three-dimensional models mimicking AD, none of them have taken all these pathophysiological features into account; thus, finding the right model may be complicated. This paper reviews the literature on the different reconstructed epidermis models of AD as well as their relevance. We focused our attention on both the defect of the epidermal barrier and the inflammation linked to the immune system.
As the outermost physical barrier of an organism, the skin is diurnally exposed to UV rays (UVRs). Recent studies have revealed that the skin exhibits a circadian rhythm in various functions, and this oscillation is disturbed and reset via a strong environmental cue, the UVRs. However, a molecular link between circadian perturbation by UVRs and UVR-induced cellular responses has not been investigated. We identified tissue inhibitor of metalloproteinase (TIMP)-3 as a novel circadian locomotor output cycles kaput (CLOCK)-dependent diurnal gene by using a CLOCK-knockdown strategy in human keratinocytes. Among dozens of identified transcripts down-regulated by CLOCK knockdown, TIMP3 displayed a rhythmic expression in a CLOCK-dependent manner, in which the expression of matrix metalloproteinase (MMP)-1 and inflammatory cytokines, such as TNF-α, chemokine (C-X-C motif) ligand (CXCL)-1, and IL-8, were inversely regulated. Upon UVB exposure, the expression of CLOCK and TIMP3 was down-regulated, which led to an up-regulation of secretion of MMP1 and TNF-α proteins and in the transcription of CXCL1 and IL-8via CCAAT-enhancer binding protein (C/EBP)-α. UVB-induced TNF-α secretion increased further or decreased by knockdown or overexpression of TIMP3, respectively, as well as by CLOCK As a novel CLOCK-dependent diurnal gene, TIMP3 inhibits the expression of inflammatory cytokines that are up-regulated by UV-irradiation in human keratinocytes. Thus, our work suggests a molecular link between circadian perturbation by UVRs and UVR-induced inflammation.-Park, S., Kim, K., Bae, I.-H., Lee, S. H., Jung, J., Lee, T. R., Cho, E.-G. TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.
Atopic dermatitis (AD) is a complex inflammatory skin condition that is not fully understood. Epidermal barrier defects and Th2 immune response dysregulations are thought to play crucial roles in the pathogenesis of the disease. A vicious circle takes place between these alterations, and it can further be complicated by additional genetic and environmental factors. Studies investigating in more depth the etiology of the disease are thus needed in order to develop functional treatments. In recent years, there have been significant advances regarding in vitro models reproducing important features of AD. However, since a lot of models have been developed, finding the appropriate experimental setting can be difficult. Therefore, herein, we review the different types of in vitro models mimicking features of AD. The simplest models are two-dimensional culture systems composed of immune cells or keratinocytes, whereas three-dimensional skin or epidermal equivalents reconstitute more complex stratified tissues exhibiting barrier properties. In those models, hallmarks of AD are obtained, either by challenging tissues with interleukin cocktails overexpressed in AD epidermis or by silencing expression of pivotal genes encoding epidermal barrier proteins. Tissue equivalents cocultured with lymphocytes or containing AD patient cells are also described. Furthermore, each model is placed in its study context with a brief summary of the main results obtained. In conclusion, the described in vitro models are useful tools to better understand AD pathogenesis, but also to screen new compounds in the field of AD, which probably will open the way to new preventive or therapeutic strategies.
Background
Inadequate representation of the human tissue environment during a preclinical screen can result in inaccurate predictions of compound effects. Consequently, pharmaceutical investigators are searching for preclinical models that closely resemble original tissue for predicting clinical outcomes.
Methods
The current research aims to compare the impact of using serum-free medium instead of complete culture medium during the last step of psoriatic skin substitute reconstruction. Skin substitutes were produced according to the self-assembly approach.
Results
Serum-free conditions have no negative impact on the reconstruction of healthy or psoriatic skin substitutes presented in this study regarding their macroscopic or histological appearances. ATR-FTIR results showed no significant differences in the CH2 bands between psoriatic substitutes cultured with or without serum, thus suggesting that serum deprivation did not have a negative impact on the lipid organization of their stratum corneum. Serum deprivation could even lead to a better organization of healthy skin substitute lipids. Percutaneous analyses demonstrated that psoriatic substitutes cultured in serum-free conditions showed a higher permeability to hydrocortisone compared to controls, while no significant differences in benzoic acid and caffeine penetration profiles were observed.
Conclusions
Results obtained with this 3D-psoriatic skin substitute demonstrate the potential and versatility of the model. It could offer good prediction of drug related toxicities at preclinical stages performed in order to avoid unexpected and costly findings in the clinic.
General significance
Together, these findings offer a new approach for one of the most important challenges of the 21st century, namely, prediction of drug toxicity.
Context: Increasing incidence and impact of inflammatory diseases have encouraged the search of new pharmacological strategies to face them. Licorice has been used to treat inflammatory diseases since ancient times in China.
Objective: To summarize the current knowledge on anti-inflammatory properties and mechanisms of compounds isolated from licorice, to introduce the traditional use, modern clinical trials and officially approved drugs, to evaluate the safety and to obtain new insights for further research of licorice.
Methods: PubMed, Web of Science, Science Direct and ResearchGate were information sources for the search terms ‘licorice’, ‘licorice metabolites’, ‘anti-inflammatory’, ‘triterpenoids’, ‘flavonoids’ and their combinations, mainly from year 2010 to 2016 without language restriction. Studies were selected from Science Citation Index journals, in vitro studies with Jadad score less than 2 points and in vivo and clinical studies with experimental flaws were excluded.
Results: Two hundred and ninety-five papers were searched and 93 papers were reviewed. Licorice extract, 3 triterpenes and 13 flavonoids exhibit evident anti-inflammatory properties mainly by decreasing TNF, MMPs, PGE2 and free radicals, which also explained its traditional applications in stimulating digestive system functions, eliminating phlegm, relieving coughing, nourishing qi and alleviating pain in TCM. Five hundred and fifty-four drugs containing licorice have been approved by CFDA. The side effect may due to the cortical hormone like action.
Conclusion: Licorice and its natural compounds have demonstrated anti-inflammatory activities. More pharmacokinetic studies using different models with different dosages should be carried out, and the maximum tolerated dose is also critical for clinical use of licorice extract and purified compounds.
Figure 1. Impact of IL-4 and IL-13 on histology, epidermal thickness, and skin surface pH of normal and filaggrin knockdown skin equivalents. (a) Representative histologic staining of untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Bar = 100 μm. (b) Epidermal thickness of untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Values are given as mean ± SEM. n = 3–4. (c) Skin equivalent surface pH values. Values are given as mean ± SEM. n = 3. Plus sign indicates statistical significance over FLG– untreated: +++P ≤ 0.001.
This study provides the scientific basis for the anti-inflammatory effects of licorice extract in a t-BHP (tert-butyl hydrogen peroxide)-induced liver damage model and the effects of its ingredients, glycyrrhizic acid (GA), liquiritin (LQ) and liquiritigenin (LG), in a lipopolysaccharide (LPS)-stimulated microglial cell model. The GA, LQ and LG inhibited the LPS-stimulated elevation of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interleukin (IL)-6 in BV2 (mouse brain microglia) cells. Furthermore, licorice extract inhibited the expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the livers of t-BHP-treated mice models. This result suggested that mechanistic-based evidence substantiating the traditional claims of licorice extract and its three bioactive components can be applied for the treatment of inflammation-related disorders, such as oxidative liver damage and inflammation diseases.
Atopic dermatitis is a chronic inflammatory skin disease with defects in the epidermal barrier. In a cohort of African-American children, a FLG2 nonsense mutation has been associated with the disease. In the epidermis of European patients, the expression of filaggrin-2, the filaggrin-related protein encoded by FLG2, is decreased. To describe the function of filaggrin-2 and evaluate the impact of its deficiency, its expression was downregulated using lentivirus-mediated shRNA interference in a three-dimensional reconstructed human epidermis (RHE) model. This resulted in parakeratosis and a compact stratum corneum, presence of abnormal vesicles inside the corneocytes, increased pH and reduced amounts of free amino acids at the RHE surface, leading to increased sensitivity to UVB radiations. The expression of differentiation markers was slightly modified. However, we observed reduced proteolytic processing of corneodesmosin, hornerin and filaggrin in parallel with reduced amounts of caspase-14 and bleomycin hydrolase. Our data demonstrated that filaggrin-2 is important for a proper cornification and a functional stratum corneum. Its downregulation in atopic patients may be involved in the disease-associated epidermis impairment.
Atopic dermatitis is a chronic inflammatory skin disorder characterized by defects in the epidermal barrier and keratinocyte differentiation. The expression of filaggrin, a protein thought to play a major role in the function of the epidermis, is down-regulated. However, the impact of this deficiency on keratinocytes is not really known. This was investigated using lentivirus-mediated shRNA interference in a three-dimensional reconstructed human epidermis (RHE) model, in the absence of other cell types than keratinocytes. Similarly to what is known for atopic skin, the experimental filaggrin down-regulation resulted in hypogranulosis, a disturbed corneocyte intracellular matrix, reduced amounts of natural moisturizing factor components, an increased permeability and UV-B sensitivity of the RHE and impaired keratinocyte differentiation, at the mRNA and protein levels. In particular, the amounts of two filaggrin-related proteins and one protease involved in the degradation of filaggrin, bleomycin hydrolase, were lower. In addition, caspase-14 activation was reduced. These results demonstrate the importance of filaggrin for the stratum corneum properties/functions. They indicate that filaggrin down-regulation in the epidermis of atopic patients, either acquired or innate, may be directly responsible for some of the disease-related alterations in the epidermal differentiation program and epidermal barrier function.Journal of Investigative Dermatology accepted article preview online, 18 June 2014; doi:10.1038/jid.2014.259.
Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are up-regulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (TNF-α) cytokines on epidermal morphogenesis, proliferation, differentiation and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31 and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.Journal of Investigative Dermatology accepted article preview online, 11 February 2014; doi:10.1038/jid.2014.83.
High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. Advances in targeting HMGB1 represent a major challenge to improve the treatment of acute/chronic inflammation.
This study is aimed at verifying whether the inhibition of HMGB1 through dipotassium glycyrrhizate (DPG) is a good strategy to reduce intestinal inflammation.
Human colon adenocarcinoma cell line, HT29, human epithelial colorectal adenocarcinoma, Caco2, and murine macrophage cell line, RAW 264.7, were cultured to investigate the effect of DPG on the secretion of HMGB1. Acute colitis was induced in C57BL/6 mice through administration of 3% dextran sodium sulphate (DSS); a combined treatment with DSS and 3 or 8 mg/kg/day DPG was used to investigate the effects of DPG on intestinal inflammation. Animals were euthanized at seventh day and colonic samples underwent molecular and histological analyses.
DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1. Moreover, DPG significantly decreases the severity of DSS-induced colitis in mice. Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4. Finally, HMGB1, abundantly present in the feces of mice with DSS-induced colitis, is strongly reduced by DPG.
HMGB1 is an early pro-inflammatory cytokine and an active protagonist of mucosal gut inflammation. DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation. Thus, we reason that DPG could represent an innovative tool for the management of human intestinal inflammation.
Cutaneous homeostasis and defenses are maintained by permanent cross-talk among particular epidermal keratinocytes and immune cells residing or recruited in the skin, through the production of cytokines. If required, a coordinated inflammatory response is triggered, relayed by specific cytokines. Due to numerous reasons, troubles in the resolution of this phenomenon could generate a cytokine-mediated vicious circle, promoting skin chronic inflammation, the most common being atopic dermatitis and psoriasis. In this paper, we discuss the biological effects of cytokine on keratinocytes, more particularly on specific or shared cytokines involved in atopic dermatitis or psoriasis. We report and discuss monolayer or 3D in vitro models of keratinocytes stimulated by specific sets of cytokines to mimic atopic dermatitis or psoriasis. IL-22, TNFa, IL-4, and IL-13 combination is able to mimic an "atopic dermatitis like" state. In psoriasis lesions, over expression of IL-17 is observed whereas IL-4 and IL-13 were not detected; the replacement of IL-4 and IL-13 by IL-17 from this mix is able to mimic in vitro a "psoriasis like" status on keratinocytes. We conclude that specific cytokine environment deregulation plays a central role on skin morphology and innate immunity, moving towards specific pathologies and opening the way to new therapeutic strategies.
Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed in keratinocytes of the epidermis. We previously showed that AQP3-mediated transport of water and glycerol is involved in keratinocyte migration and proliferation, respectively. However, the involvement of AQP3 in epidermal hyperplasia in skin diseases, such as atopic dermatitis (AD), is unknown. In this study, we found significantly increased AQP3 transcript and protein expression in the epidermis of human AD lesions. The upregulation of AQP3 expression in human keratinocytes by transfection with human AQP3 DNA plasmid was associated with increased cellular glycerol and ATP, as well as increased cell proliferation. Among several cytokines and chemokines produced in the skin, CCL17, which is highly expressed in AD, was found to be a strong inducer of AQP3 expression and enhanced keratinocyte proliferation. In mouse AD models, AQP3 was strongly overexpressed in the epidermis in wild-type mice. Epidermal hyperplasia was reduced in AQP3-deficient mice, with a decreased number of proliferating keratinocytes. These results suggest the involvement of AQP3 in epidermal hyperplasia by a mechanism involving upregulated AQP3 expression and consequent enhancement of keratinocyte proliferation.
The pathogenesis of spongiosis, which is a well-known hallmark of acute eczema, is not fully understood. We sought to clarify the mechanism for the influx of tissue fluid into the epidermis and the loss of cohesion between keratinocytes in acute eczema that result in spongiosis. We first demonstrated increased intercellular accumulation of hyaluronan (HA) in the spongiotic epidermis by immunochemical staining using hyaluronic-acid-binding protein (HABP) and augmented hyaluronan synthase 3 (HAS3) mRNA expression by spongiotic keratinocytes using in situ hybridization. We also showed that the epidermis where the intercellular space was strongly stained with HABP showed weaker expression of membrane E-cadherin. Next, we demonstrated--by a sandwich assay using HABP, real-time PCR, and flow cytometry--that, among various cytokines, only IL-4, IL-13, and IFN-gamma increased HA production, enhanced HAS3 mRNA expression, and decreased membrane E-cadherin expression by normal human epidermal keratinocytes in both low- and high-Ca media. Finally, we demonstrated IL-4, IL-13, their combination, and IFN-gamma could induce intercellular space widening of the epidermis with increased HA accumulation and decreased E-cadherin expression in the organotypic culture. These results suggest that the augmented production of HA and the decreased E-cadherin expression by keratinocytes stimulated with IL-4/IL-13 or IFN-gamma cause spongiosis in acute eczema.
Plasma tumour necrosis factor-alpha (TNF-alpha) was measured in 15 children with atopic dermatitis, 13 children with bronchial asthma, and 11 healthy controls. Plasma TNF-alpha concentration was increased in atopic dermatitis and the magnitude of the increase was correlated with the severity of the dermatitis but TNF-alpha concentration was not increased in bronchial asthma. A significant correlation was found between plasma TNF-alpha and plasma histamine concentrations in atopic dermatitis. The data suggest that the overproduction of TNF-alpha is associated with increased plasma histamine concentration, and might play a part in the pathophysiological mechanism of atopic dermatitis.
Eosinophil accumulation is a prominent feature of allergic inflammatory reactions, such as those occurring in the lung of the allergic asthmatic, but the endogenous chemoattractants involved have not been identified. We have investigated this in an established model of allergic inflammation, using in vivo systems both to generate and assay relevant activity. Bronchoalveolar lavage (BAL) fluid was taken from sensitized guinea pigs at intervals after aerosol challenge with ovalbumin. BAL fluid was injected intradermally in unsensitized assay guinea pigs and the accumulation of intravenously injected 111In-eosinophils was measured. Activity was detected at 30 min after allergen challenge, peaking from 3 to 6 h and declining to low levels by 24 h. 3-h BAL fluid was purified using high performance liquid chromatography techniques in conjunction with the skin assay. Microsequencing revealed a novel protein from the C-C branch of the platelet factor 4 superfamily of chemotactic cytokines. The protein, "eotaxin," exhibits homology of 53% with human MCP-1, 44% with guinea pig MCP-1, 31% with human MIP-1 alpha, and 26% with human RANTES. Laser desorption time of flight mass analysis gave four different signals (8.15, 8.38, 8.81, and 9.03 kD), probably reflecting differential O-glycosylation. Eotaxin was highly potent, inducing substantial 111In-eosinophil accumulation at a 1-2 pmol dose in the skin, but did not induce significant 111In-neutrophil accumulation. Eotaxin was a potent stimulator of both guinea pig and human eosinophils in vitro. Human recombinant RANTES, MIP-1 alpha, and MCP-1 were all inactive in inducing 111In-eosinophil accumulation in guinea pig skin; however, evidence was obtained that eotaxin shares a binding site with RANTES on guinea pig eosinophils. This is the first description of a potent eosinophil chemoattractant cytokine generated in vivo and suggests the possibility that similar molecules may be important in the human asthmatic lung.
Eosinophils (Eos) and fibroblasts are known to play a major role in the pathogenesis of bronchial asthma and fibrotic lung disease. Therefore, we investigated whether Th1 and Th2 cytokines stimulate the production of Eo-activating chemokines by lung fibroblasts. Analyses of the culture supernatant using multiple steps of high-performance liquid chromatography demonstrated that interleukin (IL)-4 preferentially stimulates lung fibroblasts to secrete a peak of eosinophil chemotactic activity (ECA) which, upon N-terminal analyses, showed similar sequence to eotaxin, whereas interferon (IFN)-gamma had negligible effect on the release of this chemokine. In contrast, tumor necrosis factor (TNF)-alpha stimulated lung fibroblasts to release two peaks of activity that were found to correspond to eotaxin and regulated on activation, normal T cells expressed and secreted (RANTES), respectively. Interestingly, IL-4 synergized with TNF-alpha to increase greatly the production of three biochemically distinct eotaxin forms. In contrast, IFN-gamma synergized with TNF-alpha to increase RANTES production. Neither IL-2, IL-5, IL-6 nor IL-10 had an effect on lung fibroblasts' capacity to express or release eotaxin and RANTES. Upon appropriate cytokine stimulation, lung fibroblasts were also found to express messenger RNA for monocyte chemotactic protein (MCP)-3 and MCP-4 but not eotaxin-2. However, no ECA like MCP-3 or MCP-4 was detected. These observations suggest that the release of Th1 or Th2 cytokines in the lung tissue polarizes lung fibroblasts to produce either RANTES or eotaxin as major Eo attractants.
Glycyrrhiza glabra L. has been used in herbal medicine for skin eruptions, including dermatitis, eczema, pruritus and cysts. The effect of licorice extract as topical preparation was evaluated on atopic dermatitis. The plant was collected and extracted by percolation with suitable solvent. The extract was standardized, based on Glycyrrhizinic acid by using a titrimetry method. Different topical gels were formulated by using different co-solvents. After standardizing of topical preparations, the best formulations (1% and 2%) were studied in a double-blind clinical trial in comparison with base gel on atopic dermatitis over two weeks (30 patients in each group). Propylene glycol was the best co-solvent for the extract and Carbopol 940 as gelling agent showed the best results in final formulations. The quantity of glycyrrhizinic acid was determined 20.3% in the extract and 19.6% in the topical preparation. Two percent licorice topical gel was more effective than 1% in reducing the scores for erythema, oedema and itching over two weeks (p<0.05). The results showed that licorice extract could be considered as an effective agent for treatment of atopic dermatitis.
Tissue-engineered skin is now a reality. For patients with extensive full-thickness burns, laboratory expansion of skin cells to achieve barrier function can make the difference between life and death, and it was this acute need that drove the initiation of tissue engineering in the 1980s. A much larger group of patients have ulcers resistant to conventional healing, and treatments using cultured skin cells have been devised to restart the wound-healing process. In the laboratory, the use of tissue-engineered skin provides insight into the behaviour of skin cells in healthy skin and in diseases such as vitiligo, melanoma, psoriasis and blistering disorders.
Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells (DC) and can initiate allergic inflammation. The factors inducing the production of human TSLP are not known. In this study, we show that proinflammatory (TNF-alpha or IL-1alpha) and Th2 (IL-4 or IL-13) cytokines synergized to induce the production of TSLP in human skin explants. TSLP production in situ was restricted to epidermal keratinocytes of the suprabasal layer. TSLP production could not be inhibited by factors regulating Th2 inflammation, such as IL-10, TGF-beta, or IFN-gamma. Cytokine-treated skin culture supernatants induced the maturation of blood CD11c(+) DC in a TSLP-dependent manner. Our data provide the first evidence of TSLP induction and subsequent DC activation in human skin. Blocking TSLP-inducing cytokines could represent a novel strategy for the treatment of allergic diseases.
Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease prevalent worldwide. This study investigated the effects of glycyrrhizin, an extract of licorice root, on the well-established model of 2,4-dinitrochlorobenzene-induced AD-like symptoms in mice. The severity of dermatitis, histopathological changes, serum IgE levels, changes in expression of high-mobility group box 1 (HMGB1), the receptor for advanced glycation end products (RAGE), nuclear factor (NF)-κB and inflammatory cytokines were evaluated. Treatment with glycyrrhizin inhibited the HMGB1 signaling cascade and ameliorated the symptoms of AD. Furthermore, in an in vitro study, the expression of RAGE was detected in a mouse mast cell line, P815 cells, and rmHMGB1 was found to be a potent inducer of mast cell activation by increasing Ca2+ influx, upregulating the CD117 and activating NF-κB signaling; these effects were also inhibited by glycyrrhizin. These findings implicate HMGB1 in the pathogenesis of AD and suggest that GL could be an effective therapeutic approach for cutaneous inflammation.
Psoriasis is a complex chronic immune-mediated inflammatory cutaneous disease associated with the development of inflammatory plaques on the skin. Studies proved that the disease results from a deregulated interplay between skin keratinocytes, immune cells and the environment leading to a persisting inflammatory process modulated by pro-inflammatory cytokines and activation of T cells. However, a major hindrance to study the pathogenesis of psoriasis more in depth and subsequent development of novel therapies is the lack of suitable pre-clinical models mimicking the complex phenotype of this skin disorder. Recent advances in and optimization of three-dimensional skin equivalent models have made them attractive and promising alternatives to the simplistic monolayer cultures, immunological different in vivo models and scarce ex vivo skin explants. Moreover, human skin equivalents are increasing in complexity level to match human biology as closely as possible. Here, we critically review the different types of three-dimensional skin models of psoriasis with relevance to their application potential and advantages over other models. This will guide researchers in choosing the most suitable psoriasis skin model for therapeutic drug testing (including gene therapy via siRNA molecules), or to examine biological features contributing to the pathology of psoriasis. However, the addition of T cells (as recently applied to a de-epidermized dermis-based psoriatic skin model) or other immune cells would make them even more attractive models and broaden their application potential. Eventually, the ultimate goal would be to substitute animal models by three-dimensional psoriatic skin models in the pre-clinical phases of anti-psoriasis candidate drugs.
Impact statement
The continuous development of novel in vitro models mimicking the psoriasis phenotype is important in the field of psoriasis research, as currently no model exists that completely matches the in vivo psoriasis skin or the disease pathology. This work provides a complete overview of the different available in vitro psoriasis models and suggests improvements for future models. Moreover, a focus was given to psoriatic skin equivalent models, as they offer several advantages over the other models, including commercial availability and validity. The potential and reported applicability of these models in psoriasis pre-clinical research is extensively discussed. As such, this work offers a guide to researchers in their choice of pre-clinical psoriasis model depending on their type of research question.
Aim: To estimate the Anti-inflammatory effects of Glycyrrhiza glabra extract. Background: There is an increasing demand for herbal medicines, health products, pharmaceuticals. Glycyrrhiza glabra inn is a plant used in traditional medicine across the world for its ethnopharmacological value. It is found to contain important phytoconstituents such as glycyrrhizin, glycyrrhizinic acid, glabrin A and B and isoflavones. It is effectively used as anti-inflammatory, anti-bacterial, anti-fungal, anti-diabetic, anti-viral, anti-ulcer, antitussive, anti-oxidant, skin whitening, anti-diuretic agent.anti-inflamatory drugs such as aspirin, Ketorolac (Toradol), Celecoxib (Celebrex), have various side effects such as vomiting,nausea,constipation,diarrhea,reduced appetite,headache,dizziness,rash, and drowsiness. The most serious side effects are ulcers, bleeding, kidney failure, and, rarely, liver failure. Methodology: The anti-inflammatory activity of Glycyrrhiza glabra was screened by protein denaturation assay using aspirin as control. Result: Glycyrrhiza glabra showed a very good anti-inflammatory activity when it was screened by protein denaturation assay using aspirin as control.
Atopic dermatitis (AD) skin is characterized by over-expression of interleukin (IL)-4, IL-13 and IL-25. When methyl-β-cyclodextrin (MβCD) treatment preceded exposure to these interleukins, combination of both treatments was found to mimic hallmarks of AD in vitro, such as barrier weakening, histological alterations and typical signaling responses in a reconstructed human epidermis (RHE). However, the respective role of each IL and whether any of them is critical when combined with MβCD treatment was unknown. Therefore, this work aimed to distinguish RHE responses after exposure to MβCD and each one of the three IL reported to mimic typical features of AD. IL-4 incubation preceded by MβCD was found responsible for altered histology, as well as for barrier alterations, evidenced by electrical resistance and dye permeation measurements. This combination further decreased loricrin (LOR) immunoreactivity, whereas mainly IL-25, combined to MβCD treatment, was able to downregulate filaggrin (FLG) mRNA level. Carbonic anhydrase II (CA2) and hyaluronan synthase 3 (HAS3), two other markers up-regulated in AD, were also induced when MβCD treatment was followed by IL-4, whilst the expression of neural epidermal growth factor-like 2 (NELL2) was up-regulated by paired IL-4 and IL-13. In conclusion, multiple features of AD were found in this in vitro model mainly when treatment of RHE by IL-4 was conducted after preliminary MβCD incubation.
Atopic dermatitis (AD) is a common chronic pruritic inflammatory skin disease resulting from a complex interplay between alteration of the epidermal barrier and abnormal activation of a Th2 immune response, creating a vicious circle as both components induce the other. In this study, we challenged reconstructed human epidermis (RHE) by plasma membrane cholesterol depletion, inducing epidermal reactions that notably include production of TSLP, a cytokine which promotes Th2 differentiation. In absence of immune system, RHE were first cholesterol-depleted, then treated with cytokines highly detected in vivo in AD epidermis: IL-4, IL-13 and IL-25. In such conditions, histological analysis of RHE revealed spongiosis in the tissue and concomitant disappearance of the granular layer, two major characteristics of AD epidermis. Simultaneously, the epidermal barrier function was altered together with expression of AD-specific genes. Thus, both challenging keratinocytes by cholesterol depletion and subsequent exposure to AD-specific interleukins act together to more closely mimic AD lesions than each treatment alone. Such conditions appear useful for any preclinical analysis of epidermal AD lesions and for screening potential therapeutics acting at this level.
Licorice is one of the oldest and most frequently used herbs in traditional Chinese medicine. It contains more than 20 triterpenoids and 300 flavonoids. In recent years, a lot of studies have reported that the active compounds isolated from licorice possess antitumor, antimicrobial, antiviral, anti-inflammatory, immunoregulatory, and several other activities that contribute to the recovery and protection of the nervous, alimentary, respiratory, endocrine, and cardiovascular systems. In this paper, nine different pharmacological activities of licorice are summarized. The active compounds responsible for these pharmacological activities, the molecular mechanisms, and in vivo and in vitro studies are listed in detail. Furthermore, the clinical therapeutics and toxicity studies of licorice are also discussed. We hope this work can provide a basis for further studies concerning with the safe and effective use of licorice.
Georg Thieme Verlag KG Stuttgart · New York.
Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this AD environment, pro-inflammatory cytokines are up-regulated, promoting a vicious circle of inflammation. Although several 3-dimensional (3D) in vitro models mimicking AD have been published, no study presents a fully characterized and controlled model of AD-related inflammation. In this paper, we aim to develop and characterize - from the morphological to the molecular level - a compromised reconstructed epidermis (RE) mimicking AD-related inflammation in vitro. For this purpose, normal human keratinocytes (NHK) were used to generate RE, treated or not with an inflammatory cocktail (Poly I:C, TNF-α, IL-4 and IL-13). Our results show that the inflammatory cocktail induces some modifications observed in AD patients: (1) it leads to spongiosis; (2) it alters early and terminal differentiation proteins; (3) it increases TSLP and IL-8 secretion by keratinocytes and (4) it results in a specific gene expression pattern. These results demonstrate that the inflammatory context contributes to the morphological, functional and transcriptomic changes observed in AD skins. As a result, this compromised RE model shares some characteristics with those found in AD skin and thus can be used as a relevant tool for screening formulations and drugs for the treatment of AD. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
While skin is readily available for sampling and direct studies of its constituents, an important intermediate step is to design in vitro and/or in vivo models to address scientific or medical questions in dermatology and skin biology. Pioneered more than 30 years ago, human skin equivalents have been refined with better cell culture techniques and media, together with sophisticated cell biology tools including genetic engineering and cell reprogramming. Human skin equivalents mimic key elements of human skin biology and have been instrumental in demonstrating the importance of cell-cell interactions in skin homeostasis and the role of a complex cellular microenvironment to coordinate epidermal proliferation, differentiation and pigmentation. Human skin equivalents have a wide field of applications from cell biology to dermocosmetics, modeling diseases, drug development, skin aging, pathophysiology and regenerative medicine. In this article we critically review the major current approaches to reconstruct organotypic skin models and their application with a particular emphasis on skin biology and pathophysiology of skin disorders. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Atopic dermatitis (AD) and psoriasis are the two most common chronic inflammatory skin diseases. Both of these diseases have distinct clinical findings and specific inflammatory cell infiltrates. Previous reports have focused individually on one or two chemokine genes or gene products in the lesions of both skin diseases. However, they have not captured the complex gene expression that must occur to induce specific cellular infiltrates in the skin lesions of these two diseases. DNA microarray studies allow the simultaneous comparison of thousands of messenger RNAs which may identify the disease-specific pattern of tissue inflammatory responses.MethodsRNA was extracted from skin biopsies of six AD and seven psoriasis patients, and analyzed using Hu-U95Av. GeneChip microarrays. To confirm GeneChip results, real-time PCR of selected genes were performed.ResultsIn AD skin, a total of 18 genes including the CC-chemokines, CCL-13/MCP-4, CCL-18/PARC and CCL-27/CTACK showed a statistically significant, greater than two-fold increase of gene expression compared to psoriasis. In psoriasis skin, a total of 59 genes including CCL-4/MIP-1β, CCL-20/MIP-3α, CXCL-2/GRO-β, CXCL-8/IL-8 and CXCR2/IL-8R showed a greater than two-fold increase of gene expression compared to AD skin. Real-time PCR confirmed several of these GeneChip results.Conclusions
These results show a very distinctive gene expression pattern in AD as compared to psoriasis that may explain the specific inflammatory cell infiltrates observed in these disorders, i.e. Th2 cells, eosinophils and mast cells in AD; Th1 cells and neutrophils in psoriasis.
Atopic dermatitis (AD) is a chronic inflammatory skin condition with complex etiology that is dependent upon interactions between the host and the environment. Acute skin lesions exhibit the features of a Th2-driven inflammatory disorder, and many patients are highly atopic. The skin barrier plays key roles in immune surveillance and homeostasis, and in preventing penetration of microbial products and allergens. Defects that compromise the structural integrity or else the immune function of the skin barrier play a pivotal role in the pathogenesis of AD. This article provides an overview of the array of molecular building blocks that are essential to maintaining healthy skin. The basis for structural defects in the skin is discussed in relation to AD, with an emphasis on filaggrin and its genetic underpinnings. Aspects of innate immunity, including the role of antimicrobial peptides and proteases, are also discussed.
Background
Staphylococcus aureus (S. aureus) is found on the skin of approximately 90% of patients with atopic dermatitis and approximately 20% of apparently healthy subjects. S. aureus induces keratinocytes and immune cells to secrete immunoregulatory factors that cause epidermal barrier dysfunction in atopic skin. Objective: This study examined factors that cause epidermal permeability barrier dysfunction in skin colonized by S. aureus. Methods: We examined the effect of S. aureus on keratinocyte differentiation in the stratum corneum (SC) of in vivo skin, normal human keratinocytes (NHKs) and a reconstructed human epidermis (RHE) model. The fold change in expression of the terminal differentiation markers and the level of secreted cytokines were investigated. Results: The SC displayed decreased expression of keratin 10 (KRT 10). NHKs treated with S. aureus extracts increased expression of interleukin (IL)-6 and significantly reduced expression of the terminal differentiation markers KRT 1, KRT 10, Loricrin (LOR), and filaggrin (FLG); however, the expression of basal layer markers (KRT 5, KRT 14) remained unchanged. Treatment of NHKs with an anti-IL-6 antibody in combination with IL-6 or the S. aureus extracts inhibited the decrease in KRT 10 mRNA or protein expression. After the RHEs were exposed to the S. aureus extracts, KRT 1 and KRT 10 protein levels decreased. Conclusions: These findings suggest that S. aureus inhibits the terminal differentiation of keratinocytes by stimulating IL-6 secretion.
Substantial progress has been achieved over the last few decades in the development of skin equivalents to model the skin as an organ. However, their static culture still limits the emulation of essential physiological properties crucial for toxicity testing and compound screening. Here, we describe a dynamically perfused chip-based bioreactor platform capable of applying variable mechanical shear stress and extending culture periods. This leads to improvements of culture conditions for integrated in vitro skin models, ex vivo skin organ cultures and biopsies of single hair follicular units.
Background:
Atopic dermatitis (AD) is a common dermatosis that highly impairs a patient's quality of life. The recent discovery that epidermal barrier defects caused by an aberrant differentiation process of keratinocytes are comparably important to the well-characterized changes in immune response patterns attributed a crucial role to the keratinocytes. Fibroblasts are able to alter proliferation and differentiation of keratinocytes, but their role in AD is not yet fully understood.
Objective:
We sought to determine the role of fibroblasts in skin proliferation and differentiation in patients with AD.
Methods:
We used human 3-dimensional organotypic skin cultures consisting of atopic fibroblasts and healthy keratinocytes, as well as healthy fibroblasts and atopic keratinocytes, and compared them with their controls. The expression of differentiation markers in these organotypic cultures were analyzed by using immunohistology and quantitative RT-PCR. Furthermore, the fundamental role of fibroblast-secreted leukemia inhibitory factor was assessed by using small interfering RNA-mediated knockdown cultures.
Results:
We observed that atopic fibroblasts influence the proliferation of keratinocytes and the terminal differentiation process, resulting in an in vivo-like morphology of AD. Subsequently, healthy fibroblasts were able to restore the structural deficits of the epidermis consisting of atopic keratinocytes. Partially, these effects were due to a reduced expression of the differentiation-associated cytokine leukemia inhibitory factor by atopic fibroblasts.
Conclusion:
These data demonstrate that fibroblasts and the modulation of fibroblast-derived factors might be new therapeutic targets for the alleviation of AD.
Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knock-down model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.
Chemokines play a key role in inflammatory diseases. The aim of this study was to estimate chemokine RANTES in the sera of patients with atopic dermatitis (AD) and to analyze the correlation between RANTES serum level and the immunological and clinical parameters of the disease. Serum levels of RANTES (ELISA; R&D Systems), total IgE and specific IgE (FEIA; Pharmacia CAP System) were estimated in 24 patients with AD, 28 patients with pollinosis (PL) and 22 healthy nonatopic subjects (HC). The division of the AD group into a pure AD (pAD) subgroup, without a coexisting respiratory allergy, and a subgroup of patients with AD and a respiratory allergy (AD+AO) was done according to Wütrich. Levels of RANTES were higher in the AD group than in the HC group and the PL group. RANTES levels did not differ among subgroups with various clinical scores and between the pAD and AD+AO subgroups. There were no correlations between levels of RANTES and total IgE. Significant positive correlations between serum levels of RANTES and Dermatophagoides farinae and cat dander-specific IgE were found in the AD group. We conclude that the serum level of chemokine RANTES differs patients with AD from patients with PL. The increase of RANTES concentration in the serum of patients with AD depends neither on a clinical picture nor an IgE system.
Both the immune system and the epidermis likely have an important role in the pathogenesis of atopic dermatitis (AD). The objective of the present study was to develop a human skin equivalent model exhibiting morphologic and molecular characteristics of AD in a controlled manner. Skin equivalents generated from normal adult human keratinocytes were stimulated with type 2 T-helper cell (Th2) cytokines IL-4 and IL-13, and morphologic features and gene expression of the epidermis were studied. Th2 cytokines induced intercellular edema similar to spongiotic changes observed in lesional AD as assessed at histopathologic analysis and electron microscopy. Furthermore, genes known to be specifically expressed in epidermis of patients with AD such as CAII and NELL2 were induced. In contrast, expression of psoriasis-associated genes such as elafin and hBD2 was not changed. Th2 cytokines caused DNA fragmentation in the keratinocytes, which could be inhibited by the caspase inhibitor Z-VAD, which suggests that apoptosis was induced. In addition, up-regulation of the death receptor Fas was observed in keratinocytes after Th2 cytokine stimulation. IL-4 and IL-13 induced phosphorylation of the signaling molecule STAT6. It was concluded that the skin equivalent model described herein may be useful in investigation of the epidermal aspects of AD and for study of drugs that act at the level of keratinocyte biology.
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.
In this study we addressed the regulatory function of primary human keratinocytes in the interaction with autologous T cells and monocytes.
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.
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.
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.
The aim of this study was to evaluate the possibility of using liposomes for skin delivery of dipotassium glycyrrhizinate (KG), an anti-inflammatory agent employed in treating acute and chronic dermatitis, and of formulating such liposomes in an oil-in-water emulsion (O/W). KG had emulsifying properties and the possibility of producing elastic liposomes was verified. Liposomes containing soya lecithin (PC) or hydrogenated soya lecithin (HPC) mixed with KG in w/w ratios of 2:1, 4:1 or 8:1 were prepared by the solvent evaporation method and then passed through a high pressure homogeniser. Liposome size and entrapment efficiency were determined and the interaction between KG and HPC was investigated using differential scanning calorimetry (DSC). Transepidermal permeation through intact pig skin and skin deposition of KG from liposomes and O/W emulsion containing liposomes were assessed and compared with values for aqueous control solutions. No marked differences were observed between PC and HPC liposomes. Liposome sizes ranged from 90 to 120 nm. Entrapment efficiency depended on the lipid:KG ratio; the maximum efficiency was obtained at 4:1 w/w. KG interacted with liposomes disrupting and fluidising the lipid bilayer, forming elastic liposomes able to penetrate through membrane pores of diameter much smaller than their own diameter. The liposome structure was maintained when dispersed in an O/W emulsion. The skin fluxes were less than the HPLC detection limit for all systems, while skin deposition increased 4.5-fold compared with aqueous solutions when KG was formulated in liposomes.
Wound healing can be problematic in several clinical settings because of massive tissue injury (burns), wound healing deficiencies (chronic wounds), or congenital conditions and diseases. Engineered skin substitutes have been developed to address the medical need for wound coverage and tissue repair. Currently, no engineered skin substitute can replace all of the functions of intact human skin. A variety of biologic dressings and skin substitutes have however contributed to improved outcomes for patients suffering from acute and chronic wounds. These include acellular biomaterials and composite cultured skin analogs containing allogeneic or autologous cultured skin cells.
We recently demonstrated that glycyrrhizin (GL) and its derivatives down-regulate TNFalpha- and IL-4-induced eotaxin 1 production by the human fetal lung fibroblast line HFL-1 at protein or mRNA levels. In particular, the GL derivative hetero-30-OH-GL (3beta-[(2-O-beta-D-glucopyranuronosyl-beta-D-glucopyranuronosyl)oxy]-olean-11,13(18)-dien-30-ol) showed marked inhibition of eotaxin 1 production with less cytotoxicity than 18beta-GL. To identify the molecular mechanism of this effect, we focused on the inhibition of the transcriptional factors NF-kappaB and signal transducer and activator of transcription 6 (STAT6), which regulate eotaxin 1 gene activation. STAT6 phosphorylation and translocation of phospho-STAT6 from cytosol to nuclei were slightly inhibited by 18beta-GL and significantly inhibited by hetero-30-OH-GL. While IkappaBalpha degradation and translocation of NF-kappaB p65 to nuclei were not significantly affected by either compound, the stability of eotaxin-1 mRNA was decreased with hetero-30-OH-GL. In addition, eotaxin 1 promoter activity was markedly inhibited by hetero-30-OH-GL. Electrophoretic mobility shift assay (EMSA) confirmed these results. Thus, hetero-30-OH-GL significantly inhibited eotaxin 1 expression by the selective inhibition of IL-4 signal transduction as well as by enhanced mRNA degradation.
High-mobility group box 1 protein (HMGB1) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGB1 inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGB1 chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence studies indicate that glycyrrhizin binds directly to HMGB1 (K(d) approximately 150 microM), interacting with two shallow concave surfaces formed by the two arms of both HMG boxes. Our results explain in part the anti-inflammatory properties of glycyrrhizin, and might direct the design of new derivatives with improved HMGB1-binding properties.
Differential in situ cytokine gene expression in acute versus chronic atopic dermatitis
- Q Hamid
- M Boguniewicz
- D Y Leung
Study on mechanisms of antiinflammatory action of glycyrrhetinic acid
- M Kerube
Engineered skin substitutes: practices and potentials
- D M Supp
- S T Boyce
Distinct patterns of gene expression in the skin lesions of atopic dermatitis and psoriasis: a gene microarray analysis
- I Nomura
- B Gao
- M Boguniewicz
- M A Darst
- J B Travers
- D Y Leung