Tape-stripped Skin Research Articles

Biomarker Mapping on Skin Tape Strips Using MALDI Mass Spectrometry Imaging.

Keratinocyte organization and biochemistry are important in forming the skin's protective barrier. Intrinsic and extrinsic factors can affect skin barrier function at the cellular and molecular levels. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging, a technique which combines both molecular aspects and histological details, has proven to be a valuable method in various disciplines including pharmacology, dermatology and cosmetology. It typically requires ex vivo samples, prepared following frozen tissue sectioning. This paper demonstrates the feasibility of performing MALDI analysis on tape strips collected non-invasively on skin. The aim is to obtain molecular imaging of corneocytes on tapes towards novel biological insights. Tapes were collected from two skin sites (volar forearm and cheek) of human volunteers. Ten molecules relating to skin barrier function were detected with a single mode of acquisition at high spatial resolution with a 7T MALDI-Fourier transform ion cyclotron resonance (FTICR) instrument. The method sensitivity was adequate to create molecular maps which could be overlaid on transmission microscopy images of the same area of the tape. Analysis of the molecular distributions from tapes at the two skin sites was consistent with the known skin properties of the two sites, confirming the validity of the observations. Hierarchical clustering analysis was used to differentiate corneocyte populations based on their molecular profiles. Furthermore, morphological analysis provided a new way of considering statistical populations of corneocytes on the same tape, rather than measuring a single averaged value, providing additional useful information relating to their structure-function relationship.

Modeling of Drug Diffusion Based on Concentration Profiles in Healthy and Damaged Human Skin

Based on experimental drug concentration profiles in healthy as well as tape-stripped ex vivo human skin, we model the penetration of the antiinflammatory drug dexamethasone into the skin layers by the one-dimensional generalized diffusion equation. We estimate the position-dependent free-energy and diffusivity profiles by solving the conjugated minimization problem, in which the only inputs are concentration profiles of dexamethasone in skin at three consecutive penetration times. The resulting free-energy profiles for damaged and healthy skin show only minor differences. In contrast, the drug diffusivity in the first 10 μm of the upper skin layer of damaged skin is 200-fold increased compared to healthy skin, which reflects the corrupted barrier function of tape-stripped skin. For the case of healthy skin, we examine the robustness of our method by analyzing the behavior of the extracted skin parameters when the number of input and output parameters are reduced. We also discuss techniques for the regularization of our parameter extraction method.

Side-by-Side Comparison of Skin Biopsies and Skin Tape Stripping Highlights Abnormal Stratum Corneum in Atopic Dermatitis

Skin biopsies are commonly used for the assessment of skin pathology in various skin diseases, including atopic dermatitis (AD). However, because of the invasive nature of skin biopsies, many patients, particularly children, decline participation. This can lead to potential subject sampling bias as data could be skewed toward more severe, older patients who are willing to have biopsies. Recently, researchers have begun studying the skin with a minimal, noninvasive technique using skin tape stripping (STS) to profile the epidermal transcriptome, proteins, and lipids in the skin. However, side-by-side comparisons of skin biopsies with STS have not been done to assess epidermal penetration. Therefore, 20 STS were collected from the volar surface of forearms from healthy nonatopic subjects and patients with AD, following this skin biopsies were collected from adjacent nontaped and taped areas of the skin. Using hematoxylin and eosin staining and immunostaining, we demonstrated that 20 STS reached the upper granular layer of the epidermis. Additionally, we found that the expression of terminal differentiation markers in samples from STS procedure positively correlated with the expression level of these markers in matching skin biopsies. Therefore, STS is a noninvasive and reliable approach to evaluate the expression of skin terminal differentiation markers, which are defective in AD skin.

Langerhans cells mediate the skin-induced tolerance to ovalbumin via Langerin in a murine model.

Epicutaneous sensitization is an important route of immunization for allergens in atopic diseases; however, studies have also shown that application with protein on theintact skin induces antigen-specific tolerance. Langerhans cells (LCs) play an immunosuppressive role in several inflammatory skin diseases and mouse models, and the role of LCs in the skin-induced tolerance is not fully understood. Langerin-DTA mice that were deficient in LCs were utilized to produce the model of skin-induced tolerance to ovalbumin (OVA). Binding of Langerin to OVA was analyzed by enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence. Homozygous Langerin-DTR mice that were deficient in Langerin were introduced to assess the role of Langerin in the skin-induced tolerance. Application with OVA ontothe intact, but not tape-stripped, skin attenuated the production of OVA-specific IgE, IgG1, and IgG2a induced by subsequent subcutaneous immunization with OVA, and the inhibitory effects were abolished in Langerin-DTA mice. In contrast to the tape-stripped skin, the intact skin induced the production of IL-10 by LCs in draining lymph node after application with OVA. Langerin could bind OVA, and homozygous Langerin-DTR mice demonstrated similar humoral and cellular immune responses in the model of skin-induced tolerance compared to wide-type mice. Our data suggested that LCs were critical in the intact skin-induced tolerance to protein antigen via Langerin, and LCs might be targeted via Langerin to regulate the immune responses in systemic and (or) skin inflammatory diseases.

Ingestion of Casein Hydrolysate Induces Oral Tolerance and Suppresses Subsequent Epicutaneous Sensitization and Development of Anaphylaxis Reaction to Casein in Mice

Background: Casein is the most dominant causal allergen in cow’s milk allergy (CMA). Casein hydrolysates are frequently applied in infant formulas for children with a risk or history of CMA. However, there is limited information on the oral tolerance-inducing ability of casein hydrolysates. Objectives: The aim of this study was to investigate whether the ingestion of casein hydrolysate induces tolerance to casein, ultimately preventing subsequent epicutaneous sensitization and development of an anaphylaxis reaction. Methods: BALB/c mice were orally administered casein or a casein hydrolysate (CNH) via the drinking water and were then epicutaneously sensitized by repeated exposure of casein on tape-stripped skin. Sensitization was assessed by basophil activation tests, the serum levels of casein-specific antibodies, and cytokine production from casein-stimulated spleen and mesenteric lymph node (MLN) cells. Occurrence of an anaphylaxis reaction was evaluated by measuring rectal temperature and the plasma level of mouse mast cell protease-1 (mMCP-1) after oral casein challenges. The T cell population in the spleen and MLN was assessed by flow cytometry. Intestinal mast cells and basophils were analyzed histologically. Results: Sensitization and anaphylaxis reaction to casein were significantly suppressed in casein- or CNH-fed mice compared to controls. Prior ingestion of casein or CNH had no effect on the population of regulatory T cells and activated T cells in lymphoid tissues. Intestinal basophils increased by the epicutaneous sensitization of casein, which was suppressed in casein- or CNH-fed mice. Although the increase in the plasma level of mMCP-1 after oral challenge was suppressed in casein- or CNH-fed mice, there was no change in the number of intestinal mast cells. Conclusion: Prior ingestion of casein or CNH induced oral tolerance and suppressed subsequent epicutaneous sensitization and development of systemic anaphylaxis to casein.

Skin Lipid Abnormalities in Patients with a History of Eczema Herpeticum Indicate the Involvement of the Sphingosine‐1‐Phosphate Signaling System in HSV Viral Replication

RationaleAtopic dermatitis (AD) with eczema herpeticum (EH) can result in systemic infections and can be life‐threatening. The purpose of this study was to identify differences in non‐lesional skin between AD with a history of recurrent EH (ADEH+), AD without EH (ADEH−), and non‐atopic (NA) controls to gain insight into mechanisms involved in EH pathogenesis using a novel minimally invasive skin tape strip (STS) methodology and mass spectrometric approaches.MethodsSTS layers 5–6 from 13 NA, 13 ADEH−, and 15 ADEH+ adult subjects were collected from non‐lesional skin. AD subjects were balanced by gender, age and disease severity; STS were subjected to lipidomic and filaggrin breakdown products analyses by liquid chromatography tandem mass spectrometry (LC‐MS/MS). For the in vitro experiments, differentiated primary human keratinocytes were transfected with control or sphingosine‐1‐phosphate (S1P) lyase (SGPL), sphingosine kinase‐1 (SPHK1), or sphingosine kinase‐2 (SPHK2) siRNA for 48h and infected with HSV‐1 (0.1 MOI). The effect of enzyme silencing on sphingolipidome was evaluated via the LC‐MS/MS. Viral replication was assessed in 24h with qPCR and plaque assay.ResultsADEH+, as compared to ADEH− and NA, subjects had a significantly increased proportion of short‐chain over long‐chain NS‐ceramides (sphingosine N‐acylated with non‐hydroxy fatty acids) (p=0.002), and increased levels of sphingomyelins (p=0.006). In contrast to ADEH−, ADEH+ subjects demonstrated increased level of free C18‐ and C17‐sphingosines (p=0.007 and 0.052, correspondingly) that suggests upregulated skin sphingosine‐1‐phosphate (S1P) metabolism and signaling. A comparison of the effect of silencing of major enzymes that directly regulate S1P levels (SPHK1, SPHK2, SGPL) in primary human keratinocytes in vitro revealed that only the silencing of SGPL resulted in increased viral replication after keratinocyte inoculation with HSV‐1 (Mean±SE, 0.55±0.09 vs. 0.25±0.05 for S1PL vs. control siRNA transfected keratinocytes, p<0.01, as detected by qPCR).ConclusionsSTS from ADEH+ subjects and in vitro studies indicate involvement of the S1P signaling system in EH and suggest novel potential therapeutic targets to treat EH.Support or Funding InformationThis study was funded by the Atopic Dermatitis Research Network supported by NIH/NIAID grant U19 AI117673.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of lipid fluidity likely contributes to the fructose/xylitol-induced acceleration of epidermal permeability barrier recovery.

We previously showed that topical application of hexoses such as fructose accelerates barrier recovery after disruption. We also showed that various hexoses and polyols interact with phospholipid and alter the phase transition temperature. Thus, we hypothesized that the improvement of barrier recovery by hexoses and polyols might be related to the interaction with phospholipid. Here, we tested this idea by examining the effects of xylitol (a component of some skin-care products) and fructose on lipid dynamics in an epidermal-equivalent model at the single-cell level by means of two-photon microscopy after staining with Laurdan, a fluorescent dye sensitive to the physical properties of its membrane environment. First, we confirmed that topical application of xylitol aqueous solution on tape-stripped human skin accelerated barrier recovery. Then, we examined changes of lipid fluidity in the epidermal-equivalent model after application of water or an aqueous solution of xylitol or fructose. Application of xylitol and/or fructose increased the lipid fluidity in the uppermost part of the stratum granulosum layer, compared to treatment with water alone, and accelerated the exocytosis of lamellar bodies to the intercellular domain between stratum corneum and stratum granulosum. Our results support the idea that the improvement of epidermal barrier homeostasis upon topical application of xylitol or fructose is due to increased lipid fluidity in the uppermost layer of the stratum granulosum, which enables accelerated release of lipid from the stratum granulosum, thereby improving the lamellar structure and accelerating epidermal permeability barrier recovery.

Dissolving Microneedle Systems for the Oral Mucosal Delivery of Triamcinolone Acetonide to Treat Aphthous Stomatitis

A microneedle (MN) oral mucosal patch system was prepared to increase its adhesive strength, to improve the delivery of triamcinolone acetonide (TA) into the mucosa, and to protect the adhesive layer from mouth movement and saliva. The MN oral mucosal patch system consists of an adhesive layer containing TA and a backing layer. The adhesive layer consists of a base with a thickness of 50 μm, 300 μm long MNs, and 18% TA. After patches were attached to the tape-stripped porcine skin for a predetermined period of time, the adhesion of the patches was observed. TA was extracted from the skin, and the amount of TA was measured using high-performance chromatography. When the adhesive layer was applied for 10 s, the adhesive strength was 1.69 N mm/cm2 with microneedles and 0.41 N mm/cm2 without microneedles. An oral mucosal patch with MNs (TA MN-AL) delivered 42% more TA into the skin than an oral mucosal patch without MNs (TA AL). Also, as attachment time increased, the amount of TA delivered was significantly greater using a TA MN-AL compared to a TA AL. The addition of MNs can increase the adhesion of the oral mucosal patch to the mucosa and thus improve the efficiency of drug delivery.

Digging skin deep

Allergy Disrupted epithelial barriers are thought to be central to the development of allergic disorders such as atopic dermatitis, which is commonly associated with food allergy. Leung et al. performed repeated skin tape stripping on lesional and nonlesional skin of pediatric atopic dermatitis subjects, some of whom also had food allergies. Among other parameters, the authors measured lipids, barrier integrity, and the microbiome. Nonlesional skin from atopic dermatitis patients had different characteristics depending on whether the patient also had a food allergy. The findings could help in the development of food allergy biomarkers. Sci. Transl. Med. 11 , aav2685 (2019).

The nonlesional skin surface distinguishes atopic dermatitis with food allergy as a unique endotype.

Skin barrier dysfunction has been reported in both atopic dermatitis (AD) and food allergy (FA). However, only one-third of patients with AD have FA. The purpose of this study was to use a minimally invasive skin tape strip sampling method and a multiomics approach to determine whether children with AD and FA (AD FA+) have stratum corneum (SC) abnormalities that distinguish them from AD without FA (AD FA-) and nonatopic (NA) controls. Transepidermal water loss was found to be increased in AD FA+. Filaggrin and the proportion of ω-hydroxy fatty acid sphingosine ceramide content in nonlesional skin of children with AD FA+ were substantially lower than in AD FA- and NA skin. These abnormalities correlated with morphologic changes in epidermal lamellar bilayer architecture responsible for barrier homeostasis. Shotgun metagenomic studies revealed that the nonlesional skin of AD FA+ had increased abundance of Staphylococcus aureus compared to NA. Increased expression of keratins 5, 14, and 16 indicative of hyperproliferative keratinocytes was observed in the SC of AD FA+. The skin transcriptome of AD FA+ had increased gene expression for dendritic cells and type 2 immune pathways. A network analysis revealed keratins 5, 14, and 16 were positively correlated with AD FA+, whereas filaggrin breakdown products were negatively correlated with AD FA+. These data suggest that the most superficial compartment of nonlesional skin in AD FA+ has unique properties associated with an immature skin barrier and type 2 immune activation.

Skin tape stripping: Which layers of the epidermis are removed?

Skin tape stripping: Which layers of the epidermis are removed?

Monitoring Drug Crystallization in Percutaneous Penetration Using Localized Nanothermal Analysis and Photothermal Microspectroscopy.

Drug crystallization on and in the skin has been reported following application of topical or transdermal formulations. This study explored novel probe-based approaches including localized nanothermal analysis (nano-TA) and photothermal microspectroscopy (PTMS) to investigate and locate drug crystals in the stratum corneum (SC) of porcine skin following application of simple ibuprofen (IBU) formulations. We also conducted in vitro skin permeation studies and tape stripping. The detection of drug crystals in the SC on tape strips was confirmed using localized nano-TA, based on the melting temperature of IBU. The melting of IBU was also evident as indicated by a double transition and confirmed the presence of drug crystals in the SC. The single point scans of PTMS on the tape strips allowed collection of the photothermal FTIR spectra of IBU, confirming the existence of drug crystals in the skin. The combined methods also indicated that drug crystallized in the SC at a depth of ∼4-7 μm. Future studies will examine the potential of these techniques to probe crystallization of other commonly used actives in topical and transdermal formulations.

Potential of iontophoresis as a drug delivery method for midazolam in pediatrics

Drug delivery to the neonatal and premature pediatric populations is very challenging. This research assessed the potential of delivering midazolam by transdermal iontophoresis as an alternative strategy in pediatric therapy. In vitro experiments used intact and tape-stripped porcine skin as models for the skin barrier function of full-term and premature newborns, respectively. Midazolam transdermal transport was significantly enhanced by applying higher currents, increasing the formulation pH, and optimizing the drug's mole fraction in the vehicle. When the skin barrier was decreased to half of its baseline competence, the passive permeation of midazolam increased by approximately 60-fold; and complete stratum corneum removal led to an additional 20-fold enhancement in permeation. Iontophoresis retained control of the drug transport trough partially compromised skin. However, a very high passive contribution undermined the iontophoretic control when the barrier was fully compromised. Overall, midazolam delivery could be rate-controlled by iontophoresis in most circumstances, and therapeutically useful fluxes could be achieved.

Expression and function of the ectopic olfactory receptor OR10G7 in patients with atopic dermatitis

Ectopic olfactory receptors (ORs) are found in the skin, but their expression and biological function in normal skin and skin form patients with atopic dermatitis (AD) are unknown. We sought to characterize the expression of ORs in the skin and assess OR-mediated biological responses of primary human keratinocytes in the presence of odorant ligands. OR expression was examined by using whole-transcriptome sequencing of skin tape strips collected from patients with AD and healthy control (HC) subjects. OR10G7 and filaggrin 1 (FLG-1) expression was analyzed by using RT-PCR and immunostaining in skin biopsy specimens and primary human keratinocytes from patients with AD and HC subjects. ATP and cyclic AMP production by control and OR10G7 small interfering RNA-transfected keratinocytes in response to odorant stimulation with acetophenone and eugenol was assessed. A total of 381 OR gene transcripts were detected in the skin samples, with the greatest OR expression detected in the skin tape strips corresponding to the upper granular layer of the skin. OR10G7 expression was significantly increased in skin biopsy specimens from patients with AD compared with those from HC subjects (P=.01) and inversely correlated with FLG-1 expression (P=.009). OR10G7 expression was greatest in undifferentiated keratinocytes from patients with AD and was downregulated with progressive differentiation. Primary human keratinocytes produced ATP, an essential neurotransmitter in sensory pathways, in response to acetophenone and eugenol, odorants previously identified as potential ligands for this receptor. This response was abolished in OR10G7 small interfering RNA-transfected keratinocytes. OR10G7 is expressed at significantly greater levels in undifferentiated keratinocytes from patients with AD compared with HC subjects. OR10G7 is likely involved in transmission of skin-induced chemosensory responses to odorant stimulation, which might modulate differential nociceptive responses in AD skin.

Formulation of Natural Oil Nano-Emulsions for the Topical Delivery of Clofazimine, Artemisone and Decoquinate.

The aim of this study was to formulate nano-emulsions comprising natural oils and the active pharmaceutical ingredients (APIs) clofazimine (CLF), artemisone (ATM) and decoquinate (DQ) in order to determine effectiveness of the nano-emulsions for topical delivery of the APIs. The APIs alone do not possess suitable physicochemical properties for topical drug delivery. Nano-emulsions were formulated with olive and safflower oils encapsulating the APIs. Skin diffusion and tape stripping studies were performed. By using the lactate dehydrogenase (LDH) assay, in vitro toxicity studies were carried out on immortalized human keratinocytes (HaCaT) cell line to determine cytotoxicities due to the APIs and the nano-emulsions incorporating the APIs. The nano-emulsions were effective in delivering the APIs within the stratum corneum-epidermis and the epidermis-dermis, were non-cytotoxic towards HaCaT cell lines (p< 0.05) and inhibited Mycobacterium tuberculosis in vitro. Natural oil nano-emulsions successfully deliver CLF, ATM and DQ and in principle could be used as supplementary topical treatment of cutaneous tuberculosis (CTB). Graphical Abstract ᅟ.

Study on the development of wax emulsion with liquid crystal structure and its moisturizing and frictional interactions with skin

The moisturizing and frictional interactions between emulsion and skin have a significant influence on the moisturizing efficacy and sensory properties of cosmetics. To deeply understand the interactions, we developed a kind of wax emulsion with liquid crystal structure and studied their interactions with dry, semi-moist and fully-moist skin. The moisturizing interaction was studied by using skin hydration test and tape stripping analysis, and it was found that with the increasing of liquid crystal content in the emulsion, the skin hydration level increased, desquamation index decreased and number of cutaneous lines increased, suggesting the increased moisturizing effect and barrier function. The frictional interaction was studied by finger friction test, and it was found that firstly during and after the application of emulsion on skin, the frictional interaction followed the stribeck-curve rule of lubrication. Secondly, when changing the water content of the substrate skin, the frictional behavior of the whole system showed a bell-curve behavior. Both skin hydration and water amount in the interfacial film played role in the frictional interaction between emulsion and skin, and the frictional interaction mechanism between wax emulsion and finger skin with different type of skin was proposed. This was the first time to study and discuss the effect of skin moist status on the moisturizing and frictional interactions between cosmetic emulsion and skin. The findings were significant for the design of high quality personal care products.

Advancement in Atopic Dermatitis Research Through the Use of a Novel Skin Tape Strip Mass Spectrometry Based Processing Protocol

Allergic skin diseases, including Atopic Dermatitis (AD), severely impair the quality of life of affected patients. AD development is linked to a damaged skin barrier function that can precede clinical AD manifestations by many months. Currently there are no effective tools to predict the development of AD or to characterize the efficacy of applied therapy. Therefore, novel approaches to advance mechanistic understanding of AD as well as clinical management of AD and related skin diseases are needed.We have developed a novel protocol that allows the assessment of skin stratum corneum lipid species and filaggrin breakdown products using minimally invasive tape stripping in combination with liquid chromatography tandem mass spectrometry. Our approach requires only two tape strips to provide quantitative data on multiple lipid species as well as on the degradation products of filaggrin, a major skin protein responsible for skin barrier, pH and moisturizing properties. The hallmark of our approach is in the ability to normalize targeted mass spectrometric data per sample total protein content that was never done before. Our approach also allows to use the same sample for proteomics studies. We have applied our novel protocol to evaluate the difference between lesional and non‐lesional stratum corneum of AD subjects and to compare AD subjects with healthy individuals. The comparison of stratum corneum from 25 healthy adult subjects and 30 AD adult patients using our methodology confirmed previously known information about the shift towards short chain ceramide species in AD lesional skin and also revealed novel factors associated with AD development. Thus, total levels of sphingomyelins, and especially those with short chain fatty acids, were found to be increased several fold in atopic lesional skin. Furthermore, lysophosphatidylcholines demonstrated a profound redistribution towards the prevalence of short chain species not only in lesional but also in non‐lesional skin of AD subjects. These novel findings suggest a global shift in fatty acid biosynthesis that might be triggered by hyperactivated type 2 immune response. In fact, the RNAseq analysis performed on stratum corneum from same subjects revealed the downregulation of expression of fatty acid elongases ELOVL3 and ELOVL6 that may explain the observed global redistribution of the long chain and short chain lipids. Furthermore, the modeling of the effect of type 2 cytokines on sphingolipids using human differentiated keratinocytes in vitro confirmed the ability of type 2 cytokines IL‐4 and IL‐13 to provoke similar changes in sphingolipids. Our current ongoing studies further validate the power of the developed methodology and the value of lysophosphatidylcholines as biomarkers of atopic disease.In conclusion, skin tape stripping in conjunction with our unique processing protocol and targeted mass spectrometric methodology opens novel possibilities in clinical research by unveiling novel biomarkers of skin diseases and by allowing clinical studies in the skin using minimally invasive methodology.Support or Funding InformationNIH/NIAID grant U19 AI117673, NIH/NIAMS grant R01 AR41256This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In-vitro model for assessing glucose diffusion through skin

Pig ear skin membrane-covered glucose biosensor based on oxygen electrode has been assessed as a tool to evaluate glucose penetration through skin in-vitro. For this, glucose oxidase (GOx) was immobilised on oxygen electrode and covered with the skin membrane. Exposing this electrode to the solution of glucose resulted in glucose penetration though skin membrane, its oxidation catalysed by GOx, consumption of O2 and decrease of the current of the oxygen electrode. By processing the biosensor responses to glucose, we found that glucose penetration through 250 µm thick skin membrane is slow; 90% of steady-state current response was reached in 32( ± 22) min. Apparent diffusion coefficient for glucose in skin was found to be equal to 0.15( ± 0.07)* 10−6 cm2 s−1. This value is 45 times lower than glucose diffusion coefficient in water. Tape-stripping of stratum corneum (SC) allows considerably faster glucose penetration. The electrodes covered with tape-stripped skin reached 90% of steady-state current response in 5.0(± 2.7) min. The theoretical estimate of glucose flux through SC was considered exploiting four-pathway theory of transdermal penetration. Theoretical flux values were more that three orders lower than measured experimentally. This high discrepancy might indicate that glucose penetration through healthy human skin could be even slower, allowing much lower flux, than it was found in our study for skin membranes from pig ears.

The optimal age for epicutaneous sensitization following tape-stripping in BALB/c mice

BackgroundDirect contact of food proteins with eczematous lesions is thought to be the main cause of epicutaneous sensitization. To further investigate the development and pathogenesis of food allergy in vivo, a good mouse model of epicutaneous sensitization is needed. However, a fundamental problem in that regard is that the optimal age for epicutaneous sensitization of mice is unknown. In this study, we attempted to elucidate that optimal age. MethodsDorsal skin of wild-type BALB/c female mice (1, 3, 8 and 24 weeks old) was shaved, depilated and tape-stripped. A Finn chamber containing a 20-μl-aliquot of 20-mg/ml (OVA) was applied to the tape-stripped skin on 3 consecutive days/week, for 3 weeks. The body temperature was measured after intraperitoneal OVA challenge. Serum OVA-specific IgE titers and OVA-induced cytokine production by spleen cells were measured by ELISA. Dendritic cells (DCs) that migrated to the draining lymph nodes were quantified by FITC-labeled OVA and flow cytometry. The mRNA expression levels in the dorsal skin were measured by qPCR. ResultsA significant age-dependent body temperature decline was observed after OVA challenge. The serum OVA-specific IgE titer, OVA-induced cytokine production (i.e., IL-4, IL-5 and IL-13) by spleen cells, and number of FITC-OVA-engulfing DCs increased with age. In addition, mRNA for IL-33, but not TSLP or IL-25, was significantly induced in the skin by tape-stripping and increased with age. ConclusionsTwenty-four-week-old mice showed the greatest DC migration, Th2 polarization, IgE production and body temperature decline. Skin-derived IL-33 is likely to play key roles in those changes.

Minimally invasive skin tape strip RNA sequencing identifies novel characteristics of the type 2–high atopic dermatitis disease endotype

Expression profiling of skin biopsy specimens has established molecular features of the skin in patients with atopic dermatitis (AD). The invasiveness of biopsies has prevented their use in defining individual-level AD pathobiological mechanisms (endotypes) in large research studies. We sought to determine whether minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. We sampled nonlesional and lesional skin tape strips and biopsy specimens from white adult patients with AD (18 male and 12 female patients; age [mean±SE], 36.3±2.2years) and healthy control subjects (9 male and 16 female subjects; age [mean±SE], 34.8±2.2years). AmpliSeq whole-transcriptome sequencing was performed on extracted RNA. Differential expression, clustering/pathway analyses, immunostaining of skin biopsy specimens, and clinical trait correlations were performed. Skin tape expression profiles were distinct from skin biopsy profiles and better sampled epidermal differentiation complex genes. Skin tape expression of 29 immune and epidermis-related genes (false discovery rate<5%) separated patients with AD from healthy subjects. Agnostic gene set analyses and clustering revealed 50% of patients with AD exhibited a type 2 inflammatory signature (type 2-high endotype) characterized by differential expression of 656 genes, including overexpression of IL13, IL4R, CCL22, CCR4 (log2 fold change=5.5, 2.0, 4.0, and 4.1, respectively) and at a pathway level by TH2/dendritic cell activation. Both expression and immunostaining of skin biopsy specimens indicated this type 2-high group was enriched for inflammatory, type 2-skewed dendritic cells expressing FcεRI. The type 2-high endotype group exhibited more severe disease by using both the Eczema Area and Severity Index score and body surface area covered by lesions. Minimally invasive expression profiling of nonlesional skin reveals stratification in AD molecular pathology by type 2 inflammation that correlates with disease severity.