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Atopic dermatitis (AD) is characterized by abnormal skin lipids that are largely driven by hyperactivated type 2 immune responses. The antibody to the α-subunit of interleukin (IL)-4 receptor, dupilumab, was recently approved to treat AD and demonstrated strong efficacy. However, the role of dupilumab therapy in the regulation of skin barrier structure and function has not been fully explored.We have evaluated the content of lipids and transepidermal water loss (TEWL) in lesional and non-lesional skin of adults and adolescents with moderate-to-severe AD over the course of 16-week treatment with dupilumab and compared those values with that of matched healthy volunteers.Dupilumab treatment provided a significant decrease in TEWL in AD lesions, lowering it almost to the levels seen in the skin of healthy subjects. Blocking IL-4/IL-13 signaling with dupilumab normalized lipid composition (decreased levels of ceramides with non-hydroxy fatty acids and C18-sphingosine and increased the level of esterified omega-hydroxy fatty acid-containing ceramides) and increased ceramide chain length in lesional as well as non-lesional stratum corneum of AD patients. Partial changes for these parameters were already observed after 2 weeks, with a full response achieved after 8 weeks of dupilumab treatment.Inhibition of IL-4/IL-13 signaling by dupilumab allows restoration of skin lipid composition and barrier function in patients with moderate-to-severe AD.

Life-threatening viral diseases such as eczema herpeticum (EH) and eczema vaccinatum (EV) occur in5% of individuals with atopic dermatitis (AD). The diagnosis of AD, however, excludes all individuals with AD from smallpox vaccination.We sought to identify circulatory and skin lipid biomarkers associated with EH and EV.Stratum corneum and plasma samples from 15 subjects with AD and a history of EH, 13 age- and gender-matched subjects with AD and without EH history, and 13 healthy nonatopic (NA) controls were analyzed by liquid chromatography tandem mass spectrometry for sphingolipid content. Sphingosine-1-phosphate (S1P) and ceramide levels were validated in plasma samples from the Atopic Dermatitis Vaccinia Network/Atopic Dermatitis Research Network repository (12 NA, 12 AD, 23 EH) and plasma from 7 subjects with EV and 7 matched subjects with AD. S1P lyase was downregulated in human primary keratinocytes to evaluate its effect on herpes simplex virus 1 (HSV-1) replication in vitro.The stratum corneum of patients with EH demonstrated significantly higher levels of free sphingoid bases than those in patients who were NA, indicating enhanced sphingolipid turnover in keratinocytes (P .05). Plasma from 2 independent cohorts of patients with EH had a significantly increased S1P/ceramide ratio in subjects with EH versus those with AD and or who were NA (P .01). The S1P level in plasma from subjects with EV was twice the level in plasma from subjects with AD (mean = 1,533 vs 732 pmol/mL; P .001). Downregulation of S1P lyase expression with silencing RNA led to an increased S1P level and doubled HSV-1 titer in keratinocytes.Our data point to long-term abnormalities in the S1P signaling system as a biomarker for previous disseminated viral diseases and a potential treatment target in recurring infections.

Type I interferon kappa (IFNκ) is selectively expressed in human keratinocytes. Herpes simplex virus-1 (HSV-1) is a human pathogen that infects keratinocytes and causes lytic skin lesions. Whether IFNκ plays a role in keratinocyte host defense against HSV-1 has not been investigated. In this study, we found that IFNκ mRNA expression was induced by addition of recombinant IFNκ and poly (I:C); and its expression level was significantly greater than IFNa2, IFNb1, and IFNL1 in both undifferentiated and differentiated normal human epidermal keratinocytes (NHEKs) under resting and stimulation conditions. Although IFNe was expressed at a relatively higher level than other IFNs in resting undifferentiated NHEK, its expression level did not change after stimulation with recombinant IFNκ and poly (I:C). HSV-1 infection inhibited gene expression of IFNκ and IFNe in NHEK. Silencing IFNκ in NHEK led to significantly enhanced HSV-1 replication in both undifferentiated and differentiated NHEK compared to scrambled siRNA-transfected cells, while the addition of recombinant IFNκ significantly reduced HSV-1 replication in NHEK. In addition, we found that IFNκ did not regulate protein expression of NHEK differentiation markers. Our results demonstrate that IFNκ is the dominant type of IFNs in keratinocytes and it has an important function for keratinocytes to combat HSV-1 infection.

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.

Lipids in the stratum corneum of atopic dermatitis (AD) patients differ substantially in composition from healthy subjects. We hypothesized that hyperactivated type 2 immune response alters AD skin lipid metabolism. We have analyzed stratum corneum lipids from nonlesional and lesional skin of AD subjects and IL-13 skin-specific Tg mice. We also directly examined the effects of IL-4/IL-13 on human keratinocytes in vitro. Mass spectrometric analysis of lesional stratum corneum from AD subjects and IL-13 Tg mice revealed an increased proportion of short-chain (N-14:0 to N-24:0) NS ceramides, sphingomyelins, and 14:0–22:0 lysophosphatidylcholines (14:0–22:0 LPC) with a simultaneous decline in the proportion of corresponding long-chain species (N-26:0 to N-32:0 sphingolipids and 24:0–30:0 LPC) when compared with healthy controls. An increase in short-chain LPC species was also observed in nonlesional AD skin. Similar changes were observed in IL-4/IL-13–driven responses in Ca2+-differentiated human keratinocytes in vitro, all being blocked by STAT6 silencing with siRNA. RNA sequencing analysis performed on stratum corneum of AD as compared with healthy subjects identified decreased expression of fatty acid elongases ELOVL3 and ELOVL6 that contributed to observed changes in atopic skin lipids. IL-4/IL-13 also inhibited ELOVL3 and ELOVL6 expression in keratinocyte cultures in a STAT6-dependent manner. Downregulation of ELOVL3/ELOVL6 expression in keratinocytes by siRNA decreased the proportion of long-chain fatty acids globally and in sphingolipids. Thus, our data strongly support the pathogenic role of type 2 immune activation in AD skin lipid metabolism.

BACKGROUND: Expression profiling of skin biopsies has established molecular features of the skin in atopic dermatitis (AD). Invasiveness of biopsies has prevented their use in defining individual level AD pathobiological mechanisms (endotypes) in large research studies. OBJECTIVE: To determine if minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. METHODS: We sampled non-lesional and lesional skin tape strips and biopsies from adult Caucasian subjects AD patients (18 males, 12 females; age (Mean±SE) 36.3±2.2 yrs) and healthy controls (9 males, 16 females; age (Mean±SE) 34.8±2.2 yrs). Ampliseq whole transcriptome sequencing was performed on extracted RNA. Differential expression, clustering/pathway analyses, immunostaining of skin biopsies, and clinical trait correlations were performed. RESULTS: 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 (FDR

Background A subset of patients with atopic dermatitis (AD) is prone to disseminated herpes simplex virus (HSV) infection (ie, atopic dermatitis with a history of eczema herpeticum [ADEH+]). Biomarkers that identify ADEH+ are lacking. Objective We sought to search for novel ADEH+ gene signatures in PBMCs. Methods An RNA-sequencing approach was applied to evaluate global transcriptional changes by using PBMCs from patients with ADEH+ and patients with atopic dermatitis without a history of eczema herpeticum (ADEH−). Candidate genes were confirmed by means of quantitative PCR or ELISA. Results PBMCs from patients with ADEH+ had distinct changes to the transcriptome when compared with those from patients with ADEH− after HSV-1 stimulation: 792 genes were differentially expressed at a false discovery rate of less than 0.05 (ANOVA), and 15 type I and type III interferon genes were among the top 20 most downregulated genes in patients with ADEH+. We further validated that IFN-α and IL-29 mRNA and protein levels were significantly decreased in HSV-1–stimulated PBMCs from patients with ADEH+ compared with those from patients with ADEH– and healthy subjects. Ingenuity Pathway Analysis demonstrated that the upstream regulators of type I and type III interferons, interferon regulatory factor (IRF) 3 and IRF7, were significantly inhibited in patients with ADEH+ based on the downregulation of their target genes. Furthermore, we found that gene expression of IRF3 and IRF7 was significantly decreased in HSV-1–stimulated PBMCs from patients with ADEH+. Conclusions PBMCs from patients with ADEH+ have a distinct immune response after HSV-1 exposure compared with those from patients with ADEH−. Inhibition of the IRF3 and IRF7 innate immune pathways in patients with ADEH+ might be an important mechanism for increased susceptibility to disseminated viral infection.

In chronic nonhealing wounds, the healing process is disrupted and wounds are often infected with bacteria. About 85% of lower extremity amputations in diabetes are attributed to deep infection of foot ulcers. Therefore, infection control is critical for wound care. In this study, we analyzed lipid composition of Chamaecyparis obtusa extract, and we describe the wound-healing properties of its combination of 10 major lipid components. A 10-lipid mixture up-regulated HBD-3 and LL-37 through the olfactory receptor 2AT4 and induced phosphorylation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinases in primary human keratinocytes. In addition, the 10-lipid mixture had direct bactericidal effects against Staphylococcus aureus and Streptococcus pyogenes and protected against staphylococcal α-toxin–induced keratinocyte cell death. In an animal model, the 10-lipid mixture accelerated skin wound healing and was also effective in healing wounds superinfected with S. aureus. We suggest that the 10-lipid mixture, because of its wound-healing and antimicrobial properties, can be beneficial for wound treatment.

The epidermis serves as a critical protective barrier between the internal and external environment of the human body. Its remarkable barrier function is established through the keratinocyte (KC) terminal differentiation program. The transcription factors specifically regulating terminal differentiation remain largely unknown. Using a RNA-sequencing (RNA-seq) profiling approach, we found that forkhead box c 1 (FOXC1) was significantly up-regulated in human normal primary KC during the course of differentiation. This observation was validated in human normal primary KC from several different donors and human skin biopsies. Silencing FOXC1 in human normal primary KC undergoing differentiation led to significant down-regulation of late terminal differentiation genes markers including epidermal differentiation complex genes, keratinization genes, sphingolipid/ceramide metabolic process genes and epidermal specific cell-cell adhesion genes. We further demonstrated that FOXC1 works down-stream of ZNF750 and KLF4, and upstream of GRHL3. Thus, this study defines FOXC1 as a regulator specific for KC terminal differentiation and establishes its potential position in the genetic regulatory network.

It is estimated that 1 billion people around the world are vitamin D deficient. Vitamin D deficiency has been linked to various inflammatory diseases. However, the mechanism by which vitamin D reduces inflammation remains poorly understood. In this study, we investigated the inhibitory effects of physiologic levels of vitamin D on LPS-stimulated inflammatory response in human blood monocytes and explored potential mechanisms of vitamin D action. We observed that two forms of the vitamin D, 1,25(OH)2D3, and 25(OH)D3, dose dependently inhibited LPS-induced p38 phosphorylation at physiologic concentrations, IL-6 and TNF-α production by human monocytes. Upon vitamin D treatment, the expression of MAPK phosphatase-1 (MKP-1) was significantly upregulated in human monocytes and murine bone marrow-derived macrophages (BMM). Increased binding of the vitamin D receptor and increased histone H4 acetylation at the identified vitamin D response element of the murine and human MKP-1 promoters were demonstrated. Moreover, in BMM from MKP1−/− mice, the inhibition of LPS-induced p38 phosphorylation by vitamin D was completely abolished. Vitamin D inhibition of LPS-induced IL-6 and TNF-α production by BMM from MKP-1−/− mice was significantly reduced as compared with wild-type mice. In conclusion, this study identified the upregulation of MKP-1 by vitamin D as a novel pathway by which vitamin D inhibits LPS-induced p38 activation and cytokine production in monocytes/macrophages.

Background Atopic dermatitis (AD) is a common inflammatory skin disease. A subset of patients with AD are susceptible to disseminated herpes simplex virus (HSV) infection, a complication termed eczema herpeticum (ADEH+). The immune mechanisms causing ADEH+ remain elusive. Using RNA sequencing, we recently found that ankyrin repeat domain 1 (ANKRD1) was significantly induced in human PBMCs upon HSV-1 stimulation, and its induction in patients with ADEH+ was significantly reduced compared with that seen in AD patients without a history of eczema herpeticum (ADEH−). Objective We sought to validate ANKRD1 gene expression in nonatopic (NA) subjects, patients with ADEH−, and patients with ADEH+ and to delineate the biological function of ANKRD1 and the signaling pathway or pathways involved. Methods Purification of human PBMCs, monocytes, B cells, dendritic cells, T cells, and natural killer cells; RNA extraction and quantitative RT-PCR; small interfering RNA technique; co-immunoprecipitation; and Western blot assays were used. Results ANKRD1 expression was significantly reduced in PBMCs from patients with ADEH+ after HSV-1 stimulation compared with PBMCs from patients with ADEH−. We found that the induction of ANKRD1 by HSV-1 and multiple pattern recognition receptor agonists are mediated by inflammatory cytokines. Silencing ANKRD1 gene expression in antigen-presenting cells led to increased viral load and reduced IFNB1 and IL29 production. Using co-immunoprecipitation methods, we demonstrated that ANKRD1 formed protein complexes with interferon regulatory factor (IRF) 3 and IRF7, which are important transcription factors regulating signaling transduction of pattern recognition receptors. Overexpression of ANKRD1 enhanced the IRF3-mediated signaling pathways. Conclusion ANKRD1 is involved in IRF3-mediated antiviral innate immune signaling pathways. Its reduced expression in patients with ADEH+ might contribute to the pathogenesis of ADEH+.

The epidermis serves as a critical protective barrier between the internal and external environment of the human body. Its remarkable barrier function is established through the keratinocyte (KC) terminal differentiation program. The transcription factors specifically regulating terminal differentiation remain largely unknown. Using a RNA-sequencing (RNA-seq) profiling approach, we found that forkhead box c 1 (FOXC1) was significantly up-regulated in human normal primary KC during the course of differentiation. This observation was validated in human normal primary KC from several different donors and human skin biopsies. Silencing FOXC1 in human normal primary KC undergoing differentiation led to significant down-regulation of late terminal differentiation genes markers including epidermal differentiation complex genes, keratinization genes, sphingolipid/ceramide metabolic process genes and epidermal specific cell-cell adhesion genes. We further demonstrated that FOXC1 works down-stream of ZNF750 and KLF4, and upstream of GRHL3. Thus, this study defines FOXC1 as a regulator specific for KC terminal differentiation and establishes its potential position in the genetic regulatory network.

The effects of serum vitamin D status on atopy, steroid requirement, and functional responsiveness to corticosteroids in children versus adults with asthma have not been studied systematically.We sought to explore the age-specific effects of vitamin D in asthmatic patients.Serum vitamin D levels were examined in a prospective study of adults and children (102 healthy control subjects and 103 asthmatic patients). PBMCs were cultured for 3 hours with or without 100 nmol/L dexamethasone, and the expression of corticosteroid-regulated genes was detected by using real-time PCR. Serum IgE levels were measured, and information about asthmatic patients' steroid requirements was collected.Deficient serum vitamin D levels (20 ng/mL) were found in 47.6% of asthmatic patients and 56.8% of healthy control subjects, with means ± SDs of 20.7 ± 9.8 and 19.2 ± 7.7 ng/mL, respectively. In multivariate regression models a significant positive correlation between serum vitamin D levels and the expression of vitamin D-regulated targets, cytochrome P450, family 24, subfamily a (cyp24a) expression by PBMCs (P = .0084, pediatric asthma group only) and serum LL-37 levels (P = .0006 in the pediatric group but P = .0067 in the adult asthma group), was found. An inverse association between vitamin D and serum IgE levels was observed in the pediatric (P = .006) asthma group. Serum vitamin D level (P = .05), as well as PBMC cyp24a expression (P = .0312), demonstrated a significant inverse relationship with daily inhaled corticosteroid dose in the pediatric asthma group only. Cyp24a expression in PBMCs correlated positively with in vitro suppression of TNF-α by dexamethasone (P = .05) and IL-13 (P = .0094) in PBMCs in the pediatric asthma group only.This study demonstrated significant associations between serum vitamin D status and steroid requirement and in vitro responsiveness to corticosteroids in the pediatric but not the adult asthma group. Vitamin D was also related to IgE levels in children but not in adults.

Background A subset of patients with atopic dermatitis (AD) is prone to disseminated herpes simplex virus (HSV) infection, i.e. eczema herpeticum (ADEH+). Biomarkers that identify ADEH+ are lacking.