Atopic Dermatitis Complicated by Recurrent Eczema Herpeticum Is Characterized by Multiple, Concurrent Epidermal Inflammatory Endotypes.

A subgroup of patients with atopic dermatitis (AD) suffers from recurrent, disseminated herpes simplex virus skin infection, termed eczema herpeticum. To determine the transcriptional mechanisms of the skin and immune system pathobiology that underlie development of AD with eczema herpeticum (ADEH), we performed RNA-sequencing analysis of nonlesional skin (epidermis, dermis) from AD patients with and without a history of ADEH (ADEH+, n = 15; ADEH-, n = 13) along with healthy controls (n = 15). We also performed RNA sequencing on participants' plasmacytoid dendritic cells infected in vitro with herpes simplex virus 1. ADEH+ patients exhibited dysregulated gene expression, limited in the dermis (14 differentially expressed genes) and more widespread in the epidermis (129 differentially expressed genes). ADEH+-upregulated epidermal differentially expressed genes were enriched in type 2 cytokine (IL4R , CCL22, CRLF2, IL7R), interferon (CXCL10, ICAM1, IFI44, IRF7), and IL-36γ (IL36G) inflammatory gene pathways. All ADEH+ participants exhibited type 2 cytokine and inteferon endotypes, and 87% were IL36G-high. In contrast, these endotypes were more variably expressed among ADEH- participants. ADEH+ skin also had dysregulated epidermal differentiation complex gene expression of the late-cornified envelope, S100A, and small proline-rich gene families, which are involved in skin barrier function and antimicrobial activities. Plasmacytoid dendritic cell transcriptional responses to herpes simplex virus 1 infection were unaltered by ADEH status. The study concluded that the pathobiology underlying ADEH+ risk is associated with a unique, multifaceted epidermal inflammation that accompanies dysregulation of epidermal differentiation complex genes. These findings will help direct future studies that define how these inflammatory patterns may drive risk of eczema herpeticum in AD.

SLC38A5 aggravates DC-mediated psoriasiform skin inflammation via potentiating lysosomal acidification.

Amino acid (aa) metabolism is closely correlated with the pathogenesis of psoriasis; however, details on aa transportation during this process are barely known. Here, we find that SLC38A5, a sodium-dependent neutral aa transporter that counter-transports protons, is markedly upregulated in the psoriatic skin of both human patients and mouse models. SLC38A5 deficiency significantly ameliorates the pathogenesis of psoriasis, indicating a pathogenic role of SLC38A5. Surprisingly, SLC38A5 is almost exclusively expressed in dendritic cells (DCs) when analyzing the psoriatic lesion and mainly locates on the lysosome. Mechanistically, SLC38A5 potentiates lysosomal acidification, which dictates the cleavage and activation of TLR7 with ensuing production of pro-inflammatory cytokines such as interleukin-23 (IL-23) and IL-1ß from DCs and eventually aggravates psoriatic inflammation. In summary, this work uncovers an auxiliary mechanism in driving lysosomal acidification, provides inspiring insights for DC biology and psoriasis etiology, and reveals SLC38A5 as a promising therapeutic target for treating psoriasis.

Atopic dermatitis complicated by recurrent eczema herpeticum is characterized by multiple, concurrent epidermal inflammatory endotypes.

BACKGROUND: A subgroup of atopic dermatitis (AD) patients suffer from recurrent, disseminated herpes simplex virus (HSV) skin infections, termed eczema herpeticum (EH), which can be life-threatening and contribute to AD morbidity. The pathobiology underlying ADEH is unknown. OBJECTIVE: To determine transcriptional mechanisms of skin and immune system pathobiology that underlie ADEH disease. METHODS: We performed whole transcriptome RNA-sequencing of non-lesional skin samples (epidermis, dermis) of AD patients with (ADEH + , n=15) and without (ADEH - , n=13) recurrent EH history, and healthy controls (HC, n=15). We also performed RNA-sequencing on plasmacytoid dendritic cells (pDCs) collected from these participants and infected in vitro with HSV-1. Differential expression, gene set enrichment, and endotyping analyses were performed. RESULTS: ADEH + disease was characterized by dysregulation in skin gene expression, which was limited in dermis (differentially expressed genes [DEGs]=14) and widespread in epidermis (DEGs=129). ADEH + -upregulated epidermal DEGs were enriched in type 2 cytokine (T2) ( IL4R, CCL22, CRLF2, IL7R ), interferon ( CXCL10, ICAM1, IFI44 , and IRF7) , and IL-36γ ( IL36G ) inflammatory pathway genes. At a person-level, all ADEH + participants exhibited T2 and interferon endotypes and 87% were IL36G-high. In contrast, these endotypes were more variably expressed among ADEH - participants. ADEH + patient skin also exhibited dysregulation in epidermal differentiation complex (EDC) genes within the LCE, S100 , and SPRR families, which are involved in skin barrier function, inflammation, and antimicrobial activities. pDC transcriptional responses to HSV-1 infection were not altered by ADEH status. CONCLUSIONS: ADEH + pathobiology is characterized by a unique, multi-faceted epidermal inflammation that accompanies dysregulation in the expression of EDC genes. Key Messages: AD patients with a history of recurrent EH exhibit molecular skin pathobiology that is similar in form, but more severe in degree, than in AD patients without this complication. Non-lesional skin of ADEH + patients concurrently exhibits excessive type 2 cytokine, interferon, and IL-36γ-driven epidermal inflammation. Expression of these inflammatory skin endotypes among ADEH + patients is associated with dysregulation in expression of epidermal differentiation complex genes involved in barrier function, inflammation, and antimicrobial activity. Capsule Summary: AD patients with a history of recurrent disseminated HSV-1 skin infections form a unique molecular skin endotype group that concurrently exhibits type 2 cytokine, interferon, and IL-36γ-driven skin inflammation, accompanied by dysregulation in expression of epidermal differentiation complex genes involved in barrier function, inflammation, and antimicrobial activity.

Staphylococcus aureus causes aberrant epidermal lipid composition and skin barrier dysfunction.

BACKGROUND: Staphylococcus (S) aureus colonization is known to cause skin barrier disruption in atopic dermatitis (AD) patients. However, it has not been studied how S. aureus induces aberrant epidermal lipid composition and skin barrier dysfunction. METHODS: Skin tape strips (STS) and swabs were obtained from 24 children with AD (6.0 ± 4.4 years) and 16 healthy children (7.0 ± 4.5 years). Lipidomic analysis of STS samples was performed by mass spectrometry. Skin levels of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) were evaluated. The effects of MSSA and MRSA were evaluated in primary human keratinocytes (HEKs) and organotypic skin cultures. RESULTS: AD and organotypic skin colonized with MRSA significantly increased the proportion of lipid species with nonhydroxy fatty acid sphingosine ceramide with palmitic acid ([N-16:0 NS-CER], sphingomyelins [16:0-18:0 SM]), and lysophosphatidylcholines [16:0-18:0 LPC], but significantly reduced the proportion of corresponding very long-chain fatty acids (VLCFAs) species (C22-28) compared to the skin without S. aureus colonization. Significantly increased transepidermal water loss (TEWL) was found in MRSA-colonized AD skin. S. aureus indirectly through interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, and IL-33 inhibited expression of fatty acid elongase enzymes (ELOVL3 and ELOVL4) in HEKs. ELOVL inhibition was more pronounced by MRSA and resulted in TEWL increase in organotypic skin. CONCLUSION: Aberrant skin lipid profiles and barrier dysfunction are associated with S. aureus colonization in AD patients. These effects are attributed to the inhibition of ELOVLs by S. aureus-induced IL-1ß, TNF-α, IL-6, and IL-33 seen in keratinocyte models and are more prominent in MRSA than MSSA.

Whole genome sequencing identifies novel genetic mutations in patients with eczema herpeticum.

BACKGROUND: Eczema herpeticum (EH) is a rare complication of atopic dermatitis (AD) caused by disseminated herpes simplex virus (HSV) infection. The role of rare and/or deleterious genetic variants in disease etiology is largely unknown. This study aimed to identify genes that harbor damaging genetic variants associated with HSV infection in AD with a history of recurrent eczema herpeticum (ADEH+). METHODS: Whole genome sequencing (WGS) was performed on 49 recurrent ADEH+ (≥3 EH episodes), 491 AD without a history of eczema herpeticum (ADEH-) and 237 non-atopic control (NA) subjects. Variants were annotated, and a gene-based approach (SKAT-O) was used to identify genes harboring damaging genetic variants associated with ADEH+. Genes identified through WGS were studied for effects on HSV responses and keratinocyte differentiation. RESULTS: Eight genes were identified in the comparison of recurrent ADEH+to ADEH-and NA subjects: SIDT2, CLEC7A, GSTZ1, TPSG1, SP110, RBBP8NL, TRIM15, and FRMD3. Silencing SIDT2 and RBBP8NL in normal human primary keratinocytes (NHPKs) led to significantly increased HSV-1 replication. SIDT2-silenced NHPKs had decreased gene expression of IFNk and IL1b in response to HSV-1 infection. RBBP8NL-silenced NHPKs had decreased gene expression of IFNk, but increased IL1b. Additionally, silencing SIDT2 and RBBP8NL also inhibited gene expression of keratinocyte differentiation markers keratin 10 (KRT10) and loricrin (LOR). CONCLUSION: SIDT2 and RBBP8NL participate in keratinocyte's response to HSV-1 infection. SIDT2 and RBBP8NL also regulate expression of keratinocyte differentiation genes of KRT10 and LOR.

Interferon Kappa Is Important for Keratinocyte Host Defense against Herpes Simplex Virus-1.

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.

Interferon Kappa Is Up-Regulated in Psoriasis and It Up-Regulates Psoriasis-Associated Cytokines in vivo.

PURPOSE: There is increased type I interferon signature in psoriasis patients. Interferon-kappa (IFN-κ) is a member of type I interferon family that is constitutively expressed by keratinocytes. In this study, we investigate whether IFN-κ is involved in psoriasis etiology. PATIENTS AND METHODS: Twenty healthy individuals, 20 psoriasis vulgaris patients and 10 atopic dermatitis (AD) were included for this study. Immunohistochemistry staining, normal human epidermal keratinocytes (NHEK) culture, Ca2Cl-induced differentiation, quantitative reverse transcription (qRT-PCR), ELISA and murine experiments were performed. RESULTS: We found IFN-κ protein expression was extremely low in the epidermis of normal skin, but it was significantly increased in the suprabasal layers of epidermal keratinocytes in psoriatic skin lesions. However, its expression in the skin lesions of AD was similar to normal skin. Additionally, IFN-κ protein was detected in sera from psoriasis patients, but not in sera from normal subjects and AD. We further investigated the regulation of IFNk gene expression in NHEK. We found that IFNk was significantly induced by types of nucleic acid pathogen recognition receptor (PRR) agonists in NHEK. While its expression was significantly induced by itself and IFN-γ, it was inhibited by type 2 immunity cytokines IL4 and IL13; other inflammatory cytokines including IL1 super-family members and IL17A did not alter its expression. Addition of recombinant IFN-κ did not affect keratinocytes differentiation. Using the murine experimental model, we demonstrated that subcutaneous administration of recombinant IFN-κ did not increase skin thickness, but significantly increased the transcription of TNFA and IL17A in mice skin. CONCLUSION: Increased IFN-κ in psoriasis may be caused by injured cells-released nucleic acids, increased IFN-γ and self-activation. Its enhancement may contribute to the etiology of the disease by enhancing TNFA and IL17A gene expression.

The Solute Carrier Transporter SLC15A3 Participates in Antiviral Innate Immune Responses against Herpes Simplex Virus-1.

The innate immune response is the first line defense against viral infections. Novel genes involved in this system are continuing to emerge. SLC15A3, a proton-coupled histidine and di-tripeptide transporter that was previously found in lysosomes, has been reported to inhibit chikungunya viral replication in host cells. In this study, we found that SLC15A3 was significantly induced by DNA virus herpes simplex virus-1(HSV-1) in monocytes from human peripheral blood mononuclear cells. Aside from monocytes, it can also be induced by HSV-1 in 293T, HeLa cells, and HaCaT cells. Overexpression of SLC15A3 in 293T cells inhibits HSV-1 replication and enhances type I and type III interferon (IFN) responses, while silencing SLC15A3 leads to enhanced HSV-1 replication with reduced IFN production. Moreover, we found that SLC15A3 interacted with MAVS and STING and potentiated MAVS- and STING-mediated IFN production. These results demonstrate that SLC15A3 participates in anti-HSV-1 innate immune responses by regulating MAVS- and STING-mediated signaling pathways.

A data mining paradigm for identifying key factors in biological processes using gene expression data.

A large volume of biological data is being generated for studying mechanisms of various biological processes. These precious data enable large-scale computational analyses to gain biological insights. However, it remains a challenge to mine the data efficiently for knowledge discovery. The heterogeneity of these data makes it difficult to consistently integrate them, slowing down the process of biological discovery. We introduce a data processing paradigm to identify key factors in biological processes via systematic collection of gene expression datasets, primary analysis of data, and evaluation of consistent signals. To demonstrate its effectiveness, our paradigm was applied to epidermal development and identified many genes that play a potential role in this process. Besides the known epidermal development genes, a substantial proportion of the identified genes are still not supported by gain- or loss-of-function studies, yielding many novel genes for future studies. Among them, we selected a top gene for loss-of-function experimental validation and confirmed its function in epidermal differentiation, proving the ability of this paradigm to identify new factors in biological processes. In addition, this paradigm revealed many key genes in cold-induced thermogenesis using data from cold-challenged tissues, demonstrating its generalizability. This paradigm can lead to fruitful results for studying molecular mechanisms in an era of explosive accumulation of publicly available biological data.

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

BACKGROUND: 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. OBJECTIVE: We sought to determine whether minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. METHODS: 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 ± 2.2 years) and healthy control subjects (9 male and 16 female subjects; age [mean ± SE], 34.8 ± 2.2 years). 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. 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 (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. CONCLUSION: Minimally invasive expression profiling of nonlesional skin reveals stratification in AD molecular pathology by type 2 inflammation that correlates with disease severity.

Ankyrin repeat domain 1 regulates innate immune responses against herpes simplex virus 1: A potential role in eczema herpeticum.

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+.

Correction: Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation.

[This corrects the article DOI: 10.1371/journal.pone.0167392.].

The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Inhibits Expression of SUMO/Sentrin-Specific Peptidase 6 To Facilitate Establishment of Latency.

Kaposi's sarcoma-associated herpesvirus (KSHV), which belongs to the Gammaherpesviridae, typically displays two different phases in its life cycle, the latent phase and the lytic phase. Latency-associated nuclear antigen (LANA), the primary viral product during latency, has been reported to bind to a series of cellular gene promoters to modulate gene transcription. To systemically elucidate the cellular genes regulated by LANA, we identified genome-wide LANA binding sites by chromatin immunoprecipitation coupled with sequencing (ChIP-seq). We stratified ChIP-seq data and found that LANA might be involved in the macromolecule catabolic process. Specifically, we found and verified that LANA could directly bind to the promoter of the SUMO/sentrin-specific peptidase 6 (SENP6) gene in vivo and in vitro LANA could repress SENP6 promoter activity in a dose-dependent manner in a reporter gene assay. LANA expression was sufficient to inhibit endogenous SENP6 expression at both the RNA and protein levels. Moreover, SENP6 overexpression in KSHV-infected cells reduced LANA at the protein level. Mechanistically, we found that SENP6 could interact with LANA and reduce the formation of sumoylated LANA, which relies on the desumoylation ability of SENP6. During de novo infection, SENP6 overexpression would decrease the abundance of LANA and enhance viral gene expression, which would hamper the establishment of latency. Taken together, these data suggest that KSHV-encoded LANA could inhibit SENP6 expression to regulate the abundance of itself, which may play an important role in controlling the establishment of latency.IMPORTANCE LANA, as a key latent protein produced by KSHV, is responsible for episome persistence and regulates viral reactivation. In the present study, our results demonstrated that LANA could bind to the promoter region of the SENP6 gene and inhibit SENP6 expression while the regulated SENP6 could in turn modulate the abundance of LANA through desumoylation. This delicate regulation may provide important insights to explain the abundance of LANA during KSHV latency.

Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation.

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.

Genetic and epigenetic studies of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory disease caused by the complex interaction of genetic, immune and environmental factors. There have many recent discoveries involving the genetic and epigenetic studies of AD. METHODS: A retrospective PubMed search was carried out from June 2009 to June 2016 using the terms "atopic dermatitis", "association", "eczema", "gene", "polymorphism", "mutation", "variant", "genome wide association study", "microarray" "gene profiling", "RNA sequencing", "epigenetics" and "microRNA". A total of 132 publications in English were identified. RESULTS: To elucidate the genetic factors for AD pathogenesis, candidate gene association studies, genome-wide association studies (GWAS) and transcriptomic profiling assays have been performed in this period. Epigenetic mechanisms for AD development, including genomic DNA modification and microRNA posttranscriptional regulation, have been explored. To date, candidate gene association studies indicate that filaggrin (FLG) null gene mutations are the most significant known risk factor for AD, and genes in the type 2 T helper lymphocyte (Th2) signaling pathways are the second replicated genetic risk factor for AD. GWAS studies identified 34 risk loci for AD, these loci also suggest that genes in immune responses and epidermal skin barrier functions are associated with AD. Additionally, gene profiling assays demonstrated AD is associated with decreased gene expression of epidermal differentiation complex genes and elevated Th2 and Th17 genes. Hypomethylation of TSLP and FCER1G in AD were reported; and miR-155, which target the immune suppressor CTLA-4, was found to be significantly over-expressed in infiltrating T cells in AD skin lesions. CONCLUSIONS: The results suggest that two major biologic pathways are responsible for AD etiology: skin epithelial function and innate/adaptive immune responses. The dysfunctional epidermal barrier and immune responses reciprocally affect each other, and thereby drive development of AD.

Targeted deep sequencing identifies rare loss-of-function variants in IFNGR1 for risk of atopic dermatitis complicated by eczema herpeticum.

BACKGROUND: A subset of atopic dermatitis is associated with increased susceptibility to eczema herpeticum (ADEH+). We previously reported that common single nucleotide polymorphisms (SNPs) in the IFN-γ (IFNG) and IFN-γ receptor 1 (IFNGR1) genes were associated with the ADEH+ phenotype. OBJECTIVE: We sought to interrogate the role of rare variants in interferon pathway genes for the risk of ADEH+. METHODS: We performed targeted sequencing of interferon pathway genes (IFNG, IFNGR1, IFNAR1, and IL12RB1) in 228 European American patients with AD selected according to their eczema herpeticum status, and severity was measured by using the Eczema Area and Severity Index. Replication genotyping was performed in independent samples of 219 European American and 333 African American subjects. Functional investigation of loss-of-function variants was conducted by using site-directed mutagenesis. RESULTS: We identified 494 single nucleotide variants encompassing 105 kb of sequence, including 145 common, 349 (70.6%) rare (minor allele frequency <5%), and 86 (17.4%) novel variants, of which 2.8% were coding synonymous, 93.3% were noncoding (64.6% intronic), and 3.8% were missense. We identified 6 rare IFNGR1 missense variants, including 3 damaging variants (Val14Met [V14M], Val61Ile, and Tyr397Cys [Y397C]) conferring a higher risk for ADEH+ (P = .031). Variants V14M and Y397C were confirmed to be deleterious, leading to partial IFNGR1 deficiency. Seven common IFNGR1 SNPs, along with common protective haplotypes (2-7 SNPs), conferred a reduced risk of ADEH+ (P = .015-.002 and P = .0015-.0004, respectively), and both SNP and haplotype associations were replicated in an independent African American sample (P = .004-.0001 and P = .001-.0001, respectively). CONCLUSION: Our results provide evidence that both genetic variants in the gene encoding IFNGR1 are implicated in susceptibility to the ADEH+ phenotype.

Identification of novel gene signatures in patients with atopic dermatitis complicated by eczema herpeticum.

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.

IL-25 enhances HSV-1 replication by inhibiting filaggrin expression, and acts synergistically with Th2 cytokines to enhance HSV-1 replication.

Atopic dermatitis (AD) is characterized by epidermal barrier defects and recurrent microbial skin infections. AD patients with a history of eczema herpeticum (ADEH+) have more severe skin disease and more highly T helper type 2 (Th2)-polarized immune responses as compared with uncomplicated AD (ADEH-). However, the mechanisms linking epidermal barrier defects and viral skin infection are not well understood. Recently, it has been reported that interleukin-25 may play a role in augmenting Th2 responses. We examined protein expression of IL-25 in the skin biopsies from normal subjects (n=10), ADEH- (n=18), ADEH+ (n=7), and psoriasis (n=9). IL-25 expression was increased in the skin from ADEH-, ADEH+, and psoriasis as compared with normal skin, and was significantly greater in lesional ADEH+ skin than in lesional ADEH- skin. Importantly, we demonstrated that IL-25 enhances herpes simplex virus (HSV)-1 and vaccinia virus replication by inhibiting filaggrin expression, and IL-25 acts synergistically with IL-4 and IL-13 to enhance HSV-1 replication in vitro. In contrast, IFN-γ inhibited HSV-1 replication in vitro. In addition, we demonstrate that filaggrin is a critical protein to inhibit HSV-1 replication because filaggrin small interfering RNA knockdown enhances HSV-1 replication in vitro. Filaggrin breakdown products, however, inhibited HSV-1 replication in vitro.

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

BACKGROUND: The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. OBJECTIVE: We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. METHODS: Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. RESULTS: We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. CONCLUSIONS: The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease.