Signaling sphingolipids are biomarkers for atopic dermatitis prone to disseminated viral infections.

BACKGROUND: Life-threatening viral diseases such as eczema herpeticum (EH) and eczema vaccinatum (EV) occur in <5% of individuals with atopic dermatitis (AD). The diagnosis of AD, however, excludes all individuals with AD from smallpox vaccination. OBJECTIVES: We sought to identify circulatory and skin lipid biomarkers associated with EH and EV. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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+.

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.

Specificity protein 1 is pivotal in the skin's antiviral response.

BACKGROUND: Previous studies have found specificity protein (Sp) 1 transcription factor in the viral replication machinery and postulated that Sp1 was required for viral replication in host cells. OBJECTIVES: We investigated the role of Sp1 in the skin's antiviral responses from the perspective of host defense and its biological relevance in patients with atopic dermatitis and a history of eczema herpeticum (ADEH(+)). METHODS: Small interfering RNA duplexes were used to knock down Sp1 in keratinocytes. The expression of vaccinia virus (VV), herpes simplex virus 1, and other genes were evaluated by real-time PCR, or combined with Western blot and immunohistofluorescence staining. A total of 106 human subjects participated in this study. RESULTS: Both VV and herpes simplex virus 1 replication were enhanced in Sp1 knocked-down keratinocytes. Sp1 gene expression was significantly decreased in ADEH(+) subjects compared with patients with atopic dermatitis without a history of eczema herpeticum and nonatopic subjects (P < .0001) and inversely correlated with VV DNA copy number in human skin explants incubated with VV in vitro (partial correlation r = -0.256; P = .009). Gene profiling revealed that the antiviral genes, double-stranded RNA-dependent protein kinase (PKR) and 2'5'-oligoadenylate synthetase 2 (OAS2), were significantly downregulated in Sp1-silenced keratinocytes. Gene expression of PKR and OAS2 was also significantly decreased in skin biopsies from ADEH(+) subjects compared with patients with atopic dermatitis without a history of eczema herpeticum and nonatopic subjects. IFN-γ augmented the antiviral capacity of Sp1-silenced keratinocytes. CONCLUSION: Specificity protein 1 knockdown enhances viral replication in keratinocytes by downregulating gene expression of PKR and OAS2. Sp1 deficiency in ADEH(+) patients may contribute to their increased propensity to disseminated skin viral infections. IFN-γ augmentation may be a potential treatment for ADEH(+) patients.

The signal transducer and activator of transcription 6 gene (STAT6) increases the propensity of patients with atopic dermatitis toward disseminated viral skin infections.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with increased susceptibility to recurrent skin infections. OBJECTIVE: We sought to determine why a subset of patients with AD have an increased risk of disseminated viral skin infections. METHODS: Human subjects with AD with a history of eczema herpeticum (EH) and various control groups were enrolled. Vaccinia virus (VV) expression was measured by means of PCR and immunofluorescent staining in skin biopsy specimens from each study group after incubation with VV. Transgenic mice with a constitutively active signal transducer and activator of transcription 6 gene (STAT6) were characterized for response to VV skin inoculation. Genotyping for 10 STAT6 single nucleotide polymorphisms (SNPs) was performed in a white patient sample (n = 444). RESULTS: VV gene and protein expression were significantly increased in the skin of patients with EH compared with other subject groups after incubation with VV in vitro. Antibody neutralization of IL-4 and IL-13 resulted in lower VV replication in patients with a history of EH. Mice that expressed a constitutively active STAT6 gene compared with wild-type mice had increased mortality and satellite lesion formation after VV skin inoculation. Significant associations were observed between STAT6 SNPs and EH (rs3024975, rs841718, rs167769, and rs703817) and IFN-γ production. The strongest association was observed for a 2-SNP haplotype (patients with AD with a history of EH vs patients with AD without a history of EH, 24.9% vs 9.2%; P = 5.17 × 10(-6)). CONCLUSION: The STAT6 gene increases viral replication in the skin of patients with AD with a history of EH. Further genetic association studies and functional investigations are warranted.

Human atopic dermatitis complicated by eczema herpeticum is associated with abnormalities in IFN-γ response.

BACKGROUND: The basis for increased susceptibility of patients with atopic dermatitis (AD) to develop disseminated viral skin infections such as eczema herpeticum (AD with a history of eczema herpeticum, ADEH(+)) is poorly understood. OBJECTIVE: We sought to determine whether subjects with AD prone to disseminated viral skin infections have defects in their IFN responses. METHODS: GeneChip profiling was used to identify differences in gene expression of PBMCs from patients with ADEH(+) compared with patients with AD without a history of eczema herpeticum (ADEH(-)) and nonatopic controls. Key differences in protein expression were verified by enzyme-linked immunosorbent spot assay and/or ELISA. Clinical relevance was further demonstrated by a mouse model of disseminated viral skin infection and genetic association analysis for genetic variants in IFNG and IFNGR1 and ADEH among 435 cases and controls. RESULTS: We demonstrate by global gene expression analysis selective transcriptomic changes within the IFN superfamily of PBMCs from subjects with ADEH(+) reflecting low IFN-γ and IFN-γ receptor gene expression. IFN-γ protein production was also significantly lower in patients with ADEH(+) (n = 24) compared with patients with ADEH(-) (n = 20) and nonatopic controls (n = 20). IFN-γ receptor knockout mice developed disseminated viral skin infection after epicutaneous challenge with vaccinia virus. Genetic variants in IFNG and IFNGR1 single nucleotide polymorphisms (SNPs) were significantly associated with ADEH (112 cases, 166 controls) and IFN-γ production: a 2-SNP (A-G) IFNGR1 haplotype (rs10457655 and rs7749390) showed the strongest association with a reduced risk of ADEH+ (13.2% ADEH(+) vs 25.5% ADEH(-); P = .00057). CONCLUSION: Patients with ADEH(+) have reduced IFN-γ production, and IFNG and IFNGR1 SNPs are significantly associated with ADEH(+) and may contribute to an impaired immune response to herpes simplex virus.

Inhibition of S100A11 gene expression impairs keratinocyte response against vaccinia virus through downregulation of the IL-10 receptor 2 chain.

BACKGROUND: The mechanism that predisposes patients with atopic dermatitis (AD) to disseminated vaccinia viral (VV) skin infection after smallpox vaccination is unknown. We have demonstrated that expression of S100A11, a calcium-binding protein involved in keratinocyte differentiation, is downregulated in AD. OBJECTIVE: We investigated whether inhibiting expression of S100A11 increased VV replication in human keratinocytes and the mechanism by which S100A11 affects the innate immune response of keratinocytes. METHODS: Small interfering RNA duplexes were used to reduce gene expression of S100A11 in keratinocytes. VV replication was evaluated by real-time PCR and viral plaque assay. VV cytopathic effect was assessed by crystal violet staining. Affymetrix GeneChip assay was used to compare gene expression profiles. Real time PCR, Western blotting, and immunohistochemistry staining assay were used to evaluate gene expression in keratinocytes and AD skin biopsies. RESULTS: Keratinocytes with deficient S100A11 expression supported increased VV replication and manifested augmented VV cytopathic effects. Gene microarray analysis revealed that the IL-10 receptor 2 chain (IL-10R2), which binds IFN-lambdas, was downregulated by 2.26-fold in S100A11-silenced keratinocytes. IL-10R2 expression was found to be decreased in skin biopsies from patients with acute AD (mean, 25.21 +/- 5.25; n = 20) compared with skin from normal healthy subjects (mean, 137.1 +/- 34.46; n = 19; P < .01). Furthermore, deficient S100A11 gene expression significantly impaired IL-29 (IFN-lambda1) responsiveness (2' 5'-oligoadenylate synthetase and Myxovirus [influenza virus] resistance induction) and its anti-VV effects in keratinocytes. CONCLUSIONS: Inhibition of S100A11 gene expression impairs the ability of keratinocytes to control VV replication via downregulation of IFN-lambda receptor IL-10R2.

Skin Wound Healing Is Accelerated by a Lipid Mixture Representing Major Lipid Components of Chamaecyparis obtusa Plant Extract.

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.

Ceragenins: a class of antiviral compounds to treat orthopox infections.

Eczema vaccinatum is a potentially fatal, disseminated viral skin infection that develops in individuals with atopic dermatitis after exposure to the vaccinia virus (VV). Despite advances in modern medicine, there are few options for those suffering from disseminated VV infections. Ceragenins (CSAs) are synthetic antimicrobial compounds designed to mimic the structure and function of endogenous antimicrobial peptides (AMPs). We show that CSA-13 exhibits potent antiviral activity against VV by (1) direct antiviral effects against VV; and (2) stimulating the expression of endogenous AMPs with known antiviral activity against VV. In addition, we show that a topical application of CSA-13 penetrates the skin and reduces subsequent satellite lesion formation. This suggests that treatment with CSA-13 may be an intervention for individuals with a disseminated VV skin infection.

Macrophage inflammatory protein 3alpha deficiency in atopic dermatitis skin and role in innate immune response to vaccinia virus.

BACKGROUND: Patients with atopic dermatitis (AD) are prone to disseminated viral skin infections and therefore are not vaccinated against smallpox because of potential complications. Macrophage inflammatory protein 3alpha (MIP-3alpha) is a C-C chemokine expressed by keratinocytes that exhibits antimicrobial activity against bacteria and fungi; however, its role in antiviral innate immunity is unknown. OBJECTIVE: Evaluate the level of MIP-3alpha in AD skin and its role in the innate immune response to vaccinia virus (VV). METHODS: Macrophage inflammatory protein 3alpha levels were evaluated using real-time RT-PCR, immunodot-blot, and immunohistochemistry. The antiviral activity of MIP-3alpha was determined using a standard viral plaque assay. RESULTS: Macrophage inflammatory protein 3alpha gene expression was significantly (P < .01) decreased in AD skin (0.21 +/- 0.05 ng MIP-3alpha/ng glyceraldehyde-3-phosphate dehydrogenase) compared with psoriasis skin (0.67 +/- 0.13). This was confirmed at the protein level using immunohistochemistry. We further demonstrate that T(H)2 cytokines downregulate MIP-3alpha expression. The importance of MIP-3alpha in the innate immune response against VV was established by first demonstrating that MIP-3alpha exhibits activity against VV. Second, VV replication was significantly increased (P < .01) in keratinocytes treated with an antibody to neutralize MIP-3alpha. CONCLUSION: The current study demonstrates that MIP-3alpha exhibits antiviral activity against VV and demonstrates the importance of MIP-3alpha in the innate immune response against VV. In addition, AD skin is deficient in MIP-3alpha, in part because of the overexpression of T(H)2 cytokines in AD skin. CLINICAL IMPLICATIONS: MIP-3alpha deficiency in AD skin contributes to patients' increased propensity toward eczema vaccinatum. Increasing MIP-3alpha or neutralizing T(H)2 cytokines could prevent adverse reactions in patients with AD after smallpox vaccination.

Cytokine milieu of atopic dermatitis skin subverts the innate immune response to vaccinia virus.

Atopic dermatitis (AD) is associated with eczema vaccinatum (EV), a disseminated viral skin infection that follows inoculation with vaccinia virus (VV). This study examined whether AD skin can control VV replication, and the role of IL-4 and IL-13 in modulating the human cathelicidin LL-37, an antimicrobial peptide that kills VV. AD skin exhibited increased VV replication and decreased LL-37 expression compared to normal or psoriasis skin. IL-4/IL-13 enhanced VV replication while downregulating LL-37 in VV-stimulated keratinocytes. Neutralizing IL-4/IL-13 in AD skin augmented LL-37 and inhibited VV replication. Cathelicidins were induced via toll-like receptor-3 and were inhibited by IL-4/IL-13 through STAT-6. Skin from cathelicidin-deficient mice exhibited reduced ability to control VV replication. Exogenous LL-37 controlled vaccinia viral replication in infected keratinocytes and AD skin explants. The current study demonstrates that Th2 cytokines enhance VV replication in AD skin by subverting the innate immune response against VV in a STAT-6-dependent manner.

Cathelicidin deficiency predisposes to eczema herpeticum.

BACKGROUND: The cathelicidin family of antimicrobial peptides is an integral component of the innate immune response that exhibits activity against bacterial, fungal, and viral pathogens. Eczema herpeticum (ADEH) develops in a subset of patients with atopic dermatitis (AD) because of disseminated infection with herpes simplex virus (HSV). OBJECTIVE: This study investigated the potential role of cathelicidins in host susceptibility to HSV infection. METHODS: Glycoprotein D was measured by means of real-time RT-PCR as a marker of HSV replication in skin biopsy specimens and human keratinocyte cultures. Cathelicidin expression was evaluated in skin biopsy specimens from patients with AD (n = 10) without a history of HSV skin infection and from patients with ADEH (n = 10). RESULTS: The cathelicidin peptide LL-37 (human cathelicidin) exhibited activity against HSV in an antiviral assay, with significant killing (P < .001) within the physiologic range. The importance of cathelicidins in antiviral skin host defense was confirmed by the observation of higher levels of HSV-2 replication in cathelicidin-deficient (Cnlp-/-) mouse skin (2.6 +/- 0.5 pg HSV/pg GAPDH, P < .05) compared with that seen in skin from their wild-type counterparts (0.9 +/- 0.3). Skin from patients with ADEH exhibited significantly (P < .05) lower levels of cathelicidin protein expression than skin from patients with AD. We also found a significant inverse correlation between cathelicidin expression and serum IgE levels (r2 = 0.46, P < .05) in patients with AD and patients with ADEH. CONCLUSION: This study demonstrates that the cathelicidin peptide LL-37 possesses antiviral activity against HSV and demonstrates the importance of variable skin expression of cathelicidins in controlling susceptibility to ADEH. Additionally, serum IgE levels might be a surrogate marker for innate immune function and serve as a biomarker for which patients with AD are susceptible to ADEH. CLINICAL IMPLICATIONS: A deficiency of LL-37 might render patients with AD susceptible to ADEH. Therefore increasing production of skin LL-37 might prevent herpes infection in patients with AD.

Structure-function relationships among human cathelicidin peptides: dissociation of antimicrobial properties from host immunostimulatory activities.

Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by d- and l-amino acid forms of cathelicidin and correlated with membrane permeability, suggesting that highly structure-specific binding to a cell surface receptor is not likely. However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions.

Complement activation in a model of chronic fatigue syndrome.

BACKGROUND: A need exists to identify biological markers in chronic fatigue syndrome (CFS). OBJECTIVE: To use an exercise and/or allergen challenge to induce the symptoms of CFS and to identify a biological marker that correlates with these symptoms. METHODS: Patients with CFS (n = 32) and age-matched, normal control patients (n = 29) exercised for 20 minutes on a stationary bike at 70% of their predicted max work load (Watts). Patients from each group with positive skin test results were also challenged with intranasally administered relevant allergens. Symptoms were recorded for 2 weeks before and 1 week after each challenge, using 3 different instruments. Blood samples were taken before, and 0, 1, 6, and 24 hours after challenges. Levels of complement split products, cell-associated cytokines, and eosinophilic cationic protein were measured. Mean preexercise and postexercise symptom scores were evaluated for each group. RESULTS: Exercise challenge induced significant increases of the complement split product C4a, but not C3a or C5a, at 6 hours after exercise only in the CFS group (P <.01), regardless of allergy status. Mean symptom scores were significantly increased after exercise through the use of a daily diary (P <.03) and a weekly diary (P <.01) for the CFS group only. Mean scores for the Multidimensional Fatigue Inventory categories "reduced activity" and "mental fatigue" were significantly increased in the CFS group only (P <.04 and P <.02, respectively). CONCLUSIONS: Exercise challenge may be a valuable tool in the development of diagnostic criteria and tests for CFS. Establishment of a role for complement activation products as markers or participants in production of illness require further study.

Selective killing of vaccinia virus by LL-37: implications for eczema vaccinatum.

Possible bioterrorism with smallpox has led to the resumption of smallpox (vaccinia virus) immunization. One complication, eczema vaccinatum, occurs primarily in patients with atopic dermatitis (AD). Skin lesions of patients with AD, but not psoriasis, is deficient in the cathelicidin antimicrobial peptide (LL-37) and human beta-defensin-2 (HBD-2). We hypothesized that this defect may explain the susceptibility of patients with AD to eczema vaccinatum. The Wyeth vaccine strain of vaccinia virus was incubated with varying concentrations of human (LL-37) and murine (CRAMP) cathelicidins, human alpha-defensin (HBD-1, HBD-2), and a control peptide. Outcomes included quantification of viral PFU, vaccinia viral gene expression by quantitative real-time RT-PCR, and changes in virion structure by transmission electron microscopy. CRAMP knockout mice and control animals were inoculated by skin pricks with 2 x 10(5) PFU of vaccinia and examined daily for pox development. Physiologic amounts of human and murine cathelicidins (10-50 micro M), but not human defensins, which had antibacterial activity, resulted in the in vitro reduction of vaccinia viral plaque formation (p < 0.0001), vaccinia mRNA expression (p < 0.001), and alteration of vaccinia virion structure. In vivo vaccinia pox formation occurred in four of six CRAMP knockout animals and in only one of 15 control mice (p < 0.01). These data support a role for cathelicidins in the inhibition of orthopox virus (vaccinia) replication both in vitro and in vivo. Susceptibility of patients with AD to eczema vaccinatum may be due to a deficiency of cathelicidin.