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.
Background Patients with atopic dermatitis (AD) with a history of eczema herpeticum have increased staphylococcal colonization and infections. However, whether Staphylococcus aureus alters the ...outcome of skin viral infection has not been determined. Objective We investigated whether S aureus toxins modulated host response to herpes simplex virus (HSV) 1 and vaccinia virus (VV) infections in normal human keratinocytes (NHKs) and in murine infection models. Methods NHKs were treated with S aureus toxins before incubation of viruses. BALB/c mice were inoculated with S aureus 2 days before VV scarification. Viral loads of HSV-1 and VV were evaluated by using real-time PCR, a viral plaque-forming assay, and immunofluorescence staining. Small interfering RNA duplexes were used to knockdown the gene expression of the cellular receptor of α-toxin, a disintegrin and metalloprotease 10 (ADAM10). ADAM10 protein and α-toxin heptamers were detected by using Western blot assays. Results We demonstrate that sublytic staphylococcal α-toxin increases viral loads of HSV-1 and VV in NHKs. Furthermore, we demonstrate in vivo that the VV load is significantly greater ( P < .05) in murine skin inoculated with an α-toxin–producing S aureus strain compared with murine skin inoculated with the isogenic α-toxin–deleted strain. The viral enhancing effect of α-toxin is mediated by ADAM10 and is associated with its pore-forming property. Moreover, we demonstrate that α-toxin promotes viral entry in NHKs. Conclusion The current study introduces the novel concept that staphylococcal α-toxin promotes viral skin infection and provides a mechanism by which S aureus infection might predispose the host toward disseminated viral infections.
Capsule Summary Human ORAI1 polymorphisms were associated with susceptibility to CSU, and the South Han Chinese CSU who carry rs3741595C allele have reduced responsiveness to nonsedating ...H1-antihistamines.
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.
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-λs, 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-λ1) 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-λ receptor IL-10R2.
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.
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.
To search for novel ADEH+ gene signatures, a RNA-sequencing (RNA-seq) approach was applied to evaluate global transcriptional changes of peripheral blood mononuclear cells (PBMCs) between ADEH+ and ...AD without a history of EH (ADEH-).
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