Ex Vivo Infection of Human Skin Models with Herpes Simplex Virus 1: Accessibility of the Receptor Nectin-1 during Formation or Impairment of Epidermal Barriers Is Restricted by Tight Junctions.

Herpes simplex virus 1 (HSV-1) must overcome epidermal barriers to reach its receptors on keratinocytes and initiate infection in human skin. The cell-adhesion molecule nectin-1, which is expressed in human epidermis, acts as an efficient receptor for HSV-1 but is not within reach of the virus upon exposure of human skin under nonpathological conditions. Atopic dermatitis skin, however, can provide an entry portal for HSV-1 emphasizing the role of impaired barrier functions. Here, we explored how epidermal barriers impact HSV-1 invasion in human epidermis and influence the accessibility of nectin-1 for the virus. Using human epidermal equivalents, we observed a correlation of the number of infected cells with tight-junction formation, suggesting that mature tight junctions prior to formation of the stratum corneum prevent viral access to nectin-1. Consequently, impaired epidermal barriers driven by Th2-inflammatory cytokines interleukin 4 (IL-4) and IL-13 as well as the genetic predisposition of nonlesional atopic dermatitis keratinocytes correlated with enhanced infection supporting the impact of functional tight junctions for preventing infection in human epidermis. Comparable to E-cadherin, nectin-1 was distributed throughout the epidermal layers and localized just underneath the tight-junctions. While nectin-1 was evenly distributed on primary human keratinocytes in culture, the receptor was enriched at lateral surfaces of basal and suprabasal cells during differentiation. Nectin-1 showed no major redistribution in the thickened atopic dermatitis and IL-4/IL-13-treated human epidermis in which HSV-1 can invade. However, nectin-1 localization toward tight junction components changed, suggesting that defective tight-junction barriers make nectin-1 accessible for HSV-1 which enables facilitated viral penetration. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a widely distributed human pathogen which productively infects epithelia. The open question is which barriers of the highly protected epithelia must the virus overcome to reach its receptor nectin-1. Here, we used human epidermal equivalents to understand how physical barrier formation and nectin-1 distribution contribute to successful viral invasion. Inflammation-induced barrier defects led to facilitated viral penetration strengthening the role of functional tight-junctions in hindering viral access to nectin-1 that is localized just underneath tight junctions and distributed throughout all layers. We also found nectin-1 ubiquitously localized in the epidermis of atopic dermatitis and IL-4/IL-13-treated human skin implying that impaired tight-junctions in combination with a defective cornified layer allow the accessibility of nectin-1 to HSV-1. Our results support that successful invasion of HSV-1 in human skin relies on defective epidermal barriers, which not only include a dysfunctional cornified layer but also depend on impaired tight junctions.

Viral metagenomic identification of a novel anellovirus in blood sample of a child with atopic dermatitis.

Here, using viral metagenomics, a novel anellovirus with strain name HuAV-zj-ad1 was detected in blood sample from a child with atopic dermatitis. The complete genome sequence of HuAV-zj-ad1 was determined and fully characterized. The circular genome of HuAV-zj-ad1 is 2841 nt in length and includes four polyprotein ORFs. Phylogenetic analysis and pairwise sequence comparisons based on the amino acid sequences of ORF1, ORF2, ORF3, ORF4 indicated that HuAV-zj-ad1 belonged to a novel species within the genus Betatorquevirus. Polymerase chain reaction screening results showed this anellovirus was not present 50 blood samples from normal children. Whether this novel species of anellovirus has association with a certain disease needs further study.

Herpes simplex virus 1 proteins can induce skin inflammation in an atopic dermatitis-like mouse model.

Herpes simplex virus type 1 (HSV-1) can induce in certain individuals with atopic dermatitis (AD) severe cutaneous infections that can spread throughout the entire body, a condition named as AD complicated by eczema herpeticum (ADEH). It has been recently found that ADEH patients can produce specific IgE against HSV-1 proteins, which may contribute to lower protection against HSV-1. However, little is known about the capacity of these HSV-1 proteins to produce an inflammatory response at the skin level. In this study, using a mouse model of AD-like dermatitis, three HSV-1 proteins (glycoprotein D -gD-, glycoprotein B -gB- and VP22) were applied on tape-stripped back skin mice in three exposures periods. Ovalbumin (OVA) and 0.9% NaCl were used as positive and negative controls, respectively. Skin samples were obtained for analysis of specific cell components of skin infiltration. The results showed that the viral protein gD induced a statistically significant increase in the number of dermal infiltrating CD3+, CD4+ cells and mast cells compared with the negative control group. gD was also able to induce epidermal thickening and epidermal infiltration of T cells closely related to the one produced in mice sensitized with OVA. However, VP22 and gB contributed to a lesser extent to skin inflammation. These results showed that proteins from HSV-1, especially gD, can have per se an important T cell and mast cell-driven inflammatory potential at the skin level.

Filaggrin deficiency promotes the dissemination of cutaneously inoculated vaccinia virus.

BACKGROUND: Eczema vaccinatum is a life-threatening complication of smallpox vaccination in patients with atopic dermatitis (AD) characterized by dissemination of vaccinia virus (VV) in the skin and internal organs. Mutations in the filaggrin (FLG) gene, the most common genetic risk factor for AD, confer a greater risk for eczema herpeticum in patients with AD, suggesting that it impairs the response to cutaneous viral infections. OBJECTIVE: We sought to determine the effects of FLG deficiency on the response of mice to cutaneous VV inoculation. METHODS: VV was inoculated by means of scarification of unsensitized skin or skin topically sensitized with ovalbumin in FLG-deficient flaky tail (ft/ft) mice or wild-type (WT) control mice. The sizes of primary and satellite skin lesions were measured, and hematoxylin and eosin staining was performed. VV genome copy numbers and cytokine mRNA levels were measured by using quantitative PCR. RESULTS: VV inoculation in unsensitized skin of ft/ft mice, independent of the matted hair mutation, resulted in larger primary lesions, more abundant satellite lesions, heavier viral loads in internal organs, greater epidermal thickness, dermal cellular infiltration, and higher local Il17a, Il4, Il13, and Ifng mRNA levels than in WT control mice. VV inoculation at sites of topical ovalbumin application amplified all of these features in ft/ft mice but had no detectable effect in WT control mice. The number of satellite lesions and the viral loads in internal organs after cutaneous VV inoculation were significantly reduced in both unsensitized and topically sensitized ft/ftxIl17a(-/-) mice. CONCLUSION: FLG deficiency predisposes to eczema vaccinatum. This is mediated primarily through production of IL-17A.

HPV16 E7 expression in skin induces TSLP secretion, type 2 ILC infiltration and atopic dermatitis-like lesions.

Atopic dermatitis is a common pruritic and inflammatory skin disorder with unknown etiology. Most commonly occurring during early childhood, atopic dermatitis is associated with eczematous lesions and lichenification, in which the epidermis becomes hypertrophied resulting in thickening of the skin. In this study, we report an atopic dermatitis-like pathophysiology results in a murine model following the expression of the high-risk human papillomavirus (HPV) 16 oncoprotein E7 in keratinocytes under the keratin 14 promoter. We show that HPV16 E7 expression in the skin is associated with skin thickening, acanthosis and light spongiosis. Locally, HPV16 E7-expressing skin secreted high levels of thymic stromal lymphopoietin (TSLP) and contained increased numbers of innate lymphoid cells (ILCs). High levels of circulating immunoglobulin E were associated with increased susceptibility to skin allergy in a model of cutaneous challenge, and to airway bronchiolar inflammation, enhanced airway goblet cell metaplasia and mucus production in a model of atopic march. Surprisingly, skin pathology occurred independently of T cells and mast cells. Thus, our findings suggest that the expression of a single HPV oncogene in the skin can drive the onset of atopic dermatitis-like pathology through the induction of TSLP and type 2 ILC infiltration.

Disseminated coxsackievirus A6 affecting children with atopic dermatitis.

Coxsackievirus A6 (CV-A6) is an emerging pathogen that has in recent years been associated with atypical hand, foot and mouth disease. This manifests as a generalized papular or vesicular eruption, which may be associated with fever and systemic disturbance. We report a series of six children presenting to a single centre in the UK with disseminated CV-A6 infection on a background of atopic dermatitis (AD). Our patients exhibited a widespread papular or vesicular eruption in association with exacerbation of AD. Several of our cases mimicked eczema herpeticum, but the extent was more generalized, and individual lesions were discrete rather than clustered and were less circumscribed in character. This series highlights that CV-A6 infection may be encountered in the UK, and should be considered in the differential diagnosis of an acute exacerbation of AD, particularly in children.

Transcutaneous yellow fever vaccination of subjects with or without atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common inflammatory skin disease with a global prevalence ranging from 3% to 20%. Patients with AD have an increased risk for complications after viral infection (eg, herpes simplex virus), and vaccination of patients with AD with live vaccinia virus is contraindicated because of a heightened risk of eczema vaccinatum, a rare but potentially lethal complication associated with smallpox vaccination. OBJECTIVE: We sought to develop a better understanding of immunity to cutaneous viral infection in patients with AD. METHODS: In a double-blind randomized study we investigated the safety and immunogenicity of live attenuated yellow fever virus (YFV) vaccination of nonatopic subjects and patients with AD after standard subcutaneous inoculation or transcutaneous vaccination administered with a bifurcated needle. Viremia, neutralizing antibody, and antiviral T-cell responses were analyzed for up to 30 days after vaccination. RESULTS: YFV vaccination administered through either route was well tolerated. Subcutaneous vaccination resulted in higher seroconversion rates than transcutaneous vaccination but elicited similar antiviral antibody levels and T-cell responses in both the nonatopic and AD groups. After transcutaneous vaccination, both groups mounted similar neutralizing antibody responses, but patients with AD demonstrated lower antiviral T-cell responses by 30 days after vaccination. Among transcutaneously vaccinated subjects, a significant inverse correlation between baseline IgE levels and the magnitude of antiviral antibody and CD4(+) T-cell responses was observed. CONCLUSIONS: YFV vaccination of patients with AD through the transcutaneous route revealed that high baseline IgE levels provide a potential biomarker for predicting reduced virus-specific immune memory after transcutaneous infection with a live virus.

IL-10 suppresses IL-17-mediated dermal inflammation and reduces the systemic burden of Vaccinia virus in a mouse model of eczema vaccinatum.

Individuals with atopic dermatitis (AD) are susceptible to a severe, potentially fatal, systemic infection and inflammatory response following exposure to Vaccinia virus (VV). IL-10 acts both as an inducer of Th2 responses and as a regulator of T cell activation. It has been shown to limit skin inflammation elicited by contact sensitizers. AD exacerbations have been associated with decreased IL-10 function. We used IL-10(-/-) mice to test the role of the cytokine in VV immunity. They exhibited larger primary lesions and increased cutaneous neutrophil infiltration compared to wild-type (WT) counterparts. This was associated with enhanced production of IL-17A, IL-17F and CXCL2. Paradoxically, despite intact adaptive immune responses, tissue viral burdens were increased in IL-10(-/-) mice. These findings suggest that IL-10 is important in limiting skin inflammation induced by VV and that abnormal IL-17-driven neutrophil recruitment at the primary infection site in the skin results in increased systemic viral dissemination.

Distinct prophage gene profiles of Staphylococcus aureus strains from atopic dermatitis patients and healthy individuals.

Staphylococcus aureus strains exhibit varying associations with atopic dermatitis (AD), but the genetic determinants underpinning the pathogenicity are yet to be fully characterized. To reveal the genetic differences between S. aureus strains from AD patients and healthy individuals (HE), we developed and employed a random forest classifier to identify potential marker genes responsible for their phenotypic variations. The classifier was able to effectively distinguish strains from AD and HE. We also uncovered strong links between certain marker genes and phage functionalities, with phage holin emerging as the most pivotal differentiating factor. Further examination of S. aureus gene content highlighted the genetic diversity and functional implications of prophages in driving differentiation between strains from AD and HE. The HE group exhibited greater gene content diversity, largely influenced by their prophages. While strains from both AD and HE universally housed prophages, those in the HE group were distinctively higher at the strain level. Moreover, although prophages in the HE group exhibited variously higher enrichment of differential functions, the AD group displayed a notable enrichment of virulence factors within their prophages, underscoring the important contribution of prophages to the pathogenesis of AD-associated strains. Overall, prophages significantly shape the genetic and functional profiles of S. aureus strains, shedding light on their pathogenic potential and elucidating the mechanisms behind the phenotypic variations in AD and HE environments. IMPORTANCE: Through a nuanced exploration of Staphylococcus aureus strains obtained from atopic dermatitis (AD) patients and healthy controls (HE), our research unveils pivotal genetic determinants influencing their pathogenic associations. Utilizing a random forest classifier, we illuminate distinct marker genes, with phage holin emerging as a critical differential factor, revealing the profound impact of prophages on genetic and pathogenic profiles. HE strains exhibited a diverse gene content, notably shaped by unique, heightened prophages. Conversely, AD strains emphasized a pronounced enrichment of virulence factors within prophages, signifying their key role in AD pathogenesis. This work crucially highlights prophages as central architects of the genetic and functional attributes of S. aureus strains, providing vital insights into pathogenic mechanisms and phenotypic variations, thereby paving the way for targeted AD therapeutic approaches and management strategies by demystifying specific genetic and pathogenic mechanisms.

Vaccinia virus inoculation in sites of allergic skin inflammation elicits a vigorous cutaneous IL-17 response.

Eczema vaccinatum (EV) is a complication of smallpox vaccination occurring in patients with atopic dermatitis. In affected individuals, vaccinia virus (VV) spreads through the skin, resulting in large primary lesions and satellite lesions, and infects internal organs. BALB/c mice inoculated with VV at sites of Th2-biased allergic skin inflammation elicited by epicutaneous ovalbumin (OVA) sensitization exhibited larger primary lesions that were erosive, more satellite lesions, and higher viral loads in skin and internal organs than mice inoculated in saline-exposed skin, unsensitized skin, or skin sites with Th1-dominant inflammation. VV inoculation in OVA-sensitized skin induced marked local expression of IL-17 transcripts and massive neutrophil infiltration compared to VV inoculation in saline-exposed skin. Treatment with anti-IL-17 decreased the size of primary lesions, numbers of satellite lesions, and viral loads. Addition of IL-17 promoted VV replication in skin explants. These results suggest that IL-17 may be a potential therapeutic target in EV.

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.

Marked differences in Betapapillomavirus DNA and antibody prevalence between patients with psoriasis and those with atopic dermatitis.

BACKGROUND: Recent studies revealed that Betapapillomavirus (betaPV) infections are highly prevalent. Skin diseases such as psoriasis, characterized by keratinocyte hyperproliferation, and atopic dermatitis (AD), dominated by cutaneous inflammation, might have an impact on viral life cycle and immune response induction. OBJECTIVES: To investigate whether betaPV infection is different in psoriasis and AD. METHODS: Twenty-seven patients with psoriasis and 17 with AD were included for betaPV genotyping using eyebrow hairs, and for seroresponse determination. RESULTS: BetaPV DNA was found significantly more often in patients with psoriasis than in those with AD (100% vs. 81%, P=0·022) and the mean number of betaPV types was higher (4·8 vs. 2·1 types, P=0·002). In contrast, the seroprevalence in patients with AD was significantly higher compared with that in patients with psoriasis (88% vs. 56%, P=0·023). Type-specific concordance of serological response to the betaPV type detected in eyebrow hairs was 27% in patients with psoriasis and 47% in those with AD (P=0·019). CONCLUSIONS: We speculate that the condition of the skin and the immunological state of the patients have an important impact on the life cycle of betaPV.

The clinical spectrum and therapeutic challenge of eczema herpeticum.

We present 5 cases of eczema herpeticum in patients with severe recalcitrant atopic dermatitis to illustrate the range of possible clinical findings and supporting laboratory data that can obscure the diagnosis and complicate treatment. Major issues include: the need for aggressive laboratory evaluation (molecular diagnostics, viral cultures, skin biopsy, serology); the possibility of recurrent or chronic infection; the value of empiric antiviral therapy; the possibility of infection with thymidine-kinase resistant strains; and the importance of achieving control over the dermatitis with nonmyelosuppressive immunomodulating agents such as intravenous immunoglobulin or interferon gamma for dermatitis that requires systemic treatment.

Association between varicella zoster virus infection and atopic dermatitis in early and late childhood: a case-control study.

BACKGROUND: Wild-type varicella zoster virus infection (WTVZV) early in childhood has been shown to protect against the development of asthma and atopy. OBJECTIVE: To determine whether WTVZV in childhood protects against atopic dermatitis (AD). METHODS: This retrospective, practice-based, case-control study randomly sampled 256 children and adolescents (age 1-18 years) with AD and 422 age-matched healthy controls from 2005 to 2007. Observations were made before the a priori hypothesis. RESULTS: (1) A single episode of WTVZV in childhood is associated with decreased odds ratio (OR) of developing AD (conditional logistic regression; OR, 0.55; 95% CI, 0.34-0.89; P = .01). (2) When using intervals for age corresponding to bimodal distribution of age of WTVZV infection, the effects of WTVZV infection are significant when occurring at age 0 to 8 years (OR, 0.56; 95% CI, 0.35-0.90; P = .02), but not at 8 to 18 years (OR, 0.50; 95% CI, 0.19-1.31; P = .16). Considering 5-year intervals has similar findings. (3) WTVZV is associated with decreased odds of moderate AD (multinomial logistic regression; OR, 0.08, 95% CI, 0.04-0.15; P < .0001) or severe AD (OR, 0.04; 95% CI, 0.01-0.13; P < .0001). (4) WTVZV in children is associated with prolonged AD-free survival (Kaplan-Meier; median, 15.3 years; 95% CI, 10.9-18.0) compared with controls (median, 7.5 years; 95% CI, 4.8-11.9; log-rank test, P < .0001). (5) Children with WTVZV, compared with vaccine, who eventually develop AD require fewer pediatrician sick visits for management of AD (logistic regression; OR, 0.17; 95% CI, 0.06-0.51; P = .001). CONCLUSION: WTVZV in childhood protects up to 10 years of age against AD, delays onset of AD symptoms, and decreases AD severity and office visits.

Recent insights into atopic dermatitis and implications for management of infectious complications.

Atopic dermatitis (AD) is a common complex disease that frequently follows a chronic relapsing course and affects the quality of life of patients and families in a significant manner. New insights into the pathophysiology of AD point to an important role of structural abnormalities in the epidermis combined with immune dysregulation. Patients with AD have a unique predisposition to colonization or infection by a number of microbial organisms, most notably Staphylococcus aureus and herpes simplex virus. A multipronged approach directed at healing or protecting the skin barrier and addressing the immune dysregulation is necessary to improve the likelihood of successful outcomes.

Vaccinia virus-specific molecular signature in atopic dermatitis skin.

BACKGROUND: Eczema vaccinatum (EV), a disseminated viral skin infection, is a life-threatening complication of vaccinia virus (VV) inoculation in patients with atopic dermatitis (AD) and is thought to be associated with a defective innate immune response. However, the precise mechanism or mechanisms and key factor or factors of EV are unknown. OBJECTIVE: Given that patients with psoriasis, another inflammatory skin disorder, are not susceptible to EV, we compared the global transcriptional response of skin to VV in healthy subjects, patients with psoriasis, and patients with AD, focusing on AD-specific genes. We hypothesized that differences in the transcriptional response to VV between patients with AD and patients with psoriasis or healthy subjects would identify a defective pathway or pathways that might be associated with the development of EV. METHODS: Gene expression profiling of sham-treated and VV-treated unaffected skin explants from patients with AD (n = 12), patients with psoriasis (n = 12), or healthy subjects (n = 13) were generated with U133_Plus2 (54,613 probe sets) GeneChips and analyzed with the GCOS_1.4/SAM_2.1/MAPPFinder_2.0 pipeline. RESULTS: Sixty-seven genes were significantly affected by VV in AD skin but not in psoriatic and healthy skin. Genes associated with defense response, response to wounding, and immune response were the most affected by VV in AD skin. All genes in these ontologies were downregulated, including the innate immunity genes leukotriene B(4) receptor (LTB4R), orosomucoid 1 (ORM1), coagulation factor II (thrombin) receptor (F2R), complement component 9 (C9), and LPS-binding protein (LBP). These findings were confirmed by means of real-time PCR and validated by means of PubMatrix analysis. ORM1, Toll-like receptor 4 (TLR4), and NLR family pyrin domain containing 1 (NLRP1) genes were also linked to AD severity. CONCLUSION: This study identified groups of innate immunity genes that are associated with the aberrant response of AD skin to VV and represent potential targets for EV pathogenesis.

Atopic dermatitis associated with HPV 38 lower leg verrucosis.

We describe a 45-year-old woman with atopic dermatitis who presented with a progressive, papular eruption over her anterior lower legs that began 30 years earlier. Biopsy with polymerase chain reaction analysis revealed lichenified plane warts harboring human papillomavirus 38 DNA. No signs or family history of epidermodysplasia verruciformis existed. Local trauma from shaving, barrier dysfunction, and decreased cell immunity and interferon production associated with atopic dermatitis are likely responsible for this presentation of lower leg verrucosis.