Herpes Simplex Virus 1 Can Bypass Impaired Epidermal Barriers upon Ex Vivo Infection of Skin from Atopic Dermatitis Patients

Abstract

To infect its human host, herpes simplex virus 1 (HSV-1) must overcome the protective barriers of skin and mucosa. Here, we addressed whether pathological skin conditions can facilitate viral entry via the skin surface and used ex vivo infection studies to explore viral invasion in atopic dermatitis (AD) skin characterized by disturbed barrier functions. Our focus was on the visualization of the onset of infection in single cells to determine the primary entry portals in the epidermis. After ex vivo infection of lesional AD skin, we observed infected cells in suprabasal layers indicating successful invasion in the epidermis via the skin surface which was never detected in control skin where only sample edges allowed viral access. The redistribution of filaggrin, loricrin, and tight-junction components in the lesional skin samples suggested multiple defective mechanical barriers. To dissect the parameters that contribute to HSV-1 invasion, we induced an AD-like phenotype by adding the Th2 cytokines interleukin 4 (IL-4) and IL-13 to healthy human skin samples. Strikingly, we detected infected cells in the epidermis, implying that the IL-4/IL-13-driven inflammation is sufficient to induce modifications allowing HSV-1 to penetrate the skin surface. In summary, not only did lesional AD skin facilitate HSV-1 penetration but IL-4/IL-13 responses alone allowed virus invasion. Our results suggest that the defective epidermal barriers of AD skin and the inflammation-induced altered barriers in healthy skin can make receptors accessible for HSV-1. IMPORTANCE Herpes simplex virus 1 (HSV-1) can target skin to establish primary infection in the epithelium. While the human skin provides effective barriers against viral invasion under healthy conditions, a prominent example of successful invasion is the disseminated HSV-1 infection in the skin of atopic dermatitis (AD) patients. AD is characterized by impaired epidermal barrier functions, chronic inflammation, and dysbiosis of skin microbiota. We addressed the initial invasion process of HSV-1 in atopic dermatitis skin to understand whether the physical barrier functions are sufficiently disturbed to allow the virus to invade skin and reach its receptors on skin cells. Our results demonstrate that HSV-1 can indeed penetrate and initiate infection in atopic dermatitis skin. Since treatment of skin with IL-4 and IL-13 already resulted in successful invasion, we assume that inflammation-induced barrier defects play an important role for the facilitated access of HSV-1 to its target cells.

Keywords: HSV-1; IL-4/IL-13; atopic dermatitis; epidermal barriers; human skin; viral entry.

FIG 1

HSV-1 penetration in AD skin shaves. (a) Schematic illustrating ex vivo infection of AD skin shaves. HE-stained sections visualize samples with characteristics of commencing (AD5) and acute eczema (AD7). (b to i) After infection with HSV-1 at a multiplicity of infection (MOI) of ca. 100 PFU/cell, cross sections of control (C) and AD skin shaves with DAPI (blue) as nuclear counterstain are shown. Collagen VII (colVII; red) depicts the basement membrane. Scale bars, 50 μm. (b) K10/K14 immunostainings indicate differentiation defects in the eczematic AD skin, including expanded suprabasal layers and nuclei in the stratum corneum. (c) VP5-positive virus particles (green) (arrowheads) at (C2) or below (C1) the control skin surface at 6 h p.i. F-actin (red) depicts the cell morphology. AD skin with virus particles in the stratum corneum containing cell nuclei. Dashed lines indicate sample borders. (d) VP5-positive virus particles (green) underneath the granular layer as shown in the magnification at 16 h p.i. (e) Transmission light (TL) visualizes sample edges with no ICP0-expressing cells at 6 h p.i. (f) At 16 h p.i., nuclear (open arrowhead) and cytoplasmic (arrows) ICP0-expressing cells (green) at the sample edges are shown. (g) ICP0-expressing cells (green) in the granular layer and (h) throughout the epidermis. (i) Nuclear ICP4-expressing cells (green) throughout the epidermis.

FIG 2

Characterization of HSV-1 penetration in AD skin shaves. (a) Costaining of Ki67 and ICP0 indicates very few proliferating cells with infection. (b) Quantification of Ki67-positive cells is shown for each sample and demonstrates variable numbers in AD (n = 8) compared to control (n = 3) skin shaves. (c) Costaining of CD1a (red) and ICP0 (green) shows no infected Langerhans cells. (d) qRT-PCR demonstrates variable nectin-1 transcript levels in AD (n = 7) compared to control (n = 3) skin shaves. Nectin-1 expression is shown for each infected AD sample.

FIG 3

Redistribution of barrier components in AD skin. (a) Immunostainings of skin shaves show redistributed filaggrin (flg) and loricrin (lor) in AD compared to control (C) skin with DAPI (blue) as nuclear counterstain. Transmission light (TL) visualizes the morphology of the stratum corneum (SC) and the stratum granulosum (SG). (b) Immunostainings of AD skin shaves show redistribution of ZO-1, occludin (ocln), and claudin-1 (cldn-1) in the suprabasal layers compared to control shaves. Dashed lines represent the basement membrane. Scale bars, 50 μm. (c) Quantification of fluorescence shows intensities in control skin shaves (dark blue) (n = 3) and breast skin (light blue) (n = 3) versus AD skin shaves with detectable (black) (n = 3 to 5) or undetectable apical infection (gray) (n = 1 to 3). In AD skin shaves, filaggrin staining intensities are significantly lower in the SG in the stratum granulosum and the corneum (SG/SC). The redistribution of ZO-1 to the stratum spinosum led to significantly higher intensities the stratum spinosum (SS) led to significantly compared to control shaves. The discontinuous claudin-1 stainings in AD skin shaves showed significantly lower intensities in the stratum granulosum (SG) compared to control shaves. P value (*) ≤ 0.05.

FIG 4

HSV-1 entry in IL-4/IL-13-treated human skin. (a) Schematic illustrating IL-4/IL-13-treatment of full-thickness human skin (n = 5) followed by ex vivo infection and analyses of infected cells. Epidermal whole mounts were prepared after infection by dispase II treatment to show the distribution of the infected cells in the basal layer. Localization of the immunostainings at the edge or middle of the cross sections is indicated with dotted boxes (red). (b) HE staining visualizes discrete spongiosis after IL-4/IL-13-treatment of abdominal skin sample for 3 days. (c) After infection of skin with HSV-1 at ca. 100 PFU/cell for 24 h, cross sections show ICP0-expressing cells (green) only at edges after mock-treatment. Single infected (green) suprabasal cells and infected cell layers are visible after IL-4/IL-13-treatment. DAPI (blue) serves as a nuclear counterstain, and collagen VII (colVII; red) depicts the basement membrane. Dashed lines represent the apical sample border. (d) Scheme of epidermal whole mount prepared 24 h p.i. showing the distribution of infected cells. Immunostainings of epidermal whole mounts from IL-4/IL-13-treated skin visualize ICP0-expressing single cells (green) (2, 3) and cell clusters at the hair follicles (4), in the interfollicular epidermis (1, 5), and at the sample edge (6). K14 stainings depict basal keratinocytes. (e) Quantification of infected areas in mock-treated (seven replicates from three individuals) and IL-4/IL-13-treated human skin (ten replicates from four individuals). *, P ≤ 0.05. (f) Uninfected hair follicle of mock-treated skin 24 h p.i. (g) Cross sections show comparable filaggrin (red) distribution in mock- and IL-4/IL-13-treated skin. Transmission light (TL) visualizes the skin morphology. Scale bars, 50 μm.

FIG 5

Schematic representation of how HSV-1 penetrates human skin under pathological conditions. Schematic illustrating the structure of human epidermal layers with respective markers. The stratum corneum (SC) and the tight junctions (TJs) form protective barriers to inhibit virus invasion via the skin surfaces of healthy individuals. Atopic dermatitis (AD) skin characterized by epidermal thickening, disturbed SC barriers (dotted lines), and impaired TJs (dotted boxes) offers the virus access to keratinocytes throughout the epidermis to initiate infection. After IL-4/IL-13-treatment of human skin, infected cells are detected which might result from an impaired stratum corneum (SC) and dysfunctional TJs.