Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin

<div><p>The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103⁺ dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3⁺ dermal DCs (thought to be equivalent to murine CD103⁺ dermal DCs) and DC-SIGN⁺ DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3⁺ dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 <i>in vitro</i> also underwent apoptosis and were taken up by similarly isolated BDCA3⁺ dermal DCs and DC-SIGN⁺ cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.</p></div

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting

HSV-1 infected LCs in human skin.

<p>(A) LCs in the epidermis of mock or HSV-1 infected inner foreskin explants. The right panel quantifies density of total LCs in the epidermis of mock and HSV-1 infected explants at 24 hr p.i. in 20 representative fields per sample at 60x magnification. n = 3, mean ± SEM, *p<0.05. (B) ICP27 expression of the HSV-1 infected LC emigrated into the dermis of the inner foreskin explant. ICP27: immediate early protein of HSV, D: dermis. Representative result of 3 different donors is shown. (C) LCs in the primary penile herpetic lesion. E: epidermis, gD1: HSV-1 glycoprotein D. Representative result of 2 different donors. Scale bar indicates 20 μm. Maximum projections of Z-series are presented.</p

Fate of human LCs infected with HSV-1.

<p>Inner foreskin explants (A, B, C, D & E) or isolated skin LCs (F) were infected with v-UL37GFP for 24 hr (A, D, & E), 48 hr (B & C), or 18 hr (F). (A) LCs in the dermis of inner foreskin explants. Dotted line represents basement membrane. (B) Comparison of LCs migrating into the dermis in mock and infected inner foreskin explants expressed as % LCs per total no. of dermal cells. n = 6, mean ± SEM, *p<0.05. (C) Proportion of LCs in the dermis of inner foreskin explants expressing GFP, n = 3, mean ± SEM, ***p<0.001. (B) (C) LCs with or without GFP expression were quantified in 20 representative fields per sample at 60x magnification. (D) (E) Infected LCs in the dermis of inner foreskin explants were tested for the expression of a maturation marker CD80 (D) or an apoptosis marker caspase 3 (E). (F) Infected LCs isolated from abdominal skin were examined for the expression of caspase 3. Right-hand panels show quantification of each marker for (D), (E), (F) as in (B) and (C), n = 3, mean ± SEM, ***p<0.001. Maximum projections of Z-series are presented (D, E & F). E: epidermis, D: dermis. Scale bar indicates 15 μm.</p

Migration and/or interaction of HSV infected human LCs with dermal DCs.

<p>(A) Migration of LCs and dermal DCs out of skin after topical HSV application to inner foreskin explants. (B) Summary diagram of interaction between HSV infected LCs and dermal DCs in human skin.</p

CLEC9A expression by BDCA3⁺ dermal DCs in HSV-1 infected foreskin explants.

<p>Inner foreskin explants were infected with or without v-UL37GFP for 48 hr. (A) Representative image from 3 different donors is shown. D: dermis. Scale bar indicates 15 μm. (B) CLEC9A⁺BDCA3⁺ cells were quantified in 20 representative fields per sample at 60x magnification from 3 donors, mean ± SEM, p***<0.001.</p

Interaction of HSV-1 infected LCs with DC-SIGN⁺ dermal cells (dermal DCs/macrophages).

<p>(A) (B) (C) Foreskin explants, 48 hr p.i. (A) LCs and DC-SIGN⁺ dermal cells interacted in clusters. (B) Proportion of clusters GFP⁺LC/DC-SIGN⁺ dermal cells containing >10 cells, n = 3, mean ± SEM, p***>0.001. (C) DC-SIGN⁺ dermal cells with or without GFP expression were quantified in 20 representative fields per sample at 60x magnification from 3 separate samples. Mean ± SEM, p***>0.001 (D) Primary penile herpetic lesion, blue: DAPI, orange: langerin, red: DC-SIGN, green: gD1. E: epidermis, D: dermis. The dotted line represents the basement membrane. Maximum projections of Z-series are presented (A & D). Scale bar in (A) indicates 15 μm and scale bars in (D) indicate 50 μm (white) and 15 μm (yellow), respectively.</p