Fig 1.
Schemes of experimental systems used to examine LCs and dermal DC interaction
(A) Serial sections of a primary herpetic lesion stained by H & E (upper panels) and IF (lower panels). Blue: DAPI, green: HSV-1 glycoprotein D, orange: langerin, red: DC-SIGN. E: epidermis, D: dermis, S: section. Dotted line indicates basement membrane. Scale bar indicates 50 μm. (B) Scheme showing infection and processing the ex vivo inner foreskin tissue explant culture. (C) Scheme showing isolation of epidermal LCs and dermal DCs from abdominal skin.
Fig 2.
HSV-1 infected LCs in human skin.
(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.
Fig 3.
Fate of human LCs infected with HSV-1.
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.
Fig 4.
Migration of HSV infected LCs into the dermis and their subsequent interaction with BDCA3+ dermal DCs in clusters.
(A) (B) (C) (D) LCs and BDCA3+ dDCs in the dermis of foreskin explants at 24 hr p.i. (A) & 48 hr p.i. (B, C, & D), yellow arrows: GFP+langerin+ cells, green box: GFP+langerin+ cells, yellow box: GFP+BDCA3+ cells, pink box: GFP/langerin+BDCA3+ dermal DCs. (C) Proportion of GFP+LC/BDCA3+ DC clusters with >10 cells, n = 3, mean ± SEM, p***<0.001. (D) Proportion of BDCA3+ dermal DCs expressing GFP, n = 3, mean ± SEM, p***<0.001. BDCA3+ cells with or without GFP expression were quantified in 20 representative fields per sample at 60x magnification. (E) (F) Isolated skin LCs and BDCA3+ dermal DCs: LCs were pulsed with v-UL37GFP for 2 hr, incubated for 12 hr then washed before co-culturing with BDCA3+ dermal DCs for another 6 hr. Images were acquired by maximal intensity Z projection (A, B, E & F) and orthogonal views with the xz and yz planes (F). Scale bar indicates 20 μm. Images are representative from 3 donors.
Fig 5.
CLEC9A expression by BDCA3+ dermal DCs in HSV-1 infected foreskin explants.
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.
Fig 6.
Interaction of HSV-1 infected LCs with DC-SIGN+ dermal cells (dermal DCs/macrophages).
(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.
Fig 7.
Emigration of DCs from inner foreskin explants with or without HSV infection.
Inner foreskin explants were cultured for 72 hr in 24 transwell plates (membrane with 5 μm diameter pore-size) with or without v-UL37GFP. Tissues were placed in the upper chamber and allogeneic PBMCs were placed in the bottom chamber as shown in Fig 1B. After 72 hr, cells were collected from bottom chambers and labelled for flow cytometry. (A) Cells were gated on viable cells (PI-) then CD45+HLA-DR+ cells before identifying different subsets of DCs migrated out of tissues to the bottom chambers. (B) The percentage of viable CD45+HLA-DR+ cells co-expressing langerin and BDCA3 or DC-SIGN and the percentage of individual emigrated cells in the bottom wells of mock or infected samples are shown. Repeated measures ANOVA was used to test statistical significance, n = 3, mean ± SEM, p*<0.05.
Fig 8.
Migration and/or interaction of HSV infected human LCs with dermal DCs.
(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.