Fig 1.
Development of an explant infection model to investigate the initial events during HSV epidermal infection.
(A) Vaxxas HD-MAPs were pre-treated with HSV1-GFP (1x108 PFU/mL) and then applied to inner foreskin tissue via a magnetic applicator. Tissue was cultured for 24 hours at 37°C and snap-frozen in OCT. (B-C) Cryosectioned 24-hour HSV1-GFP infected inner foreskin was labelled with (B) rabbit anti-human GFP primary and donkey anti-rabbit Alexa Fluor (AF)-488 secondary (green), mouse anti-human CD11c primary and donkey anti-mouse AF647 (red) and goat anti-human Langerin primary and donkey anti-goat AF755 secondary (blue) antibodies and DAPI nuclear stain (grey). (C) Anti-GFP (green), HSV1 DNA via RNAscope (red) and DAPI nuclear stain (grey). HSV1 infection spreads outwards from punctures as indicated by the yellow arrows. Scale bars = 100 μm.
Table 1.
Summary of all genital tissue samples treated with HSV1-GFP via pre-treatment of high-density microarray patches*.
Fig 2.
Intraepithelial microneedle penetration of greater than 30% of the epidermis is required to establish detectable intra-epidermal infection in anogenital tissue.
(A-D) Cryosectioned 24-hour HSV1-GFP infected inner foreskin was labelled by RNAscope to detect HSV1 DNA (red), anti-GFP antibody (green) and DAPI nuclear stain (grey). Scale bars = 100 μm unless indicated otherwise. (A) HSV1 infected keratinocytes of the mid-epidermis, but not upper layers, as indicated by HSV1 DNA. (B) HSV1 infection cascade is shown with HSV1 DNA only detected in lower epidermis, and both DNA and GFP detected in upper layers. (C) Single virions or small aggregates of particles (right inset: zoomed in image of smaller particles) of HSV1 DNA observed within the puncture region (dotted line; left inset: zoomed in image in different Z plane) indicated by the yellow arrow at 100x magnification, imaged using the Olympus VS200 Slidescanner. (D) Nuclei of infected cells show various infectious states within keratinocytes; nuclear globular DNA alone (green arrow), nuclear globular DNA with cytoplasmic GFP (white arrows), single punctate virions and cytoplasmic GFP (yellow arrow). (E) A table of averages for the measurements of all HSV1+ explants infected with HSV1 including; the average number of HSV1+ regions per mm of epidermis, proportion of HSV1+ area, and average size of the HSV1+ foci in width (μm) and in number of cells (n = 5). (F) 24-hour HSV1-GFP infected inner foreskin samples were classified as HSV- (no expression of GFP or DNA) or HSV+ (expressing GFP and/or DNA) (i) the puncture depth and (ii) the puncture depth proportional to the total epidermal thickness were measured. Data presented as mean ± S.D. (HSV-: n = 3, HSV+: n = 4). * = p<0.05, determined by unpaired parametric t tests with Welch’s correction assuming unequal variances.
Fig 3.
Epidermal MNP subsets interact with HSV1-infected keratinocytes and become productively infected.
(A and B) Cryosectioned 24-hour HSV1-GFP infected inner foreskin from two samples labelled by RNAscope to detect HSV1 DNA (red), anti-GFP (green), anti-CD11c (blue) and anti-Langerin (magenta) antibodies and DAPI nuclear stain (grey). (A) Yellow arrows indicate HSV1 DNA+ LCs, white arrows indicate HSV1 DNA+ Epi DCs. Scale bars = 20 μm unless indicated otherwise. (B) Yellow arrows indicate HSV1 DNA surrounding a langerin+CD11c- LC and localised to the nucleus. Scale bars = 10 μm. (C) High resolution Z-stack with orthogonal view inset showing the colocalisation of DAPI (blue) and HSV1 DNA (red) overlapping to appear magenta, in a langerin+ (yellow) LC. Image acquired on Olympus VS200 Slide Scanner at 100x magnification. Scale bars = 10 μm. (D) Pair-wise comparisons of the mean densities of LCs in HSV- (black circles) and HSV+ (magenta circles) regions, and Epi DCs in HSV- (black circles) and HSV+ (blue circles) regions. * = p<0.05, determined by paired t tests with Wilcoxon’s matched pairs signed rank test. (E) Percentage of HSV DAPI+DNA+ and DAPI+DNA- Epi DCs of total DCs within foci presented as mean ± S.D. (n = 3). * = p<0.05, determined by paired parametric t tests.
Fig 4.
Nectin-1 staining is decreased in HSV1-infected keratinocytes, but enhanced as a collar in surrounding uninfected cells.
(A) Cryosectioned 24-hour HSV1-GFP infected inner foreskin from two samples labelled by RNAscope to detect HSV1 DNA (red), anti-GFP (green), mouse anti-human Nectin-1 primary conjugated in house to Cyanine5 (Lumiprobe) and mouse anti-human Plakoglobin (PG, cyan) primary followed by donkey anti-mouse AF755 secondary antibodies and DAPI nuclear stain (grey). Scale bars = 100 μm. (B) Pixel intensity of nectin-1 within HSV1-GFP+ or negative epidermal regions within the same tissue presented as mean ± standard deviation (n = 3). * = p<0.05, determined by an unpaired t test. (C) 24h HSV1-GFP infected HaCaT cells with arrows indicating ICP27+GFP+Nectin-1+ (orange), ICP27-GFP+Nectin-1+ (white) and ICP27-GFP-Nectin-1+ (magenta) cells. Scale bars = 100 μm unless stated otherwise. (D) HaCaT cells cultured for 24 hours with either serum-free medium only (mock), HSV1 or HSV1-GFP (MOI 0.1), pre-treated with Foscarnet (1 mg/mL) for 1 hour (and remaining in the culture thereafter), or treated with UV-inactivated HSV1-GFP (UV-HSV1-GFP). Cells were labelled with anti-gD (green; when treated with untagged HSV1), anti-ICP27 (red) and anti-Nectin-1 (magenta) antibodies and DAPI nuclear stain (grey). Yellow arrows indicate gD-/lowICP27+Nectin-1- cells. Scale bars = 100 μm. (E) The average area (mm2) and maximum pixel intensity for (i) nectin-1 and (ii) gD expression in mock, and HSV1 vs HSV1 + Foscarnet treated HaCaT cells, n = 2 and iii) nectin-1 and iv) GFP expression in mock, HSV1-GFP and UV-HSV1-GFP treated HaCaT cells. * = p<0.05 determined by an ordinary one-way ANOVA with Tukey’s multiple comparisons test.
Fig 5.
HaCaT keratinocytes produce inflammatory chemokines in response to HSV1 infection, which increase nectin-1 redistribution.
(A) HaCaT cells cultured for 12 or 24 hours with either serum-free medium (mock) or HSV1-GFP (MOI 1). Supernatants were analysed for an array of cytokines and chemokines via the LEGENDplex Assay (BioLegend). Concentrations (pg/mL) of detected cytokines and chemokines are displayed as mean ± S.D. (n = 4). * = p<0.05 determined by an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (B) HaCaT cells were treated with serum-free media (mock) or CCL3, CCL5, CXCL1, CXCL5, CXCL9, CXCL10, IL-6 and IL-8 at 10, 50 or 100 pg/mL for 6 hours and labelled with anti-Nectin-1 (magenta) antibody and DAPI nuclear stain (grey). Fold change in the percentage of the area that is nectin-1+, and relative to the mock, is displayed as mean ± S.D. (CCL3: n = 4, all others: n = 3). * = p<0.05, ** = p<0.01, *** = p<0.001, determined by repeated measures one-way ANOVA with Dunnett’s multiple comparisons or single unpaired parametric t tests. (C) Representative images of HaCaT cells treated with the concentrations required for optimal response are displayed. Scale bars = 100 μm. (D) HaCaT cells were treated with serum-free media (mock) or CCL3 or IL-8 at 10 pg/mL for 2 and 6 hours. RNA was extracted and RT-qPCR was performed using primers to nectin-1 and GAPDH. The fold change in nectin-1 expression by IL-8 and CCL3 treatment normalised to GAPDH is shown, displayed as mean ± S.D. (n = 2). Statistical analysis to assess data significance was determined by repeated measures two-way ANOVA (Treatment x Time) with Dunnett’s multiple comparisons test.