Active neutrophil responses counteract Candida albicans burn wound infection of ex vivo human skin explants

Burn wounds are highly susceptible sites for colonization and infection by bacteria and fungi. Large wound surface, impaired local immunity, and broad-spectrum antibiotic therapy support growth of opportunistic fungi such as Candida albicans, which may lead to invasive candidiasis. Currently, it remains unknown whether depressed host defenses or fungal virulence drive the progression of burn wound candidiasis. Here we established an ex vivo burn wound model, where wounds were inflicted by applying preheated soldering iron to human skin explants, resulting in highly reproducible deep second-degree burn wounds. Eschar removal by debridement allowed for deeper C. albicans penetration into the burned tissue associated with prominent filamentation. Active migration of resident tissue neutrophils towards the damaged tissue and release of pro-inflammatory cytokine IL-1β accompanied the burn. The neutrophil recruitment was further increased upon supplementation of the model with fresh immune cells. Wound area and depth decreased over time, indicating healing of the damaged tissue. Importantly, prominent neutrophil presence at the infected site correlated to the limited penetration of C. albicans into the burned tissue. Altogether, we established a reproducible burn wound model of candidiasis using ex vivo human skin explants, where immune responses actively control the progression of infection and promote tissue healing.

Surgery and sample production were done by a commercial provider (a). The prepared skin biopsies were embedded in a solid matrix within overhanging inserts (b). Culture medium was added to wells on arrival. All samples had a silicone ring surrounding the top keratinocyte layer of the skin, with the purpose of allowing improved topical application of compounds to the surface of the skin. Skin tissues were received on "day 0", burnt (c), infected and depending on the experiment, supplemented with neutrophils. Samples were processed on day 1 and 6 post-infection (d) for histological staining (e; PAS, H&E in order to analyze wound area, wound depth, tissue injury), immunohistochemical staining (HMGB1 in order to analyze tissue viability) or immunofluorescent staining (NE in order to analyze neutrophil infiltration) and analysis of cytokine production (IL-1β, IL-6, IL-8).
Representative images are shown (N=5, n=6; N = number of donors, n = number of technical replicates). Unburned, burned, and burned-infected samples were stained applying PAS (left, a -c) for a histological overview, DAPI for localization of nuclei (blue) and AF647 labelled immunofluorescence staining (pink) for neutrophil elastase (NE) (right, d -f). Unburned control samples present with normal skin structure (a). The burned area has a darker purple color (b, c) than the surrounding tissue and C. albicans fungal cell walls stain magenta (c). Defined signals of NE localize tissue-resident inactive neutrophils, while diffuse signals indicate release of NE by activated neutrophils.

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In unburned skin (d) neutrophils are present in dermal tissues whereas in burned (e) and burned-infected (f) skin neutrophils cluster at the epidermis.
All samples were fixed at day 6. Dotted lines present the burned area. Rectangles indicate the region enlarged in Fig. 5. For number of skin donors and technical replicates see Table S1. Representative images are shown. Fig. 6).

Fig. S4. Neutrophil supplementation to the media induces wound healing of burned skin tissue explants (Supplement to
Unburned, burned, and burned-infected samples were stained applying PAS (left, a-c) for a histological overview, DAPI for localization of nuclei (blue) and AF647 labelled immunofluorescence staining (pink) for neutrophil elastase (NE) (right, d-f). Unburned control samples present with normal skin structure (a). The burned area has a darker purple color (b, c) than the surrounding tissue and C. albicans fungal cell walls stain magenta (c). Defined signals of NE localize tissue-resident inactive neutrophils, while diffuse signals indicate release of NE by activated neutrophils.
Following neutrophil supplementation larger clusters of neutrophils are present in the lower dermal layers of 6 unburned skin tissue (d). Due to burning (e) or burning and infection (f) neutrophils migrate to the upper dermal layers and cluster at the site of damage and infection.
All samples were fixed at day 6. Dotted lines present the burned area whereas rectangles indicate the region enlarged in Fig. 6. For number of skin donors and technical replicates see Table S1. Representative images are shown.
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