MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

AbstractAfter allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure. The skin equivalent consisted of a reconstructed epidermis containing primary differentiated keratinocytes and CD1a+ MUTZ-LC on a primary fibroblast-populated dermis. Skin equivalents were exposed to a panel of allergens and irritants. Topical exposure to sub-toxic concentrations of allergens (nickel sulfate, resorcinol, cinnamaldehyde) and irritants (Triton X-100, SDS, Tween 80) resulted in LC migration out of the epidermis and into the dermis. Neutralizing antibody to CXCL12 blocked allergen-induced migration, whereas anti-CCL5 blocked irritant-induced migration. In contrast to allergen exposure, irritant exposure resulted in cells within the dermis becoming CD1a−/CD14+/CD68+ which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell in the dermis. This phenotypic switch was blocked with anti-IL-10. Mechanisms previously identified as being involved in LC activation and migration in native human skin could thus be reproduced in the in vitro constructed skin equivalent model containing functional LC. This model therefore provides a unique and relevant research tool to study human LC biology in situ under controlled in vitro conditions, and will provide a powerful tool for hazard identification, testing novel therapeutics and identifying new drug targets

Comparison of cell density between full-thickness skin and gingiva (per 100μm tissue cross-section).

<p>Comparison of cell density between full-thickness skin and gingiva (per 100μm tissue cross-section).</p

Phenotypic analysis of migratory dendritic cell (DC) subsets from skin and gingiva.

<p>Chemotaxis, maturation and differentiation-associated marker expression on DC subsets 1–5 from skin vs. gingiva, shown per indicated subset (*P<0.05, n = 4–9 for skin and gingiva). Explants (6mm diameter) from skin and gingiva were taken and cultured floating in medium for 48h, after which they were discarded and migrated DC harvested, stained and analysed by flowcytometry.</p

Dendritic cell (DC) marker expression, density and distribution over full-thickness human gingiva or skin.

<p>(A) CD1a staining of representative full-thickness skin (left panel) and gingiva (right panel) biopsies. Red dotted lines denote full-area epithelial surface (left panel, used for quantitation as shown in Fig 1B upper panel) and 100 μm wide full-thickness cross-section (right panel, used for quantitation as shown in Fig 1B lower panel, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180333#sec002" target="_blank">Materials and Methods</a>). (B) Quantitation of CD1a⁺ DC according to total epithelium area (upper panel) or epithelium area in 100 μm wide full-thickness cross-section (lower panel), see areas denoted by red dotted lines in Fig 1A for the respective definitions (n = 10). The number of positively stained cells in the epidermis, dermis, mucosal epithelium and subjacent lamina propria, were assessed for each sample per 100 μm² tissue. * P<0.05, ***P<0.001. (C) Representative staining of indicated additional DC maturation/differentiation markers shows distribution between epithelial and underlying connective tissue layers in skin and gingival biopsies (n = 10).</p

Dendritic cell (DC) subset definition and distribution.

<p>Dendritic cell (DC) subset definition and distribution according to CD1a and CD14 expression in CD11c^hi DC migrated from human skin or gingiva. Explants (6mm diameter) from skin and gingiva were taken and cultured floating in medium for 48h, after which they were discarded and migrated DC harvested, stained and analysed by flowcytometry. (A) Flow cytometry dot plots with gates denoting five migrated DC subsets (numbered 1 to 5) in skin and gingiva. (B) Frequency distribution of the five subsets among migrated DC (n = 9).</p

Inflammatory cytokine release profile of skin vs. gingival explants.

<p>Shown in pg/ml and measured in supernatants of skin and gingiva explants (6 mm diameter, 3mm depth) after 48 h of culture in 1 ml volume. IL-8, IL-6, IL-1β, IL-10 and TNFα were all significantly higher in the gingiva-conditioned cultures, whereas IL-12p70 levels were below the detection limit for both skin and gingiva (*P<0.05, **P<0.05; n = 3 skin, n = 3 gingiva).</p

Monoclonal antibodies used for immunohistochemical staining.

<p>Monoclonal antibodies used for immunohistochemical staining.</p

Intradermal Delivery of TLR Agonists in a Human Explant Skin Model: Preferential Activation of Migratory Dendritic Cells by Polyribosinic-Polyribocytidylic Acid and Peptidoglycans

TLR agonists are attractive candidate adjuvants for therapeutic cancer vaccines as they can induce a balanced humoral and T cell-mediated immune response. With a dense network of dendritic cells (DCs) and draining lymphatics, the skin provides an ideal portal for vaccine delivery. Beside direct DC activation, TLR agonists may also induce DC activation through triggering the release of inflammatory mediators by accessory cells in the skin microenvironment. Therefore, a human skin explant model was used to explore the in vivo potential of intradermally delivered TLR agonists to stimulate Langerhans cells and dermal DCs in their natural complex tissue environment. The skin-emigrated DCs were phenotyped and analyzed for T cell stimulatory capacity. We report that, of six tested TLR-agonists, the TLR2 and -3 agonists peptidoglycan (PGN) and polyribosinic-polyribocytidylic acid (Poly I:C) were uniquely able to enhance the T cell-priming ability of skin-emigrated DCs, which, in the case of PGN, was accompanied by Th1 polarization. The enhanced priming capacity of Poly I:C-stimulated DCs was associated with a strong upregulation of appropriate costimulatory molecules, including CD70, whereas that of PGN-stimulated DCs was associated with the release of a broad array of proinflammatory cytokines. Transcriptional profiling further supported the notion that the PGN- and Poly I:C-induced effects were mediated through binding to TLR2/nucleotide-binding oligomerization domain 2 and TLR3/MDA5, respectively. These data warrant further exploration of PGN and Poly I:C, alone or in combination, as DC-targeted adjuvants for intradermal cancer vaccines. The Journal of Immunology, 2013, 190:3338-3345