Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells

Abstract

Together with keratinocytes (KCs) and the dense network of Langerhans cells (LCs), the epidermis is an ideal portal for vaccine delivery. Pattern recognition receptor agonists, in particular polyinosinic-polycytidylic acid [p(I:C)], are promising adjuvant candidates for therapeutic vaccination to generate protective T-cell immunity. Here we established an ex vivo skin explant model to study the expression and activation of double-stranded RNA (dsRNA)-sensing pattern recognition receptors in LCs and KCs in human skin. Whereas KCs expressed all known dsRNA sensing receptors at a constitutive and inducible level, LCs exclusively expressed melanoma differentiation-associated protein 5 (MDA5) in untreated skin and freshly isolated cells. Comparative assessments of downstream signaling pathways induced by p(I:C) revealed distinct mitochondrial antiviral-signaling protein, IFN-regulatory factor 3, and NF-κB activation in LCs and KCs. Consequently, p(I:C) treatment of LCs significantly induced IFN-α and IFN-β mRNA expression, while in KCs an up-regulation of IFN-β and TNF-α mRNA was detectable. Stimulation of LCs with specific ligands revealed that not the TLR3- but only the MDA5-specific ligand induced IFN-α2, IFN-β, and TNF-α cytokines, but no IL-6 and -8. In KCs, both ligands induced production of high IL-6 and IL-8 levels, and low IFN-α2 and IFN-β levels, indicating that different dsRNA-sensing receptors and/or downstream signaling pathways are activated in both cell types. Our data suggest that MDA5 may be an attractive adjuvant target for epicutaneous delivery of therapeutic vaccines with the goal to target LCs.-Tajpara, P., Schuster, C., Schön, E., Kienzl, P., Vierhapper, M., Mildner, M., Elbe-Bürger, A. Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.

Keywords: downstream signaling; nuclear translocation; skin; tape stripping; therapeutic vaccination.

Figure 1

Rhodamine (RH)-labeled p(I:C) is taken up by epidermal cells. A) Hematoxylin and eosin staining of paraffin-embedded sections from healthy human skin (middle) indicates efficient removal of stratum corneum in tape-stripped skin (right) compared to untreated skin (left). Scale bars, 50 µm. B) One representative strip disc with corneocytes is shown (left). Protein estimation of each strip disc was analyzed with bicinchoninic acid protein assay. Data are represented as means ± sem of measurements obtained with 4 different donors (middle). Schematic representation of p(I:C) application (right). C) Immunofluorescence staining of cryostat section from stripped cultured skin showing uptake of RH-labeled low MW p(I:C) (red) in epidermal cells. Shown is single representative of 3 biopsy samples from different donors. Scale bars, 10 µm. Arrows in inset show RH⁺ KCs. D, E) Representative images of primary KCs (60% confluency) and sorted CD207⁺ LCs (300 cells/slot) showing uptake of RH-labeled p(I:C) at 24 and 2 h, respectively. Data are from 1 of 3 similar experiments from different donors. Nuclei were counterstained with Hoechst (blue). Scale bars, 20 µm. F, G) Freshly isolated and sorted KCs and LCs from same donor were cultured for 48 h with unlabeled, low MW p(I:C) (+) or left untreated (−). Indicated cytokine concentrations from supernatants were determined with LegendPlex Bead Array. Results are expressed as means ± sem of duplicate cultures from 1 experiment, representative of 2 with different donors.

Figure 2

Removal of stratum corneum and topical p(I:C) treatment leads to strong up-regulation of maturation marker CD83 on LCs. A, C) Merged images of epidermal sheets with CD83, CD207 (to visualize LCs), and nuclear [Hoechst (H)] staining 24 and 48 h after removal of stratum corneum and with application of p(I:C) or left untreated and culture. Scale bars, 50 µm. B, D) Bar graphs show percentages of CD83⁺CD207⁺ LCs among total CD207⁺ LCs in untreated (UT) epidermal sheets before culture and at 24 and 48 h for indicated treatments. Values are means ± sem of results from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated, cultured group; 1-way ANOVA test. Strp, stripped.

Figure 3

LCs express MDA5 but not TLR3, RIG-I, and PKR in situ. A) Immunofluorescence double staining for markers indicated was performed on cryostat sections of untreated skin. Arrows denote MDA5⁺CD207⁺ LCs; white dotted line indicates basal membrane. B) Image of untreated epidermal sheet showing MDA5⁺CD207⁺ LCs (inset). C) Application of MDA5-specific blocking peptide revealed antibody specificity. Nuclei were counterstained with Hoechst (H). Scale bars, 50 µm.

Figure 4

LCs down-regulate MDA5 on skin barrier perturbation and p(I:C) activation on culture. Immunofluorescence double labeling of cryostat sections (TLR3, PKR, RIG-I) as well as epidermal sheets (MDA5) 48 h after culture of indicated skin explant groups. Representative merged figures with Hoechst (H) nuclear staining are shown from 3 donors in each group. Percentages in merged figures represent means ± sem of results from 3 independent experiments; MDA5⁺CD207⁺ LCs among total CD207⁺ LCs are shown. Tape-stripped (Strp) and p(I:C) group revealed not only less MDA5⁺CD207⁺ LCs but also much weaker MDA5 signal in LCs than respective control groups. Scale bars, 50 µm.

Figure 5

dsRNA signaling pathways are functional in LCs and KCs. Subcellular localization of MAVS, IRF3, and p65 as well as induction of IFNs and proinflammatory cytokine TNF-α in primary KCs and LCs on culture in medium or stimulation with p(I:C) was assessed at indicated time points. A, B) Distinct aggregation of MAVS as well as translocation of IRF3 and p65 from cytoplasm into nucleus was observed in stimulated KCs and LCs. Nuclear counterstain was performed with Hoechst. Localization was examined with confocal microscopy. Shown is single representative staining of 3 independent experiments. Scale bars, 5 µm. C, D) Fold change of IFN-α, IFN-β, and TNF-α mRNA expression of primary KCs (C) and sorted LCs (D) cultured for 24 h without or with p(I:C) is shown. Results expressed as means ± sem are from 6 independent experiments with 6 different donors. *P < 0.05, **P < 0.01 compared to unstimulated cultured group.