Hallmarks of Atopic Skin Mimicked In Vitro by Means of a Skin Disease Model Based on FLG Knock-down

Abstract

Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knockdown model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.

Atopic Dermatitis Studies through In Vitro Models

“…Using siRNA as well but in HSE analyzed after 7 days of epidermis reconstitution, Mildner et al (99) have described increased permeability of the epidermal barrier to the lucifer yellow dye, hypogranulosis, enhanced activation of caspase-3 after UVB irradiation, but no alteration of lipid composition or of keratinocyte differentiation in the knockdown tissue. While using an shRNA procedure to also suppress FLG expression in a RHE model grown for 11 days, Pendaries and collaborators (100) reported hypogranulosis and increased barrier permeability of the lucifer yellow dye, together with a reduced number of keratinocyte layers, reduced cornified layer thickness, and decreased levels of the NMF, potentially responsible for the increased UV sensitivity already reported by Kuchler and colleagues (98). However, unlike Mildner and co-workers (99), alterations were reported in the mRNA and protein expression of components deeply involved in the epidermal differentiation process (100).…”

“…Organotypic skin models containing knockdown of FLG in keratinocytes have been set up. Two studies have analyzed FLG knockdown in HSE by means of siRNAs (98, 99). Kuchler et al (87) showed that FLG silencing performed in keratinocytes used to produce HSE grown at the air–liquid interface for 14 days, disturbed the development of the stratum corneum and also induced spongiosis.…”

Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair

“…After a second incubation, primary human keratinocytes were added on top of the collagen matrix. On the next day, the construct was lifted to the air-liquid interface and medium was changed to a differentiation medium 64. After 2 days, we induced a wound by scratching the epidermal layer with a needle.…”

Suitability of skin integrity tests for dermal absorption studies in vitro

“…To avoid an insufficient number of valid skin samples for the entire study, we recommend a combined use of the binary standard test TEWL in advance of an experiment -which is able to identify the majority of defect skin samples without pretreatment of the skin samples -and the outlined continuous ISTD approach -to evaluate effects observed during the absorption experiment. Since good correlations were observed for all skin preparation types (excised human, reconstructed human and excised rat skin), the ISTD approach is probably transferable to diseased skin or reconstructed diseased skin (Kuechler et al, 2011;Oji et al, 2010) as well.…”