Type 2 cytokine-dependent skin barrier regulation in personalized 2D- and 3D skin models of atopic dermatitis: A pilot study. Type 2 cytokine-dependent skin barrier regulation in personalized 2D- and 3D skin models of atopic dermatitis: A pilot study

Keratinocytes from healthy donors stimulated with type 2 cytokines are often used to experimentally study atopic dermatitis (AD) inflammatory responses. Due to potential intrinsic alterations, it seems favorable to use keratinocytes from AD patients. Keratinocytes isolated from hair follicles offer a non-invasive approach to investigate AD-derived keratinocytes. To evaluate whether such AD-derived keratinocytes are suitable to mimic AD inflammatory responses we compared hair follicle-derived keratinocytes from healthy donors and AD patients in a type 2 cytokine environment. Stimulation of AD-derived keratinocytes with IL-4 and IL-13 induced higher expression changes of AD-associated markers as compared to healthy keratinocytes. The combination of IL-4 and IL-13 generally induced highest expression changes, but IL-13 alone also induced significant changes of AD-specific markers. Similar to the 2D cultures, IL-4/IL-13 stimulation of 3D skin models generated with AD-derived keratinocytes modulated the expression of several AD-relevant factors. Whole transcriptome analysis revealed that IL-4 and IL-13 acted similarly on these 3D skin models. Histologically, IL-13 alone and in combination with IL-4 increased epidermal spongiosis, a histological hallmark of AD skin. Taken together, our pilot study suggests that hair follicle-derived keratinocytes from AD patients represent a useful model system to study AD-related inflammation in a personalized in vitro model. Overall design: Organotypic 3D skin models generated from hair follicle-derived human keratinocytes from two atopic dermatitis patients were left unstimulated or stimulated with Th2 cytokines IL-4, IL-13 and IL-4+IL-13. Two stimulations were done for each donor and each condition.

来自健康供者的角质形成细胞经2型细胞因子刺激后，常被用于特应性皮炎（atopic dermatitis, AD）炎症应答的实验研究。由于潜在的内在特性改变，使用特应性皮炎患者来源的角质形成细胞似乎更为适宜。从毛囊中分离得到的角质形成细胞为研究特应性皮炎来源的角质形成细胞提供了一种非侵入性的方法。

为评估此类特应性皮炎来源的角质形成细胞是否适于模拟特应性皮炎的炎症应答，我们在2型细胞因子环境下，对比了健康供者与特应性皮炎患者的毛囊来源角质形成细胞。与健康供者来源的角质形成细胞相比，用白细胞介素4（IL-4）和白细胞介素13（IL-13）刺激特应性皮炎来源的角质形成细胞，可诱导更多与特应性皮炎相关的标志物表达变化。白细胞介素4与白细胞介素13联合刺激通常可诱导最显著的表达变化，但单独使用白细胞介素13也可诱导特应性皮炎特异性标志物的显著表达变化。

与二维培养体系相似，用特应性皮炎来源的角质形成细胞构建的三维皮肤模型经白细胞介素4/白细胞介素13刺激后，可调控多种与特应性皮炎相关的因子的表达。全转录组分析显示，白细胞介素4与白细胞介素13对这些三维皮肤模型的作用模式相似。组织学分析表明，单独使用白细胞介素13以及其与白细胞介素4联合使用，均可增加表皮海绵水肿——这是特应性皮炎皮肤的组织学标志性特征。

综上，本预实验表明，特应性皮炎患者来源的毛囊角质形成细胞可作为一种实用的模型体系，在个性化体外模型中研究特应性皮炎相关炎症。

实验设计概况：从2名特应性皮炎患者的毛囊中分离得到人角质形成细胞，以此构建器官型三维皮肤模型，将其分为未刺激组与经2型辅助性T细胞（type 2 helper T cell, Th2）细胞因子刺激组，刺激因子包括IL-4、IL-13以及IL-4+IL-13。每名供者的每种处理条件均设置2次重复。