Claudin targeting as an effective tool for directed barrier modulation of the viable epidermis,Annals of the New York Academy of Sciences

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Claudin targeting as an effective tool for directed barrier modulation of the viable epidermis
Annals of the New York Academy of Sciences ( IF 4.1 ) Pub Date : 2022-08-22 , DOI: 10.1111/nyas.14879
Laura-Sophie Beier _{1,

2} , Ayk Waldow ₁ , Saeed Khomeijani Farahani ₁ , Roman Mannweiler ₁ , Sabine Vidal-Y-Sy ₃ , Johanna M Brandner ₃ , Jörg Piontek ₁ , Dorothee Günzel ₁

Affiliation

Tight junction (TJ) formation is vital for epidermal barrier function. We aimed to specifically manipulate TJ barriers in the reconstructed human epidermis (RHE) by claudin-1 and -4 knockdown (KD) and by claudin-binding fusion proteins of glutathione S-transferase and modified C-terminal fragments of Clostridium perfringens enterotoxin (GST-cCPE). Impedance spectroscopy and tracer permeability imaging were employed for functional barrier assessment and investigation of claudin contribution. KD of claudin-1, but not claudin-4, impaired the paracellular barrier in vitro. Similarly, claudin-binding GST-cCPE variants weakened the paracellular but not the stratum corneum barrier. Combining both TJ targeting methods, we found that claudin-1 targeting by GST-cCPE after claudin-4 KD led to a marked decrease in paracellular barrier properties. Conversely, after claudin-1 KD, GST-cCPE did not further impair the barrier. Comparison of GST-cCPE variants with different claudin-1/claudin-4 affinities, NHS-fluorescein tracer detection, and immunostaining of RHE paraffin sections showed that GST-cCPE variants bind to extrajunctional claudin-1 and -4, which are differentially distributed along the stratum basale–stratum granulosum axis. GST-cCPE binding blocks these claudins, thereby specifically opening the paracellular barrier of RHE. The data indicate a critical role for claudin-1 in regulating paracellular permeability for ions and small molecules in the viable epidermis. Claudin targeting is presented as a proof-of-concept for precise barrier modulation.

中文翻译：

Claudin 靶向作为活表皮定向屏障调节的有效工具

紧密连接 (TJ) 的形成对于表皮屏障功能至关重要。我们旨在通过 claudin-1 和 -4 敲低 (KD) 以及谷胱甘肽S-转移酶的 claudin 结合融合蛋白和产气荚膜梭状芽孢杆菌肠毒素 (GST ) 修饰的 C 末端片段来特异性操纵重建人表皮 (RHE) 中的 TJ 屏障-cCPE）。阻抗谱和示踪剂渗透成像被用于功能屏障评估和密蛋白贡献的研究。claudin-1 而非 claudin-4 的 KD在体外损害了细胞旁屏障. 同样，紧密连接蛋白结合的 GST-cCPE 变体削弱了细胞旁屏障而不是角质层屏障。结合这两种 TJ 靶向方法，我们发现在 claudin-4 KD 后 GST-cCPE 靶向 claudin-1 导致细胞旁屏障特性显着降低。相反，在 claudin-1 KD 之后，GST-cCPE 没有进一步损害屏障。比较具有不同 claudin-1/claudin-4 亲和力的 GST-cCPE 变体、NHS-荧光素示踪剂检测和 RHE 石蜡切片的免疫染色表明，GST-cCPE 变体与 extrajunctional claudin-1 和 -4 结合，它们沿不同方向分布基底层-颗粒层轴。GST-cCPE 结合可阻断这些密蛋白，从而特异性打开 RHE 的细胞旁屏障。数据表明 claudin-1 在调节活表皮中离子和小分子的细胞旁通透性方面起着关键作用。Claudin 目标被呈现为精确屏障调制的概念验证。

更新日期：2022-08-22

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