Claudin targeting as an effective tool for directed barrier modulation of the viable epidermis

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.