Claudin-1 decrease impacts epidermal barrier function in atopic dermatitis lesions dose-dependently

Abstract

The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with insideout and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1β expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci. In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment. Tight junctions (TJs) are an important component of the complex epidermal barrier system. They are localized in the stratum granulosum (SG) of the epidermis and provide mechanical barrier function to ions and solutes of different molecular sizes 1-4. The transmembrane protein claudin-1 (Cldn-1) is a major component of TJs 5. It is also found outside of TJs in basal and suprabasal layers of the epidermis 2,5. Mice with a complete Cldn-1 knockout (KO) die at the first day of birth due to increased transepidermal water loss (TEWL) 5. They develop TJs leaky to a molecular tracer (Biotin-556) 5 , and a highly water permeable stratum corneum (SC) 6. Human subjects lacking Cldn-1 suffer from the Neonatal Ichthyosis-Sclerosing Cholangitis (NISCH) syndrome which includes an ichthyosiform skin phenotype 7. An archetypical disease of epidermal barrier dysfunction is atopic dermatitis (AD) 8. Cldn-1 single nucleotide polymorphisms were linked to AD in some cohorts 9-11 , but not in others 11,12. Using immunostaining-intensity measurements and western blot analyses, reduced Cldn-1 levels were found in lesional AD skin 13-16. For non-lesional skin, divergent observations were described. Some authors found decreased mRNA and immunointensity levels 10 , while others observed no alteration of Cldn-1 immunointensity and western-blot levels 14,16 .

Skin Barriers in Dermal Drug Delivery: Which Barriers Have to Be Overcome and How Can We Measure Them?

“…In AD lesional skin there is a strong downregulation of claudin-1 [ 73 , 74 , 75 ] and, depending on the cohort investigated, no, mild or medium downregulation in non-lesional skin [ 74 , 75 , 76 , 77 ]. Concerning barrier function, there is strong impairment of TJ barrier in lesional and no impairment in AD non-lesional skin with no or only mild downregulation of claudin-1 [ 74 ], reflecting the dose dependency of TJ barrier function on claudin-1 levels [ 74 , 78 ]. There is a slight upregulation of occludin and claudin-4 in non-lesional AD skin [ 74 , 75 ] while there is an upregulation of occludin and an altered localization of claudin-4 in lesional skin [ 74 ].…”

“…BV: blood vessel, HF: hair follicle, SbG: sebaceous gland, SwG: sweat gland. ( A ) from [ 122 ], ( C ) from [ 123 ], ( F ) from [ 74 ], ( G ) from [ 124 ].…”

“…It is commonly used in monolayer cell cultures grown on transwell filters [ 60 , 125 ] applying “chopstick” electrodes or EndOhm chambers. In recent years it was also used for 3D cultures of reconstructed human epidermis/skin [ 74 , 84 , 126 , 127 ] or human skin [ 128 ]. While this is a good tool to measure overall barrier function to ions, which means comprising transcellular and paracellular barrier as well as barrier of the SC and the viable epidermis including TJs, it is not able to distinguish between these barriers.…”

“…Topical application of usually fluorescent dyes, such as Lucifer yellow and fluorescein isothocyanates (FITCs) of different sizes are used to measure kinetics of dye permeation in 2D cell cultures but also in reconstructed human epidermis and in skin [ 60 , 74 , 125 , 128 , 150 ] to determine skin barrier function. To this end, specimens are taken from the acceptor/basal compartment and measured using fluorescent readers.…”

“…To investigate TJ-related barrier function, the simultaneous analysis of molecular tracers which can be primarily or secondarily detected by fluorescence microscopy and that were injected into the dermis (or applied to the basal compartment of 3D cell cultures), in combination with labelling of distinct TJ proteins by antibody-linked immunofluorescence, have frequently been used in skin samples of different species, applying normal fluorescence microscopy or confocal laser scanning microscopy (CLSM) [ 64 , 65 , 74 , 97 , 109 , 169 , 170 , 171 ]. This method which is called “biotinylation assay” when using EZ-Link TM -Sulfo-NHS-LC-Biotin (Biotin-SH) gives insight into barrier function of TJs in interfollicular epidermis of healthy and diseased/knock-out skin [ 64 , 65 , 74 , 169 , 170 ], HFs [ 97 , 171 ] and glands [ 109 ]. This method warrants high molecular resolution of the localization of the stop of tracers of different sizes (up to now 556 Da (“Biotin-SH”), 1500 Da, 5000 Da, 31 kDa) in correlation with the TJ barrier.…”