Human α1-antitrypsin (AAT) is an abundant acute phase glycoprotein performing anti-protease, anti-apoptotic and immunomodulatory functions. Purified human plasma AAT used as a specific therapy for patients with lung emphysema and panniculitis due to inherited AAT deficiency, and it is beneficial in treating of other disorders. This therapy administered intravenously although other routes of administration are tested. Herein, we examined the transdermal application of native or recombinant AAT by using epiCS®, the 3D human epidermis equivalent reconstructed from human primary epidermal keratinocytes. Topically applied AAT protein (50µl, prepared in Hank`s balance solution, HBSS) freely diffused across epidermis layers in a concentration and time-dependent manner. We concluded that within 18 hours topically added 0.2 mg AAT well penetrates the stratum corneum and can be detected inside the cytosol of keratinocytes. Importantly, treatments with AAT did not induce obvious morphological changes in keratinocyte layers. Next, we supplemented culture medium with 100µg/ml of combined bacterial lipopolysaccharide (LPS) and peptidoglycan (PNG) mixture and incubated epiCS for 18h with or without the topical application of 0.2 mg AAT. Under these conditions, LPS/PNG triggered a strongly activation of epidermis model. Even though AAT exhibited a limited capacity to neutralize the effect of LPS/PNG, it lowered expression of IL-18 and IL-8, and preserved levels of filaggrin, an important protein for maintaining the epidermal barrier integrity. Our results suggest that the transdermal route for delivering AAT is worth exploration. If successful, this approach may offer easy-to-use therapy with AAT.
Overall design: In this study, we used the 3D human epiCS reconstructed from the normal human primary epidermal keratinocytes, which resemble human epidermis containing a basement membrane, proliferating keratinocytes and a stratum corneum with an intact barrier function. Our aim was to study a free diffusion of AAT across epidermis. epiCS were treated with various concentrations of AAT added topically or into the culture medium (basolaterally) for different time points up to 48 h. The control epiCS we treated by adding the same volume of Hank`s balance salt solution (HBSS cat nr. 14025092, ThermoFisher Scientific, Germany) as for the AAT treatments. Additionally, epiCS were cultured for 18 h in the medium containing 100µg/ml of combined bacterial lipopolysaccharide (LPS, Sigma-Aldrich, Darmstadt, Germany) and peptidoglycan (PGN, stock solution prepared in 0.1 mg/ml in sterile water and few seconds sonicated; Sigma-Aldrich) mixture with and without topical application of 0.2 mg of native or recombinant AAT. At the end of experiment, we collected culture supernatants and cells for further studies.
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