Durotimi Dina: Development of Human Skin Equivalents with Inducible Ceramide Depletion for In Vitro Modeling of Lipid Impairment

(Nemes and Steinert, 1999). The stratum corneum relies on an intricate network of proteins and lipids, including the lipid lamellar bilayer (LLB) and cornified lipid envelope (Feingold and Elias, 2014). The LLB confers the water-impermeable properties of the epidermis, and the cornified lipid envelope acts as a sealant for the cohesion of the corneocytes and a scaffold for the LLB (Akiyama, 2017; Elias et al, 2014; Nemes and Steinert, 1999; Van Smeden et al, 2014; Wertz et al, 1989). These lipid-based structures consist of ceramides, cholesterol, and fatty acids, with ceramides making up the greatest fraction by mass (Kendall et al, 2017).

The specific class of ω-esterified ultralong-chain ceramides, also known as the acylceramides (acylCers), are key for the molecular organization and subsequent function of the LLB and cornified lipid envelope, and the enzyme ceramide synthase 3 (CerS3) is uniquely required for acylCer synthesis (Kihara, 2016; Rabionet et al, 2014). To date, 5 species of ω-esterified hydroxy ceramides have been identified: Cer[EOH], Cer[EOP], Cer[EOS], Cer[EODS], and Cer[EOSD]. Their unique chemical structure, namely their esterification to linoleic acid, is responsible for the formation and organization of the LLB (Behne et al, 2000; Breiden and Sandhoff, 2014). Both Cer[EOS] and Cer[EOH] play important roles in the long-periodicity-phase lamellar organization of the LLB, and it has been shown that in the absence of Cer[EOS], almost no long periodicity phase is formed (Bouwstra et al, 2001, 2000, 1998). Overall deficiencies in ceramides can compromise the barrier function of the skin and contribute to numerous skin conditions, ranging from dry skin to severe ichthyoses (Coderch et al, 2003; Sahle et al, 2015; Schreiner et al, 2000; Shen et al, 2018), and long-periodicity-phase malformation directly correlates with an inadequate epidermal barrier and a dry skin phenotype (Bouwstra et al, 2002; Imokawa et al, 1991; Schreiner et al, 2000).

CerS3 deficiency in mice leads to a loss of ultralong-chain ceramides and subsequent fatality shortly after birth (Jennemann et al, 2012). Variation in the CERS3 gene in humans affect barrier function and can cause specific forms of autosomal recessive congenital ichthyoses (Elias et al, 1984; Oji et al, 2010). For example, the codon exchange variant (p.Trp15Arg) results in moderate lamellar ichthyosis with a reduction in long-chain acylCers (Eckl et al, 2013), and similar findings have been reported for other variants leading to Cers3 deficiency (Radner et al, 2013). Thus, Cers3 and acylCers appear to be crucial for the lipid barrier function of the skin.

Human skin equivalents (HSEs) that replicate the 3-dimensional (3D) structure and function of the skin in vitro are powerful tools for investigating human-specific disease mechanisms and, in a few cases, have been applied to lipid-associated diseases. For example, HSEs constructed using patient-derived keratinocytes have been used to replicate disease phenotypes of CerS3 deficiency in autosomal recessive congenital ichthyoses (Eckl et al, 2013). HSEs have also been used to model harlequin ichthyosis using keratinocytes with engineered knock out of the lipid transporter ABCA12 (Enjalbert et al, 2020). Importantly, HSEs are also compatible with lipidomic analysis through mass spectrometry (Thakoersing et al, 2013) and can be used to replicate key differences in the lipid composition of the epidermis in diseases, such as atopic dermatitis (Danso et al, 2017). However, despite these advances and the importance of lipids in the skin barrier, there are limited experimental models that support precise modulation of lipid synthesis.

Because lipid deficiencies can cause a spectrum of conditions with varying degrees of severity, the ability to precisely tune the amount and composition of lipids within an HSE model would be advantageous for in vitro modeling of human lipid disorders and testing drugs and skincare products. In this study, we developed an inducible HSE model of ceramide deficiency using engineered keratinocytes with doxycycline (Dox)-inducible knockdown (KD) of CerS3. We focused on CerS3 as a proof of concept because it is a critical regulator of ceramide synthesis in the epidermis and is solely responsible for the synthesis of the acylCer class of lipids. We show that reduction of CerS3 expression in keratinocytes results in a global depletion of polar lipids within the epidermis of the HSE as well as a lower abundance of specific classes of ceramides and a reduction in ceramide chain length. Moreover, we demonstrate the reversibility of inducible CerS3 KD and ability to study the kinetics of lipid recovery within the HSEs after acute ceramide depletion. Together, these findings establish a robust and tunable methodology for modeling lipid impairment in human skin.

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