Linoleic acid (LA) is an essential omega-6 polyunsaturated fatty acid (PUFA) derived from the diet. Sebocytes, whose primary role is to moisturise the skin, process free fatty acids (FFAs) to produce the lipid-rich sebum. Importantly, like other sebum components such as palmitic acid (PA), LA and its derivative arachidonic acid (AA) are known to modulate sebocyte functions. Given the different roles of PA, LA and AA in skin biology, the aim of this study was to assess the specificity of sebocytes for LA and to dissect the different roles of LA and AA in regulating sebocyte functions. Using RNA sequencing, we confirmed that gene expression changes in LA-treated sebocytes were largely distinct from those induced by PA. LA, but not AA, regulated the expression of genes related to cholesterol biosynthesis, androgen and nuclear receptor signalling, keratinisation, lipid homeostasis and differentiation. In contrast, a set of mostly down-regulated genes involved in lipid metabolism and immune functions overlapped in LA- and AA-treated sebocytes. Lipidomic analyses revealed that the changes in the lipid profile of LA-treated sebocytes were more pronounced than those of AA-treated sebocytes, suggesting that LA may serve not only as a precursor of AA but also as a potent regulator of sebaceous lipogenesis, which may not only influence the gene expression profile but also have further specific biological relevance. In conclusion, we have shown that sebocytes are able to respond selectively to different lipid stimuli and that LA-induced effects can be both AA-dependent and independent. Our findings allow for the consideration of LA application in the therapy of sebaceous gland-associated inflammatory skin diseases such as acne, where lipid modulation and selective targeting of AA metabolism are potential treatment options.

Atopic dermatitis (AD) is a composite disease presenting disruption of the skin permeability barrier (SPB) in the stratum corneum (SC). Recent evidence supports derangement of the sebaceous gland (SG) activity in the AD pathomechanisms. The objective of this study was to delineate profiles of both sebaceous and epidermal lipids and of aminoacids from SG‐rich (SGR) and SG‐poor (SGP) areas in AD. Both sebum and SC were sampled from SGR areas, while SC was sampled also from SGP areas in 54 adult patients with AD, consisting of 34 and 20 subjects, respectively with and without clinical involvement of face, and in 44 age and sex‐matched controls. Skin biophysics were assessed in all sampling sites. Disruption of the SBP was found to be associated with dysregulated lipidome. Abundance of sapienate and lignocerate, representing, respectively, sebum and the SC type lipids, were decreased in sebum and SC from both SGR and SGP areas. Analogously, squalene was significantly diminished in AD, regardless the site. Extent of lipid derangement in SGR areas was correlated with the AD severity. The abundance of aminoacids in the SC from SGR areas was altered more than that determined in SGP areas. Several gender‐related differences were found in both controls and AD subgroups. In conclusion, the SG activity was differently compromised in adult females and males with AD, in both SGR and SGP areas. In AD, alterations in the aminoacidome profiles were apparent in the SGR areas. Lipid signatures in association with aminoacidome and skin physical properties may serve the definition of phenotype clusters that associate with AD severity and gender.

Sebum is a lipid‐rich mixture secreted by the sebaceous gland (SG) onto the skin surface. By penetrating through the epidermis, sebum may be involved in the regulation of epidermal and dermal cells in both healthy and diseased skin conditions. Saturated and monounsaturated fatty acids (FAs), found as free FAs (FFAs) and in bound form in neutral lipids, are essential constituents of sebum and key players of the inflammatory processes occurring in the pilosebaceous unit in acne‐prone skin. Little is known on the interplay among uptake of saturated FFAs, their biotransformation, and induction of proinflammatory cytokines in sebocytes. In the human SG, palmitate (C16:0) is the precursor of sapienate (C16:1n‐10) formed by insertion of a double bond (DB) at the Δ6 position catalysed by the fatty acid desaturase 2 (FADS2) enzyme. Conversely, palmitoleate (C16:1n‐7) is formed by insertion of a DB at the Δ9 position catalysed by the stearoyl coenzyme A desaturase 1 (SCD1) enzyme. Other FFAs processed in the SG, also undergo these main desaturation pathways. We investigated lipogenesis and release of IL‐6 and IL‐8 pro‐inflammatory cytokines in SZ95 sebocytes in vitro after treatment with saturated FFAs, that is, C16:0, margarate (C17:0), and stearate (C18:0) with or without specific inhibitors of SCD1 and FADS2 desaturase enzymes, and a drug with mixed inhibitory effects on FADS1 and FADS2 activities. C16:0 underwent extended desaturation through both SCD1 and FADS2 catalysed pathways and displayed the strongest lipoinflammatory effects. Inhibition of desaturation pathways proved to enhance lipoinflammation induced by SFAs in SZ95 sebocytes. Palmitate (C16:0), margarate (C17:0), and stearate (C18:0) are saturated fatty acids that induce different arrays of neutral lipids (triglycerides) and dissimilar grades of inflammation in sebocytes.

Derangement of the epidermal barrier lipids and dysregulated immune responses are key pathogenic features of atopic dermatitis (AD). The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signaling axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D epidermal equivalents. Tofacitinib, a low-molecular-mass JAK inhibitor, was used to screen for JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4 and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitoleic acid and caused depletion of triglycerides, in association with altered phosphatidylcholine profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very-long-chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by the halted homeostasis of FA metabolism.

Atopic dermatitis (AD) is a chronic inflammatory disorder presenting a dysregulated immune response and derangement of the epidermal barrier lipids. The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signal axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D-epidermal equivalents. We used tofacitinib, a low-molecular-mass JAK inhibitor, to screen for the JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4, and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase, and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitate and caused a depletion of triglycerides. This depletion was associated with altered PC profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very long chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by halted homeostasis of FA metabolism.

The lipid composition of the epidermis plays a critical role in the skin’s barrier function, and defects in lipid synthesis or assembly can cause a spectrum of skin diseases, ranging from dry skin to severe ichthyoses. The aim of this study was to develop an in vitro model of human skin with tunable inhibition of lipid synthesis. Human N/TERT keratinocytes were engineered to express doxycycline-inducible short hairpin RNAs targeting ceramide synthase 3, which is essential for synthesis of ultralong-chain ceramides and skin barrier function. We show that 3-dimensional human skin equivalents with induced knockdown of ceramide synthase 3 display normal stratification and terminal differentiation but have reduced Nile red staining for polar lipids. Further analysis of the lipidome by mass spectrometry confirmed a significant reduction in specific classes of ceramides and ceramide chain length in the ceramide synthase 3–depleted human skin equivalents. We also show that ceramide synthase 3 knockdown is reversible upon removal of doxycycline and can be used to study recovery and repair of epidermal lipids. Together, these findings provide an overall strategy for genetically regulating the lipid composition within human skin models and establish a tunable in vitro model of ceramide deficiency.