The maintenance of a functional lipid distribution within the epidermis, a structure with a rapid directional turnover of differentiating keratinocytes (KC) requires extreme precision. Synthesis and metabolism must be fully coordinated with the controlled cell death in terminally differentiating keratinocytes and also in skin appendages that provide the surface lipids.
While the basal layer of the epidermis, and cultured keratinocytes are made up mostly of phospholipids (70%), cholesterol (Chol, 13%), triacylglycerides (TAG, 11%), terminal differentiation of KC drastically changes the lipid composition. In the granular layer, the last stratum that contains nucleated keratinocytes, intracellular organelles termed lamellar bodies are formed in which glucosylceramides, phospholipids and sphingomyelin are stored.
These are metabolized to produce the final stratum corneum (SC) lipids after co-exocytosis with the enzymes involved, yielding a mixture of free fatty acids (FFA, 40%), cholesterol and ceramides (Cer) (both roughly 30%). The FFA are mostly saturated and contribute to the acidity of the stratum corneum.
The stratum corneum lipids form the lipid matrix or lipid envelope, a flexible connection of low water permeability between the corneocytes, the rigid remnantns of the terminally differentiated keratinocytes. The spacial ordering of barrier lipids is essential for functioning of the epidermal barrier, and investigating this requires specialized analytic techniques.
The skin lipidome in development and homeostasis
From the last trimester of fetal development to birth the human skin is covered by a protective layer termed vernix caseosa (VC), a mixture of water, proteins and lipids that also contributes to the initial formation of the stratum corneum. To comprehensively address which lipids, in addition to ceramides, could provide the barrier and hypothetical signaling functions of VC, Checa et al. analyzed VC from 156 children classified as pre-term, full-term and post term.
Sphingolipids on the one hand and oxylipins plus endocannabinoids on the other hand, were analyzed in two separate ESI-UPLC-MS/MS protocols (triple quadrupole detectors). One key finding of the study was that the ceramides/sphingomyelin ratio (only Cer[NS], the non-hydroxylated fatty acid/sphingosine backbone class, were analyzed) in VC significantly increased with gestational age, which, as the authors point out, may be required for dry-adaptation post partum.
The authors could also detect the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in VC, which have known signaling bioactivity in keratinocytes, as AEA regulates keratinocyte differentiation, and both mediators affect lipogenesis in epidermal cells. Additionally, 2-AG appears to be a proresolving factor for wound healing. Together these newly discovered bioactive lipids present in VC are compatible with functions of VC beyond being a mere barrier surrogate at birth.
Ultraviolet irradiation and chemical skin stress
As the skin is the body’s outermost organ, it is constantly exposed to high oxygen levels, ultraviolet (UV) radiation, and environmental stress, such as pollution. Several groups have thus studyied the effect of these exogenous factors on the lipid composition of the skin, and how these modifications in turn may affect skin biology. External insult can cause enzymatic and nonenzymatic (free radical or singlet oxygen mediated) oxidative modification or nitration or chlorination of cutaneous lipids.
Cholesterol, phospholipids and squalene are targets for nonenzymatic lipid oxidation and can yield bioactive products. Stress induced activation of the epidermal lipoxygenases (ALOXE3 and ALOX12B), dermal lipoxygenases and cyclooxygenase produce a wide variety of fatty acid derived mediators, most prominently eicosanoid species. Also endocannabinoid levels (via PLC activation) and ceramides are UV responsive, and the reader is referred to excellent recent reviews on that topic.
Skin inflammation, skin sensitization and therapeutic interventions
Glucocorticosteroids (GC), by far the most common class of drugs used in treatment and management of various inflammatory skin diseases cause, as side effects, atrophy of the skin and epidermal barrier defects. Ropke and colleagues performed lipidomics with two separate UHPLCToF-MS approaches for FFA and glycero-/sphingolipids, respectively.
Analysis was performed on epidermal lipid extracts from tape strips collected from healthy volunteers after a 28-day treatment period, with once daily applications of clobetasol (high potency GC) or betamethasone (medium potency GC) ointments at the site of collection. Both corticoids reduced stratum corneum thickness and increased trans-epidermal water loss, each an indicator of reduced barrier function. This was associated with a significant reduction of the epidermal specific, omega hydroxyl chain esterified ceramides.
Furthermore, a significant decrease of ceramides with total chain lengths of C40 to C43 was observed among all other ceramide classes except Cer[NS] and Cer[AS], which remained unchanged. The analysis showed a decrease in triacylglycerols and in saturated free fatty acids, but these two findings were dominant only in clobetasol (the super potent GC-) treated areas.
Acne: The appendage of the skin with the most active lipid synthetic machinery is the sebaceous gland, which is usually found associated with a hair follicle. This gland produces the sebum, the major contribution to skin surface lipids, a mixture of triglycerides, diglycerides, free fatty acids, wax esters, squalene, cholesterol esters and cholesterol that are released in a holocrine secretion process during terminal differentiation of sebocytes This appendage is also the site where acne develops, a common pathology with strong involvement of lipids and lipid derived signaling mediators.
One study investigated the sebum lipids isolated with adsorbent tapes in a cohort of patients classified into three severity grades plus unaffected controls. Using a HPLC-TOF/MS protocol, the authors could identify diglyceride species that correlate with acne severity. These could, as the authors state, arise as a result of increased triglyceride hydrolysis, or from incomplete triglyceride synthesis, and are in line with association of DG levels with inflammation.
Pappas and colleagues specifically investigated the ceramide composition in acne skin using a UPLC/ ESI-MS/MS method. The special benefit of this study is that the composition was monitored over the course of a year with several sampling timepoints, together with trans-epidermal water loss (TEWL) measurement and assessment of acne severity. The authors found that in acne affected skin Cer[NH]
and Cer[AH] species were most prominently reduced. This effect was strongest in the cold season and correlated with the highest TEWL. Ceramide species with 18-carbon 6-hydroxysphingosine bases appeared to be most significantly reduced and are thus candidates for markers of barrier function.
Psoriasis: Psoriasis is a chronic inflammatory skin disease that affects roughly 3% of the adult population worldwide, and poses a severe psychological burden for the affected, as it most commonly manifests as disfiguring erythematous plaques. It is driven by pathogenic T cells which produce IL-17 in response to IL-23, and is amplified by inflammatory responses in the surrounding epidermal keratinocytes.
The co-morbidities that have been observed in most epidemiological studies include vascular and metabolic diseases, both being chronic inflammatory states with a prominent contribution of lipid mediators to pro-and anti-inflammatory events that contribute to the pathogenesis. The example of psoriasis underlines the need to perform lipidomic analyses to achieve full interpretation of conventional transcriptomic and proteomic strategies in complex diseases.
Atopic dermatitis: Lipidomics also enhance our understanding of atopic dermatitis, the second major inflammatory skin disease, affecting 7% to 10% of the population. A dysfunctional barrier of the epidermis is most likely causative – or at least heavily correlated – with this disease, with an early onset usually before the age of five years.
As composition and ordering of lipids in the differentiated strata of the epidermis is a main determinant for the barrier function and its loss in AD, lipidanalyses have high potential in uncovering the mechanisms behind both the barrier defect and the disease. Recent findings from transcriptomics support lipid involvement in the etiology of AD, as lipid synthesis and -metabolizing enzymes appear to be even more important at the onset of the disease than barrier proteins.
Translational potential in dietary and skin care intervention and future trends
Lipidomic analyses are becoming increasingly valuable in the evaluation of skin care applications and dietary interventions directed at the skin, beginning from analysis of formulations to evaluation of effectiveness of active ingredients and most importantly yielding mechanistic insights to skin biology beyond the strictly clinical or basic scientific context. In a study that investigated the effect of TiO2 nanoparticles which are widely used in cosmetics, on macrophage biology, Chen and colleagues combined several omics techniques.
The lipidomic aspect yielded evidence that TiO2 induced decrease of cardiolipin, a lipid important for maintenance of the mitochondrial electron transport chain, and thus an effect of TiO2 on phagocytic cells, warranting further investigation. Lipidomics were used to define the components of Andiroba oil, a traditional remedy from the brasilian Amazon with potential as skin care ingredient.
Author: Florian Grubera, Christopher Kremslehnera, Marie-Sophie Narzta