The impact of recent advances in lipidomics and redox lipidomics on dermatological research

Free Radical Biology and Medicine 144 (2019) 256–265

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The impact of recent advances in lipidomics and redox lipidomics on dermatological research

T

Florian Grubera,b,∗, Christopher Kremslehnera,b, Marie-Sophie Narzta,b a b

Department of Dermatology, Medical University of Vienna, Austria Christian Doppler Laboratory for the Biotechnology of Skin Aging, Vienna, Austria

ABSTRACT

Dermatological research is a major beneficiary of the rapidly developing advances in lipid analytic technology and of bioinformatic tools which help to decipher and interpret the accumulating big lipid data. At its interface with the environment, the epidermis develops a blend of lipids that constitutes the epidermal lipid barrier, essential for the protection from water loss and entry of dangerous noxae. Apart from their structural role in the barrier, novel intra- and inter-cellular signaling functions of lipids and their oxidation products have been uncovered in most cutaneous cell types over the last decades, and the discovery rate has been boosted by the advent of high resolution and –throughput mass spectrometric techniques. Our understanding of epidermal development has benefited from studies on fetal surface lipids, which appear to signal for adaptation to desiccation post partum, and from studies on the dynamics of epidermal lipids during adjustment to the atmosphere in the first months of life. At birth, external insults begin to challenge the skin and its lipids, and recent years have yielded ample insights into the dynamics of lipid synthesis and –oxdiation after UV exposure, and upon contact with sensitizers and irritants. Psoriasis and atopic dermatitis are the most common chronic inflammatory skin diseases, affecting at least 3% and 7% of the global population, respectively. Consequently, novel (redox-) lipidomic techniques have been applied to study systemic and topical lipid abnormalities in patient cohorts. These studies have refined the knowledge on eicosanoid signaling in both diseases, and have identified novel biomarkers and potential disease mediators, such as lipid antigens recognized by psoriatic T cells, as well as ceramide species, which specifically correlate with atopic dermatitis severity. Both biomarkers have yielded novel mechanistic insights. Finally, the technological progress has enabled studies to be performed that have monitored the consequences of diet, lifestyle, therapy and cosmetic intervention on the skin lipidome, highlighting the translational potential of (redox-) lipidomics in dermatology.

1. Introduction 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%) [1,2]. 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

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permeability between the corneocytes, the rigid remnantns of the terminally differentiated keratinocytes [3]. The spacial ordering of barrier lipids is essential for functioning of the epidermal barrier, and investigating this requires specialized analytic techniques [2]. An important step for the formation of this lipid envelope is the esterification of hydroxylated ceramides to involucrin and other corneocyte proteins by transglutaminases. These keratinocyte derived lipids blend with the sebum lipids, a mixture of TAG (45%), wax esters (WE, 25%), squalene (12%) and FFA (10%) which derive from holocrine secretion of terminally differentiating cells of the sebaceous gland (SG), a lipid producing skin appendage that is usually connected to the hair follicle, forming the “pilosebaceous unit” [4]. The epidermis also contains a network of Langerhans cells and dendritic antigen presenting cells that constitute the first immunological barrier against the environment. In addition, the basal layer of human epidermis harbors melanocytes, the pigment producing cells that supplement the surrounding KC with UVprotective melanin, stacked in vesicles called melanosomes. Below the basal membrane of the epidermis lies the dermal compartment of the skin, containing fibroblasts, microvessels and cells of the immune system. The lipid composition of dermal cells is usually rich in

Corresponding author. Department of Dermatology, Medical University of Vienna, Austria. E-mail address: [email protected] (F. Gruber).

https://doi.org/10.1016/j.freeradbiomed.2019.04.019 Received 30 January 2019; Received in revised form 1 April 2019; Accepted 15 April 2019 Available online 18 April 2019 0891-5849/ © 2019 Elsevier Inc. All rights reserved.

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phospholipids (75%), with cholesterol, di-and triacylglycerols, FFA and sphingomyelin levels all below 10%, but the exact composition depending on the specific cell type. Activation of the innate and the adaptive immune system and metabolic perturbations can further unbalance the lipid equilibrium within the skin. For this reason, and also because of the diversity of the lipid distribution within the stratified architecture of the skin, sampling and analytics are a demanding task, which is not yet covered by a “one shot” comprehensive protocol that covers quantification or imaging of all lipid classes and their modifications or adducts. Samples for (redox-) lipidomics can be collected noninvasively from the surface with sebum adsorbent patches (e.g. Sebutape) [5,6]. A mild method to sample corneocytes in increasing depth, is tape stripping with special adhesive tapes (e.g. D-squame), from which lipids for analytics can be successfully recovered. Applying subsequent -epidermal water loss (TEWL) measurements at the site of sampling the concurrent loss of barrier function can be determined Sadowski et al. [7] have recently used shotgut lipidomics to thoroughly compare the change in lipid composition of stratum corneum after tape stripping versus scraping of sample material with a scalpel. was Cyanoacrylate (superglue) stripping is a less mild method that has also been successfully applied for barrier lipid analytics [8]. In earlier decades, successful in vivo lipid extraction was done with a glass tube and organic solvents [9]. Interstitial fluid lipids can be sampled from suction blisters, or, in a controlled fashion over several hours, by open flow microperfusion (OFM). The species detected indicate a primarily epidermal origin of suction blister fluid [10–12]. Lipids can also be extracted from the enzymatically separated dermal and epidermal compartments of skin biopsies, and from explant- and organotypic culture systems, which are increasingly used for genetic and translational studies on the lipid composition of the skin in a three dimensional setting [13,14]. Several recent technical advances in analysis of lipids have boosted our knowledge of the skin lipidome in homeostasis and disease. Firstly, the refinement of shotgun lipidomic methods, which allow acquisition of mass spectra in high throughput without a chromatographic separation step, was made possible by high resolution instruments and massive data processing capabilities. The resulting complex spectra have been successfully applied to the analysis of cutaneous lipids [7,15,16]. While being more time consuming, methods involving chromatographic separation still show higher sensitivity for less abundant or novel lipid species, and quantification of structurally similar sub-classes of skin lipids such as the ceramides [17,18]. Both shotgunand chromatographic methods have also recently been refined for the study of lipid oxidation in biological samples, including the skin [19–21], and recent excellent reviews cover this specific topic [22–25], as well as skin lipid analytics in general [2,3,26–28]. We havehere compiled a selection of very recent advances that translate lipidomic and redoxlipidomic technology to dermatological basic- and clinical research questions. Our focus is on the skin lipidome in development and homeostasis, in environmental and irritant stress, in acne, and in atopic dermatitis and psoriasis, as representatives of the major inflammatory skin diseases.

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 [30]. Together these newly discovered bioactive lipids present in VC are compatible with functions of VC beyond being a mere barrier surrogate at birth. And indeed, a recent study by Boiten and colleagues demonstrates that a formulation based on known components of VC (whether endocannabinoids are included is not specified) can promote barrier recovery after tape stripping in healthy volunteers and alters lipid synthesis patterns. Using LC/MS, the authors showedthat disruption-induced synthesis of ceramides with a total chain length of 34 carbons was attenuated by the VC formulation, and esterified omega-acyl-ceramides (Cer[EO]) were increased. SC lipid organization was thus favorably changed to facilitate accelerated barrier restoration [31] (Fig. 1, top left). The post-natal adaptation to the atmosphere is conceivably one of the major alterations the developing skin must achieve. For example, Michael-Jubeli and colleagues [32] used high temperature gas chromatography-mass spectrometry to investigate how the skin surface lipids (SSL) change from birth until the sixth month post partum, and developed a new method to reduce the amount of data, correct for inter-assay variations and integrate the data into a single analysis matrix. This so-called clustering-based preprocessing method (CB-PPM) is based on measuring mathematical distance between the mass spectra including all m/z values and retention times. They then searched for compounds that were significantly changed from less than 40 days of age more than 85 days. They found squalene, waxes, triglycerides (with 49–52 carbons and two C16 acyls), cholesterol and cholesteryl esters (C14-C18), palmitic-, sapienic- and oleic acid to be dominant in the samples taken before day 40. Interestingly, they observed an increase in cholesteryl esters with C20 to C25 in the period from day 85 onward, pointing to esterification of cholesterol with epidermal fatty acids. This supports the hypothesis that, for a short postnatal period, the epidermis compensates for insufficient sebaceous lipid production, thereby maintaining the barrier function of the skin. Ceramides, the major constituents of the adult epidermal lipids and the most abundant stratum corneum lipids, were the subject of a study by Jia et al. [33]. The authors applied a targeted normal phase HPLC method, coupled with dynamic MRM-MS, to identify and quantify ceramides within the twelve subclasses. From tape strip samples collected at the lower inner forearm the authors could achieve quantitative data on 483 ceramide species from healthy individuals split in two age groups, children (0–13 yr; n = 32) and adults (22–28 yr; n = 24). The ceramide composition of the samples could be used to differentiate clearly between children and adults, but interestingly not between the sexes. Among the discriminating ceramides, those with alpha-hydroxy fatty acids were the most frequent (Fig. 1, bottom left). The ceramide composition was also in the focus of a study by Kendall and colleagues [34], where the authors investigated how supplementation with n-3 polyunsaturated fatty acids (PUFA) would affect structural ceramides, most prominently the omega hydroxylated, epidermis specific lipid species. In addition, bioactive sphingolipid signaling compounds in epidermis and dermis were quantified. As several of the long chain polyunsaturated fatty acids (PUFA) required for assembly of the skin's lipid barrier are provided systemically rather than being synthesized within the epidermis [35], changes in PUFA supplementation were hypothesized to affect both barrier and signaling lipids. Earlier studies [10,36] had shown that supplementation with n-3 PUFA in cutaneous cells acts in an anti-inflammatory manner, most likely by affecting the bouquet of PUFA derived lipid mediators. In this study Kendall and colleagues [34] supplemented skin explants with EPA or DHA for six days, then applied a targeted UPLC ESI MS/MS lipidomic approach (positive mode, MRM),

2. 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 [29]. 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 257

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where epidermis and dermis were analyzed separately after mechanical separation. The supplementation affected the dermal ceramides more than the epidermal ones, and while the levels of the omega hydroxylated ceramides were unaffected, several dermal non-hydroxy fatty acid/sphingosine base ceramide (Cer[NS]) and non-hydroxy fatty acid/ dihydrosphingosine base ceramide (Cer[NDS]) species were elevated upon EPA supplementation. The best studied signaling sphingolipid (S1P) was not regulated by supplementation, but two ceramide– 1-phosphate (C1P) species, N(16)S(18)C1P and N(18)S(18)C1P were significantly increased in the epidermis of EPA treated explants. The observed changes in the Cer species could, as the authors suggest, be due to specific hydrolysis of sphingomyelin precursors or de novo synthesis. While no biological function is known for the dermal ceramide species regulated by EPA, the elevation in epidermal C1Ps may contribute to the anti-inflammatory effect of EPA supplementation. The most comprehensive quantitative lipidomics of surface and SC lipids performed so far was done by Sadowski and colleagues [7], who sampled skin lipidomes of 104 subjects, from 14 different body sites and in different sampling depths. The analytics were performed with a high-throughput shotgun MS/MS method for all classes except cholesterol which was assayed after acetylation in a separate run. This work established adhesive tape stripping as the most practical method for SC sample generation, and demonstrated that one typical tape stripping disc yielded enough material (roughly 62 μg) for analysis and that the tape did not affect extraction and analytics. The method showed a quantification limit for most lipid classes in the low picomolar range and a linear dynamic range of about tow orders of magnitude for several ceramide, DAG, cholesterol, TAG and CE classes. This study defined with precision which sebum derived lipid species (TAG and DAG) dominated the outermost layers of the differentiated epidermis, and showed that their content decreased to a plateau within the first five to seven stripping layers while the keratinocyte-derived ceramide profiles were less variable with respect to SC sampling depth. They also found that among the surface lipids shorter cholesterol ester species (CE (10:0), (11:1), (14:0) as typical examples) derive from the sebum, whereas different, longer chain

species (predominantly CE (18:1) and CE (18:2)) were keratinocyte derived (Fig. 1, top right). They could also show that the sampling site strongly determines the lipid profile and that again is dependent on the sebaceous lipids. The cheek and forehead, which are rich in sebaceous glands, showed the overall highest lipid content, and displayed a unique lipid composition distinct from all other body parts, in hierarchical clustering analyses of the lipid patterns. Furthermore, the authors could asses the inter-individual variabilities of the lipidomes sampled at the forearm of 65 females and 39 males of different ages, and found a marked age dependent decrease in TAG and DAG, and increase in cholesterol in female skin, less so in men, whereas ceramide levels were constant. This gender dependent decrease could be due to age related changes in hormonal regulation of sebum production, as the authors point out (Fig. 1, top right). These findings were extended in a study by Ludovici and colleagues [37] which investigated how the site and density of sebaceous glands would affect the composition of stratum corneum lipids and thereby the lipid component of the epidermal barrier. From a small cohort of eight donors the stratum corneum lipids from forearm, chest and forehead were sampled and analyzed with both an untargeted negative mode HPLC/ESI-TOF-MS and a targeted one for free fatty acids and cholesterol sulfate. Cholesterol and squalene were quantified by GC-MS. Apart from defining a set of 52 lipid species that allow differentiating the SC lipidome from the three anatomical sites, this study yielded the finding that there is indeed an effect of SG density on the composition of epidermis derived lipids. Especially epidermal cholesterol sulfate abundance was positively correlated with sebum derived lipid species (e.g. squalene), whereas other epidermal fatty acids (e.g. 24:0) were not positively correlated. As the epidermal cholesterol sulfate cycle plays a crucial role in regulating the permeability barrier [38] this finding provides a model for sebum regulating barrier function. Mass spectrometric analysis methods of cholesterol sulfate and other sulfate based lipids in fluids cells (including skin cells) were very recently described by Dias and colleagues [39] (Fig. 1, bottom right).

Fig. 1. Lipidomics of fetal, childhood and young adult skin. Left: Vernix caseosa is the lipid rich protective layer covering the skin of newborns. (Cer) ceramides, (FA) fatty acids, (TAG) triacylgylerides, (DAG) diacylglycerides, (CE) cholesterol esters. Right: lipidomic analysis of tape strip stratum corneum (SC) samples differentiates between sebum-derived surface lipids and lipids deriving from differentiating keratinocytes. Sebaceous gland (SG) density at different anatomical sites affects abunance or metabolization of stratum corneum lipids (lignoceric acid, FFA 24:0; cholesterol sulfate, CHS).

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3. Ultraviolet irradiation and chemical skin stress

recovery of the cells post exposure. Previously our group and others observed that at comparable fluencies, the oxidation of PUFA contained in phospholipids is much more efficient than oxidation of the same PUFA in the free form. One explanation may be that the presence of the FA within the phospholipid causes amplification of singlet oxygen generation and subsequent oxidation of the esterified fatty acid [45]. A recent study performed by our lab underlines the high susceptibility of KC phospholipids to UVA exposure. We used cultured human primary keratinocytes and assessed changes to the “oxidizedPC-lipidome”, the mRNA transcriptome and the proteome elicited by UVA (in our case the longer wavelength UVA-1, 40 J/cm2) REF. In an initial HPLC-MS/MS screening assay, which we had established earlier in fibroblasts irradiated with UVA [46], we observed significant elevation of 173 OxPC species immediately after irradiation, including non-enzymatic oxidation products such as PC-hydroperoxides and hydroxides, POVPC, PONPC, PGPC and PAzPC. In a second round using high resolution equipment, we identified several lipid species to be regulated by UV, among them two PC with C1 carbonyls and 20:3 Lyso-PC, the latter having been described as elevated in oncogene induced senescence. The conclusion from this multi omic study was that even at the high UVA-1 fluence of 40 J/cm2 the cells recover within 24 h, insofar as they limit the amount of highly reactive carbonyl containing lipids. The mRNA and protein data suggest that besides the NRF2 antioxidant response and UPR, the transcriptional regulator NUPR1 also takes part in the protective response, and that its depletion through modification by reactive oxidized lipids may be the mechanism of downstream transcriptional activation [19] (Fig. 2, left). Dietary intervention to increase systemic or even cutaneous omega-3 to omega-6 fatty acid ratios and thereby lower pro-inflammatory cyclooxygenase/lipoxygenase metabolites has long been a research topic. Lipidomic profiling permits the analysis of studies on the metabolite profile, for instance in environmental stress settings, as reported by Pilkington and colleagues [47]. In this randomized controlled study on dietary supplementation with eicosapentaenoic acid (EPA, 12 weeks), the authors found that dermal EPA relative to AA was more than doubled at the endpoint. The test subjects were exposed to UV at a four-fold minimal erythema dose (4MED) with UVA plus UVB before and after supplementation. Eicosanoids were then determined in skin suction blisters and biopsies with a HPLC-MS/MS targeted method. The supplementation could lower the basal and UVinduced ratios of prostaglandin E2 to prostaglandin E3 (PGE2: PGE3), and of 12-hydroxy-eicosatetraenoic acid to 12-hydroxy-eicosapentaenoic acid (12-HETE:12-HEPE) towards the less inflammatory EPA metabolites. The long term biological consequences of such a shift are however not well studied. Work from our own group showed that part of the anti-inflammatory effect of n-3 PUFA supplementation in cutaneous cells may depend on the redox sensitive transcription factor NRF2. DHA supplementation led to increased levels of DHA-containing phospholipids and these were strongly susceptible to UVAmediated oxidation. In the absence of NRF2 the DHA supplementation resulted in increased inflammatory gene expression after UV exposure [36]. Finally, the oxidized lipid platelet activating factor (PAF, which itself and its mimics are UV inducible), can trigger the release of microvesicles from UVB irradiated keratinocytes, as shown recently [48]. Thus not only proteins, but theoretically also bioactive oxidized lipids present in the microvesicle membrane, could exert a systemic signaling function as a response to UV, a mechanism that will be of interest in further studies (Fig. 2, right).

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 [40–42]. Here we will merely discuss the very recent data on stress induced changes to the (ox-) lipidome of the skin. Dalmau and colleagues investigated the effect of acute and chronic solar simulator exposure on cultured primary keratinocytes, with twice weekly irradiation with 0.7 J/cm 2 of UVA in combination with 25 mJ/cm2 of UVB for up to four weeks for the chronic group. Lipids were analyzed in two separate orthogonal acceleration time-of-flight (oa-TOF) LC/MS runs, one for sphingolipids and one for all other detectable lipids, respectively [43]. In the response to acute irradiation (one single irradiation) they found a decrease in the sphingomyelin to glucosylceramide ratio, and a strong increase in phospholipids, especially (34:0) and (36:2) phosphatidylcholines, whereas phosphatidyl-inositoles were decreased. Furthermore, triacylglyceride (48:5) was elevated after acute exposure. The chronic irradiation (in total 8 exposures over four weeks) resulted in elevation of long chain ceramides and a decrease of sphingomyelin, as a result of the sphingomyelinase activation which the authors had determined. Most prominently, a strong increase in lysophosphatidylcholine species was observed after chronic exposure. Together, the authors argue that the lipid composition after chronic exposure may present an early KC differentiation lipid signature which contains protein kinase C (PKC) agonistic lipids (Fig. 2, top left). A specific investigation of fatty acids and their metabolites was performed by Leung et al. in HaCaT cells exposed to two fluences of UVA (5 J/cm2 and 20 J/cm2, respectively). Using a HPLC-MS/MS technique (triple quadrupole, negative mode) and deuterated standards for normalization of many of the analyzed metabolites, they found little effect of UVA on n-6 PUFA and their nonenzymatic oxidation products immediately after exposure [44]. Only a minimal effect on lipoxygenase (LOX) products (8-HETE and 12-HETE) and on cyclooxygenase (COX)-mediated DHA metabolites was observed, and most species returned to baseline after 24 h, while specific F Isoprostanes (15-F2t-IsoP) were even suppressed post treatment. At 20 J/ cm2 the elevation of metabolite levels did not reach significance immediately after exposure, however arachidonic acid (AA) as the precursor of eicosanoids was significantly decreased. Interestingly, at 24 h post exposure the authors detected an elevation of AA, n-3 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), together with a reduction of adrenic acid (AdA) and eicosapentaenoic acid (EPA). The authors conclude that HaCaT cells require 24 h to return to PUFA homeostasis and that elevation of DHA synthesis may favor

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Fig. 2. Lipidomics and redoxlipidomics of skin and cultured skin cells exposed to ultraviolet radiation induced stress. (SM) sphingomyelin, (PI) phosphatidylinositol, (PC) phosphatidylcholine, (TG) triglycerides, (Cer) ceramides, (LysoPC) lysophosphatidylcholine, (AA) arachidonic acid, (F-isoP) F-isoprostanes, (DPA) docosapentaenoic acid, (DHA) docosahexaenoic acid, (AdA) adrenic acid, (EPA) eicosapentaenoic acid, (OxPL) oxidized phospholipids, (PL-OOH) phospholipid hydroperoxide, (PLOH) phospholipid hydroxide, (PGE2) prostaglandin E−2, (HETE) hydroxyeicosatetraenoic acid, (HEPE) hydroxyeicosapentaenoic acid, (DHA-PL) phospholipids with esterified DHA, UVA (ultraviolet-A radiation; 320–400 nm), UVB (ultraviolet B radiation; 280–320 nm).

4. Skin inflammation, skin sensitization and therapeutic interventions

MS methods the authors could identify 18:1/18:1 phosphatidylcholine (PC) as inducible by LPS and decreased by BC. Furthermore, they found the phosphatidylethanolamine (PE) species (18:0/18:1) and (18:0/20:4) was decreased by DNFB and BC but not LPS, and the PS species (16:0/18:1) was decreased by BC only, whereas PS (18:0/18:1) was decreased by BC but induced by the other stressors [51]. This study was followed by others which compared DNFB induced phospholipid profiles to those elicited by the respiratory allergen hexamethylene di-isocyanate (HDI) and the irritant methyl salicylate (MESA) in monocytic cells. Here, the authors found phosphatidylinositol PI (38:4) was induced and PC (e34:1) and PI (34:1) were reduced by all three treatments, whereas treatment with DNFB exclusively led to an upregulation of PC (36:2) and cardiolipin (70:6 and 70:4). MESA application generally decreased PE and 16:0 free fatty acids (FFA). Other potential markers are also provided in the study [52]. Apart from addressing basic scientific questions with respect to the differential regulation of these lipid species, these studies provide us with candidate biomarkers that can be used in evaluating the potential sensitization and irritation properties of chemicals. It should not be left unmentioned, that the involvement of lipids themselves, as adjuvants co-administered with allergens -e.g. peanut lipids [53] needs future investigation (Fig. 3, left).

Glucocorticosteroids (GC), by far the most common class of drugs used in treatment and management of various inflammatory skin diseases [49] cause, as side effects, atrophy of the skin and epidermal barrier defects. Ropke and colleagues [50] 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. The authors conclude that supplementation with long chain ceramides may counteract treatment-induced atrophy and restore impaired barrier function (Fig. 3, top left). Skin sensitization and allergic contact dermatitis are promising fields for development of reliable predictive biomarkers. Santinha and colleagues REF have analyzed phospholipid profiles of keratinocytes after exposure to the sensitizing chemical dinitrofluorobenzene (DNFB), the toll-like receptor (TLR) agonist lipopolysaccharide (LPS) and the irritant benzalkoniumchloride (BC) in cell culture. Using TLC-ESI-MS, HPLC-MS and –MS/

5. 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. 260

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This appendage is also the site where acne develops, a common pathology with strong involvement of lipids and lipid derived signaling mediators [4]. 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 (DG 30:1, 31:1, 32:1 and 34:1) [6]. 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 [54] (Fig. 3, right). Zhou and colleagues performed UPLC-QTOF-MS on lipids isolated from lesional or control skin in a cohort of 35 acne patients and 35 controls. From 874 lipids species identified they could establish that 36 could discriminate between patients and controls. The major differences were elevated levels of several phosphatidylserine species, a reduction in ceramide chain length, an elevation of unsaturated fatty acids (EPA) and a reduction of prenol- and saccharolipids [55]. This study is notable for demonstrating the significant regulation ofseveral species that had not been described previously in the context of skin biology. As the authors state, the elevation of phosphatidylserines may have functional relevance as “eat me” signals to phagocytes, the first example of a potential lipid danger associated molecular pattern (DAMP) in acne pathology (Fig. 3, bottom right).

6. 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 [56]. 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 [57]. The example of psoriasis underlines the need to perform lipidomic analyses to achieve full interpretation of conventional transcriptomic and proteomic strategies in complex diseases. Several studies report changes in the expression of lipid transport proteins (reviewed in [58]) and lipid metabolizing enzymes in Psoriasis [59]. As a consequence of these events on protein levels, a dysregulation of downstream lipid mediators which could contribute to etiology -or temporary resolution-of psoriasis appears feasible. Using an untargeted UPLC-MS/MS method Zeng et al. analyzed the plasma lipids of 45 healthy individuals and 45 psoriasis patients [60]. They identified seventeen lipids, most prominently lysophosphocholines (LPC (16:0) & (18:0)) and phosphatidic acid (PA) which were elevated in psoriasis, whereas PC (18:0/18:1) and PI (16:0/ 16:0) & (16:0/16:1) were reduced (Fig. 4, top left). In a similar study, the lipidome of peripheral blood plasma, and lesional and nonlesional punch biopsy tissue from psoriatic skin was compared to samples isolated from healthy individuals [61]. Using targeted LC-MS/MS on lipids isolated from plasma samples the authors identified a significant increase of PUFA (omega-3 as well as omega-6) and the linoleic acid derived - oxidation products 9- and 13-hydroxyoctadecadienoic acid (9- and 13- HODE). At the same time, the

Fig. 3. Lipidomics of skin and cultured skin cells exposed to chemical sensitizers and -irritant stress. (KC) keratinocyte, (GC) glucocorticoid, (DNFB) 2,4-dinitrofluorobenzene, (BC) benzalkonium chloride, (ω-OH Cer) omega –hydroxylated ceramide, (TG) triglyceride, (FA) fatty acid, (FFA) free fatty acid, PC (phosphatiylcholine), PS (phosphatidylserine), (CL) cardiolipin, (PE) phosphatidylethanolamine, (DG) diacylglycerol, Cer[NH] ceramide class with non-hydroxy fatty acids and 6-hydroxy-4-sphingenines, Cer[AH] ceramide class with α-hydroxy fatty acids and 6-hydroxy-4-sphingenine, (EPA) eicosapentaenoic acid. 261

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Fig. 4. Lipidomics of Psoriasis and Atopic Dermatitis (AD). (PA) phosphatidic acid, (LPC) lysophosphatidylcholines, (PC) phosphatidylcholine, (PI) phosphatidylinositol, (PUFA) polyunsaturated fatty acid, (HODE) hydroxyoctadecadienoic acid, (HETE) hydroxyeicosatetraenoic acid, (Oxo-ODE) oxo-octadecadienoic acid, (LTB4) leukotriene B4, (TXB2) thromboxane B2, (Lyso PE) lysophosphatidylethanolamine, (DHET) dihydroxyicosatrienoic acid, Cer[AP] ceramide class with α-hydroxy fatty acids and 4-hydroxysphinganine, Cer[AS] ceramide class with α-hydroxy fatty acids and 4-sphingenines, Cer[NS], ceramide class with non-hydroxy fatty acids and 4sphingenines, (SCORAD) severity scoring of AD.

that are enriched in lesional skin of psoriasis patients [62]. A further recent hint on lipid involvement in psoriasis etiology is that lipid scavenger receptor CD-5 like (CD5L/AIM) is required to suppress the pathogenicity of Th17 cells [63]. In future, these may permit targeted treatments that combine insights from metabolism research, lipid biology and immunology, but may also allow (lipid) dietary interventions that might be beneficial for psoriasis patients [64] (Fig. 4, left).

plasma of psoriasis patients showed elevated levels of 7-hydroxycholesterol. Conversely, the levels of glutathione were decreased in the psoriasis plasma samples. A subsequent analysis of free and total (esterified plus free) FA and FA oxidation products in skin samples identified 8- and 12- hydroxy-eicosatetraenoic acids, 9- and 13 hydroxy-octadecadienoic acids as well as 9-, and 13- oxo-octadecadienoic acids as elevated in their free form in psoriasis as compared to healthy skin. The net effect on inflammation regulation in psoriasis exerted by the observed mediators is difficult to predict, as mediators with documented pro-inflammatory (9-HODE, 12-HETE)- but also anti-inflammatory (13-HODE, 15-HETE) properties were similarly elevated. The same group performed a second study using a targeted search for pro-resolving lipid mediators in a smaller patient cohort. Here, they showed docosahexaenoic acid (DHA-) derived resolvin D5, and (15-R) lipoxins A4 and B4 (derived from arachidonic acid) to be elevated in psoriatic lesional samples. However, the unoxidized PUFA giving rise to these mediators were likewise strongly elevated in the samples. The authors plotted the ratio of the DHA and EPA derived specialized proresolving mediators (SPM) to leukotrienes and prostaglandins, showing an elevation of the total SPMs. Rudimentary functional data from human keratinocytes exposed to resolvin D1, one of the SPMs, suggest TNFα antagonistic activity for this mediator in keratinocytes [57]. Together, these recent (metabo-) lipidomic studies are a first step in identifying lipid contributions to psoriasis, and apparently major breakthrough findings are imminent. The recent discovery of a phospholipase (PLA2G4D) as psoriasis “autoantigen” will initiate further investigation on psoriasis lipids. Cheung et al. found that PLA2G4D activity transferred from mast cells via exosomes to Langerhans cells results in the formation of (as of yet not clearly defined) antigens presented by CD1a, which are specifically recognized by psoriatic T cells

7. 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 [3,65], lipid analyses 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 [66]. A study by Huang and colleagues has used a non-targeted metabolomic and a targeted approach to study eicosanoids in childhood AD, comparing sera from unaffected controls and two AD patient cohorts with normal and high immunoglobulin E (IgE) levels, respectively [67]. In both AD patient cohorts elevation in leukotriene B4 (LTB4), thromboxane 2 (TXB2), prostaglandins, hydroxyl eicosatetraenoic and hydroxyl octadecadienoic acids (mostly from lipoxygenase and cyclooxygenase pathways) were observed, as compared to controls. The serum 262

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levels of TXB2 were higher in the high IgE group, whereas several HETEs and HODEs were lower in the high IGE groups. Interestingly, 19HETE and 20-HETE which are synthetized by cytochrome P450 (Cyp), were found elevated only in the normal IgE group. The metabolomic approach also yielded evidence on elevated Cyp metabolites in AD with normal IgE. The patients with high IgE however, showed energy metabolism and amino acid metabolism anomalies. This approach yielded useful combinatorial biomarkers to distinguish between the two AD groups, namely carnitine (18:2) and lysophosphatidyl-ethanolamine (18:2) from the metabolomics analysis, and TXB2, as well as 11, 12 -dihydroxyeicosatrienoic acid (DHET) from the targeted eicosanoid analysis, respectively. In a recent study by Töröcsik and colleagues that had low sample number but included tissue lipids in addition to the serum lipids, the elevation of TBX2 and 13-HODE in AD patient serum was confirmed. Further a significant reduction in DHA and a trend to decreased AA, EPA and LA levels was found. In the skin, however, the AA levels were elevated and the ratios of free n3/n6-PUFA were decreased. Further, the HETE levels were elevated in AD patient skin, most prominently in affected tissue. While ALOX5 expression was not elevated, the 12/15 lipoxygenases ALOX12B and ALOXE3 were elevated in AD skin and ALOX12B protein expression could be shown in KC and infiltrated cells. The 12-LOX metabolites were evenly elevated in unaffected and affected skin, whereas the sum of 15-LOX metabolites was highly elevated only in affected skin. COX1 was elevated in affected and unaffected skin, whereas prostaglandin E synthase was a factor that could differentiate between disease states, as it was strongly decreased in non-affected skin but elevated in affected skin. The ratio of the sum of known pro-inflammation to the sum of pro-resolution mediators showed a gradual increase from healthy via non-affected to affected AD tissue, and was also strongly elevated in the patient sera. Last, the sum of known PPAR (gamma and alpha) ligands was elevated in affected vs. healthy skin, whereas the expression of the receptors was significantly decreased (to 50% for PPARalpha and to 20% for PPARgamma [68]. In an organotypic model of AD, elevated AA and 12HETE, a PPARgamma agonist, appear to contribute to inflammation in AD and to in turn disturb differentiation, thereby reinforcing the pathologic state [69]. A study focusing on the barrier lipids rather than the inflammatory mediators used a targeted UPLC-MS/MS approach to quantify ceramides and FFA species in stratum corneum samples from 10 patients and 10 controls [70]. The authors found that Cer[AP] (alpha hydroxy FA conjugated to phytosphingosine) of C22 to C32 chain length and long fatty acid chain lengths were significantly decreased in AD unaffected skin, and even significantly further decreased in affected skin, as compared to healthy controls. The same was found for saturated FA from C22 to C28, and the authors conclude that normalization of the MS data to cholesterol rather than to protein allowed the better distinction between the groups. Since several of the dysregulated ceramides are negatively correlated with TEWL [71], these lipid markers also have functional relevance beyond being potential disease surveillance tools. In a very recent study by Shen et al. [72] who profiled the ceramides with the targeted NP-HPLC-dMRM-MS mentioned above [33] the authors could correlate Cer[AS] and Cer[NS] significantly to SCORAD grading of disease severity in children. These studies add to earlier findings (reviewed in [27]) on ceramide aberrations in AD, stressing that EOS ceramides are reduced in AD (and thereby the linking of the cornified lipid envelope to involucrin and other barrier proteins) [73], and levels of specific C34 ceramide being elevated, which apparently causes impaired organization of the lipid envelope [74] (Fig. 4, right).

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 [75]. Lipidomics were used to define the components of Andiroba oil, a traditional remedy from the brasilian Amazon with potential as skin care ingredient [76]. Dietary supplementation can actually alter lipid composition of the skin, and this has major potential for health and skin aesthetics, and further applications of lipidomics and other analyses to study such interventions are reviewed in [77]. For future development of skin photobiology and skin aging research the analytic developments in detection and imaging of lipid adducts and protein carbonylaton will be of increasing relevance [78]. The ability to track the “fate” of reactive lipids identified in lipidomic studies of skin stress (such as POVPC) to their protein adducts [79] boosts the predictive power of lipidomics data on functional cell biological consequences. The next major revolution of lipid analysis for dermatology will be the advent of high resolution mass imaging techniques that will permit a major step forward in investigating and understanding structure/function contributions of lipids throughout the skin and its appendages. The lipid mass imaging process has started with MALDI-MS imaging [80] and is constantly being refined (e.g. by combination with micro x ray fluorescence imaging [81]). It has now reached the subcellular level through combination of an ion beam with time-of-flight secondary ion mass spectrometry (TOF-SIMS) [82,83] and further development of such technology will, in our opinion, boost progress in skin biology. Acknowledgements F.G., M.S.N. and C.K. are grateful for financial support by the Federal Ministry for Digital and Economic Affairs of Austria and the National Foundation for Research, Technology and Development of Austria to the Christian Doppler Laboratory for Biotechnology of Skin Aging. The illustrative histology samples were provided from the collection of the Department of Dermatology of the Medical University of Vienna, which have been collected in studies approved by the local ethics committee and conducted in accordance with the Declaration of Helsinki principles; participants or their legal representatives gave their written informed consent. Ethic committee vote numbers 004/2011 and 1149/2011. We thank Heidemarie Rossiter for proofreading. References [1] K.R. Feingold, P.M. Elias, Role of lipids in the formation and maintenance of the cutaneous permeability barrier, Biochim. Biophys. Acta 1841 (2014) 280–294. [2] J. van Smeden, M. Janssens, G.S. Gooris, J.A. Bouwstra, The important role of stratum corneum lipids for the cutaneous barrier function, Biochim. Biophys. Acta 1841 (2014) 295–313. [3] J. van Smeden, J.A. Bouwstra, Stratum corneum lipids: their role for the skin barrier function in healthy subjects and atopic dermatitis patients, Curr. Probl. Dermatol. 49 (2016) 8–26. [4] C.C. Zouboulis, M. Picardo, Q. Ju, I. Kurokawa, D. Torocsik, T. Biro, et al., Beyond acne: current aspects of sebaceous gland biology and function, Rev. Endocr. Metab. Disord. 17 (2016) 319–334. [5] P. Clarys, A. Barel, Quantitative evaluation of skin surface lipids, Clin. Dermatol. 13 (1995) 307–321. [6] E. Camera, M. Ludovici, S. Tortorella, J.L. Sinagra, B. Capitanio, L. Goracci, et al., Use of lipidomics to investigate sebum dysfunction in juvenile acne, J. Lipid Res. 57 (2016) 1051–1058. [7] T. Sadowski, C. Klose, M.J. Gerl, A. Wojcik-Maciejewicz, R. Herzog, K. Simons, et al., Large-scale human skin lipidomics by quantitative, high-throughput shotgun mass spectrometry, Sci. Rep. 7 (2017) 43761. [8] E.M. Ropke, W. Augustin, H. Gollnick, Improved method for studying skin lipid samples from cyanoacrylate strips by high-performance thin-layer chromatography, Skin Pharmacol. 9 (1996) 381–387.

8. 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 263

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