Distribution of Bioactive Lipid Mediators in Human Skin

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Distribution of Bioactive Lipid Mediators in Human Skin Alexandra C Kendall, Suzanne M Pilkington, Karen A Massey, Gary Sassano, Lesley E Rhodes, Anna Nicolaou

Cite this article as: Alexandra C Kendall, Suzanne M Pilkington, Karen A Massey, Gary Sassano, Lesley E Rhodes, Anna Nicolaou, Distribution of Bioactive Lipid Mediators in Human Skin, Journal of Investigative Dermatology accepted article preview 10 February 2015; doi: 10.1038/jid.2015.41. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Received 7 August 2014; revised 17 December 2014; accepted 23 January 2015; Accepted article preview online 10 February 2015

© 2015 The Society for Investigative Dermatology

Distribution of bioactive lipid mediators in human skin

Alexandra C. Kendall1, Suzanne M. Pilkington2, Karen A. Massey3, Gary Sassano4, Lesley E. Rhodes2, Anna Nicolaou1*

1

Manchester Pharmacy School and 2Dermatology Centre, Institute of Inflammation and

Repair, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, UK, 3School of Pharmacy and Centre for Skin Sciences, School of Life Sciences, University of Bradford, Bradford, UK, 4Safety and Environmental Assurance Centre, Unilever, Sharnbrook, MK44 1LQ, UK.

*Corresponding author: Professor Anna Nicolaou, Manchester Pharmacy School, The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK. Tel: +44 (0) 161 2752374; Email: [email protected]

The work was carried out in Bradford, West Yorkshire, UK.

Short title: Cutaneous bioactive lipids

Abbreviations: AA, arachidonic acid; AEA, N-arachidonoyl ethanolamide; 2-AG, 2-arachidonoyl glycerol; ALAE, N-alpha-linolenoyl ethanolamide; CB, cannabinoid receptor; COX, cyclooxygenase; CYP, cytochrome P450; DGLA, dihomo-gamma-linolenic acid; DHA, docosahexaenoic acid; 1

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DHEA, N-docosahexaenoyl ethanolamide; EPA, eicosapentaenoic acid; EPEA: Neicosapentaenoyl ethanolamide; GC, gas chromatography; HDHA, hydroxydocosahexaenoic acid; HEPE, hydroxyeicosapentaenoic acid; HETE, hydroxyeicosatetraenoic acid; HETrE, hydroxyeicosatrienoic acid; HFA, hydroxy fatty acid; HODE, hydroxyoctadecadienoic acid; HPLC, high performance liquid chromatography; LA, linoleic acid; LC-MS/MS, liquid chromatography tandem mass spectrometry; LEA, N-linoleoyl ethanolamide; LOX, lipoxygenase; LT, leukotriene; NAE, N-acyl ethanolamide; OA, oleic acid; OEA, N-oleoyl ethanolamide; PA, palmitic acid; PEA, N-palmitoyl ethanolamide; PLA2, phospholipase A2; PLD, phospholipase D; PPAR, peroxisome proliferator-activated receptor; PUFA, polyunsaturated fatty acid; Rv, resolvin; SEA, N-stearoyl ethanolamide; S1P, sphingosine-1phosphate; S1P1, sphingosine-1-phosphate receptor 1; SPE, solid phase extraction; TP, thromboxane receptor; TX, thromboxane.

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Abstract Skin produces bioactive lipids that participate in physiological and pathological states, including homeostasis, induction, propagation and resolution of inflammation. However, comprehension of the cutaneous lipid complement, and contribution to differing roles of the epidermal and dermal compartments, remains incomplete. We assessed the profiles of eicosanoids, endocannabinoids, N-acyl ethanolamides and sphingolipids, in human dermis, epidermis, and suction blister fluid. We identified 18 prostanoids, 12 hydroxy-fatty acids, 9 endocannabinoids and N-acyl ethanolamides, 21 non-hydroxylated ceramides and sphingoid bases, several demonstrating significantly different expression in the tissues assayed. The array of dermal and epidermal fatty acids were reflected in the lipid mediators produced, while similarities between lipid profiles in blister fluid and epidermis indicated a primarily epidermal origin of suction blister fluid. Supplementation with omega-3 fatty acids ex vivo showed that their action is mediated through perturbation of existing species and formation of other anti-inflammatory lipids. These findings demonstrate the diversity of lipid mediators involved in maintaining tissue homeostasis in resting skin, and hint at their contribution to signalling, cross-support and functions of different skin compartments. Profiling lipid mediators in biopsies and suction blister fluid can support studies investigating cutaneous inflammatory responses, dietary manipulation, and skin diseases lacking biomarkers and therapeutic targets.

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INTRODUCTION Skin is rich in lipids that not only contribute to formation and maintenance of the epidermal barrier, but also perform critical roles in membrane structure and the functions of cutaneous cells. Bioactive lipid mediators are derived from complex membrane lipids and are produced upon request and in response to environmental and signalling stimuli. They contribute to both skin physiology and pathology, with evidence of involvement in psoriasis, dermatitis, acne, wound healing and UV responses (reviewed in (Kendall and Nicolaou, 2013)). Dermal and epidermal compartments have diverse roles whilst supporting each other with nutrients and transcellular signals (Edmondson et al, 2003; Yamaguchi et al, 2005; Yu et al, 2011), and exhibit considerable inter-family cross-talk (Fig.1). Thus the lipid complement of human skin and its compartments demand definition, to assist understanding of skin biology, and to facilitate diverse applications including assessment of novel treatment targets in skin disorders. Of particular relevance because of their wide range of potential activities in the skin are the eicosanoid, endocannabinoid, N-acyl ethanolamide, and sphingolipid families. The eicosanoids are oxygenated metabolites of the 20-carbon polyunsaturated fatty acids (PUFA) arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3) and dihomogamma linolenic acid (DGLA; 20:3n-6). This family comprises the cyclooxygenase (COX)-derived prostaglandins (PG), thromboxanes (TX) and prostacyclin (PGI2), the lipoxygenase (LOX)derived leukotrienes (LT), lipoxins, E-series resolvins and hydroxy fatty acids (HFA) including hydroxyeicosatetraenoic acids (HETE) and hydroxyeicosapentaenoic acids (HEPE), derived from LOX and cytochrome P450 (CYP)-mediated reactions. Oxygenation of linoleic acid (LA, 18:2n-6) gives rise to octadecanoids such as hydroxyoctadecanoic acids (HODE), while docosahexaenoic acid (DHA, 22:6n-3) is the precursor of docosanoids such as D-series resolvins 4

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and protectins and hydroxydocosahexaenoic acids (HDHA) (reviewed in (Massey and Nicolaou, 2011; Nicolaou, 2013)). A number of PG, HETE, HODE and LT have been identified in human skin and contribute to keratinocyte proliferation, melanocyte dendricity, photocarcinogenesis, allergy and inflammation (Honma et al, 2005; Kendall and Nicolaou, 2013; Rhodes et al, 2009; Satoh et al, 2006; Scott et al, 2004; Ziboh et al, 2000). The endocannabinoids N-arachidonoyl ethanolamide (anandamide; AEA) and 2arachidonoyl glycerol (2-AG) are also derivatives of AA and function as endogenous lipid ligands of the two cannabinoid receptors (CB), CB1 and CB2 that are expressed throughout the skin in keratinocytes, melanocytes, fibroblasts, sebocytes and hair follicles (Dobrosi et al, 2008; McPartland, 2008; Pucci et al, 2012; Stander et al, 2005). Cell and organ culture systems indicate endocannabinoids are released by, and alter the functions of, various skin cells (Czifra et al, 2012; Dobrosi et al., 2008; Pucci et al., 2012; Sugawara et al, 2012; Toth et al, 2011), and they have been implicated in several cutaneous pathologies (Kupczyk et al, 2009). A range of Nacyl ethanolamides (NAE) can derive from membrane PUFA. Their relevance to cutaneous biology is illustrated by the activity of N-palmitoyl ethanolamide (PEA), which has been implicated in suppression of mast cell degranulation and inflammatory cytokine release, and explored for the treatment of atopic eczema (De Filippis et al, 2011; Eberlein et al, 2008; Petrosino et al, 2010). Sphingolipids are amides of sphingoid bases and an array of complex species can be derived through addition of fatty acids and head groups. Species present in the skin include free sphingoid bases and hundreds of ceramide species, as well as their phosphorylated versions (Masukawa et al, 2008; Rabionet et al, 2014; t'Kindt et al, 2012; van Smeden et al, 2011). The long chain omega-esterified ceramides found in the stratum corneum are considered structural 5

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lipids pivotal for the integrity of the epidermal barrier, and recent studies have revealed their diversity of structure and physiological functions (Breiden and Sandhoff, 2014; Iwai et al, 2012; Janssens et al, 2012; Masukawa et al., 2008; van Smeden et al., 2011). In contrast, nonhydroxylated ceramides and sphingoid bases are important mediators of immune cell regulation, with roles in homeostasis and inflammation (Chalfant and Spiegel, 2005; Uchida, 2014), and require further investigation to elucidate their contributions in the skin. Dermis and epidermis exhibit distinct cell populations, enzyme profiles and roles, and consequently lipid mediator expression could differ between the compartments. While epidermis comprises keratinocytes, Langerhans cells, melanocytes and Merkel cells, dermis contains fibroblasts, immune cells, hair follicles, sweat glands, blood vessels and sensory nerves, and provides skin with elasticity and resistance to mechanical stress. Historically, epidermis has been considered more important in terms of lipid biology because of the lipid-rich nature of the epidermal barrier. However, dermis also displays considerable activity and provides biochemical support to the epidermis, with mediator cross-talk between compartments: e.g. the epidermis exhibits impaired chain elongation of essential fatty acids and depends on the dermis for local production of long chain PUFA including AA, EPA and DHA (Chapkin et al, 1986), while dermally-derived 15-HETE impairs epidermal 12-LOX activity and dermally-produced PGE2 stimulates keratinocyte proliferation (Conconi et al, 1996; Kragballe et al, 1986). Despite increased awareness of the role of lipid mediators in cutaneous physiology and disease, there is insufficient information on individual lipid mediator species and their variation through the skin. To address this, we undertook comprehensive analysis of (i) eicosanoids and related species, (ii) endocannabinoids, (iii) N-acyl ethanolamides, and (iv) non-hydroxylated ceramides and free sphingoid bases, in human skin tissue and blister fluid, using mass 6

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spectrometry-based targeted mediator lipidomics assays. Our aim was to assess the production, range and variation of cutaneous lipid metabolites, including the contributions of dermal and epidermal compartments, in humans. This valuable information can provide insights into the mode of action of bioactive lipids, including the anti-inflammatory omega-3 fatty acids, and assist understanding of cutaneous biology, with potential for wide application in skin disease and its treatment.

RESULTS Eicosanoids in human dermis, epidermis and blister fluid Eighteen species of prostanoids and 12 HFA were identified and quantified (Fig.2A; fold changes from dermal to epidermal expression are presented in Supplementary Table S1). The AA-derived prostaglandins PGE2, PGD2, PGF2 and PGI2 (as its stable metabolite 6-keto PGF1α) were found in all tissues tested, with prevalence significantly higher in the epidermis (P=0.0003, P=0.003, P=0.003, P=0.00004 respectively, compared with dermis); TXA2 (measured as its stable metabolite TXB2) and PGJ2 were found predominantly in the epidermis. The EPA-derived PGE3, PGD3, PGF3

and DGLA-derived PGE1 and PGD1, although identified in both

compartments were higher in epidermis. Higher epidermal prevalence was also observed for all keto- and dihydro-keto PG (e.g. 15-keto-PGE2 and 13,14-dihydro-15-keto-PGE2); these derive from tissue specific catabolic reactions reducing the bioactivity of primary PG (Tai et al, 2002). A large number of LOX-derived HFA products of DGLA, AA, EPA, LA and DHA were found in both dermis and epidermis (HETrE, HETE, HEPE, HODE and HDHA, respectively). Interestingly, the LA-derived 9-HODE and 13-HODE were the predominant species in both 7

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compartments, although found at higher levels in the epidermis (P=0.015, P=0.045, respectively) with 9-HODE at mean concentrations of 326.9 ± 88.2 pg/mg protein in dermis and 2728.5 ± 548.8 pg/mg protein in epidermis, and 13-HODE at 350.6 ± 83.5 pg/mg protein in dermis and 1416.7 ± 201.5 pg/mg protein in epidermis. While 13-HODE is considered a 15-LOX product, 9HODE can be produced by a partially-completed COX-2 reaction (Laneuville et al, 1995), its levels reflecting the high COX activity observed in both skin compartments. Concentrations of the 15-LOX products 15-HETrE and 15-HETE were not significantly different between dermis and epidermis (Fig.2A; P=0.75, P=0.38, respectively). Although two more 15-LOX products were identified (EPA-derived 15-HEPE and DHA-derived 17-HDHA), these were not found in all skin samples and their levels were close to the assay detection limit (20 pg and 10 pg on the column, respectively) (Massey and Nicolaou, 2013). The 5-LOX-derived product of AA, 5HETE, was found at a very low concentration (≤34.7 pg/mg protein) in both epidermis and dermis. The 12-LOX products 12-HETE, 12-HEPE, 13-HDHA and 14-HDHA were significantly higher in epidermis (P=0.002, P=0.027, P=0.012, P=0.04 respectively) with 12-HETE being one of the predominant mediators found in both skin compartments (127.3 ± 10.5 pg/mg protein in dermis and 570.4 ± 54.3 pg/mg protein in epidermis). Epidermis had higher concentrations of 8and 11-HETE, products of either oxidation or cutaneous CYP isoforms (Rhodes et al., 2009). The profile and level of eicosanoids found in suction blister fluid was similar to that in epidermis, though with differences in some species (Fig.2A): 15-deoxy-Δ12,14 PGJ2 was found only in blister fluid while 13,14 dihydro PGE1, PGJ2, PGD3 and PGF2α, all found in the dermis and epidermis, appeared to be minor species of blister fluid with concentrations close to the method detection limit (0.5-5 pg on the column) (Massey and Nicolaou, 2013).

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Endocannabinoids and N-acyl ethanolamides in human dermis, epidermis and blister fluid Nine endocannabinoids and NAE species derivatives of palmitic acid (PA; C16:0), alphalinolenic acid (ALA; C18:3n-3), linoleic acid (LA; C18:2n-6), oleic acid (OA; C18:1n-9), stearic acid (SA; C18:0), EPA, AA and DHA were quantified. Although a larger number of NAE were detected, only those accurately identified and quantified based on the availability of commercial standards are reported. Both dermis and epidermis expressed all species measured, mostly with comparable expression (Fig.2A). Derivatives of AA, EPA and DHA, namely: AEA, eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA), were significantly higher in epidermis than dermis (P=0.016, P=0.004, P=0.004, respectively) (Fig.2A and Supplementary Table S1). Finally, all these metabolites were also present in blister fluid, in ratios similar to the epidermis.

Ceramides and sphingoid bases in the dermis, epidermis and blister fluid A huge diversity of cutaneous ceramides derives from the different sphingoid bases and acyl chains (Janssens et al., 2012; Mizutani et al, 2009; Rabionet et al., 2014; t'Kindt et al., 2012; van Smeden et al., 2011). Initial screening for ceramides for which information on specific transition ions suitable for mass spectrometric analysis was available, was based on the data reported by Masukawa et al (2008). Analysis of whole skin lipid extracts, indicated the presence of ceramides with sphingosine, dihydrosphingosine, phytosphingosine, and 6-hydroxy sphingosine bases, and non-hydroxy, alpha-hydroxy or ester-linked omega hydroxy fatty acids, belonging to 11 of the 15 ceramide families currently identified (Rabionet et al., 2014), namely: CER[NS], CER[AS], CER[NH], CER[AH], CER[NP], CER[AP], CER[NDS], CER[ADS], CER[EOP], CER[EOS] and CER[EOH]. Lack of commercially available appropriate synthetic standards did not permit identification of individual species. We therefore focused on non-hydroxylated 9

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ceramides of the CER[NS] family and phosphorylated ceramides, sphingoid bases and phosphorylated base species that could be analysed using 17-carbon-containing ceramides as internal standards (Fig.2B and Supplementary Table S1). Further to their role in epidermal barrier function, the CER[NS] family includes bioactive medium-chain (40-48-carbon) nonhydroxylated ceramides involved in cutaneous inflammation (Uchida, 2014). Overall, we identified two sphingoid bases, 18-carbon sphingosine (C18 S) and 18carbon dihydrosphingosine (C18 DS), and their phosphorylated forms 18-carbon sphingosine-1phosphate (C18 S1P) and 18-carbon dihydrosphingosine-1-phosphate (C18 DS1P). We also found 13 CER[NS] ceramides and four phosphorylated ceramides with long-chain non-hydroxy fatty acids (22-26 carbons) and sphingoid bases with 16-24 carbons. With the exception of phosphorylated ceramides that were expressed at similar levels, the epidermis showed greater expression of all these sphingolipids than the dermis. However, high inter-individual variability meant there was limited statistical significance, with only two ceramide species, CER[N(26)S(16)] and CER[N(24)S(20)] (P=0.01, P=0.005, respectively), two phosphorylated bases (C18S1P, C18DS1P, P=0.024 and P=0.018, respectively) and one sphingoid base (C18DS, P=0.003) being statistically significantly higher in epidermis (Fig.2B). Of the sphingolipid species found in blister fluid, the majority were in similar proportions to the dermis and epidermis (Fig.2B). However, some species were at noticeably low levels in the blister fluid, including C18S and the largest ceramides measured (CER[N(26)S(16)], CER[N(24)S(21)], CER[N(25)S(22)] and CER[N(24)S(24)]).

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Contribution of cutaneous fatty acids The fatty acid precursors of the eicosanoids and NAE reported here comprise 77.1 % and 61.9 % of fatty acids in dermis and epidermis, respectively (Fig.3D). Other, non-precursor, fatty acids make up the remainder (Supplementary Table S2). Despite similar proportions of LA in dermis and epidermis (10.7 % and 9.6 % of total fatty acids, respectively), the epidermis demonstrated higher levels of the LA-derived 9- and 13-HODE compared to dermis (69.7 % and 50.3 % of total HFA detected in the dermis and epidermis, respectively) and this was reflected in the composition of blister fluid (65.2 % of total HFA detected in blister fluid) (Fig.3B). The eicosanoid precursors AA and EPA contributed in all classes of mediators detected. AA was found at a higher concentration in epidermis than dermis (2.7 % versus 0.7 % of total, respectively) and this was directly reflected in the higher abundance of AEA in epidermis and blister fluid (13.1 % and 11.2 %, respectively) compared with dermis (8.3 %). In all cases AEA and EPEA levels were lower than DHEA (Fig.3C). Although cutaneous DHA was detected at very low levels in epidermis and dermis (0.2 % and 0.5 %, respectively) and was only a minor contributor of epidermal, but not dermal, HFA (Fig.3B), it was detected as DHEA in dermis, epidermis and blister fluid (11.7 % ,16.5 % and 14.6 %, respectively) (Fig.3C). The high levels of dermal OA (44.8 % of total fatty acids) were reflected in the higher abundance of OEA (18.3 % of total dermal NAE), while PA, which was the most abundant fatty acid in skin, contributed as PEA to almost 40% of the NAE detected in dermis, epidermis and blister fluid (Fig.3C).

Manipulation of lipid mediators by omega-3 PUFA supplementation ex vivo Exogenous provision of EPA did not have a statistically significant effect on the levels of COXderived PGE2 and LOX-derived 12-HETE in dermis or epidermis, although it induced the formation of PGE3 and 12-HEPE, two less inflammatory eicosanoids that can attenuate PGE211

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and 12-HETE mediated activities (Fig. 4A-D). Conversely, DHA appeared to inhibit PGE2 with concomitant stimulation of 12-HETE production, suggesting the diversion of cutaneous AA from COX to LOX-mediated metabolism. Both EPA and DHA stimulated the production of AEA, EPEA and DHEA in epidermis and dermis, indicating that their anti-inflammatory activities may be mediated, at least in part, via the endocannabinoid system (Fig.4E-F). Finally, the formation of 18-HEPE, 17-HDHA and 14-HDHA post-supplementation, shows that human skin can produce the biochemical precursors of the anti-inflammatory and protective lipids resolvins (RvE and RvD) and maresins (Fig.4C-D) (Serhan, 2014).

DISCUSSION Our findings demonstrate the ability of both dermis and epidermis to produce an array of bioactive lipids that may act locally or be transported and contribute to cross-talk between these skin compartments. It is noteworthy that despite differences in their physiology, dermis and epidermis produce the same mediators, although significant differences in the levels and ratios of certain species highlight their varying requirements. Higher production of AA, DGLA and EPA-derived epidermal prostanoids can be attributed to increased expression and/or activity of COX isoforms and prostanoid synthases, and agrees with a higher epidermal concentration of AA (Fig.3) as well as reports of higher expression of the constitutive COX-1 in the epidermis (Kragballe et al., 1986). The presence of deactivated prostanoids such as 15-keto-PGE2 and 13,14-dihydro-15-keto PGE2, shows that the levels of potent proinflammatory PG such as PGE2 are under active control, a step crucial for successful resolution of inflammation. Although the prostanoids found in blister fluid matched 12

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the epidermal profile (Fig.2A), some minor epidermal species were not detected suggesting that skin biopsy samples are more appropriate when investigating low abundance lipid mediators. Interestingly, PGJ2 was found in the epidermis, but not blister fluid, whilst its metabolite 15deoxy-∆12,14 PGJ2, was found at high levels in blister fluid but not in the epidermis or dermis. Since 15-deoxy-∆12,14 PGJ2 is believed to act as a PPARγ agonist involved in anti-inflammatory proresolving signalling, its presence in blister fluid could potentially be linked to a cutaneous healing response to the trauma involved in raising suction blisters (Surh et al, 2011). The epidermal expression of HFA was also higher, with LA- and AA-derived species being the predominant mediators detected (Fig.2A). High production of 12-HETE suggests increased expression/activity of 12-LOX and agrees with reports of three different isozymes highly expressed in the epidermis (Boeglin et al, 1998; Muller et al, 2002; Takahashi et al, 1993). 12-HETE has proinflammatory and neutrophil chemotactic properties indicating the potential involvement of 12-LOX in inflammatory skin disease (e.g. psoriasis) (Baer et al, 1995; Fogh et al, 1993). While the LA-derived 15-LOX product 13-HODE was found at higher levels in the epidermis, the AA-derived 15-HETE and DGLA-derived 15-HETrE were present at similar levels in both compartments, albeit at lower concentrations, in accordance with the much lower levels of their precursor fatty acids (Fig.3). 15-LOX products have anti-inflammatory activities and may have a specific role in the cross-talk of dermis and epidermis, as demonstrated by the regulation exhibited by dermal 15-HETE on epidermal 12-LOX activity (Kragballe et al., 1986; Yoo et al, 2008). Additionally, hair follicles, found in the dermis, are known to express high levels of a 15S-LOX (Brash et al, 1997) that preferentially metabolises AA over LA. Although the abdominal skin used in this study was not densely populated with hair, the presence of hair follicles in the dermis could possibly explain why 15-HETE was present at comparable 13

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levels in the dermis and epidermis but 13-HODE was not. Finally, the 5-LOX product 5-HETE was found at very low levels in both skin compartments, suggesting a low cutaneous expression of 5-LOX. This supports current understanding that 5-LOX-derived leukotrienes, found in inflammatory skin disease such as psoriasis, are derived from infiltrating immune cells rather than resident cells (Ford-Hutchinson, 1993; Sadik et al, 2014). The significantly higher epidermal levels of the endocannabinoid AEA, and the NAE species EPEA and DHEA, are of interest, indicating their potential roles in homeostatic processes in resting skin. AEA has been reported to regulate keratinocyte proliferation, sebum production and melanogenesis (Conconi et al., 1996; Dobrosi et al., 2008; Pucci et al., 2012). It is noteworthy that although all HFA derivatives of DHA contributed less than 2% of total class species, DHEA was one of the main NAE detected (11–16% of total; Fig.3C). DHEA is an endocannabinoid-like molecule exhibiting weak affinities for cannabinoid receptors (Kim and Spector,

2013).

EPEA

and

DHEA

have

anti-inflammatory

properties,

reducing

lipopolysaccharide (LPS)-induced interleukin (IL)-6 and monocyte chemotactic protein (MCP)-1 production in adipocytes, and LPS-induced ˙NO production in macrophages (Satoh et al., 2006; Uchida, 2014), and may operate similar anti-inflammatory effects in the skin. The saturated NAE species, PEA and OAE, derivatives of palmitic (PA) and oleic (OA) acid, respectively, were found at equally high level in both dermis and epidermis. PEA is known to suppress cutaneous mast cell activity, and may confer anti-inflammatory activity (Mizutani et al., 2009; Wang and Ueda, 2009). OEA is known to affect food intake and the sleep-wake cycle, but its role in skin is unknown (Honma et al., 2005). Supplementation with exogenous EPA and DHA showed that their cutaneous metabolism alters the profile of dermal and epidermal lipids, stimulating production of anti-inflammatory species including COX- and LOX-derived products, and NAE 14

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(Fig.4). This provides information on how skin can respond to nutritional interventions and suggests that n-3PUFA can also create a protective and pro-resolving environment, as supported by the identification of biochemical precursors for protectins and resolvins in the skin. Ceramides are components of the stratum corneum involved in epidermal barrier function, particularly the omega-esterified species with long acyl chains, with more than 300 species identified to date (Behne et al, 2000; Iwai et al., 2012; Rabionet et al., 2014; van Smeden et al., 2011). In this study, we focus our attention particularly on the family of non-hydroxylated medium-chain ceramides that can be found in cellular membranes, and notably, we demonstrate the presence of 21 species in epidermis as well as in dermis and blister fluid. This suggests their wider involvement in epidermal and dermal function, where they may mediate activities including regulation of apoptosis and inflammation (Maceyka and Spiegel, 2014; Stiban et al, 2008). Blister fluid showed lower expression of the longest ceramides measured, possibly indicating impaired release of these species during the blister formation. Further work is indicated to elucidate the prevalence of these non-hydroxylated species in epidermal and dermal cells, and explain the intriguing finding of their comparable abundance in both skin compartments. Phosphorylated ceramides were also present in dermis and epidermis at equally high concentrations, suggesting that they originate from cellular membranes and are not restricted to the differentiated epidermis. This is indicative of an active signalling role in both compartments that may include the ceramide-1-phosphate and sphingosine-1-phosphate mediated activation of cutaneous PLA2 (Chalfant and Spiegel, 2005). Ceramide-1-phosphate has also been recently reported to be involved in wound healing through acting in concert with eicosanoid production (Wijesinghe et al, 2014), and sphingosine-1-phosphate is a modulator of cutaneous immunity 15

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(Herzinger et al, 2007). In this study we focused on the more common 18-carbon sphingosine and dihydrosphingosine bases, and found that the free sphingoid base C18DS, known to promote keratinocyte differentiation (Paragh et al, 2008), demonstrated increased expression in the epidermis (Fig.2B). Future investigations could include analysis of the less abundant sphingosines and dihydrosphingosines with other carbon chain-lengths, as well as the phytosphingosine and 6-hydroxy sphingosine bases. A limitation of the present study is the method used to separate dermis from epidermis, i.e. physical separation. The commonly-used methods for their separation along the dermoepidermal junction rely on long incubation with salts or enzymes, or rapid transition from high to low temperatures, to degrade the basement membrane, and are inappropriate for this study’s purposes as they can induce oxidation or thermal degradation of the lipid mediators (Oakford et al, 2011; Zhang et al, 2003). Although physical separation of the dermis and epidermis resulted in slight dermal contamination of the epidermis, care was taken that the less cellular dermis would remain completely free of any epidermal component. Following analysis, it was found that differences in lipid expression between the two compartments were reliably identified. Overall, this study shows that dermis produces a wide range of lipid mediators and this production may be important biochemical support for the epidermis, acting as an intermediary between the epidermal interface with the external environment and the subcutaneous circulatory and lymphatic systems. To our knowledge, this is the most complete analysis of bioactive lipid mediators in human skin to be reported. Profiles of lipid mediators can support the characterisation of COX, LOX and CYP isoforms, whose activity and pattern of expression depends on post translational modifications and may contribute to cutaneous disorders (Aguiar et al, 2005; Kuhn and O'Donnell, 2006; Mbonye and Song, 2009). We have also shown an ability 16

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to manipulate the balance of bioactive lipids in favour of the protective omega-3-derived species, with implications for their mode of action in skin health and disease. Finally, direct comparison of lipid mediators found in suction blister fluid, a widely used technique for sampling cutaneous intercellular fluid, with skin lipid profiles, provides confirmatory information on study design to explore the potency and importance of lipid mediators in skin biology.

MATERIALS AND METHODS Tissue Samples Biobank skin: Skin samples were obtained from a biobank (Ethical Tissue, University of Bradford, Bradford, UK) with full ethical approval (Leeds East Research Ethics Committee reference 07/H1306/98+5). Skin was provided by 8 healthy donors (6 female, 2 male; 30-60 years; white Caucasian), undergoing elective abdominoplastic surgery, delivered to the biobank within 1 hour of the operation. The tissue was washed and the adipose layer removed. Punch biopsies (3 mm) were cut, snap frozen and stored at -80ºC, or were cultured ex vivo. Ex vivo culture was performed in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin and 1.4 mM Ca2+(Promocell, Heidelberg, Germany), with or without the addition of EPA or DHA (50 µM) (Sigma Aldrich, Poole, UK), for 3 days (Tavakkol et al, 1999). For lipidomics assays three punch biopsies per donor were used. Prior to analysis, skin was divided into dermis and epidermis (on ice, by scalpel, with the aid of visual inspection at 40X magnification). Whilst epidermal samples demonstrated minor contamination with dermal tissue, care was taken that dermal tissue was not contaminated with epidermal tissue.

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Skin suction blister fluid and punch biopsies: Healthy human volunteers (n=8; all females; 28-56 years; white Caucasian) were recruited by the Photobiology Unit, Dermatology Centre, Salford Royal Hospital, Manchester, UK. Ethical approval was obtained from the North Manchester Research Ethics Committee (reference 08/H1006/79). Written informed consent was obtained from participants and the study adhered to Declaration of Helsinki principles. Volunteers provided 5mm skin punch biopsies and suction blisters from buttock skin for these analyses. Punch biopsy dermis and epidermis were separated as described above, and samples used for total fatty acid analysis. Suction blistering was performed using suction cups with a 1 cm central aperture as described previously (Rhodes et al., 2009). Skin blister fluid was aspirated with a 23gauge needle, snap-frozen in liquid nitrogen and stored at -80ºC.

Eicosanoid extraction and analysis Skin samples (30-60 mg) and blister fluid samples (40-90 µl) were extracted using ice-cold 15% (v/v) methanol solution. 12-HETE-d8 and PGB2-d4 (Cayman Chemicals, Ann Arbor, MI, USA) were used as internal standards, as published before (Masoodi et al, 2008; Masoodi and Nicolaou, 2006; Massey and Nicolaou, 2013).+The extracts were semi-purified by SPE cartridges (C18-E; Phenomenex, Macclesfield, UK); the assay recovery was estimated at 9698%. LC/ESI-MS/MS was performed on an HPLC pump (Waters Alliance 2695) coupled to an electrospray ionisation triple quadrupole mass spectrometer (Quattro Ultima, Waters, Elstree, Hertfordshire, UK). Results are expressed as pg/mg protein (skin) or pg/ml (blister fluid), estimated using calibration lines covering the range 0.02-20 ng/ml. Detailed description of the experimental protocol is provided as Supplementary Information (SM1).

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Endocannabinoid and NAE extraction and analysis Skin samples (30-60 mg) and blister fluid samples (40-90 µl) were extracted using ice-cold 2:1 (v/v) chloroform/methanol. Arachidonoyl EA-d8 and 2-arachidonoyl glycerol-d8 (Cayman Chemicals) were used as internal standards. Lipid extracts were analysed by LC/ESI-MS/MS. Recovery was estimated at 91%. Results are expressed as pg/mg protein (skin) or pg/ml (blister fluid), estimated using calibration lines covering the range 0.02-50 ng/ml. Detailed description of the experimental protocol is provided as Supplementary Information (SM2).

Sphingolipid extraction and analysis Skin samples (30-60 mg) and blister fluid samples (40-90 µl) were extracted using ice-cold isopropanol:water:ethyl acetate (30:10:60; v/v/v). The following internal standards were used: C17 S (used for C18 S), C17 DS (used for C18 DS), C17 S1P (used for C18 S1P), C17 DS1P (used for C18 DS1P), d18:1/12:0 C1P (used for all C1P species) and C25 Cer (used for all CER[NS] species) (Ceramide/Sphingoid Internal Standard Mixture I, Avanti Polar Lipids, Alabaster, Alabama, USA). The resulting lipid extracts were analysed by LC/ESI-MS/MS (Bielawski et al, 2006; Kelly et al, 2011). The assay recovery was estimated at 75 -79%. Results are expressed as pmol/g protein (skin) or pmol/ l (blister fluid), relative to the appropriate internal standard (covering the range 0.005-8.0 nmol/ml). Detailed description of the experimental protocol is provided as Supplementary Information (SM3).

Fatty acid analysis Fatty acids were analysed by gas chromatography (GC) in dermis and epidermis as previously described (Pilkington et al, 2014). Results are expressed as percentage of total fatty acids. 19

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Detailed description of the experimental protocol is provided as Supplementary Information (SM4).

Protein content During lipid extractions, protein pellets were retained for analysis of protein content using a standard Bradford protein assay kit (Bio-Rad, Hemel Hempstead, UK) (Bradford, 1976). Proteins were extracted using 1 M NaOH and analysed within the linear range of the assay to ensure accuracy.

Statistical analysis Statistical analyses of lipid mediator expression were performed using repeated measures ANOVAs with Greenhouse-Geisser corrections and Bonferroni post-hoc tests. Analyses were conducted using SPSS 20 software and P<0.05 was considered significant.

CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGEMENTS This work was funded by Unilever as part of its ongoing program developing novel non-animal approaches for assessing consumer safety. We thank Andrew Healey, Analytical Centre, University of Bradford and Wayne Burrill, Ethical Tissue, University of Bradford, for excellent technical support. 20

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Kupczyk P, Reich A, Szepietowski JC (2009). Cannabinoid system in the skin – a possible target for future therapies in dermatology. Exp Dermatol 18: 669-79. Laneuville O, Breuer DK, Xu N, et al (1995). Fatty acid substrate specificities of human prostaglandin-endoperoxide H synthase-1 and -2. Formation of 12-hydroxy-(9Z, 13E/Z, 15Z)octadecatrienoic acids from alpha-linolenic acid. J Biol Chem 270: 19330-6. Maceyka M, Spiegel S (2014). Sphingolipid metabolites in inflammatory disease. Nature 510: 58-67. Masoodi M, Mir AA, Petasis NA, et al (2008). Simultaneous lipidomic analysis of three families of bioactive lipid mediators leukotrienes, resolvins, protectins and related hydroxy-fatty acids by liquid chromatography/electrospray ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom 22: 75-83. Masoodi M, Nicolaou A (2006). Lipidomic analysis of twenty-seven prostanoids and isoprostanes by liquid chromatography/electrospray tandem mass spectrometry. Rapid Commun Mass Spectrom 20: 3023-9. Massey KA, Nicolaou A (2011). Lipidomics of polyunsaturated-fatty-acid-derived oxygenated metabolites. Biochem Soc Trans 39: 1240-6. Massey KA, Nicolaou A (2013). Lipidomics of oxidized polyunsaturated fatty acids. Free Radic Biol Med 59: 45-55. Masukawa Y, Narita H, Shimizu E, et al (2008). Characterization of overall ceramide species in human stratum corneum. J Lipid Res 49: 1466-76. Mbonye UR, Song I (2009). Posttranscriptional and posttranslational determinants of cyclooxygenase expression. BMB Rep 42: 552-60. McPartland JM (2008). Expression of the endocannabinoid system in fibroblasts and myofascial tissues. J Bodyw Mov Ther 12: 169-82. Mizutani Y, Mitsutake S, Tsuji K, et al (2009). Ceramide biosynthesis in keratinocyte and its role in skin function. Biochimie 91: 784-90. Muller K, Siebert M, Heidt M, et al (2002). Modulation of epidermal tumor development caused by targeted overexpression of epidermis-type 12S-lipoxygenase. Cancer Res 62: 4610-6. Nicolaou A (2013). Eicosanoids in skin inflammation. Prostaglandins Leukot Essent Fatty Acids 88: 131-8. Oakford ME, Dixon SV, August S, et al (2011). Migration of immunocytes across the basement membrane in skin: The role of basement membrane pores. J Invest Dermatol 131: 1950-3. 23

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Paragh G, Schling P, Ugocsai P, et al (2008). Novel sphingolipid derivatives promote keratinocyte differentiation. Exp Dermatol 17: 1004-16. Petrosino S, Cristino L, Karsak M, et al (2010). Protective role of palmitoylethanolamide in contact allergic dermatitis. Allergy 65: 698-711. Pilkington SM, Rhodes LE, Al-Aasswad NM, et al (2014). Impact of EPA ingestion on COXand LOX-mediated eicosanoid synthesis in skin with and without a pro-inflammatory UVR challenge report of a randomised controlled study in humans. Mol Nutr Food Res 58: 580-90. Pucci M, Pasquariello N, Battista N, et al (2012). Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor. J Biol Chem 287: 15466-78. Rabionet M, Gorgas K, Sandhoff R (2014). Ceramide synthesis in the epidermis. Biochim Biophys Acta 1841: 422-34. Rhodes LE, Gledhill K, Masoodi M, et al (2009). The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases. FASEB J 23: 3947-56. Sadik CD, Sezin T, Kim ND (2014). Leukotrienes orchestrating allergic skin inflammation. Exp Dermatol 22: 705-9. Satoh T, Moroi R, Aritake K, et al (2006). Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor. J Immunol 177: 2621-9. Scott G, Leopardi S, Printup S, et al (2004). Proteinase-activated receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2 on melanocyte dendricity. J Invest Dermatol 122: 1214-24. Serhan CN (2014). Pro-resolving lipid mediators are leads for resolution physiology. Nature 510: 92-101. Stander S, Schmelz M, Metze D, et al (2005). Distribution of cannabinoid receptor 1 (CB1) and 2 (CB2) on sensory nerve fibers and adnexal structures in human skin. J Dermatol Sci 38: 17788. Stiban J, Caputo L, Colombini M (2008). Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins. J Lipid Res 49: 625-34. Sugawara K, Biro T, Tsuruta D, et al (2012). Endocannabinoids limit excessive mast cell maturation and activation in human skin. J Allergy Clin Immunol 129: 726-38 e8.

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Surh YJ, Na HK, Park JM, et al (2011). 15-Deoxy-Delta(1)(2),(1)(4)-prostaglandin J(2), an electrophilic lipid mediator of anti-inflammatory and pro-resolving signaling. Biochem Pharmacol 82: 1335-51. t'Kindt R, Jorge L, Dumont E, et al (2012). Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry. Anal Chem 84: 403-11. Tai HH, Ensor CM, Tong M, et al (2002). Prostaglandin catabolizing enzymes. Prostaglandins Other Lipid Mediat 68-69: 483-93. Takahashi Y, Reddy GR, Ueda N, et al (1993). Arachidonate 12-lipoxygenase of platelet-type in human epidermal cells. J Biol Chem 268: 16443-8. Tavakkol A, Varani J, Elder JT, et al (1999). Maintenance of human skin in organ culture: role for insulin-like growth factor-1 receptor and epidermal growth factor receptor. Arch Dermatol Res 291: 643-51. Toth BI, Dobrosi N, Dajnoki A, et al (2011). Endocannabinoids modulate human epidermal keratinocyte proliferation and survival via the sequential engagement of cannabinoid receptor-1 and transient receptor potential vanilloid-1. J Invest Dermatol 131: 1095-104. Uchida Y (2014). Ceramide signaling in mammalian epidermis. Biochim Biophys Acta 1841: 453-62. van Smeden J, Hoppel L, van der Heijden R, et al (2011). LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery. J Lipid Res 52: 1211-21. Wang J, Ueda N (2009). Biology of endocannabinoid synthesis system. Prostaglandins Other Lipid Mediat 89: 112-9. Wijesinghe DS, Brentnall M, Mietla JA, et al (2014). Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts. J Lipid Res 55: 1298309. Yamaguchi Y, Hearing VJ, Itami S, et al (2005). Mesenchymal-epithelial interactions in the skin: aiming for site-specific tissue regeneration. J Dermatol Sci 40: 1-9. Yoo H, Jeon B, Jeon MS, et al (2008). Reciprocal regulation of 12- and 15-lipoxygenases by UV-irradiation in human keratinocytes. FEBS Lett 582: 3249-53. Yu C, Fedoric B, Anderson PH, et al (2011). Vitamin D(3) signalling to mast cells: A new regulatory axis. The international journal of biochemistry & cell biology 43: 41-6. Zhang X-J, Chinkes DL, Wolfe RR (2003). Measurement of protein metabolism in epidermis and dermis. Am J Physiol Endocrinol Metab 284: E1191-E201. 25

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Ziboh VA, Miller CC, Cho Y (2000). Metabolism of polyunsaturated fatty acids by skin epidermal enzymes: generation of antiinflammatory and antiproliferative metabolites. Am J Clin Nutr 71: 361s-6s.

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Figure legends Figure 1. Schematic outline of bioactive lipid mediator production and related signalling events. AA, arachidonic acid; AEA, arachidonoyl ethanolamide; AP-1, activator protein 1; BLT, leukotriene B4 receptor; CB, cannabinoid receptor; COX, cyclooxygenase; C1P, ceramide-1phosphate; DHA, docosahexaenoic acid; DP, prostaglandin D2 receptor; EP, prostaglandin E2 receptor; EPA, eicosapentaenoic acid; FA, fatty acid; FA-EA, fatty acid ethanolamide; G2A, G protein-coupled

receptor

132;

GPR55,

G

protein-coupled

receptor

55;

HDHA,

hydroxydocosahexaenoic acid; HETE, hydroxyeicosatetraenoic acid; IP, prostacyclin receptor; LOX, lipoxygenase; LT, leukotriene; LX, lipoxin; NF-κB, nuclear factor of kappa-light-chainenhancer in B-cells; PD, protectin; PEA, palmitoyl ethanolamide; PG, prostaglandin; PLA2, phospholipase A2; PLD, phospholipase D; PPAR, peroxisome proliferator-activated receptor; PUFA, polyunsaturated fatty acid; Rv, resolvin; S1P, sphingosine-1-phosphate; S1P1, sphingosine-1-phosphate receptor 1; TP, thromboxane receptor; TRPV, transient receptor potential vanilloid; TX, thromboxane.

Figure 2. Expression of eicosanoids, endocannabinoids and N-acyl ethanolamides (a), and ceramides, phosphorylated ceramides and sphingoid bases (b), in human dermis, epidermis and blister fluid. All lipid mediators were analysed by LC-MS/MS. The arachidonic acidderived PGE2, 12-HETE, AEA and 2-AG, and C18 S and its derivatives C18 S1P, CER[N(22)S(18)] and d18:1/16:0 C1P are provided as example structures of the different classes of lipid mediators presented in this figure. Data for prostaglandins (PG), prostacyclin (PGI2) (measured as the stable derivative 5-keto PGF1 ), thromboxanes (TX), hydroxy fatty acids, endocannabinoids and N-acyl ethanolamides (NAE) are expressed as pg/mg tissue protein or 27

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pg/ml blister fluid. Data for ceramides, phosphorylated ceramides and sphingoid bases are expressed as pmol/g protein or pmol/ l blister fluid (dermis and epidermis; n=8 donors; labelled 1-8) and pg/ml blister fluid (n=3 donors, labelled 9-11). *P<0.05, **P<0.01 and ***P<0.001 when comparing dermis to epidermis. C18S: 18-carbon sphingosine (S); C18DS: 18-carbon dihydrosphingosine (DS); C18S1P: 18carbon

sphingosine-1-phosphate;

C18DS1P:

18-carbon

dihydrosphingosine-1-phosphate;

Ceramides derivatives of sphingosine (S) with a non-hydroxy fatty acid (N) are named according to the number of carbons of the base (e.g. 16, 18 etc) and fatty acid (e.g. 22, 24, etc). Phosphorylated ceramides are denoted by the base (d18:1 representing sphingosine and d18:0 representing dihydrosphingosine) and the fatty acid (e.g. 16:0 represents palmitic acid).

Figure 3. Contribution of cutaneous fatty acid precursors of lipid mediator populations found in dermis, epidermis and suction blister fluid. Prostanoids (a), hydroxy fatty acids (b) and N-acyl ethanolamides (c) (as quantified in Figure 2) are shown as % of total mediators detected in each tissue, together with the % abundance of their precursor fatty acid (d) in dermis and epidermis. Data are expressed as a % of total lipid mediators detected in the dermis and epidermis (n=8 donors), % of total lipid mediators detected in the blister fluid (n=3 donors) or as % of total fatty acids detected in dermis and epidermis (n=5 donors). AA, arachidonic acid; ALA, α-linolenic acid; DGLA, dihomo-gamma-linolenic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; LA, linoleic acid; OA, oleic acid; PA, palmitic acid; SA, stearic acid.

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Figure 4. Effect of omega-3 PUFA supplementation on the production of cutaneous lipid mediators ex vivo. Skin was treated for 3 days with EPA or DHA (50 M). PGE2 and PGE3 extracted from dermis (a) and epidermis (b), 12-HETE, 12-HEPE, 18-HEPE, 17-HDHA and 14HDHA extracted from dermis (c) and epidermis (d), AEA, EPEA and DHEA extracted from dermis (e) and epidermis (f), and analysed by LC-MS/MS. Data are expressed as pg/mg tissue protein (n=3 biopsies). *P<0.05, **P<0.01 and ***P<0.001 when comparing all data to control. PG, prostaglandin; HETE, hydroxyeicosatetraenoic acid; HEPE, hydroxyeicosapentaenoic acid; HDHA, hydroxydocosahexaenoic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; AEA, N-arachidonoyl ethanolamide; EPEA, N-eicosapentaenoyl ethanolamide; DHEA, Ndocosahexaenoyl ethanolamide.

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SUPPLEMENTARY MATERIAL

Supplementary Methods: SM1, SM2, SM3, SM4. Supplementary Table S1. Supplementary Table S2.

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© 2015 The Society

© 2015 The Society

© 2015 The Society