Thematic Review Series: Skin Lipids. Antimicrobial lipids at the skin surface

The skin surface represents our interface with the external environment, and as such, is our first line of defense against microbial colonization and infection. Lipids at the skin surface are thought to underlie at least part of an antimicrobial barrier. Some of these lipids are synthesized in the epidermis and are carried to the surface as cells differentiate, whereas others are secreted onto the surface from the sebaceous glands. One such group, free sphingoid bases, are known to have broad antimicrobial activity, and our previous studies demonstrate their presence at the skin surface. Free sphingoid bases may be generated by enzymatic hydrolysis of epidermal ceramides. In addition, our preliminary results demonstrate potent antibacterial activity associated with two specific fatty acids derived from sebaceous triglycerides. Most remarkably, one of these fatty acids (sapienic acid, C16:1Δ6), in combination with a low concentration of ethanol, is very effective against methicillin-resistant Staphylococcus aureus (MRSA). In fact, this combination was far more effective than mupirocin with or without ethanol. Mupirocin is a “gold standard” for activity against MRSA. The skin surface represents our interface with the external environment, and as such, is our first line of defense against microbial colonization and infection. Lipids at the skin surface are thought to underlie at least part of an antimicrobial barrier. Some of these lipids are synthesized in the epidermis and are carried to the surface as cells differentiate, whereas others are secreted onto the surface from the sebaceous glands. One such group, free sphingoid bases, are known to have broad antimicrobial activity, and our previous studies demonstrate their presence at the skin surface. Free sphingoid bases may be generated by enzymatic hydrolysis of epidermal ceramides. In addition, our preliminary results demonstrate potent antibacterial activity associated with two specific fatty acids derived from sebaceous triglycerides. Most remarkably, one of these fatty acids (sapienic acid, C16:1Δ6), in combination with a low concentration of ethanol, is very effective against methicillin-resistant Staphylococcus aureus (MRSA). In fact, this combination was far more effective than mupirocin with or without ethanol. Mupirocin is a “gold standard” for activity against MRSA. INNATE IMMUNE MECHANISMSThe innate immune system in mammals and other vertebrates plays a number of crucial roles (1Germain R.N. The art of the probable: system control in the adaptive immune system.Science. 2001; 293: 240-245Crossref PubMed Scopus (160) Google Scholar, 2Medzhitov R. Janeway Jr., C. 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Biology and clinical relevance of naturally occurring antimicrobial peptides.J. Allergy Clin. Immunol. 2002; 110: 823-831Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 30Burtenshaw J.M. The mechanisms of self disinfection of the human skin and its appendages.J. Hyg. (Lond.). 1942; 42: 184-209Crossref PubMed Scopus (53) Google Scholar, 31Gell G. Drake D. Wertz P.W. Antimicrobial effects of epithelial lipids.J. Dent. Res. 1993; 72: 399Google Scholar, 32Gell G. Drake D.R. Wertz P.W. Peridex and lauric acid exhibit a synergistic effect on Streptococcus mutans..J. Dent. Res. 1995; 74: 76Google Scholar, 33Wertz P.W. Downing D.T. Free sphingosine in porcine epidermis.Biochim. Biophys. Acta. 1989; 1002: 213-217Crossref PubMed Scopus (43) Google Scholar). The purpose of this review is to summarize what is known about antimicrobial lipids found at the skin surface. Some points will be illustrated with our own preliminary results.In recent years, much attention has focused on the epithelial production of antimicrobial cationic peptides as part of an innate immune system, an ancient first line of defense in all surface epithelia (29Gallo R.L. Murakami M. Ohtake T. Zaiou M. Biology and clinical relevance of naturally occurring antimicrobial peptides.J. Allergy Clin. Immunol. 2002; 110: 823-831Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar).Burtenshaw showed in the 1940s that lipid extracts from the skin surface have the ability to kill Staphylococcus aureus in vitro, and it was thought that free fatty acids were the active agent (30Burtenshaw J.M. The mechanisms of self disinfection of the human skin and its appendages.J. Hyg. (Lond.). 1942; 42: 184-209Crossref PubMed Scopus (53) Google Scholar); however, this proposition was not extensively tested with fatty acids actually found at the human skin surface until recently (31Gell G. Drake D. Wertz P.W. Antimicrobial effects of epithelial lipids.J. Dent. Res. 1993; 72: 399Google Scholar, 32Gell G. Drake D.R. Wertz P.W. Peridex and lauric acid exhibit a synergistic effect on Streptococcus mutans..J. Dent. Res. 1995; 74: 76Google Scholar). In addition to a potential role for antimicrobial fatty acids, studies have demonstrated significant levels of free sphingosines, dihydrosphingosines, and 6-hydroxysphingosines in the stratum corneum (33Wertz P.W. Downing D.T. Free sphingosine in porcine epidermis.Biochim. Biophys. Acta. 1989; 1002: 213-217Crossref PubMed Scopus (43) Google Scholar, 34Stewart M.E. Downing D.T. Free sphingosines of human skin include 6-hydroxysphingosine and unusually long-chain dihydrosphingosines.J. Invest. Dermatol. 1995; 105: 613-618Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Several studies have demonstrated that these long-chain bases are potent antimicrobials (35Bibel D.J. Miller S.J. Brown B.E. Pandey B.B. Elias P.M. Shinefield H.R. Aly R. Antimicrobial activity of lipids from normal and essential fatty acid-deficient mice.J. Invest. Dermatol. 1989; 92: 632-638Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 36Bibel D.J. Aly R. Shinefield H.R. Antimicrobial activity of sphingosines.J. Invest. Dermatol. 1992; 98: 269-273Abstract Full Text PDF PubMed Scopus (142) Google Scholar, 37Bibel D.J. Aly R. Shah S. Shinefield H.R. Sphingosines: antimicrobial barriers of the skin.Acta Derm. Venereol. 1993; 73: 407-411PubMed Google Scholar, 38Bibel D.J. Aly R. Shinefield H.R. Topical sphingolipids in antisepsis and antifungal therapy.Clin. Exp. Dermatol. 1995; 20: 395-400Crossref PubMed Scopus (49) Google Scholar, 39Payne C.D. Ray T.L. Downing D.T. Cholesterol sulfate protects Candida albicans from inhibition by sphingosine in vitro..J. Invest. Dermatol. 1996; 106: 549-552Abstract Full Text PDF PubMed Scopus (16) Google Scholar).The chemical structures of the antimicrobial lipids known to present at the human skin surface are given in Fig. 1 .COMPOSITION OF LIPIDS AT THE SKIN SURFACEIn epidermis, lipids accumulate with increasing cellular differentiation (40Gray G.M. Yardley H.J. Different populations of pig epidermal cells: isolation and lipid composition.J. Lipid Res. 1975; 16: 441-447Abstract Full Text PDF PubMed Google Scholar, 41Squier C.A. Wertz P.W. Cox P. Thin-layer chromatographic analyses of lipids in different layers of porcine epidermis and oral epithelium.Arch. Oral Biol. 1991; 36: 647-653Crossref PubMed Scopus (31) Google Scholar). Much of the accumulating lipid is packaged in the form of small membranous organelles known as lamellar granules (42Landmann L. The epidermal permeability barrier.Anat. Embryol. (Berl.). 1988; 178: 1-13Crossref PubMed Scopus (178) Google Scholar, 43Madison K.C. Sando G.N. Howard E.J. True C.A. Gilbert D.C. Swartzendruber D.C. Wertz P.W. Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the Golgi.J. Investig. Dermatol. Symp. Proc. 1998; 3: 80-86Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 44Freinkel R.K. Traczyk T.N. Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis.J. Invest. Dermatol. 1985; 85: 295-298Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 45Grayson S. Johnson-Winegar A.G. Wintraub B.U. Isseroff R.R. Epstein E.H. Elias P.M. Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization.J. Invest. Dermatol. 1985; 85: 289-294Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 46Wertz P.W. Downing D.T. Freinkel R.K. Traczyk T.N. Sphingolipids of the stratum corneum and lamellar granules of fetal rat epidermis.J. Invest. Dermatol. 1984; 83: 193-195Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The lamellar granules are round to ovoid in shape and consist of a unit-bounding membrane surrounding one or several stacks of lamellar disks. Isolated lamellar granules contain abundant phospholipids, cholesterol, and glycolipids (43Madison K.C. Sando G.N. Howard E.J. True C.A. Gilbert D.C. Swartzendruber D.C. Wertz P.W. Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the Golgi.J. Investig. Dermatol. Symp. Proc. 1998; 3: 80-86Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 44Freinkel R.K. Traczyk T.N. Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis.J. Invest. Dermatol. 1985; 85: 295-298Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 45Grayson S. Johnson-Winegar A.G. Wintraub B.U. Isseroff R.R. Epstein E.H. Elias P.M. Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization.J. Invest. Dermatol. 1985; 85: 289-294Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 46Wertz P.W. Downing D.T. Freinkel R.K. Traczyk T.N. Sphingolipids of the stratum corneum and lamellar granules of fetal rat epidermis.J. Invest. Dermatol. 1984; 83: 193-195Abstract Full Text PDF PubMed Scopus (127) Google Scholar). A number of hydrolytic enzymes are also associated with the granules (44Freinkel R.K. Traczyk T.N. Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis.J. Invest. Dermatol. 1985; 85: 295-298Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 45Grayson S. Johnson-Winegar A.G. Wintraub B.U. Isseroff R.R. Epstein E.H. Elias P.M. Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization.J. Invest. Dermatol. 1985; 85: 289-294Abstract Full Text PDF PubMed Scopus (167) Google Scholar). Near the boundary between the stratum corneum and the granular layer, the contents of the lamellar granules are exocytosed into the intercellular spaces. The hydrolytic enzymes act on the phospholipids to produce ceramide from sphingomyelin and glucosylceramides and a mixture of saturated fatty acids and monounsaturated cholesterol esters from the phosphoglycerides. Ceramides, cholesterol, and free fatty acids are the principal lipids of the stratum corneum (40Gray G.M. Yardley H.J. Different populations of pig epidermal cells: isolation and lipid composition.J. Lipid Res. 1975; 16: 441-447Abstract Full Text PDF PubMed Google Scholar, 41Squier C.A. Wertz P.W. Cox P. Thin-layer chromatographic analyses of lipids in different layers of porcine epidermis and oral epithelium.Arch. Oral Biol. 1991; 36: 647-653Crossref PubMed Scopus (31) Google Scholar, 47Law S. Wertz P.W. Swartzendruber D.C. Squier C.A. Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy.Arch. Oral Biol. 1995; 40: 1085-1091Crossref PubMed Scopus (144) Google Scholar, 48Wertz P.W. The nature of the epidermal barrier: biochemical aspects.Adv. Drug Deliv. Rev. 1996; 18: 283-294Crossref Scopus (82) Google Scholar).When the contents of the lamellar granules are extruded into the intercellular spaces, the keratin filaments inside the cell condense under the influence of the histidine-rich protein filaggrin, and the cells become extremely flattened. All of the internal organelles are degraded. Simultaneously, a thick band of protein is deposited at the periphery of the corneocyte (48Wertz P.W. The nature of the epidermal barrier: biochemical aspects.Adv. Drug Deliv. Rev. 1996; 18: 283-294Crossref Scopus (82) Google Scholar). This peripheral protein becomes a polymerized cross-linked protein layer through the formation of both disulfide linkages and isopeptide linkages and is known as the cornified envelope. In epidermis, ω-hydroxyceramide molecules derived from the bounding membrane of the lamellar granule become covalently attached to the outer surface of the cornified envelope (50Wertz P.W. Downing D.T. Covalently bound omega-hydroxyceramide in the stratum corneum.Biochim. Biophys. Acta. 1987; 917: 108-111Crossref PubMed Scopus (163) Google Scholar, 51Wertz P.W. Madison K.C. Downing D.T. Covalently bound lipids of human stratum corneum.J. Invest. Dermatol. 1989; 91: 109-111Crossref Scopus (179) Google Scholar, 52Swartzendruber D.C. Wertz P.W. Kitko D.J. Madison K.C. Downing D.T. Molecular models of the intercellular lipid lamellae in mammalian stratum corneum.J. Invest. Dermatol. 1989; 92: 251-257Abstract Full Text PDF PubMed Scopus (297) Google Scholar). This covalently bound lipid consists mainly of 30- through 34-carbon ω-hydroxyacids amide-linked to sphingosine bases. Owing to the unusual length of the ω-hydroxyceramide molecule, this lipid layer has the dimensions of a typical bilayer (50Wertz P.W. Downing D.T. Covalently bound omega-hydroxyceramide in the stratum corneum.Biochim. Biophys. Acta. 1987; 917: 108-111Crossref PubMed Scopus (163) Google Scholar, 52Swartzendruber D.C. Wertz P.W. Kitko D.J. Madison K.C. Downing D.T. Molecular models of the intercellular lipid lamellae in mammalian stratum corneum.J. Invest. Dermatol. 1989; 92: 251-257Abstract Full Text PDF PubMed Scopus (297) Google Scholar). Thus, epidermal stratum corneum consists of an array of extremely flat, keratin-filled cells bounded by a cornified envelope and embedded in a lipid matrix. This structure provides a permeability barrier that prevents desiccation.The fatty acids in the stratum corneum are saturated, straight-chained, and mostly 20–28 carbons in length. The ceramides of the stratum corneum are structurally diverse and include all combinations of normal and α-hydroxyacids amide-linked to sphingosines and dihydrosphingosines, phytosphingosines and 6-hydroxysphingosines (53Ponec M. Weerheim A. Lankhorst P. Wertz P.W. New acylceramide in native and reconstructed epidermis.J. Invest. Dermatol. 2003; 120: 581-588Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In addition, there are acylceramides with 30- through 34-carbon-long ω-hydroxyacids amide-linked to long-chain bases and bearing linoleic acid ester-linked to the ω-hydroxyl group. These acylceramides are derived from analogous acylglucosylceramides associated with the lamellar granules and are thought to be of major importance for the physical organization of lipids in the stratum corneum. In addition, the aforementioned ω-hydroxyceramides on the cornified envelope are derived from acylglucosylceramide precursors. A shorthand nomenclature system has been introduced (54Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghidoni R. Ceramide composition of the psoriatic scale.Biochim. Biophys. Acta. 1993; 1182: 147-151Crossref PubMed Scopus (375) Google Scholar) in which N, A, or O indicates amide-linked normal fatty acid, amide-linked α-hydroxyacid, and amide-linked ω-hydroxyacid, respectively. S, P, and H indicate the presence of sphingosine, phytosphingosine, and 6-hydroxysphingosine, respectively. Sphingosine and dihydrosphingosine usually occur together. The presence of ester-linked fatty acid is indicated with the prefix E. Thus, for example, the acylceramide containing sphingosine bases would be represented CER EOS.In addition to the major structural lipids, the stratum corneum contains free sphingosine bases. Concentrations of about 5 mg total long-chain base per gram of dry stratum corneum have been reported (55Law S. Squier C.A. Wertz P.W. Free sphingosines in oral epithelium.Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1995; 110: 511-513Crossref PubMed Scopus (13) Google Scholar). The free long-chain bases found here mainly range from 16 through 20 carbons in length and include sphingosines, dihydrosphingosines, and 6-hydroxysphingosines (33Wertz P.W. Downing D.T. Free sphingosine in porcine epidermis.Biochim. Biophys. Acta. 1989; 1002: 213-217Crossref PubMed Scopus (43) Google Scholar, 34Stewart M.E. Downing D.T. Free sphingosines of human skin include 6-hydroxysphingosine and unusually long-chain dihydrosphingosines.J. Invest. Dermatol. 1995; 105: 613-618Abstract Full Text PDF PubMed Scopus (38) Google Scholar). There are also some unusually long chain dihydrosphingosines (34Stewart M.E. Downing D.T. Free sphingosines of human skin include 6-hydroxysphingosine and unusually long-chain dihydrosphingosines.J. Invest. Dermatol. 1995; 105: 613-618Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Free sphingoid bases in the stratum corneum are produced through the action of ceramidases on ceramides. Ceramidases are found in both the viable portion of the epidermis and in the stratum corneum (56Wertz P.W. Downing D.T. Ceramidase activity in porcine epidermis.FEBS Lett. 1990; 268: 110-112Crossref PubMed Scopus (50) Google Scholar). In addition, a sphingosine gradient exists, with higher sphingosine concentrations in the stratum corneum (55Law S. Squier C.A. Wertz P.W. Free sphingosines in oral epithelium.Comp. Biochem. Physiol. B Biochem. Mol. 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