ORIGINAL ARTICLE

Quantitative Proteomics of the Human Skin Secretome Reveal a Reduction in Immune Defense Mediators in Ectodermal Dysplasia Patients Marc Burian1, Ana Velic2,KatarinaMatic2, Stephanie Gu¨nther1, Beatrice Kraft1, Lena Gonser1, Stephan Forchhammer1, Yvonne Tiffert3, Christian Naumer3, Michael Krohn3, Mark Berneburg4, Amir S. Yazdi1, Boris Macˇek2 and Birgit Schittek1

In healthy human skin host defense molecules such as antimicrobial peptides (AMPs) contribute to skin immune homeostasis. In patients with the congenital disease ectodermal dysplasia (ED) skin integrity is disturbed and as a result patients have recurrent skin infections. The disease is characterized by developmental abnormalities of ectodermal derivatives and absent or reduced sweating. We hypothesized that ED patients have a reduced skin immune defense because of the reduced ability to sweat. Therefore, we performed a label-free quantitative proteome analysis of wash solution of human skin from ED patients or healthy individuals. A clear-cut difference between both cohorts could be observed in cellular processes related to immunity and host defense. In line with the extensive underrepresentation of proteins of the immune system, dermcidin, a sweat-derived AMP, was reduced in its abundance in the skin secretome of ED patients. In contrast, proteins involved in metabolic/ catabolic and biosynthetic processes were enriched in the skin secretome of ED patients. In summary, our proteome profiling provides insights into the actual situation of healthy versus diseased skin. The systematic reduction in immune system and defense-related proteins may contribute to the high susceptibility of ED patients to skin infections and altered skin colonization. Journal of Investigative Dermatology (2015) 135, 759–767; doi:10.1038/jid.2014.462; published online 4 December 2014

INTRODUCTION sweat glands (Schittek et al., 2001), hair bulb cells (Muller Human skin represents the largest organ of our body and et al., 2003), and sebocytes (Nagy et al., 2006; Lee et al., provides the epithelial barrier to the external environment. 2008), are also able to produce AMPs. These AMPs either Although the skin is continuously challenged by an immense directly kill the pathogens or activate immune cells in the sub- number of potential pathogens, it rarely develops infections. epithelial layers to clear invading pathogens (Schroder and This is achieved by an effective skin innate immune response Harder, 2006; Weindl et al., 2007). It has been shown that in in healthy people. Essential defense mediators in the skin are some human skin diseases recurrent skin infections are inflammatory cytokines, chemokines, and antimicrobial pep- associated with reduced AMP production by keratinocytes tides (AMPs), which are expressed constitutively or after an (Schittek et al., 2008; Yamasaki and Gallo, 2008; Zanger inflammatory stimulus, mainly by keratinocytes in the stratum et al., 2009; Simanski et al., 2010; Zanger et al., 2010; corneum. In addition, cells present in the skin, such as Schittek, 2011). neutrophils (Gallo and Nakatsuji, 2011), mast cells (Di Sweat produced by eccrine sweat glands, which are Nardo et al., 2003), T cells (Agerberth et al.,2000),eccrine distributed over the whole body, is constantly secreted and covers the skin’s surface. Peptides processed from the dermci- 1Department of Dermatology, Eberhard-Karls-University Tu¨bingen, Tu¨bingen, din precursor are the most abundant AMPs in eccrine sweat Germany; 2Proteome Center Tu¨bingen, Eberhard-Karls-University Tu¨bingen, with a broad spectrum of antimicrobial activity (Schittek et al., 3 Tu¨bingen, Germany; B.R.A.I.N AG, Biotechnology Research And Information 2001). We showed previously that patients with atopic derma- Network, Zwingenberg, Germany and 4Department of Dermatology, University of Regensburg, Regensburg, Germany titis, who are characterized by aberrant Staphylococcus aureus Correspondence: Marc Burian or Birgit Schittek, Department of Dermatology, colonization of the skin, have a reduced amount of dermcidin- Eberhard-Karls-University Tu¨bingen, Liebermeisterstrae 25, D-72076 derived peptides in their sweat. This correlated with an Tu¨bingen, Germany. E-mail: [email protected] or impaired ability of sweat to eradicate bacteria on the skin of [email protected] these patients (Rieg et al., 2005; Schittek, 2011). This indicates Abbreviations: AMP, antimicrobial peptide; ED, ectodermal dysplasia; HED, that eccrine sweat does not only have a function in hypohidrotic ED temperature regulation but also in the skin immune defense. Received 10 April 2014; revised 9 September 2014; accepted 11 September 2014; accepted article preview online 27 October 2014; published online 4 The capability of thermoregulation and infection defense by December 2014 sweat is severely limited in patients suffering from ectodermal

& 2015 The Society for Investigative Dermatology www.jidonline.org 759 M Burian et al. Quantitative Proteomics of the Skin Secretome

dysplasia (ED) (Schneider et al., 2011). ED is a complex group of inherited disorders that share common developmental abnormalities of two or more of the following: hair, teeth, nails, sweat glands, and other ectodermal structures. Until 61 383 100 now, more than 160 different ED symptoms have been described clinically and around 30 are known to be Healthy involved in ED pathogenesis (Irvine, 2009). The most frequent ED patients individuals form of ED is hypohidrotic ED (HED), also called the Christ– Siemens–Touraine syndrome, which can be inherited in an 12 X-linked, autosomal recessive or autosomal dominant manner 11 (Mikkola, 2009; Knaudt et al., 2012). HED is characterized by 10 sparse scalp hair, few and conical teeth, and absent or 9 reduced sweating due to missing or nonfunctional sweat 8 glands (Mikkola and Thesleff, 2003). Patients generally 7 present with clinical symptoms resembling atopic dermatitis, 6 such as dry skin, erythema, eczema, bacterial skin infection, 5 mostly with S. aureus, increased fragility of the skin and 4 hyperkeratosis (Knaudt et al., 2012). On a molecular level, –Log t -test P -value 3 HED has been associated with various X-linked mutations of 2 ectodysplasin, which is a tumor necrosis factor ligand within 1 the nuclear factor B essential modulator (NEMO) pathway. An 0

intact NEMO regulatory protein appears to be critical for both –2 –1.5 –1 –0.5 0 0.5 1 1.5 2 ectodermal development and proper immune function t-test difference signaling (Schimke et al., 2013). In the present study, we used a label-free quantitative Figure 1. Label-free mass spectrometry analyses of pooled skin wash solution from 3 6 healthy volunteers and 3 6 hypohidrotic ectodermal dysplasia proteomic approach to determine the relative abundance of (HED) patients. (a) Venn diagram showing the overlap between the numbers of proteins present on the skin of healthy individuals and HED proteins quantitatively identified from healthy individuals and HED patients. patients. As eccrine sweat is a rich source of functionally Of the 383 overlapping proteins statistically significant differentially expressed important cellular proteins, we hypothesized that patients who proteins (Benjamini Hochberg false discovery rateo0.05) are shown in the suffer from HED have a different protein spectrum on the skin volcano plot. (b) Volcano plot analysis reveals that 81 proteins are significantly compared with healthy individuals. Their missing or reduced differentially expressed. Among the 81 proteins, 42 proteins were significantly ability to sweat might influence the clinical skin symptoms, elevated in HED patients (indicated by red dots), whereas 39 proteins were significantly decreased in HED patients (indicated by green dots). such as recurrent skin infections. Especially, the AMP dermci- ED, ectodermal dysplasia; HED, hypohidrotic ectodermal dysplasia. din, which is only produced by eccrine sweat glands, should be present in limited amounts in ED patients. We provide, to our knowledge previously unreported, a global analysis of the not significantly differentially expressed are present in composition of secreted or shedded proteins on the skin in Supplementary Table S2 online. healthy individuals and ED patients. Biological significance of identified proteins RESULTS To understand the functional significance of the identified Global proteome characterization of the secretome of healthy proteins, we performed analyses of ontology for bio- and ED skin logical function classification using the ‘‘Database for annota- For a comparative analysis of proteins found on the skin of tion, visualization, and integrated discovery’’ (DAVID) healthy individuals versus HED patients, we performed a (DAVID Bioinformatics Resources 6.7, National Institute of label-free quantitative proteomic analysis of samples of skin Allergy and Infectious Diseases (NIAID), NIH, Bethesda, MD) wash solution from three independent replicates of a pool from that systematically identifies functional classes enriched in the each six ED patients and six healthy volunteers, respectively. gene lists (Dennis et al., 2003; Huang da et al., 2009). The In total, 544 proteins could be identified. Of the 544 proteins, analysis revealed major differences in the proteins that were 61 proteins were exclusively identified in healthy volunteers, differentially or exclusively expressed in healthy volunteers whereas 100 proteins were uniquely identified in ED patients. (Figure 2a) or ED patients (Figure 2b). Cellular processes A total of 383 proteins were identified in both ED patients and significantly enriched in the secretome of healthy individuals healthy volunteers (Figure 1a). Of the 383 overlapping mainly included categories related to immunity and host proteins, 81 proteins were significantly differentially expressed defense (55%), proteolysis (26%) and development (17%). (Benjamini Hochberg false discovery rateo0.05). Among The main category of immunity and host defense included those 81 proteins, 42 proteins were elevated in their abun- proteins significantly enriched in the subcategories of defense dance in ED patients, whereas 39 proteins were decreased in response (P ¼ 1.19E–05), inflammatory response (P ¼ 1.31E– ED patients (Figure 1b). A list of all identified and quantified 03), response to stress (P ¼ 4.10E–03), and wounding proteins is present in Supplementary Table S1 online. Proteins (P ¼ 5.44E–03) (Figure 2a). Tissue development represents

760 Journal of Investigative Dermatology (2015), Volume 135 M Burian et al. Quantitative Proteomics of the Skin Secretome

Healthy individuals ED patients

Developmental Cellular Cellular Regulation of processes regulation Cellular metabolic Developmental Localization & apoptosis 2% 2% regulation processes processes transport 3% <1% <1% 3% Proteolysis 17% Response to 1% stress/ wounding 11% Metabolic/ catabolic Immunity & processes host defense 40% Proteolysis 55% 26% Biosynthetic processes 25%

Nucleic acid metabolic processes 13%

Defense response Carbohydrate catabolic process Inflammatory response Glucose catabolic process Alcohol metabolic process Response to stress Hexose catabolic process Response to wounding Glycolysis Response to stimulus Monosaccharide catabolic process Response to external stimulus Monosaccharide catabolic process Alcohol catabolic process 0123456 Hexose metabolic process –Log (P) 10 Carbohydrate metabolic process Glucose metabolic process Catabolic process Generation of precursor...

0 246810

–Log10 (P)

Figure 2. analysis for biological functions of proteins identified in the human skin secretome. Gene ontology analysis for biological functions of proteins identified in skin wash solutions of healthy individuals (a) and of HED patients (b) using Database for annotation, visualization, and integrated discovery software (DAVID). (a) Cellular processes represented by the proteins, which were exclusively and significantly elevated in healthy individuals. (b)Cellular processes represented by the proteins, which were exclusively and significantly elevated in HED patients. The bars represent log10(P) values, where P represents the significance of each cellular process being enriched by exclusively monitored proteins and differentially expressed proteins. ED, ectodermal dysplasia; HED, hypohidrotic ectodermal dysplasia. the most enriched subcategory included in developmental Proteins involved in tissue development are reduced in the skin processes (1.92E–03; data not shown). secretome of ED patients With exception of the functional categories response to ED patients are impaired in several ectodermally derived stress and response to wounding, none of these cellular structures such as the skin, hair, nails, teeth, and sweat glands. processes found in healthy individuals were significantly Accordingly, in the skin secretome of healthy individuals, we enriched in the secretome of ED patients (Figure 2b). In ED observed an enrichment of proteins involved in developmen- patients, proteins of cellular processes related to metabolism/ tal processes, which is rigorously reduced in ED patients catabolism (40%), biosynthetic processes (25%), and (Figure 2a and b). Especially, the categories epidermis and nucleic acid metabolism (13%) were significantly enriched ectoderm development were significantly reduced in ED (Figure 2b). The group of proteins in the functional cluster patients (P ¼ 2.46E–03 and P ¼ 3.45E–03 in healthy indivi- metabolic/catabolic processes included proteins in the sub- duals vs. P ¼ 4.57E–02 and P ¼ 5.78E–02 in ED patients). categories of carbohydrate, glucose, alcohol, hexose, and Thereby, the P-values imply the significance of being monosaccharide metabolism/catabolism (Figure 2b). The cate- enriched, which is almost 10-fold lower in ED patients (see gory of genes involved in biosynthetic processes included also Supplementary Table S3 and S4 online). By zooming into mainly the subcategories of different metabolic processes the single protein level multiple proteins involved in epider- (organic and carboxylic acid metabolic process, cellular mal differentiation and morphogenesis, e.g., kallikrein-7, ketone, and amine metabolic process), as well as nitrogen kallikrein-5, protein-glutamine gamma-glutamyltransferase, compound metabolic process (data not shown). Therefore, the hornerin, and isoform 4 of extracellular matrix protein 1, were category of biosynthetic processes (25%) overlaps with the either exclusively or significantly enriched in the skin secre- category of metabolic/catabolic processes (40%) and cellular tome of healthy individuals (Figure 4a and b; Supplementary metabolic processes (3%), indicating that in ED patients Figure S2 online). In contrast, kallikrein-8 and the fatty acid– mainly proteins involved in metabolic and catabolic activity binding protein, both are known to be highly expressed in are overrepresented. psoriatic skin, were significantly enriched in ED patients

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Proteins exclusively monitored in ED patients

Dystrophin (P11532) Phosphoglycerate mutase 1 (P18669)

Keratin, type II cuticular Hb2 (Q9NSB4) Glutathione S-transferase P (P09211)

Keratin, type II cuticular Hb5 (P78386) Oligoribonuclease, mitochondrial (Q9Y3B8)

Keratin, type II cutoskeletal 73 (Q86Y46) Extracellular (Q9GZZ8)

Phosphoglycerate kinase 1 (P00558) Heme-binding protein 2 (Q9Y5Z4)

Keratin, type I cuticular Ha5 (Q92764) Elongation factor 2 (P13639)

Keratin, type I cuticular HA3-II (Q14525) Alpha-1-acid glycoprotein 2 (P19652)

Isoform 2 of glucose-6-phosphate isomerase (P06744-2) Phosphoglucomutase-2 (Q96G03)

Coactosin-like protein (Q14019) Rho GDP-dissociation inhibitor 2 (P52566)

Galectin-related protein (Q3ZCW2) UPF0556 protein (Q969H8)

Keratin, type I cuticular Ha6 (076013) Keratin, type II cytoskeletal 4 (P19013)

Nuclear transport factor 2 (P61970) Junction plakoglobin (P14923)

N(G),N(G)-dimethylarginine dimethylaminohydrolase 2 (O95865) Ras-related protein Rab-7a (P51149)

Ribosyldihydronicotinamide dehydrogenase [quinone] (P16083) NADH-cytochrome b5 reductase 2 (Q6BCY4)

Cystatin-B (P04080) DNA damage-binding protein 1 (Q16531)

0 2x1007 4x1007 6x1007 8x1007 0 2x1006 4x1006 6x1006 8x1006 1x1007 LFQ intensity LFQ intensity

Proteins significantly elevated in ED patients

Keratin, type II cuticular Hb3 (P78385) Glutathione synthetase (P48637) Triosephosphate isomerase (P6074) Purine nucleoside phosphorylase (P00491) Lipocalin-1 (P31025) Malate dehydrogenase, mitochondrial (P40926) Gamma-glutamylcycotransferase (O75223) 78 kDa glucose-regulated protein (P11021) Alpha-amylase 1 (P04745) Coatomer subunit beta (P35606) Arginase-1 (P05089) Isoform 3 of interleukin-1 receptor antagonist protein (P18510-3) Fatty acid-binding protein, epidermal (Q01469) D-tyrosyl-tRNA(Tyr) deacylase 1 (Q8TEA8) Alpha-enolase (P0733) Isoform 2 of kallikrein-8 (O60259-2) Serotransfrrin (P02787) Cystatin-SN (P01037) Catalase (P04040) Profilin-1 (P07737) Serpin B8 (P50452) Cystatin-SA (P09228) Phosphatidyethanolamine-binding protein 1 (P30086) lsoform 3 of malate dehydrogenase, cytoplasmic (P40925-3) Cystatin-S (P01036) Endonuclease domain-containing 1 protein (O94919) Transaldolase (P37837) WW domain-binding protein 2 (O969T9) Serpin B13 (Q9UIV8) ADP-sugar pyrophosphatase (A6NFX8) Hemoglobin subunit alpha (P69905) Keratin, type II cytoskeletal 1b (Q7Z794) Plasminogen activator inhibitor 2 (P05120) Protein S100-A6 (P06703) Leukocyte elastase inhibitor (P30740) Alpha-2-HS-glycoprotein (C9JV77) PITH domain-containig protein 1(Q9GZP4) Filamin-A (P21333) Saposin-D (B1AVU8) Calmodulin (H0Y7A7 07 07 07 0 2x1008 4x1008 6x1008 0 1x10 2x10 3x10 LFQ intensity LFQ intensity

Figure 3. The most intense proteins identified in the skin secretome of ED patients. The top 30 most intense proteins exclusively monitored in HED patients (a)or the top 40 most intense proteins significantly elevated in HED patients (b) identified by label-free LC-MS/MS analyses. Blue bars represent the protein intensity from healthy individuals and red bars represent the protein intensity from HED patients. Mean±SD. ED, ectodermal dysplasia; HED, hypohidrotic ED; LC-MS/MS, liquid chromatography–mass spectrometry; LFQ, label-free quantification.

(Figure 3b). Furthermore, an increased amount of several types azurocidin, and mucin-7), or have a role in the humoral of keratins could be observed for ED patients (Figure 3a and immune response (i.e., complement component C7, several Ig b). Thus, the high abundance of proteins involved in tissue molecules; Figure 4a and Supplementary Figure S2 online). development in healthy individuals indicates an intact Among the top 40 most abundant proteins significantly renewal of the healthy skin, whereas the presence of proteins elevated in healthy individuals compared with ED patients normally found in psoriatic skin, together with several types of (Figure 4b) again several proteins were involved in local host keratins might be due to the dry skin in ED patients, which is defense. As already noted for proteins exclusively present in often clinically characterized with erythema, wrinkling, ero- healthy individuals (Figure 4a), proteins with antimicrobial sions, increased fragility of skin, or plantar hyperkeratosis activity and/or proteolytic activity were significantly enriched in (Knaudt et al., 2012). the secretome of healthy skin (dermcidin, cathepsin D, lysozyme C, ribonuclease 7, and S100 proteins A8 and A9; Figure 4b). In Proteins involved in host defense are reduced in the skin accordance with the proteomic data (Figures 4b and 5f), we secretome of ED patients could validate by ELISA that the AMP ribonuclease 7 was Considering the 45 most abundant proteins exclusively identi- significantly elevated in healthy individuals compared with ED fied in healthy individuals, it is striking that many identified patients (including all types of ED forms; P ¼ 0.004; Figure 5g). proteins were related to cellular processes involved in immunity and host defense. Several of the identified proteins are proteases Dermcidin is specifically reduced in the secretome of ED (i.e., antileukoproteinase, neutrophil elastase, carboxypeptidase, patients and ), AMPs/proteins (i.e., bactericidal/permeability- In the proteomic analysis, we found that the AMP dermcidin is increasing fold–containing family A member 1, bactericidal/ significantly underrepresented in wash solutions of ED patients permeability-increasing fold–containing family B member 1, compared with healthy individuals (median of label-free

762 Journal of Investigative Dermatology (2015), Volume 135 M Burian et al. Quantitative Proteomics of the Skin Secretome

Proteins exclusively monitored in healthy individuals

BPI fold-containing family A member 1 (Q9NP55) Podocalyxin (F5GWY5)

UPF0762 protein C6orf58 (Q6P5S2) Mammaglobin-A (Q6NX70)

Centriolin (Q7Z7A1) Ig lambda chain V-II region NIG-84 (P04209)

BPI fold-containing family B member 1 (Q8TDL5) Chitotriosidase-1 (Q13231)

Integrator complex subunit 7 (Q9NVH2) Azurocidin (P20160)

Antileukoproteinase (P03973) Mucin-7 (Q8TAX7)

Neutrophil elastase (P08246) Probable carboxypeptidase PM20D1 (Q6GTS8)

Isoform 3 of kallikrein-11 (Q9UBX7-3) IgGFc-binding protein (Q9Y6R7)

Lipocalin-15 (Q6UWW0) Protein CREG1 (O75629)

Ig heavy chain V-II region ARH-77 (P06331) Macrophage-capping protein (P40121)

Glucosylceramidase (P04062) Isoform 2 of clusterin (P10909-2)

Isoform 3 of L-lactate dehydrogenase A chain (P00338-3) CD44 antigen (P16070)

Probable histidine--tRNA ligase, mitochondrial (P49590) Isoform 2 of acid ceramidase (Q13510-2)

Cathepsin L2 (O60911) Retinoid-inducible serine carboxypeptidase (Q9HB40)

L-lactate dehydrogenase C chain (P07864) Interleukin-37 (Q9NZH6)

0 1×1007 2×1007 3×1007 4×1007 0 1×1006 2×1006 3×1006 4×1006 5×1006 LFQ intensity LFQ intensity

Proteins significantly elevated in healthy individuals

Prolactin-inducible protein (P12273) Kallikrein-7 (P49862) Isoform 2 of annexin (P07355-2) Cysteine-rich secretory protein 3 (J3KPA1) Dermcidin (P81605) Ly6/PLAUR domain-containing protein 3 (O95274) Protein S100-A8 (P05109) Phospholipase B-like 1 (Q6P4A8) Secretoglobin family 1D member 2 (O95969) Carboxypeptidase E (P16870) Desmocollin-1 (Q08554) Ly6/PLAUR domain-containing protein 5 (Q6UWN5) Lysosome-associated membrane glycoprotein 1 (P11279) Lysosomal protective protein (P10619) Protein S100-A9 (P06702) Kallikrein-5 (Q9Y337) Desmoglein-1 (Q02413) Isoform 2 of lysosomal pro-X carboxypeptidase (P42785-2) Serpin A12 (Q8IW75) Protein-glutamine gamma-glutamyltransferase K (P22735) Cathepsin D (P07339) Ly-6/neurotoxin-like protein 1 (Q9BZG9) Ganglioside GM2 activator (P17900) Ribonuclease 7 (Q9H1E1) Lysozyme C (P61626) Carboxypeptidase M (P14384) Carboxypeptidase A4 (Q9UI42) Suprabasin (Q6UWP8) Toll-interacting protein (Q9H0E2) Isoform 2 of Ig mu chain C region (P01871-2) Sialate O-acetylesterase (Q9HAT2) Zymogen granule protein 16 homolog B (Q96DA0) Leucine-rich alpha-2-glycoprotein (P02750) (P07858) Isoform 2 of delta-aminolevulinic acid dehydratase (P13716-2) Alpha-2-macroglobulin-like protein 1 (A8K2U0) Peroxisomal multifunctional enzyme type 2 (P51659) Hornerin (Q86YZ3) Nicastrin (Q92542)

05×1008 5×1009 2×1009 05×1007 2×1007 3×1007 4×1007 5×1007 Protein intensity ED patients LFQ intensity LFQ intensity Protein intensity healthy individuals

Figure 4. The most intense proteins identified in the skin secretome of healthy individuals. The top 30 most intense proteins exclusively monitored in healthy individuals (a) and the top 40 most intense proteins significantly elevated in healthy individuals (b) identified by label-free LC-MS/MS analyses. Blue bars represent the protein intensity from healthy individuals and red bars represent the protein intensity from HED patients. Mean±SD. BPI, bactericidal/permeability-increasing; ED, ectodermal dysplasia; HED, hypohidrotic ED; LC-MS/MS, liquid chromatography–mass spectrometry; LFQ, label-free quantification. quantification intensity in healthy individuals 1.18 108 and dermcidin might influence the development of skin infections. median of label-free quantification intensity in ED patients To investigate whether dermcidin alone will lead to a reduc- 3.04 106 (Figure 5a)). We could validate this result in wash tion in colony-forming units, mouse skin was epicutaneously solutions of the 18 patients used for proteomic analysis by infected with S. aureus and treated either with a dermcidin ELISA (Sagawa et al., 2003) (Figure 5b). To validate these containing ointment or with a placebo twice daily. A sig- findings in a larger cohort, we determined the concentration of nificant reduction in colony-forming units could be observed dermcidin in wash solution of human skin of 51 healthy 24 hours after the beginning of treatment with a dermcidin-1- individuals and 46 ED patients (including all types of ED containing ointment (30 mgml 1) compared with mice treated forms) by ELISA. Whereas skin wash solutions in both groups with a placebo. This effect could be observed either for did not differ in their total protein content (P ¼ 0.3551; bacteria that were loosely attached to the epidermal surface, Figure 5c), the abundance of dermcidin-derived peptides as well as for bacteria that were invaded into the skin in ED patients was significantly reduced (P ¼ o0.0001; (Figure 6). Seventy-two hours after the beginning of treatment Figure 5d). Furthermore, we could show that the amount of again a reduction in colony-forming units could be observed. dermcidin-derived peptides is independent of the total protein However, this decrease in S. aureus cells did not reach concentrationinbothcohorts(Figure5e).Insummary,using statistical significance (Figure 6). Thus, dermcidin in the form two quantitative methods we show that dermcidin is specifi- of an ointment led to a reduction in S. aureus load, which cally reduced in the skin secretome of ED patients. might be a new therapeutic option to prevent skin infections in ED patients. Treatment of mice with a dermcidin containing ointment reduces S. aureus colonization DISCUSSION As ED patients frequently suffer from bacterial skin infections Cutaneous hallmarks of ED are depigmentation, scaling, mainly caused by S. aureus, we speculate that the level of atrophy, or hyperkeratosis (Itin et al., 1993b). Furthermore,

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***

*** )

1.5x1008 –1 0.1

10x1008 0.01

5.0x1007 LFQ intensity (DCD-peptides in µ gml 10 0.0 0.001

ED patients Healthy volunteers Log ED patients Healthy volunteers

ns

) *** ) –1

–1 100 0.1

0.01

10

0.001 (total protein in µ gml 10 (DCD-peptides in µ gml 10

Log 1 0.0001 ED patients Healthy volunteers Log ED patients Healthy volunteers

) 0.10 –1

0.08

0.06

0.04

0.02

DCD-peptides in skin wash solution ( µ gml 0.00 01020 30 40 50 60 Total protein concentration in skin wash solution (µgml–1)

** **

8.0x106 ) 1 000 –1

6.0x106 100

4.0x106 10 LFQ intensity 2.0x106 (RNase 7 in µ gml 10 Log 0 1 ED patients Healthy volunteers ED patients Healthy volunteers

Figure 5. Quantitative analysis of the concentration of antimicrobial peptides in wash solution of human skin. Quantitative analysis of the concentration of dermcidin (DCD)-derived peptides in wash solution of human skin by label-free LC-MS/MS analysis (a) and by ELISA (b–e). (a) Protein intensity of dermcidin of pooled skin wash solution from 3 6 healthy volunteers and 3 6 HED patients. ***Po0.001. (b) Concentration of dermcidin in wash solution of human skin of the 36 individuals analyzed in the label-free LC-MS/MS analyses determined by ELISA. ***Po0.001. (c) Total protein concentration in skin wash solutions of 46 ED patients and 51 healthy individuals. (d) Concentration of dermcidin in wash solution of human skin of 46 ED patients and 51 healthy individuals. (e) Individual values for the concentration of dermcidin versus the total protein concentration of either healthy volunteers (filled squares) or ED patients (filled triangles). (f) Protein intensity of RNase 7 of pooled skin wash solution from 3 6 healthy volunteers and 3 6 HED patients. **Po0.001–0.01. (g) Concentration of RNase 7 in wash solution of human skin of 18 ED patients and 18 healthy individuals determined by ELISA. Mean±SD. ED, ectodermal dysplasia; HED, hypohidrotic ectodermal dysplasia; LC-MS/MS, liquid chromatography–mass spectrometry; LFQ, label-free quantification; NS, not significant; RNase 7, ribonuclease 7.

the body hair is often diminished (Itin et al., 1993a) and sweat healthy individuals. Our analyses by label-free liquid glands are reduced or totally absent (Itin and Fistarol, 2004; chromatography–mass spectrometry were paralleled by a Schneider et al., 2011). Using a label-free quantitative quantitative ELISA using an antibody directed against dermci- proteome analysis we compared the abundance of din. Our investigations revealed a specifically reduced secre- proteins present on human skin from HED patients and tion of dermcidin, because total protein concentration did not

764 Journal of Investigative Dermatology (2015), Volume 135 M Burian et al. Quantitative Proteomics of the Skin Secretome

1,000 1,000,000 * **

100,000 NS NS 100 10,000 –1 –1 1,000 CFUml CFUml 10 100

10

1 1 24 hours 72 hours 24 hours 72 hours

CFU recovered from mouse skin treated CFU recovered from mouse skin treated with DCD-1 containing ointment with placebo

Figure 6. Treatment of C57BL/6 mice with a dermcidin (DCD)-1-containing ointment reduces S. aureus infection. Bacterial counts were determined from recovered mouse skin 24 and 72 hours after the beginning of treatment either with a DCD-1-containing ointment (red squares) or with a placebo (blue squares). Bacteria loosely attached to the epidermal surface were recovered simply by washing (a) and bacteria invaded into the skin’s surface were recovered by scraping (b). Horizontal lines represent median amounts of colony-forming units (CFUs). Statistically significant differences between the treatment groups are indicated: *Po0.01–0.05; **Po0.001–0.01; NS, not significant.

differ in wash solutions of human skin from ED patients versus evaluate the abundance of proteins on human skin derived healthy individuals. This is in analogy to patients with atopic from eccrine sweat glands. dermatitis, who are characterized by aberrant S. aureus Several proteomic studies demonstrated that normal sweat colonization of the skin, having a reduced amount of obtained from healthy volunteers contains proteolytic dermcidin-derived peptides in their sweat. This lack of anti- enzymes, AMPs, as well as the proinflammatory cytokines microbial defense correlated with an impaired ability of sweat IL-1 and IL-8 (Dai et al., 2013). We could not find IL-8 in the to eradicate bacteria on the skin of these patients (Rieg et al., secretome of healthy individuals or ED patients, indicating 2005; Schittek, 2011). In contrast, Rieg and colleagues that this interleukin is not very abundant on human skin. recently reported in a correlation study that patients with Interestingly, the only cytokine significantly differentially recurrent S. aureus skin and soft tissue infections and healthy expressed was IL-37 (Figure 4a), which is known to suppress people have a comparable level of dermcidin-derived peptides innate inflammatory and immune responses by reducing IL-1a in their sweat (Rieg et al., 2014). The high abundance of and IL-6 among others (Sharma et al., 2008; Nold et al., dermcidin in the secretome of healthy individuals, which is 2010). IL-1a,IL-36b, and IL-36g have been identified, known to be exclusively expressed by eccrine sweat glands in however, without significantly different expression the skin (Rieg et al., 2004; Schittek, 2012), suggests that a (Supplementary Table S2 online). majority of the identified proteins in the skin secretome of In the skin secretome of ED patients, a decrease in proteins healthy individuals are derived from eccrine sweat. Indeed, involved in developmental processes was observed. Especially many proteins we identified have been described to be present for the categories ectoderm and tissue development a in human eccrine sweat as for example cathepsin D and rigorous reduction could be observed. This is in line with the prolactin-inducible protein (Baechle et al., 2006; Raiszadeh fact that ED patients have defects in their ectodermally derived et al., 2012). Importantly, most of the proteins in the skin structures (Itin and Fistarol, 2004). By zooming into the single secretome of healthy individuals were enriched in cellular protein level, proteins normally found in psoriatic skin were processes involved in immune defense and underrepresented highly expressed, indicating a disturbed renewal of the skin. in the skin secretome of ED patients. In particular, proteins in HED is often associated with an immunodeficiency syn- this functional group were mainly involved in local infection drome with defective Ig production and bacterial skin infec- defense, as for example the humoral immune response (Igs and tions. Indeed, Ig levels are reduced in the skin secretome of ED complement factors), protease activity, or antimicrobial patients, and these patients frequently suffer from S. aureus activity. This underlines the importance of host defense skin infections (data not shown). In many HED patients, the molecules that are constantly present on the skin surface. disease is caused by mutations in genes encoding proteins These mediators provide a protective environment resulting in involved in the activation of NF-kB), a transcription factor efficient constitutive defense against infections. To our pivotal for innate and adaptive immune responses, cell knowledge previously unreported, this study describing the adhesion, , apoptosis, and ectodermal develop- secretome of human skin gives a snapshot of proteins on the ment (Schimke et al., 2013). It seems that in healthy epidermal barrier. In addition, the comparison of the individuals the NF-kB signaling pathway is of importance for secretome of healthy individuals with that of ED patients expression of genes involved in immune responses, which is suffering from reduced sweating enables us to indirectly lacking in ED patients. By this, the proteins involved in

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immune responses are underrepresented in ED patients. As it techniques in the field of clinical medicine. One potential is known that ED is caused among others by mutations of outcome of this study is that identification of a more global genes encoding for adhesion (Itin and Fistarol, 2004; South, proteomic composition in normal skin may serve as the basis 2004), we found a significant reduction in proteins involved in for characterizing and comparing the skin proteomes from a cell–cell adhesion and cell–matrix adhesion in ED patients variety of disease states, which may lead to a more complete (desmocollin-1, desmocollin-2, desmoglein-1, CD44 antigen, understanding of the pathology of the disease eventually and the Ly6/PLAUR domain–containing protein 3) (Figures 4a helping to develop new therapeutic options. and b). Desmosomal cadherins are important for the adhesion of neighboring cell membranes. Furthermore, proteins MATERIALS AND METHODS involved in the epidermis and tissue development are over- Study population and patients represented in healthy individuals, which might be a sign of Wash solution of human skin was collected from 46 ED patients (18 epidermal renewal. female and 28 male) registered in an ED network (Selbsthilfegruppe ED patients are clinically characterized by dry skin with Ektodermale Dysplasie e.V., Aichtal, Germany) and from 51 healthy erythema, wrinkling, erosions, bacterial infection, increased individuals (22 female and 29 male). The study was approved by the fragility of skin, or plantar hyperkeratosis (Knaudt et al., 2012). local ethics (713/2013BO2), and all study participants gave written They share many aspects with xerosis and mild-to-moderate informed consent. From each donor, wash solution was taken from atopic dermatitis. Because of the hyperkeratosis it is not both hands and from one forearm and subsequently pooled. Samples astonishing that especially different types of keratins are were frozen at 20 1C until further analysis. overrepresented in the skin secretome of ED patients. Keratins are known to be involved in the differentiation and Collection of wash solution of human skin and processing survival of keratinocytes but also in inflammation and wound Wash solution from each skin area was taken by incubation of the healing (Roth et al., 2012; Lessard et al., 2013). The skin with 3 ml of 1:10 diluted phosphate-buffered saline for 2 minutes overrepresentation of keratins in ED patients might either be restricting the liquid flow by a truncated collection tube of B5.7 cm2. a symptom of the dry and erosive skin and hyperkeratosis or Subsequently, the wash solution was mixed with protease inhibitors are functionally relevant in mediating cutaneous immuno- (Roche complete, Mannheim, Germany). Wash samples from human suppression. Together with the lower abundance of AMPs and skin were centrifuged for 5 minutes at 5,000 r.p.m. and supernatant proteases in the secretome of ED patients the disturbed keratin decanted in a new collection tube. For analysis, samples were profile might make the skin prone for infections. Many AMPs lyophilized and the lyophilisate was solved in 400 mlH20. From each have to be post-proteolytically processed into their mature sample the protein concentration was determined by the bicincho- form by proteases to gain full activity. For example cathepsin ninic acid test (Pierce, Bonn, Germany) as described by the D, which is known to cleave dermcidin into shorter peptides, manufacturer. For proteome analysis, 100 ml of the dissolved lyophi- was similar to dermcidin itself, significantly elevated in its lisate of 3 6 HED patients and 3 6 healthy individuals, respec- abundance on healthy skin. tively, were used and pooled. The external application of the DCD-1 ointment on S. aureus infected mouse skin clearly demonstrated a reduction Gene ontology in the bacterial load. Thus, indicating on a functional level To determine gene ontological annotations for selected proteins, we that DCD is an essential determinant of cutaneous immune used the DAVID software (Dennis et al., 2003; Huang da et al., homeostasis. DCD in the form of an ointment might be a new 2009). The list of all identified categories is present in Supplementary therapeutic option to alleviate and/or prevent skin infections in Tables S2 and S3 online. ED patients. On the basis of the proteomic data, it would be desirable to develop a DCD-1 ointment containing additional Statistical methods AMPs/factors to limit S. aureus skin infection not only in ED Statistical analysis was performed with the Prism 5.0 package patients but also in patients with atopic dermatitis. As (GraphPad Software, San Diego, CA). DCD and total protein con- ribonuclease 7 in addition to dermcidin was significantly centrations, determined by ELISA and the bicinchoninic acid assay, reduced in ED patients, as demonstrated by two independent were compared using two-sample t-tests. Po0.05 was considered to quantitatively approaches, this potent antimicrobial ribonu- be statistically significant. clease active especially against S. aureus (Harder and Schroder, 2002) might be another missing factor that favors CONFLICT OF INTEREST skin infections in ED patients. The authors state no conflict of interest. In summary, we describe a noninvasive method for the determination of proteins in the skin secretome of healthy ACKNOWLEDGMENTS We thank all healthy volunteers and ED patients for their support by providing individuals and patients suffering from ED. Using the liquid us the skin washing fluids and Birgit Sauer for expert technical assistance. This chromatography–mass spectrometry approach, we identified a work was supported by grants from the DFG (SFB766, 510/8-1, 510/7-1) and comprehensive protein profile composed of proteins related to by the Centre for Rare Diseases. cellular processes such as immunity and host defense in healthy individuals and metabolic/catabolic and biosynthetic SUPPLEMENTARY MATERIAL processes in ED patients. Our investigations underscore the Supplementary material is linked to the online version of the paper at http:// power of proteomics to utilize quantitative molecular www.nature.com/jid

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