ORIGINAL ARTICLE

Rosacea Is Characterized by a Profoundly

Diminished Skin Barrier
Barbara Medgyesi1,2,3,7, Zsolt Dajnoki1,2,7, Gabriella Béke1,2, Krisztián Gáspár1,2,
Imre Lo}rinc Szabó1,2, Eszter Anna Janka2, Szilárd Póliska4, Zoltán Hendrik5, Gábor Méhes5,
}csik2, Tamás Bı́ró6, Anikó Kapitány1,2,8 and Andrea Szegedi1,2,8
Dániel Töro

Rosacea is a common chronic inflammation of sebaceous glanderich facial skin characterized by severe skin
dryness, elevated pH, transepidermal water loss, and decreased hydration levels. Until now, there has been no
thorough molecular analysis of permeability barrier alterations in the skin of patients with rosacea. Thus, we
aimed to investigate the barrier alterations in papulopustular rosacea samples compared with healthy seba-
ceous glanderich skin, using RNA sequencing analysis (n ¼ 8). Pathway analyses by Cytoscape ClueGO
revealed 15 significantly enriched pathways related to skin barrier formation. RT-PCR and immunohisto-
chemistry were used to validate the pathway analyses. The results showed significant alterations in barrier
components in papulopustular rosacea samples compared with sebaceous glanderich skin, including the
cornified envelope and intercellular lipid lamellae formation, desmosome and tight junction organizations,
barrier alarmins, and antimicrobial peptides. Moreover, the barrier damage in papulopustular rosacea was
unexpectedly similar to atopic dermatitis; this similarity was confirmed by immunofluorescent staining. In
summary, besides the well-known dysregulation of immunological, vascular, and neurological functions, we
demonstrated prominent permeability barrier alterations in papulopustular rosacea at the molecular level,
which highlight the importance of barrier repair therapies for rosacea.
Journal of Investigative Dermatology (2020) 140, 1938e1950; doi:10.1016/j.jid.2020.02.025

INTRODUCTION persistent erythema, telangiectasia, papules, pustules, pla-

Rosacea is a common chronic immune-mediated inflamma- ques, edema, and phyma (Gallo et al., 2018b). The four
tory skin disease of unknown cause (Buhl et al., 2015; Gallo major clinical subtypes of rosacea are erythemato-
et al., 2018a; Steinhoff et al., 2011). Rosacea mainly affects telangiectatic, phymatous, ocular, and papulopustular rosa-
sebaceous glanderich (SGR) skin regions, particularly the cea (PPR) (Crawford et al., 2004; Gallo et al., 2018b).
central face, nose, chin, and forehead of light-skinned peo- Although rosacea occurs in SGR skin regions, affected
ple, aged 30e50 years (Buechner, 2005). The prevalence of skin of patients is frequently observed as severely dry and
rosacea in Europe is 2e10% (Gallo et al., 2018b). Clinical sensitive by dermatologists. Moreover, several functional
features of rosacea include flushing (transient erythema), studies indicate that the skin permeability barrier is
dysfunctional in rosacea. The lesional skin of patients is
characterized by significantly increased pH and trans-
1
Division of Dermatological Allergology, Department of Dermatology, epidermal water loss, whereas skin hydration levels are
Faculty of Medicine, University of Debrecen, Debrecen, Hungary;
2
Department of Dermatology, Faculty of Medicine, University of Debrecen,
significantly decreased (Darlenski et al., 2013; Powell and
Debrecen, Hungary; 3Doctoral School of Clinical Immunology and Ni Raghallaigh, 2011). Although recent studies demon-
Allergology, University of Debrecen, Debrecen, Hungary; 4Genomic strate that a dysfunctional barrier is capable of either initi-
Medicine and Bioinformatics Core Facility, Department of Biochemistry ating or augmenting inflammatory skin diseases, barrier
and Molecular Biology, Faculty of Medicine, University of Debrecen,
Debrecen, Hungary; 5Department of Pathology, Faculty of Medicine, damage in the skin of patients with rosacea has not been
University of Debrecen, Debrecen, Hungary; and 6Department of thoroughly investigated.
Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Therefore, in this study, in the lesional skin of patients
Hungary
suffering from PPR, we characterized the major groups of
7
These authors contributed equally to this work. molecules involved in permeability barrier formation
8
These authors contributed equally to this work. including cornified envelope and intercellular lipid lamellae
Correspondence: Andrea Szegedi, Division of Dermatological Allergology, formation, desmosome and tight junction organizations,
Department of Dermatology, Faculty of Medicine, University of Debrecen, barrier alarmins, and antimicrobial peptides (AMPs). We
Hungary, 4032, Nagyerdei street 98, Debrecen, Hungary. E-mail: aszegedi@
med.unideb.hu performed whole transcriptomic analysis using RNA
Abbreviations: AD, atopic dermatitis; AMP, antimicrobial peptide; CDSN, sequencing (RNASeq) and confirmed our results with quan-
corneodesmosin; DEG, differentially expressed gene; DSG1, desmoglein 1; titative real-time reverse transcriptaseePCR and immuno-
KLK, kallikrein; IHC, immunohistochemistry; PPR, papulopustular rosacea; histochemistry (IHC). In addition, we compared PPR barrier
RNASeq, RNA sequencing; sfTSLP, short form thymic stromal lymphopoietin;
damage to that of atopic dermatitis (AD), another inflam-
SGP, sebaceous glandepoor; SGR, sebaceous glanderich; Th, T helper type
matory skin disease with a well-described dysfunctional skin
Received 9 December 2019; revised 17 January 2020; accepted 7 February
2020; accepted manuscript published online 19 March 2020; corrected proof barrier. Because different healthy skin regions have unique
published online 19 April 2020 immune and barrier characteristics, exclusively SGR skin was

ª 2020 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. This is
1938 Journal of Investigative Dermatology (2020), Volume 140 an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 B Medgyesi et al.
Skin Barrier Alterations of PPR

used as a healthy control for comparison with PPR specimens differentially expressed (e.g., epithelium development,
(Béke et al., 2018; Dajnoki et al., 2017; Jenei et al., 2019). morphogenesis of an epithelium, epithelial cell differentia-
According to our results, all major components of the skin tion, and epithelial cell proliferation) (Figure 1c,
permeability barrier in patients with PPR were severely Supplementary Table S2).
affected and were unexpectedly similar to that of affected AD
skin. To confirm the similarities between the two inflamma- Pathway analysis 2. Next, we performed a second pathway
tory skin diseases, we performed immunofluorescent staining enrichment analysis to determine the specific functions of
on selected molecules important in the formation of the DEGs; thus, a stricter analytical approach was applied and
permeability skin barrier. Our findings also highlight the the up- and downregulated DEGs were analyzed as two
importance of skin barrier restoring therapies for the man- different clusters by ClueGO (the detailed parameters of the
agement of rosacea. Based on our results, we recommend the analysis can be found in the Materials and Methods section).
incorporation of skin barrier targeted therapies into clinical This analytical approach revealed 426 significantly enriched
guidelines for rosacea, similar to the recommendations for terms and/or pathways (Supplementary Table S3) and most of
AD. them (291) belonged to innate and adaptive immune mech-
anisms (e.g., T helper type [Th] 17 cell differentiation, toll-
RESULTS like receptor cascades, T-cell selection, and neutrophil
RNASeq reveals prominent barrier differences between PPR migration). In addition, several terms were involved in
and SGR skin samples cellular and metabolic functions (63 terms; e.g., transport
along microtubule, exocytosis of specific granule membrane
Heatmap, principal component analysis. To identify the in- proteins, and response to cAMP) and took part in vasculari-
depth differences in gene expression patterns between SGR zation (2 terms; positive regulation of vasculature develop-
and PPR skin samples, RNASeq analysis was performed on ment and positive regulation of angiogenesis) and the
lysates of eight healthy SGR skin samples and eight PPR nervous system (21 terms; e.g., axonogenesis, glial cell dif-
samples. The PPR and SGR sample groups were clearly ferentiation, and axon guidance) were differentially
separated in the heatmap as well as in the principal expressed (Supplementary Table S3). Notably, 15 terms were
component analysis of the RNASeq data (Figure 1a and b). found to be related to skin barrier function (e.g., keratiniza-
The following statistical cut-off level was applied: a minimum tion, cornification, and tight junction) (Figure 1d and e,
of 1.5-fold alteration (fold change; higher or lower levels in Supplementary Table S3). Hereafter, we focused on the skin
PPR over SGR) in the average expression of the given mo- barrier-related pathways (Figure 1d).
lecular transcript in all donors at P < 0.05 statistical signifi-
cance. A total of 5,136 genes were differentially expressed in RT-qPCR and IHC validation confirm the significant
PPR compared with SGR skin. Of the differentially expressed alterations in the major skin barrier components in PPR
genes (DEGs), 3,133 genes showed higher expression, To gain further insight into the permeability barrier differ-
whereas 2,003 genes were expressed at lower levels in PPR ences in PPR versus healthy SGR skin, we examined the
as compared with SGR (Supplementary Table S1). expression of genes belonging to the major groups of skin
barrier molecules by RT-qPCR. These major groups included
Pathway analysis 1. To identify the function of the DEGs, (i) cornified envelope formation (FLG, KRT1, KRT10, LCE1D,
multiple bioinformatics analyses were performed using the LCE1F, LOR, SPRR1A, SPRR2A, TGM1, TGM3, and TGM5),
Cytoscape ClueGO bioinformatics tool (Bindea et al., 2009). (ii) intercellular lipid lamellae formation (ABCA12), (iii)
First, to identify the general biological function of DEGs, we desmosome organization (CDH1, corneodesmosin [CDSN],
performed a pathway enrichment analysis on all DEGs with desmoglein 1 [DSG1], DSC1, and PKP1), (iv) corneocyte
fold change  1.5. To identify the significantly enriched (P  desquamation (kallikrein [KLK]5, KLK7, and KLK14), (v) tight
0.05) terms and/or pathways with global functions, the junction formation (CLDN1, CLDN16, CLDN23, and
following criterion was applied: all terms contain at least 50 OCLN), (vi) barrier alarmins (KRT6, KRT16, and KRT17), and
genes from our input gene set (the detailed parameters of the (vii) AMPs (S100A7, S100A8, S100A9, DEFB4B, LCN2, and
analysis can be found in the Materials and Methods section). cathelicidin). Furthermore, we also assessed the expression of
By using the above approach, 1,239 significantly enriched selected molecules at the protein level by using IHC.
terms were found by ClueGO. Unsurprisingly, the identified
terms were mostly involved in cellular/metabolic functions Cornified envelope formation. First, we focused on inves-
(e.g., ion transport, lipid biosynthetic process, and transferase tigating the mRNA levels of molecules composing the cor-
activity) and innate (e.g., response to external stimulus, nified envelope in healthy SGR and PPR skin samples. Using
response to stress, cytokine secretion, defense response to RT-qPCR, we verified the RNASeq results. Most structural
bacterium, NOD-like receptor signaling pathway, and com- molecules (KRT1, KRT10, FLG, LOR, LCE1D, and LCE1F)
plement activation) and adaptive (e.g., T-cell activation, were downregulated in PPR samples, whereas SPRR1A and
leukocyte migration, TNF production, IFN-gamma produc- SPRR2A mRNA levels were higher in the diseased samples
tion, and chemokine signaling pathway) immune mecha- compared with the healthy controls (Table 1, Supplementary
nisms. In addition, genes taking part in vascularization (e.g., Figure S1). The differences were statistically significant in all
regulation of vasculature development and angiogenesis) and cases, except for LCE1D, LCE1F, and SPRR1A. Enzymes
the nervous system (e.g., nervous system development) were crucial for peptide cross-linking (TGM1 and TGM3) were
differentially expressed (Figure 1c, Supplementary Table S2). expressed at similar levels in PPR and SGR samples, except
Most importantly, 24 terms related to skin barrier were also for TGM5. TGM5 was significantly downregulated in PPR

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 B Medgyesi et al.
Skin Barrier Alterations of PPR

a b
Legend - Hierarchical Combined Tree on status (Non-averaged)
Color range
Legend - PCA Scores
Color by status
−6.8 0 3.4 6.8
Condition
60

40

Component 2 (9.31%)
20

–20

–40

–60

–150 –100 –50 0 50 100 150

Component 1 (51.49%)

Rosacea Healthy SGR

Condition
status
PPR1
PPR2
PPR3
PPR4
PPR5
PPR6
PPR7
PPR8
SGR1
SGR2
SGR3
SGR4
SGR5
SGR6
SGR7
SGR8

Rosacea Healthy SGR

c Nervous system-
related terms
3.7% (46)
Immune system-
related terms Other terms
19% (235) 8% (102)
Vasculature-
related terms
0.4% (5)
Cellular functions/ Skin barrier-
metabolism-related terms related terms
67% (827) 1.9% (24)

Figure 1. RNASeq analyses revealed significant skin barriererelated differences between PPR and healthy SGR skin samples. (a) Heat map was created by
analyzing genes showing significantly different expression (P < 0.05) between SGR (n ¼ 8) and PPR (n ¼ 8) skin. (b) Principal component analysis generated by
StrandNGS software could distinguish the two sample groups unambiguously. (c) The distribution of significantly enriched terms based on their functions on the
basis of the first-round enrichment analyses of significantly differentially expressed genes with FC > 1.5 between SGR and PPR by Cytoscape and ClueGO
(www.cytoscape.org). (d) Barrier-related significantly enriched terms revealed by Cytoscape and ClueGO between SGR and PPR in the second pathway analysis
of significantly differentially expressed genes with FC > 1.5. (e) Representative barrier-related terms visualized by ClueGO. CASP, caspase; CDSN,
corneodesmosin; FC, fold change; Nr., number; PPR, papulopustular rosacea; RNASeq, RNA sequencing; SGR, sebaceous glanderich.

samples compared with SGR samples (Table 1, ABCA12 was significantly downregulated in the diseased
Supplementary Figure S1). specimens (Table 1, Supplementary Figure S1).
Next, we analyzed cornified envelope components at the
protein level using IHC. LOR and KRT1 protein levels were Desmosome organization. The gene expression levels of
significantly lower in PPR than SGR skin, whereas FLG levels desmosome components (DSG1, DSC1, CDSN, PKP1, and
were similar in the sample groups (Table 1, Figure 2). Among CDH1) were examined by RT-qPCR. We found that all
the previously mentioned enzymes, no significant differences investigated molecules were highly and significantly down-
in TGM5 were detected between healthy SGR and PPR regulated in PPR specimens, except for CDSN. CDSN mRNA
samples (Table 1, Figure 2). levels were not significantly different between the two groups
(Table 1, Supplementary Figure S1). To validate our results at
the protein level, we also examined two junction compo-
Intercellular lipid lamellae formation. Among molecules nents, DSG1 and CDSN, by IHC. DSG1 protein levels were
with a pivotal role in composing intercellular lipid lamellae, significantly lower in PPR compared with SGR samples,
the gene expression level of ABCA12 was assessed by RT- whereas CDSN protein was expressed at a similar level in the
qPCR in PPR and SGR samples. According to our results, two sample groups (Table 1, Figure 2).

1940 Journal of Investigative Dermatology (2020), Volume 140

 B Medgyesi et al.
Skin Barrier Alterations of PPR

d % Associated Genes Nr. Associated Genes

Plasma membrane resorption 43,81% 46
Late envelope proteins bind cornified envelope: CDSN 43,40% 46
CASP14 cleaves filaggrin 42,99% 46
CDSN binds the cornified envelope 41,38% 36
Reinforcement of the Cornified Envelope 40,70% 35
Lamellar bodies bind the early cornified envelope 40,70% 35
Formation of the cornified envelope 40,31% 52
Cornification 35,25% 43
Cell junction organization 29,67% 27
Keratinization 24,77% 53
Keratinocyte differentiation 24,04% 88
Epidermal cell differentiation 23,81% 105
Skin development 23,81% 120
Morphogenesis of a branching epithelium 23,21% 55
Tight junction 20,47% 35

0% 5% 10% 15% 20% 25% 30% 35% 40% 45%

Downregulated genes Upregulated genes

Figure 1. Continued

Corneocyte desquamation. We measured mRNA levels for Therefore, we investigated the gene expression levels of these
skin barriererelated enzymes having a crucial role in barrier alarmins. According to our results, the mRNA levels of
desquamation (KLK5, KLK7, and KLK14). The expression KRT6, KRT16, and KRT17 were significantly upregulated in
levels for KLK5, KLK7, and KLK14 were similar in PPR versus PPR compared with SGR (Table 1, Supplementary Figure S2).
SGR samples (Table 1, Supplementary Figure S2). We Immunostaining of KRT6 confirmed our mRNA data. KRT6
immunostained for KLK5 in healthy SGR and PPR samples, was almost absent from SGR skin but highly expressed in PPR
and no significant differences between PPR and SGR were (Table 1, Figure 2).
detected (Table 1, Figure 2).
AMPs. We also examined the gene expression levels for
Tight junction formation. The mRNA levels of tight junc-
key components of the immunological barrier, including the
tion components (CLDN1, CLDN16, CLDN23, and OCLN)
following AMPs: S100A7, S100A8, S100A9, DEFB4B, LCN2,
were measured. The tight junction molecules were signifi-
and cathelicidin. All of the investigated AMPs were signifi-
cantly downregulated in PPR samples compared with healthy
cantly upregulated in PPR samples compared with healthy
SGR skin (Table 1, Supplementary Figure S2). To confirm our
SGR skin samples (Table 1, Supplementary Figure S2). To
results at the protein level, we measured CLDN1 by IHC and
confirm that the increased mRNA levels resulted in increased
detected significantly lower levels in PPR than SGR samples
protein, we measured the protein levels of S100A8 and
(Table 1, Figure 2).
LCN2. S100A8 and LCN2 protein levels were significantly
Barrier alarmins. KRT6, KRT16, and KRT17 are highly higher in PPR samples compared with controls (Table 1,
induced in response to an epidermal barrier breach. Figure 2).

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Skin Barrier Alterations of PPR

B Medgyesi et al.
Journal of Investigative Dermatology (2020), Volume 140

Table 1. Comparison of Barrier Components in PPR and SGR Skin

RNASeq (PPR vs SGR) qRT-PCR (PPR vs SGR) IHC (PPR vs SGR) ROS vs HC literature AD vs HC literature

protein/ protein/
Variable P-Value FC P-Value FC1 P-Value FC1 mRNA data (Ref.) mRNA data (Ref.)

Cornified envelope
formation
FLG NS 2.60E-03 2.95Y NS NS mRNA (Deng et al., 2019) Y prot. and (Brunello, 2018;
mRNA De Benedetto et al., 2011;
Ghosh et al., 2015;
Pellerin et al., 2013;
Sugiura et al., 2005)
KRT1 3.28E-03 2.86Y <1.00E-4 2.20Y 4.80E-03 2.22Y # Y prot. and mRNA Y prot. and (Sugiura et al., 2005;
KRT10 3.28E-03 2.52Y 9.00E-04 2.75Y Nd NS mRNA (Deng et al., 2019) mRNA Totsuka et al., 2017)
LCE1D 2.32E-03 3.57Y 3.42E-02 1.97Y Nd # Y mRNA Y mRNA (De Benedetto et al., 2011)
LCE1F NS NS Nd # NS mRNA
LOR 8.65E-03 2.95Y 1.16E-02 2.03Y 2.11E-02 1.81Y NS mRNA (Deng et al., 2019) Y mRNA (De Benedetto et al., 2011;
Ghosh et al., 2015;
Sugiura et al., 2005)
SPRR1A 1.17E-02 3.26[ NS Nd # NS mRNA [ mRNA (Sugiura et al., 2005)
SPRR2A 1.63E-03 8.64[ 2.59E-02 4.96[ Nd [ mRNA [ mRNA (De Benedetto et al., 2011;
Sugiura et al., 2005)
TGM1 NS NS Nd # NS mRNA no available data
TGM3 NS NS Nd # NS mRNA no available data
TGM5 NS NS NS # NS prot. and mRNA no available data
Intercellular lipid
lamellae formation
ABCA12B NS 1.00E-03 2.09Y Nd # Y mRNA no available data
Corneodesmosome
organization
CDH1 3.57E-02 1.38Y 5.10E-03 1.60Y Nd # Y mRNA no available data
CDSN 3.57E-02 1.92Y NS NS # NS mRNA no available data
DSC1 1.13E-03 2.49Y 1.50E-03 4.03Y Nd # Y mRNA Y prot. (Totsuka et al., 2017)
DSG1 2.32E-03 1.85Y <1.00E-4 5.76Y 1.60E-03 2.86Y # Y prot. and mRNA
PKP1 1.63E-03 2.79Y 5.00E-04 2.19Y Nd # Y mRNA no available data
Corneocyte
desquamation
KLK5 8.65E-03 1.69Y NS Nd [ exp. and (Deng et al., 2019; NS prot. (Brunello, 2018;
act. Yamasaki and Gallo, 2011) Rawlings and Voegeli, 2013)
KLK7 NS NS Nd # NS mRNA NS prot. (Brunello, 2018;
Igawa et al., 2017)
KLK14 NS NS Nd # NS mRNA NS prot. and (Brunello, 2018;
mRNA Rawlings and Voegeli, 2013)
(continued )
 Table 1. Continued
RNASeq (PPR vs SGR) qRT-PCR (PPR vs SGR) IHC (PPR vs SGR) ROS vs HC literature AD vs HC literature

protein/ protein/
Variable P-Value FC P-Value FC1 P-Value FC1 mRNA data (Ref.) mRNA data (Ref.)
Tight junction
formation
CLDN1 7.78E-04 2.22Y 1.30E-03 3.75Y 2.67E-02 1.91Y Y prot. and (Deng et al., 2019) Y mRNA (De Benedetto et al., 2011)
mRNA
CLDN16 1.17E-02 1.68Y <1.00E-4 3.44Y Nd # Y mRNA NS mRNA
CLDN23 1.13E-03 3.28Y 1.26E-02 2.56Y Nd # Y mRNA Y mRNA
OCLN NS <1.00E-4 3.04Y Nd # Y mRNA NS prot. and
mRNA
Barrier alarmins
KRT6A 4.57E-03 5.03[ 3.70E-03 4.92[ 1.00E-03 109.28[ # [ prot. and mRNA [ mRNA (Ghosh et al., 2015;
KRT16 1.57E-02 2.50[ 6.00E-04 9.76[ Nd # [ mRNA [ mRNA Sugiura et al., 2005)
KRT17 8.65E-03 4.16[ 1.28E-02 3.26[ Nd # [ mRNA [ mRNA (Sugiura et al., 2005)
AMPs
DEFB4B 7.78E-04 84.35[ 3.82E-02 62.35[ Nd # [ mRNA NS mRNA (Sugiura et al., 2005)
CAMP 4.58E-02 2.91[ 3.44E-02 7.90[ Nd [ prot. and (Yamasaki et al., 2007) controversial (Ballardini et al., 2009;
mRNA Gambichler et al., 2008;
Patra et al., 2018)
LCN2 3.28E-03 6.93[ 3.15E-02 3.46[ 4.60E-03 3.08[ # [ prot. and mRNA no available data
S100A7 1.13E-03 19.30[ < 1.00E-4 19.68[ Nd # [ mRNA [ mRNA (De Benedetto et al., 2011;
S100A8 7.78E-04 24.75[ 2.00E-04 21.75[ 4.54E-02 2.20[ # [ prot. and mRNA [ mRNA Sugiura et al., 2005)
S100A9 7.78E-04 29.64[ 7.00E-04 18.57[ Nd # [ mRNA [ mRNA
Abbreviations: act., activity; AD, atopic dermatitis; CAMP, cathelicidin; CDSN, corneodesmosin; DSG1, desmoglein 1; exp., expression; FC, fold change; HC, healthy control; IHC, immunohistochemistry; KLK,
kallikrein; nd, not determined; NS, not significant; PPR, papulopustular rosacea; prot., protein; Ref., reference; RNASeq, RNA sequencing; ROS, rosacea; SGR, sebaceous glanderich.
Boxes represent the studied seven major groups of barrier composing molecules. The first column contains the molecules that have been investigated in our study. The second, third, and fourth columns summarize
our findings according to the applied methods: RNASeq, qRT-PCR and IHC, respectively. Eight samples were examined in each group regarding all the investigated molecules.
Available literature data about papulopustular rosacea regarding all the investigated molecules, as well as our present findings from column 1e3 are compiled in Column 5. In this column, # represents that to our
knowledge our study provides novel, previously unreported data, whereas the absence of # represents preexisting literature data. Available literature data about atopic dermatitis regarding all the investigated
molecules are referenced in column 6. Comparison of column 5 and 6 highlights the similarities between the barrier alterations of these two skin disorders.
Statistical analyses between protein and mRNA levels were determined by two-sample t-test. Bold type indicates data with significant differences.
1
Arrows indicate the direction of significant changes.

Skin Barrier Alterations of PPR

B Medgyesi et al.
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1943
 B Medgyesi et al.
Skin Barrier Alterations of PPR

SGR PPR FLG

5

Epidermal FLG levels

4

FLG 2

0
SGR skin PPR skin
(n=8) (n=8)
LOR
8

Epidermal LOR levels

*
6

4
LOR
2

0
Cornified SGR skin PPR skin
(n=8) (n=8)
envelope
Epidermal KRT1
formation 30 **

Epidermal KRT1 levels

20

KRT1 10

0
SGR skin PPR skin
(n=8) (n=8)
Epidermal TGM5
15

Epidermal TGM5 levels

10

TGM5
5

0
SGR skin PPR skin
(n=8) (n=8)
Epidermal CDSN
15

Epidermal CDNS levels

10

CDSN
5

Desmosome SGR skin PPR skin

(n=8) (n=8)
organization Epidermal DSG1

Epidermal DSG1 levels

20
*
15

DSG1 10

0
SGR skin PPR skin
(n=8) (n=8)
Epidermal CLDN1
Epidermal CLDN1 levels

20
*
15
Tight junction
CLDN1 10
formation
5

0
SGR skin PPR skin
(n=8) (n=8)
Epidermal KRT6
30
Epidermal KRT6 levels

***

20
Barrier alarmin KRT6
10

0
SGR skin PPR skin
(n=8) (n=8)
Epidermal S100A8
Epidermal S100A8 levels

50
*
40

S100A8 30
20
10
0
Antimicrobial SGR skin PPR skin
peptides (n=8) (n=8)
Epidermal LCN2
20 *
Epidermal LCN2 levels

15
LCN2
10

0
SGR skin PPR skin
(n=8) (n=8)

Figure 2. Prominent differences in the protein expression levels of skin barriererelated molecules between PPR and healthy SGR skin samples. Representative
images for immunostaining and quantification of epidermal levels of FLG, LOR, KRT1, TGM5, CDSN, DSG1, CLDN1, KRT6, S100A8, and LCN2 in SGR and
PPR skin sections. The graphs show the mean  95% confidence interval of measured protein levels (*P < 0.05; **P < 0.01; ***P < 0.001, as determined by
two-sample t-test). CDSN, corneodesmosin; DSG1, desmoglein 1; PPR, papulopustular rosacea; SGR, sebaceous glanderich. Bar ¼ 50 mm.

1944 Journal of Investigative Dermatology (2020), Volume 140

 B Medgyesi et al.
Skin Barrier Alterations of PPR

Figure 3. Highly similar barrier

alteration occurs in PPR and AD. PPR
and AD, as well as their respective
control samples (SGR and SGP), were
immunostained. Representative
images for immunofluorescent
staining of epidermal levels of LOR,
DSG1, KRT6, and S100A8 in SGR,
PPR, SGP, and AD skin sections. AD,
atopic dermatitis; DSG1,
desmoglein1; PPR, papulopustular
rosacea; SGP, sebaceous glandepoor;
SGR, sebaceous glanderich. Bar ¼ 50
mm.

Disrupted skin barrier features of PPR are similar to that of permeability barrier alterations (Darlenski et al., 2013;
AD Powell and Ni Raghallaigh, 2011). However, no detailed
analysis of skin barrier disruption in rosacea at the molecular
Literature search. As barrier alterations in PPR seemed to
level has been conducted so far. In the present study, we
be similar to that of lesional AD skin, we reviewed the
performed whole transcriptomic analysis (RNASeq) of PPR
literature on AD barrier damage and compared barrier al-
skin samples, and gene expression profiles of diseased skin
terations of the two disorders (Table 1). According to the
were compared with that of healthy SGR skin. According to
literature search, the skin barrier alterations were similar in
our findings, all major components of the skin barrier are
PPR and AD skin compared with the respective healthy
severely altered in PPR.
control skin (SGR and sebaceous glandepoor [SGP]).
An intact skin barrier is essential for maintaining homeo-
Immunofluorescent staining. To confirm similarities in skin stasis as it protects the body against external agents and mi-
barrier changes between PPR and AD, immunofluorescent crobes and provides a waterproof cover. According to the
staining was performed in PPR and AD samples and the literature, stratum corneum, the outermost layer of the
respective SGR and SGP skin specimens. According to the epidermis, and tight junctions are considered the most
immunostaining results, a cornified envelope structural important components of the skin permeability barrier
molecule (LOR) and key components of desmosomes (DSG1) (Egawa and Kabashima, 2016; Egawa and Kabashima, 2018).
and tight junctions (CLDN1) were highly downregulated in Molecules forming the stratum corneum can be further
PPR and AD samples compared with controls (Figure 3). In divided into four groups: cornified envelope formation,
contrast, the barrier alarmin KRT6 and the AMP S100A8 were intercellular lipid lamellae formation, corneodesmosome
almost absent from healthy skin types and were expressed at organization, and corneocyte desquamation (Egawa and
high levels in PPR and AD skin specimens (Figure 3). Kabashima, 2016; Egawa and Kabashima, 2018).
Notably, the expression patterns of these proteins were Cornified envelope is built up from several types of intra-
similar in PPR and AD skin (Figure 3). cellular structure proteins, including FLG, LOR, LCE, and
SPRR proteins; envoplakin; periplakin; involucrin; and KRT
DISCUSSION filaments that are derived from keratohyalin granules
Clinical features and functional studies on the affected facial (Carregaro et al., 2013; Egawa and Kabashima, 2016; Koch
skin of patients with rosacea indicate the presence of et al., 2000; Palmer et al., 2006). These proteins undergo

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Skin Barrier Alterations of PPR

cross-linking, which is catalyzed by TGM1, TGM3, and focused on mRNA expression levels of cornified envelope
TGM5 (Eckert et al., 2005). According to our results, FLG, components, including KRT10, FLG, and LOR in different
LOR, KRT1, KRT10, and LCE1D were significantly down- rosacea subtypes (Deng et al., 2019). They also studied the
regulated, whereas SPRR2A was significantly upregulated in tight junction protein CLDN1 and could detect significantly
PPR skin compared with healthy SGR skin. The significant lower gene expression and protein levels of CLDN1 in PPR
decrease in LOR and KRT1 levels was also confirmed at the samples compared with controls, in agreement with our re-
protein level. Regarding the expression of TGMs, no signifi- sults. Regarding the mRNA levels of KRT10 and LOR, the
cant difference was detected either at mRNA or protein authors of this study did not find significant differences when
levels. comparing PPR to controls, and protein levels were not
Lipid lamellae formation is also a critical event in the assessed (Deng et al., 2019). The expression of KLK5 in ro-
development of intact stratum corneum, in which lip- sacea has been studied previously by another workgroup,
oxygenases (ALOX12B and ALOXB3) and lipid transporters and its mRNA and protein levels were demonstrated to be
(ABCA12) are essential to lipid synthesis and transport (Iwai significantly increased, in parallel with elevated enzyme
et al., 2012; Kelsell et al., 2005; Krieg and Fürstenberger, activity; however, the authors did not specify which rosacea
2014). In our study, ABCA12 was significantly down- subtype had been assessed (Morizane et al., 2010; Yamasaki
regulated at the mRNA level in PPR compared with controls. and Gallo, 2011). In our study, in PPR, we could not detect
Another component of the stratum corneum is the significantly elevated KLK5 mRNA levels, but protein levels
desmosome apparatus, which is responsible for cell adhesion and enzyme activity were not assessed. Among AMPs, only
of corneocytes (Egawa and Kabashima, 2016; Egawa and the expression of cathelicidin was assessed previously in
Kabashima, 2018). Based on our findings, desmosomes are PPR, whereas data regarding hBD-2 is available only in
highly disrupted in PPR, as demonstrated by the significantly ocular rosacea (Gökçnar et al., 2019; Morizane et al., 2010;
decreased gene expression levels of CDH1, CDSN, DSC1, Yamasaki and Gallo, 2011). In line with these previous re-
DSG1, and PKP1. The significant decrease in DSG1 was also sults, in our study, cathelicidin and hBD-2 were significantly
confirmed at the protein level by IHC. upregulated in PPR as compared with SGP skin at the mRNA
In the stratum corneum, corneocytes are shed as part of an level.
event called desquamation, which is primarily regulated by As a whole, our in-depth molecular biological investiga-
KLKs (KLK5, KLK7, and KLK14) via a pH-dependent proteo- tion, using whole transcriptomic and bioinformatics analyses,
lytic cascade (Brattsand et al., 2005; Rippke et al., 2004). In qRT-PCR, and quantitative IHC, indicate that all major
this study, we detected similar gene expression levels of components of the permeability skin barrier are severely
KLK5, KLK7, and KLK14 in lesional PPR compared with disrupted in PPR, which may significantly contribute to dis-
healthy skin; however, the enzymes’ protein levels and ac- ease pathophysiology. Barrier alterations have gained far-
tivity were not examined. reaching importance in recent years. In AD, it has been
In addition to the stratum corneum, tight junctions are also proven that barrier disruption is able to induce skin inflam-
key components for the integrity of the skin barrier by sealing mation, and, in this manner, can be considered as an initiator
adjacent keratinocytes in the stratum granulosum and acting of disease pathogenesis. From this point of view, it is quite
as a barrier for water and solutes. Tight junctions are interesting that we could detect highly similar barrier alter-
composed of transmembrane proteins, particularly the ations in PPR and AD. According to our literature search and
CLDNs and OCLN (Kirschner et al., 2010; Yokouchi et al., results, these similarities included the altered expression of
2015). In our study, mRNA levels for CLDN1, CLDN16, cornified envelope components, desmosome and tight junc-
CLDN23, and OCLN were significantly decreased in PPR tion molecules, barrier alarmins, and AMPs. Moreover, we
compared with controls. The significant decrease in CLDN1 could confirm some of these similarities at the protein level
was confirmed at the protein level by IHC. by immunofluorescent staining of selected molecules. The
In parallel with the stratum corneum and tight junction demonstrated similarity between the barrier disruptions in
components, we focused on investigating two additional PPR and AD is surprising because, up until now, mainly the
groups of molecules, key barrier alarmins and AMPs, because differences in clinical features and immune composition of
the expression levels of these molecules are tightly connected these diseases have been highlighted (Figure 4).
with the level of permeability barrier structural components Although it is well known that barrier dysfunction is a
(Borkowski and Gallo, 2011; Zhang et al., 2019). In our potent initiator of inflammation in AD, on the basis of our
study, all investigated barrier alarmins (KRT6A, KRT16, and results, we cannot determine the role of barrier alterations in
KRT17) and AMPs (cathelicidin, hBD-2, LCN2, S100A7, rosacea, if it is the result or the cause of inflammation. Barrier
S100A8, and S100A9) were significantly upregulated in PPR damage may occur because of the Th1/Th17 type inflam-
skin compared with controls. The significantly higher pres- mation that is characteristic in rosacea, and IL-17A recently
ence of LCN2, S100A8, and KRT6 in PPR was confirmed at has been shown to downregulate FLG, LOR, and KRT10
the protein level. mRNA levels in organotypic three-dimensional skin equiva-
To our knowledge, until now, only one study has investi- lents (Pfaff et al., 2017). In contrast, it is also possible that
gated rosacea samples at a whole transcriptomic level. In this dysfunctional barrier is the initiating factor of rosacea
previous study, the authors focused on examining the development. Although barrier alteration is an adjuvant fac-
immunological characteristics of different rosacea subtypes, tor for Th2 type inflammation in AD, the reason why similar
and no data were published on barrier components (Buhl barrier disruption may precipitate Th1/Th17 type inflamma-
et al., 2015). Another study, performed by Deng et al., tion in rosacea could be that AD has an SGP region

1946 Journal of Investigative Dermatology (2020), Volume 140

 B Medgyesi et al.
Skin Barrier Alterations of PPR

a
SGP skin
SGR skin
Th17(β)
(no sfTSLP, low DC, Th17(β
Th17(β)
(sfTSLP, higher noninflammatory
and Treg count)
Th17(β)
DC, Th17(
Th17(ββ) and Treg counts,
IL10/IL17 milieu)
Different homeostatic
immune milieu of distinct
skin regions

b
Due to the unique immune
milieu, permeability barrier Barrier dysfunction of SGP: Barrier dysfunction of SGR:
alterations of distinct skin IfTSLP ↑, IL-33 ↑, IL-25 ↑ sfTSLP ↓, AMPs ↑
regions may initiate different
types of immune responses
and immune-mediated
AD PPR
diseases
disease of SGP skin disease of SGR skin

Immune response: Immune response:

Th2/Th22, IL-4, IL-13, IL-5 ↑ Th1/Th17(23), IFN-γ, IL-17

c
Although in AD and PPR Major differences of atopic dermatitis and papulopustular rosacea
permeability barrier
alteration is similar, the Atopic dermatitis Papulopustular rosacea
different types of immune flushing, persistent erythema,
inflammation result major papules, plaques, papulosus
Clinical features teleangiectasia, papules and
distinction between the infiltration, scaling, excoriations
pustules
two diseases
usually in childhood, less
frequently in adolescence or Age at onset in adulthood
adulthood
mainly on SGP skin Localisation on SGR skin
Th2/Th22 cells, DCs, proliferation Th1/Th17 cells, DCs, high
of KCs, high eosinophil and low Histology macrophage, mast cell and
neutrofil count neutrofil counts
high inflammatory lfTSLP loss of homeostatic sfTSLP
expression in AD skin expression in PPR

TARC+ inflammatory DCs TARC-DCs

Key players of
Th2/Th22 cells: IL-4/IL-13/IL-5/ pathomechanism Th1/Th17 T cells: IL-17/IFNγ
IL-22 /TNFα
low TLR2 expression high TLR2 expression
low AMP level high AMP level

Figure 4. The possible role of permeability barrier alteration in the pathogenesis of PPR. Until now, when comparing AD and PPR, mainly the differences in
their clinical and immunological characteristics were pronounced by dermatologists. However, based on our results, these two skin disorders can be considered
from another point of view, because both are characterized by severe and notably similar skin barrier damage, although being developed on distinct skin areas.
We propose that the prominently different steady-state immune and barrier features of (a) the skin regions (SGR and SGP skin) where these diseases prefer to
localize can influence (b) the barrier damageeconnected inflammation differently, leading to (c) region-specific immune-mediated skin diseases and explaining
the prominent distinctions between AD and PPR. AD, atopic dermatitis; AMP, antimicrobial peptide; DC, dendritic cell; KC, keratinocyte; lfTSLP, long form
thymic stromal lymphopoietin; sfTSLP, short form thymic stromal lymphopoietin; PPR, papulopustular rosacea; SGP, sebaceous glandepoor; SGR, sebaceous
glanderich; Th, T helper; Th17(b), noninflammatory Th17 cell; Th17(23), inflammatory Th17 cell; TLR2, toll-like receptor 2; Treg, regulatory T cell.

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 B Medgyesi et al.
Skin Barrier Alterations of PPR

preference, whereas rosacea localizes exclusively on SGR autoclaved metal beads (Qiagen). RNA concentration and purity were
skin. SGR and SGP skin areas have prominently different measured and cDNA was synthesized using the High Capacity cDNA
homeostatic immune and barrier characteristics (Béke et al., Archive Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA).
2018; Dajnoki et al., 2017). Whereas SGR skin regions
RT-qPCR
dispose significantly higher AMP levels, noninflammatory
RT-PCR was carried out in triplicate using pre-designed MGB assays
Th17(b) and regulatory T-cell counts, and constitutive
(Thermo Fisher Scientific). The assessed TaqMan Gene Expression
expression of homeostatic short form thymic stromal lym-
assays are listed in Supplementary Materials and Methods. All re-
phopoietin (sfTSLP), SGP skin is characterized by low AMP
actions were performed with an ABI PRISM 7000 Sequence Detec-
levels and T-cell counts, without TSLP presence under steady-
tion System. Relative mRNA levels were calculated using the 2-DDCt
state (Béke et al., 2018; Dajnoki et al., 2017). These features
method normalized to the expression of PPIA mRNA.
of the skin seem to be very similar to that of another barrier,
namely the gut, where the regional immune-related differ- RNASeq analysis
ences are well known. Th17(b) cells are enriched and sfTSLP Complementary DNA library for RNASeq was generated from 1 mg
is expressed in the small intestine, in contrast to the colon, total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San
where Th17(b) cells are absent and sfTSLP is present only in Diego, CA) according to the manufacturer’s protocol. A single read
the proximal part (Iliev et al., 2009; Rimoldi et al., 2005). It 50 base pair sequencing run was performed on Illumina HiScan SQ
has been raised that these unique immune and barrier char- instrument (Illumina), and 16e18 million reads per sample were
acteristics of different gut sections can lead to distinct types of obtained. Sequenced reads were aligned to Human Genome v19
immune-mediated diseases. In Crohn’s disease, the loss of using TopHat and Cufflinks algorithms and bam files were generated.
sfTSLP from the small intestine can promote the activation of StrandNGS software was used for further statistical analysis. To
inflammatory Th1/Th17 cells (Iliev et al., 2009; Rimoldi et al., identify statistically significant gene expression patterns, nonpara-
2005), whereas in ulcerative colitis, significantly increased metric WilcoxoneManneWhitney test was used. Library prepara-
inflammatory long form TSLP levels initiate Th2 type tions, sequencing, and data analysis were performed at the Genomic
inflammation in the colon (Fornasa et al., 2015). On the basis Medicine and Bioinformatics Core Facility of University of Debre-
of these findings, we hypothesize that similar barrier alter- cen. RNA sequencing data of our samples were deposited to the
ations of SGR and SGP skin regions, because of their different Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/
homeostatic immune and barrier milieu, initiate distinct sra), under accession numbers PRJNA421246 and PRJNA592080.
immune-mediated skin diseases with unique clinical features
Pathway analyses
driven by different Th subsets (AD on SGP and PPR on SGR
Multiple bioinformatics analyses were performed by Cytoscape
areas) (Figure 4). Notably, De Benedetto et al. already raised
ClueGO bioinformatics tool. Pathway enrichment analyses were
the possibility that barrier defects can lead to the production
performed on all DEGs with fold change  1.5. The details of our
of keratinocyte-derived mediators including pro-Th2 and pro-
analytical approach can be found in the Supplementary Materials
Th17 types that affect the characteristics of Th response (De
and Methods.
Benedetto et al., 2012).
To determine if PPR barrier damage is the initiator of the dis- Immunohistochemistry
ease or a consequence of manifested inflammation, the exact For IHC analyses, paraffin-embedded sections from patients and
time of barrier disruption should be studied and a detailed healthy controls were deparaffinized. Heat-induced antigen retrieval
analysis of the perilesional and/or nonlesional skin of patients was performed. Sections were stained with primary antibodies listed
with PPR should be performed in the future. Moreover, analo- in Supplementary Materials and Methods. Subsequently, anti-mouse/
gous experiments are needed in other subtypes of rosacea. rabbit horseradish peroxidaseeconjugated secondary antibody
In summary, our results unambiguously prove the presence (Biogenex, Fremont, CA) was employed. Staining was detected with
of severe barrier alterations in the facial skin of patients with the Vector NovaRed Kit (Vector Laboratories, Burlingame, CA).
PPR; thus, we suggest that skin barrier restoring therapies Sections were counterstained with methylene green and digitized by
should be incorporated into clinical guidelines for rosacea Whole Slide Imaging, and Pannoramic Viewer software was used for
management, similar to that of AD. the evaluation of the degree of staining of the slides.

Statistical analysis
MATERIALS AND METHODS Data distribution was analyzed by Kolmogorov-Smirnov test.
Skin biopsies Because our data showed normal distribution, two groups of sam-
2
Skin punch biopsies (0.5e1 cm ) were taken from normal SGR skin ples were compared statistically by two-sample t-test. Differences
sites of eight healthy individuals (mean age  SD: 56.25  13.11 between the groups were demonstrated using mean  95% confi-
years) undergoing plastic surgery and from lesional skin of eight dence interval. P-values < 0.05 were considered statistically sig-
patients with PPR (mean age  SD: 59.38  13.59 years) nificant (*P < 0.05; **P < 0.01; ***P < 0.001). Statistical data was
(Supplementary Table S4). Written informed consent according to analyzed using GraphPad Prism v6 (GraphPad Software Inc., La
the Declaration of Helsinki principles was obtained by all in- Jolla, CA) and SPSS 25 (SPSS package for Windows, Chicago, IL).
dividuals before participating in the study. The study was approved Additional details are provided in the Supplementary Information
by the local ethics committee of the University of Debrecen. online.

RNA isolation and reverse transcription Data availability statement

Samples were homogenized in Tri reagent (Sigma-Aldrich, St. Louis, The data that support the findings of this study are available from the
MO) with Tissue Lyser (Qiagen, Hilden, Germany) using previously corresponding author upon reasonable request.

1948 Journal of Investigative Dermatology (2020), Volume 140

 B Medgyesi et al.
Skin Barrier Alterations of PPR

ORCIDs De Benedetto A, Rafaels NM, McGirt LY, Ivanov AI, Georas SN, Cheadle C,
Barbara Medgyesi: https://orcid.org/0000-0001-8800-7693 et al. Tight junction defects in patients with atopic dermatitis. J Allergy Clin
Zsolt Dajnoki: https://orcid.org/0000-0002-7787-1002 Immunol 2011;127:773e86.e1e7.
Gabriella Béke: https://orcid.org/0000-0003-3113-2287 Deng Z, Chen M, Xie H, Jian D, Xu S, Peng Q, et al. Claudin reduction
Krisztián Gáspár: https://orcid.org/0000-0001-6530-7052 may relate to an impaired skin barrier in rosacea. J Dermatol 2019;46:
Imre L} orinc Szabó: https://orcid.org/0000-0002-9628-4372 314e21.
Eszter Anna Janka: https://orcid.org/0000-0003-0724-5281 Eckert RL, Sturniolo MT, Broome AM, Ruse M, Rorke EA. Transglutaminase
Szilárd Póliska: https://orcid.org/0000-0002-9722-251X function in epidermis. J Invest Dermatol 2005;124:481e92.
Zoltán Hendrik: https://orcid.org/0000-0001-8546-2148
Gábor Méhes: https://orcid.org/0000-0003-2503-7451 Egawa G, Kabashima K. Multifactorial skin barrier deficiency and atopic
Dániel Tör}ocsik: https://orcid.org/0000-0002-6094-6266 dermatitis: essential topics to prevent the atopic march. J Allergy Clin
Tamás Bı́ró: https://orcid.org/0000-0002-3770-6221 Immunol 2016;138:350e8.e1.
Anikó Kapitány: https://orcid.org/0000-0003-4540-5258 Egawa G, Kabashima K. Barrier dysfunction in the skin allergy. Allergol Int
Andrea Szegedi: https://orcid.org/0000-0003-2109-9014 2018;67:3e11.
Fornasa G, Tsilingiri K, Caprioli F, Botti F, Mapelli M, Meller S, et al. Di-
CONFLICT OF INTEREST
chotomy of short and long thymic stromal lymphopoietin isoforms in in-
The authors state no conflict of interest.
flammatory disorders of the bowel and skin. J Allergy Clin Immunol
2015;136:413e22.
ACKNOWLEDGMENTS
Gallo RL, Granstein RD, Kang S, Mannis M, Steinhoff M, Tan J, et al.
The publication is supported by Hungarian Research Grant (NKFIH K-128250
Rosacea comorbidities and future research: the 2017 update by the
and NKFIH PD-131689) and the GINOP-2.3.2-15-2016-00050 and EFOP-
National Rosacea Society Expert Committee. J Am Acad Dermatol
3.6.1-16-2016-00022 projects. The project is cofinanced by the European
2018a;78:167e70.
Union and the European Regional Development Fund and the European
Social Fund. This project was supported by the János Bolyai Research Gallo RL, Granstein RD, Kang S, Mannis M, Steinhoff M, Tan J, et al. Standard
Scholarship of the Hungarian Academy of Sciences (AK and DZ) and is also classification and pathophysiology of rosacea: the 2017 update by the
supported by the ÚNKP-18-4 New National Excellence Program of the National Rosacea Society Expert Committee. J Am Acad Dermatol
Ministry of Human Capacities (AK) and the ÚNKP-19-4 New National 2018b;78:148e55.
Excellence Program of the Ministry for Innovation and Technology (AK and Gambichler T, Skrygan M, Tomi NS, Othlinghaus N, Brockmeyer NH,
DZ). Altmeyer P, et al. Differential mRNA expression of antimicrobial peptides
and proteins in atopic dermatitis as compared to psoriasis vulgaris and
SUPPLEMENTARY MATERIAL healthy skin. Int Arch Allergy Immunol 2008;147:17e24.
Supplementary material is linked to the online version of the paper at www. Ghosh D, Ding L, Sivaprasad U, Geh E, Biagini Myers J, Bernstein JA,
jidonline.org, and at https://doi.org/10.1016/j.jid.2020.02.025. et al. Multiple transcriptome data analysis reveals biologically relevant
atopic dermatitis signature genes and pathways. PLOS ONE 2015;10:
e0144316.
REFERENCES
Gökçnar NB, Karabulut AA, Onaran Z, Yumus‚ak E, Budak Yldran FA. Elevated
Ballardini N, Johansson C, Lilja G, Lindh M, Linde Y, Scheynius A, et al.
tear human neutrophil peptides 1e3, human beta defensin-2 levels and
Enhanced expression of the antimicrobial peptide LL-37 in lesional skin of
conjunctival cathelicidin LL-37 gene expression in ocular rosacea. Ocul
adults with atopic eczema. Br J Dermatol 2009;161:40e7.
Immunol Inflamm 2019;27:1174e83.
Béke G, Dajnoki Z, Kapitány A, Gáspár K, Medgyesi B, Póliska S, et al.
Igawa S, Kishibe M, Minami-Hori M, Honma M, Tsujimura H, Ishikawa J,
Immunotopographical differences of human skin. Front Immunol 2018;9:
et al. Incomplete KLK7 secretion and upregulated LEKTI expression un-
424.
derlie hyperkeratotic stratum corneum in atopic dermatitis. J Invest Der-
Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, et al. matol 2017;137:449e56.
ClueGO: a Cytoscape plug-in to decipher functionally grouped gene
Iliev ID, Spadoni I, Mileti E, Matteoli G, Sonzogni A, Sampietro GM, et al.
ontology and pathway annotation networks. Bioinformatics 2009;25:
Human intestinal epithelial cells promote the differentiation of tolerogenic
1091e3.
dendritic cells. Gut 2009;58:1481e9.
Borkowski AW, Gallo RL. The coordinated response of the physical and
Iwai I, Han H, den Hollander L, Svensson S, Ofverstedt LG, Anwar J,
antimicrobial peptide barriers of the skin. J Invest Dermatol 2011;131:
et al. The human skin barrier is organized as stacked bilayers of
285e7.
fully extended ceramides with cholesterol molecules associated
Brattsand M, Stefansson K, Lundh C, Haasum Y, Egelrud T. A proteolytic with the ceramide sphingoid moiety. J Invest Dermatol 2012;132:
cascade of kallikreins in the stratum corneum. J Invest Dermatol 2005;124: 2215e25.
198e203.
Jenei A, Dajnoki Z, Medgyesi B, Gáspár K, Béke G, Kinyó Á, et al. Apocrine
Brunello L. Atopic dermatitis. Nat Rev Dis Primers 2018;4:2. gland-rich skin has a non-inflammatory IL-17-related immune milieu, that
Buechner SA. Rosacea: an update. Dermatology 2005;210:100e8. turns to inflammatory IL-17-mediated disease in hidradenitis suppurativa.
J Invest Dermatol 2019;139:964e8.
Buhl T, Sulk M, Nowak P, Buddenkotte J, McDonald I, Aubert J, et al. Mo-
lecular and morphological characterization of inflammatory infiltrate in Kelsell DP, Norgett EE, Unsworth H, Teh MT, Cullup T, Mein CA, et al. Mu-
rosacea reveals activation of Th1/Th17 pathways. J Invest Dermatol tations in ABCA12 underlie the severe congenital skin disease harlequin
2015;135:2198e208. ichthyosis. Am J Hum Genet 2005;76:794e803.
Carregaro F, Stefanini AC, Henrique T, Tajara EH. Study of small proline-rich Kirschner N, Houdek P, Fromm M, Moll I, Brandner JM. Tight junctions form a
proteins (SPRRs) in health and disease: a review of the literature. Arch barrier in human epidermis. Eur J Cell Biol 2010;89:839e42.
Dermatol Res 2013;305:857e66. Koch PJ, de Viragh PA, Scharer E, Bundman D, Longley MA, Bickenbach J,
Crawford GH, Pelle MT, James WD. Rosacea: I. Etiology, pathogenesis, and et al. Lessons from loricrin-deficient mice: compensatory mechanisms
subtype classification. J Am Acad Dermatol 2004;51:327e41:quiz 42e4. maintaining skin barrier function in the absence of a major cornified en-
velope protein. J Cell Biol 2000;151:389e400.
Dajnoki Z, Béke G, Kapitány A, Mócsai G, Gáspár K, Rühl R, et al. Sebaceous
gland-rich skin is characterized by TSLP expression and distinct immune Krieg P, Fürstenberger G. The role of lipoxygenases in epidermis. Biochim
surveillance which is disturbed in rosacea. J Invest Dermatol 2017;137: Biophys Acta 2014;1841:390e400.
1114e25. Morizane S, Yamasaki K, Kabigting FD, Gallo RL. Kallikrein expression and
Darlenski R, Kazandjieva J, Tsankov N, Fluhr JW. Acute irritant threshold cathelicidin processing are independently controlled in keratinocytes by
correlates with barrier function, skin hydration and contact hypersensitivity calcium, vitamin D(3), and retinoic acid. J Invest Dermatol 2010;130:
in atopic dermatitis and rosacea. Exp Dermatol 2013;22:752e3. 1297e306.
De Benedetto A, Kubo A, Beck LA. Skin barrier disruption: a requirement for Palmer CN, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, et al.
allergen sensitization? J Invest Dermatol 2012;132:949e63. Common loss-of-function variants of the epidermal barrier protein filaggrin

www.jidonline.org 1949
 B Medgyesi et al.
Skin Barrier Alterations of PPR

are a major predisposing factor for atopic dermatitis. Nat Genet 2006;38: Sugiura H, Ebise H, Tazawa T, Tanaka K, Sugiura Y, Uehara M, et al. Large-
441e6. scale DNA microarray analysis of atopic skin lesions shows overexpression
Patra V, Mayer G, Gruber-Wackernagel A, Horn M, Lembo S, Wolf P. Unique of an epidermal differentiation gene cluster in the alternative pathway and
profile of antimicrobial peptide expression in polymorphic light eruption lack of protective gene expression in the cornified envelope. Br J Dermatol
lesions compared to healthy skin, atopic dermatitis, and psoriasis. Photo- 2005;152:146e9.
dermatol Photoimmunol Photomed 2018;34:137e44. Totsuka A, Omori-Miyake M, Kawashima M, Yagi J, Tsunemi Y. Expression of
Pellerin L, Henry J, Hsu CY, Balica S, Jean-Decoster C, Méchin MC, et al. keratin 1, keratin 10, desmoglein 1 and desmocollin 1 in the epidermis:
Defects of filaggrin-like proteins in both lesional and nonlesional atopic possible downregulation by interleukin-4 and interleukin-13 in atopic
skin. J Allergy Clin Immunol 2013;131:1094e102. dermatitis. Eur J Dermatol 2017;27:247e53.
Pfaff CM, Marquardt Y, Fietkau K, Baron JM, Lüscher B. The psoriasis- Yamasaki K, Di Nardo A, Bardan A, Murakami M, Ohtake T, Coda A, et al.
associated IL-17A induces and cooperates with IL-36 cytokines to control Increased serine protease activity and cathelicidin promotes skin inflam-
keratinocyte differentiation and function. Sci Rep 2017;7:15631. mation in rosacea. Nat Med 2007;13:975e80.
Powell FC, Ni Raghallaigh S. Interventions for ’rosacea’. Br J Dermatol Yamasaki K, Gallo RL. Rosacea as a disease of cathelicidins and skin innate
2011;165:707e8. immunity. J Investig Dermatol Symp Proc 2011;15:12e5.
Rawlings AV, Voegeli R. Stratum corneum proteases and dry skin conditions. Yokouchi M, Kubo A, Kawasaki H, Yoshida K, Ishii K, Furuse M, et al.
Cell Tissue Res 2013;351:217e35. Epidermal tight junction barrier function is altered by skin inflammation,
but not by filaggrin-deficient stratum corneum. J Dermatol Sci 2015;77:
Rimoldi M, Chieppa M, Salucci V, Avogadri F, Sonzogni A, Sampietro GM, 28e36.
et al. Intestinal immune homeostasis is regulated by the crosstalk between
epithelial cells and dendritic cells. Nat Immunol 2005;6:507e14. Zhang X, Yin M, Zhang LJ. Keratin 6, 16 and 17-critical barrier alarmin
molecules in skin wounds and psoriasis. Cells 2019;8.
Rippke F, Schreiner V, Doering T, Maibach HI. Stratum corneum pH in atopic
dermatitis: impact on skin barrier function and colonization with Staphy-
lococcus aureus. Am J Clin Dermatol 2004;5:217e23.
This work is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0
Steinhoff M, Buddenkotte J, Aubert J, Sulk M, Novak P, Schwab VD, et al.
Clinical, cellular, and molecular aspects in the pathophysiology of rosacea. International License. To view a copy of this license, visit
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1950 Journal of Investigative Dermatology (2020), Volume 140

 B Medgyesi et al.
Skin Barrier Alterations of PPR

SUPPLEMENTARY MATERIALS AND METHODS random primers and SuperScript II reverse transcriptase (Life
Patients and healthy controls Technologies). Following this step, second strand cDNA was
Skin punch biopsies (0.5e1 cm2) were taken from lesional synthesized, double-stranded cDNA was end repaired and 30
facial skin of eight patients with papulopustular rosacea and ends adenylated, and Illumina index adapters were ligated.
from sebaceous glanderich (SGR) skin of eight healthy in- After adapter ligation enrichment, PCR was performed to
dividuals after obtaining written informed consent, according amplify adapter ligated cDNA fragments. Fragment size dis-
to the Declaration of Helsinki principles (Supplementary tribution and molarity of libraries were checked on Agilent
Table S4). The study was approved by the local ethics com- BioAnalyzer DNA1000 chip (Agilent Technologies, Santa
mittee of University of Debrecen, Hungary. Clara, CA). Concentrations of RNASeq libraries were set to 10
nM and five libraries were pooled together before
Processing of skin biopsy specimens
sequencing. A single read 50 base pair sequencing run was
All biopsies were cut into two pieces. For immunohisto-
performed on Illumina HiScan SQ instrument (Illumina), and
chemistry, samples were formalin-fixed and paraffin-
16e18 million reads per sample were obtained. CASAVA
embedded, and for quantitative RT-PCR, samples were
software was used for pass filtering and demultiplexing pro-
stored in RNAlater (Qiagen, Hilden, Germany) at 70  C
cess. Sequenced reads were aligned to Human Genome v19
until RNA isolation.
using TopHat and Cufflinks algorithms and bam files were
RNA isolation and reverse transcription generated. StrandNGS software was used for further statistical
All samples were homogenized in Tri reagent solution analysis. Bam files were imported and normalized using
(Sigma-Aldrich, St. Louis, MO) with Tissue Lyser (Qiagen) DESeq algorithm. To identify statistically significant gene
using previously autoclaved metal beads (Qiagen). The con- expression patterns between conditions, nonparametric
centration and purity of the RNA were measured by means of WilcoxoneManneWhitney test was used.
NanoDrop spectrophotometer (Thermo Fisher Scientific, Library preparations, sequencing, and data analysis were
Bioscience, Waltham, MA), and its quality was checked using performed at the Genomic Medicine and Bioinformatics Core
an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Facility of University of Debrecen.
Clara, CA). For RT-PCR, cDNA was synthesized from the A part of our RNASeq data (SGR1eSGR6 samples) has
isolated RNA using the High Capacity cDNA Archive Kit been previously published and deposited to the Sequence
(Invitrogen, Life Technologies, Carlsbad, CA). Read Archive database (https://www.ncbi.nlm.nih.gov/sra),
under accession number PRJNA421246, whereas SGR7,
RT-qPCR
SGR8, and PPR1ePPR8 samples are available in the
RT-PCR was carried out in triplicate using pre-designed MGB
Sequence Read Archive database (https://www.ncbi.nlm.nih.
assays ordered from Applied Biosystems (Life Technologies).
gov/sra), under accession number PRJNA592080.
The following TaqMan Gene Expression assays were used:
PPIA (Hs99999904_m1), FLG (Hs00856927_g1), KRT1
Pathway analyses
(Hs00196158_m1), KRT6A (Hs01699178_g1), KRT10
To identify the function of the aforementioned differentially
(Hs00166289_m1), KRT16 (Hs00373910_g1), KRT17
expressed genes (DEGs), multiple bioinformatics analyses
(Hs00356958_m1), LCE1D (Hs04224967_gH), LCE1F
were performed by Cytoscape ClueGO bioinformatics tool
(Hs00820275_sH), LOR (Hs01894962_s1), CLDN1
using Gene Ontology Biological Process, Immune System
(Hs00221623_m1), CLDN16 (Hs00198134_m1), CLDN23
Process, and Molecular Function; Kyoto Encyclopedia of
(Hs01013638_s1), OCLN (Hs00170162_m1), CDH1
Genes and Genomes and Kyoto Encyclopedia of Genes and
(Hs01023895_m1), DSC1 (Hs00245189_m1), CDSN
Genomes Compound; Reactome Pathways; and Reactome
(Hs00169911_m1), DSG1 (Hs00355084_m1), TGM1
Reactions databases.
(Hs00165929_m1), TGM3 (Hs00162752_m1), TGM5
First, to identify the general biological function of DEGs,
(Hs00909973_m1), KLK5 (Hs01548153_m1), KLK7
we performed a pathway enrichment analysis on all DEGs
(Hs00192503_m1), KLK14 (Hs00983577_m1), SPRR1A
with fold change  1.5. To reveal the significantly enriched (P
(Hs00954595_s1), SPRR2A (HS03046643_s1), ABCA12
 0.05) terms and pathways, the following criterion was
(Hs00292421_m1), PKP1 (Hs00240873_m1), S100A7
applied: all terms should have contained at least 50 genes
(Hs00161488_m1), S100A8 (Hs00374264_g1), S100A9
from our input gene set.
(Hs00610058_m1), LCN2 (Hs01008571_m1), DEFB4B
Then, we performed a second, stricter pathway enrichment
(hBD-2) (Hs00175474_m1), TSLP (Hs00263639_m1), and
analysis to find out the function of barrier-related significant
LL37 (CAMP) (Hs00189038_m1). All reactions were per-
DEGs with fold change  1.5 in more detail; thus, a different
formed with an ABI PRISM 7000 Sequence Detection System.
analytical approach was applied. Up- and downregulated
Relative mRNA levels were calculated using the 2-DDCt
DEGs were subjected as two different clusters to a more
method normalized to the expression of PPIA mRNA.
detailed pathway enrichment analysis by ClueGO. According
RNA sequencing (RNASeq) analysis to the fact that most DEGs were upregulated (approximately
Complementary DNA library for RNASeq was generated 61%), the criteria of the analysis were different in the cases of
from 1 mg total RNA using TruSeq RNA Sample Preparation up- and downregulated gene sets. Regarding the cluster of
Kit (Illumina, San Diego, CA) according to the manufacturer’s upregulated genes, terms should have contained at least 30
protocol. Briefly, poly-A tailed RNAs were purified by genes from our input gene set and at least 20% of all genes
oligodT-conjugated magnetic beads and fragmented on 94  C characteristic to each term, whereas the cluster of down-
for 8 minutes, then first strand cDNA was transcribed using regulated genes required at least 18 genes from our input

www.jidonline.org 1950.e1
 B Medgyesi et al.
Skin Barrier Alterations of PPR

gene set and at least 12% of all genes characteristic to each Subsequently, the following horseradish
term. This approach allowed us not only to reveal signifi- peroxidaseeconjugated secondary antibodies were
cantly enriched specific terms and pathways but also made us employed: anti-mouse/rabbit (Biogenex, Fremont, CA).
capable of easily distinguishing and visualizing up- and Before and after incubating with antibodies, washing of
downregulated DEGs belonging to each terms. Regarding the samples was performed for 5 minutes, 3 times in each step.
statistical approach of the enrichment analyses by Cytoscape, Staining was detected with the Vector NovaRed Kit (Vector
a P-value < 0.05 and kappa coefficient of 0.4 were consid- Laboratories, Burlingame, CA). Sections were counterstained
ered as threshold values and correction was performed by with methylene green, dehydrated, and covered with a glass
BenjaminieHochberg test. coverslip. The detection of one protein was carried out on all
sections in parallel at the same time to enable us to evaluate
Immunohistochemistry comparable protein levels. Positive, Ig, and isotype controls
For immunohistochemistry analyses, paraffin-embedded were also used to normalize staining against all proteins
sections from patients and healthy controls were deparaffi- (mouse IgG2a Kappa [Covalab, Villeurbanne, France] and
nized. Heat-induced antigen retrieval was performed, and rabbit immunoglobulin fraction [Sigma-Aldrich]).
sections were preprocessed with H2O2 for 10 minutes. Sec- The sections were digitized using Whole Slide Imaging
tions were stained with primary antibodies against human technology in the Department of Pathology, and the Pan-
CDSN (rabbit polyclonal IgG [HPA054184], Sigma-Aldrich), noramic Viewer software was used for the evaluation of the
human CLDN1 (rabbit polyclonal IgG [ab15098], Abcam, degree of staining of the slides.
Cambridge, United Kingdom), human DSG1 (rabbit poly- Statistical analysis
clonal IgG [NBP1-84567], Novus Biologicals, Centennial, Data distribution was analyzed by Kolmogorov-Smirnov test.
CO), human FLG (mouse monoclonal IgG [ab218862], Because our data showed normal distribution, two groups of
Abcam), human KRT1 (rabbit monoclonal IgG [ab185628], samples were compared statistically by two-sample t-test.
Abcam), human KRT6 (mouse monoclonal IgG [ab18586], Differences between the groups were demonstrated using
Abcam), human lipocalin/NGAL (rabbit polyclonal IgG [PA5- mean  95% confidence interval. P-values < 0.05 were
32476], Invitrogen), human LOR (rabbit monoclonal IgG considered statistically significant (*P < 0.05; **P < 0.01;
[NBP133610], Novus Biologicals), human S100A8 (rabbit ***P < 0.001). Statistical data was analyzed using GraphPad
polyclonal IgG [PA532476], Invitrogen), and human TGM5 Prism v6 (GraphPad Software Inc., La Jolla, CA) and SPSS 25
(rabbit polyclonal IgG [ab133786], Abcam). (SPSS package for Windows, Chicago, IL).

1950.e2 Journal of Investigative Dermatology (2020), Volume 140

 B Medgyesi et al.
Skin Barrier Alterations of PPR

a
Cornified envelope formation
FLG LOR KRT1 KRT10

KRT10 rel.gene expression

KRT1 rel.gene expression
**

LOR rel.gene expression

FLG rel.gene expression

0.5 0.8 20 15
* *** ***
0.4 0.6 15
10
0.3
0.4 10
0.2
5
0.1 0.2 5

0.0 0.0 0 0
SGR skin ROS SGR skin ROS SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=8) (n=8) (n=8) (n=8) (n=7) (n=8)

LCE1D LCE1F SPRR1A SPRR2A

SPRR1A rel.gene expression

SPRR2A rel.gene expression

LCE1D rel.gene expression

LCE1F rel.gene expression

1.0 0.08 0.10 0.6 *

0.8 0.06 0.08
0.4
0.6 0.06
0.04
0.4 0.04
0.2
0.2 0.02 0.02
0.0 0.00 0.00 0.0
SGR skin ROS SGR skin ROS SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=7) (n=8) (n=8) (n=8) (n=8) (n=8)

TGM1 TGM3 TGM5

TGM1 rel.gene expression

TGM3 rel.gene expression

0.20 0.08 TGM5 rel.gene expression 0.015

0.15 0.06
0.010
0.10 0.04
0.005
0.05 0.02

0.00 0.00 0.000

SGR skin ROS SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=8) (n=8) (n=8) (n=8)

b c
Intercellular lipid Desmosome organization
lamellae formation CDH1 CDSN DSC1
ABCA12 rel.gene expression

ABCA12
CDSN rel.gene expression
CDH1 rel.gene expression

DSC1 rel.gene expression

0.15 0.4 0.6 0.6 **
** **
0.3
0.10 0.4 0.4
0.2
0.05 0.2 0.2
0.1

0.00 0.0 0.0 0.0

SGR skin ROS SGR skin ROS SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=8) (n=8) (n=8) (n=8) (n=8) (n=8)

DSG1 PKP1
DSG1 rel.gene expression

PKP1 rel.gene expression

0.8 *** 0.8 ***

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=8) (n=8)

Supplementary Figure S1. Significantly altered expression of the components of cornified envelope, intercellular lamellae formation, and desmosome
organization in ROS examined by qRT-PCR. (a) Cornified envelope. (b) Intercellular lamellae formation. (c) Desmosome organization. The graphs show the
mean  95% confidence interval of measured mRNA transcript levels (*P < 0.05; **P < 0.01; ***P < 0.001, as determined by two-sample t-test). CDSN,
corneodesmosin; DSG1, desmoglein 1; ROS, papulopustular rosacea; SGR, sebaceous glanderich.

www.jidonline.org 1950.e3
 B Medgyesi et al.
Skin Barrier Alterations of PPR

a
Corneocyte desquamation
KLK5 KLK7 KLK14

KLK14 rel.gene expression

KLK5 rel.gene expression

KLK7 rel.gene expression

0.3 0.5 0.010
0.4 0.008
0.2
0.3 0.006
0.2 0.004
0.1
0.1 0.002
0.0 0.0 0.000
SGR skin ROS SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=8) (n=8) (n=8) (n=8)

b
Tight junction formation
CLDN1 CLDN16 CLDN23 OCLN
CLDN16 rel.gene expression

CLDN23 rel.gene expression

CLDN1 rel.gene expression

OCLN rel.gene expression

0.5 0.0006 0.015 0.05
*** *** ***
0.4 * 0.04
0.0004 0.010
0.3 0.03
0.2 0.02
0.0002 0.005
0.1 0.01
0.0 0.0000 0.000 0.00
SGR skin ROS SGR skin ROS SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=7) (n=8) (n=8) (n=8) (n=8) (n=8)

c
Barrier alarmins
KRT6A KRT16 KRT17
KRT6A rel.gene expression

KRT16 rel.gene expression

KRT17 rel.gene expression

4
** 1.5 3 **
3
***
1.0 2
2
0.5 1
1

0 0.0 0
SGR skin ROS SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=7) (n=8) (n=8) (n=8)

d
Antimicrobial peptides
CAMP DEFB4B LCN2
DEFB4 rel.gene expression
CAMP rel.gene expression

LCN2 rel.gene expression

0.0020 0.5 0.6

* * *
0.0015 0.4
0.4
0.3
0.0010
0.2
0.2
0.0005 0.1
0.0000 0.0 0.0
SGR skin ROS SGR skin ROS SGR skin ROS
(n=6) (n=8) (n=8) (n=8) (n=8) (n=8)

S100A7 S100A8 S100A9

S100A7 rel.gene expression

S100A8 rel.gene expression

S100A9 rel.gene expression

6 5 8
*** *** ***
4 6
4
3
4
2
2
1 2

0 0 0
SGR skin Ros SGR skin ROS SGR skin ROS
(n=8) (n=8) (n=8) (n=8) (n=8) (n=8)

Supplementary Figure S2. Significantly altered expression of the components of corneocyte desquamation, tight junctions, barrier alarmins, and antimicrobial
peptides in ROS examined by qRT-PCR. (a) Corneocyte desquamation. (b) Tight junctions. (c) Barrier alarmins. (d) Antimicrobial peptides. The graphs show the
mean  95% confidence interval of measured mRNA transcript levels (*P < 0.05; **P < 0.01; ***P < 0.001, as determined by two-sample t-test). CAMP,
cathelicidin; ROS, papulopustular rosacea; SGR, sebaceous glanderich.

1950.e4 Journal of Investigative Dermatology (2020), Volume 140

 B Medgyesi et al.
Skin Barrier Alterations of PPR

Supplementary Table S4. Characteristics of the

Studied Skin Samples of Healthy Controls and Patients
with PPR
SGR skin samples (n [ 8)

Healthy individuals Sex Age Localization

SGR1 F 77 Hairy scalp

SGR2 M 62 Mandibula
SGR3 F 57 Nose
SGR4 F 61 Nose
SGR5 F 42 Scapula
SGR6 F 38 Chin
SGR7 M 47 Face
SGR8 M 66 Face
Mean age  SD 56.25  13.11
Rosacea skin samples (n [ 8)

PPR individuals Sex Age Localization

PPR1 F 36 Forehead
PPR2 F 53 Nose
PPR3 F 60 Chin
PPR4 M 79 Nose
PPR5 M 71 Nose
PPR6 F 69 Forehead
PPR7 F 57 Forehead
PPR8 F 50 Chin
Mean age  SD 59.38  13.59
Abbreviations: F, female; M, male; PPR, papulopustular rosacea; SGR,
sebaceous glanderich.

www.jidonline.org 1950.e5