ArticlePDF Available

Abstract and Figures

Background: Petrolatum is a common moisturizer often used in the prevention of skin infections after ambulatory surgeries and as a maintenance therapy of atopic dermatitis (AD). However, the molecular responses induced by petrolatum in the skin have never been assessed. Objective: We sought to define the cutaneous molecular and structural effects induced by petrolatum. Methods: Thirty-six healthy subjects and 13 patients with moderate AD (mean SCORAD score, 39) were studied by using RT-PCR, gene arrays, immunohistochemistry, and immunofluorescence performed on control skin, petrolatum-occluded skin, and skin occluded with a Finn chamber only. Results: Significant upregulations of antimicrobial peptides (S100A8/fold change [FCH], 13.04; S100A9/FCH, 11.28; CCL20/FCH, 8.36; PI3 [elafin]/FCH, 15.40; lipocalin 2/FCH, 6.94, human β-defensin 2 [DEFB4A]/FCH, 4.96; P < .001 for all) and innate immune genes (IL6, IL8, and IL1B; P < .01) were observed in petrolatum-occluded skin compared with expression in both control and occluded-only skin. Application of petrolatum also induced expression of key barrier differentiation markers (filaggrin and loricrin), increased stratum corneum thickness, and significantly reduced T-cell infiltrates in the setting of "normal-appearing" or nonlesional AD skin, which is known to harbor barrier and immune defects. Conclusions: Petrolatum robustly modulates antimicrobials and epidermal differentiation barrier measures. These data shed light on the beneficial molecular responses of petrolatum in barrier-defective states, such as AD and postoperative wound care.
Content may be subject to copyright.
Petrolatum: Barrier repair and antimicrobial responses
underlying this ‘‘inert’’ moisturizer
Tali Czarnowicki, MD,
a
* Dana Malajian, BA,
a,b
* Saakshi Khattri, MD,
a,c
* Joel Correa da Rosa, PhD,
a,d
Riana Dutt, ScB,
a,c
Robert Finney, MD,
e
Nikhil Dhingra, MD,
c
Peng Xiangyu, MSc,
a,c
Hui Xu, MSc,
a,c
Yeriel D. Estrada, BS,
c
Xiuzhong Zheng, MSc,
a
Patricia Gilleaudeau, NP,
a
Mary Sullivan-Whalen, NP,
a
Mayte Suar
ez-Fari~
nas, PhD,
a,c,g,h,i
Avner Shemer, MD,
f
James G. Krueger, MD, PhD,
a
and Emma Guttman-Yassky, MD, PhD
a,c
New York, NY, Philadelphia,
Pa, and Tel Aviv, Israel
Background: Petrolatum is a common moisturizer often used in
the prevention of skin infections after ambulatory surgeries and
as a maintenance therapy of atopic dermatitis (AD). However,
the molecular responses induced by petrolatum in the skin have
never been assessed.
Objective: We sought to define the cutaneous molecular and
structural effects induced by petrolatum.
Methods: Thirty-six healthy subjects and 13 patients with
moderate AD (mean SCORAD score, 39) were studied by using
RT-PCR, gene arrays, immunohistochemistry, and
immunofluorescence performed on control skin, petrolatum-
occluded skin, and skin occluded with a Finn chamber only.
Results: Significant upregulations of antimicrobial peptides
(S100A8/fold change [FCH], 13.04; S100A9/FCH, 11.28;
CCL20/FCH, 8.36; PI3 [elafin]/FCH, 15.40; lipocalin 2/FCH,
6.94, human b-defensin 2 [DEFB4A]/FCH, 4.96; P< .001 for all)
and innate immune genes (IL6,IL8, and IL1B;P< .01) were
observed in petrolatum-occluded skin compared with
expression in both control and occluded-only skin. Application
of petrolatum also induced expression of key barrier
differentiation markers (filaggrin and loricrin), increased
stratum corneum thickness, and significantly reduced T-cell
infiltrates in the setting of ‘‘normal-appearing’’ or nonlesional
AD skin, which is known to harbor barrier and immune defects.
Conclusions: Petrolatum robustly modulates antimicrobials and
epidermal differentiation barrier measures. These data shed
light on the beneficial molecular responses of petrolatum in
barrier-defective states, such as AD and postoperative wound
care. (J Allergy Clin Immunol 2015;nnn:nnn-nnn.)
Key words: Petrolatum, moisturizer, occlusion, patch tests,
antimicrobial peptides, innate immunity, atopic dermatitis, barrier,
skin surgeries
Petrolatum, available since 1872,
1
is a widely used moisturizer
consisting mainly of long-chain aliphatic hydrocarbons.
2
It has
been shown to decrease transepidermal water loss (TEWL) in
healthy
3
and irritated
4,5
human skin. Although petrolatum is
formally classified as an occlusive, it has also been described
using the broader term ‘‘moisturizer,’ (a category that includes
occlusives, as well as humectants and emollients), which reflects
this therapeutic quality.
6
Petrolatum has been proposed to protect against postambula-
tory surgical skin infections and is widely used after minor
surgical procedures.
7
A large randomized trial of postoperative
ambulatory surgery patients found petrolatum to be equivalent
to bacitracin, a topical antibiotic commonly used in the
prevention of infections.
8
Importantly, petrolatum rarely induces
allergic contact dermatitis (ACD) reactions
9
and has never been
reported to cause contact anaphylaxis, whereas bacitracin was
shown to induce ACD in up to 13% of patients
10
and has caused
contact anaphylaxis in several cases.
11-15
However, the molecular
changes induced by petrolatum are unknown.
Lesional skin of patients with atopic dermatitis (AD) exhibits
immune and barrier defects with increased penetration of small
molecules.
16-19
This results in increased prevalence of ACD and
microbial colonization/infection in the population with AD.
20,21
Despite similar bacterial colonization in patients with AD and
those with psoriasis,
22
another common inflammatory skin
disease, significantly higher rates of skin infection were observed
only in patients with AD.
23
These differences were postulated to
result in part from significantly lower antimicrobial peptide
(AMP) responses in AD lesions compared with psoriatic
lesions.
22,24
In adult and pediatric patients with AD, moisturizers
have been shown to improve AD disease severity,
25
TEWL,
26
and
From
a
the Laboratory for Investigative Dermatology and
d
the Center for Clinical and
Translational Science, The Rockefeller University, New York;
b
Columbia University
College of Physicians and Surgeons, New York; the Departments of
c
Dermatology,
g
Population Health Science and Policy, and
h
Genetics and Genomics Science and
i
the Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine
at Mount Sinai, New York;
e
the Department of Dermatology, Jefferson Medical Col-
lege, Philadelphia; and
f
the Department of Dermatology, Tel-Hashomer Hospital, Tel
Aviv.
*These authors contributed equally to this work.
T.C. was cosponsored bythe Center for Basic and Translational Research on Disorders of
the Digestive System through the generosity of the Leona M. and Harry B. Helmsley
Charitable Trust. D.M. was supported by the American Dermatological Association’s
Medical Student Fellowship. J.G.K. was supported by grant no. 5UL1RR024143-02
from the National Center for Research Resources (NCRR), a component of the Na-
tional Institutes of Health (NIH), and the NIH Roadmap for Medical Research.
E.G.-Y. was supported by the Dermatology Foundation Physician Scientist Career
Development Award and by a CTSA grant from the Rockefeller University.
Disclosure of potential conflict of interest: J. G. Krueger has received personal fees from
and been supported by Pfizer, Janssen, Lilly, Merck, Novartis, Kadmon, Dermira,
Boehringer, and BMS; has been supported by Amgen, Innovaderm, Kyowa, and
Parexel; and has received personal fees from Serono, BiogenIdec, Delenex, AbbVie,
Sanofi, Baxter, Xenoport, and Kineta. E. Guttman-Yasskyis a board member for Sanofi
Aventis, Regeneron, Stiefel/GlaxoSmithKline, MedImmune, Celgene, Anacor, and
Leo Pharma; has received consultancy fees from Regeneron, Sanofi Aventis,
Medimmune, Celgene, Steifel/GlaxoSmithKline, Celsus, BMS, Amgen, and Drais;
and has received research support from Regeneron, Celgene, BMS, and Janssen. The
rest of the authors declare that they have no relevant conflicts of interest.
Received for publication June 24, 2015; revised August 9, 2015; accepted for publication
August 21, 2015.
Corresponding author: Emma Guttman-Yassky, MD, PhD, Department of Dermatology,
Icahn School of Medicine at Mount Sinai Medical Center, 5 E 98th St, New York, NY
10029. E-mail: eguttman@rockefeller.edu.
0091-6749/$36.00
Ó2015 American Academy of Allergy, Asthma & Immunology
http://dx.doi.org/10.1016/j.jaci.2015.08.013
1
Abbreviations used
ACD: Allergic contact dermatitis
AD: Atopic dermatitis
AHR: Aryl hydrocarbon receptor
AMP: Antimicrobial peptide
DC: Dendritic cell
DEG: Differentially expressed gene
FCH: Fold change
FLG: Filaggrin
HBD2: Human b-defensin 2
H&E: Hematoxylin and eosin
IHC: Immunohistochemistry
LCN2: Lipocalin 2
LOR: Loricrin
PI3: Peptidase inhibitor 3/elafin
QC: Quality control
SC: Stratum corneum
TEWL: Transepidermal water loss
skin capacticance,
27
as well as to reduce rates of Staphylococcus
aureus colonization.
28
Emollients have recently been shown to
effectively prevent AD development in high-risk newborns.
29,30
This study aims to uncover the molecular responses triggered
by petrolatum application that can improve clinical and barrier
measures, ultimately reducing infections in patients with
barrier-disrupted conditions, such as AD, and after cutaneous
surgeries. In 2 separate cohorts totaling 49 patients, we detected
significant upregulation of key AMPs and innate immune genes in
biopsy specimens from skin occluded with petrolatum compared
with occlusion alone and control (or nonlesional AD) skin.
Occlusion with petrolatum also resulted in altered epidermal
structure and increased expression of terminal differentiation
proteins, including filaggrin (FLG) and loricrin (LOR), which
were particularly evident when petrolatum was applied to
‘normal-appearing’’ or nonlesional AD skin.
METHODS
Patients’ characteristics and skin samples
This study included 2 cohorts under institutional review board–approved
protocols. The first cohort included 29 patients (18 female/11 male patients;
age, 19-62 years [median, 38 years]; 13 with AD/16 without AD; mean
SCORAD score, 39). These patients had petrolatum (‘‘White petrolatum’’;
Dynarex, Israel; NDC #67777-211-01) applied for 48 hours under occlusion
with a Finn chamber, with biopsy specimens taken at 72 hours (24 hours after
removal of petrolatum). A total of 2 biopsy specimens were obtained from this
cohort: petrolatum-occluded and control (nonoccluded or nonlesional in
patients with AD) skin. In patients with AD, all patches were applied on
uninvolved skin; lesional/eczematous skin biopsies were not involved in this
study. The petrolatum used in this study contained white petrolatum USP
100% wt/wt. White petrolatum often contains small amounts of antioxidants
to prevent discoloration but does not contain antimicrobial preservatives.
To evaluate occlusion effects, as well as to determine the time point of
maximal gene induction, we enrolled a second cohort including 20 healthy
volunteers (8 female/12 male subjects; age, 18-80 years [median, 50.5 years]).
These subjects had biopsy specimens taken from control skin, skin occluded
with petrolatum under a Finn chamber, and skin undergoing occlusion alone
(under a Finn chamber). For assessing the time point of maximal gene
induction, the first 5 volunteers in the second cohort had 5 biopsies performed:
control, occlusion-only and occlusion with petrolatum at 48 hours, and
occlusion-only and occlusion with petrolatum at 72 hours skin. After analysis
of preliminary kinetic data and to minimize the number of skin biopsies, the
remaining 15 volunteers had only 3 biopsies: control skin, occlusion at
72 hours, and petrolatum at 72 hours (a flow chart of the 2 cohorts is shown in
Fig 1).
Immunostaining and immunofluorescence
Immunohistochemistry (IHC) was performed on frozen tissue sections by
using anti-human mAbs against S100A8/A9, lipocalin 2 (LCN2), CCL20,
FLG, and LOR (see Table E1 in this article’s Online Repository at
www.jacionline.org). Immunofluorescence staining for neutral lipids with
Nile Red was performed, as previously reported.
31,32
Epidermal thickness
and positive cells per millimeter were quantified for IHC with ImageJ
V1.42 software (National Institutes of Health, Bethesda, Md), and
immunofluorescence was imaged with MetaView software (Visitron Systems,
Puchheim, Germany).
33
For further details, see the Methods section in this
article’s Online Repository at www.jacionline.org.
Quantitative RT-PCR and gene arrays
RNA was extracted for RT-PCR and gene arrays with EZ-PCR Core
Reagents (Life Technologies, Grand Island, NY), and custom primers were
generated (see Table E2 in this article’s Online Repository at www.jacionline.
org for primers and probes).
16,24,34-38
Expression values were normalized to
human acidic ribosomal protein (hARP). Human HGU133Plus2.0 GeneChip
probe arrays (Affymetrix, Santa Clara, Calif) were used for gene
arrays.
16,24,34-38
Total RNA was extracted with the Qiagen miRNeasy Mini
Kit (Qiagen, Valencia, Calif), and DNA was removed with the Qiagen
RNAse-free DNAse Set. Total RNA (50 ng) was reverse transcribed and
amplified with Ovation Whole Blood Solution from NuGen (San Carlos,
Calif). The labeled target was fragmented and hybridized to probe arrays by
using the Encore Biotin Module from NuGen. For further details, see the
Methods section in this article’s Online Repository.
Statistical analysis
The 2 previously described cohorts (cohort 1, 29 subjects; cohort 2, 20
subjects) were analyzed separately. Quality control (QC) of microarray gene
expression was carried out with the standard QC metrics from Quality Control
R package. Expression values from arrays and RT-PCR were modeled by using
linear mixed-effect models from the limma and nlme packages in R,
respectively. For microarray data, differentially expressed genes (DEGs) in
petrolatum were identified by the significance of moderated tstatistics after
Benjamini-Hochberg adjustment for multiple hypotheses and with a fold
change (FCH) of greater than 2. Analysis of RT-PCR gene expression
consisted of fitting the mixed-effects model followed by pairwise comparisons
among skin types: petrolatum-occluded, occluded-only, and normal skin
(cohort 2). The significance of the differences was displayed in bar plots.
The subgroup analysis for patients with and without AD (cohort 1) was carried
out by including in the mixed-effects model skin type (petrolatum-occluded or
normal skin), AD personal history (yes/no), and their interaction as fixed
effects. A random intercept was adjusted for each subject. The same approach
used for RT-PCR was applied to IHC counts (cohort 1) under the assumption
of normality. Estimating contrasts by using restricted maximum likelihood
assessed the significance of the differences between specified groups. Heat
maps were built based on the McQuitty algorithm and Euclidean distances.
More specific details can be found in the Methods section this article’s Online
Repository.
RESULTS
Our study included 2 cohorts. The first contained 29 subjects
who had biopsy specimens taken after 72 hours from control
skin (or nonlesional skin for those with AD) and skin
occluded with petrolatum for 48 hours. The second cohort
included 20 healthy volunteers with biopsy specimens taken
J ALLERGY CLIN IMMUNOL
nnn 2015
2CZARNOWICKI ET AL
after 72 hours from control, occlusion with petrolatum, and
occlusion-only sites (Fig 1).
Occlusion with petrolatum induces tissue
expression of innate immune genes
To investigate the general effects of petrolatum (in both AD and
non-AD skin), we first compared control skin versus petrolatum
patch-tested skin in cohort 1. Gene-expression profiling detected
51 DEGs in petrolatum-occluded compared with control
skin (FCH, >2; false discovery rate, <0.05). The DEGs in
petrolatum-occluded compared with control skin are presented
in a heat map (Fig 2 and Table I).
Several IL-17– and IL-22–regulated AMPs
24
were signifi-
cantly induced by petrolatum. These include human b-defensin
2 (HBD2)/DEFB4A (FCH, 4.96), LCN2 (FCH, 5.42), peptidase
inhibitor 3/elafin (PI3; FCH, 4.94), S100A8 (FCH, 2.02),
S100A9 (FCH, 7.19), CXCL1 (FCH, 3.25), and CXCL2 (FCH,
2.61; P< .05 for all). Certain T-cell and dendritic cell (DC)
markers showed mostly minor increases in petrolatum-occluded
skin (CD2, CD28, CD1b, and CCL19, a ligand of CCR7 that
marks lymphoid structures in skin).
39
Petrolatum upregulates AMPs more than occlusion
alone
To investigate whether genomic effects are specifically related
to petrolatum rather than occlusion, we recruited an additional
cohort of 20 healthy volunteers (cohort 2, Fig 1) from whom
petrolatum-occluded, occlusion-only, and control skin biopsy
specimens were obtained.
Fig 3 presents modulation of the IL-17/IL-22–induced AMPs
and selected immune genes by using RT-PCR in skin biopsy spec-
imens from petrolatum under occlusion, occlusion-only, and
control skin. Although some significant increases were observed
with occlusion alone, occlusion with petrolatum induced more
extensive and significant increases in AMP (ie, S100A7/A8/A9/
A12, LCN2, elafin/PI3, CCL20, and cathelicidin/LL37; P< .05
for all; Fig 3) and innate immune gene (ie, IL1B,IL6,IL8, and
TNFA;P< .01 for all) levels (Fig 3). Significant upregulation of
IL17,IL22, and both the p19 and p40 subunits of IL23, a known
activator of the T
H
17 pathway,
40,41
was observed in petrolatum
compared with both occluded and control skin, supporting our
microarray data from the initial cohort. This is in contrast to
IFNG (Fig 3), which showed similar expression (P>.1) in control,
occlusion-only, and petrolatum-occluded skin.
FIG 1. Study design. Two cohorts were enrolled in this study. Cohort 1 included 29 (13 patients with AD and
16 subjects without AD) subjects, and cohort 2 included 20 healthy volunteers. Patch tests were applied for
48 hours in cohort 1 and 72 hours in cohort 2, and biopsy specimens were obtained at 72 hours. Five
subjects from cohort 2 had another 3 biopsy specimens taken at 48 hours.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 3
In the first cohort petrolatum occlusion occurred for 48 hours,
with biopsies at 72 hours, whereas in the second cohort we
performed a preliminary time-effect study on the first 5 recruited
subjects to evaluate for the highest gene inductions. Although
some effects were present at 48 hours, maximal induction of
AMPs was observed at 72 hours of occlusion with petrolatum (see
Fig E1 in this article’s Online Repository at www.jacionline.org).
Therefore in the additional 15 patients in this cohort, all
subsequent biopsy specimens were taken at the 72-hour time
point only.
Increased AMP protein expression determined by
using IHC
Hematoxylin and eosin (H&E) staining and IHC for select
AMPs (S100A8/A9, LCN2, and CCL20) was performed in
control, occluded-only, and petrolatum-occluded skin biopsy
specimens obtained from patients in cohort 2.
Both occluded and petrolatum-occluded compared with
control skin demonstrated thickening of the stratum corneum
(SC), as seen by using H&E in Fig 4,A-C, and Fig E2,A, in this
article’s Online Repository at www.jacionline.org, with a nearly
2-fold increase in thickness seen with petrolatum compared
with control skin (41 mm in petrolatum vs 32 mm in occlusion
vs 24 mm in control, P< .01 for all comparisons; see Fig E2,
A). Nile Red immunofluorescence staining for neutral lipids
showed widening of the spaces between lamellar bodies most
strikingly with petrolatum occlusion, probably accounting for
the thickened SC observed with H&E staining (see Fig E2,B-D).
Increased epidermal staining with the antimicrobial markers
S100A8/A9, LCN2, and CCL20 was observed only in
petrolatum-occluded skin (Fig 4,D-L) and was most noticeable
for S100A8/A9 (Fig 4,D-F).
Neutrophil counts were measured by using neutrophil
elastase staining, with no significant increases in neutrophil
counts in either occluded-only or petrolatum-occluded skin
compared with control skin to rule out infection (rather than
colonization) as a potential inducer of AMPs in petrolatum-
occluded skin (see Fig E3 in this article’s Online Repository at
www.jacionline.org).
Differential genomic inductions by petrolatum in
patients with AD versus subjects without AD
Because cohort 1 included both patients with AD and subjects
without AD (n 529, Fig 1), we sought to evaluate whether
petrolatum exerts differential effects in these groups. Thus we
compared gene and protein expression separately among control
(nonoccluded, nonlesional) and petrolatum-occluded skin in both
populations without and with AD. Control skin in the AD group
refers to nonlesional skin. Control skin was very different in the
2 populations. In patients with AD, there were large increases
in expression of many T
H
2 pathway genes (ie, IL4,IL13,IL10,
and CCL26). AMP (S100A8-9, LCN2, and PI3) expression
FIG 2. Unsupervised hierarchic clustering of DEGs across petrolatum-occluded and control skin samples
(red, upregulated; blue, downregulated). Expression of the AMPs HBD2/DEFB4A, LCN2, elafin/PI3, S100A8,
S100A9, CXCL1, and CXCL2 was increased significantly by petrolatum occlusion. The right column shows
FCHs of petrolatum compared with control values. **P< .05 and ***P< .01.
J ALLERGY CLIN IMMUNOL
nnn 2015
4CZARNOWICKI ET AL
TABLE I. DEGs in petrolatum versus control skin through gene arrays
Probe set Symbol Gene name
lgFCH_
Petrolatum
FCH_
Petrolatum
pvals_
Petrolatum fdrs
StatusFCH2FDR0.05_
Petrolatum
1553505_at A2ML1 Alpha-2-macroglobulin-like 1 1.07 2.1 1.28E-05 0.00455785 1
206561_s_at AKR1B10 Aldo-keto reductase family 1, member B10
(aldose reductase)
1.53 2.89 4.89E-07 0.00057312 1
214490_at ARSF Arylsulfatase F 1.44 2.72 1.01E-08 6.10E-05 1
207367_at ATP12A ATPase, H1/K1transporting, nongastric, alpha
polypeptide
2.01 4.04 5.45E-05 0.01081601 1
210538_s_at BIRC3 Baculoviral IAP repeat containing 3 1.11 2.16 9.51E-13 3.46E-08 1
227736_at C10orf99 Chromosome 10 open reading frame 99 1.51 2.85 3.42E-05 0.00816811 1
209924_at CCL18 Chemokine (C-C motif) ligand 18 (pulmonary and
activation-regulated)
1.39 2.61 0.00013771 0.01750246 1
210072_at CCL19 Chemokine (C-C motif) ligand 19 1.13 2.19 2.27E-08 9.17E-05 1
207861_at CCL22 Chemokine (C-C motif) ligand 22 1.25 2.38 5.35E-06 0.00252604 1
206749_at CD1B CD1b molecule 2.03 4.08 2.62E-11 4.76E-07 1
205831_at CD2 CD2 molecule 1.05 2.08 1.22E-06 0.00106969 1
206545_at CD28 CD28 molecule 1.18 2.26 9.38E-05 0.01483149 1
205043_at CFTR Cystic fibrosis transmembrane conductance
regulator (ATP-binding cassette sub-family C,
member 7)
21.15 22.22 9.15E-05 0.01466372 21
213060_s_at CHI3L2 Chitinase 3-like 2 1.54 2.9 9.61E-08 0.00018377 1
214596_at CHRM3 Cholinergic receptor, muscarinic 3 21.21 22.32 2.63E-05 0.00703566 21
210140_at CST7 Cystatin F (leukocystatin) 1.07 2.1 1.48E-05 0.00489151 1
204470_at CXCL1 Chemokine (C-X-C motif) ligand 1 (melanoma
growth stimulating activity, alpha)
1.7 3.25 8.69E-08 0.0001832 1
209774_x_at CXCL2 Chemokine (C-X-C motif) ligand 2 1.38 2.61 4.73E-06 0.00229236 1
207356_at DEFB4A Defensin, beta 4A 2.31 4.96 0.00010607 0.01570483 1
239586_at FAM83A Family with sequence similarity 83, member A 1.11 2.15 0.00019116 0.02153566 1
1559603_at GPR12 G protein–coupled receptor 12 21.07 22.09 5.45E-05 0.01081601 21
224997_x_at H19 H19, imprinted maternally expressed transcript
(non-protein coding)
21.06 22.08 0.00027757 0.02634261 21
223541_at HAS3 Hyaluronan synthase 3 1.4 2.65 8.04E-05 0.01365243 1
220322_at IL36G Interleukin 36, gamma 1.38 2.6 0.00011757 0.0164361 1
224328_s_at LCE3D Late cornified envelope 3D 1.65 3.14 5.40E-05 0.01081601 1
212531_at LCN2 Lipocalin 2 2.44 5.42 2.11E-07 0.00033392 1
232504_at LOC285628 Uncharacterized LOC285628 1.09 2.13 5.32E-08 0.0001522 1
228648_at LRG1 Leucine-rich alpha-2-glycoprotein 1 1.45 2.74 5.92E-05 0.01137823 1
207339_s_at LTB Lymphotoxin beta (TNF superfamily, member 3) 1.05 2.07 5.61E-07 0.00062026 1
202018_s_at LTF Lactotransferrin 2.65 6.26 1.35E-08 7.03E-05 1
235672_at MAP6 Microtubule-associated protein 6 21.05 22.07 9.18E-06 0.0036279 21
204580_at MMP12 Matrix metallopeptidase 12 (macrophage elastase) 2.41 5.32 5.63E-07 0.00062026 1
212768_s_at OLFM4 Olfactomedin 4 1.8 3.49 1.31E-05 0.0046275 1
41469_at PI3 Peptidase inhibitor 3, skin-derived 2.3 4.94 4.90E-08 0.0001522 1
202988_s_at RGS1 Regulator of G-protein signaling 1 1.35 2.55 2.64E-07 0.00038338 1
1553454_at RPTN Repetin 21.03 22.04 0.00014025 0.01764038 21
205916_at S100A7 S100 calcium binding protein A7 1.22 2.34 2.04E-07 0.00033392 1
232170_at S100A7A S100 calcium binding protein A7A 3.1 8.58 5.88E-07 0.00062913 1
214370_at S100A8 S100 calcium binding protein A8 1.02 2.02 8.85E-08 0.0001832 1
203535_at S100A9 S100 calcium binding protein A9 2.85 7.19 4.33E-09 3.15E-05 1
205241_at SCO2 SCO2 cytochrome c oxidase assembly protein 1.03 2.04 2.92E-05 0.00742997 1
202376_at SERPINA3 Serpin peptidase inhibitor, clade A (alpha-1
antiproteinase, antitrypsin), member 3
1.2 2.29 0.00053038 0.03658212 1
211906_s_at SERPINB4 Serpin peptidase inhibitor, clade B (ovalbumin),
member 4
2.33 5.02 6.75E-06 0.00285278 1
229151_at SLC14A1 Solute carrier family 14 (urea transporter),
member 1 (Kidd blood group)
21.38 22.61 7.63E-06 0.00315037 21
208539_x_at SPRR2D Small proline-rich protein 2D 1.87 3.66 0.00013187 0.01736658 1
236119_s_at SPRR2G Small proline-rich protein 2G 1.72 3.29 5.26E-05 0.01075143 1
218960_at TMPRSS4 Transmembrane protease, serine 4 1.33 2.51 1.46E-05 0.00489151 1
221627_atTRIM10 Tripartite motif containing 10 1.4 2.64 1.43E-05 0.00487377 1
205844_at VNN1 Vanin 1 1.72 3.3 2.72E-06 0.00161902 1
227174_at WDR72 WD repeat domain 72 21.16 22.24 2.45E-07 0.00037126 21
218810_at ZC3H12A Zinc finger CCCH-type containing 12A 1.25 2.37 8.98E-11 1.09E-06 1
FDR, False discovery rate.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 5
levels were largely similar in non-AD and nonlesional AD
skin (Fig 5,A). Furthermore, we evaluated modulation
of genomic expression in the non-AD and AD groups
compared with their individual control subjects. Overall,
similar trends of genomic induction were observed in both
groups, but greater increases in AMP (S100A7-9, LCN2,
CCL20, and PI3) and immune gene (IL12/IL23p40,IL23p19,
IL22,IFNG, and IL17F) levels were seen in subjects without
AD (Fig 5,B). Importantly, no further increases of T
H
2 markers
were observed with petrolatum occlusion in patients with AD
(Fig 5,C).
Occlusion with petrolatum improves epidermal
differentiation
To asses epidermal differentiation changes, we performed
staining with H&E and the terminal differentiation markers FLG
and LOR on petrolatum-occluded and unoccluded skin of patients
with AD and subjects without AD (Fig 6).
As evident by using H&E staining (Fig 6,A-D), nonlesional
AD compared with non-AD skin displayed parakeratosis and
focal disruption of the granular layer. Petrolatum-occluded AD
skin demonstrated a renewed continuity of the granular layer
and restoration of orthokeratosis. Similarly, FLG and LOR
expression was inconsistent and pale in nonlesional AD skin,
with intensified and continuous expression after petrolatum
occlusion (Fig 6,G,H,K, and L). Non-AD skin also
showed increased expression of FLG and LOR in the
petrolatum-occluded compared with control skin (Fig 6,E,F,I,
and J), but the change was less evident than in the AD group
(Fig 6,E-L).
Suppression of cellular infiltrates after petrolatum
occlusion in patients with AD
We measured infiltrates of CD3
1
CD8
1
T cells and CD1c
1
DCs in nonlesional and petrolatum-occluded skin of patients
with AD and subjects without AD (see Fig E4 in this article’s
Online Repository at www.jacionline.org). Significant reductions
in CD3
1
and CD8
1
T-cell counts and CD1c
1
DC counts with
petrolatum were observed mostly (except CD8
1
) in AD but not
in non-AD skin when compared with respective control values
(P< .05 for all).
DISCUSSION
This study is the first to identify alterations in AMP expression
with the application of petrolatum, a substance previously
presumed to have no activity in the nucleated layers of the
epidermis.
5
Our findings provide novel insights into the molecular
responses induced by an over-the-counter moisturizer frequently
used by the public for everything from routine wound care to
epistaxis.
42
It has been estimated that 14 million skin excisions or biopsies
are performed annually in the United States.
43
After a pivotal
randomized controlled trial showing equivalent postoperative
infection rates for petrolatum and topical antibiotic ointment in
ambulatory surgery patients,
8
69.4% of Mohs surgeons
recommend petrolatum for wound care after routine procedures.
7
This recommended change in practice is due to several factors,
such as higher cost,
22
increased rates of ACD,
44
and even cases
of contact anaphylaxis
11-15
with use of topical antibiotic
ointments, such as bacitracin. The induction of various AMPs,
innate immune genes, and restoration of barrier responses by
FIG 3. Expression of AMPs, innate immune genes, and major cytokines, as determined by using RT-PCR, in
control, occlusion-only, and occlusion with petrolatum skin. Mean expression estimates normalized to
hARP are represented in a (log
2
[expression/hARP]) scale. Data were grouped according to the major
immune pathways represented. Increased expression of significant AMPs, innate immune genes, and
cytokines was observed with petrolatum occlusion. Pvalues are indicated if significant. *P< .1, **P< .05,
and ***P< .01.
J ALLERGY CLIN IMMUNOL
nnn 2015
6CZARNOWICKI ET AL
petrolatum, as shown in this study, provides a plausible
explanation for its efficacy in reducing postoperative infections.
In general, most AMPs are cationic, with an amphipathic
structure that allows for interaction with microbial membranes,
leading to pore formation and release of cytosolic
components.
45-47
This mechanism is used by most petrolatum-
induced AMPs in our study, including LL-37
48
and HBD2.
49,50
LL-37 is one of the most well-characterized AMPs,
51
forming
an a-helix to disrupt both bacterial membranes and viral
envelopes.
48,52
Its precursor, cathelicidin, was recently shown to
be upregulated in dermal adipocytes on infection with S aureus,
53
pointing to its importance in innate defense against this
pathogen.
54,55
Consistent with our results, LL-37 also upregulates
innate immune genes, including IL6,IL8, and IL10.
48
Conversely,
HBD2/DEFB4A has potent activity against gram-negative
bacteria, including Escherichia coli and Pseudomonas
aeruginosa, but is less effective against gram-positive
organisms.
56
Additionally, calprotectin, the antimicrobial
heterodimeric complex formed by S100A8 and S100A9, exerts
effects against S aureus,Staphylococcus epidermidis,
57
gram-
negative bacteria (E coli,Klebsiella species, and Capnocyto-
phaga sputigena),
57,58
Borellia burgdorferi,
59
and Candida
albicans.
60
Our results show that levels of the S100A8/A9
complex are remarkably increased in petrolatum-occluded skin,
FIG 4. Representative H&E (A-C) and IHC staining of S100A8/A9 (D-F), lipocalin/LCN2 (G-I), and CCL20
(J-L) in control, occluded, and petrolatum-occluded skin samples, demonstrating increased expression of
AMPs in petrolatum-occluded skin (histologic magnification 310). Note also increased thickness of the
SC in H&E-stained samples (Fig 4, A-C), with intercellular clefts most notable in petrolatum-occluded skin
(Fig 4, C).
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 7
FIG 5. Heat map showing average RT-PCR expression values for control and petrolatum AD and non-AD
groups. A, Mean RT-PCR expression values in control non-AD and control AD skin. Large T
H
2 pathway gene
(ie, IL4,IL13,IL10, and CCL26) expression increases in control skin were observed in patients with AD. Band
C, Mean RT-PCR expression values in control and petrolatum non-AD (Fig 5, B) and AD (Fig 5, C) skin,
showing greater increases in AMP (S100A7-9, LCN2, CCL20, and PI3) and immune gene (IL12/IL23p40,
IL23p19,IL22,IFNG, and IL17F) expression in subjects without AD. FCH values are shown in the columns
to the right.*P< .1, **P< .05, and ***P< .01.
FIG 6. Representative staining of H&E (A-D), LOR (E-H), and FLG (I-L) in control skin and petrolatum-
occluded skin from patients with AD and subjects without AD. AD skin shows parakeratosis and focal
disruptions of the granular layer Fig 6, C), with restoration of orthokeratosis with petrolatum (Fig 6, D).
Weak and discontinuous LOR (Fig 6, G) and FLG (Fig 6, K) staining was observed in control AD skin, with
increased intensity and restoration of continuous expression of both markers after occlusion with
petrolatum (Fig 6, Hand L; histologic magnification 310).
J ALLERGY CLIN IMMUNOL
nnn 2015
8CZARNOWICKI ET AL
as determined by using IHC, and that psoriasin/S100A7, which
acts against E coli
61
and as an ‘‘alarmin’’ to amplify the skin’s
inflammatory response to pathogens,
62
also shows robust
mRNA expression increases with petrolatum.
The IL-17 and IL-22 axes are responsible for fighting
cutaneous infections in various disease states through induction
of AMPs and innate immune genes.
63-66
The AMPs found to be
upregulated by petrolatum in our skin-profiling study were
reported to be induced by IL-17 alone (HBD2/DEFB4A, LCN2,
CCL20, and PI3)
24,67,68
or both IL-17 and IL-22 (S100A7,
S100A8, S100A9, and S100A12).
69
IL-17A, IL-22 and IL-23 (a
known activator of the T
H
17/T
H
22 pathways)
40,41,70
cytokines
were also significantly upregulated with petrolatum occlusion
compared with both occlusion alone and control skin or
nonlesional AD skin, providing a potential explanation for
increased AMP levels.
Innate immune genes (ie, IL8) were also significantly
upregulated in petrolatum-occluded skin, which is consistent
with their modulation by the T
H
17 pathway.
66,71
Importantly,
petrolatum does not induce broad upregulation of all immune
axes but rather is selective for T
H
17 and T
H
22 pathways, as
suggested by the relative lack of induction of T
H
2 cytokines
(IL-13, IL-5, and IL-31) with petrolatum, particularly in the
setting of nonlesional AD skin.
Because the nonlesional AD skin in our study already
expressed increased levels of T
H
2 cytokines and chemokines
(IL-5, IL-10, and CCL26), as previously reported,
16
we evaluated
for differential gene modulation by petrolatum on patients with
AD versus healthy subjects. Overall, lower magnitude responses
were observed and fewer immune genes were differentially
expressed in the petrolatum-occluded AD group compared with
those in subjects without AD. Attenuation of genomic responses
in patients with AD has also been observed in biopsy specimens
from positive patch test skin reactions to common allergens,
such as nickel and fragrance,
72
suggesting an overall immune
hyporesponsiveness in the setting of AD. It has been proposed
that differential immune skewing and impaired T-cell priming
in patients with AD can mitigate responses to certain cutaneous
stimuli.
33,73
The changes observed with petrolatum are greatly dependent
on baseline skin characteristics. An altered microbiome,
74,75
reduced AMP levels,
24
cytokine axis deviation,
35
and a defective
barrier
68,76-78
are characteristic of AD and render these patients
more susceptible to recurrent cutaneous infections, especially in
comparison with psoriasis, a disease characterized by T
H
17
skewing and increased AMP levels.
21,79,80
Although our patients
with AD mounted significant AMP responses with petrolatum
occlusion, their responses were lesser in magnitude than those
of the subjects without AD, possibly because of the upregulation
of T
H
2 markers, which have been shown to inhibit AMP
production in nonlesional AD skin.
22,24
In addition to its
inhibitory effect on AMPs,
12
the T
H
2 overexpression seen in
nonlesional AD skin
32,81
has also been suggested to reduce
FLG mRNA expression and other structural components of
corneocytes.
82-86
Therefore the lack of T
H
2 upregulation by
petrolatum might be a major benefit when considering the
potential use of petrolatum in patients with AD.
In addition to T
H
2 skewing, a T
H
1 deficiency is also present
in AD skin,
87,88
which has been associated with a lack of
protective immunity against cutaneous viral infections, including
eczema herpeticum.
89,90
Our data show that petrolatum boosts the
T
H
1/IFN-gresponses in patients with AD, potentially
ameliorating a defective antiviral mechanism in patients who
are prone to recurrent viral skin infections. Additionally,
petrolatum occlusion resulted in reduced cellularity and strong
induction of AMPs in AD skin, although to a lesser extent than
in non-AD skin.
Patients with AD harbor structural barrier abnormalities,
including reduced secretion of lamellar bodies in the SC,
76
altered
function of the cornified cell envelope,
31
and changes in
composition of epidermal lipids.
78,91,92
Petrolatum occlusion
displayed marked changes in SC structure, with widening of the
intercellular spaces leading to increased SC thickness. In
accordance with our data, a prior report by Ghadially et al
5
showed that petrolatum application induces cleft formation
(intercellular petrolatum clumps) in the SC, leading to SC
thickening, improved barrier function, and reduced TEWL.
Future functional studies will need to determine the effect of
petrolatum on barrier function, although recovery of consistent
FLG and LOR expression throughout the epidermis of patients
with AD suggests improvement in barrier integrity.
Emollient use has recently been shown to significantly reduce
rates of AD development in high-risk newborns.
93,94
Simpson
et al
29
found that prophylactic emolliation reduced the relative
risk of AD development by 50% in high-risk neonates, whereas
a concurrent study also found a significant reduction in AD
development with daily moisturizers in a Japanese population
of high-risk neonates.
30
Although initial proposed mechanisms
for the beneficial effect of emollients focused on barrier repair
and decreased TEWL, recent preliminary data showed that early
emolliation leads to an altered skin microbiome and skin pH in
high-risk newborns.
95
Given our results showing increases in
AMP levels, innate immune gene expression, and barrier
characteristics, we offer a possible explanation for the preventive
effect of emollients, which potentially alter the innate and
adaptive immune profiles in axes relevant to protection against
infections,
96-100
AMPs, and barrier profile, ultimately changing
the natural history of AD development in high-risk newborns.
The mechanism by which petrolatum mediates the above
molecular and structural changes is yet to be determined.
Recently, Van den Bogaard
101
showed that coal tar, an ancient
skin product used in the treatment of AD,
102
mediates its effects
through activation of the aryl hydrocarbon receptor (AHR)
pathway. Although it is likely that most aryl hydrocarbons are
separated from petrolatum during the process of its extraction
from petroleum, trace amounts might still be present and might
therefore mediate its effect through the AHR. Interestingly,
AHR has been shown to transcriptionally regulate T
H
17 and
T
H
22 cells, increasing IL-17 and IL-22 expression.
103,104
This
is in accordance with our results, which demonstrate significant
induction of IL-17– and IL-22–regulated AMPs. Furthermore, a
comparison of genes modulated by coal tar with genes modulated
through petrolatum in our study showed that there are 2
overlapping genes, TGM1 (transglutaminase 1) and IVL
(involucrin), induced by both products (data not shown). Thus a
possible mechanism for the effects of petrolatum might be
AHR pathway activation, although this is speculative and requires
further study.
Our study has several limitations. First, the presence of FLG or
other skin barrier mutations might affects responses to petrolatum
application. However, skin genotyping was beyond the scope of
this study and should be addressed in the future.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 9
Second, for practical reasons, we assessed petrolatum under
occlusion only. Because petrolatum is more commonly used
without occlusion, future studies will be required to determine
molecular and barrier alterations induced by a nonoccluded
petrolatum.
Lastly, future studies will need to assess and compare not only
the long-term effect of emollients in high-risk newborns
29
but also
the ability of different moisturizer categories to improve barrier
function and AMP production. This is particularly important in
preventing development of AD lesions and reverting the
‘abnormal’’ nonlesional AD phenotype
16,105
back to a normal
skin pattern.
Clinical implications: Because prophylactic moisturizers pre-
vent AD development in high-risk newborns, our data can be
further explored to evaluate the differential effects of various
moisturizers on disease prevention, barrier function, and skin
infections.
REFERENCES
1. Chesebrough RA, inventor. Improvement in products from petroleum. US patent
US127568. June 4, 1872.
2. Jakasa I, Kezic S, Boogaard PJ. Dermal uptake of petroleum substances. Toxicol
Lett 2015;235:123-39.
3. Lod
en M. The increase in skin hydration after application of emollients with
different amounts of lipids. Acta Derm Venereol 1992;72:327-30.
4. Lod
en M, Barany E. Skin-identical lipids versus petrolatum in the treatment of
tape-stripped and detergent-perturbed human skin. Acta Derm Venereol 2000;
80:412-5.
5. Ghadially R, Halkier-Sorensen L, Elias PM. Effects of petrolatum on stratum
corneum structure and function. J Am Acad Dermatol 1992;26:387-96.
6. Rawlings AV, Canestrari DA, Dobkowski B. Moisturizer technology versus
clinical performance. Dermatol Ther 2004;17(suppl 1):49-56.
7. Nijhawan RI, Smith LA, Mariwalla K. Mohs surgeons’ use of topical emollients
in postoperative wound care. Dermatol Surg 2013;39:1260-3.
8. Smack DP, Harrington AC, Dunn C, Howard RS, Szkutnik AJ, Krivda SJ, et al.
Infection and allergy incidence in ambulatory surgery patients using white
petrolatum vs bacitracin ointment. A randomized controlled trial. JAMA 1996;
276:972-7.
9. Zug KA, Pham AK, Belsito DV, DeKoven JG, DeLeo VA, Fowler JF Jr, et al.
Patch testing in children from 2005 to 2012: results from the North American
contact dermatitis group. Dermatitis 2014;25:345-55.
10. Fraki JE, Peltonen L, Hopsu-Havu VK. Allergy to various components of
topical preparations in stasis dermatitis and leg ulcer. Contact Dermatitis 1979;
5:97-100.
11. James WD. Use of antibiotic-containing ointment versus plain petrolatum during
and after clean cutaneous surgery. J Am Acad Dermatol 2006;55:915-6.
12. Greenberg K, Espinosa J, Scali V. Anaphylaxis to topical bacitracin ointment. Am
J Emerg Med 2007;25:95-6.
13. Saryan JA, Dammin TC, Bouras AE. Anaphylaxis to topical bacitracin zinc
ointment. Am J Emerg Med 1998;16:512-3.
14. Schechter JF, Wilkinson RD, Del Carpio J. Anaphylaxis following the use of
bacitracin ointment. Report of a case and review of the literature. Arch Dermatol
1984;120:909-11.
15. Vale MA, Connolly A, Epstein AM, Vale MR. Bacitracin-induced anaphylaxis.
Arch Dermatol 1978;114:800.
16. Suar
ez-Fari~
nas M, Tintle SJ, Shemer A, Chiricozzi A, Nograles K, Cardinale I,
et al. Nonlesional atopic dermatitis skin is characterized by broad terminal
differentiation defects and variable immune abnormalities. J Allergy Clin
Immunol 2011;127:954-64, e1-4.
17. Holm EA, Wulf HC, Thomassen L, Jemec GB. Instrumental assessment of atopic
eczema: validation of transepidermal water loss, stratum corneum hydration,
erythema, scaling, and edema. J Am Acad Dermatol 2006;55:772-80.
18. Jakasa I, Verberk MM, Esposito M, Bos JD, Kezic S. Altered penetration of
polyethylene glycols into uninvolved skin of atopic dermatitis patients. J Invest
Dermatol 2007;127:129-34.
19. Winge MC, Hoppe T, Berne B, Vahlquist A, Nordenskjold M, Bradley M, et al.
Filaggrin genotype determines functional and molecular alterations in skin of pa-
tients with atopic dermatitis and ichthyosis vulgaris. PLoS One 2011;6:e28254.
20. Malajian D, Belsito DV. Cutaneous delayed-type hypersensitivity in patients with
atopic dermatitis. J Am Acad Dermatol 2013;69:232-7.
21. Czarnowicki T, Krueger JG, Guttman-Yassky E. Skin barrier and immune
dysregulation in atopic dermatitis: an evolving story with important clinical
implications. J Allergy Clin Immunol Pract 2014;2:371-81.
22. Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, et al.
Endogenous antimicrobial peptides and skin infections in atopic dermatitis.
N Engl J Med 2002;347:1151-60.
23. Christophers E, Henseler T. Contrasting disease patterns in psoriasis and atopic
dermatitis. Arch Dermatol Res 1987;279(suppl):S48-51.
24. Guttman-Yassky E, Lowes MA, Fuentes-Duculan J, Zaba LC, Cardinale I,
Nograles KE, et al. Low expression of the IL-23/Th17 pathway in atopic
dermatitis compared to psoriasis. J Immunol 2008;181:7420-7.
25. Miller DW, Koch SB, Yentzer BA, Clark AR, O’Neill JR, Fountain J, et al.
An over-the-counter moisturizer is as clinically effective as, and more
cost-effective than, prescription barrier creams in the treatment of children with
mild-to-moderate atopic dermatitis: a randomized, controlled trial. J Drugs
Dermatol 2011;10:531-7.
26. Pennick G, Harrison S, Jones D, Rawlings AV. Superior effect of isostearyl
isostearate on improvement in stratum corneum water permeability barrier
function as examined by the plastic occlusion stress test. Int J Cosmet Sci
2010;32:304-12.
27. Matsumoto T, Yuasa H, Kai R, Ueda H, Ogura S, Honda Y. Skin capacitance in
normal and atopic infants, and effects of moisturizers on atopic skin. J Dermatol
2007;34:447-50.
28. Seite S, Flores GE, Henley JB, Martin R, Zelenkova H, Aguilar L, et al.
Microbiome of affected and unaffected skin of patients with atopic
dermatitis before and after emollient treatment. J Drugs Dermatol 2014;13:
1365-72.
29. Simpson EL, Chalmers JR, Hanifin JM, Thomas KS, Cork MJ, McLean WH,
et al. Emollient enhancement of the skin barrier from birth offers effective atopic
dermatitis prevention. J Allergy Clin Immunol 2014;134:818-23.
30. Horimukai K, Morita K, Narita M, Kondo M, Kitazawa H, Nozaki M, et al.
Application of moisturizer to neonates prevents development of atopic dermatitis.
J Allergy Clin Immunol 2014;134:824-30.e6.
31. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged
epidermal permeability barrier. Structural, functional, and lipid biochemical
abnormalities in humans and a senescent murine model. J Clin Invest 1995;95:
2281-90.
32. Guttman-Yassky E, Suar
ez-Fari~
nas M, Chiricozzi A, Nograles KE, Shemer A,
Fuentes-Duculan J, et al. Broad defects in epidermal cornification in atopic
dermatitis identified through genomic analysis. J Allergy Clin Immunol 2009;
124:1235-44.e58.
33. Dhingra N, Shemer A, Correa da Rosa J, Rozenblit M, Fuentes-Duculan J, Gittler
JK, et al. Molecular profiling of contact dermatitis skin identifies allergen-
dependent differences in immune response. J Allergy Clin Immunol 2014;134:
362-72.
34. Suar
ez-Fari~
nas M, Dhingra N, Gittler J, Shemer A, Cardinale I, de Guzman
Strong C, et al. Intrinsic atopic dermatitis shows similar TH2 and higher TH17
immune activation compared with extrinsic atopic dermatitis. J Allergy Clin
Immunol 2013;132:361-70.
35. Gittler JK, Shemer A, Suar
ez-Fari~
nas M, Fuentes-Duculan J, Gulewicz KJ, Wang
CQ, et al. Progressive activation of T(H)2/T(H)22 cytokines and selective
epidermal proteins characterizes acute and chronic atopic dermatitis. J Allergy
Clin Immunol 2012;130:1344-54.
36. Nograles KE, Zaba LC, Shemer A, Fuentes-Duculan J, Cardinale I, Kikuchi T,
et al. IL-22-producing ‘‘T22’’ T cells account for upregulated IL-22 in atopic
dermatitis despite reduced IL-17-producing TH17 T cells. J Allergy Clin
Immunol 2009;123:1244-52.e2.
37. Guttman-Yassky E, Lowes MA, Fuentes-Duculan J, Whynot J, Novitskaya I,
Cardinale I, et al. Major differences in inflammatory dendritic cells and their
products distinguish atopic dermatitis from psoriasis. J Allergy Clin Immunol
2007;119:1210-7.
38. Tintle S, Shemer A, Suar
ez-Fari~
nas M, Fujita H, Gilleaudeau P, Sullivan-Whalen
M, et al. Reversal of atopic dermatitis with narrow-band UVB phototherapy and
biomarkers for therapeutic response. J Allergy Clin Immunol 2011;128:583-93,
e1-4.
39. Bose F, Petti L, Diani M, Moscheni C, Molteni S, Altomare A, et al. Inhibition of
CCR7/CCL19 axis in lesional skin is a critical event for clinical remission
induced by TNF blockade in patients with psoriasis. Am J Pathol 2013;183:
413-21.
40. Yin X, Cheng H, Zhang R, Fan X, Zhou F, Jiang L, et al. Combined effect of five
single nucleotide polymorphisms related to IL23/Th17 pathway in the risk of
psoriasis. Immunogenetics 2014;66:215-8.
J ALLERGY CLIN IMMUNOL
nnn 2015
10 CZARNOWICKI ET AL
41. Li CW, Lu HG, Chen de H, Lin ZB, Wang de Y, Li TY. In vivo and in vitro studies
of Th17 response to specific immunotherapy in house dust mite-induced allergic
rhinitis patients. PLoS One 2014;9:e91950.
42. Burton MJ, Doree CJ. Interventions for recurrent idiopathic epistaxis
(nosebleeds) in children. Cochrane Database Syst Rev 2004;(1):CD004461.
43. Fleischer AB Jr, Feldman SR, White RE, Leshin B, Byington R. Procedures for
skin diseases performed by physicians in 1993 and 1994: analysis of data from the
National Ambulatory Medical Care Survey. J Am Acad Dermatol 1997;37:
719-24.
44. Gette MT, Marks JG Jr, Maloney ME. Frequency of postoperative allergic contact
dermatitis to topical antibiotics. Arch Dermatol 1992;128:365-7.
45. Jenssen H, Hamill P, Hancock RE. Peptide antimicrobial agents. Clin Microbiol
Rev 2006;19:491-511.
46. Bowdish DM, Hancock RE. Anti-endotoxin properties of cationic host defence
peptides and proteins. J Endotoxin Res 2005;11:230-6.
47. Hancock RE. Peptide antibiotics. Lancet 1997;349:418-22.
48. Schauber J, Gallo RL. Antimicrobial peptides and the skin immune defense
system. J Allergy Clin Immunol 2009;124(suppl 2):R13-8.
49. Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O,
et al. The structure of human beta-defensin-2 shows evidence of higher order
oligomerization. J Biol Chem 2000;275:32911-8.
50. White SH, Wimley WC, Selsted ME. Structure, function, and membrane
integration of defensins. Curr Opin Struct Biol 1995;5:521-7.
51. Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M, Liden S, Wigzell H,
et al. The expression of the gene coding for the antibacterial peptide LL-37 is
induced in human keratinocytes during inflammatory disorders. J Biol Chem
1997;272:15258-63.
52. Braff MH, Gallo RL. Antimicrobial peptides: an essential component of the skin
defensive barrier. Curr Top Microbiol Immunol 2006;306:91-110.
53. Zhang LJ, Guerrero-Juarez CF, Hata T, Bapat SP, Ramos R, Plikus MV, et al.
Innate immunity. Dermal adipocytes protect against invasive Staphylococcus
aureus skin infection. Science 2015;347:67-71.
54. Krishna S, Miller LS. Innate and adaptive immune responses against
Staphylococcus aureus skin infections. Semin Immunopathol 2012;34:261-80.
55. Ryu S, Song PI, Seo CH, Cheong H, Park Y. Colonization and infection of the
skin by S. aureus: immune system evasion and the response to cationic
antimicrobial peptides. Int J Mol Sci 2014;15:8753-72.
56. Harder J, Bartels J, Christophers E, Schroder JM. A peptide antibiotic from
human skin. Nature 1997;387:861.
57. Brandtzaeg P, Gabrielsen TO, Dale I, Muller F, Steinbakk M, Fagerhol MK. The
leucocyte protein L1 (calprotectin): a putative nonspecific defence factor at
epithelial surfaces. Adv Exp Med Biol 1995;371A:201-6.
58. Miyasaki KT, Bodeau AL, Murthy AR, Lehrer RI. In vitro antimicrobial activity
of the human neutrophil cytosolic S-100 protein complex, calprotectin, against
Capnocytophaga sputigena. J Dent Res 1993;72:517-23.
59. Lusitani D, Malawista SE, Montgomery RR. Calprotectin, an abundant cytosolic
protein from human polymorphonuclear leukocytes, inhibits the growth of
Borrelia burgdorferi. Infect Immun 2003;71:4711-6.
60. Murthy AR, Lehrer RI, Harwig SS, Miyasaki KT. In vitro candidastatic properties
of the human neutrophil calprotectin complex. J Immunol 1993;151:6291-301.
61. Glaser R, Harder J, Lange H, Bartels J, Christophers E, Schroder JM.
Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli
infection. Nat Immunol 2005;6:57-64.
62. Hegyi Z, Zwicker S, Bureik D, Peric M, Koglin S, Batycka-Baran A, et al.
Vitamin D analog calcipotriol suppresses the Th17 cytokine-induced
proinflammatory S100 ‘‘alarmins’’ psoriasin (S100A7) and koebnerisin
(S100A15) in psoriasis. J Invest Dermatol 2012;132:1416-24.
63. Niebuhr M, Mamerow D, Heratizadeh A, Satzger I, Werfel T. Staphylococcal
alpha-toxin induces a higher T cell proliferation and interleukin-31 in atopic
dermatitis. Int Arch Allergy Immunol 2011;156:412-5.
64. Wolk K, Mitsui H, Witte K, Gellrich S, Gulati N, Humme D, et al. Deficient
cutaneous antibacterial competence in cutaneous T-cell lymphomas: role of
Th2-mediated biased Th17 function. Clin Cancer Res 2014;20:5507-16.
65. Dhingra N, Suar
ez-Fari~
nas M, Fuentes-Duculan J, Gittler JK, Shemer A, Raz A,
et al. Attenuated neutrophil axis in atopic dermatitis compared to psoriasis
reflects TH17 pathway differences between these diseases. J Allergy Clin
Immunol 2013;132:498-501.e3.
66. Isailovic N, Daigo K, Mantovani A, Selmi C. Interleukin-17 and innate immunity
in infections and chronic inflammation. J Autoimmun 2015;60:1-11.
67. Shen F, Hu Z, Goswami J, Gaffen SL. Identification of common transcriptional
regulatory elements in interleukin-17 target genes. J Biol Chem 2006;281:
24138-48.
68. Goupil M, Cousineau-Cote V, Aumont F, Senechal S, Gaboury L, Hanna Z, et al.
Defective IL-17- and IL-22-dependent mucosal host response to Candida
albicans determines susceptibility to oral candidiasis in mice expressing the
HIV-1 transgene. BMC Immunol 2014;15:49.
69. Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M,
et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and
cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006;
203:2271-9.
70. Fujita H. The role of IL-22 and Th22 cells in human skin diseases. J Dermatol Sci
2013;72:3-8.
71. Reynolds JM, Angkasekwinai P, Dong C. IL-17 family member cytokines:
regulation and function in innate immunity. Cytokine Growth Factor Rev 2010;
21:413-23.
72. Correa da Rosa J, Malajian D, Shemer A, Rozenblit M, Dhingra N, Czarnowicki
T, et al. Patients with atopic dermatitis have attenuated and distinct contact
hypersensitivity responses to common allergens in skin. J Allergy Clin Immunol
2015;135:712-20.
73. Dhingra N, Gulati N, Guttman-Yassky E. Mechanisms of contact sensitization
offer insights into the role of barrier defects vs. intrinsic immune abnormalities
as drivers of atopic dermatitis. J Invest Dermatol 2013;133:2311-4.
74. Warner JA, McGirt LY, Beck LA. Biomarkers of Th2 polarity are predictive of
staphylococcal colonization in subjects with atopic dermatitis. Br J Dermatol
2009;160:183-5.
75. Park HY, Kim CR, Huh IS, Jung MY, Seo EY, Park JH, et al. Staphylococcus
aureus colonization in acute and chronic skin lesions of patients with atopic
dermatitis. Ann Dermatol 2013;25:410-6.
76. Elias PM, Wakefield JS. Mechanisms of abnormal lamellar body secretion and the
dysfunctional skin barrier in patients with atopic dermatitis. J Allergy Clin
Immunol 2014;134:781-91.e1.
77. Elias PM, Hatano Y, Williams ML. Basis for the barrier abnormality in atopic
dermatitis: outside-inside-outside pathogenic mechanisms. J Allergy Clin
Immunol 2008;121:1337-43.
78. van Smeden J, Janssens M, Gooris GS, Bouwstra JA. The important role of
stratum corneum lipids for the cutaneous barrier function. Biochim Biophys
Acta 2014;1841:295-313.
79. Sa SM, Valdez PA, Wu J, Jung K, Zhong F, Hall L, et al. The effects of IL-20
subfamily cytokines on reconstituted human epidermis suggest potential roles
in cutaneous innate defense and pathogenic adaptive immunity in psoriasis.
J Immunol 2007;178:2229-40.
80. Nomura I, Goleva E, Howell MD, Hamid QA, Ong PY, Hall CF, et al. Cytokine
milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of
innate immune response genes. J Immunol 2003;171:3262-9.
81. Beck LA, Thaci D, Hamilton JD, Graham NM, Bieber T, Rocklin R, et al.
Dupilumab treatment in adults with moderate-to-severe atopic dermatitis. N
Engl J Med 2014;371:130-9.
82. Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Morel F.
IL-22 inhibits epidermal differentiation and induces proinflammatory gene
expression and migration of human keratinocytes. J Immunol 2005;174:
3695-702.
83. Nograles KE, Zaba LC, Guttman-Yassky E, Fuentes-Duculan J, Suar
ez-Fari~
nas
M, Cardinale I, et al. Th17 cytokines interleukin (IL)-17 and IL-22 modulate
distinct inflammatory and keratinocyte-response pathways. Br J Dermatol 2008;
159:1092-102.
84. Howell MD, Kim BE, Gao P, Grant AV, Boguniewicz M, DeBenedetto A, et al.
Cytokine modulation of atopic dermatitis filaggrin skin expression. J Allergy Clin
Immunol 2009;124(suppl 2):R7-12.
85. Pellerin L, Henry J, Hsu CY, Balica S, Jean-Decoster C, Mechin MC, et al.
Defects of filaggrin-like proteins in both lesional and nonlesional atopic skin.
J Allergy Clin Immunol 2013;131:1094-102.
86. Seltmann J, Roesner LM, von Hesler FW, Wittmann M, Werfel T. IL-33 impacts
on the skin barrier by downregulating the expression of filaggrin. J Allergy Clin
Immunol 2015;135:1659-61.e4.
87. Gros E, Petzold S, Maintz L, Bieber T, Novak N. Reduced IFN-gamma receptor
expression and attenuated IFN-gamma response by dendritic cells in patients with
atopic dermatitis. J Allergy Clin Immunol 2011;128:1015-21.
88. Czarnowicki T, Gonzalez J, Shemer A, Malajian D, Xu H, Zheng X, et al. Severe
atopic dermatitis is characterized by selective expansion of circulating TH2/TC2
and TH22/TC22, but not TH17/TC17, cells within the skin-homing T-cell
population. J Allergy Clin Immunol 2015;136:104-15.e7.
89. Mathias RA, Weinberg A, Boguniewicz M, Zaccaro DJ, Armstrong B, Schneider
LC, et al. Atopic dermatitis complicated by eczema herpeticum is associated with
HLA B7 and reduced interferon-gamma-producing CD81T cells. Br J Dermatol
2013;169:700-3.
90. Bin L, Edwards MG, Heiser R, Streib JE, Richers B, Hall CF, et al. Identification
of novel gene signatures in patients with atopic dermatitis complicated by eczema
herpeticum. J Allergy Clin Immunol 2014;134:848-55.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 11
91. van Smeden J, Janssens M, Kaye EC, Caspers PJ, Lavrijsen AP, Vreeken RJ, et al.
The importance of free fatty acid chain length for the skin barrier function in
atopic eczema patients. Exp Dermatol 2014;23:45-52.
92. Janssens M, van Smeden J, Gooris GS, Bras W, Portale G, Caspers PJ, et al.
Increase in short-chain ceramides correlates with an altered lipid organization
and decreased barrier function in atopic eczema patients. J Lipid Res 2012;53:
2755-66.
93. Macharia WM, Anabwani GM, Owili DM. Effects of skin contactants on evolution
of atopic dermatitis in children: a case control study. Trop Doct 1991;21:104-6.
94. Simpson EL, Berry TM, Brown PA, Hanifin JM. A pilot study of emollient
therapy for the primary prevention of atopic dermatitis. J Am Acad Dermatol
2010;63:587-93.
95. Glatz M, Polley E, Simpson E, Kong H. Emollient therapy alters skin barrier and
microbes in infants at risk for developing atopic dermatitis [abstract]. J Invest
Dermatol 2015;135(suppl):S31.
96. Sonnenberg GF, Fouser LA, Artis D. Border patrol: regulation of immunity,
inflammation and tissue homeostasis at barrier surfaces by IL-22. Nat Immunol
2011;12:383-90.
97. Eyerich S, Eyerich K, Cavani A, Schmidt-Weber C. IL-17 and IL-22: siblings, not
twins. Trends Immunol 2010;31:354-61.
98. Ishigame H, Kakuta S, Nagai T, Kadoki M, Nambu A, Komiyama Y, et al.
Differential roles of interleukin-17A and -17F in host defense against muco-
epithelial bacterial infection and allergic responses. Immunity 2009;30:108-19.
99. Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR, et al. IL-17
is essential for host defense against cutaneous Staphylococcus aureus infection in
mice. J Clin Invest 2010;120:1762-73.
100. Liu SY, Sanchez DJ, Aliyari R, Lu S, Cheng G. Systematic identification of type I
and type II interferon-induced antiviral factors. Proc Natl Acad Sci U S A 2012;
109:4239-44.
101. van den Bogaard EH, Bergboer JG, Vonk-Bergers M, van Vlijmen-Willems IM,
Hato SV, van der Valk PG, et al. Coal tar induces AHR-dependent skin barrier
repair in atopic dermatitis. J Clin Invest 2013;123:917-27.
102. Slutsky JB, Clark RA, Remedios AA, Klein PA. An evidence-based review of the
efficacy of coal tar preparations in the treatment of psoriasis and atopic dermatitis.
J Drugs Dermatol 2010;9:1258-64.
103. Quintana FJ, Basso AS, Iglesias AH, Korn T, Farez MF, Bettelli E, et al. Control
of T(reg) and T(H)17 cell differentiation by the aryl hydrocarbon receptor. Nature
2008;453:65-71.
104. Trifari S, Kaplan CD, Tran EH, Crellin NK, Spits H. Identification of a
human helper T cell population that has abundant production of interleukin 22
and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol 2009;10:
864-71.
105. Suar
ez-Fari~
nas M, Ungar B, Correa da Rosa J, Ewald DA, Rozenblit M, Gonzalez
J, et al. RNA sequencing atopic dermatitis transcriptome profiling provides
insights into novel disease mechanisms with potential therapeutic implications.
J Allergy Clin Immunol 2015;135:1218-27.
J ALLERGY CLIN IMMUNOL
nnn 2015
12 CZARNOWICKI ET AL
METHODS
IHC
Biopsy specimens were frozen in optimal cutting temperature (O.C.T.)
medium, and cryostat tissue sections of all patients were stained with
hematoxylin (Fisher, Fair Lawn, NJ) and eosin (Shandon, Pittsburgh, Pa).
IHC was performed with purified anti-human mAbs. For IHC, biotin-labeled
horse anti-mouse secondary antibodies (Vector Laboratories, Burlingame,
Calif) were used to detect the primary antibodies, and the staining signal was
developed with chromogen 3-amino-9-ethylcarbazole (Sigma-Aldrich, St
Louis, Mo). Table E1 provides details on antibodies used for IHC. Epidermal
thickness and positive cells per millimeter were manually quantified by using
computer-assisted image analysis software (ImageJ 1.42; National Institutes
of Health, Bethesda, Md).
Gene array analysis
RNA was extracted, and Affymetrix Human U133Plus 2.0 arrays
(Affymetrix, Santa Clara, Calif) were used. Briefly, we used human
HGU133Plus2.0 GeneChip probe arrays (Affymetrix) containing
approximately 54,000 probe sets to assess expression of more than 47,000
transcripts, including approximately 38,500 genes and unigenes. Total RNA
was extracted from tissues frozen in liquid nitrogen by using the Qiagen
miRNeasy Mini Kit (Qiagen, Valencia, Calif). DNA was removed by using
on-column DNAse digestion with the Qiagen RNAse-free DNAse Set. Total
RNA (100 ng) was reverse transcribed and amplified with Ovation Whole
Blood Solution from NuGen (San Carlos, Calif). The labeled target was
fragmented and hybridized to probe arrays with the Encore Biotin Module
from NuGen. The chips were washed, stained with streptavidin-
phycoerythrin, and scanned (HP GeneArray Scanner; Hewlett-Packard
Company, Palo Alto, Calif). On each chip, the human housekeeping genes
b-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as
controls. Suite 5.0 software normalized expression levels using these controls.
RT-PCR analysis
The TaqMan RT-PCRreaction was performed with EZ-PCR Core Reagents
(Life Technologies, Grand Island, NY), according to the manufacturer’s
directions. The primers and probes for each gene are listed in Table E2 and
were designed by Life Technologies. hARP, a housekeeping gene, was used
to normalize each sample and each gene. The primer sequences used were
generated with the Primer Express algorithm (version 1.0; Applied
Biosystems, Foster City, Calif) by using published genetic sequences
(National Center for Biotechnology Information/PubMed), as follows:
hARP forward, CGCTGCTGAACATGCTCAA; hARP reverse, TGTCGAA
CACCTGCTGGATG; and hARP probe, 6-FAM- TCCCCCTTCTCC
TTTGGGCTGG-TAMRA (Gene Bank accession no. NM-001002). Please
see Table E2 for details on primers and probes used. Data were analyzed
and quantified by using software provided with the Applied Biosystems
PRISM 7700 (Sequence Detection Systems, version 1.7).
Statistical methods
Statistical methods were applied to 2 different cohorts that were
analyzed separately. The first cohort had IHC, microarrays, and RT-PCR
expressions taken from petrolatum-occluded and matched control skin
from 29 patients. Data and their description are available on the Gene
Expression Omnibus GSE60028 database repository. The second cohort
contains RT-PCR gene expressions taken from control, occluded, and
petrolatum-occluded skin measured in a set of 20 healthy volunteers. In this
cohort gene expressions were normalized to hARP and multiplied by 10
5
.
For the first 5 volunteers of this cohort, RT-PCR expressions were evaluated
from 5 biopsy specimens: control, occlusion-only and occlusion with
petrolatum at 48 hours, and occlusion-only and occlusion with petrolatum at
72 hours skin.
Microarray and RT-PCR analysis for the first cohort aimed to identify genes
that were differentially expressed in petrolatum-occluded skin when
compared with control skin. QC of microarray was carried out with standard
QC metrics from Quality Control R package. Log
2
-expressions were modeled
by using linear mixed-effects models, with skin type as the fixed effect and a
random effect for each patient. For microarray gene expression, the limma
package in R version 3.1.0 was used to identify a set of 51 genes with FCHs
of greater than 2 and a false discovery rate of less than 0.05 (Table I).
These DEGs are shown in the heat map in Fig 2, followed by the estimated
FCHs. The parameters for the hierarchic clustering in this heat map are as
follows: McQuitty algorithm, Euclidean distance, and standardization by
rows using the function heatmap.2 in R package. Subgroup analysis for
patients with AD and subjects without AD was carried out for a subset of
52 genes in which expressions were measured by using RT-PCR. Specification
of the mixed-effects model considered as fixed effects: skin type
(petrolatum-occluded or normal skin), AD personal history (yes/no), and their
interaction. This model also included a random intercept for each subject. The
package nlme was used for gene-wise estimation of the mean log
2
-transformed
expression and its SEM, which were associated with the following groups:
control AD, control non-AD, petrolatum AD, and petrolatum non-AD.
Restricted maximum likelihood estimated the log
2
FCHs for specific
contrasts. A heat map with the same parameters as described before was
created to illustrate the differences between the 4 groups. Blocks of interest
in this heat map, followed by estimated FCHs and their statistical significance,
are shown in Fig 5. This mixed-effects modeling approach for subgroup
analysis was also applied to IHC data, assuming normal distribution for cell
counts. The contrasts of interest in IHC are the ones that compare cell counts
in petrolatum-occluded with those in normal skin within the AD and non-AD
groups.
Analysis for the second cohort consisted of fitting a linear mixed-effects
model to log
2
-transformed RT-PCR expressions of 17 genes (AMPs, innate
immune, and cytokines). This model has only fixed effects for skin type
(petrolatum-occluded, occluded, or normal skin) and a random effect for
each subject. Pairwise comparisons after estimating the model parameters
were evaluated with the use of the estimable function, which is part of the
gmodels R package. The evaluation of differences between mean
log
2
-transformed RT-PCR expressions in the 5 different biopsy specimens
taken from the first 5 volunteers was performed with ANOVA for
repeated measures as implemented in Prism Version 6.0 software (GraphPad
Software, La Jolla, Calif). Post hoc comparisons were carried out with paired
ttests adjusting Pvalues for multiple hypotheses. The results are displayed in
Fig E1.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 12.e1
FIG E1. RT-PCR log
2
expressions normalized to hARP for 5 key AMPs. To assess the point of maximum gene
induction for petrolatum and occlusion, we compared gene expression at different time points. S100A7,
LCN2,S100A8, and PI3 all showed significant increases in expression from baseline at 72 hours of occlusion
with petrolatum but not at 48 hours. Bar plots represent means 6SDs. *P< .1, **P< .05, and ***P< .01.
J ALLERGY CLIN IMMUNOL
nnn 2015
12.e2 CZARNOWICKI ET AL
FIG E2. A, Thickness (in micrometers) of the SC in control, occlusion-only, and occlusion with petrolatum
skin. Occluded and petrolatum-occluded SC showed significant thickness increases compared with
baseline, and petrolatum also significantly increased SC thickness compared with occlusion alone.
B-D, Immunofluorescence staining with Nile Red for neutral lipids in control (Fig E2, B), occlusion-only
(Fig E2, C), and occlusion with petrolatum skin in a representative subject (Fig E2, D). Intercellular lipid clefts
are observed after occlusion with petrolatum, which, in Fig E2, Band C, were not observed in control and
occlusion-only skin, probably accounting for the increased SC thickness observed. Bar plots represent
means 6SEMs. **P< .05 and ***P< .01.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 12.e3
FIG E3. Neutrophil elastase cell counts of control, occluded, and
petrolatum-occluded skin. There were no statistically significant increases
in neutrophil elastase–positive infiltrates in occluded or petrolatum-
occluded skin. Bar plots represent means 6SEMs.
J ALLERGY CLIN IMMUNOL
nnn 2015
12.e4 CZARNOWICKI ET AL
FIG E4. Cell counts for CD8, dendritic cell lysosome-associated membrane glycop rotein (DC-LAMP), CD11c,
CD3, CD1a, CD1c, maltose-binding protein (MBP), and Langerin for control and occlusion with petrolatum
skin in patients with AD and subjects without AD, demonstrating significantly decreased CD8 infiltrates for
patients with AD and subjects without AD and decreases in CD1c and CD3 for patients with AD only. Bar
plots represent means 6SEMs. *P< .1 and **P< .05.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 12.e5
TABLE E1. Antibodies used for IHC
Antibody Vendor Clone Dilution
S100A8/A9 Abcam 27E10 1:20
LCN2 Abcam 5G5 1:100
CCL20 LifeSpan BioSciences B5917 1:20
FLG Acris FLG01 1:100
LOR Abcam polyclonal 1:200
Neutrophil elastase Dako NP57 1:200
Nile Red*Sigma-Aldrich N3013 500 mg/mL
*Used for immunofluorescence.
J ALLERGY CLIN IMMUNOL
nnn 2015
12.e6 CZARNOWICKI ET AL
TABLE E2. Primers and probes for RT-PCR
Gene Primer/probe
IL1B Hs01555410_m1
IL8 Hs00174103_m1
IL6 Hs00985639_m1
TNFA Hs01113624_g1
IFNA1 Hs00855471_g1
HBD2/DEFB4 Hs00823638_m1
IL13 Hs00174379_m1
IFNG Hs00989291_m1
IL17A Hs00174383_m1
IL22 Hs01574154_m1
IL23A/IL23p19 Hs00900828_g1
IL12B/IL23p40 Hs01011518_m1
CCL27/CTACK Hs00171157_m1
CXCL9 Hs00171065_m1
CCL20 Hs01011368_m1
PI3/elafin Hs00160066_m1
LCN2 Hs01008571_m1
LL37 Hs00189038_1
S100A7 Hs01923188_u1
S100A8 Hs00374264_g1
S100A9 Hs00610058_m1
S100A12 Hs00942835_g1
TSLP Hs00263639_m1
TSLP, Thymic stromal lymphopoietin.
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
CZARNOWICKI ET AL 12.e7
... Therefore, Dexpanthenol-containing preparations stand as an alternative to petroleum jelly [17], which forms a thick occlusive protective layer with the objective of water loss prevention [18]. Several publications, as well as the manufacturers of some CO 2 laser devices, recommend the use of petroleum jelly to protect skin tissue from exposure to air until it is completely healed [13,17,19,20]. ...
... Although scientific literature on using petroleum jelly as a moisturizer is scarce, it is widely used for wound healing, especially after minor surgical procedures. Its main properties are occlusion, thereby blocking transepidermal water loss and keeping the skin moisturized [18,21]. ...
... All the procedures were bilateral, with each side randomized to receive either Dexpanthenol-containing product or control (petroleum jelly). Petroleum jelly was chosen as the positive control product because of its inert nature as a physical barrier [17,18]. Dexpanthenol-containing spray, a water-based product, was used after non-ablative laser resurfacing, laser depilation, and chemical peel, and Dexpanthenol-containing cream was used after ablative facial laser. ...
Article
Full-text available
Moisturizers are commonly prescribed after laser and chemical peel aesthetic procedures, but the evidence regarding their efficacy and safety of such use is scarce. We conducted four single-blind, three-week, controlled studies to evaluate the efficacy and safety of topical Dexpanthenol-containing products (Bepantol® spray and Bepantol® cream) using petroleum jelly as a positive control. Skin recovery was assessed after four aesthetic procedures: (1) non-ablative facial laser resurfacing, (2) laser depilation on the external genital and inguinal regions, (3) chemical peel on the external genital and inguinal regions, and (4) ablative facial laser resurfacing. Efficacy was assessed through transepidermal water loss (TEWL) combined with clinical assessment of the skin by the investigators and the participants. In studies (1) and (4), the erythema intensity was evaluated by measuring dermal temperature with a thermal imaging camera. Safety was assessed through adverse event reporting and acceptability through a questionnaire. Dexpanthenol-containing products significantly decreased TEWL and dermal temperature, therefore maintaining skin integrity, promoting its recovery, and reducing erythema. No statistical differences with the positive control were observed. In addition, Dexpanthenol-containing products were well appreciated by the participants from a sensory perspective. These findings suggest that these Dexpanthenol-containing products are adequate for post-procedural care in aesthetic dermatology.
... Therefore, individually and empirically adapted skin care using emollients or moisturizers to complement the control of inflammation still remains the only approach to improve barrier function, dryness and water loss and is qualified as basis therapy 35 . In line with this concept, even a simple product such as petrolatum has been shown to modulate the antimicrobial and epidermal barrier function 47 . After initial promising results using such emollients and moisturizers in the prevention of AD in newborns at high risk 48,49 , a more recent report has questioned this strategy 50,51 . ...
Article
Full-text available
Updated review and critical appraisal of current the pipeline therapeutic pipeline for atopic dermatitis. Discussion on value of precision medicine approach in this complex disease
... Skincare should be included in the treatment modalities. Infants should undergo such treatments one or two times per day in order to promote the shedding of the stratum corneum and increase hydration [32]. A common complication of HI is also the necrosis of the digits. ...
Article
Full-text available
Harlequin ichthyosis (HI) is a life-threatening genetic disorder that largely affects the skin of infants. HI is the most severe form of the autosomal recessive disorder known as ichthyosis. It is caused by mutations in the A12 cassette (lipid-transporter adenosine triphosphate-binding cassette A12). Neonates affected by this disease are born with specific morphological characteristics, the most prominent of which is the appearance of platelet keratotic scales separated by erythematous fissures. The facial features include eclabium, ectropion, a distinct flattened nose, and dysplastic ears. A common finding among those with HI is impaired skin barrier function. The purpose of the present narrative review is to assess the most recent literature regarding the management of HI. Emphasis is given to surgical management and consultation, to the indications for timing and surgical intervention, to the risks that are presented with surgery, and to the details of the surgical procedure itself. Management of HI requires a multidisciplinary team of experts, and specific guidelines are needed in order for the risks to be minimized and viability to be increased.
Article
Atopic dermatitis (AD) is a common chronic pruritic inflammatory cutaneous disease. AD is characterized by intense pruritus and enormous clinical heterogeneity. Treatment goals are to improve skin lesions and minimize exacerbations and symptom burden. Currently, topical corticosteroids (TCS) and topical calcineurin inhibitors (TCI) are still considered the main topical therapies in disease treatment. However, despite being very effective, TCS and TCI are not recommended for continuous long-term use, due to potential safety issues. Although research in AD has focused primarily on systemic drugs, more than 20 new topical compounds are under development to treat the disease. This review aims to provide a synthesized summary of the current knowledge about AD topical treatment, echoing existing gaps and coming research trends. The available data seems promising, with some drugs already approved (ruxolitinib being the most recent), and several are in an advanced stage of development and will soon be available for treatment of mild to moderate disease, namely tapinarof, difamilast, and roflumilast. However, longer and larger prospective studies are needed to assess the long-term efficacy and safety of these new compounds and evaluate their benefits over current treatments.
Article
Hyperkeratotic flexural erythema (HKFE), also termed granular parakeratosis (GP), is a rare skin condition thought to be linked to a skin barrier dysfunction process, however the exact cause of this is yet to be determined. Management options are varied, with no consensus on treatment. Several previous reports have recorded successful treatment with amoxycillin‐clavulanic acid combination. We propose the use of oral doxycycline in addition to topical coconut oil compound as a treatment option in therapy resistant HKFE.
Article
Objective Provide a review of atopic dermatitis management, focusing on optimizing topical therapy, creating a stepwise approach for treatment plans, and guide when to start systemic therapy. Data Sources PubMed search of English-language articles regarding atopic dermatitis in all ages. Study Selection Articles on the subject matter were selected and reviewed. Results Topical corticosteroids are first-line treatment for managing atopic dermatitis. Topical nonsteroidal agents, calcineurin inhibitors, crisaborole, and recently ruxolitinib, that cause no cutaneous atrophy are options for reducing use of topical corticosteroids, including on sensitive sites. Emerging topical agents are in clinical trials. Proactive management, with continued application 2-3 times weekly of a mid-potency topical corticosteroid or tacrolimus, may maintain control for clear or almost clear, localized sites of dermatitis that rapidly recur when topical anti-inflammatory medication is stopped. If topical therapy alone cannot control disease and quality of life is impacted, re-evaluation to confirm diagnosis, manage comorbid conditions, address compliance and patient-specific concerns, and optimize topical therapy must be undertaken before deciding to advance to systemic medication. Dupilumab, an interleukin-4 receptor inhibitor, has become first-line systemic therapy, given its efficacy and safety, allowing long-term treatment without laboratory monitoring. Other biologics and Janus kinase inhibitors are emerging as alternatives that could eliminate the need for immunosuppressants with their higher risks. Conclusion Several options are now available for topical treatment. A stepwise approach is needed to consider alternative therapies and diagnoses before advancing to systemic treatment, but the safety of newer immunomodulators will lower the threshold for more aggressive intervention.
Chapter
The aim of this chapter is to provide an overview of basic and tailored topical moisturisers and discuss how and why they form the backbone for the management of psoriasis. Our discussion begins by describing the main characteristics of psoriasis and by indicating how alterations in the skin’s integrity and barrier function contribute to the initial development of psoriasis and subsequent changes in psoriasis phenotype. Next, we address the evolution of topical moisturisers to ever more sophisticated and beneficial products, and describe the key biophysical effects exerted on the psoriatic skin by their active ingredients, as well as the myriad benefits offered by fundamental and specialty ingredients. Furthermore, we delineate how topical moisturiser formulation modalities can help to improve compromised skin barrier function and to alleviate the symptoms of psoriasis, cosmetically and/or therapeutically as well as discuss the associated concerns and challenges encountered along the way.
Chapter
Moisturizers help regulate the skin barrier and continue to be the foundation of maintenance treatment for atopic dermatitis. Though a tremendous variety of moisturizers at difference price points exist on the market, studies do not show that one moisturizer or moisturizer ingredient is significantly better than another in patients with atopic dermatitis.
Article
Introduction: The vulnerable skin of neonates and infants is still developing anatomically and functionally and more susceptible to skin barrier disruption. The current consensus paper explores challenges in caring for neonates and infants' skin, skincare use and evaluates the role of ceramides (CERs) containing cleansers and moisturizers. Evidence aquisition: A panel of eight clinicians who treat neonates and infants developed a consensus paper on new-born and infant skin barrier integrity and CERs-containing skincare importance. The consensus process consisted of a modified Delphi technique. The selected information from the literature searches, coupled with the panel's opinion and experience, was used to adopt statements to provide clinical data for paediatric dermatologists, dermatologists, and paediatric healthcare providers who treat neonates and infants. Evidence synthesis: Increasingly, evidence supports skincare starting early in life, recognizing the benefits of ongoing daily use of non-alkaline cleansers and moisturizers to maintain skin barrier function. Skincare for neonates and infants should be safe, effective, and fragrance as well as sensitizing agent-free. Skincare with CERs may benefit the stratum corneum's lipid and water content. Conclusions: When applied from birth onwards, gentle cleansers and moisturizers containing barrier lipids help maintain the protective skin barrier and soothe the skin with long-term moisturizing benefits.
Article
Rectal prolapse (RP) is a common clinical condition in mice, that does not have a recognized or documented standard ofcare. At our institution, an average of 240 mice develop RP each year. Our practice has been to recommend euthanasia uponidentifying a RP based on its appearance as a painful or distressful condition. This study aimed to assess treatment options that would maintain the RP mucosa and allow mice to reach their study endpoint, and to evaluate the perception of this condition as a painful or distressful event. This study used 120 mice with spontaneous RP, concurrently assigned to ongoing research protocols. Mice were randomly assigned to 1 of 3 treatment groups: petroleum jelly, lidocaine jelly, or no treatment. Fecal samples were collected for pathogen testing, and all mice received an initial base score, followed by weekly blind scores. Upon euthanasia, RP tissue was collected for histopathology. Of the 120 mice identified with RP, 47 mice were breeders; 28% successfully produced 22 additional litters after developing RP. Seventy-three were nonbreeders, with 92% reaching their research study endpoint. No statistically significant differences were detected between the 3 treatment groups based on gross mucosal health, pain and distress, or histopathology. In this study, none of the mice in any group were euthanized based on the RP endpoint scoring criteria. These findings demonstrate that treatment is unnecessary for RP, and mice with RP did not show signs of pain or distress. In adherence to the 3Rs, this study supports animal number reduction and clinical refinement, allowing mice with RPs to reach their intended research study endpoints or produce additional litters.
Article
Full-text available
Background Emollients are a mainstay of treatment in atopic dermatitis (AD), a disease distinguished by skin bacterial dysbiosis. However, changes in skin microbiota when emollients are used as a potential AD preventative measure in infants remain incompletely characterized. Results We compared skin barrier parameters, AD development, and bacterial 16S ribosomal RNA gene sequences of cheek, dorsal and volar forearm samples from 6-month-old infants with a family history of atopy randomized to receive emollients (n = 11) or no emollients (controls, n = 12). The emollient group had a lower skin pH than the control group. The number of bacterial taxa in the emollient group was higher than in the control group at all sites. The Streptococcus salivarius proportion was higher in the emollient versus control groups at all sites. S. salivarius proportion appeared higher in infants without AD compared to infants with AD. A decrease in S. salivarius abundance was further identified in a separate larger population of older children demonstrating an inverse correlation between AD severity at sampling sites and S. salivarius proportions. Conclusions The decreased skin pH and the increased proportion of S. salivarius after long-term emollient use in infants at risk for developing AD may contribute to the preventative effects of emollients in high-risk infants.
Article
Full-text available
Atopic dermatitis (AD) and psoriasis represent contrasting poles of the T(H)1 versus T(H)2 paradigm. Both diseases have been associated with increased numbers of dendritic cells (DCs) in the skin, but the similarities and differences in DC populations need to be established. We aimed to characterize the specific DC subsets, as well as chemokine and cytokine environment in chronic AD compared with psoriasis. Skin biopsies were obtained from patients with acute exacerbation of chronic AD (n = 18), psoriasis (n = 15), and healthy volunteers (n = 15) for microarray analysis, RT-PCR, immunohistochemistry, and double-label immunofluorescence. Myeloid DCs upregulate CCL17 and CCL18 in AD, as opposed to TNF-alpha and inducible nitric oxide synthase (iNOS) in psoriasis. In our study, we identified cells phenotypically identical to the inflammatory dendritic epidermal cells in the dermis in both diseases, although to a lesser extent in psoriasis. We found substantially higher numbers of dermal CCL22 producing plasmacytoid DCs in AD. The thymic stromal lymphopoietin receptor showed significantly higher expression in AD, whereas the thymic stromal lymphopoietin ligand was upregulated more in psoriasis. There are major differences in myeloid and plasmacytoid subsets of cutaneous DCs and the chemokine/cytokine environment between AD and psoriasis. Distinct subsets within the CD11c(+) population may influence polarization through the production of regulatory mediators, including iNOS, TNF, CCL17, and CCL18. Plasmacytoid DCs may also influence T(H)2 polarization, having a more important role in AD than previously appreciated. Dermal inflammatory dendritic cells in AD and TNF and iNOS-producing DCs in psoriasis, and/or their regulatory products, may be potential targets for future therapeutic interventions.
Article
Interleukin 17 (IL-17) includes several cytokines among which IL-17A is considered as one of the major pro-inflammatory cytokine being central to the innate and adaptive immune responses. IL-17 is produced by unconventional T cells, members of innate lymphoid cells (ILCs), mast cells, as well as typical innate immune cells, such as neutrophils and macrophages located in the epithelial barriers and characterised by a rapid response to infectious agents by recruiting neutrophils as first line of defence and inducing the production of antimicrobial peptides. Th17 responses appear pivotal in chronic and acute infections by bacteria, parasites, and fungi, as well as in autoimmune and chronic inflammatory diseases, including rheumatoid arthritis, psoriasis, and psoriatic arthritis. The data discussed in this review cumulatively indicate that innate-derived IL-17 constitutes a major element in the altered immune response against self antigens or the perpetuation of inflammation, particularly at mucosal sites. New drugs targeting the IL17 pathway include brodalumab, ixekizumab, and secukinumab and their use in psoriatic disease is expected to dramatically impact our approach to this systemic condition. Copyright © 2015 Elsevier Ltd. All rights reserved.