Petrolatum: Barrier repair and antimicrobial responses
underlying this ‘‘inert’’ moisturizer
Tali Czarnowicki, MD,
* Dana Malajian, BA,
* Saakshi Khattri, MD,
* Joel Correa da Rosa, PhD,
Riana Dutt, ScB,
Robert Finney, MD,
Nikhil Dhingra, MD,
Peng Xiangyu, MSc,
Hui Xu, MSc,
Yeriel D. Estrada, BS,
Xiuzhong Zheng, MSc,
Patricia Gilleaudeau, NP,
Mary Sullivan-Whalen, NP,
Avner Shemer, MD,
James G. Krueger, MD, PhD,
and Emma Guttman-Yassky, MD, PhD
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,
Petrolatum, available since 1872,
is a widely used moisturizer
consisting mainly of long-chain aliphatic hydrocarbons.
been shown to decrease transepidermal water loss (TEWL) in
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.
Petrolatum has been proposed to protect against postambula-
tory surgical skin infections and is widely used after minor
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.
Importantly, petrolatum rarely induces
allergic contact dermatitis (ACD) reactions
and has never been
reported to cause contact anaphylaxis, whereas bacitracin was
shown to induce ACD in up to 13% of patients
and has caused
contact anaphylaxis in several cases.
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
This results in increased prevalence of ACD and
microbial colonization/infection in the population with AD.
Despite similar bacterial colonization in patients with AD and
those with psoriasis,
another common inﬂammatory skin
disease, signiﬁcantly higher rates of skin infection were observed
only in patients with AD.
These differences were postulated to
result in part from signiﬁcantly lower antimicrobial peptide
(AMP) responses in AD lesions compared with psoriatic
In adult and pediatric patients with AD, moisturizers
have been shown to improve AD disease severity,
the Laboratory for Investigative Dermatology and
the Center for Clinical and
Translational Science, The Rockefeller University, New York;
College of Physicians and Surgeons, New York; the Departments of
Population Health Science and Policy, and
Genetics and Genomics Science and
the Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine
at Mount Sinai, New York;
the Department of Dermatology, Jefferson Medical Col-
lege, Philadelphia; and
the Department of Dermatology, Tel-Hashomer Hospital, Tel
*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: email@example.com.
Ó2015 American Academy of Allergy, Asthma & Immunology
ACD: Allergic contact dermatitis
AD: Atopic dermatitis
AHR: Aryl hydrocarbon receptor
AMP: Antimicrobial peptide
DC: Dendritic cell
DEG: Differentially expressed gene
FCH: Fold change
HBD2: Human b-defensin 2
H&E: Hematoxylin and eosin
LCN2: Lipocalin 2
PI3: Peptidase inhibitor 3/elaﬁn
QC: Quality control
SC: Stratum corneum
TEWL: Transepidermal water loss
as well as to reduce rates of Staphylococcus
Emollients have recently been shown to
effectively prevent AD development in high-risk newborns.
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.
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
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.
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,
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).
Expression values were normalized to
human acidic ribosomal protein (hARP). Human HGU133Plus2.0 GeneChip
probe arrays (Affymetrix, Santa Clara, Calif) were used for gene
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.
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
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
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
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).
Petrolatum upregulates AMPs more than occlusion
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
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.
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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
Increased AMP protein expression determined by
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
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
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
4CZARNOWICKI ET AL
TABLE I. DEGs in petrolatum versus control skin through gene arrays
Probe set Symbol Gene name
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
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
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
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,
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
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),
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.
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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
were observed with petrolatum occlusion in patients with AD
Occlusion with petrolatum improves epidermal
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
Suppression of cellular inﬁltrates after petrolatum
occlusion in patients with AD
We measured inﬁltrates of CD3
T cells and CD1c
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
T-cell counts and CD1c
DC counts with
petrolatum were observed mostly (except CD8
) in AD but not
in non-AD skin when compared with respective control values
(P< .05 for all).
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
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
It has been estimated that 14 million skin excisions or biopsies
are performed annually in the United States.
After a pivotal
randomized controlled trial showing equivalent postoperative
infection rates for petrolatum and topical antibiotic ointment in
ambulatory surgery patients,
69.4% of Mohs surgeons
recommend petrolatum for wound care after routine procedures.
This recommended change in practice is due to several factors,
such as higher cost,
increased rates of ACD,
and even cases
of contact anaphylaxis
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
[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
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
This mechanism is used by most petrolatum-
induced AMPs in our study, including LL-37
LL-37 is one of the most well-characterized AMPs,
an a-helix to disrupt both bacterial membranes and viral
Its precursor, cathelicidin, was recently shown to
be upregulated in dermal adipocytes on infection with S aureus,
pointing to its importance in innate defense against this
Consistent with our results, LL-37 also upregulates
innate immune genes, including IL6,IL8, and IL10.
HBD2/DEFB4A has potent activity against gram-negative
bacteria, including Escherichia coli and Pseudomonas
aeruginosa, but is less effective against gram-positive
Additionally, calprotectin, the antimicrobial
heterodimeric complex formed by S100A8 and S100A9, exerts
effects against S aureus,Staphylococcus epidermidis,
negative bacteria (E coli,Klebsiella species, and Capnocyto-
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).
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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
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
8CZARNOWICKI ET AL
as determined by using IHC, and that psoriasin/S100A7, which
acts against E coli
and as an ‘‘alarmin’’ to amplify the skin’s
inﬂammatory response to pathogens,
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.
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)
or both IL-17 and IL-22 (S100A7,
S100A8, S100A9, and S100A12).
IL-17A, IL-22 and IL-23 (a
known activator of the T
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
petrolatum does not induce broad upregulation of all immune
axes but rather is selective for T
17 and T
22 pathways, as
suggested by the relative lack of induction of T
(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
2 cytokines and chemokines
(IL-5, IL-10, and CCL26), as previously reported,
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,
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
The changes observed with petrolatum are greatly dependent
on baseline skin characteristics. An altered microbiome,
reduced AMP levels,
cytokine axis deviation,
and a defective
are characteristic of AD and render these patients
more susceptible to recurrent cutaneous infections, especially in
comparison with psoriasis, a disease characterized by T
skewing and increased AMP levels.
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
2 markers, which have been shown to inhibit AMP
production in nonlesional AD skin.
In addition to its
inhibitory effect on AMPs,
2 overexpression seen in
nonlesional AD skin
has also been suggested to reduce
FLG mRNA expression and other structural components of
Therefore the lack of T
2 upregulation by
petrolatum might be a major beneﬁt when considering the
potential use of petrolatum in patients with AD.
In addition to T
2 skewing, a T
1 deﬁciency is also present
in AD skin,
which has been associated with a lack of
protective immunity against cutaneous viral infections, including
Our data show that petrolatum boosts the
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,
function of the corniﬁed cell envelope,
and changes in
composition of epidermal lipids.
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
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.
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.
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
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
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
showed that coal tar, an ancient
skin product used in the treatment of AD,
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
22 cells, increasing IL-17 and IL-22 expression.
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
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.
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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
Lastly, future studies will need to assess and compare not only
the long-term effect of emollients in high-risk newborns
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
back to a normal
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
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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.
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 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
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
-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
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
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
Analysis for the second cohort consisted of ﬁtting a linear mixed-effects
model to log
-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
-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
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FIG E1. RT-PCR log
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.
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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.
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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.
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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.
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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.
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TABLE E2. Primers and probes for RT-PCR
TSLP, Thymic stromal lymphopoietin.
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