Control of the Physical and Antimicrobial
Skin Barrier by an IL-31−IL-1 Signaling
Network
This information is current as
of June 18, 2017.
Kai H. Hänel, Carolina M. Pfaff, Christian Cornelissen,
Philipp M. Amann, Yvonne Marquardt, Katharina Czaja,
Arianna Kim, Bernhard Lüscher and Jens M. Baron
J Immunol published online 4 March 2016
http://www.jimmunol.org/content/early/2016/03/04/jimmun
ol.1402943
http://www.jimmunol.org/content/suppl/2016/03/04/jimmunol.140294
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Supplementary
Material
Published March 4, 2016, doi:10.4049/jimmunol.1402943
The Journal of Immunology
Control of the Physical and Antimicrobial Skin Barrier by an
IL-31–IL-1 Signaling Network
Kai H. Hänel,*,†,1,2 Carolina M. Pfaff,*,†,1 Christian Cornelissen,*,†,3 Philipp M. Amann,*,4
Yvonne Marquardt,* Katharina Czaja,* Arianna Kim,‡ Bernhard L€
uscher,†,5 and
Jens M. Baron*,5
he skin is the largest organ of the human body and forms
an indispensable barrier to protect against penetration by
environmental pathogens, allergens, or irritants. Another
major function of the skin is to inhibit transepidermal water loss
(TEWL) and thus minimize dehydration (1, 2). Inflammatory skin
diseases like atopic dermatitis (AD), a highly pruritic skin disorder, are characterized by impairment of skin barrier function, increased skin surface pH, and allergen priming, as well as
decreased hydration of the stratum corneum (3–5). In addition,
enhanced susceptibility to infections (e.g., by Staphylococcus
aureus) has been observed in patients with AD with an overall
altered skin microbiota (6–8). Mechanisms underlying this attenuation of skin barrier functions and the contribution of inflammatory cytokines to this phenotype are incompletely understood.
At the genetic level, impaired skin barrier function can be linked
to reduction or loss of expression of the structural protein profilaggrin.
T
It is extensively modified and processed and fulfills essential functions in the formation of the skin barrier. In addition, processed
profilaggrin enhances moisturization and contributes to the acidity of
the epidermis, both important for sustaining the integrity of the skin
barrier (1, 9, 10). Profilaggrin is processed to filaggrins, which are
cross-linked to and induce bundling of keratin filaments. This together with other proteins and enzymatic activities substantially
augments the mechanical stability of keratin filaments (2, 9, 11).
Thus, filaggrin is central to the development of an efficient skin
barrier in combination with other processes that result in the generation of the cornified envelope (2, 3, 5, 12, 13), including the
strengthening of cell–cell contacts by the maturation of desmosomes
to corneodesmosomes (3, 5, 8, 9, 14, 15), the formation of an lipid
envelope (8, 9, 16, 17), and the production of an antimicrobial
barrier by synthesizing antimicrobial peptides (AMPs) and fatty
acids (9, 16, 18–21).
*Department of Dermatology and Allergology, Medical School, RWTH Aachen University, 52074 Aachen, Germany; †Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, 52074 Aachen, Germany; and
‡
Department of Dermatology, College of Physicians and Surgeons, Columbia University, New York, NY 10032
The sequences presented in this article have been submitted to the National Center
for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.
nih.gov/geo/) under accession number GSE76880.
1
K.H.H. and C.M.P. are cofirst authors.
2
Current address: Chiltern International, Bad Homburg, Germany.
3
Current address: Novartis Pharma, N€urnberg, Germany.
4
Current address: Department of Dermatology, Stadt- und Landkreis Hospital Heilbronn, Heilbronn, Germany.
5
B.L. and J.M.B. are cosenior authors.
ORCIDs: 0000-0001-7983-657X (C.C.); 0000-0002-1824-1318 (K.C.); 0000-00034609-1182 (A.K.); 0000-0002-1174-6946 (J.M.B.).
Received for publication November 21, 2014. Accepted for publication February 8,
2016.
This work was supported by Deutsche Forschungsgemeinschaft (BA 1803/7-1) and
the START program of the Medical School of RWTH Aachen University (to C.C.,
B.L., and J.M.B.).
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402943
Address correspondence and reprint requests to Prof. Bernhard L€uscher or Prof. Jens
M. Baron, Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany (B.L.) or Department of Dermatology and Allergology, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen,
Germany (J.M.B.). E-mail addresses: [email protected] (B.L.) or jensmalte.
[email protected] (J.M.B.)
The online version of this article contains supplemental material.
Abbreviations used in this article: AD, atopic dermatitis; AMP, antimicrobial peptide;
CsSSE, cell-sorted skin equivalent; 3D, three-dimensional; hBD, human b-defensin;
HDF, human dermal fibroblast; HPRT, hypoxanthine guanine phosphoribosyl transferase; NHEK, normal human epithelial keratinocyte; qRT-PCR, quantitative RTPCR; rh, recombinant human; TEWL, transepidermal water loss.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
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Atopic dermatitis, a chronic inflammatory skin disease with increasing prevalence, is closely associated with skin barrier defects. A cytokine related to disease severity and inhibition of keratinocyte differentiation is IL-31. To identify its molecular targets, IL-31–dependent
gene expression was determined in three-dimensional organotypic skin models. IL-31–regulated genes are involved in the formation of
an intact physical skin barrier. Many of these genes were poorly induced during differentiation as a consequence of IL-31 treatment,
resulting in increased penetrability to allergens and irritants. Furthermore, studies employing cell-sorted skin equivalents in SCID/NOD
mice demonstrated enhanced transepidermal water loss following s.c. administration of IL-31. We identified the IL-1 cytokine network
as a downstream effector of IL-31 signaling. Anakinra, an IL-1R antagonist, blocked the IL-31 effects on skin differentiation. In addition
to the effects on the physical barrier, IL-31 stimulated the expression of antimicrobial peptides, thereby inhibiting bacterial growth on
the three-dimensional organotypic skin models. This was evident already at low doses of IL-31, insufficient to interfere with the physical
barrier. Together, these findings demonstrate that IL-31 affects keratinocyte differentiation in multiple ways and that the IL-1 cytokine
network is a major downstream effector of IL-31 signaling in deregulating the physical skin barrier. Moreover, by interfering with IL31, a currently evaluated drug target, we will have to consider that low doses of IL-31 promote the antimicrobial barrier, and thus a
complete inhibition of IL-31 signaling may be undesirable. The Journal of Immunology, 2016, 196: 000–000.
2
study will likely be relevant when considering interfering with IL31 function.
Materials and Methods
Primary cell culture and skin equivalents
Normal human epithelial keratinocytes (NHEKs) and human dermal fibroblasts (HDFs) were prepared from sterile human skin samples (approved
by the ethic committee of the Medical School of the RWTH Aachen
University) and cultivated under regular cell-culture conditions. Organotypic skin equivalents of NHEKs and HaCaT-IL31RA cells were constructed as described previously (1, 41), cultured over a period of up to 10 d
at the air–liquid interphase, and treated with recombinant human (rh)IL-31
(PeproTech, Hamburg, Germany) or rhIL-1a (PeproTech). The reconstructs were harvested, cut into pieces, and either fixed according to a
standardized protocol for routine histology or embedded in Tissue Tec (O.
C.T.) compound (Sakura Finetek, Zoeterwoude, the Netherlands) for cryosectioning. Parts of the cultures were stored in RNA later (Ambion/Applied
Biosystems, Darmstadt, Germany) for RNA isolation.
HaCaT cells with inducible IL-31RA
HaCaT cells were obtained from N. Fusenig (German Cancer Research
Center, Heidelberg, Germany) (50). IL-31RA was expressed stably in
HaCaT cells using a tet-inducible lentiviral expression vector as described
previously (1).
Cell-sorted skin equivalents
Cell-sorted skin equivalents (CsSSEs) were based on protocols as described
previously (12). Briefly, 48 h before implantation, 4–6-wk-old SCID/NOD
mice were fed with doxycycline (2 mg/ml) provided in the drinking water
supplemented with 5% sucrose. On day 21, two silicon chambers were
implanted onto the muscle fascia of the mouse back. Twenty-four hours
later, a cell slurry of 106 cells of each HDFs and HaCaT cells (chamber 1)
or HDFs and HaCaT-IL31RA cells (chamber 2) were seeded into the
chambers. From days 4 to 7, 20 mg rhIL-31 in 50 ml PBS or 50 ml PBS for
control were applied s.c. under the skin equivalents. On day 6, the
chambers were removed followed by TEWL measurements on day 7. Mice
were sacrificed and skin equivalents were either embedded in paraffin or
cryoconserved for further investigation.
Skin barrier analysis
For skin barrier analysis, fluorescently labeled recombinant timothy grass
pollen major allergen phl p1 (Biomay, Vienna, Austria) was applied
topically on 7-d-old HaCaT-IL31RA organotypic skin equivalents for 45
min and the luminescence measured. To analyze the vulnerability of an
organotypic skin equivalent to the penetration by irritating agents, 7-d-old
HaCaT-IL31RA organotypic skin equivalents were treated topically with
0.2% SDS for 40 min, and 24 h later, the expression of IL1A was analyzed
by quantitative RT-PCR (qRT-PCR), and the IL-1a release was measured
by ELISA (IL-1a: DY200; R&D Systems, Wiesbaden, Germany).
Penetration assay
HaCaT models were treated with 10 mmol Lucifer Yellow/Biocytin (L6950;
Invitrogen) from the basolateral side at room temperature for 1 h and
subsequently processed for cryoconservation and sectioning.
TEWL analysis in cell-sorted skin equivalents
TEWL was measured using a Tewameter TM210 (Courage+Khazaka,
Cologne, Germany) according to the protocol of the manufacturer.
Ex vivo explants
Ex vivo skin explants were prepared from sterile human skin samples
(approved by the ethics committee of the Medical School of the RWTH
Aachen University). Subcutaneous fat was carefully removed, and skin
samples were rinsed three times in sterile PBS containing penicillin,
streptomycin, and amphotericin B (Biochrom, Berlin, Germany). Eightmillimeter punch biopsies were taken from these skin samples and
placed on the bottom of a polycarbonate membrane insert (3-mm pore size;
Nunc, Rochester, NY). Then inserts were placed in six-well plates, and
skin grafts were cultured at the air–liquid interphase and kept in the cell
culture incubator at 37˚C with 5% CO2. The medium consisting of equal
volumes of DMEM (Life Technologies, Carlsbad, CA) and KGM (Lonza)
supplemented with 5% FCS, 50 mg/ml L-ascorbic acid (Sigma-Aldrich),
and a calcium concentration of 1.2 mmol with and without 100, 500, or
1000 ng/ml rhIL-31 (PeproTech) was changed daily for 4 d.
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AD and other common dermatologic and allergologic diseases
are associated with loss-of-function mutations in FLG, which
encodes profilaggrin, further supporting the key role of this protein
in skin barrier formation (22–24). FLG mutations are strongly
associated with AD, although only about half of the heterozygotes
develop clinical disease (10, 25). Interestingly, the acute lesional
skin of these patients with AD carrying FLG mutations exhibits
lower levels of filaggrin expression as compared with the clinically unaffected skin of the same patients. These findings suggest
that additional factors may contribute to the regulation of filaggrin
expression and the AD phenotype (1, 26, 27). Indeed, AD is associated with the deregulated expression of various cytokines,
including IL-4, IL-13, IL-22, IL-25, and IL-31, which are known
to modulate the expression of structural proteins implicated in the
formation of the skin barrier (9, 28–30). IL-31 belongs to the IL-6
family of proinflammatory cytokines and signals through heterodimeric receptors composed of the oncostatin M receptor and the
IL-31–specific receptor IL-31RA (30–32). IL-31 expression is
increased in lesions and serum of patients with AD and correlates
with disease severity (30, 33–36). Furthermore, distinct haplotypes of the IL31 gene are associated with AD (27, 37, 38).
In morphological studies employing human three-dimensional
(3D) skin equivalents, we recently demonstrated that IL-31 disturbs the differentiation of keratinocytes, interferes with filaggrin
expression, and weakens the lipid envelope formation (1, 9, 24, 39,
40). In this study, we report on our studies to clarify further the
molecular and functional consequences of IL-31 signaling in
keratinocytes. Primary normal keratinocytes express very low
levels of IL-31RA, which can be induced by IFN-g (33–36, 41).
However, such treatment has additional consequences, including
cell cycle arrest, making it difficult to distinguish between IFN-g– and
IL-31–specific effects. To avoid interference by IFN-g stimulation, we used a donor-independent reproducible system applying
HaCaT cells expressing the IL-31RA receptor (1). We demonstrate that IL-31 influences the formation of the skin barrier in
multiple ways. In addition to deregulating the expression of
structural proteins, including filaggrin, IL-31 represses enzymes
and proteins involved in filaggrin processing and the formation of
desmosomes, whereas genes encoding tight junction proteins were
not affected. The net result is a weakening of the physical barrier.
As downstream effector of IL-31 signaling, we identified the IL-1
cytokine network. Through this network, IL-31 also enhances the
antimicrobial barrier by inducing the expression of AMPs, including human b-defensin (hBD)-2 and -3 and members of the
S100 Ca 2+ -binding protein family (S100A8, S100A9, and
S100A12).
These findings suggest that IL-31 is a key player in the pathogenesis of AD. Furthermore, we uncovered a positive effect of
IL-31 on the antimicrobial barrier function of the skin. IL-31
stimulates the expression of AMPs with a broad spectrum of antimicrobial activities against skin pathogens, including S. aureus.
Of note is that these latter effects are measurable in response to
low concentrations of IL-31, which are insufficient to affect the
physical barrier. Thus, IL-31 appears to exert bifunctional effects,
with low doses promoting the antimicrobial barrier but high doses
additionally impairing the physical barrier. Low levels of IL-31
may be produced by different skin-resident immune cells, whereas
high levels are associated with different inflammatory skin diseases, including AD, allergic contact dermatitis, and prurigo
nodularis (30), but also with tumors such as mastocytosis and
cutaneous T cell lymphoma (42–45), and with pruritus in many
pathological conditions (46–49). Thus, these findings suggest that
IL-31 is an emerging therapeutic target. The differential effects of
low and high doses of IL-31 on the skin barrier described in this
IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER
The Journal of Immunology
Analysis of gene expression using exon expression arrays
For gene expression analysis, HaCaT-IL31RA organotypic skin equivalents
were stimulated with rhIL-31 (100 ng/ml; PeproTech) for 2, 8, 24, or 48 h.
mRNA was then purified and analyzed on GeneChip Human Exon 1.0 ST
arrays as reported previously (1, 51). Data visualization and analysis were
performed with GeneSpring GX software (Agilent Technologies, Böblingen,
Germany). The primary data have been assigned the Gene Expression
Omnibus accession number GSE76880 (http://www.ncbi.nlm.nih.gov/geo/).
Western blotting
Cells were lysed in RIPA buffer (10 mmol Tris/HCl [pH 7.4], 150 mmol
NaCl, 1% Nonidet P-40, 1% deoxycholic acid, 0.1% SDS, and 0.5%
Trasylol) containing a protease inhibitor mixture (Proteobloc; Fermentas,
Waltham, MA), on ice (52). The lysates were sonicated twice for 30 s on
ice and cleared by centrifugation. Proteins were separated using 10–12%
SDS-PAGE and then blotted on nitrocellulose membranes for the detection
of selected proteins using specific Abs. Abs were purchased from either
Cell Signaling Technology (p-STAT3, p-p38, p65, p-p65, and IkBa) or
Santa Cruz Biotechnology (STAT3 and p38; Santa Cruz, CA). Abs specific
for tubulin and actin were purchased from Sigma-Aldrich and MP Biomedicals, respectively.
For the analysis of antimicrobial activity, HaCaT-IL31RA organotypic skin
equivalents were stimulated with or without rhIL-31 for 10 d. Cultures of S.
aureus stably expressing GFP (ATCC29213) (53) were grown in LuriaBertani medium containing 20 mg/ml chloramphenicol overnight and then
diluted 1:10 for an additional 2 h until an OD of 1 was reached. The
bacteria were diluted 1:100 in Luria-Bertani and applied topically onto the
3D skin equivalents. The models were harvested directly or incubated at
37˚C and 5% CO2 for 8 or 16 h. The models were cut into three parts. One
part was used for immunohistochemistry, the other two parts were lysed,
and genomic DNA or RNA was isolated. RNA was isolated using the
NucleoSpin RNA II Kit (Macherey-Nagel, Dren, Germany) according to
the manufacturer’s instructions for Gram-positive bacteria. cDNA was
synthesized with the SuperScript VILO cDNA Synthesis Kit (Invitrogen).
Genomic DNA was prepared using the peqGOLD Bacterial DNA Kit
(Macherey-Nagel). GFP DNA and cDNA was quantified by real-time PCR
using the TaqMan system and Assay-on-Demand gene expression products
for GFP (Mr03989638_mr; Applied Biosystems). The real-time PCR reactions were performed with the TaqMan Gene Expression Master Mix
(Applied Biosystems). Genomic DNA measurements were performed in
duplicates, and cDNA was analyzed in triplicates. For determining the
relative bacterial growth, the changes of threshold cycle values for GFP
from 0 to 8 or 16 h were calculated. The values given in Fig. 9 are 2DCT
(i.e., fold change compared with the 0-h values). The experiments were
performed in triplicates.
all measurements were performed in triplicates in separate reaction wells.
Statistical significance was evaluated by using the two-sided Student t test
on all of the experiments as indicated in the figure legends.
Light microscopy and immunofluorescence
For light microscopy and immunofluorescence analyses of the 3D skin
models, 4-mm cryosections were processed as described previously (1, 54).
The following Abs specific for the indicated proteins were used: filaggrin
(sc-66192; Santa Cruz Biotechnology), S100A7 (MCA5253Z; AbD
Serotec, D€usseldorf, Germany), hBD-2 (ab63982; Abcam, Cambridge,
U.K.), and IL-1a (ab9614; Abcam); and the DNA was stained with DAPI
(Applichem, Darmstadt, Germany).
Flow cytometry
Cultured HaCaT-IL31RA cells were washed with PBS/EDTA and dissociated by addition of trypsin/EDTA. To inactivate the trypsin, culture
medium containing 10% FCS was added, and then the cells were washed
with PBS. For analysis of the surface expression of IL-1R1 and IL-1R2, the
cells were fixed in 3.7% paraformaldehyde for 20 min, washed, blocked
with PBS/1% BSA, and incubated with receptor-specific Abs IL-1R1 (ab
40774; Abcam) and IL-1R2 (ab89159; Abcam) at room temperature for 30
min. The cell-surface fluorescence intensity was measured by adding
fluorescently labeled secondary Abs on an FACS Canto flow cytometry
system (BD Biosciences, Franklin Lakes, NJ).
ELISA
Cells were treated as specified in the figure legends and lysed in RIPA buffer
as described before or the supernatants were collected (55). The samples
were either directly applied to the ELISA or diluted in the provided dilution buffer and measured according to the manufacturer’s recommendations (IL-1a: DY200, R&D Systems; IL-1b: DY201, R&D Systems;
hBD-2: 900-K172, PeproTech; hBD-3, S100A7, S100A9, and S100A12:
Cloud-Clone, Houston, TX).
RNA preparation, reverse transcription, and qRT-PCR
The RNeasy Mini Kit (Qiagen, Hilden, Germany) was used for total RNA
extraction, according to the manufacturer’s instruction, and residual genomic DNA was removed by DNaseI (Qiagen) digestion. A total of 1 mg
RNA was reverse transcribed into cDNA by using the QuantiTect reverse
transcription kit (Qiagen) and analyzed by quantitative real-time PCR by
using the Corbett RotorGene system (Qiagen). The real-time PCR reactions were performed with the SensiFAST SYBR Kit (Bioline, Luckenwalde,
Germany). All primer pairs used were QuantiTect primer assays (Qiagen)
except the primer pairs for hypoxanthine guanine phosphoribosyl transferase (HPRT) (forward, 59-TGACACTGGCAAAACAATGCA-39 and
reverse, 59-GGTCCTTTTCACCAGCAAGCT-39) and OSMRb (forward,
59-GTGTGGGTGCTTCTCCTGCTTC-39 and reverse, 59-TCTGTGCTAATGACTGTGCTTG-39). All measurements were performed in duplicates.
The relative quantification was calculated by using the comparative
cycle threshold method and normalized to HPRT. In case of organotypic
3D models, the tissues were mechanically disrupted and homogenized by
using tissue lyzer (Qiagen). Total RNA was extracted with Nucleo Spin
RNA II (Macherey-Nagel) according to the manufacturer’s protocol.
Purified RNA was reverse transcribed by using High Capacity RNA-tocDNA Master Mix (Applied Biosystems, Foster City, CA). TaqMan experiments were carried out on an ABI PRISM 7300 sequence detection
system (Applied Biosystems) using Assay-on-Demand gene expression
products (Applied Biosystems) for FLG (Hs00418578_m1), S100A7
(Hs00161488_m1), and DEFB4A (Hs00823638_m1) according to the
manufacturer’s recommendations. An Assay-on-Demand product for
HPRT mRNA (Hs99999909_m1) was used as an internal reference to
normalize the target transcripts. For FLG, S100A7, DEFB4A, and HPRT,
FIGURE 1. IL-31 deregulates the expression of genes associated with the
physical skin barrier in organotypic HaCaT-IL31RA 3D models. (A) qRT-PCR
analysis of the indicated genes in HaCaT-IL31RA cells stimulated with rhIL-31;
mean values 6 SD; n = 3. (B and C) qRT-PCR analysis of a 10-d HaCaTIL31RA 3D model; mean values 6 SD; n = 3. The p values were calculated
using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001.
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Analysis of antimicrobial activity
3
4
Sera from patients suffering from AD were collected (approved by the
ethics committee of the Medical School of the RWTH Aachen University)
and stored at 280˚C. IL-31 serum concentrations were determined by
ELISA (Human IL-31 DuoSet DY2824E; R&D Systems). ELISA plates
were precoated with anti-human IL-31 Ab, blocked with PBS/1% BSA,
and incubated for 2 h with serum samples. The samples were either directly applied to the ELISA or diluted in PBS/1% BSA. Detection was
performed according to the manufacturer’s recommendations.
Results
IL-31 controls the expression of genes associated with the
physical skin barrier
FIGURE 2. IL-31 weakens the skin barrier. (A) HaCaT-IL31RA 3D
models were stimulated with or without rhIL-31. Fluorescently labeled phl
p1 was applied topically for 45 min. Histological sections were H&E
stained, and the location of phl p1 (green) was determined by fluorescence
microscopy. Scale bars, 200 mm (H&E) and 100 mm (fluorescence). (B)
Biotin (green) was applied basolaterally for 60 min. DNA was labeled
with DAPI (blue). Scale bars, 100 mm. (C) 3D models were treated
with 0.2% SDS and/or 100 ng/ml rhIL-31 for 40 min. IL-1a release and
IL1A mRNA expression were measured after 24 h. Mean values of two
experiments. n.d., not detected.
kallikrein-like peptidase 7, two important proteases involved in the
processing of filaggrin during keratinocyte differentiation, were
repressed (Supplemental Fig. 1B) (56, 60, 61). These effects were
verified in HaCaT-IL31RA monolayer cultures (Fig. 1A) and in
HaCaT-IL31RA organotypic skin equivalents stimulated with IL-31
(Fig. 1B). Although the findings were reproduced in the 3D models,
the gene expression pattern was more complex in the monolayer
cultures, suggesting that observations made in the latter have to be
interpreted with caution. This may relate to the lack of appropriate
cellular interactions and differentiation-associated effects. Unlike
genes expressing desmosomal proteins, tight junction genes were
not deregulated (Fig. 1C). Together, these findings are consistent
with the IL-31–induced defect in skin differentiation observed
previously (1, 30) and suggest defects in the physical barrier due to
reduced expression of structural proteins and corneodesmosomes.
IL-31 impairs skin barrier function
We assessed the integrity of the epidermal barrier upon IL-31
treatment. We incubated a 3D model with fluorescently labeled
recombinant timothy grass pollen major allergen (phl p1) (4). The
allergen did not penetrate the control organotypic epidermis,
whereas it was enriched in deeper layers upon IL-31 treatment
(Fig. 2A). Moreover, biotin applied basolaterally accumulated in
the stratum corneum only when the model was pretreated with IL31 (Fig. 2B) (6). The former and latter findings are consistent with
FIGURE 3. IL-31 promotes transepidermal water loss. (A) Macroscopic
pictures of a 6-d-old CsSSE. Mice were fed with doxycycline and s.c.
treated with 20 mg rhIL-31 at days 4 to 7. (B) Histological sections of
CsSSE were stained with H&E, for filaggrin (green), the DNA with DAPI
(blue), and lipids with nile red. Scale bars, 200 mm (H&E) and 100 mm
(immunofluorescence). (C) TEWL was measured on 7-d-old CsSSEs;
mean values 6 SD; n = 3. The p values were calculated using Student t
test. (D) Human ex vivo skin explants were cultivated with and without
100, 500, or 1000 ng/ml rhIL-31 and harvested at day 4. Histological
sections were stained for filaggrin (green). *p , 0.05.
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IL-31 disturbs keratinocyte differentiation in organotypic 3D skin
models with either NHEKs or HaCaT-IL31RA cells (HaCaT cells
expressing the IL-31RA under the control of doxycycline) (1). We
analyzed gene expression in organotypic 3D models of HaCaTIL31RA cells in response to IL-31 after 2, 8, 24, or 48 h. IL-31
treatment resulted in altered expression of a broad range of genes
(Supplemental Table I), including many that encode differentiationassociated structural proteins (Supplemental Fig. 1A). Moreover,
genes encoding desmosomal proteins and filaggrin-processing enzymes were repressed (Supplemental Fig. 1B). Most notably, the
genes encoding desmoglein 1 and 4 but also desmocollin 1 and 2 and
corneodesmosin were downregulated. Desmoglein 1 and desmocollin
1 are essential components of corneodesmosomes and targets of
proteases important for skin desquamation (1, 56–58). Corneodesmosin is covalently linked to the cornified envelope (6, 59).
Moreover, the expression of the genes encoding caspase-14 and
IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER
The Journal of Immunology
NOD mice do not have T and B cells, we stained the sections for
F4/80, a marker found on macrophages and other myeloid cells.
Indeed, this revealed some infiltrating myeloid cells; however, no
differences between the HaCaT and HaCaT-IL31RA models were
observed (data not shown). To expand on these findings, we used
human ex vivo skin explants that were treated with increasing
doses of IL-31 in culture (Fig. 3D). Filaggrin expression was reduced when 1 mg/ml IL-31 was applied for 4 d. Together, these
findings suggest strongly that the profound effect of IL-31 on the
gene expression program of keratinocytes results in an impairment
of the physical skin barrier.
IL-31 activates the IL-1 signaling network
IL-1a is a proinflammatory cytokine secreted by inflammatory
cells upon activation of the inflammasome (similar to IL-1b) (62,
63). Physiological levels of IL-1a enhance barrier formation and
wound healing following mechanical injury (16, 25, 64–67),
promote the synthesis of epidermal lipids and the formation of
lamellar bodies, and induce the upregulation of genes and proteins
associated with cell adhesion, proliferation, and epidermal differentiation in keratinocytes (16, 19, 25). In contrast, dysregulation of the IL-1 signaling network has been associated with
different skin diseases, including AD (22). Enhanced expression
FIGURE 4. IL-31 stimulates the IL-1 signaling network. (A) qRT-PCR analysis of the indicated genes in HaCaT-IL31RA cells stimulated with rhIL-31;
mean values 6 SD; n = 3. (B and C) IL-1a and IL-1b proteins were analyzed from cell lysates and supernatants by ELISA; mean values 6 SD; n = 3. (D)
Histological sections of HaCaT-IL31RA cells stained for IL-1a (green) and for DNA (blue). Scale bar, 200 mm. (E) IL-1a in the supernatant of 10-d HaCaTIL31RA 3D models; mean values 6 SD; n = 3. (F) IL-1a levels in 26 human sera: 14 with IL-31 below and 12 with IL-31 .4.1 ng/ml were compared. (G)
HaCaT-IL31RA cells were treated with IL-31 for 4 h. In addition, the indicated kinases were blocked with the following inhibitors: JAK inhibitor I (100 nmol);
JNK inhibitor II (20 mmol); SB202190, selective for p38 MAPKs (20 mmol); U0126, selective for ERKs (20 mmol); and wortmannin, selective for PI3K
kinases (500 nmol). The inhibitors were added 1 h prior to stimulation with IL-31. The expression of the indicated genes were analyzed using qRT-PCR; mean
values 6 SD; n = 3. The p values were calculated using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
defects in the stratum corneum and in cellular adhesion, respectively, as expected from the reduced expression of relevant genes
(Fig. 1) (1). The IL-31–treated models were also more vulnerable
to the application of irritants. Topical treatment with the irritant
SDS increased expression and release of IL-1a in a fully developed
3D model pretreated with IL-31 compared with untreated controls
(Fig. 2C) (11, 16, 25). Moreover, IL-31 was sufficient to induce
IL1A mRNA expression and release of the cytokine (Fig. 2C).
To determine the impact of IL-31 on skin differentiation in vivo,
we developed an epidermal mouse model based on a CsSSE as
described (12). In this in vivo skin reconstitution assay, we compared the morphology, filaggrin expression, lipid layer, and TEWL
of HaCaT keratinocytes with HaCaT-IL31RA cells seeded together with HDFs in chambers placed on the back of SCID/NOD
mice (Fig. 3A). Both models were injected s.c. with rhIL-31 daily.
HaCaT-IL-31RA cells exhibited a profound defect in epidermal
development with a reduction in filaggrin expression and a reduced nile red staining (Fig. 3B), resulting in disturbed barrier
formation as shown by increased TEWL (Fig. 3C). The TEWL
values in the control models were higher compared with normal
skin of healthy individuals (typically 20–22 g/m2 3 h), most
likely due to incomplete barrier formation at the time of analysis.
We noted that some immune cells infiltrated the models. As SCID/
5
6
IL-1a interferes with keratinocyte differentiation
Treatment of HaCaT-IL31RA and NHEK organotypic skin models
with IL-1a inhibited filaggrin mRNA and protein expression
comparable to IL-31 and interfered with keratinocyte differentiation (Fig. 5A, 5B). The IL-31 effects on filaggrin expression and
keratinocyte differentiation were blocked by anakinra, a potent IL1R antagonist that inhibits the binding of IL-1 to IL-1R1 (28),
indicating that IL-1a mediates at least part of the response to IL31 (Fig. 5B, 5C). Anakinra alone was also slightly increasing
filaggrin expression, possibly as a result of repressing basal ac-
FIGURE 5. IL-1a is a downstream effector of IL-31 signaling.
(A) NHEK (one representative experiment) and HaCaT-IL31RA 3D
models were stimulated with rhIL31 and rhIL-1a, and FLG expression
was measured by qRT-PCR. (B)
NHEK 3D models were treated with
rhIL-1a, rhIL-31, and anakinra
(Ana). Histological sections were
stained with H&E for filaggrin
(green) and the DNA with DAPI
(blue). Scale bars, 200 mm (H&E)
and 100 mm (immunofluorescence).
(C) FLG expression in NHEK 3D
models was analyzed by qRT-PCR;
mean values 6 SD; n = 3. (D) The
indicated signaling molecules were
analyzed on Western blots. The p
values were calculated using Student t test. *p , 0.05, ***p , 0.001.
tivities of the IL-1 network (Fig. 5B, 5C). We verified the functionality of anakinra by monitoring the induction of IL-31/IL-1a–
induced signaling pathways (Fig. 5D). Anakinra efficiently
blocked the activation of the MAPK p38 by both cytokines and the
phosphorylation of p65–NF-kB by IL-1a, but had no effect on IL31–induced STAT3 activation. This supports the concept of an
important role of IL-1a downstream of IL-31 for keratinocyte
differentiation.
IL-31 regulates the antimicrobial barrier dependent on IL-1a
activation
In addition to effects on various genes encoding proteins associated
with barrier function, our array data indicated that several genes
encoding AMPs were deregulated in response to IL-31 (Supplemental
Fig. 1E), including genes encoding S100 Ca2+-binding proteins and
human b-defensin-2 (hBD2/DEFB4A) and -3 (hBD3/DEFB103A).
These genes are known IL-1a targets in keratinocytes grown in
monolayers (19). We verified the induction of S100A7, S100A8,
S100A9, S100A12, DEFB103A, and DEFB4A in HaCaT-IL31RA
monolayer cultures (Fig. 6A). The expression of all genes increased
strongly over time. Moreover, IL-31 stimulated the release of these
AMPs and the expression of S100A7 and S100A9 in 3D organotypic
models of HaCaT-IL31RA cells (Fig. 6B, 6C). The AMP encoding
genes were also induced by IL-1a in HaCaT-IL31RA cells, as indicated previously for S100A7 and DEFB4A (19), albeit less efficiently when compared with the response to IL-31 (Fig. 7).
Anakinra reduced or inhibited the effects of both IL-1a and IL-31,
providing further support for IL-1a being a downstream effector of
IL-31.
Bifunctional effects of IL-31 on the epidermal barrier
IL-31 prevents the formation of the physical barrier but stimulates
the antimicrobial barrier, at least in part by activating IL-1 signaling. To evaluate whether the two effects can be separated, we
titrated IL-31 and analyzed skin differentiation. A dose of 10 ng/ml
IL-31 was enough to prevent filaggrin expression in organotypic
HaCaT-IL31RA models (Fig. 8A). Lower doses had minor or no
effects on the expression of FLG and INV and genes encoding
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of IL-1a (e.g., artificially by intradermal injection) promotes an
inflammatory skin phenotype (25). We observed that IL-31 stimulated the expression of IL1A and IL1B, the genes encoding the
IL-1R subunits IL1R1 and IL1R2, and the natural IL-1R antagonist
IL1RN, as well as several genes of IL-1R–associated proteins
(Fig. 4A, Supplemental Fig. 1D). Consistent with the mRNA data,
FACS analysis of HaCaT-IL31RA cells revealed an increase of IL1R1 and IL-1R2 cell-surface expression (Supplemental Fig. 2),
enhanced expression of IL-1a and IL-1b upon IL-31 stimulation
(Fig. 4B), and their release when treated with nigericin (Fig. 4C).
In the 3D model, IL-31 increased the expression (Fig. 4D) and the
release of IL-1a (Fig. 4E). In contrast, IL-1b could not be detected
in the supernatant of the 3D model (not shown), consistent with
the findings in HaCaT-IL31RA monolayer cells (Fig. 4B). Additional signals might be required for IL-1b release. An interaction
between IL-31 and IL-1a was also apparent when these cytokines
were compared in sera of 26 patients with AD. High IL-31 levels
(.4.1 ng/ml) correlated with high IL-1a levels (Fig. 4F). The
specificity of induction of the IL1A gene was addressed by using
different kinase inhibitors. PI3K kinases, p38, and ERKs were
essential to induce the IL1A gene (Fig. 4G). In contrast to these
findings, the induction of the expression of IL20 was dependent on
the JAK kinase, p38, and ERK pathways (Fig. 4G), comparable to
previous findings (1). This suggested that IL-1 signaling pathways
are potential downstream mediators of IL-31 and that the regulation of the IL-1 network is distinct from how IL20 and IL24 are
controlled.
IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER
The Journal of Immunology
7
doses have a profound effect on the physical barrier. The latter
likely antagonizes enhancement of the antimicrobial barrier.
Discussion
desmosomal proteins (Fig. 8B, 8C), and were insufficient to prevent differentiation (Fig. 8A). However, 1 ng/ml IL-31 was sufficient to enhance the expression of IL1A and IL1B and of the
genes encoding several antimicrobial peptides (S100A8, S100A9,
S100A12, DEFB103A, and DEFB4A) in HaCaT-IL31RA cells
(Fig. 8D). To address the functional relevance of induced AMP
expression, we cocultivated bacteria with HaCaT-IL31RA 3D
models. Antimicrobial activity against the pathogen S. aureus was
evident both at low (1 ng/ml) and high (100 ng/ml) concentrations
of IL-31 compared with untreated control (Fig. 9). In organotypic
HaCaT-IL31RA models, the addition of S. aureus inflicted severe
damage to the skin model, resulting in the destruction of the
epidermis and infiltration of bacteria into the dermis (Fig. 9A–C).
This was efficiently prevented when the models were treated with
IL-31. Quantification of the S. aureus–associated GFP fluorescence demonstrated a significant reduction of infiltrating bacteria
(Fig. 9D). In agreement with these observations, the analysis of
genomic GFP sequences as well as GFP mRNA indicated that
bacterial growth was substantially inhibited (Fig. 9E, 9F). Thus,
these findings demonstrate that even low doses of IL-31 have a
bacteriostatic effect on S. aureus, most likely due to the induction
of several AMPs. Taken together, these findings demonstrate that
low doses of IL-31 enhance the expression of genes encoding
AMPs, thus promoting the antimicrobial barrier, whereas higher
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FIGURE 6. IL-31 regulates the expression of AMPs. (A) qRT-PCR
analysis of the indicated genes stimulated with rhIL-31; mean values 6
SD; n = 3. (B) The indicated proteins were measured in the supernatants of
3D models by ELISA; mean values 6 SD; n = 3. (C) Histological sections
of HaCaT-IL-31RA were stained with H&E for the indicated proteins
(green) and the DNA with DAPI (blue). Scale bar, 100 mm. The p values
were calculated using Student t test. *p , 0.05, **p , 0.01.
AD is a common chronic inflammatory disease characterized by a
defect in keratinocyte differentiation and skin barrier formation
(27). Consequences of the impaired skin barrier are TEWL and
increased percutaneous penetration of allergens and pathogens,
thereby promoting inflammation that can be further aggravated by
secondary infections. Alterations in genes encoding key structural
components for proper keratinocyte differentiation and skin barrier formation are associated with barrier dysfunction. A seminal
finding was the identification of FLG mutations in patients with
ichthyosis vulgaris and AD (9, 24, 39, 40). Also, cytokines, including IL-31, are dysregulated in AD and postulated to participate in disease progression (29). Serum IL-31 expression is
increased and correlates with disease severity in these patients
(33–36). IL-31 is produced by immune cells that can either infiltrate the skin or are resident in skin (30). Recently, it was shown
that IL-4, a prominent cytokine in AD, induces IL-31 expression
in Th1 cells (42) and downregulates FLG expression (46). It is
possible that IL-31 is a downstream effector of IL-4 in controlling
keratinocyte differentiation. Previously, we demonstrated that IL31 inhibits keratinocyte proliferation and differentiation without
effects on apoptosis, with FLG being a repressed target of IL-31
signaling (1). However, it remained open whether FLG is one of
few genes that are deregulated in response to IL-31 or whether this
cytokine has a broader effect on skin differentiation. Moreover, it
was unclear whether IL-31 signaling was directly responsible for
affecting FLG expression.
We observed that IL-31 interferes with the expression of many
genes associated with skin barrier formation in 3D organotypic
models of HaCaT-IL31RA cells (Supplemental Table I), whereas
only a few genes were deregulated in monolayers (1). Indeed,
many genes encoding proteins associated with corneodesmosomes,
such as desmogleins or desmocolleins, with the formation of
terminally differentiated corneocytes and with the production of
natural moisturizing factors, including filaggrin and its processing
enzymes caspase-14 and kallikrein-like peptidase 7, were
deregulated in response to IL-31 (Fig. 1). Altogether, our array
data identified ∼570 genes that were altered .2-fold in response
to IL-31. Our results indicate that IL-31 has a profound effect on
the gene expression program that defines keratinocyte differentiation. The functional consequences of this reprogramming were
confirmed by measuring keratinocyte differentiation in 3D organotypic and CsSSE models and by assessing the integrity of the
skin barrier. The poor differentiation, reduced expression of late
differentiation markers, increased penetration of biotin and phl p1
allergen into 3D models, high vulnerability to skin irritants, and
enhanced TEWL all point to a severe disturbance of the physical
and functional properties of the skin barrier (Figs. 2, 3). Thus, IL31 has multidimensional effects on keratinocyte behavior. In
contrast to recent findings in which histamine treatment of 3D
organotypic skin models results in a disturbed barrier formation
and a downregulation of many differentiation-associated factors,
including desmosomal and tight junction proteins (6), IL-31
showed a more selective effect. In particular, no altered expression of genes associated with tight junctions was measured. This
argues for different signaling pathways controlling different aspects of the differentiation process in keratinocytes.
Of particular interest to us was the observation that many genes
encoding components of the IL-1 signaling network were affected
by IL-31, including increased IL-1a release (Fig. 4), supporting
the concept that IL-1 signaling is downstream of IL-31. The
8
IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER
FIGURE 7. Regulation of AMPs by IL-31 is dependent on IL-1a induction. The indicated genes were
analyzed by qRT-PCR. HaCaT-IL31RA cells were
treated for 72 h; mean values 6 SD; n = 3. The p values
were calculated using Student t test. *p , 0.05, **p ,
0.01, ***p , 0.001. Ana, anakinra.
expression of IL-1a leads to the development of spontaneous inflammatory skin lesions in mice (72). In contrast to these findings,
the intracutaneous administration of IL-1a was shown to improve
epidermal barrier function (16, 25) and results in the upregulation
of genes associated with cell adhesion, proliferation, and differentiation (19). Our findings support the inhibitory role of IL-1a on
keratinocyte differentiation and suggest that IL-1 signaling is an
important mediator of the effects of IL-31 (Fig. 5), suggesting that
FIGURE 8. Bifunctional role of IL-31 in human skin
barrier formation. (A) HaCaT-IL31RA 3D models were
stimulated with rhIL-31 for 10 d, and histological
sections were stained with H&E or for filaggrin (green)
and DNA with DAPI (blue). Scale bars, 200 mm (H&E)
and 100 mm (immunofluorescence). (B and C) qRTPCR analysis of the indicated genes in 10-d 3D HaCaTIL31RA models stimulated with rhIL-31; mean values 6
SD; n = 3. (D) qRT-PCR analysis of the indicated genes in
HaCaT-IL31RA cells stimulated with rhIL-31; mean values 6 SD; n = 3. The p values were calculated using
Student t test. **p , 0.01, ***p , 0.001.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
proinflammatory IL-1 signaling network is known to be an important mediator of innate immunity (62). Overactivation of the
network, for example, due to mutations in inflammasome components or as a result of IL-1RA deficiency can lead to autoinflammatory diseases with severe skin involvement (68, 69).
Indeed, increased secretion of IL-1a has been observed in lesional
skin of patients with psoriasis (70). Exposure of keratinocytes to
IL-1a provokes hyperkeratosis (71). Keratinocyte-specific over-
The Journal of Immunology
9
this cascade of cytokines controls skin differentiation. Moreover,
these signaling pathways offer entry sites for crosstalk with other
signals that might influence differentiation. It is also of interest
that FLG loss-of-function mutations are associated with enhanced
IL-1a and IL-1b expression (63). This finding suggests that the
lack of filaggrin stimulates IL-1 signaling that in turn may aggravate the consequences of the genetic defect.
IL-31 also modulated the expression of genes encoding different
AMPs (Fig. 5). These effects were at least in part dependent on the
activation of the IL-1 signaling network as they were inhibited by
anakinra (Figs. 5, 7). IL-1a has been known to regulate AMP
expression in several cell types, including keratinocytes (25, 64–
67, 73). Expression and secretion of AMPs by keratinocytes
represent the first line of defense against cutaneous pathogens (18,
20). Corneocytes are embedded in an acidic lipid envelope that
largely inhibits the growth of various microbes (3, 5, 74, 75).
There are at least three groups of AMPs expressed in the skin. In
the first group, AMPs like hBD-1 are constitutively expressed (7,
8, 18). Members of the second group that includes psoriasin/
S100A7 are present in normal skin and further induced in response to skin inflammation and wounding (2, 76). Members of
the third group, for example, hBD-2, hBD-3, and LL-37, are only
detectable in inflamed skin (2, 77–79). Increased amounts of the
AMPs psoriasin, RNase-7, hBD-2, and hBD-3 were found in
lesional and nonlesional skin and fluids obtained in washings from
the skin of AD patients compared with healthy controls (2, 5, 80–
84). This finding seems to be at odds with the observations of
increased superinfection with bacteria in patients suffering from
AD (5, 8, 9, 85, 86). However, the increased availability of AMPs
in these washings may not reflect the total amounts. More likely,
the reduced or the absence of a lipid barrier in these patients
prevents the attachment of AMPs to the epidermal layer and thus
allows more efficient extraction. Furthermore, the reduction of
desmosomal proteins and the reduction of filaggrin and keratins 1
and 10 weaken the skin barrier, leading to an increased penetration
of bacteria into the skin. This is further complicated by the fact
that persistent, relapsing, and difficult-to-treat S. aureus infections
are associated with the formation of a slow-growing small colony
variant phenotype (87, 88). Bacteria associated with small colony
variant phenotype show reduced susceptibility toward AMPs (9,
18, 20, 21, 89). Of note also is that different cytokines associated
with the complex cytokine milieu of AD lesions may have antagonistic effects on the expression of AMPs by keratinocytes. For example, the Th2 cytokines IL-4, IL-13, and IL-33 have been shown to
inhibit the induction of AMPs by TNF-a and IFN-g (90). Thus, the
positive consequence of IL-31 on the antimicrobial barrier, as
reported in this study, is most certainly modified by other factors,
with many potentially reverting or antagonizing the IL-31 effect.
We have observed concentration-dependent differences in the
effects of IL-31 on the barrier formation and the induction of AMPs
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FIGURE 9. IL-31 promotes the
antimicrobial defense. HaCaTIL31RA 3D models (10 d) were
treated with or without IL-31 as indicated. Subsequently, S. aureus–
expressing GFP were applied to the
apical surface and incubated for the
times displayed. The models were
harvested and prepared for stainings
and DNA or RNA extraction. (A)
Sections were stained with H&E.
(B) Gram staining of histological
sections. (C) S. aureus was visualized in sections using an S. aureus–
specific Ab (red). The DNA was
stained with DAPI (blue). Scale
bars, 100 mm (A–C). (D) Quantification of S. aureus fluorescence,
which was measured from three
sections using the ImageJ software
(National Institutes of Health). (E)
DNA was prepared and GFP measured using quantitative PCR, relative bacterial growth was calculated,
and mean values 6 SD of three experiments are shown. (F) GFP
mRNA was measured using qRTPCR. The relative bacterial growth
was calculated, and mean values 6
SD of three experiments measured
in triplicates are displayed. The p
values were calculated using Student t test. *p , 0.05, **p , 0.01,
***p , 0.001.
10
the IL-31 and the IL-1 signaling networks promote a feedback loop
that can aggravate a proinflammatory environment. Our results also
indicate that pharmacological modulation of IL-31 and IL-1a
activity in AD and possibly other skin diseases might be therapeutically beneficial.
Acknowledgments
We thank Juliane L€uscher-Firzlaff for helpful discussions during the early
phase of the project, Bernd Denecke and Sebastian Huth for help with
bioinformatics, and Oleg Krut and Lothar Rink for the staphylococcus
aureus strain stably expressing GFP.
Disclosures
The authors have no financial conflicts of interest.
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(Figs. 6–8). Low doses of IL-31 promote the antibacterial barrier
without compromising the physical barrier. We suggest that this is
a potentially important physiological role of IL-31. This is supported by the fact that the IL-31RA is upregulated by S. aureus
a-toxin and staphylococcal enterotoxin B in monocytes (91),
suggesting a role in innate immunity. Furthermore, PBMCs of
patients with AD secrete significantly more IL-31 compared with
those of healthy controls upon stimulation with a-toxin and
staphylococcal enterotoxin B (26, 92). This regulation seems to be
specific, and IL31 RNA is not upregulated in response to herpes
simplex or influenza virus infection (30). The expression of IL31RA in keratinocytes is also stimulated by the inflammatory
cytokine IFN-g and activation of the TLR2 receptor (30, 93). The
activation of the IL-31 signaling network by different bacteria and
the subsequent production and release of different AMPs by
keratinocytes is of clinical interest. It is known that eccrine sweat,
a complex mixture of minerals, proteins, and proteolytic enzymes,
contains IL-31. Although sweat is not harmful to healthy skin, it
can aggravate the lesions of patients with AD. Sweat stimulates
IL-1b and IL-1RA expression in keratinocytes, possibly due to the
presence of IL-31, and also contains IL-1a and IL-1b (33–36, 94).
The presence of these cytokines is consistent with their antimicrobial function as described in this study. Our findings suggest
that the induction of IL-1a by IL-31 and the subsequent upregulation of AMPs are independent of the IL-31–induced barrier
disruption that includes repression of many factors associated with
the physical barrier (Figs. 1, 2). The different immune cells that
reside in the skin or are recruited into the skin by diverse signaling
mechanisms are integrated into a complex network of cells that
crosstalk at multiple levels. For example, the production and secretion of AMPs by keratinocytes can augment secretion of IL-31
by mast cells (95), and AMPs have also been described to function
as a chemoattractant of neutrophils and monocytes (96, 97). This
may, on the one hand, further alter the differentiation of keratinocytes and thus promote the pathogenesis of diseases such as AD
and, on the other hand, promote the antimicrobial barrier.
Recently, an outside-inside, then back to outside pathogenic
mechanism in AD was postulated, expanding on the previous view
that AD is largely a disease of immunologic etiology (98). The
authors suggested that primary inherited barrier abnormalities in
AD ultimately stimulate downstream paracrine mechanisms that
could further compromise skin barrier function, driving an
outside-inside-outside pathogenic loop in AD. This model suggests a cyclic interplay between inherited barrier abnormalities
and exogenous and endogenous stressors with key functions of
different secreted signaling molecules. Our findings endorse this
model. Inherited barrier abnormalities allow enhanced uptake of
allergens and Ags, which results in increased IL-31 expression, as
well as other factors, in immune cells, including Th2 cells, and
enhanced IL-31RA expression on keratinocytes (1). This promotes
the induction of the IL-1 signaling network and subsequent
downstream effects that further affect barrier function. Moreover,
IL-1 modulates, in addition to keratinocytes, many immune cells
that will most likely further influence skin function. Thus, multiple loops of skin barrier–dependent effects and immunological
responses can be envisaged. We suggest that the IL-31–IL-1 signaling network identified in this study functions as one loop,
supporting the outside-inside-outside pathogenic model in AD.
In summary, our study reveals bifunctional effects of IL-31 on
the physical and the antimicrobial skin barrier. The reduction of the
physical barrier mediated by genetic factors or a deregulated cytokine milieu, in which these two are likely to cooperate, results in
the increased penetration with allergens and pathogens. It is likely
that in individuals genetically susceptible to AD, dysregulation of
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