- Email: [email protected]
Aryl Hydrocarbon Receptor in Keratinocytes Is Essential for Murine Skin Barrier Integrity
Accepted Manuscript Aryl hydrocarbon receptor in keratinocytes is essential for murine skin barrier integrity Katharina Haas, Heike Weighardt, René Deenen, Karl Köhrer, Björn Clausen, Sonja Zahner, Petra Boukamp, Wilhelm Bloch, Jean Krutmann, Charlotte Esser PII:
S0022-202X(16)32107-8
DOI:
10.1016/j.jid.2016.06.627
Reference:
JID 444
To appear in:
The Journal of Investigative Dermatology
Received Date: 1 March 2016 Revised Date:
21 June 2016
Accepted Date: 28 June 2016
Please cite this article as: Haas K, Weighardt H, Deenen R, Köhrer K, Clausen B, Zahner S, Boukamp P, Bloch W, Krutmann J, Esser C, Aryl hydrocarbon receptor in keratinocytes is essential for murine skin barrier integrity, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.06.627. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Aryl hydrocarbon receptor in keratinocytes is essential for murine skin barrier integrity Katharina Haas1, Heike Weighardt1,3, René Deenen2, Karl Köhrer2, Björn Clausen4, Sonja Zahner6, Petra Boukamp1, Wilhelm Bloch5, Jean Krutmann1, Charlotte Esser1,#
2
BMFZ Genomics & Transcriptomics Laboratory University of Düsseldorf Universitätsstrasse 1 40225 Düsseldorf GERMANY
3
M AN U
LIMES University of Bonn Carl-Troll-Str. 3 53115 Bonn GERMANY
SC
IUF- Leibniz Research Institute for Environmental Medicine Auf’m Hennekamp 50 40225 Düsseldorf GERMANY
RI PT
1
4
5
TE D
Institute for Molecular Medicine University Medical Center of the Johannes Gutenberg-University Mainz Obere Zahlbacher Strasse 67 55131 Mainz GERMANY
6
EP
Institute of Cardiovascular Research and Sport Medicine, German Sport University Cologne, Am Sportpark Müngersdorf 6 50933 Cologne GERMANY
#
AC C
Division of Developmental Immunology La Jolla Institute for Allergy & Immunology, 9420 Athena Circle, La Jolla, CA 92037 USA Address Correspondence to Prof. Charlotte Esser IUF- Leibniz Research Institute for Environmental Medicine Auf’m Hennekamp 50 40225 Düsseldorf GERMANY phone: +49 211 3389 253 fax: +49 211 3389 226 [email protected]
Keywords: Aryl hydrocarbon receptor, skin barrier, indole-3-carbinol, mouse, microbiome 1
ACCEPTED MANUSCRIPT short title: Aryl hydrocarbon receptor and skin barrier
abbreviations:
AhR-KO, AhR-deficient mouse; AhR∆K5, keratinocyte-specific AhR-deficient;
SC
AhR∆LC Langerhans cell-specific AhR-deficient; ARNT, AhR nuclear translocator;
I3C, indole-3-carbinol; KC, keratinocyte;
TE D
LC, Langerhans cell;
M AN U
FICZ, 6-formylindolo[3,2]b-carbazol; DRE, dioxin-responsive elements
RI PT
AhR, aryl hydrocarbon receptor;
OTU, operational taxonomic unit;
AC C
EP
TEWL, transepidermal water loss;
2
ACCEPTED MANUSCRIPT
Abstract The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in adaptive cell functions, and is highly active in the epidermis. AhR ligands can accelerate keratinocyte
RI PT
differentiation, but the precise role of AhR in the skin barrier is unknown. Our study showed that transepidermal water loss (TEWL), a parameter of skin barrier integrity, is high in AhR-deficient (AhRKO) mice. Experiments with conditionally AhR-deficient mouse lines identified keratinocytes as the primary cell population responsible for high TEWL. Electron microscopy showed weaker inter-cellular connectivity in the epidermis of keratinocytes in AhR-KO mice, and gene expression analysis
SC
identified many barrier-associated genes as AhR targets. Moreover, AhR-deficient mice had higher inter-individual differences in their microbiome. Interestingly, removing AhR ligands from the diet of
M AN U
wild-type mice mimicked AhR deficiency with respect to the impaired barrier; conversely, re-addition of the plant-derived ligand indole-3-carbinol (I3C) rescued the barrier deficiency even in aged mice. Our results suggested that functional AhR expression is critical for skin barrier integrity and that AhR represents a molecular target for the development of novel therapeutic approaches for skin barrier
AC C
EP
TE D
diseases, including by dietary intervention.
3
ACCEPTED MANUSCRIPT
Introduction
The skin is the outer barrier of the body, which conserves internal fluids and protects against chemical, physical, and biological threats such as UV damage, toxins, and pathogenic microbes (Elias and Choi, 2005). In this context, the aryl hydrocarbon receptor (AhR) is an important player in skin integrity and immunity (Esser et al., 2013). Numerous proteins and lipids shape skin barrier function on the structural level, and skin maintenance is well regulated by calcium levels and cytokines
RI PT
(Matsui and Amagai, 2015;Esser C, 2014;Proksch et al., 2008;Nemes and Steinert, 1999). AhR is a latent transcription factor, which initiates transcription from dioxin responsive elements (DREs) in the promoter regions of target genes, including genes of the epidermal barrier (Furue et al.,
2015;Sutter et al., 2011;Abel and Haarmann-Stemmann, 2010). Many chemicals can activate AhR.
SC
They include environmental polycyclic aromatic hydrocarbons, coal tar for medical use,
phytochemicals, ultraviolet B (UVB) photoproducts, and products from commensal and pathogenic microorganisms such as skin-residing yeasts (Rannug et al., 1987;Fritsche et al., 2007;Gaitanis et al.,
M AN U
2008;Denison et al., 2011;Moura-Alves et al., 2014;van den Bogaard et al., 2013;Hou et al., 2013). The signaling outcome of AhR is additionally shaped by interference with other signaling pathways such as EGFR, MAPK, NFkB, β-catenin, or STATs in different inflammatory contexts (HaarmannStemmann et al., 2015).
Keratinocytes (KC), fibroblasts, melanocytes, and skin immune cells express AhR at high levels (Esser
TE D
et al., 2013), and AhR is involved in their specific cell functions (Esser et al., 2013;Nguyen et al., 2013;Abel and Haarmann-Stemmann, 2010;Bock and Kohle, 2006). AhR controls the expression of KC genes involved in epidermal differentiation, e.g. Filaggrin and Loricrin (van den Bogaard et al., 2015;Sutter et al., 2011;Sutter et al., 2009;Loertscher et al., 2001) and the KC produced pro-
EP
inflammatory cytokine IL-1β (Greenlee et al., 1985;Sutter et al., 1991). KC respond to persistent AhR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by proliferating and
AC C
secreting cytokines (Greenlee et al., 1985;Puhvel et al., 1982;Panteleyev and Bickers, 2006). In utero exposure to TCDD accelerates the formation of an abnormal epidermal barrier of the fetus with “leaky” tight junctions at the stratum granulosum (Muenyi et al., 2014;Sutter et al., 2011;Loertscher et al., 2002). Mice engineered to have a constitutively active AhR in KC suffer from itching and inflammatory skin lesions (Tauchi et al., 2005). Finally, therapeutic AhR activation to repair skin barrier has been used for atopic dermatitis (van den Bogaard et al., 2013), and may dampen inflammation in psoriasis (Di Meglio et al., 2014). It is currently unknown, however, how lack of AhR or reduced AhR ligand availability affects skin barrier function and the profile of skin barrier genes. We investigated skin barrier functions in mice with impaired AhR signaling, either due to genetic AhR deficiency or due to low levels of AhR ligands in the diet. We demonstrated that AhR deficiency
4
ACCEPTED MANUSCRIPT
affects skin integrity, repair capacity, barrier gene expression, and the skin microbiome. Moreover, we established that dietary availability of AhR ligands affects the skin barrier, and that dietary supplementation with AhR ligands can therapeutically improve the skin barrier.
RI PT
Results Enhanced transepidermal water loss (TEWL) in AhR-deficient (AhR-KO) mice reveals barrier impairment
The stratum corneum protects the body from TEWL. We measured TEWL kinetics in full AhR-KO, cell-
SC
keratinocyte-specific AhR-deficient (AhR∆K5) and Langerhans cell-specific AhR-deficient (AhR∆LC) mice after mechanically removing (tape-stripping) the upper layers of the stratum corneum. Genotypes differed slightly for the removed stratum corneum among the mice, but this did not
M AN U
significantly correlate to TEWL differences (supplementary Figure S1). TEWL measurements were performed in the mornings to level potential influences of circadian rhythms (LeFur et al., 2001). In 10-week-old AhR-KO mice, TEWL values increased significantly compared to wild-type (WT) littermate controls, and did not return fully to baseline within 24 hours (Figure 1a). In AhR∆K5 mice, results were similar, albeit the difference between AhR-KO and control mice was weaker (Figure 1b). In contrast, no difference in TEWL was observed in AhR∆LC mouse lines (Figure 1c), indicating that
TE D
Langerhans cells (LC) do not contribute to skin barrier function upon mechanical stress. Structural proteins and skin enzymes can change after acute barrier disruption (de Koning et al., 2012;Sextius et al., 2010;Stachowitz et al., 2002). In both mice and humans it is known that aged skin
EP
loses its capacity to repair itself, a defect linked to impaired stratum corneum acidity (Choi et al., 2007;Ghadially et al., 1995). We therefore analyzed TEWL in 9-month-old and 18-month-old mice
AC C
(lifespan of mice is 2-3 years) (Figure 1d-i). As expected for aged mice, they exhibited an increased basal TEWL. Upon tape stripping, 18-month-old WT mice showed an even higher initial increase of TEWL than the 9-month-old WT mice (compare Figures 1d and 1g). Interestingly, the difference in TEWL between WT mice and AhR-KO mice disappeared with increasing age (Figures 1d-f, and 1g-i). This suggests that with respect to skin barrier/TEWL, the AhR-KO mice represented a premature aging phenotype. Of note, epidermal AhR expression increased with age, possibly reflecting an attempt to compensate for barrier deficiency (supplementary Figure S2). Together, our data indicated that AhR expression, at least in keratinocytes, is critical for resistance to TEWL.
5
Skin Histology EM microscopy
ACCEPTED MANUSCRIPT
Epidermal thickness did not differ between AhR-KO, AhR∆K5, and WT mice (supplementary Figure S3). Using electron microscopy we analyzed skin sections for changes in KC integrity, inflammatory infiltrates, and stratum corneum layer appearance (Figure 2). In both WT and AhR-KO samples, unstressed epidermis looked normal (Figures 2a and b). The stratum corneum of mechanically stressed skin in WT mice had the same number of layers as that of AhR-KO mice (Figures 2c and d).
RI PT
Vital epidermis in WT mice appeared activated 6 hours after mechanical stress, with more loose contacts between KC than in unstressed skin, clearly affecting the adherens junctions (Figures 2c and e). In contrast, the epidermis in AhR-KO mice contained dead cells and had wider intercellular spaces than in the WT mice skin upon stress (Fig. 2d,f). The basal membrane was occasionally wider with
SC
respect to the ratio of the lamina densa and lamina rara (not shown). Overall, the epidermis of young AhR-KO mice appeared more strongly stressed upon challenge by tape stripping than the epidermis
M AN U
of WT mice.
Microarrays reveal AhR-mediated gene expression profile changes specific for barrier function We performed microarrays from WT and AhR-KO skin samples (i) from “un-taped” mice and (ii) from skin samples 6 and 24 hours after tape stripping. The skin's gene expression profile of un-taped WT mice differed significantly from that of the AhR-KO mice (P<0.05) in 2,810 genes, with an approximately equal share of up-regulation versus down-regulation. The heat map of the microarray
TE D
demonstrates the striking difference in overall gene expression between AhR-KO mice and WT mice (Figure 3a; see also principal component analysis in supplementary Figure S4). However, only nine genes differed significantly >3-fold (Table 1). As we have shown in previous work, AhR-KO mice have a strongly reduced frequency of invariant Vγ5+ γδ T cells in their skin (Kadow et al., 2011).
EP
Consequently, expression of T cell genes Trc-Vg5, CD3e, RIKENA630038E17, and Cd7 was low in AhRKO mice epidermis. A literature search showed that all of the other >3-fold down-regulated genes
AC C
(Ptgs2, Mfge8, Krt16, and Sprr1b) are related to skin functions. We verified their modulation by quantitative real time-PCR (not shown). Only Pyy, a gene of unknown skin function, was expressed higher in AhR-KO mice than in WT mice. Interestingly, Ptgs2, Mfge8, Krt16, and other genes have DRE motifs in their promoters (Sun et al., 2004), and are therefore potential targets for AhR-mediated gene transcription. However, while the presence of DRE motifs is suggestive, they may not be functional. Six hours after tape stripping, 3,927 genes differed, but only 13 genes >3-fold (Table 1). At this time point, all genes (except the T cell genes) were expressed stronger in the AhR-KO mice than in the WT mice (Table 1, bold numbers). In particular, keratin6A, 6B, 16, and Il1b were upregulated strongly in AhR-KO mice compared to WT mice, indicating stress. Only a few of the modulated genes had DREs,
6
ACCEPTED MANUSCRIPT
suggesting secondary responses at the 6-hour time point. Also at 24 hours, keratin genes and Il1b were still higher in AhR-KO mice than in WT mice. Additional genes were up-regulated at this time as well, including several small proline-rich proteins, which are important for the cornified envelope. Again, only a few of the 24-hour genes had DREs in their promoter (see supplementary Table S4). We sorted all modulated genes into relevant skin and immune-related pathways by GeneOntology analysis, and then searched for genes related to barrier functions (see Figure 3b and supplementary
RI PT
Tables S2-4). In unstressed skin, expression of those genes was already lower in the epidermis of AhR-KO mice than in the epidermis of WT mice. When mice were subjected to mechanical stress, important barrier genes remained lower in AhR-KO mice than in WT mice, including occludin (13 DREs), filaggrin2, involucrin, and envoplakin (supplementary Tables S3 and S4). Together, this may
SC
reflect the reduced capability of AhR-deficient skin to cope properly with mechanical stress. The many small changes in lower expression of barrier genes in AhR-KO mice compared to WT mice
M AN U
conceivably explain the observed effects. Microbiome analysis
The skin is a habitat for microorganisms, termed the microbiome, whose composition is critical for skin health. Barrier disruption is associated with microbiome changes and growth of opportunistic pathogens(Grice and Segre, 2011). We therefore analyzed the skin microbiome of AhR-KO and WT mice. We isolated bacterial DNA from skin biopsies in order to collect a complete microbiome. Figure
TE D
4 shows the relative frequencies of prokaryotic phyla in back skin and ear skin of WT mice, AhR-KO mice and conditional AhR∆K5 mice. On the back skin, the inter-individual variation of bacterial phyla was somewhat stronger in AhR-KO mice than in WT mice. However, this was not statistically significant. The analysis of the complexity of the microbiomes (alpha diversity) and a principal
EP
component analysis confirmed these differences (supplementary Figure S6). In agreement with findings in human skin, the mice's ear skin and back skin harbored different bacterial patterns (Figure
AC C
4). In all mouse lines, the majority of detectable operational taxonomic units (OTUs) belonged to three phyla, namely Firmicutes, Proteobacteria, and Bacteroidetes. Interestingly, all AhR-KO and AhR∆K5 mice, but only one WT mouse, had at least 1% Actinobacteria. The genus Staphylococcus was very common (14% in WT mice, 53% in AhR-KO mice). Several genera, which were present at least 1% or higher on WT mice's skin (>1%), were almost lacking on AhR-KO mice's skin (supplementary Table S5a). Differences were also detectable regarding rare bacteria. For instance, Prevotella species (spec.). Actinomyces spec., and Corynebacterium spec. were more abundant in AhR-KO mice than in WT mice (supplementary Table S5b). In conclusion, the microbiome of AhR-KO mice appears more variable and complex, possibly reflecting difficulties in controlling a stable skin microflora.
7
ACCEPTED MANUSCRIPT
Removal of AhR ligands from the diet impairs barrier integrity in C57BL/6 mice within weeks Removal of AhR-ligands from the diet can mimic several AhR-deficiency effects in the gut (Li et al., 2011;Kiss et al., 2011). We therefore fed C57BL/6 mice (i.e. WT mice) either an AhR-ligand-free synthetic diet (NALD) or the same diet with an addition of 2 g/kg indole-3-carbinol (ALD) for several weeks up to months. We followed two strategies. First, the WT mice received NALD or ALD for 3 months after weaning; then we switched the diet and feeding continued for another 3 months
RI PT
(Figure 5a). The WT mice fed NALD had TEWL kinetics similar to the AhR-KO mice (on standard diet, SDT). After switching the diet of the WT mice to ALD, TEWL returned to the values normally seen in WT mice on a standard diet. As expected, WT mice fed with ALD had TEWL kinetics similar to WT mice on the standard diet. Interestingly, even after switching the diet from ALD to NALD, the barrier
SC
remained intact for many weeks, indicating a carry-over effect of the AhR-ligand in the diet (Figure 5a). In a second set of experiments, we analyzed diet effects in aged mice. Feeding intervention began in 10-week old mice and lasted for 5 months. At the end of this time, TEWL kinetics after tape-
M AN U
stripping of NALD-fed mice resembled that of AhR-KO mice, while TEWL kinetics in ALD-fed mice were similar to control WT mice on a standard diet (Figure 5b). We then put all mice on a standard diet for a month. Interestingly, at the end of this period, all mice, regardless of their former diet, displayed the high TEWL of aged mice (Figure 5b insert time point 3; compare also Figure 1d-f). Thus, when intervention started in adult mice we did not observe a carry-over effect of the ALD. To see whether we could improve barrier strength even in aged mice, we restarted NALD or ALD feeding.
TE D
Indeed, the 8-month-old mice fed with ALD showed barrier improvement, that is, a TEWL kinetic similar to young mice (compare Figure 5b, lower graph and Figure 1d-i). As expected, TEWL kinetics differed neither in the NALD-fed mice, standard diet-fed mice, nor in AhR-KO mice at old age. In conclusion, AhR ligands in the diet are critical for skin barrier, and even in old mice barrier strength
AC C
Discussion
EP
can be improved by a dietary intervention.
The latent transcription factor AhR is a receptor for small organic chemicals (Denison et al., 2011;Pohjanvirta, 2011) and relevant for many skin functions (Esser et al., 2009;Esser et al., 2013;Stockinger et al., 2014). We have shown here that both AhR expression and AhR-ligand availability in the diet are critical for skin barrier function. In our first set of experiments, we found that AhR expression is necessary for (i) constitutive expression of many barrier genes, (ii) maintenance of skin barrier function upon mechanical stress, and (iii) possibly a stable microbiome. Conditional mice showed that keratinocytes are mainly the responsible cells, with AhR in Langerhans cells playing little or no role. In AhR-deficient mice, a battery of barrier-associated genes was expressed at lower levels than in WT mice. Although the individual differences were mostly small, 8
ACCEPTED MANUSCRIPT
they conceivably synergized, resulting in the functional and histological changes we observed in the skin.. Intriguingly, a majority of the down-regulated genes, including keratins, had one or more DREs in their promoters (Table 1, supplementary Tables S1-4). The presence of DREs is congruent with control of the constitutive gene expression by the transcription factor AhR, although it is unclear to what extent the number and position of consensus DREs influence the strength of AhR-mediated transcription (Swanson, 2012;Dere et al., 2011). Daniel Nebert coined the term “AhR-gene battery”
RI PT
when he studied the coordinated AhR-mediated transcription of phase I and phase II metabolizing genes (Nebert et al., 1990;Nebert and Gonzalez, 1987). We think that AhR's action as a master-
regulator for cell-specific/function-specific gene batteries is a very important aspect. In contrast to the homeostatic situation, DREs were rare in those genes, which changed 6 or 24 hours after
SC
mechanical stress. This later gene activity most likely represented secondary and cascading effects but no direct transcriptional activation by AhR. Congruent with secondary effects, genes were also upregulated, especially at the 6-hour time-point (see heat map Figure 3a). For instance, IL1ß, stefin A
M AN U
(also known as cystatin A, coding for a cornified envelope protein), small proline-rich protein 2 (coding for precursor proteins of the cornified envelope), and Keratin 6A (known to increase in inflammation) increased in AhR-KO mice's skin. In conclusion, AhR limited expression of these genes in normal skin after mechanical stress, lack of which might have aided in the AhR-deficient mice overshooting inflammatory responses and/or acanthosis (van den Bogaard et al., 2013;Panteleyev and
TE D
Bickers, 2006;Tauchi et al., 2005).
AhR-KO mice had no obvious macroscopic signs of impaired skin health in unstressed skin even at old age (18 months). In contrast, mechanical stress in AhR-KO mice resulted in increased TEWL and changes in epidermal constitution on the microscopic level. Congruent with its role as an
EP
environmental sensor, AhR may be particularly relevant not only in situations of mechanical stress, but also for infections, sterile inflammation, and environmental exposure to UV or chemicals. For
AC C
instance, AhR-deficient mice are more susceptible to Citrobacter rodentium infection (a murine gut bacterium), dextran sodium sulfate induced colitis, or UV-induced non-melanoma skin cancer (Esser and Rannug, 2015). It will thus be interesting to analyze further skin stress factors, such as UVexposure or infections, for AhR-related consequences. Commensal bacteria, fungi, and viruses colonize the skin (Grice et al., 2008), and interact reciprocally with the skin immune system. There is a dynamic and reciprocal crosstalk between the microbiome and the immune system across the skin barrier (Gallo and Nakatsuji, 2011;Cogen et al., 2008;de Benedetto A. et al., 2012). Much of this is not understood yet, and for mice only a few skin microbiome studies are available (Scharschmidt et al., 2009;Scholz et al., 2014;Naik et al., 2015;Natsuga et al., 2016). Beyond these studies, we report here that the skin of AhR-deficient mice
9
ACCEPTED MANUSCRIPT
was colonized with more Actinobacteria and a particularly high frequency of staphylococci. Associated changes in the human skin microbiome exist for diseases such as atopic dermatitis, skin cancer, and diabetes (Williams and Gallo, 2015;Kong et al., 2012;Grice et al., 2010). However, when interpreting microbiome changes in relation to diseases, species differences have to be considered. So far, there are no reports on barrier-associated changes in the murine skin microbiome of AhR-KO mice. Our data thus add important information about skin microbiome and barrier in this species.
RI PT
AhR-KO mice lack innate γδ T cells, and their LC remain in a more immature state (Kadow et al., 2011;Jux et al., 2009). These cell types are active in the skin immune surveillance, and lack of them conceivably can affect the skin resident bacteria. We also identified in AhR-KO mice several changes in gene expression, which are important in innate immunity (e.g. IL1β, TLR9, or TSLP, see
SC
supplementary Tables 2-4).These gene products might conceivably contribute to the microbiome changes we observed. However, more work is necessary to identify the role of AhR in changes of the
M AN U
microbiome.
Various AhR-dependent adverse immune effects in the gut can be mimicked by feeding mice with a synthetic AhR-free diet (Li et al., 2011;Kiss et al., 2011). We showed here that feeding mice with this synthetic diet (which is composed of nutrients from non-plant sources, except for the purified carbohydrate fraction) impairs the skin barrier similar to AhR-deficiency. Moreover, re-addition of a single AhR ligand, I3C, to the diet restored TEWL within a short time. The skin appeared to have a
TE D
memory for AhR ligand rich food, because TEWL values remained low after switching diets, when the mice had received the chow containing I3C at a young age. Orally administered I3C converts to highaffinity AhR ligands DIM, ICZ, and other minor products (not all of them AhR ligands) in the stomach. These compounds are in equilibrium between tissues and plasma (Anderton et al., 2004;Bjeldanes et
EP
al., 1991). Standard mouse chow is made from grain oil seed and other natural plant products, with an unknown and varying content of possible AhR-ligands such as flavonoids, glucosinolates, and/or
AC C
other phytochemicals. However, our data clearly indicated (i) that dietary AhR ligands are necessary for intact skin barrier, and (ii) that an average daily dietary uptake of 250 mg/kg body weight I3C – in the absence of other ligands – is able to restore skin barrier, at least in mice. In a note of caution, this I3C dose may be toxic if consumed long-term1. Thus, it will be necessary to identify the lowest effective dose of dietary AhR-ligands for preventive approaches or therapy.
1
https://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr584/ind ex.html, accessed 01/26/2016.
10
ACCEPTED MANUSCRIPT
In conclusion, we demonstrated that AhR expression controls many barrier-associated genes of the epidermis, thereby contributing to the homeostasis of the skin. We also provided evidence that AhR is a target for preventive dietary intervention.
RI PT
Material and Methods Mice
We used B6.129-AhRtm1Bra/J (Schmidt et al., 1996), or mice with conditional deletion of AhR in KC and Langerhans cells (LC) (Zahner et al., 2011;Kadow et al., 2011;Jux et al., 2009), referred to as AhR-KO,
SC
AhR∆K5, or AhR∆LC, respectively. Animal housing was specific-pathogen free, with a 12 hour/12 hour light–dark cycle, and access to food and water ad libitum. All experiments were in accordance with
M AN U
relevant German animal welfare laws. Feeding studies
For feeding studies, mice received a synthetic diet (EF R/M AIN 76A, Ssniff, Soest, Germany) containing reduced amounts of AhR ligands (NALD), or the same diet to which 2 g/kg I3C was added (ALD, (Kiss et al., 2011)). TEWL
TE D
For TEWL measurements (Grubauer et al., 1989), mice were shaved three days before the start of the experiment. Under isoflurane anesthesia, a Tegaderm medical adhesive tape (TegadermTM, 3M Healthcare, Neuss, Germany) was applied to the skin under gentle pressure, and then removed. Mice were tape-stripped 5 times. TEWL was recorded with TEWAMETERTM 300 (Courage + Khazaka
EP
Electronic, Cologne, Germany). Electron Microscopy (EM)
AC C
Ultra-thin (70-nm) EM samples from full-skin biopsies were prepared and osmium stained as described before (Bechtel et al., 2012). Transmission electron microscopy was performed using a 902A electron microscope (Zeiss, Oberkochen, Germany). Microarrays
Total RNA from epidermal sheets was isolated and 3 replicates of biotin labeled cDNA were prepared from each experimental group and hybridized to Affymetrix Mouse Gene 2.0 ST Gene Expression Microarrays. Data analyses on Affymetrix CEL files were conducted with GeneSpring GX software (version 12.5, Agilent Technologies). Probe level signal intensities were normalized across all samples to reduce inter-array variability (Bolstad et al., 2003), and transformed to baseline. For a detailed
11
ACCEPTED MANUSCRIPT
description, see supplementary material. The array data are accessible in NCBI's Gene Expression Omnibus (Edgar et al., 2002) through GEO number GSE802732. NGS analysis AhR-KO, AHR∆K5, and wild-type littermates were separated after weaning into different cages and experimental cohorts co-housed for at least 4 weeks before the experiment (Grice et al., 2008). We isolated bacterial DNA from the full-skin biopsies and generated 16S V3 region specific amplicons by
(version 4.2.1, LifeTechnologies, Darmstadt, Germany).
RI PT
PCR. Demultiplexing and adaptor trimming of amplicons was done using TorrentSuite software
Sequence data analysis and operational taxonomic unit (OTU) clustering were performed using CLC Genomics Workbench (version 8.02, Qiagen, Hilden, Germany) and CLC Microbial Genomics Module
SC
(version 1.1, Qiagen, Hilden, Germany), respectively. OTU clustering, annotation, and taxon
abundance calculation were done using the software-provided reference database and taxonomy.
M AN U
Alpha diversity was estimated using the number of OTUs, and beta diversity was computed by UniFrac analysis (Chen et al., 2012). For more details, see supplementary Table S1 and supplementary material. Statistical evaluation
Data were analyzed using student’s t-test with GraphPadPrismTM. P values <0.05 were considered
Statistical evaluation
TE D
significant. Means ± SEM are shown.
Data were analyzed using student’s t test with GraphPadPrismTM. P values <0.05 were considered
Conflict of interest
EP
significant. Means ± SEM are shown.
AC C
The authors state no conflict of interest.
Acknowledgements
We thank Babette Martiensen, Swantje Steinwachs, Andrea Droste, and Mojgan Ghilav for technical help. We are grateful to Christiane Hammerschmidt-Kamper and Daniel Biljes for help with experiments, and Johannes Hegemann for discussion. The work in the lab of CE is supported by grants from the Deutsche Forschungsgemeinschaft, JK and HW received support from the Wissenschaftsgemeinschaft Leibniz.
2
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE80723, last accessed May 4th, 2016 12
ACCEPTED MANUSCRIPT
Reference List
Abel J, Haarmann-Stemmann T. An introduction to the molecular basics of aryl hydrocarbon receptor biology. Biol Chem 2010; 391:1235-1248.
RI PT
Anderton MJ, Manson MM, Verschoyle RD, Gescher A, Lamb JH, Farmer PB, et al. Pharmacokinetics and tissue disposition of indole-3-carbinol and its acid condensation products after oral administration to mice. Clin Cancer Res 2004; 10:5233-5241. Bechtel M, Keller MV, Bloch W, Sasaki T, Boukamp P, Zaucke F, et al. Different domains in nidogen-1 and nidogen-2 drive basement membrane formation in skin organotypic cocultures. FASEB J 2012; 26:3637-3648.
M AN U
SC
Bjeldanes LF, Kim JY, Grose KR, Bartholomew JC, Bradfield CA. Aromatic hydrocarbon responsivenessreceptor agonists generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc Natl Acad Sci U S A 1991; 88:9543-9547. Bock KW, Kohle C. Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem Pharmacol 2006; 72:393-404.
TE D
Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 2003; 19:185193. Chen J, Bittinger K, Charlson ES, Hoffmann C, Lewis J, Wu GD, et al. Associating microbiome composition with environmental covariates using generalized UniFrac distances. Bioinformatics 2012; 28:2106-2113.
EP
Choi EH, Man MQ, Xu P, Xin S, Liu Z, Crumrine DA, et al. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol 2007; 127:2847-2856.
AC C
Cogen AL, Nizet V, Gallo RL. Skin microbiota: a source of disease or defence? Br J Dermatol 2008; 158:442-455. de Benedetto A., Kubo A, Beck LA. Skin barrier disruption: a requirement for allergen sensitization? J Invest Dermatol 2012; 132:949-963. de Koning HD, van den Bogaard EH, Bergboer JG, Kamsteeg M, van Vlijmen-Willems IM, Hitomi K, et al. Expression profile of cornified envelope structural proteins and keratinocyte differentiationregulating proteins during skin barrier repair. Br J Dermatol 2012; 166:1245-1254.
13
ACCEPTED MANUSCRIPT
Denison MS, Soshilov AA, He G, DeGroot DE, Zhao B. Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol Sci 2011; 124:1-22. Dere E, Forgacs AL, Zacharewski TR, Burgoon LD. Genome-wide computational analysis of dioxin response element location and distribution in the human, mouse, and rat genomes. Chem Res Toxicol 2011; 24:494-504.
RI PT
Di Meglio P, Duarte JH, Ahlfors H, Owens ND, Li Y, Villanova F, et al. Activation of the aryl hydrocarbon receptor dampens the severity of inflammatory skin conditions. Immunity 2014; 40:989-1001.
SC
Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 2002; 30:207-210.
M AN U
Elias PM, Choi EH. Interactions among stratum corneum defensive functions. Exp Dermatol 2005; 14:719-726. Esser C: Dioxins and the immune system. In: Vohr H-W, editor. Encyclopedia of Immunotoxicology 2nd ed. (Springer, Berlin 2014).
TE D
Esser C, Bargen I, Weighardt H, Haarmann-Stemmann T, Krutmann J. Functions of the aryl hydrocarbon receptor in the skin. Semin Immunopathol 2013; 35:677-691. Esser C, Rannug A. The aryl hydrocarbon receptor (AhR) in barrier organ physiology, immunology, and toxicology. Pharmacol Rev 2015; 67:259-279.
EP
Esser C, Rannug A, Stockinger B. The aryl hydrocarbon receptor and immunity. Trends Immunol 2009; 9:447-454.
AC C
Fritsche E, Schäfer C, Calles C, Bernsmann T, Bernshausen T, Wurm M, et al. Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmatic target for ultraviolet B radiation. Proc Natl Acad Sci U S A 2007; 104:8851-8856. Furue M, Tsuji G, Mitoma C, Nakahara T, Chiba T, Morino-Koga S, et al. Gene regulation of filaggrin and other skin barrier proteins via aryl hydrocarbon receptor. J Dermatol Sci 2015; 80:83-88. Gaitanis G, Magiatis P, Stathopoulou K, Bassukas ID, Alexopoulos EC, Velegraki A, et al. AhR ligands, malassezin, and indolo[3,2-b]carbazole are selectively produced by Malassezia furfur strains isolated from seborrheic dermatitis. J Invest Dermatol 2008; 128:1620-1625. Gallo RL, Nakatsuji T. Microbial symbiosis with the innate immune defense system of the skin. J Invest Dermatol 2011; 131:1974-1980. 14
ACCEPTED MANUSCRIPT
Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest 1995; 95:2281-2290. Greenlee WF, Dold KM, Osborne R. Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human epidermal keratinocytes in culture. In Vitro Cell Dev Biol 1985; 21:509-512.
RI PT
Grice EA, Kong HH, Renaud G, Young AC, Bouffard GG, Blakesley RW, et al. A diversity profile of the human skin microbiota. Genome Res 2008; 18:1043-1050. Grice EA, Segre JA. The skin microbiome. Nat Rev Microbiol 2011; 9:244-253.
SC
Grice EA, Snitkin ES, Yockey LJ, Bermudez DM, Liechty KW, Segre JA. Longitudinal shift in diabetic wound microbiota correlates with prolonged skin defense response. Proc Natl Acad Sci U S A 2010; 107:14799-14804.
M AN U
Grubauer G, Elias PM, Feingold KR. Transepidermal water loss: the signal for recovery of barrier structure and function. J Lipid Res 1989; 30:323-333. Haarmann-Stemmann T, Esser C, Krutmann J. The Janus-faced role of aryl hydrocarbon receptor signaling in the skin: Consequences for prevention and treatment of skin disorders. J Invest Dermatol 2015; 135:2572-2576.
TE D
Hou M, Sun R, Hupe M, Kim PL, Park K, Crumrine D, et al. Topical apigenin improves epidermal permeability barrier homoeostasis in normal murine skin by divergent mechanisms. Exp Dermatol 2013; 22:210-215.
EP
Jux B, Kadow S, Esser C. Langerhans cell maturation and contact hypersensitivity are impaired in aryl hydrocarbon receptor-null mice. J Immunol 2009; 182:6709-6717.
AC C
Kadow S, Jux B, Zahner SP, Wingerath B, Chmill S, Clausen BE, et al. Aryl hydrocarbon receptor Is critical for homeostasis of invariant gamma-delta T cells in the murine epidermis. J Immunol 2011; 187:3104-3110. Kiss EA, Vonarbourg C, Kopfmann S, Hobeika E, Finke D, Esser C, et al. Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles. Science 2011; 334 :1561-1665. Kong HH, Oh J, Deming C, Conlan S, Grice EA, Beatson MA, et al. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res 2012; 22:850-859.
15
ACCEPTED MANUSCRIPT
LeFur I, Reinberg A, Lopez S, Morizot F, Mechkouri M, Tschachler E. Analysis of circadian and ultradian rhythms of skin surface properties of face and forearm of healthy women. J Invest Dermatol 2001; 117:718-724. Li Y, Innocentin S, Withers DR, Roberts NA, Gallagher AR, Grigorieva EF, et al. Exogenous Stimuli Maintain Intraepithelial Lymphocytes via Aryl Hydrocarbon Receptor Activation. Cell 2011; 147:629-640
RI PT
Loertscher JA, Lin TM, Peterson RE, Allen-Hoffmann BL. In utero exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin causes accelerated terminal differentiation in fetal mouse skin. Toxicol Sci 2002; 68:465-472.
SC
Loertscher JA, Sattler CA, Allen-Hoffmann BL. 2,3,7,8-Tetrachlorodibenzo-p-dioxin alters the differentiation pattern of human keratinocytes in organotypic culture. Toxicol Appl Pharmacol 2001; 175:121-129.
M AN U
Matsui T, Amagai M. Dissecting the formation, structure and barrier function of the stratum corneum. Int Immunol 2015; 27:269-280. Moura-Alves P, Fae K, Houthuys E, Dorhoi A, Kreuchwig A, Furkert J, et al. AhR sensing of bacterial pigments regulates antibacterial defence. Nature 2014; 512:387-392.
TE D
Muenyi CS, Carrion SL, Jones LA, Kennedy LH, Slominski AT, Sutter CH, et al. Effects of in utero exposure of C57BL/6J mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin on epidermal permeability barrier development and function. Environ Health Perspect 2014; 122:1052-1058.
EP
Naik S, Bouladoux N, Linehan JL, Han SJ, Harrison OJ, Wilhelm C, et al. Commensal-dendritic-cell interaction specifies a unique protective skin immune signature. Nature 2015; 520:104-108.
AC C
Natsuga K, Cipolat S, Watt FM. Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility. J Invest Dermatol 2016; 136:99-106. Nebert DW, Gonzalez FJ. P450 genes: structure, evolution, and regulation. Annu Rev Biochem 1987; 56:945-993. Nebert DW, Petersen DD, Fornace AJ, Jr.. Cellular responses to oxidative stress: the [Ah] gene battery as a paradigm. Environ Health Perspect 1990; 88:13-25. Nemes Z, Steinert PM. Bricks and mortar of the epidermal barrier. Exp Mol Med 1999; 31:5-19. Nguyen NT, Hanieh H, Nakahama T, Kishimoto T. The roles of aryl hydrocarbon receptor in immune responses. Int Immunol 2013; 25:335-343. 16
ACCEPTED MANUSCRIPT
Panteleyev AA, Bickers DR. Dioxin-induced chloracne--reconstructing the cellular and molecular mechanisms of a classic environmental disease. Exp Dermatol 2006; 15:705-730. Pohjanvirta R: The AH Receptor in Biology and Toxicology 1st ed. (Wiley & Sons, Hoboken, N.J. 2011).
RI PT
Proksch E, Brandner JM, Jensen JM. The skin: an indispensable barrier. Exp Dermatol 2008; 17:10631072. Puhvel SM, Sakamoto M, Ertl DC, Reisner RM. Hairless mice as models for chloracne: a study of cutaneous changes induced by topical application of established chloracnegens. Toxicol Appl Pharmacol 1982; 64:492-503.
SC
Rannug A, Rannug U, Rosenkranz HS, Winqvist L, Westerholm R, Agurell E, et al. Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances. J Biol Chem 1987; 262:15422-15427.
M AN U
Scharschmidt TC, List K, Grice EA, Szabo R, Renaud G, Lee CC et al. Matriptase-deficient mice exhibit ichthyotic skin with a selective shift in skin microbiota. J Invest Dermatol 2009; 129:2435-2442. Schmidt JV, Su GH, Reddy JK, Simon MC, Bradfield CA. Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc Natl Acad Sci U S A 1996; 93:6731-6736.
TE D
Scholz F, Badgley BD, Sadowsky MJ, Kaplan DH. Immune mediated shaping of microflora community composition depends on barrier site. PLoS One 2014; 9:e84019.
EP
Sextius P, Marionnet C, Bon FX, de La Chapelle AL, Tacheau C, Lahfa M, et al. Large scale study of epidermal recovery after stratum corneum removal: dynamics of genomic response. Exp Dermatol 2010; 19:259-268.
AC C
Stachowitz S, Alessandrini F, Abeck D, Ring J, Behrendt H. Permeability barrier disruption increases the level of serine palmitoyltransferase in human epidermis. J Invest Dermatol 2002; 119:10481052. Stockinger B, Di MP, Gialitakis M, Duarte JH. The aryl hydrocarbon receptor: multitasking in the immune system. Annu Rev Immunol 2014; 32:403-432. Sun YV, Boverhof DR, Burgoon LD, Fielden MR, Zacharewski TR. Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences. Nucleic Acids Res 2004; 32:4512-4523.
17
ACCEPTED MANUSCRIPT
Sutter CH, Bodreddigari S, Campion C, Wible RS, Sutter TR. 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases the expression of genes in the human epidermal differentiation complex and accelerates epidermal barrier formation. Toxicol Sci 2011; 124:128-137. Sutter CH, Yin H, Li Y, Mammen JS, Bodreddigari S, Stevens G, et al. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes. Proc Natl Acad Sci U S A 2009; 106:4266-4271.
RI PT
Sutter TR, Guzman K, Dold KM, Greenlee WF. Targets for dioxin genes for plasminogen activator inhibitor-2 and interleukin-1 beta. Science 1991; 254:415-418.
SC
Swanson H. Dioxin response elements and regulation of gene transcription. In: Pohjanvirta R, editor: The AH receptor in biology and toxicology. (Wiley, Hoboken, N.J. 2012) p 81-91.
M AN U
Tauchi M, Hida A, Negishi T, Katsuoka F, Noda S, Mimura J, et al. Constitutive expression of aryl hydrocarbon receptor in keratinocytes causes inflammatory skin lesions. Mol Cell Biol 2005; 25:9360-9368. van den Bogaard EH, Bergboer JG, Vonk-Bergers M, van Vlijmen-Willems IM, Hato SV, van der Valk PG, et al. Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis. J Clin Invest 2013; 123:917-927.
TE D
van den Bogaard EH, Podolsky MA, Smits JP, Cui X, John C, Gowda K, et al. Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation. J Invest Dermatol 2015; 135:1320-1328.
EP
Williams MR, Gallo RL.: The role of the skin microbiome in atopic dermatitis. Curr Allergy Asthma Rep 2015; 15:65.
AC C
Zahner SP, Kel JM, Martina CA, Brouwers-Haspels I, van Roon MA, Clausen, B. Conditional deletion of TGF-betaR1 using Langerin-Cre mice results in Langerhans cell deficiency and reduced contact hypersensitivity. J Immunol 2011; 187:5069-5076.
18
ACCEPTED MANUSCRIPT
Table 1 AHR-dependent gene changes after mechanical stress
gene symbol
number of DREsc
fold p-value changed
1
-10.89
8.16E-06
-8.62
7.66E-05
3
-4.10
4.60E-04
8
-4.04
6.93E-06
4
-3.64
1.29E-02
3
+3.39
1.46E-02
7 0 2
-3.19 -3.17 -3.04
4.52E-03 1.53E-02 1.59E-04
RIKENcDNAA630038E17gene
A630038E17Rik
Z48591
T cell receptor gamma. variable 5
Tcrg-V5 f
NM_011198
prostaglandinendoperoxidesynthase 2
Ptgs2
NM_008594
milk fat globule-EGF factor 8 protein
Mfge8
NM_008470
keratin 16. type I
Krt16
NM_145435
peptide YY
Pyy
NM_009854 NM_009265 NM_007648
CD7 antigen small proline-richprotein 1B CD3 antigen. epsilon polypeptide
TE D Cd7 f Sprr1b Cd3e f
EP
6 h after mechanical stresse
0
M AN U
BC111819
SC
before mechanical stresse
RI PT
genbank/refseqa,b gene description
keratin 6B. typ II RIKENcDNAA630038E17gene T cell receptor gamma. variable 5
Krt6b A630038E17Rik Tcrg-V5
0 1 0
+9.27 -8.92 -6.77
6.70E-03 1.92E-06 1.61E-05
NM_010357 NM_008476 NM_001082543 NM_001206684 NM_008361
glutathione S-transferase alpha 4 keratin 6A. type II stefin A1 RIKENcDNA2610528A11gene interleukin 1 beta
Gsta4 Krt6a Stfa1 2610528A11Rik Il1b
5 0 0 0 0
+6.32 +6.23 +5.14 +4.60 +3.49
1.71E-03 1.16E-02 2.77E-04 5.30E-03 2.26E-05
AC C
NM_010669 BC111819 Z48591
19
ACCEPTED MANUSCRIPT
RAB31. member RAS oncogene family CD7 antigen gasdermin C keratin 16. type I predicted pseudogene 9782
+3.39 -3.32 +3.29 +3.10 +3.06
1.02E-03 1.02E-05 1.04E-04 5.21E-03 3.41E-02
+9.68 +8.67 -7.82 +5.48 +5.20 -4.72 +4.12 +3.78 +3.72
5.58E-04 6.80E-03 2.56E-06 3.84E-03 8.59E-04 1.32E-05 2.87E-03 1.43E-02 9.09E-03
0
+3.58
1.47E-03
Rab31 Cd7 Gsdmc Krt16 Gm9782
0 7 0 4 0
2610528A11Rik Krt6b Tcrg-V5 Gsta4 Gm5416 A630038E17Rik Il1b Stfa1 Krt6a
0 0 0 5 0 1 0 0 0
24 h after mechnical stresse
RI PT
NM_133685 NM_009854 NM_031378 NM_008470 NM_001270426
RIKENcDNA2610528A11gene keratin6B T cell receptor gamma. variable 5 glutathioneS-transferase.alpha4 predicted gene 5416 RIKENcDNAA630038E17gene interleukin 1 beta stefin A1 keratin 6A
XR_142038
smallprolinerichprotein2A2|smallprolinerichprotein2A1|smallprolinerichprotein2A3
NM_009850 NM_009854 NM_173869
CD3antigen.gammapolypeptide CD7antigen stefin A2 like 1
Cd3g Cd7 Stfa2l1
1 7 0
-3.36 -3.30 +3.26
2.05E-04 3.22E-04 3.31E-03
NM_011468
smallprolinerichprotein2A1|smallprolinerichprotein2A2|smallprolinerichprotein2A3
Sprr2a1|Sprr2a2|Spr r2a3
0
+3.21
1.33E-03
TE D
M AN U
SC
NM_001206684 NM_010669 Z48591 NM_010357 NM_001082542 BC111819 NM_008361 NM_001082543 NM_008476
AC C
EP
Sprr2a2|Sprr2a1 |Sprr2a3
20
ACCEPTED MANUSCRIPT
0
NM_008491 NM_008470
lipocalin 2 keratin 16. type I
Lcn2 Krt16
0 4
NM_011468/
smallprolinerichprotein2A1|smallprolinerichprotein2A2|smallprolinerichprotein2B|smallprolinerichprotein2A3
Sprr2a1|Sprr2a2|Spr r2b|Sprr2a3
NM_011471
smallproline-richprotein 2E
Sprr2e
+3.20
RI PT
Sprr2a1|Sprr2a2|Spr r2a3
SC
NM_011468/
smallprolinerichprotein2A1|smallprolinerichprotein2A2|smallprolinerichprotein2A3
M AN U
0
0
1.37E-03
+3.16 +3.15
7.12E-03 1.32E-03
+3.10
8.24E-04
+3.04
7.66E-03
EP
GenBank sequence number www.ncbi.nlm.nih.gov/genbank/; b RefSequence number (www.ncbi.nlm.nih.gov/refseq/); c DREs were identified by sequence analysis (Sun et al., 2004;Jux et al., 2009); dhigher (+) or lower (-) gene expression: higher expression in AhR-KO verus WT is shown in bold; e Gene expression before mechanical stress (time 0), 6 or 24 hours later.
AC C
a
TE D
AhR-KO and WT littermates were mechanically stressed by tape-stripping as described in Material and Methods. Epidermis from skin biopsis was analyzed for differential gene expression on an Affymetrix microarray. Shown are gene changes in AhR-KO with a cut-off threshold >3.
21
ACCEPTED MANUSCRIPT Figure legends Figure 1 Increased TEWL in full and KC-specific AhR-deficient mice Basal TEWL and TEWL after tape-stripping of AhR-KO (a,d,g, left column), AhR∆KC (b,e,h, middle column), and AhR∆LC (c,f,i, right column). The results are shown for mice at ages 10 weeks (upper row), 9 months old (middle row), and 18 months old (lower row). (o) AhR-deficient; () WT littermates. N=7-10/group.; mean ±SEM, * P<0.05, ** P<0.01; ***P<0.001, student´s t-test
SC
RI PT
Figure 2 Electron microscopy reveals structural changes in AhR-KO epidermis Electron micrographs of back skin epidermis of WT littermates (a,c,e) and AhR-KO (b,d,f); (a,b): unstressed skin (3000x magnification). (c,d): 6 hours after tape-stripping (3000x (c) or 4000x magnification (d)). (e,f): Same samples as in c,d (12,000x magnification). Letters in the photographs indicate: N=nucleus, BM=basal membrane, C=cytoplasm, SC=stratum corneum, dC=dead cell. White arrows indicate desmosomes, red arrows indicate enlargement of KC contacts, bridged by adherens junctions. Scale bars:20 nm
TE D
M AN U
Figure 3 Gene expression profile differences of WT and AhR-KO epidermis reveal an AhR battery of barrier genes AhR-KO and WT littermates were mechanically stressed as described in Materials and Methods. Skin biopsies were analyzed for differential gene expression on an Affymetrix Mouse Gene 2.0 ST microarray. N=3 each genotype/point of time (a). Heat map of unstressed skin and skin 6 or 24 hours after tape stripping. Green: decreased signal intensity / expression; red: (increased signal intensity / expression compared to the median signal intensity across all samples). (b) Stacked bars show the number of significantly up- or down-regulated genes of a given function in AhR-KO vs. WT littermates. Genes assigned a relevant function by gene ontology were selected, and further identified for the indicated functional involvement via PubMed. Cut-off threshold >1. For full list see supplementary Tables S2, S3, and S4. Figure 4 Microbiome differences on the skin of WT versus full and KC-specific AhR-deficient mice
AC C
EP
Direct sequencing of bacterial 16S RNA from back skin epidermis of 3 WT littermates and 3 AhR-KO mice reveals relative abundance of the main bacterial phyla. (a, b) back skin habitat (c,d) ear skin habitat. KO=AhR-KO, K5KO=AhR∆K5. Figure 5 Dietary intervention by AhR ligands impairs or improves skin barrier in WT mice TEWL measurements during diet intervention with AhR-ligand free diet (NALD, red line) or with the same diet supplemented with AhR ligand-precursor I3C (ALD, green line). Standard diet fed WT (SDTWT, black square) and AhR-KO (SDT-AhR-KO, white circle) served as comparative controls. Numbers indicate time points when TEWL kinetics was measured, and graphs are labeled accordingly. (a) Feeding scheme, where diet intervention was started directly after weaning, and later switched. Total duration of feeding was 190 days (b) Feeding scheme where diet intervention was started in 10-week-old adults, discontinued and then resumed. Total duration was 237 days. Inserts for time points 1 and 3 show relative units of the area under the curve (AUC) calculated by GraphPadPrism™. N=8; *P<0.05, ** P<0.01, *** P<0.001, student’s t-test.
22
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0022-202X(16)32107-8
DOI:
10.1016/j.jid.2016.06.627
Reference:
JID 444
To appear in:
The Journal of Investigative Dermatology
Received Date: 1 March 2016 Revised Date:
21 June 2016
Accepted Date: 28 June 2016
Please cite this article as: Haas K, Weighardt H, Deenen R, Köhrer K, Clausen B, Zahner S, Boukamp P, Bloch W, Krutmann J, Esser C, Aryl hydrocarbon receptor in keratinocytes is essential for murine skin barrier integrity, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.06.627. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Aryl hydrocarbon receptor in keratinocytes is essential for murine skin barrier integrity Katharina Haas1, Heike Weighardt1,3, René Deenen2, Karl Köhrer2, Björn Clausen4, Sonja Zahner6, Petra Boukamp1, Wilhelm Bloch5, Jean Krutmann1, Charlotte Esser1,#
2
BMFZ Genomics & Transcriptomics Laboratory University of Düsseldorf Universitätsstrasse 1 40225 Düsseldorf GERMANY
3
M AN U
LIMES University of Bonn Carl-Troll-Str. 3 53115 Bonn GERMANY
SC
IUF- Leibniz Research Institute for Environmental Medicine Auf’m Hennekamp 50 40225 Düsseldorf GERMANY
RI PT
1
4
5
TE D
Institute for Molecular Medicine University Medical Center of the Johannes Gutenberg-University Mainz Obere Zahlbacher Strasse 67 55131 Mainz GERMANY
6
EP
Institute of Cardiovascular Research and Sport Medicine, German Sport University Cologne, Am Sportpark Müngersdorf 6 50933 Cologne GERMANY
#
AC C
Division of Developmental Immunology La Jolla Institute for Allergy & Immunology, 9420 Athena Circle, La Jolla, CA 92037 USA Address Correspondence to Prof. Charlotte Esser IUF- Leibniz Research Institute for Environmental Medicine Auf’m Hennekamp 50 40225 Düsseldorf GERMANY phone: +49 211 3389 253 fax: +49 211 3389 226 [email protected]
Keywords: Aryl hydrocarbon receptor, skin barrier, indole-3-carbinol, mouse, microbiome 1
ACCEPTED MANUSCRIPT short title: Aryl hydrocarbon receptor and skin barrier
abbreviations:
AhR-KO, AhR-deficient mouse; AhR∆K5, keratinocyte-specific AhR-deficient;
SC
AhR∆LC Langerhans cell-specific AhR-deficient; ARNT, AhR nuclear translocator;
I3C, indole-3-carbinol; KC, keratinocyte;
TE D
LC, Langerhans cell;
M AN U
FICZ, 6-formylindolo[3,2]b-carbazol; DRE, dioxin-responsive elements
RI PT
AhR, aryl hydrocarbon receptor;
OTU, operational taxonomic unit;
AC C
EP
TEWL, transepidermal water loss;
2
ACCEPTED MANUSCRIPT
Abstract The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in adaptive cell functions, and is highly active in the epidermis. AhR ligands can accelerate keratinocyte
RI PT
differentiation, but the precise role of AhR in the skin barrier is unknown. Our study showed that transepidermal water loss (TEWL), a parameter of skin barrier integrity, is high in AhR-deficient (AhRKO) mice. Experiments with conditionally AhR-deficient mouse lines identified keratinocytes as the primary cell population responsible for high TEWL. Electron microscopy showed weaker inter-cellular connectivity in the epidermis of keratinocytes in AhR-KO mice, and gene expression analysis
SC
identified many barrier-associated genes as AhR targets. Moreover, AhR-deficient mice had higher inter-individual differences in their microbiome. Interestingly, removing AhR ligands from the diet of
M AN U
wild-type mice mimicked AhR deficiency with respect to the impaired barrier; conversely, re-addition of the plant-derived ligand indole-3-carbinol (I3C) rescued the barrier deficiency even in aged mice. Our results suggested that functional AhR expression is critical for skin barrier integrity and that AhR represents a molecular target for the development of novel therapeutic approaches for skin barrier
AC C
EP
TE D
diseases, including by dietary intervention.
3
ACCEPTED MANUSCRIPT
Introduction
The skin is the outer barrier of the body, which conserves internal fluids and protects against chemical, physical, and biological threats such as UV damage, toxins, and pathogenic microbes (Elias and Choi, 2005). In this context, the aryl hydrocarbon receptor (AhR) is an important player in skin integrity and immunity (Esser et al., 2013). Numerous proteins and lipids shape skin barrier function on the structural level, and skin maintenance is well regulated by calcium levels and cytokines
RI PT
(Matsui and Amagai, 2015;Esser C, 2014;Proksch et al., 2008;Nemes and Steinert, 1999). AhR is a latent transcription factor, which initiates transcription from dioxin responsive elements (DREs) in the promoter regions of target genes, including genes of the epidermal barrier (Furue et al.,
2015;Sutter et al., 2011;Abel and Haarmann-Stemmann, 2010). Many chemicals can activate AhR.
SC
They include environmental polycyclic aromatic hydrocarbons, coal tar for medical use,
phytochemicals, ultraviolet B (UVB) photoproducts, and products from commensal and pathogenic microorganisms such as skin-residing yeasts (Rannug et al., 1987;Fritsche et al., 2007;Gaitanis et al.,
M AN U
2008;Denison et al., 2011;Moura-Alves et al., 2014;van den Bogaard et al., 2013;Hou et al., 2013). The signaling outcome of AhR is additionally shaped by interference with other signaling pathways such as EGFR, MAPK, NFkB, β-catenin, or STATs in different inflammatory contexts (HaarmannStemmann et al., 2015).
Keratinocytes (KC), fibroblasts, melanocytes, and skin immune cells express AhR at high levels (Esser
TE D
et al., 2013), and AhR is involved in their specific cell functions (Esser et al., 2013;Nguyen et al., 2013;Abel and Haarmann-Stemmann, 2010;Bock and Kohle, 2006). AhR controls the expression of KC genes involved in epidermal differentiation, e.g. Filaggrin and Loricrin (van den Bogaard et al., 2015;Sutter et al., 2011;Sutter et al., 2009;Loertscher et al., 2001) and the KC produced pro-
EP
inflammatory cytokine IL-1β (Greenlee et al., 1985;Sutter et al., 1991). KC respond to persistent AhR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by proliferating and
AC C
secreting cytokines (Greenlee et al., 1985;Puhvel et al., 1982;Panteleyev and Bickers, 2006). In utero exposure to TCDD accelerates the formation of an abnormal epidermal barrier of the fetus with “leaky” tight junctions at the stratum granulosum (Muenyi et al., 2014;Sutter et al., 2011;Loertscher et al., 2002). Mice engineered to have a constitutively active AhR in KC suffer from itching and inflammatory skin lesions (Tauchi et al., 2005). Finally, therapeutic AhR activation to repair skin barrier has been used for atopic dermatitis (van den Bogaard et al., 2013), and may dampen inflammation in psoriasis (Di Meglio et al., 2014). It is currently unknown, however, how lack of AhR or reduced AhR ligand availability affects skin barrier function and the profile of skin barrier genes. We investigated skin barrier functions in mice with impaired AhR signaling, either due to genetic AhR deficiency or due to low levels of AhR ligands in the diet. We demonstrated that AhR deficiency
4
ACCEPTED MANUSCRIPT
affects skin integrity, repair capacity, barrier gene expression, and the skin microbiome. Moreover, we established that dietary availability of AhR ligands affects the skin barrier, and that dietary supplementation with AhR ligands can therapeutically improve the skin barrier.
RI PT
Results Enhanced transepidermal water loss (TEWL) in AhR-deficient (AhR-KO) mice reveals barrier impairment
The stratum corneum protects the body from TEWL. We measured TEWL kinetics in full AhR-KO, cell-
SC
keratinocyte-specific AhR-deficient (AhR∆K5) and Langerhans cell-specific AhR-deficient (AhR∆LC) mice after mechanically removing (tape-stripping) the upper layers of the stratum corneum. Genotypes differed slightly for the removed stratum corneum among the mice, but this did not
M AN U
significantly correlate to TEWL differences (supplementary Figure S1). TEWL measurements were performed in the mornings to level potential influences of circadian rhythms (LeFur et al., 2001). In 10-week-old AhR-KO mice, TEWL values increased significantly compared to wild-type (WT) littermate controls, and did not return fully to baseline within 24 hours (Figure 1a). In AhR∆K5 mice, results were similar, albeit the difference between AhR-KO and control mice was weaker (Figure 1b). In contrast, no difference in TEWL was observed in AhR∆LC mouse lines (Figure 1c), indicating that
TE D
Langerhans cells (LC) do not contribute to skin barrier function upon mechanical stress. Structural proteins and skin enzymes can change after acute barrier disruption (de Koning et al., 2012;Sextius et al., 2010;Stachowitz et al., 2002). In both mice and humans it is known that aged skin
EP
loses its capacity to repair itself, a defect linked to impaired stratum corneum acidity (Choi et al., 2007;Ghadially et al., 1995). We therefore analyzed TEWL in 9-month-old and 18-month-old mice
AC C
(lifespan of mice is 2-3 years) (Figure 1d-i). As expected for aged mice, they exhibited an increased basal TEWL. Upon tape stripping, 18-month-old WT mice showed an even higher initial increase of TEWL than the 9-month-old WT mice (compare Figures 1d and 1g). Interestingly, the difference in TEWL between WT mice and AhR-KO mice disappeared with increasing age (Figures 1d-f, and 1g-i). This suggests that with respect to skin barrier/TEWL, the AhR-KO mice represented a premature aging phenotype. Of note, epidermal AhR expression increased with age, possibly reflecting an attempt to compensate for barrier deficiency (supplementary Figure S2). Together, our data indicated that AhR expression, at least in keratinocytes, is critical for resistance to TEWL.
5
Skin Histology EM microscopy
ACCEPTED MANUSCRIPT
Epidermal thickness did not differ between AhR-KO, AhR∆K5, and WT mice (supplementary Figure S3). Using electron microscopy we analyzed skin sections for changes in KC integrity, inflammatory infiltrates, and stratum corneum layer appearance (Figure 2). In both WT and AhR-KO samples, unstressed epidermis looked normal (Figures 2a and b). The stratum corneum of mechanically stressed skin in WT mice had the same number of layers as that of AhR-KO mice (Figures 2c and d).
RI PT
Vital epidermis in WT mice appeared activated 6 hours after mechanical stress, with more loose contacts between KC than in unstressed skin, clearly affecting the adherens junctions (Figures 2c and e). In contrast, the epidermis in AhR-KO mice contained dead cells and had wider intercellular spaces than in the WT mice skin upon stress (Fig. 2d,f). The basal membrane was occasionally wider with
SC
respect to the ratio of the lamina densa and lamina rara (not shown). Overall, the epidermis of young AhR-KO mice appeared more strongly stressed upon challenge by tape stripping than the epidermis
M AN U
of WT mice.
Microarrays reveal AhR-mediated gene expression profile changes specific for barrier function We performed microarrays from WT and AhR-KO skin samples (i) from “un-taped” mice and (ii) from skin samples 6 and 24 hours after tape stripping. The skin's gene expression profile of un-taped WT mice differed significantly from that of the AhR-KO mice (P<0.05) in 2,810 genes, with an approximately equal share of up-regulation versus down-regulation. The heat map of the microarray
TE D
demonstrates the striking difference in overall gene expression between AhR-KO mice and WT mice (Figure 3a; see also principal component analysis in supplementary Figure S4). However, only nine genes differed significantly >3-fold (Table 1). As we have shown in previous work, AhR-KO mice have a strongly reduced frequency of invariant Vγ5+ γδ T cells in their skin (Kadow et al., 2011).
EP
Consequently, expression of T cell genes Trc-Vg5, CD3e, RIKENA630038E17, and Cd7 was low in AhRKO mice epidermis. A literature search showed that all of the other >3-fold down-regulated genes
AC C
(Ptgs2, Mfge8, Krt16, and Sprr1b) are related to skin functions. We verified their modulation by quantitative real time-PCR (not shown). Only Pyy, a gene of unknown skin function, was expressed higher in AhR-KO mice than in WT mice. Interestingly, Ptgs2, Mfge8, Krt16, and other genes have DRE motifs in their promoters (Sun et al., 2004), and are therefore potential targets for AhR-mediated gene transcription. However, while the presence of DRE motifs is suggestive, they may not be functional. Six hours after tape stripping, 3,927 genes differed, but only 13 genes >3-fold (Table 1). At this time point, all genes (except the T cell genes) were expressed stronger in the AhR-KO mice than in the WT mice (Table 1, bold numbers). In particular, keratin6A, 6B, 16, and Il1b were upregulated strongly in AhR-KO mice compared to WT mice, indicating stress. Only a few of the modulated genes had DREs,
6
ACCEPTED MANUSCRIPT
suggesting secondary responses at the 6-hour time point. Also at 24 hours, keratin genes and Il1b were still higher in AhR-KO mice than in WT mice. Additional genes were up-regulated at this time as well, including several small proline-rich proteins, which are important for the cornified envelope. Again, only a few of the 24-hour genes had DREs in their promoter (see supplementary Table S4). We sorted all modulated genes into relevant skin and immune-related pathways by GeneOntology analysis, and then searched for genes related to barrier functions (see Figure 3b and supplementary
RI PT
Tables S2-4). In unstressed skin, expression of those genes was already lower in the epidermis of AhR-KO mice than in the epidermis of WT mice. When mice were subjected to mechanical stress, important barrier genes remained lower in AhR-KO mice than in WT mice, including occludin (13 DREs), filaggrin2, involucrin, and envoplakin (supplementary Tables S3 and S4). Together, this may
SC
reflect the reduced capability of AhR-deficient skin to cope properly with mechanical stress. The many small changes in lower expression of barrier genes in AhR-KO mice compared to WT mice
M AN U
conceivably explain the observed effects. Microbiome analysis
The skin is a habitat for microorganisms, termed the microbiome, whose composition is critical for skin health. Barrier disruption is associated with microbiome changes and growth of opportunistic pathogens(Grice and Segre, 2011). We therefore analyzed the skin microbiome of AhR-KO and WT mice. We isolated bacterial DNA from skin biopsies in order to collect a complete microbiome. Figure
TE D
4 shows the relative frequencies of prokaryotic phyla in back skin and ear skin of WT mice, AhR-KO mice and conditional AhR∆K5 mice. On the back skin, the inter-individual variation of bacterial phyla was somewhat stronger in AhR-KO mice than in WT mice. However, this was not statistically significant. The analysis of the complexity of the microbiomes (alpha diversity) and a principal
EP
component analysis confirmed these differences (supplementary Figure S6). In agreement with findings in human skin, the mice's ear skin and back skin harbored different bacterial patterns (Figure
AC C
4). In all mouse lines, the majority of detectable operational taxonomic units (OTUs) belonged to three phyla, namely Firmicutes, Proteobacteria, and Bacteroidetes. Interestingly, all AhR-KO and AhR∆K5 mice, but only one WT mouse, had at least 1% Actinobacteria. The genus Staphylococcus was very common (14% in WT mice, 53% in AhR-KO mice). Several genera, which were present at least 1% or higher on WT mice's skin (>1%), were almost lacking on AhR-KO mice's skin (supplementary Table S5a). Differences were also detectable regarding rare bacteria. For instance, Prevotella species (spec.). Actinomyces spec., and Corynebacterium spec. were more abundant in AhR-KO mice than in WT mice (supplementary Table S5b). In conclusion, the microbiome of AhR-KO mice appears more variable and complex, possibly reflecting difficulties in controlling a stable skin microflora.
7
ACCEPTED MANUSCRIPT
Removal of AhR ligands from the diet impairs barrier integrity in C57BL/6 mice within weeks Removal of AhR-ligands from the diet can mimic several AhR-deficiency effects in the gut (Li et al., 2011;Kiss et al., 2011). We therefore fed C57BL/6 mice (i.e. WT mice) either an AhR-ligand-free synthetic diet (NALD) or the same diet with an addition of 2 g/kg indole-3-carbinol (ALD) for several weeks up to months. We followed two strategies. First, the WT mice received NALD or ALD for 3 months after weaning; then we switched the diet and feeding continued for another 3 months
RI PT
(Figure 5a). The WT mice fed NALD had TEWL kinetics similar to the AhR-KO mice (on standard diet, SDT). After switching the diet of the WT mice to ALD, TEWL returned to the values normally seen in WT mice on a standard diet. As expected, WT mice fed with ALD had TEWL kinetics similar to WT mice on the standard diet. Interestingly, even after switching the diet from ALD to NALD, the barrier
SC
remained intact for many weeks, indicating a carry-over effect of the AhR-ligand in the diet (Figure 5a). In a second set of experiments, we analyzed diet effects in aged mice. Feeding intervention began in 10-week old mice and lasted for 5 months. At the end of this time, TEWL kinetics after tape-
M AN U
stripping of NALD-fed mice resembled that of AhR-KO mice, while TEWL kinetics in ALD-fed mice were similar to control WT mice on a standard diet (Figure 5b). We then put all mice on a standard diet for a month. Interestingly, at the end of this period, all mice, regardless of their former diet, displayed the high TEWL of aged mice (Figure 5b insert time point 3; compare also Figure 1d-f). Thus, when intervention started in adult mice we did not observe a carry-over effect of the ALD. To see whether we could improve barrier strength even in aged mice, we restarted NALD or ALD feeding.
TE D
Indeed, the 8-month-old mice fed with ALD showed barrier improvement, that is, a TEWL kinetic similar to young mice (compare Figure 5b, lower graph and Figure 1d-i). As expected, TEWL kinetics differed neither in the NALD-fed mice, standard diet-fed mice, nor in AhR-KO mice at old age. In conclusion, AhR ligands in the diet are critical for skin barrier, and even in old mice barrier strength
AC C
Discussion
EP
can be improved by a dietary intervention.
The latent transcription factor AhR is a receptor for small organic chemicals (Denison et al., 2011;Pohjanvirta, 2011) and relevant for many skin functions (Esser et al., 2009;Esser et al., 2013;Stockinger et al., 2014). We have shown here that both AhR expression and AhR-ligand availability in the diet are critical for skin barrier function. In our first set of experiments, we found that AhR expression is necessary for (i) constitutive expression of many barrier genes, (ii) maintenance of skin barrier function upon mechanical stress, and (iii) possibly a stable microbiome. Conditional mice showed that keratinocytes are mainly the responsible cells, with AhR in Langerhans cells playing little or no role. In AhR-deficient mice, a battery of barrier-associated genes was expressed at lower levels than in WT mice. Although the individual differences were mostly small, 8
ACCEPTED MANUSCRIPT
they conceivably synergized, resulting in the functional and histological changes we observed in the skin.. Intriguingly, a majority of the down-regulated genes, including keratins, had one or more DREs in their promoters (Table 1, supplementary Tables S1-4). The presence of DREs is congruent with control of the constitutive gene expression by the transcription factor AhR, although it is unclear to what extent the number and position of consensus DREs influence the strength of AhR-mediated transcription (Swanson, 2012;Dere et al., 2011). Daniel Nebert coined the term “AhR-gene battery”
RI PT
when he studied the coordinated AhR-mediated transcription of phase I and phase II metabolizing genes (Nebert et al., 1990;Nebert and Gonzalez, 1987). We think that AhR's action as a master-
regulator for cell-specific/function-specific gene batteries is a very important aspect. In contrast to the homeostatic situation, DREs were rare in those genes, which changed 6 or 24 hours after
SC
mechanical stress. This later gene activity most likely represented secondary and cascading effects but no direct transcriptional activation by AhR. Congruent with secondary effects, genes were also upregulated, especially at the 6-hour time-point (see heat map Figure 3a). For instance, IL1ß, stefin A
M AN U
(also known as cystatin A, coding for a cornified envelope protein), small proline-rich protein 2 (coding for precursor proteins of the cornified envelope), and Keratin 6A (known to increase in inflammation) increased in AhR-KO mice's skin. In conclusion, AhR limited expression of these genes in normal skin after mechanical stress, lack of which might have aided in the AhR-deficient mice overshooting inflammatory responses and/or acanthosis (van den Bogaard et al., 2013;Panteleyev and
TE D
Bickers, 2006;Tauchi et al., 2005).
AhR-KO mice had no obvious macroscopic signs of impaired skin health in unstressed skin even at old age (18 months). In contrast, mechanical stress in AhR-KO mice resulted in increased TEWL and changes in epidermal constitution on the microscopic level. Congruent with its role as an
EP
environmental sensor, AhR may be particularly relevant not only in situations of mechanical stress, but also for infections, sterile inflammation, and environmental exposure to UV or chemicals. For
AC C
instance, AhR-deficient mice are more susceptible to Citrobacter rodentium infection (a murine gut bacterium), dextran sodium sulfate induced colitis, or UV-induced non-melanoma skin cancer (Esser and Rannug, 2015). It will thus be interesting to analyze further skin stress factors, such as UVexposure or infections, for AhR-related consequences. Commensal bacteria, fungi, and viruses colonize the skin (Grice et al., 2008), and interact reciprocally with the skin immune system. There is a dynamic and reciprocal crosstalk between the microbiome and the immune system across the skin barrier (Gallo and Nakatsuji, 2011;Cogen et al., 2008;de Benedetto A. et al., 2012). Much of this is not understood yet, and for mice only a few skin microbiome studies are available (Scharschmidt et al., 2009;Scholz et al., 2014;Naik et al., 2015;Natsuga et al., 2016). Beyond these studies, we report here that the skin of AhR-deficient mice
9
ACCEPTED MANUSCRIPT
was colonized with more Actinobacteria and a particularly high frequency of staphylococci. Associated changes in the human skin microbiome exist for diseases such as atopic dermatitis, skin cancer, and diabetes (Williams and Gallo, 2015;Kong et al., 2012;Grice et al., 2010). However, when interpreting microbiome changes in relation to diseases, species differences have to be considered. So far, there are no reports on barrier-associated changes in the murine skin microbiome of AhR-KO mice. Our data thus add important information about skin microbiome and barrier in this species.
RI PT
AhR-KO mice lack innate γδ T cells, and their LC remain in a more immature state (Kadow et al., 2011;Jux et al., 2009). These cell types are active in the skin immune surveillance, and lack of them conceivably can affect the skin resident bacteria. We also identified in AhR-KO mice several changes in gene expression, which are important in innate immunity (e.g. IL1β, TLR9, or TSLP, see
SC
supplementary Tables 2-4).These gene products might conceivably contribute to the microbiome changes we observed. However, more work is necessary to identify the role of AhR in changes of the
M AN U
microbiome.
Various AhR-dependent adverse immune effects in the gut can be mimicked by feeding mice with a synthetic AhR-free diet (Li et al., 2011;Kiss et al., 2011). We showed here that feeding mice with this synthetic diet (which is composed of nutrients from non-plant sources, except for the purified carbohydrate fraction) impairs the skin barrier similar to AhR-deficiency. Moreover, re-addition of a single AhR ligand, I3C, to the diet restored TEWL within a short time. The skin appeared to have a
TE D
memory for AhR ligand rich food, because TEWL values remained low after switching diets, when the mice had received the chow containing I3C at a young age. Orally administered I3C converts to highaffinity AhR ligands DIM, ICZ, and other minor products (not all of them AhR ligands) in the stomach. These compounds are in equilibrium between tissues and plasma (Anderton et al., 2004;Bjeldanes et
EP
al., 1991). Standard mouse chow is made from grain oil seed and other natural plant products, with an unknown and varying content of possible AhR-ligands such as flavonoids, glucosinolates, and/or
AC C
other phytochemicals. However, our data clearly indicated (i) that dietary AhR ligands are necessary for intact skin barrier, and (ii) that an average daily dietary uptake of 250 mg/kg body weight I3C – in the absence of other ligands – is able to restore skin barrier, at least in mice. In a note of caution, this I3C dose may be toxic if consumed long-term1. Thus, it will be necessary to identify the lowest effective dose of dietary AhR-ligands for preventive approaches or therapy.
1
https://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr584/ind ex.html, accessed 01/26/2016.
10
ACCEPTED MANUSCRIPT
In conclusion, we demonstrated that AhR expression controls many barrier-associated genes of the epidermis, thereby contributing to the homeostasis of the skin. We also provided evidence that AhR is a target for preventive dietary intervention.
RI PT
Material and Methods Mice
We used B6.129-AhRtm1Bra/J (Schmidt et al., 1996), or mice with conditional deletion of AhR in KC and Langerhans cells (LC) (Zahner et al., 2011;Kadow et al., 2011;Jux et al., 2009), referred to as AhR-KO,
SC
AhR∆K5, or AhR∆LC, respectively. Animal housing was specific-pathogen free, with a 12 hour/12 hour light–dark cycle, and access to food and water ad libitum. All experiments were in accordance with
M AN U
relevant German animal welfare laws. Feeding studies
For feeding studies, mice received a synthetic diet (EF R/M AIN 76A, Ssniff, Soest, Germany) containing reduced amounts of AhR ligands (NALD), or the same diet to which 2 g/kg I3C was added (ALD, (Kiss et al., 2011)). TEWL
TE D
For TEWL measurements (Grubauer et al., 1989), mice were shaved three days before the start of the experiment. Under isoflurane anesthesia, a Tegaderm medical adhesive tape (TegadermTM, 3M Healthcare, Neuss, Germany) was applied to the skin under gentle pressure, and then removed. Mice were tape-stripped 5 times. TEWL was recorded with TEWAMETERTM 300 (Courage + Khazaka
EP
Electronic, Cologne, Germany). Electron Microscopy (EM)
AC C
Ultra-thin (70-nm) EM samples from full-skin biopsies were prepared and osmium stained as described before (Bechtel et al., 2012). Transmission electron microscopy was performed using a 902A electron microscope (Zeiss, Oberkochen, Germany). Microarrays
Total RNA from epidermal sheets was isolated and 3 replicates of biotin labeled cDNA were prepared from each experimental group and hybridized to Affymetrix Mouse Gene 2.0 ST Gene Expression Microarrays. Data analyses on Affymetrix CEL files were conducted with GeneSpring GX software (version 12.5, Agilent Technologies). Probe level signal intensities were normalized across all samples to reduce inter-array variability (Bolstad et al., 2003), and transformed to baseline. For a detailed
11
ACCEPTED MANUSCRIPT
description, see supplementary material. The array data are accessible in NCBI's Gene Expression Omnibus (Edgar et al., 2002) through GEO number GSE802732. NGS analysis AhR-KO, AHR∆K5, and wild-type littermates were separated after weaning into different cages and experimental cohorts co-housed for at least 4 weeks before the experiment (Grice et al., 2008). We isolated bacterial DNA from the full-skin biopsies and generated 16S V3 region specific amplicons by
(version 4.2.1, LifeTechnologies, Darmstadt, Germany).
RI PT
PCR. Demultiplexing and adaptor trimming of amplicons was done using TorrentSuite software
Sequence data analysis and operational taxonomic unit (OTU) clustering were performed using CLC Genomics Workbench (version 8.02, Qiagen, Hilden, Germany) and CLC Microbial Genomics Module
SC
(version 1.1, Qiagen, Hilden, Germany), respectively. OTU clustering, annotation, and taxon
abundance calculation were done using the software-provided reference database and taxonomy.
M AN U
Alpha diversity was estimated using the number of OTUs, and beta diversity was computed by UniFrac analysis (Chen et al., 2012). For more details, see supplementary Table S1 and supplementary material. Statistical evaluation
Data were analyzed using student’s t-test with GraphPadPrismTM. P values <0.05 were considered
Statistical evaluation
TE D
significant. Means ± SEM are shown.
Data were analyzed using student’s t test with GraphPadPrismTM. P values <0.05 were considered
Conflict of interest
EP
significant. Means ± SEM are shown.
AC C
The authors state no conflict of interest.
Acknowledgements
We thank Babette Martiensen, Swantje Steinwachs, Andrea Droste, and Mojgan Ghilav for technical help. We are grateful to Christiane Hammerschmidt-Kamper and Daniel Biljes for help with experiments, and Johannes Hegemann for discussion. The work in the lab of CE is supported by grants from the Deutsche Forschungsgemeinschaft, JK and HW received support from the Wissenschaftsgemeinschaft Leibniz.
2
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE80723, last accessed May 4th, 2016 12
ACCEPTED MANUSCRIPT
Reference List
Abel J, Haarmann-Stemmann T. An introduction to the molecular basics of aryl hydrocarbon receptor biology. Biol Chem 2010; 391:1235-1248.
RI PT
Anderton MJ, Manson MM, Verschoyle RD, Gescher A, Lamb JH, Farmer PB, et al. Pharmacokinetics and tissue disposition of indole-3-carbinol and its acid condensation products after oral administration to mice. Clin Cancer Res 2004; 10:5233-5241. Bechtel M, Keller MV, Bloch W, Sasaki T, Boukamp P, Zaucke F, et al. Different domains in nidogen-1 and nidogen-2 drive basement membrane formation in skin organotypic cocultures. FASEB J 2012; 26:3637-3648.
M AN U
SC
Bjeldanes LF, Kim JY, Grose KR, Bartholomew JC, Bradfield CA. Aromatic hydrocarbon responsivenessreceptor agonists generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc Natl Acad Sci U S A 1991; 88:9543-9547. Bock KW, Kohle C. Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem Pharmacol 2006; 72:393-404.
TE D
Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 2003; 19:185193. Chen J, Bittinger K, Charlson ES, Hoffmann C, Lewis J, Wu GD, et al. Associating microbiome composition with environmental covariates using generalized UniFrac distances. Bioinformatics 2012; 28:2106-2113.
EP
Choi EH, Man MQ, Xu P, Xin S, Liu Z, Crumrine DA, et al. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol 2007; 127:2847-2856.
AC C
Cogen AL, Nizet V, Gallo RL. Skin microbiota: a source of disease or defence? Br J Dermatol 2008; 158:442-455. de Benedetto A., Kubo A, Beck LA. Skin barrier disruption: a requirement for allergen sensitization? J Invest Dermatol 2012; 132:949-963. de Koning HD, van den Bogaard EH, Bergboer JG, Kamsteeg M, van Vlijmen-Willems IM, Hitomi K, et al. Expression profile of cornified envelope structural proteins and keratinocyte differentiationregulating proteins during skin barrier repair. Br J Dermatol 2012; 166:1245-1254.
13
ACCEPTED MANUSCRIPT
Denison MS, Soshilov AA, He G, DeGroot DE, Zhao B. Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol Sci 2011; 124:1-22. Dere E, Forgacs AL, Zacharewski TR, Burgoon LD. Genome-wide computational analysis of dioxin response element location and distribution in the human, mouse, and rat genomes. Chem Res Toxicol 2011; 24:494-504.
RI PT
Di Meglio P, Duarte JH, Ahlfors H, Owens ND, Li Y, Villanova F, et al. Activation of the aryl hydrocarbon receptor dampens the severity of inflammatory skin conditions. Immunity 2014; 40:989-1001.
SC
Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 2002; 30:207-210.
M AN U
Elias PM, Choi EH. Interactions among stratum corneum defensive functions. Exp Dermatol 2005; 14:719-726. Esser C: Dioxins and the immune system. In: Vohr H-W, editor. Encyclopedia of Immunotoxicology 2nd ed. (Springer, Berlin 2014).
TE D
Esser C, Bargen I, Weighardt H, Haarmann-Stemmann T, Krutmann J. Functions of the aryl hydrocarbon receptor in the skin. Semin Immunopathol 2013; 35:677-691. Esser C, Rannug A. The aryl hydrocarbon receptor (AhR) in barrier organ physiology, immunology, and toxicology. Pharmacol Rev 2015; 67:259-279.
EP
Esser C, Rannug A, Stockinger B. The aryl hydrocarbon receptor and immunity. Trends Immunol 2009; 9:447-454.
AC C
Fritsche E, Schäfer C, Calles C, Bernsmann T, Bernshausen T, Wurm M, et al. Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmatic target for ultraviolet B radiation. Proc Natl Acad Sci U S A 2007; 104:8851-8856. Furue M, Tsuji G, Mitoma C, Nakahara T, Chiba T, Morino-Koga S, et al. Gene regulation of filaggrin and other skin barrier proteins via aryl hydrocarbon receptor. J Dermatol Sci 2015; 80:83-88. Gaitanis G, Magiatis P, Stathopoulou K, Bassukas ID, Alexopoulos EC, Velegraki A, et al. AhR ligands, malassezin, and indolo[3,2-b]carbazole are selectively produced by Malassezia furfur strains isolated from seborrheic dermatitis. J Invest Dermatol 2008; 128:1620-1625. Gallo RL, Nakatsuji T. Microbial symbiosis with the innate immune defense system of the skin. J Invest Dermatol 2011; 131:1974-1980. 14
ACCEPTED MANUSCRIPT
Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest 1995; 95:2281-2290. Greenlee WF, Dold KM, Osborne R. Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human epidermal keratinocytes in culture. In Vitro Cell Dev Biol 1985; 21:509-512.
RI PT
Grice EA, Kong HH, Renaud G, Young AC, Bouffard GG, Blakesley RW, et al. A diversity profile of the human skin microbiota. Genome Res 2008; 18:1043-1050. Grice EA, Segre JA. The skin microbiome. Nat Rev Microbiol 2011; 9:244-253.
SC
Grice EA, Snitkin ES, Yockey LJ, Bermudez DM, Liechty KW, Segre JA. Longitudinal shift in diabetic wound microbiota correlates with prolonged skin defense response. Proc Natl Acad Sci U S A 2010; 107:14799-14804.
M AN U
Grubauer G, Elias PM, Feingold KR. Transepidermal water loss: the signal for recovery of barrier structure and function. J Lipid Res 1989; 30:323-333. Haarmann-Stemmann T, Esser C, Krutmann J. The Janus-faced role of aryl hydrocarbon receptor signaling in the skin: Consequences for prevention and treatment of skin disorders. J Invest Dermatol 2015; 135:2572-2576.
TE D
Hou M, Sun R, Hupe M, Kim PL, Park K, Crumrine D, et al. Topical apigenin improves epidermal permeability barrier homoeostasis in normal murine skin by divergent mechanisms. Exp Dermatol 2013; 22:210-215.
EP
Jux B, Kadow S, Esser C. Langerhans cell maturation and contact hypersensitivity are impaired in aryl hydrocarbon receptor-null mice. J Immunol 2009; 182:6709-6717.
AC C
Kadow S, Jux B, Zahner SP, Wingerath B, Chmill S, Clausen BE, et al. Aryl hydrocarbon receptor Is critical for homeostasis of invariant gamma-delta T cells in the murine epidermis. J Immunol 2011; 187:3104-3110. Kiss EA, Vonarbourg C, Kopfmann S, Hobeika E, Finke D, Esser C, et al. Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles. Science 2011; 334 :1561-1665. Kong HH, Oh J, Deming C, Conlan S, Grice EA, Beatson MA, et al. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res 2012; 22:850-859.
15
ACCEPTED MANUSCRIPT
LeFur I, Reinberg A, Lopez S, Morizot F, Mechkouri M, Tschachler E. Analysis of circadian and ultradian rhythms of skin surface properties of face and forearm of healthy women. J Invest Dermatol 2001; 117:718-724. Li Y, Innocentin S, Withers DR, Roberts NA, Gallagher AR, Grigorieva EF, et al. Exogenous Stimuli Maintain Intraepithelial Lymphocytes via Aryl Hydrocarbon Receptor Activation. Cell 2011; 147:629-640
RI PT
Loertscher JA, Lin TM, Peterson RE, Allen-Hoffmann BL. In utero exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin causes accelerated terminal differentiation in fetal mouse skin. Toxicol Sci 2002; 68:465-472.
SC
Loertscher JA, Sattler CA, Allen-Hoffmann BL. 2,3,7,8-Tetrachlorodibenzo-p-dioxin alters the differentiation pattern of human keratinocytes in organotypic culture. Toxicol Appl Pharmacol 2001; 175:121-129.
M AN U
Matsui T, Amagai M. Dissecting the formation, structure and barrier function of the stratum corneum. Int Immunol 2015; 27:269-280. Moura-Alves P, Fae K, Houthuys E, Dorhoi A, Kreuchwig A, Furkert J, et al. AhR sensing of bacterial pigments regulates antibacterial defence. Nature 2014; 512:387-392.
TE D
Muenyi CS, Carrion SL, Jones LA, Kennedy LH, Slominski AT, Sutter CH, et al. Effects of in utero exposure of C57BL/6J mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin on epidermal permeability barrier development and function. Environ Health Perspect 2014; 122:1052-1058.
EP
Naik S, Bouladoux N, Linehan JL, Han SJ, Harrison OJ, Wilhelm C, et al. Commensal-dendritic-cell interaction specifies a unique protective skin immune signature. Nature 2015; 520:104-108.
AC C
Natsuga K, Cipolat S, Watt FM. Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility. J Invest Dermatol 2016; 136:99-106. Nebert DW, Gonzalez FJ. P450 genes: structure, evolution, and regulation. Annu Rev Biochem 1987; 56:945-993. Nebert DW, Petersen DD, Fornace AJ, Jr.. Cellular responses to oxidative stress: the [Ah] gene battery as a paradigm. Environ Health Perspect 1990; 88:13-25. Nemes Z, Steinert PM. Bricks and mortar of the epidermal barrier. Exp Mol Med 1999; 31:5-19. Nguyen NT, Hanieh H, Nakahama T, Kishimoto T. The roles of aryl hydrocarbon receptor in immune responses. Int Immunol 2013; 25:335-343. 16
ACCEPTED MANUSCRIPT
Panteleyev AA, Bickers DR. Dioxin-induced chloracne--reconstructing the cellular and molecular mechanisms of a classic environmental disease. Exp Dermatol 2006; 15:705-730. Pohjanvirta R: The AH Receptor in Biology and Toxicology 1st ed. (Wiley & Sons, Hoboken, N.J. 2011).
RI PT
Proksch E, Brandner JM, Jensen JM. The skin: an indispensable barrier. Exp Dermatol 2008; 17:10631072. Puhvel SM, Sakamoto M, Ertl DC, Reisner RM. Hairless mice as models for chloracne: a study of cutaneous changes induced by topical application of established chloracnegens. Toxicol Appl Pharmacol 1982; 64:492-503.
SC
Rannug A, Rannug U, Rosenkranz HS, Winqvist L, Westerholm R, Agurell E, et al. Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances. J Biol Chem 1987; 262:15422-15427.
M AN U
Scharschmidt TC, List K, Grice EA, Szabo R, Renaud G, Lee CC et al. Matriptase-deficient mice exhibit ichthyotic skin with a selective shift in skin microbiota. J Invest Dermatol 2009; 129:2435-2442. Schmidt JV, Su GH, Reddy JK, Simon MC, Bradfield CA. Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc Natl Acad Sci U S A 1996; 93:6731-6736.
TE D
Scholz F, Badgley BD, Sadowsky MJ, Kaplan DH. Immune mediated shaping of microflora community composition depends on barrier site. PLoS One 2014; 9:e84019.
EP
Sextius P, Marionnet C, Bon FX, de La Chapelle AL, Tacheau C, Lahfa M, et al. Large scale study of epidermal recovery after stratum corneum removal: dynamics of genomic response. Exp Dermatol 2010; 19:259-268.
AC C
Stachowitz S, Alessandrini F, Abeck D, Ring J, Behrendt H. Permeability barrier disruption increases the level of serine palmitoyltransferase in human epidermis. J Invest Dermatol 2002; 119:10481052. Stockinger B, Di MP, Gialitakis M, Duarte JH. The aryl hydrocarbon receptor: multitasking in the immune system. Annu Rev Immunol 2014; 32:403-432. Sun YV, Boverhof DR, Burgoon LD, Fielden MR, Zacharewski TR. Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences. Nucleic Acids Res 2004; 32:4512-4523.
17
ACCEPTED MANUSCRIPT
Sutter CH, Bodreddigari S, Campion C, Wible RS, Sutter TR. 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases the expression of genes in the human epidermal differentiation complex and accelerates epidermal barrier formation. Toxicol Sci 2011; 124:128-137. Sutter CH, Yin H, Li Y, Mammen JS, Bodreddigari S, Stevens G, et al. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes. Proc Natl Acad Sci U S A 2009; 106:4266-4271.
RI PT
Sutter TR, Guzman K, Dold KM, Greenlee WF. Targets for dioxin genes for plasminogen activator inhibitor-2 and interleukin-1 beta. Science 1991; 254:415-418.
SC
Swanson H. Dioxin response elements and regulation of gene transcription. In: Pohjanvirta R, editor: The AH receptor in biology and toxicology. (Wiley, Hoboken, N.J. 2012) p 81-91.
M AN U
Tauchi M, Hida A, Negishi T, Katsuoka F, Noda S, Mimura J, et al. Constitutive expression of aryl hydrocarbon receptor in keratinocytes causes inflammatory skin lesions. Mol Cell Biol 2005; 25:9360-9368. van den Bogaard EH, Bergboer JG, Vonk-Bergers M, van Vlijmen-Willems IM, Hato SV, van der Valk PG, et al. Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis. J Clin Invest 2013; 123:917-927.
TE D
van den Bogaard EH, Podolsky MA, Smits JP, Cui X, John C, Gowda K, et al. Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation. J Invest Dermatol 2015; 135:1320-1328.
EP
Williams MR, Gallo RL.: The role of the skin microbiome in atopic dermatitis. Curr Allergy Asthma Rep 2015; 15:65.
AC C
Zahner SP, Kel JM, Martina CA, Brouwers-Haspels I, van Roon MA, Clausen, B. Conditional deletion of TGF-betaR1 using Langerin-Cre mice results in Langerhans cell deficiency and reduced contact hypersensitivity. J Immunol 2011; 187:5069-5076.
18
ACCEPTED MANUSCRIPT
Table 1 AHR-dependent gene changes after mechanical stress
gene symbol
number of DREsc
fold p-value changed
1
-10.89
8.16E-06
-8.62
7.66E-05
3
-4.10
4.60E-04
8
-4.04
6.93E-06
4
-3.64
1.29E-02
3
+3.39
1.46E-02
7 0 2
-3.19 -3.17 -3.04
4.52E-03 1.53E-02 1.59E-04
RIKENcDNAA630038E17gene
A630038E17Rik
Z48591
T cell receptor gamma. variable 5
Tcrg-V5 f
NM_011198
prostaglandinendoperoxidesynthase 2
Ptgs2
NM_008594
milk fat globule-EGF factor 8 protein
Mfge8
NM_008470
keratin 16. type I
Krt16
NM_145435
peptide YY
Pyy
NM_009854 NM_009265 NM_007648
CD7 antigen small proline-richprotein 1B CD3 antigen. epsilon polypeptide
TE D Cd7 f Sprr1b Cd3e f
EP
6 h after mechanical stresse
0
M AN U
BC111819
SC
before mechanical stresse
RI PT
genbank/refseqa,b gene description
keratin 6B. typ II RIKENcDNAA630038E17gene T cell receptor gamma. variable 5
Krt6b A630038E17Rik Tcrg-V5
0 1 0
+9.27 -8.92 -6.77
6.70E-03 1.92E-06 1.61E-05
NM_010357 NM_008476 NM_001082543 NM_001206684 NM_008361
glutathione S-transferase alpha 4 keratin 6A. type II stefin A1 RIKENcDNA2610528A11gene interleukin 1 beta
Gsta4 Krt6a Stfa1 2610528A11Rik Il1b
5 0 0 0 0
+6.32 +6.23 +5.14 +4.60 +3.49
1.71E-03 1.16E-02 2.77E-04 5.30E-03 2.26E-05
AC C
NM_010669 BC111819 Z48591
19
ACCEPTED MANUSCRIPT
RAB31. member RAS oncogene family CD7 antigen gasdermin C keratin 16. type I predicted pseudogene 9782
+3.39 -3.32 +3.29 +3.10 +3.06
1.02E-03 1.02E-05 1.04E-04 5.21E-03 3.41E-02
+9.68 +8.67 -7.82 +5.48 +5.20 -4.72 +4.12 +3.78 +3.72
5.58E-04 6.80E-03 2.56E-06 3.84E-03 8.59E-04 1.32E-05 2.87E-03 1.43E-02 9.09E-03
0
+3.58
1.47E-03
Rab31 Cd7 Gsdmc Krt16 Gm9782
0 7 0 4 0
2610528A11Rik Krt6b Tcrg-V5 Gsta4 Gm5416 A630038E17Rik Il1b Stfa1 Krt6a
0 0 0 5 0 1 0 0 0
24 h after mechnical stresse
RI PT
NM_133685 NM_009854 NM_031378 NM_008470 NM_001270426
RIKENcDNA2610528A11gene keratin6B T cell receptor gamma. variable 5 glutathioneS-transferase.alpha4 predicted gene 5416 RIKENcDNAA630038E17gene interleukin 1 beta stefin A1 keratin 6A
XR_142038
smallprolinerichprotein2A2|smallprolinerichprotein2A1|smallprolinerichprotein2A3
NM_009850 NM_009854 NM_173869
CD3antigen.gammapolypeptide CD7antigen stefin A2 like 1
Cd3g Cd7 Stfa2l1
1 7 0
-3.36 -3.30 +3.26
2.05E-04 3.22E-04 3.31E-03
NM_011468
smallprolinerichprotein2A1|smallprolinerichprotein2A2|smallprolinerichprotein2A3
Sprr2a1|Sprr2a2|Spr r2a3
0
+3.21
1.33E-03
TE D
M AN U
SC
NM_001206684 NM_010669 Z48591 NM_010357 NM_001082542 BC111819 NM_008361 NM_001082543 NM_008476
AC C
EP
Sprr2a2|Sprr2a1 |Sprr2a3
20
ACCEPTED MANUSCRIPT
0
NM_008491 NM_008470
lipocalin 2 keratin 16. type I
Lcn2 Krt16
0 4
NM_011468/
smallprolinerichprotein2A1|smallprolinerichprotein2A2|smallprolinerichprotein2B|smallprolinerichprotein2A3
Sprr2a1|Sprr2a2|Spr r2b|Sprr2a3
NM_011471
smallproline-richprotein 2E
Sprr2e
+3.20
RI PT
Sprr2a1|Sprr2a2|Spr r2a3
SC
NM_011468/
smallprolinerichprotein2A1|smallprolinerichprotein2A2|smallprolinerichprotein2A3
M AN U
0
0
1.37E-03
+3.16 +3.15
7.12E-03 1.32E-03
+3.10
8.24E-04
+3.04
7.66E-03
EP
GenBank sequence number www.ncbi.nlm.nih.gov/genbank/; b RefSequence number (www.ncbi.nlm.nih.gov/refseq/); c DREs were identified by sequence analysis (Sun et al., 2004;Jux et al., 2009); dhigher (+) or lower (-) gene expression: higher expression in AhR-KO verus WT is shown in bold; e Gene expression before mechanical stress (time 0), 6 or 24 hours later.
AC C
a
TE D
AhR-KO and WT littermates were mechanically stressed by tape-stripping as described in Material and Methods. Epidermis from skin biopsis was analyzed for differential gene expression on an Affymetrix microarray. Shown are gene changes in AhR-KO with a cut-off threshold >3.
21
ACCEPTED MANUSCRIPT Figure legends Figure 1 Increased TEWL in full and KC-specific AhR-deficient mice Basal TEWL and TEWL after tape-stripping of AhR-KO (a,d,g, left column), AhR∆KC (b,e,h, middle column), and AhR∆LC (c,f,i, right column). The results are shown for mice at ages 10 weeks (upper row), 9 months old (middle row), and 18 months old (lower row). (o) AhR-deficient; () WT littermates. N=7-10/group.; mean ±SEM, * P<0.05, ** P<0.01; ***P<0.001, student´s t-test
SC
RI PT
Figure 2 Electron microscopy reveals structural changes in AhR-KO epidermis Electron micrographs of back skin epidermis of WT littermates (a,c,e) and AhR-KO (b,d,f); (a,b): unstressed skin (3000x magnification). (c,d): 6 hours after tape-stripping (3000x (c) or 4000x magnification (d)). (e,f): Same samples as in c,d (12,000x magnification). Letters in the photographs indicate: N=nucleus, BM=basal membrane, C=cytoplasm, SC=stratum corneum, dC=dead cell. White arrows indicate desmosomes, red arrows indicate enlargement of KC contacts, bridged by adherens junctions. Scale bars:20 nm
TE D
M AN U
Figure 3 Gene expression profile differences of WT and AhR-KO epidermis reveal an AhR battery of barrier genes AhR-KO and WT littermates were mechanically stressed as described in Materials and Methods. Skin biopsies were analyzed for differential gene expression on an Affymetrix Mouse Gene 2.0 ST microarray. N=3 each genotype/point of time (a). Heat map of unstressed skin and skin 6 or 24 hours after tape stripping. Green: decreased signal intensity / expression; red: (increased signal intensity / expression compared to the median signal intensity across all samples). (b) Stacked bars show the number of significantly up- or down-regulated genes of a given function in AhR-KO vs. WT littermates. Genes assigned a relevant function by gene ontology were selected, and further identified for the indicated functional involvement via PubMed. Cut-off threshold >1. For full list see supplementary Tables S2, S3, and S4. Figure 4 Microbiome differences on the skin of WT versus full and KC-specific AhR-deficient mice
AC C
EP
Direct sequencing of bacterial 16S RNA from back skin epidermis of 3 WT littermates and 3 AhR-KO mice reveals relative abundance of the main bacterial phyla. (a, b) back skin habitat (c,d) ear skin habitat. KO=AhR-KO, K5KO=AhR∆K5. Figure 5 Dietary intervention by AhR ligands impairs or improves skin barrier in WT mice TEWL measurements during diet intervention with AhR-ligand free diet (NALD, red line) or with the same diet supplemented with AhR ligand-precursor I3C (ALD, green line). Standard diet fed WT (SDTWT, black square) and AhR-KO (SDT-AhR-KO, white circle) served as comparative controls. Numbers indicate time points when TEWL kinetics was measured, and graphs are labeled accordingly. (a) Feeding scheme, where diet intervention was started directly after weaning, and later switched. Total duration of feeding was 190 days (b) Feeding scheme where diet intervention was started in 10-week-old adults, discontinued and then resumed. Total duration was 237 days. Inserts for time points 1 and 3 show relative units of the area under the curve (AUC) calculated by GraphPadPrism™. N=8; *P<0.05, ** P<0.01, *** P<0.001, student’s t-test.
22
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT