Article
Reference
IL-17E (IL-25) and IL-17A differentially affect the functions of human keratinocytes
BOROWCZYK, Julia, et al.
Abstract
Our group has recently shown that keratinocyte-derived IL-17E (IL-25), one of six members of the IL-17 family, is overexpressed in lesional psoriatic skin and is involved in its pathophysiology. We show here that IL-22 enhances IL-17E production in human keratinocytes and that these cells display a complete IL-17E receptor at their surface, which expression is further induced by IL-17A, indicating a potential autocrine effect of IL-17E.
Therefore, we addressed the impact of IL-17E on the function of human primary keratinocytes. IL-17E promoted the proliferation of keratinocytes in 2D and 3D cultures and caused the concomitant up-regulation of differentiation-associated gene transcripts (e.g keratin 10), while their expression was either inhibited or not changed by IL-17A. Contrary to IL-17A, IL-17E was not involved in the induction of antimicrobial proteins. Time-lapse analysis of cell movement showed that IL-17E influences cell motility increasing both cell speed and displacement. This was associated with specific changes in the actin cytoskeleton organization and the cell-substrate adhesion. No such effects were [...]
BOROWCZYK, Julia, et al. IL-17E (IL-25) and IL-17A differentially affect the functions of human keratinocytes. The Journal of Investigative Dermatology, 2020
PMID : 31958433
DOI : 10.1016/j.jid.2019.12.013
Available at:
http://archive-ouverte.unige.ch/unige:129612
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IL-17E (IL-25) and IL-17A differentially affect the functions of human keratinocytes Julia Borowczyk, Claudia Buerger, Neschaat Tadjrischi, Justyna Drukala, Michal Wolnicki, Dawid Wnuk, Ali Modarressi, Wolf-Henning Boehncke, Nicolò Costantino Brembilla
PII: S0022-202X(20)30002-6
DOI: https://doi.org/10.1016/j.jid.2019.12.013 Reference: JID 2247
To appear in: The Journal of Investigative Dermatology Received Date: 26 July 2019
Revised Date: 18 December 2019 Accepted Date: 27 December 2019
Please cite this article as: Borowczyk J, Buerger C, Tadjrischi N, Drukala J, Wolnicki M, Wnuk D, Modarressi A, Boehncke W-H, Brembilla NC, IL-17E (IL-25) and IL-17A differentially affect the functions of human keratinocytes, The Journal of Investigative Dermatology (2020), doi: https://doi.org/10.1016/
j.jid.2019.12.013.
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© 2020 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
IL-17E (IL-25) and IL-17A differentially affect the functions of human keratinocytes
Julia Borowczyk1, Claudia Buerger2, Neschaat Tadjrischi2, Justyna Drukala3, Michal Wolnicki4, Dawid Wnuk3, Ali Modarressi5, Wolf-Henning Boehncke1,6 and Nicolò Costantino Brembilla1
1Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
2Department of Dermatology, Clinic of the Goethe-University, Frankfurt am Main, Germany
3Cell Bank, Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland
4Department of Pediatric Urology, Jagiellonian University Medical College, Cracow, Poland
5Plastic, reconstructive & aesthetic unit, University Hospitals of Geneva and School of Medicine, Geneva, Switzerland
6Division of Dermatology and Venereology, University Hospitals of Geneva, Geneva, Switzerland
CORRESPONDENCE:
Nicolò Costantino Brembilla
Department of Pathology and Immunology Centre Médicale Universitaire
Rue Michel Servet 1 CH-1211 Genève 4 Switzerland
SHORT TITLE: The role of IL-17E in epidermal homeostasis
ORCID:
Julia Borowczyk: https://orcid.org/0000-0002-4638-1596 Claudia Buerger: https://orcid.org/0000-0002-7838-197X Neschaat Tadjrischi: https://orcid.org/0000-0002-2870-8196 Justyna Drukala: https://orcid.org/0000-0001-5573-2979 Michal Wolnicki: https://orcid.org/0000-0002-7805-3503 Dawid Wnuk: https://orcid.org/0000-0002-5807-2440 Ali Modarressi: https://orcid.org/0000-0003-2984-0866
Wolf-Henning Boehncke: https://orcid.org/0000-0002-1225-7124 Nicolo Brembilla: https://orcid.org/0000-0003-1591-1790
ABSTRACT
Our group has recently shown that keratinocyte-derived IL-17E (IL-25), one of six members of the IL-17 family, is overexpressed in lesional psoriatic skin and is involved in its pathophysiology. We show here that IL-22 enhances IL-17E production in human keratinocytes and that these cells display a complete IL-17E receptor at their surface, which expression is further induced by IL-17A, indicating a potential autocrine effect of IL-17E.
Therefore, we addressed the impact of IL-17E on the function of human primary keratinocytes. IL-17E promoted the proliferation of keratinocytes in 2D and 3D cultures and caused the concomitant up-regulation of differentiation-associated gene transcripts (e.g keratin 10), while their expression was either inhibited or not changed by IL-17A. Contrary to IL-17A, IL-17E was not involved in the induction of antimicrobial proteins. Time-lapse analysis of cell movement showed that IL-17E influences cell motility increasing both cell speed and displacement. This was associated with specific changes in the actin cytoskeleton organization and the cell-substrate adhesion. No such effects were observed upon IL-17A stimulation. In summary, we identified to our knowledge previously unreported effects of IL- 17E clearly distinct from IL-17A, pointing towards an important role of IL-17E in the physiology and pathophysiology of the epidermis.
KEYWORDS: IL-17A, IL-17E, IL-25, IL-22, keratinocyte, proliferation, differentiation, migration, psoriasis, wound healing
INTRODUCTION
The skin is not a simple mechanical barrier, but a dynamic tissue ensuring an effective communication with the external environment. Sophisticated and well-coordinated
interactions between the epidermis and the underlying dermis, as well as between skin stromal cells and immune cells, ensure the homeostasis of the skin and its proper function (Hanel et al., 2013). This communication largely depends on small soluble mediators, among which cytokines are the most important.
The alteration of the expression level of one or multiple cytokines locally in the skin represents the pathogenic basis of several skin-inflammatory disorders such as psoriasis (Wittmann et al., 2014). This disease is characterized by an exaggerated Th17 lymphocyte response that leads to the overproduction of IL-17A and IL-22 cytokines among others (Boehncke and Schon, 2015). In physiological conditions, IL-17A is important to guarantee a proper innate defence against extracellular bacteria and fungi, as it induces an anti-microbial and inflammatory response (Eyerich et al., 2017). In the case of psoriasis, the abundance of IL-17A establishes an inflammatory positive feedback loop finally resulting in the hyperproliferation of keratinocytes and their altered differentiation, in addition to the augmented production of antimicrobial peptides (Boehncke and Schon, 2015, Brembilla et al., 2018, Ha et al., 2014).
IL-22, which is critical for the homeostasis of the epidermis, is thought to play an important role in psoriasis, particularly via the promotion of hyperproliferative keratinocytes (Hao, 2014).
IL-17A is the founding member of the IL-17 family, which comprises 6 distinct members: IL- 17A-F (Gaffen, 2011). These cytokines partially overlap with IL-17A with regard to amino acid sequence and function (Brembilla et al., 2018). IL-17E, also known as IL-25, possesses the lowest homology with IL-17A and is also produced by epithelial cells (Angkasekwinai et al., 2007, Johnston et al., 2013, Xu and Dong, 2017). Although initially thought to exclusively
participate to Th2 responses in the gut and lung (Angkasekwinai et al., 2007, Ballantyne et al., 2007, Caruso et al., 2009, Hvid et al., 2011, Owyang et al., 2006, Wang et al., 2014, Xu and Dong, 2017), IL-17E was recently demonstrated to be implicated in several skin inflammatory disorders. In atopic dermatitis, IL-17E was shown to impair the proper epidermal barrier formation (Deleuran et al., 2012, Hvid et al., 2011). In psoriasis, we identified that keratinocytes overproduce IL-17E, leading to inflammatory events favouring the recruitment of neutrophils (Senra et al., 2019, Senra et al., 2016). Later studies confirmed our observations and reported an autocrine function for IL-17E in mouse keratinocytes (Xu et al., 2018). Finally, in contact dermatitis IL-17E was shown to enhance Th17 cell-mediated inflammation through induction of IL-1β by dendritic cells (Suto et al., 2018).
The fact that IL-17E is implicated in the pathogenesis of skin diseases of very different aetiology suggests that its role may be much broader than previously thought. It is noteworthy that all skin-inflammatory diseases associated with an overexpression of IL-17E are also characterized by keratinocyte hyperproliferation and abnormal epidermis stratification.
Whether IL-17E may directly target the epidermis, in a manner similar or distinct from IL- 17A, remains largely unknown.
In the present study, we have thus addressed the role of IL-17E with respect to the physiological functions of keratinocytes. We show that the production of IL-17E is stimulated by IL-22 and that it affects the biology of the epidermis in a manner clearly distinct from IL- 17A. IL-17E is capable of promoting both proliferation and differentiation of keratinocytes, as well as enhancing their migration but has no effect on the production of antimicrobial peptides. These data reveal a role of IL-17E, which is independent from the activation of immune cells and relies on its ability to directly affect the epidermal homeostasis.
RESULTS
IL-22 up-regulates the production of IL-17E by keratinocytes.
In healthy subjects, IL-17E transcripts are uniformly produced by keratinocytes of all epidermal layers as assed by in-situ hybridization (Figure 1a, left panel). While IL-17E protein is accomulating within the cells of spinous and granular layers (Figure 1a, right panel). In psoriasis, keratinocytes over-produce IL-17E which is then present in all epidermal strati (Senra et al., 2016). We have thus investigated whether cytokines involved in psoriasis, i.e. IL-17A and IL-22, may influence the epidermal production of IL-17E using in vitro reconstructed human epidermis model (RHE). Interestingly, we detected that IL-17E transcripts (Figure 1b) and proteins (Figure 1c and d) were increased in cellular extract of IL- 22 treated RHE. IL-17A had only a minor effect on the expression of IL-17E in the same experimental system. These data identify IL-22 as an important inducer of IL-17E in humans.
IL-17E targets keratinocytes in an autocrine manner.
We next investigated the presence of the IL-17E receptor on human primary keratinocytes isolated from healthy individuals in 2D culture. Normal keratinocytes express both subunits of the IL-17E receptor, namely IL-17RA (common to IL-17A and other IL-17 family members) and IL-17RB (specific for IL-17E), as shown on mRNA (Figure 2a) and protein levels (Figure 2b). IL-17RB transcript (Figure 2a) and protein (Figure 2b) were specifically increased along with calcium-dependent differentiation and by IL-17A and IFN-γ stimulation, while IL-17RA levels were unchanged. Of interest, these stimuli led to a significant increase of the amount of IL-17RB subunit actually displayed at the surface of the cells, as revealed by flow cytometry analysis (Figure 2c). While IL-17RB was mostly polarized to the cell surface, IL-17RA was rather dispersed throughout the entire cell (Figure 2d). In this 2D experimental model, IL-22 was not efficiently inducing the production of neither subunits of the IL-17E
receptor. These data indicate that keratinocytes dynamically modulate the expression of the IL-17E receptor, particularly in the presence of inflammatory cytokines such as IL-17A and IFNγ.
IL-17E activates Akt/mTOR signalling pathway
Next, we evaluated the signalling events induced by IL-17E in keratinocytes and compared them to those induced by IL-17A. After 30 min of stimulation, IL-17E induced prompt activation of the mTOR pathway as measured by the phosphorylation of S6, a downstream target of mTOR, while only minimally interfering with the activation of Akt, STAT3, NF-ƙB and the MEK/ERK cascade. Interestingly, IL-17A had the opposite effects, leading to the phosphorylation of NF-ƙB while not activating the mTOR pathway (Figure 2e). In contrast, TNF-α, used as positive control, induced strong activation of each signaling molecule tested.
Thus, IL-17E seems to mediate its biological effects on keratinocytes mainly through the mTOR pathway. Taken together, these results indicate that keratinocyte are an important target of IL-17E in the skin and that IL-17E differs from the related IL-17A with respect to the signalling pathway exploited.
IL-17E is an inducer of keratinocyte proliferation
We next addressed the influence of IL-17E on keratinocyte proliferation in 2D cultures. In this setting, IL-17E induced an increase of the metabolic activity of keratinocytes reaching its optimum after 72 hours of stimulation (Figure 3a). This increase corresponded to an enhanced cell proliferation rate, as shown by crystal violet staining (Figure 3b). In line with previous reports (Ma et al., 2016, Wu et al., 2015), we also observed a slight increase in cell number after IL-17A addition, whereas IL-22 had maximal effects (Eyerich et al., 2017, Wolk et al., 2006) (Figure 3a and b). No additive or synergistic activity were observed when IL-17A and
IL-17E were added simultaneously. Subsequently, we analysed whether IL-17E influences the proliferation rate of keratinocytes when they are subjected to differentiation and stratification during RHE culture. In this setting, IL-17E was at least as effective as IL-22 in inducing cell proliferation, as reflected by the increased frequency of Ki67 positive cells located in the basal layer (Figure 3c and d, left panel). IL-17A, on the other hand, had no effect on the number of proliferating cells. Importantly, IL-17E treated 3D epidermal cultures had similar thickness when compared to control condition (Figure 3d, right panel), suggesting that it simultaneously modulates other keratinocyte functions. IL-17A and IL-17E stimulation resulted in a decreased and increased thickness, respectively. Overall, these data define IL- 17E as an important factor participating in the proliferation of keratinocytes, to an extent superior to IL-17A and comparable to the epidermal homeostatic cytokine IL-22.
IL-17E promotes the terminal differentiation of keratinocytes
IL-17A is known to affect the barrier function of the epidermis (Rabeony et al., 2014). We therefore assessed whether IL-17E may have a similar role. To study this, we exposed keratinocytes induced to differentiate by a calcium switch to IL-17E, IL-17A or IL-22. In the presence of calcium, IL-17E did not alter the differentiation process, with the exception of an increase in the transcription of loricrin (Figure 4a, right side of the loricrin panel). The addition of IL-22, and partially IL-17A, resulted instead in a global decrease of several differentiation markers i.e. keratin 10 (K10), loricrin, filaggrin, desmoglein 1, desmocollin1 (Figure 4a, right side of each graph) as determined at 72 hours by qPCR. To detect differences possibly masked by conditions that strongly force differentiation of keratinocytes, we repeated the experiment in the absence of calcium. Surprisingly, we observed a significant increase in the transcription level of several genes of interest, namely, K10, involucrin, loricrin, filaggrin and desmocollin 1 after IL-17E stimulation (Figure 4a, left side of each
graph). In this setting, IL-17A had no effect while IL-22 decreased the expression of loricrin and slightly increased the expression of involucrin. Simultaneous addition of IL-17A and IL- 17E in the absence of calcium led to an intermediate level of expression being the resultant value of the two cytokines. The effects of IL-17E were instead almost abrogated by simultaneous addition of IL-17A in the presence of calcium. To further substantiate these findings, we investigated the expression of selected markers in RHE generated in the presence of IL-17 cytokines by immunohistochemistry. In these experimental conditions, IL-17E induced a higher expression of K10 and tended to increase loricrin, but had no effect on the level of filaggrin and involucrin with respect to the control (Figure 4b). As expected, IL-17A blocked the regular differentiation of cells in RHE. In conclusion, IL-17E has the opposite effects to IL-17A and it promotes the terminal differentiation of keratinocytes in vitro.
IL-17E does not modify the ability of keratinocytes to produce antimicrobial peptides.
The production of anti-microbial peptides is an important function of keratinocytes. Since IL- 17A has been reported to efficiently induce an anti-microbial response in keratinocytes (Chiricozzi et al., 2014), we have tested whether IL-17E might function in a similar manner.
We observed that IL-17E, unlike IL-17A, was not capable of inducing the transcription of any antimicrobial peptide genes tested such as b-defensin 2, LL-37 and S100A7 independent of the culture conditions (with or w/o calcium; Figure 5a). Simultaneous addition of IL-17A and IL-17E resulted in similar transcription levels as in the presence of IL-17A alone.
IL-17E increases the expression of wound healing-associated keratins and enhances keratinocyte migration.
Finally, we investigated the possible role of IL-17E in the context of cell migration and wound healing. We found that IL-17E, unlike IL-17A, strongly induced the expression of
keratins found in hyperproliferative and healing epidermis, notably the keratin 6/16 pair, in in vitro cultured cells. In addition, IL-17E and IL-17A were both capable to increase the mRNA
level for keratin 17, a keratin characteristic of migrating keratinocytes after skin injury (Figure 5b). Simultaneous addition of IL-17A and IL-17E led to an intermediate level of expression being the resultant value of the two cytokines. Immunohistochemical analysis of RHE confirmed that IL-17E, but not IL-17A, induced a significant increase of K6 while minimally impacting K16 expression (Figure 5c). Moreover, time-lapse analysis of keratinocytes cultured in 2D showed that IL-17E significantly influences cell motility, increasing both cell speed and displacement, when compared to untreated and IL-17A-treated counterparts (Figure 6a and b). Surprisingly, IL-17E even had a slightly stronger potency in promoting keratinocyte migration than IL-22, which was used as positive control in the experiment (Figure 6a and b). In line with these results, IL-17E induced a faster wound closure in an in vitro scratch assay model when compared to IL-17A or IL-22 (Figure 6c, Supplementary video). Finally, we tested if effects induced by IL-17E on cell motility are directly reflected in changes of the actin cytoskeleton and cell-substrate adhesions by analysing the distribution of the focal adhesion marker vinculin by immunofluorescence. Of interest, we observed that individually migrating keratinocytes cultured in the presence of IL- 17E assembled smaller focal adhesions than keratinocytes cultured under control conditions or in the presence of IL-17A, suggesting their reduced maturation and faster turnover (Figure 6d). Accordingly, in the presence of IL-17E but not IL-17A the actin cytoskeleton showed a more disperse organisation with thinner actin bundles compared to controls, reflecting a migratory cell phenotype. Overall, these data indicate that IL-17E is involved in the induction of cell migration.
DISCUSSION
Recent studies documented an overexpression of IL-17E in several inflammatory skin diseases such as psoriasis, contact dermatitis and atopic dermatitis (Hvid et al., 2011, Senra et al., 2016, Suto et al., 2018). Most of these studies aimed at assessing the impact of IL-17E on cells of hematopoietic origin and underlined the general ability of this cytokine to amplify an inflammatory reaction. The hypothesis of IL-17E exhibiting additional effects rather related to the overall function of the epidermis is only starting to emerge. In this context, Xu and colleagues reported that IL-17E may target epidermal keratinocytes during skin inflammation induced by imiquimod application in mice, enhancing their proliferation and leading to the production of inflammatory mediators (Xu et al., 2018). Our study expands this topic and reveals that IL-17E significantly affects human keratinocyte biology.
An important finding in our manuscript is that IL-22, rather than IL-17A, induces the production of IL-17E in human keratincoytes. This is intriguing, since IL-17A was reported to act as an IL-17E-inducer in the mouse (Xu et al., 2018). Despite not being implicated in the IL-17E production, IL-17A is capable of inducing an increase of the surface expression of the IL-17E receptor. IL-17A and IL-22, whose levels are increased in psoriasis, might thus function as fine-tuners of the IL-17E related effects during development of the disease.
We further demonstrate that IL-17E strongly increases the metabolic activity of human primary keratinocytes and enhances their proliferation rate in both 2D and 3D culture conditions. This is particularly important, as IL-17E appears as of now to be the only member of the IL-17 family capable of significantly and directly influencing the proliferation of keratinocytes. Although IL-17A was reported to stimulate mouse keratinocyte or HaCaT cell proliferation (Ma et al., 2016, Wu et al., 2015), it had only a slight effect on primary human
keratinocyte monolayer cultures and no effect on RHEs. These effects may not be limited to the epidermis, but apply to other epithelial tissues. Notably, transgenic overexpression of IL- 17E in mice elicited epithelial hyperplasia and hypertrophy in multiple tissues including liver, heart, and lungs (Kim et al., 2002, Pan et al., 2001).
In addition, we show that IL-17E favours the process of terminal differentiation in human primary keratinocytes. This was particularly true when keratinocytes were stimulated in 2D cultures in the absence of calcium, which globally masked the effect of IL-17E. However, IL- 17E was still able to indcuce an increase in K10 level in RHEs despite the presence of high content of calcium in the medium. No other studies have comprehensively addressed the impact of IL-17E on epidermal differentiation, although IL-17E was reported to decrease the late differentiation protein filaggrin in the context of atopic dermatitis (Hvid et al., 2011, Kim et al., 2013). The discrepancy with our study might be related to the different culture conditions. We report an increase in filaggrin transcripts in cell-cell contact-stimulated differentiation but no visible changes in the calcium-induced differentiation or when RHE were cultured in thepresence of IL-17E. This latter model better reflects the in vivo situation, as it allows for the analysis of differentiation and stratification in a physiologically relevant system.
As outlined above, we found that IL-17A and IL-17E act in a distinct manner in their ability to stimulate the proliferation and differentiation of keratinocytes. These dissimilarities are equally reflected in differences in the signalling pathways. IL-17E was shown to signal through several molecules, particularly STAT3, NF-ƙB and MAP kinases in human mesenchymal cells and immune cells (Wang et al., 2015; Wong et al., 2005). The activation of STAT3 was further reported to occur in mouse primary keratinocytes (Xu et al., 2018),
while we did not observe a significant activation of this pathway when testing human keratinocytes. In addition, we report here the activation of the mTOR pathway as highlighted by the specific phosphorylation of the mTOR target S6 protein. Of interest, mTORC1 signalling is strongly induced in psoriatic lesions (Buerger et al., 2013, Burger et al., 2017).
The closure of a wound by keratinocytes is achieved by a combination of migration and proliferation of cells at the edges of the damage (Martin, 1997, Watt et al., 2006). In this regard, it is interesting to note that IL-17E is capable of promoting in an in vitro setting all functions implicated in the wound closure (proliferation, migration and differentiation). Thus, we hypothesize that the accumulated IL-17E in the granular layer of the epidermis serves as a kind of “protective reservoir”. In the case of epidermal barrier breach, the cytokine could be released from the cytoplasm of dead and damaged keratinocytes to promote all the processes indispensable for healing. The potential involvement of IL-17E in wound healing is further supported by the fact that it induces the expression of keratin 6, 16 and 17. During homeostasis, K6 is absent in the interfollicular epidermis of hairy skin whereas K16 is only detectable in keratinocytes of the basal layer. However, these are “stress-inducible” keratins and their expression is rapidly switched on after injury or in various hyperproliferative disorders, such as psoriasis (Moll et al., 2008, Paladini et al., 1996).
With regard to epidermal pathophysiological conditions, both IL-17A and IL-17E are highly overexpressed in lesional psoriatic skin. IL-17A is likely the most important factor driving psoriasis, as demonstrated by the high efficacy of anti-IL-17A targeted therapies (Yiu and Griffiths, 2016). Previously, we demonstrated that IL-17E amplifies the inflammatory loop in psoriasis by acting on immune cells (Senra et al., 2019, Senra et al., 2016). Here we document the capacity of IL-17E to target keratinocytes in an autocrine manner. The characteristic
epidermal abnormalities observed in psoriasis might thus result from the combined direct effects of both IL-17A and IL-17E cytokines. IL-17E might mediate keratinocyte hyperproliferation and upregulation of wound healing-associated keratins, while IL-17A might be more crucial for the induction of antimicrobial peptides and an abnormal epidermal differentiation.
In conclusion, we show here that IL-17E, in addition to the reported functions on immune cells, also directly targets human keratinocytes in an autocrine manner, leading to increased proliferation, differentiation and migration properties. The fact that IL-17E can directly target the epidermis could help to explain the existence of shared epidermal alterations in skin diseases presenting IL-17E overexpression but otherwise having a different pathogenesis such as psoriasis, contact dermatitis and atopic dermatitis. However, this has to be further investigated, especially in the light of potential interactions between IL-17E and other components of the respective disease-specific cytokine milieus. Our data suggest that IL-17E may also be implicated in the wound healing process and physiological functions in skin repair/regeneration. Overall, the functions of IL-17E reported here point towards an important role of this cytokine in the physiology and pathophysiology of the epidermis.
MATERIALS & METHODS
See the Supplementary Materials and Methods online for more experimental methods.
Human samples and cell culture
Skin biopsies were taken from 6 healthy adults presenting at the Department of Plastic and Reconstructive Surgery of the Geneva University Hospitals in Switzerland, or 6 children undergoing surgery at the Polish-American Children's Hospital, Krakow, Poland. This study was conducted according to the Declaration of Helsinki and approved by local ethical committee of the University Hospitals of Geneva, Switzerland, and the Jagiellonian University according to Polish law (No. 1072.6120.9.2017). Written informed consent was obtained from each individual. Briefly, human keratinocytes were isolated from biopsies after enzymatic digestion with 10 U/mL dispase (Gibco, Grand Island, NY, USA) and 0.05%
trypsin with 2 mmol/L EDTA subsequently (Sigma). Cells were cultured in serum free Keratinocyte Growth Medium 2 (Promocell, Heidelberg, Germany) in a humidified atmosphere with 5% CO2 at 37°C. NHEK/SVTERT cell line was used for signalling pathway analysis. Whenever added the concentrations of cytokines were as follow: IL-17A (100 ng/mL), IL-17E (100 ng/mL), TNF-a (20ng/mL) and IL-22 (20 ng/mL, all from R&D Systems, Minneapolis, MN, U.S.A).
In vitro Reconstructed Human Epidermis model (RHE)
For the generation of in vitro reconstructed epidermis equivalents, primary human keratinocytes were seeded onto 0.4 µm pore size inserts with PET-membranes (ThinCerts, Greiner Bio-One GmbH, Frickenhausen, Germany). First, keratinocytes were cultured in a submerged manner in fully-supplemented CnT-Prime Medium (CELLnTEC, Bern, Switzerland). Next, medium was changed to CnT-Prime 3D Barrier Medium (CELLnTEC)
and inserts were lifted to the air-liquid interface to induce cell differentiation in the presence of cytokines for 10 days.
Statistical analysis
Data are reported as mean ± standard error of the mean (SEM), with ‘n’ denoting the number of donors. Statistical analysis was performed using GraphPad Prism version 6.0 (Graphpad Software, La Jolla, CA). Nonparametric statistics (paired t-test) were used to compare populations. P-values less than 0.05 were considered statistically significant, and were indicated as follow: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.
DATA AVAILABILITY STATEMENT
No datasets were generated or analyzed during the current study.
CONFLICT OF INTEREST WHB received honoraria as advisor or invited speaker from Abbvie, Almirall, BMS, Celgene, Leo, Lilly, Novartis, UCB. The other authors have no conflict of interest related to the present manuscript to declare.
ACKNOWLEDGMENTS
We are grateful to Maria Shutova for fruitful discussions on aspects of keratinocyte motility and to Loïc Mermoud for help with a real time PCR analysis.
AUTHOR CONTRIBUTIONS Conceptualization: JB, NCB
Investigation: JB, CB, NT, JD, DW, NCB Formal analysis: JB, NCB
Methodology: JB, CB, JD Project administration: JB, NCB Visualization: JB, NCB
Writing – original draft: JB, NCB
Writing – review & editing: JB, CB, AM, JD, DW, WHB, NCB Resources: CB, AM, JD, MW, WHB
Funding acquisition: WHB, NCB Supervision: WHB, NCB
FUNDING: This work was supported by grant 310030_152680 and 310030_175470/1 from the Swiss National Science Foundation (SNSF), the Ernest Boninchi Foundation, the Société Académique de Genève and the De Reuter Foundation.
FIGURE LEGENDS
Figure 1. IL-22 enhances the production of IL-17E in reconstructed human epidermis.
a) The presence of IL-17E in the healthy skin was assessed by in-situ hybridization (mRNA level, left) and immunohistochemistry (protein level, right). Scale bar 50µ m. b) mRNA levels of IL-17E relative to the geometric mean of three housekeeping genes (GADPH, PGK1, PPIA) assessed by qPCR in cellular lysates from RHE models generated from 4 independent donors in control condition (medium only) and in the presence of IL-17A or IL-22. Grey and black dots refer to values standing below and above the detection threshold, respectively. c) Protein levels of IL-17E relative to the control condition in cell lysates of RHE generated as in b, as assessed by MesoScale technology. Values from the same donor are connected with a dotted line. d) Immunoflorescence analysis of IL-17E (red) in RHE generated as in b. One representative donor of two is shown. Scale bar 20µm.
Figure 2. IL-17E targets epidermal keratinocytes in an autocrine manner. a) mRNA levels in primary keratinocytes cultured in the presence of 2mM Ca2+ with or without the indicated stimuli relative to the control conditions (without calcium) (mean±SEM, n≥3).
Shown are significant difference vs control condition as assessed by paired-t-test, * ≤ 0.05. b) IL-17RA and IL-17RB expression in human primary keratinocytes cultured with or w/o 2mM Ca2+ and w/wo IL-17A or IFNγ as assessed by western blotting. One representative donor of three is shown. Numbers depict the quantification of western blot bands relative to actin. c) Expression of IL-17RB at the surface of keratinocytes stimulated as in b) as assessed by FACS. One representative histogram (left) and the cumulative data from 3 independent keratinocyte cultures (right) are shown. Significant difference vs control condition as assessed by paired-t-test, ** ≤ 0.01. d) Tri-dimensional view of the expression of IL-17RA (left panel) or IL-17RB (right panel) in cultured keratinocytes as assessed by confocal microscopy. An
oblique plane in z-stack images (dotted box) was selected and shown below each panel.
Nuclei were counterstained with DAPI (blue). One representative donor of three is shown. e) Detection of signalling molecules by western blotting in NHEK/SVERT keratinocytes cultured for 30 min in the presence of the indicated stimuli. One representative donor of four is shown. Numbers depict the quantification of western blot bands relative to the non- phosphorylated form of each molecule detected.
Figure 3. IL-17E enhances the proliferation rate of human primary keratinocytes. a) Metabolic activity of primary keratinocytes cultured in the presence of IL-17A, IL-17E, IL- 17A+IL-17E or IL-22 for 72 hours (mean±SEM, n=3). b) Proliferation rate measured by crystal violet staining in primary keratinocytes cultured as in (a) (mean±SEM, n=4). Culture plates stained with crystal violet dye are shown (left) c) Representative staining of Ki67 positive cells RHE in the presence of IL-17A, IL-17E or IL-22. Scale bar 100µm d) quantitative assessment of the number of Ki67+ cells (left, n=5) and the epidermal thickness (right, n=6-9) in RHE as in (c) (mean±SEM). Overall in the figure, significant differences assessed by paired-t-test: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001.
Figure 4. IL-17E stimulates the differentiation of keratinocytes. a) mRNA levels of differentiation markers relative to GAPDH assessed by qPCR in primary keratinocytes cultured in the presence of IL-17A, IL-17E, IL-17A+IL-17E or IL-22 for 72h. 2mM Ca2+
was added (right side of each graph) or not (left side of each graph) to the culture to force keratinocyte differentiation. Data from 3 independent experiments for a total of 5 to 6 different donors). b) Immunohistochemical analysis of selected differentiation markers in RHE generated in the presence of IL-17A or IL-17E. One representative donor of six is shown. Scale bar 50µm. Cumulative quantification of brown intensity is shown. In all panels,
significance differences versus control condition assessed by paired-t-test, * ≤ 0.05; ** ≤ 0.01.
Figure 5. IL-17E does not induce an anti-microbial response while increasing the expression of wound healing-associated keratins. a) mRNA levels of anti-microbial peptides relative to GADPH assessed by qPCR in primary keratinocytes cultured in the presence of IL-17A, IL-17E, IL-17A+IL-17E or IL-22 for 72h. 2mM Ca2+ was added (right side of each graph) or not (left side of each graph) to the culture to force keratinocyte differentiation. Data from 3 independent experiments for a total of 5 to 6 different donors) b) mRNA levels of keratin 6, keratin 16 and keratin 17 relative to GADPH assessed by qPCR in primary keratinocytes cultured in the presence of IL-17A, IL-17E, IL-17A+IL-17E or IL-22 for 72h. 2mM Ca2+ was added (right side of each graph) or not (left side of each graph) to the culture to force keratinocyte differentiation. Data from 3 independent experiments for a total of 5 to 6 different donors) c) Immunohistochemical analysis of keratin 6 and keratin 16 in RHE generated in the presence or absence of IL-17A or IL-17E. One representative donor of six is shown. Scale bar 50µm. Cumulative quantification of brown intensity is shown. In all panels, significance differences versus control condition assessed by paired-t-test, * ≤ 0.05;
** ≤ 0.01.
Figure 6. IL-17E enhances the migration of keratinocytes a) circular diagrams presenting the trajectories of 50 keratinocytes migrating for 1.5 hour under the influence of the indicated stimuli. One representative donor of four is shown. b) Quantitative analysis of the speed and displacement of keratinocytes cultured in presence of IL-17A, IL-17E or IL-22 (mean±SEM;
n=4, 50 cells/donor). * indicates significant difference by paired t-test: * ≤ 0.05, ** ≤ 0.01,
**** ≤ 0.0001. c) Primary keratinocytes were cultured up to confluence in the presence of the
indicated stimuli and a wound was mechanically produced 6 hours after the addition of indicated cytokines. Shown is the decrease in wound area over 10 hours in one representative experiment of two. d) Immunofluorescence staining of vinculin (red) and actin (green) in keratinocytes stimulated with IL-17A or IL-17E for 24h. Nuclei were counterstained with DAPI (blue). One representative donor of four is shown. Scale bar 20µm
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