ORIGINAL ARTICLE

IL-19 Is a Component of the Pathogenetic IL-23/IL-17 Cascade in Ellen Witte1,2, Georgios Kokolakis1,2,KatrinWitte1,2,SandraPhilipp1,2, Wolf-Dietrich Doecke3, Nina Babel4,5,BiancaM.Wittig6, Katarzyna Warszawska1, Agata Kurek1,2, Magdalena Erdmann-Keding1,2, Stefanie Kunz1,2, Khusru Asadullah3,MarshallE.Kadin7, Hans-Dieter Volk4,8, Wolfram Sterry9, Kerstin Wolk1,2,10 and Robert Sabat1,2,10

Psoriasis is a common chronic inflammatory disease with characteristic skin alterations and functions as a model of immune-mediated disorders. have a key role in psoriasis pathogenesis. Here, we demonstrated that out of 30 individually quantified cytokines, IL-19 showed the strongest differential expression between psoriatic lesions and healthy skin. Cutaneous IL-19 overproduction was reflected by elevated IL-19 blood levels that correlated with psoriasis severity. Accordingly, anti-psoriatic therapies substantially reduced both cutaneous and systemic IL-19 levels. IL-19 production was induced in by IL-17A and was further amplified by -a and IL-22. Among skin cells, keratinocytes were found to be important targets of IL-19. IL-19 alone, however, regulated only a few functions. While increasing the production of S100A7/8/9 and, to a moderate extent, also IL-1b, IL-20, C-X-C motif 8, and matrix metalloproteinase 1, IL-19 had no clear influence on the differentiation, proliferation, or migration of these cells. Importantly, IL-19 amplified many IL-17A effects on keratinocytes, including the induction of b-defensins, IL-19, IL-23p19, and T helper type 17- cell- and neutrophil-attracting . In summary, IL-19 as a component of the IL-23/IL-17 axis strengthens the IL-17A action and might be a biomarker for the activity of this axis in chronic inflammatory disorders. Journal of Investigative Dermatology (2014) 134, 2757–2767; doi:10.1038/jid.2014.308; published online 11 September 2014

INTRODUCTION dilatation of dermal papillae vessels underlie the psoriatic skin Psoriasis vulgaris (referred to as psoriasis) is a common, alterations that appear as sharply demarcated, erythematous, chronic, inflammatory skin disease frequently accompanied scaly plaques (Schon and Boehncke, 2005; Griffiths and by as well as endocrine, cardiovascular, and meta- Barker, 2007). Despite the disturbed barrier function of these bolic alterations (Schon and Boehncke, 2005; Griffiths and lesions, they are highly protected against bacterial and viral Barker, 2007; Armstrong et al., 2013). Hyperproliferation and infections, which is due to the strong local upregulation of disturbed differentiation of keratinocytes as well as growth and anti-bacterial and anti-viral (Ong et al., 2002; Wolk et al., 2013). 1Interdisciplinary Group of Molecular Immunopathology, Dermatology/ Although the pathogenesis of psoriasis is not fully under- 2 Medical Immunology, University Hospital Charite´, Berlin, Germany; Psoriasis stood, it is widely accepted that cytokines play a key role in Research and Treatment Center, University Hospital Charite´, Berlin, Germany; 3Global Biomarker, Bayer Pharma AG, Berlin, Germany; 4Berlin-Brandenburg both emergence and maintenance of psoriatic lesions. Accord- Center for Regenerative Therapies, University Hospital Charite´, Berlin, ingly, numerous immune mediators play been described as Germany; 5Department of Nephrology, University Hospital Charite´, Berlin, being elevated in the diseased skin of these patients (Sabat 6 Germany; Medical Clinic 1 (Gastroenterology, Rheumatology, Infectiology), et al., 2007). Among them are members of the IL-10 University Hospital Charite´, Berlin, Germany; 7Department of Dermatology, Boston University, Roger Williams Medical Center, Providence, Rhode Island, family—namely, IL-19, IL-20, IL-24, IL-22, IL-26, and IL-29 USA; 8Institute of Medical Immunology, University Hospital Charite´, Berlin, (Romer et al., 2003; Wolk et al., 2004; Kunz et al., 2006; Germany; 9Department of Dermatology and Allergy, University Hospital 10 Wolk et al., 2013). The cellular sources of these family Charite´, Berlin, Germany and Research Center Immunosciences, University members differ; in fact, IL-22, IL-26, and IL-29 are secreted Hospital Charite´, Berlin, Germany by T cells and innate lymphoid cells, whereas IL-24 is Correspondence: Robert Sabat, Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital preferentially produced by dendritic cells/ and Charite´, Charite´platz 1, Berlin D-10117, Germany. keratinocytes (Romer et al., 2003; Donnelly et al., 2010; E-mail: [email protected] Kumari et al., 2013; Wolk et al., 2013; Sabat et al., 2014). Abbreviations: BD, b-defensin; CXCL8, chemokine C-X-C motif ligand 8; IL-19 and IL-20 expression in psoriasis has been solely located GM-CSF, granulocyte--CSF; M-CSF, macrophage colony-stimulating in keratinocytes as impressively demonstrated by the Kragballe factor; mRNA, messenger RNA; TNF-a, tumor necrosis factor-a group (Romer et al.,2003).WiththeexceptionofIL-10,the Received 27 March 2013; revised 10 June 2014; accepted 30 June 2014; accepted article preview online 21 July 2014; published online 11 September IL-10 family members do not act on immune cells but instead 2014 target certain tissue cells (Sabat, 2010). Despite this, transgenic

& 2014 The Society for Investigative Dermatology www.jidonline.org 2757 EWitteet al. Role of IL-19 in Psoriasis

overexpression of IL-20, IL-22, and IL-24 in mice led to very of these cytokines’ action reduced cutaneous pathological similar psoriatic-like skin alterations (Blumberg et al., 2001; alterations in murine dermatitis models (Ma et al., 2008; Wolk et al., 2009a; He and Liang, 2010). Moreover, inhibition Stenderup et al., 2009; Kumari et al., 2013). This appears

IL-19 IL-1β IL-6 IL-12p35 IL-20 IL-23p19 p40 1 *** 1 1 1 1 1 1 *** *** 0.1 0.1 0.1 0.1 0.1 0.1 0.1 *** *** 0.01 0.01 0.01 0.01 0.01 0.01 0.01 **

Skin mRNA, 0.001 0.001 0.001 0.001 0.001 0.001 0.001 relative to HPRT relative

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

IL-25 IL-27p28 EBI3 IL-33 IL-36γ TNF-α M-CSF 1 1 1 1 100 1 1 ** *** *** 0.1 0.1 0.1 *** 0.1 10 0.1 0.1

0.01 0.01 *** 0.01 0.01 1 0.01 0.01 **

Skin mRNA, 0.001 0.001 0.001 0.001 0.1 0.001 0.001 relative to HPRT relative

0.0001 0.0001 0.0001 0.0001 0.01 0.0001 0.0001

IFN-γ OSM IL-4 IL-17A IL-21 IL-22 1 1 1 1 1 1

0.1 0.1 0.1 0.1 0.1 0.1 *** *** *** Healthy 0.01 0.01 *** 0.01 0.01 0.01 0.01 *** Psoriasis Skin mRNA, 0.001 0.001 0.001 0.001 0.001 0.001 relative to HPRT relative

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

IL-19 IL-19 IL-20 IL-22 Corr. with PASI 1,600 500 50 100

–1 rs P –1 *** 400 40 80 1,200 * * *** IL-19 0.653 0.000 Healthy 300 30 60 800 Psoriasis 200 20 40 IL-20 0.570 0.004 400 100 10 20

Skin , pg ml IL-22 0.624 0.001 Serum protein, pg ml 0 0 0 0

IL-19 IL-19 500 1,000 1,000 125 125 –1 400 100 100 100 100 * 300 75 * 75 10 10 * 200 50 50 *

UV therapy * 1 1 % of 0 weeks 100 *** 25 25 etanercept therapy mRNA, % of 0 wks * mRNA, % of 0 weeks

Serum protein, pg ml *** 0 0.1 0.1 0 0 0 weeks 4 weeks 0 weeks 2–5 weeks 0 weeks 0 weeks of therapy of UV-based therapy Healthy Asthma 1 week 12 week 4–6 weeks 24 weeks Serum IL-19 PASI Transplanted Crohn’s disease Crohn’s

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to be caused by the direct effects of these mediators these patients. Importantly, IL-19 blood levels positively and on the keratinocytes, leading to their disturbed differentiation most significantly correlated with the disease severity repre- and chemokine production as demonstrated by in vitro sented by the psoriasis area and severity index value studies (Wolk et al., 2006; Sa et al., 2007; Wolk et al., (Figure 1d). Virtually no systemic levels of IL-19 were found 2009a; Kumari et al., 2013). Moreover, some IL-10 in healthy participants (Figure 1c and e). Analysis of further family members might have an essential role in the immune-mediated conditions revealed that patients suffering elevated anti-microbial competence of psoriatic lesions: from asthma and Crohn’s disease, but not those after renal IL-20, IL-22, and IL-24 strongly enhance the expression transplantation, also showed increased IL-19 blood levels, of anti-bacterial proteins in keratinocytes (Wolk et al., 2004; which, however, were far from reaching those of psoriasis Sa et al., 2007; Kumari et al., 2013), whereas the elevated patients (Figure 1e). anti-viral defense of psoriatic lesions seems to be dependent Next, we investigated the influence of anti-psoriatic thera- on IL-29 (Wolk et al., 2013). In contrast to IL-20, IL-22, IL-24, pies on the IL-19 levels in the skin and blood of psoriasis and IL-29, the roles of IL-19 and IL-26 were largely patients. A pronounced drop of cutaneous IL-19 mRNA to unexplored so far. below 1% of the initial expression levels was induced by UV In this study, we compared the expression of a broad range radiation and TNF-a inhibition–based therapies (Figure 1f). of cytokines belonging to different cytokine families (including Strikingly, only a single application of ustekinumab induced a the IL-1, IL-12/IL-23, IL-10, and IL-17 families) in psoriatic marked decrease in blood IL-19 concentrations (Figure 1g). lesions and came across the outstanding upregulation of IL-19 Importantly, this decrease in blood IL-19 amounts seems to in this situation. This let us characterize the induction, target precede that of the clinical improvement represented by cells, and effects of IL-19 with special focus on its interaction psoriasis area and severity index reduction. A similar behavior with other cytokines and its classification into an important of IL-19 levels was seen in patients after UV-based combina- pathogenetic cascade. tion therapy (Figure 1g). These data demonstrate that blood IL-19 level may show therapy response before the clinically RESULTS visible improvement in skin alterations. IL-19 dominates the local and systemic cytokine milieu in psoriasis IL-17A drives keratinocyte IL-19 production In order to get a more thorough picture of the cutaneous The outstanding IL-19 production (Figure 1a and b) and its cytokine networks in psoriasis, we individually quantified location in the psoriatic lesion in the epidermis above the the messenger RNA (mRNA) expression of a broad range of dermal papillae only (Romer et al., 2003) suggested that cytokines (IL-1a,IL-1b, IL-2, IL-4, IL-6, IL-9, IL-10, IL-12p35, keratinocytes produce this cytokine in response to different p40, IL-13, IL-17A, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, mediators of the psoriatic cytokine pool. On the basis of this IL-23p19, IL-24, IL-25, IL-26, IL-27p28, IL-29, EBI3, IL-33, assumption, we first tested a range of cytokines overexpressed IL-36g, tumor necrosis factor-a (TNF-a), macrophage colony- in lesional psoriatic skin for their IL-19-inducing capacity stimulating factor (M-CSF), granulocyte-macrophage-CSF using primary human keratinocytes and epidermis models. (GM-CSF), OSM, IFN-b1, and IFN-g)inlesionalskinfrom Whereas IL-6, IL-21, IL-22, IL-24, IFN-g, TNF-a,GM-CSF,or psoriasis patients. The vast majority of analyzed mediators IL-19 itself did not virtually provoke IL-19 production, we were highly expressed in the lesions compared with skin from found a marked IL-19 production driven by IL-17A (Figure 2a healthy donors (Figure 1a and data not shown). Strikingly, and data not shown). This effect was exerted via transcrip- among all quantified cytokines, IL-19 was the most strongly tional regulation (Figure 2b). As IL-22 has been shown to upregulated mediator with expressions several thousand times synergize with IL-17A in regulating keratinocyte functions higher in psoriatic skin compared with healthy skin (Liang et al., 2006; Wolk et al., 2011), we investigated (Figure 1a). The lesional IL-19 mRNA upregulation was whether the same is true regarding the keratinocyte IL-19 accompanied by strong IL-19 protein production (Figure 1b). production. Indeed, although IL-22 itself did not clearly Moreover, the cutaneous IL-19 dominance was reflected by induce IL-19 mRNA expression or protein secretion high systemic amounts of IL-19 protein (Figure 1c). In fact, (Figure 2a and c), its presence amplified the IL-17A-induced blood concentrations of this cytokine even exceeded those of levels (Figure 2c). As anti-TNF-a therapy reduced IL-19 levels IL-20 and IL-22 by B10- and 5-fold, respectively, two in psoriatic patients (Figure 1f) and an amplifying action of cytokines known for the strongly elevated systemic levels in TNF-a regarding some IL-22 effects has been observed (Wolk

Figure 1. IL-19 is highly present in the skin and blood of psoriasis patients. (a) Expression in skin biopsies from healthy individuals (n ¼ 10) and psoriasis patients (n ¼ 16, except for IL-25 (n ¼ 10)). (b) Concentration in the supernatant of skin biopsy cultures from psoriasis patients (n ¼ 5) and healthy donors (n ¼ 4) cultured for 20 hours. (c, d) Concentration in blood serum (IL-19) and plasma (IL-20, IL-22) from healthy donors (n ¼ 13), psoriasis patients (n ¼ 25); correlation with patients’ psoriasis area and severity index (PASI) values. (e) Concentration in serum from healthy donors (n ¼ 26), asthma patients (n ¼ 17), patients with Crohn’s disease (n ¼ 20), and transplanted patients (n ¼ 20). (f) Expression in skin biopsies from psoriasis patients before/after anti-psoriatic therapies (n ¼ 3). (g) PASI values and IL-19 serum concentrations of psoriasis patients before (PASI: 10.1±0.6)/during ustekinumab therapy (n ¼ 6) (PASI after 16wks: 4.4 ±1.0) and before (PASI: 14.1±1.4)/during UV-based combination therapy (n ¼ 6; for details refer to the Materials and Methods section) (PASI after 2–5wks: 5.4±0.7). (a–c, e–g)Mean data±s.e.m. HPRT, hypoxanthine-guanine phosphoribosyltransferase; M-CSF,macrophage colony-stimulating factor; mRNA, messenger RNA; OSM, ; TNF-a, tumor necrosis factor-a.

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et al., 2009a), we investigated the influence of TNF-a on the 150

IL-22 and IL-17A effects with respect to IL-19 production. As –1 120 demonstrated in Figure 2d, the combination of TNF-a with 90 only IL-22 had no IL-19-inducing effect. However, TNF-a 60 increased the IL-17A-induced IL-19 expression (Figure 2e). Secreted IL-19 Interestingly, the highest IL-19 production was observed when protein, pg ml 30 keratinocytes were stimulated with the combination of IL-17A/ 0 contr. IFN-γ IL-17A IL-22 TNF-α IL-24 IL-22/TNF-a. 1

Keratinocytes are important IL-19 targets in the skin 0.1 Next, we questioned which cell types in the skin are targeted 0.01 by IL-19. IL-19 acts via a heterodimeric transmembrane receptor composed of IL-20R1 and IL-20R2 (Dumoutier to HPRT 0.001 et al., 2001). As a number of studies have excluded any IL-19 mRNA, relative 0.0001 expression of IL-20R1 in immune cells (Sabat, 2010), we contr. IFN-γ IL-17A IL-22 TNF-α IL-24 focused on the analysis of skin-resident tissue cells for IL-19 receptor subunit expression. As demonstrated in Figure 3, 150 1 expression of IL-20R1 and IL-20R2 was virtually absent in * * –1 120 0.1 microvascular endothelial cells and melanocytes. Although 90 dermal fibroblasts clearly expressed IL-20R2, only marginal 0.01 contr. 60 levels of IL-20R1 mRNA were detectable in these cells. In IL-17A

IL-19 mRNA, 0.001

Secreted IL-19 30 protein, pg ml contrast, keratinocytes strongly expressed both IL-19 receptor relative to HPRT components (Figure 3), which highlights these cells as impor- 0 0.0001 tant IL-19 target cells in the skin. 500 1

IL-19 regulates only a few functions of keratinocytes –1 400 0.1 Having identified keratinocytes as IL-19 target cells, we next 300 0.01 aimed to characterize the keratinocyte response to IL-19 200 stimulation. We investigated the influence of IL-19 on human IL-19 mRNA, 0.001 Secreted IL-19

protein, pg ml 100 epidermal models and conventional keratinocyte cultures relative to HPRT regarding functions known to be altered in the psoriatic skin 0 0.0001 contr. IL-22 IL-17A IL-17A + IL-22 as follows: (i) disturbed terminal differentiation, (ii) increased proliferation, (iii) increased migratory capacity, (iv) increased 500 anti-bacterial competence, and (v) production of pro-inflam-

–1 400 matory mediators. IL-19’s relative IL-22 was included in these contr. investigations as it is well known to regulate many of these 300 IL-22 functions (Sabat et al., 2014). Surprisingly, IL-19 had no 200 TNF-α IL-22 + TNF-α

Secreted IL-19 100 relevant influence on the expression of molecules with a key protein, pg ml function in keratinocyte terminal differentiation, such as 0 keratin-10, calmodulin-like 5, desmocollin-1, and kallikrein-7 (Figure 4a). Consequently, there were no histologically visible 25,000 1

signs of a disturbed differentiation in IL-19-treated epidermis –1 20,000 models (Figure 4b). In contrast and in line with our previous 15,000 studies (Wolk et al., 2006, 2009a), IL-22 altered the expression 0.1 of molecules with key functions in keratinocyte terminal 10,000 IL-19 mRNA, Secreted IL-19 protein, pg ml 5,000

differentiation and provoked acanthosis, hypogranularity, relative to HPRT and partly parakeratosis. IL-19 also had no influence on 0 0.01 keratinocyte proliferation, whereas -b had a IL-17A IL-17A + TNF-α reducing and IL-17A had a slightly inducing effect IL-17A + IL-22 IL-17A + IL-22 + TNF-α (Figure 4c). Moreover, only minimal if any influence of IL- Figure 2. T helper type 17-cytokines drive keratinocyte IL-19 production. 19 could be detected on the keratinocyte migration in IL-19 expression and concentration in the supernatant of EpiDerm cultures wounded keratinocyte monolayers, whereas EGF was highly stimulated with the indicated cytokines for 72 hours. n ¼ 3(a,c,d,e)orn ¼ 6(b). potent in this regard as expected (Barrandon and Green, Mean data±s.e.m. contr., control; HPRT, hypoxanthine-guanine 1987); (Figure 4d). However, IL-19 increased the keratinocyte phosphoribosyltransferase; mRNA, messenger RNA. production of the anti-bacterial proteins psoriasin (S100A7), S100A8, and S100A9 (Figure 5a). Interestingly, this IL-19 effect b-defensin (BD)2, BD3, lipocalin-2 , LL37/cathelicidin, and on the microbial defense was actually specific for these S100 RNAse7 (Figure 5a). IL-19 also slightly affected the expression proteins as no modulation was observed regarding of a few molecules with inflammatory properties. In fact, a

2760 Journal of Investigative Dermatology (2014), Volume 134 EWitteet al. Role of IL-19 in Psoriasis

Keratinocytes Fibroblasts Endothelial cells Melanocytes 10 10 10 10

1 1 1 1

0.1 0.1 0.1 0.1

0.01 0.01 0.01 0.01

mRNA, relative to HPRT 0.001 0.001 0.001 0.001

IL-20R1 IL-20R2

Figure 3. Keratinocytes are a major target of IL-19. Receptor subunit expression in isolated keratinocytes and dermal fibroblasts, microvascular endothelial cells, and melanocytes (n ¼ 2; mean ±range). HPRT, hypoxanthine-guanine phosphoribosyltransferase; mRNA, messenger RNA.

Keratin 10 CALML5 DSC1 KLK7 125 125 * 125 * 125 **125 125 125 125 * 100 100 100 100 100 100 100 100 contr. 75 75 75 75 75 75 75 75 IL-19 50 50 50 50 50 50 50 50 IL-22 25 25 25 25 25 25 25 25

mRNA, % of control 0 0 0 0 0 0 0 0

contr. contr. 0 h 24 h 150

100 EGF

IL-19 IL-22 50 Cell count, % of control

0 contr. IL-19 IL-19 200 200 * IL-17A IFN-β 150 150 contr. 100 100 IL-19 contr. IL-22

Epidermal 50 50

0 0 thickness, % of control Figure 4. IL-19 has no major impact on keratinocyte differentiation, proliferation, or migration. (a, b) Expression, histological appearance, and thickness of EpiDerm stimulated with the indicated cytokines for 72 hours. n ¼ 5(a), n ¼ 7(b, top), n ¼ 3(IL-19),orn ¼ 6(IL-22)(b,bottom);bar¼ 100 mm. (c)Cellcountsof keratinocytes stimulated with the indicated cytokines for 5d (n ¼ 4). Mean data±s.e.m. (a–c). (d) Confluent keratinocyte monolayers subjected to scratch wounding and subsequently stimulated with the indicated cytokines for 24 hours. Closure of wounded area monitored before (0 hours) and after (24 hours) stimulation (n ¼ 1 out of 2); bar ¼ 500 mm. CALML5, calmodulin-like 5; contr., control; DSC1, desmocollin-1; KLK7, kallikrein-7; mRNA, messenger RNA.

stimulatory effect of IL-19 was observed on the expression of IL-19 strengthens numerous IL-17A effects on keratinocytes IL-1b, IL-20, chemokine C-X-C motif ligand 8 (CXCL8), As demonstrated in Figure 1a, psoriatic lesions contain several and the extracellular matrix–degrading and migration-facilitat- different mediators that might modulate the effects of indivi- ing enzyme matrix metalloproteinase 1. No influence by dual cytokines on their target cells. Hence, we screened for IL-19 was found regarding the expression of IL-6, IL-23p19, the influence of selected cytokines (IL-6, IL-17A, IL-21, IL-22, IL-36g, GM-CSF, CXCL1, CXCL11, regenerating islet-derived IL-23, TNF-a, GM-CSF, and IFN-g) on IL-19 action. Interest- protein 3 alpha, or its own mRNA (Figure 5b and data not ingly, IL-17A clearly synergized with IL-19 (data not shown), shown). which prompted us to focus on the cooperative activity of

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BD2 BD3 LCN2 LL37 100 100 * 1,000 0.1

* 10 10 100 * 0.1

1 1 10 0.01

0.1 0.001 mRNA, relative to HPRT 0.1 1 contr. IL-19 RNAse7 S100A7 S100A8 S100A9 IL-22 0.1 1,000 * 10,000 10,000 * * * * * 0.1 100 1,000 1,000

0.01 10 100 100

1 mRNA, relative to HPRT 0.001 10 10

IL-1β IL-20 IL-23p19 IL-36γ CXCL1 CXCL8 MMP1 * 250 250 * 250 250 250 250 250 * 200 200 200 200 200 200 200 * contr. 150 150 150 150 150 150 150 IL-19 100 100 100 100 100 100 100 50 50 50 50 50 50 50 mRNA, % of control 0 0 0 0 0 0 0

Figure 5. IL-19 upregulates expression of selected mediators of anti-bacterial defense and inflammation in keratinocytes. (a, b) Expression in EpiDerm stimulated with the indicated cytokines for 72 hours. n ¼ 5(a)orn ¼ 6(b). Mean ±s.e.m. BD, b-defensin; CXCL, chemokine C-X-C motif ligand; HPRT, hypoxanthine- guanine phosphoribosyltransferase; LCN2, lipocalin-2; MMP1, matrix metalloproteinase 1.

both cytokines. Using epidermis models, we could demon- DISCUSSION strate that IL-19 increases the effect of IL-17A regarding The aim of our study was to illuminate the cytokine network in the expression of the anti-bacterial proteins BD2, BD3, and psoriatic lesions. To address this aim, the cytokine pattern may S100A7 in a synergistic manner (Figure 6a and b). Similarly, be assessed by different approaches including global screens IL-19 synergized with IL-17A to upregulate the expression (microarray, mRNA sequencing) or specific individual quanti- of IL-1b, IL-20, IL-23p19, CXCL1, CXCL8, and CCL20 fication. Using highly quantitative real-time PCR analyzing a (Figure 6a and c). In contrast, IL-19 had no influence on the broad range (n ¼ 30) of cytokines, we detected an outstanding IL-17A-induced IL-36g or lipocalin-2 expression, and IL-17A expression of the IL-10 family member IL-19, a so far under- did not promote IL-19-induced upregulation of matrix appreciated mediator. Follow-up investigations identified IL- metalloproteinase 1 mRNA (Figure 6a). Overall, most IL-17A 19 as a component of the IL-23/IL-17 cascade, meaning that effects were amplified by IL-19. This amplification IL-19 might especially contribute to the chronicity of the might be based on the synergistic/additive activation of signal pathologic interaction between immune cells and tissue cells. transducer and activator of transcription 3 and c-Jun N-term- This conclusion is drawn on the basis of the following inal kinase by both cytokines (Supplementary Figure S1 observations: First, IL-17A drives IL-19 production by kerati- online). nocytes, which is further amplified by the psoriasis key The known absence of IL-20R1, the receptor used by IL-19, cytokines IL-22 and TNF-a. As an autocrine mediator, IL-19 on immune cells (Sabat, 2010) and the here described IL-19 acts on the keratinocytes. These cells seem to be the sole effects (Figure 5b and Figure 6a–c) suggest a role of IL-19 in target cells of this cytokine in the skin as deduced from our local (cutaneous) but not systemic inflammation. Accordingly, analyses of skin tissue cells isolated from healthy donors. We the inhibition of IL-20R1 profoundly reduced the cutaneous surprisingly found that IL-19 itself has no major impact on the production of pro-inflammatory cytokines and skin thickening keratinocyte functions known to be characteristically altered in the murine DNFB model of contact dermatitis (Figure 6d), in psoriasis. A clear influence of IL-19 could be observed only whereas it had no influence on the blood levels of with respect to the production of anti-bacterial S100 proteins. pro-inflammatory cytokines in lipopolysaccharide-induced In addition, IL-19 slightly induced the production of some systemic inflammation in mice (Figure 6e). inflammatory (IL-1b, CXCL8) and regeneration-promoting

2762 Journal of Investigative Dermatology (2014), Volume 134 EWitteet al. Role of IL-19 in Psoriasis

BD2 BD3 S100A7 LCN2 RegIIIα S100A7 100,000 10,000 10,000 10,000 0.1 * contr. 10,000 1,000 1,000 * * 1,000 0.01 1,000 * * * 100 * * 100 * 100 * 0.001 100 * mRNA, % of control mRNA, % of control mRNA, % of control mRNA, % of control

10 10 10 10 to HPRT mRNA, relative 0.0001 IL-19 IL-1β IL-19 IL-20 IL-23p19 IL-36γ 1,000 0.1 1,000 1,000 1,000 * * * 0.01 * * * 100 * 100 * 100 * 100 * * 0.001 IL-17A

mRNA, % of control * * mRNA, % of control mRNA, % of control mRNA, % of control 10 to HPRT mRNA, relative 0.0001 10 10 10

GM-CSF CXCL1 CXCL8 MMP1 CCL20 1,000 1,000 1,000 1,000 3,500 IL-19 + IL-17A * * 2,800 * * * 2,100 100 100 * 100 * 100 * 1,400

700 mRNA, % of control mRNA, % of control mRNA, % of control mRNA, % of control 10 10 10 10 0

contr. IL-19 IL-17A IL-19 + IL-17A Secreted protein, % of control

TNF-α IL-1β TNF-α p40 ** **

40 100 –1 400 ** 5,000 ** ** 0.40 ** ** ** ** ** 4,000 **

–1 30 ** ** 300 ** ** 75 0.35 3,000

20 50 0.30 **** 200 2,000 10 25 100 0.25 1,000 protein, pg ml Ear homogenate Ear swelling, mm Ear swelling, 0 0 0.20 Plasma protein, pg ml 0 0 Day 0 Day 1 Day 2 Day 3

Vehicle DNFB + IgG Vehicle DNFB + IgG PBS LPS + IgG DNFB DNFB + αIL-20R1 DNFB DNFB + αIL-20R1 LPS LPS + αIL-20R1

Figure 6. IL-19 provokes a strengthened IL-17A-induced keratinocyte response. (a, b) Expression and immunohistological staining of EpiDerm stimulated with the indicated cytokines for 72 hours. n ¼ 6 or (for IL-19, IL-1b, CXCL1, and chemokine C-X-C motif ligand 8 (CXCL8)) n ¼ 5(a); and n ¼ 1outof4(b); bar ¼ 100 mm. Significant differences from the IL-19/IL-17 group are indicated. (c) Concentration in the supernatant of keratinocyte cultures stimulated with the indicated cytokines for 48 hours (n ¼ 3). (d) Concentrations of tumor necrosis factor-a (TNF-a) and IL-1b in ear homogenate and ear swelling of DNFB-sensitized and DNFB-challenged or not challenged mice (n ¼ 6 per group) treated 1 hour before challenge with anti-IL-20R1 antibody (aIL-20R1), control Ab (IgG), or phosphate-buffered saline (PBS). (e) Concentrations of TNF-a and p40 in blood plasma in intraperitoneally lipopolysaccharide (LPS)-injected mice (n ¼ 5 per group) treated 16 hours before LPS application with anti-IL-20R1 Ab, control Ab, or PBS. Mean ±s.e.m. BD, b-defensin; contr., control; CCL20, chemokine C-C motif ligand 20; CXCL1, chemokine C-X-C motif ligand 1; GM-CSF, granulocyte-macrophage colony-stimulating factor; LCN2, lipocalin-2; RegIIIa, regenerating islet- derived protein 3 alpha; mRNA, messenger RNA.

mediators (IL-20, matrix metalloproteinase 1). No influence referring to this relation as the IL-23/IL-17 cascade (Ouyang was found on keratinocyte differentiation, migration, or pro- et al., 2008). Many results point to the crucial role of this liferation, or on the production of further mediators. However, cascade in psoriasis pathogenesis. First, different studies IL-19 potentiated the action of IL-17A. This potentialization demonstrated that IL-23/lL-17A contributes to experimental might occur at the level of signal transduction. The IL-17 cutaneous inflammation (Lowes et al., 2013). Furthermore, production is driven by IL-23, providing the reason for several single nucleotide polymorphisms of IL-23-related

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have been described to be associated with the controls, as demonstrated by the Ouyang group (Sa et al., susceptibility to psoriasis (Capon et al., 2007; Nair et al., 2007), suggesting clear effects of this cytokine on 2009). Moreover, anti-p40 and the recent IL-17 system– keratinocytes. Interestingly, the IL-19 signature also differed targeting therapies are very effective in psoriasis (Leonardi from that of samples treated with its cytokine siblings IL-22 or et al.,2008;Pappet al., 2008; Miossec and Kolls, 2012). IL-24 (Sa et al., 2007). The consequences of the simultaneous presence of IL-19 Our new results regarding the effects of IL-19 on keratino- and IL-17A in psoriasis lesions may have an impact on cytes very well match both, the excessive IL-19 production in different levels of the pathogenetic process. First, both cyto- psoriatic lesions and the absence of skin alterations in IL-19- kines synergistically increase the anti-bacterial competence of transgenic mice reported as ‘‘unpublished observations from keratinocytes by upregulating the expression of b-defensins transgenic group of ZymoGenetics’’ by Parrish-Novak et al. and S100A proteins. Besides their function in preventing (Parrish-Novak et al., 2002). As described above, a major role infections, some of these proteins are reported to also have of IL-19 seems to be the amplification of the IL-17A action, chemoattractive properties, which may support the infiltration whereas in the absence of IL-17A (like in the IL-19-transgenic of immune cells into the skin and may boost the local model) no relevant alterations in tissue homeostasis are to be inflammation (Soruri et al., 2007; Wolf et al., 2008). expected by IL-19. Moreover, the directly enhanced induction of chemokines In addition to the outstanding IL-19 presence in psoriatic attracting particularly T helper type 1-cells and dendritic skin, we observed large amounts of this cytokine in the cells (CCL20) and neutrophilic granulocytes (CXCL1 and circulation of patients. Importantly, IL-19 levels highly CXCL8) by the cooperative action of IL-19 and IL-17A exceeded those of known key psoriasis cytokines and rapidly might further support this process. Third, IL-19 and IL-17A decreased upon anti-psoriatic therapy. The fact that reduction synergize with respect to the production of mediators, which of systemic IL-19 levels preceded the clinical improvement promote the maintenance and effector function of T helper of the patients might be useful for the selection and monitoring type 17-cells (IL-1b, IL-23). IL-1b has been reported to be of specific anti-psoriatic therapies, in particular the anti-IL-12/ crucial for the pathogenicity of T helper type 17-cells IL-23 and anti-IL-17 treatment. Wang et al. very recently (Brereton et al., 2009; Coccia et al., 2012), whereas IL-23 demonstrated that the psoriasis-associated Act1D10N drives their proliferation, survival, and cytokine production mutation––although accompanied by a high presence of IL- (Ouyang et al., 2008). These IL-19/IL17 effects might be 23, T helper type 17-cells, and IL-17A in the affected skin–– responsible for the persistence of IL-17-producing cells in prevented IL-17-related signal transduction and effects (Wang the inflamed skin. Fourth, IL-19 and IL-17A further cooperate et al., 2013). Therefore, a selection of psoriasis patients in the induction of IL-20, which contributes to the psoriasis- according to the activity of the IL-23/IL-17 axis by typical epidermal alterations. quantifying systemic IL-19 levels before initiation of such a The results of our study therefore extend the knowledge therapy might be advisable. about IL-19, a so far largely unexplored mediator. In fact, even In summary, our findings regarding production, target cells, more than 10 years after its discovery by Gallagher´s group and effects of IL-19 present this cytokine as a component of (Gallagher et al., 2000), the actual role of IL-19 remained the IL-23/IL-17 axis, and therefore uncovers a further part of obscure. IL-19 expression in vivo has been demonstrated in the pathogenic cascade in psoriasis that can be outlined as inflammation in murine models (Kunz et al., 2006; Wegenka follows: Having infiltrated the skin, lymphoid cells produce IL- et al., 2007; Hsing et al., 2008) and human chronic 17A and IL-22, both of which drive their individual, and inflammatory diseases (Kunz et al., 2006; Huang et al., mediate their cooperative actions. IL-22 on its own inhibits 2008; Li et al., 2008; Sakurai et al., 2008). Moreover, on the terminal differentiation, enhances the mobility of keratino- basis of the association of psoriasis, pustulosis palmoplantaris, cytes, and sensitizes these cells to the action of IL-19, IL-20, vitiligo, type-I diabetes, ulcerative colitis, and Crohn’s disease and IL-22 by increasing the production of their essential signal with single nucleotide polymorphisms or the epigenetic transduction element signal transducer and activator of tran- imprint of the IL19, IL20RA, and IL20RB genes, a role of scription 3. IL-17A acts as a major inducer of inflammatory IL-19 was suggested for these conditions (Koks et al., 2005; cytokines and chemokines attracting neutrophils, dendritic Kingo et al., 2007; Barrett et al., 2009; Kingo et al., 2010; cells, and T cells and may support keratinocyte hyperproli- Yamamoto-Furusho et al., 2011; Nimmo et al., 2012). feration (Nograles et al., 2008; Wolk et al., 2009a; Lai et al., However, the so far known effects of IL-19 on primary 2012). IL-17A, TNF-a, and IL-22 synergistically induce the human cells are limited and include the prevention of production of certain anti-bacterial proteins (Liang et al., starvation-induced and IL-6 secretion in 2006; Wolk et al., 2011), of IL-20 (Wolk et al., 2009b), and synovial cells, the inhibition of of IL-19. IL-20 potentiates the IL-22-initiated inhibition of the proliferation, migration, and -induced keratinocyte terminal differentiation, whereas IL-19 further apoptosis of vascular smooth muscle cells, as well as the strengthens the immune-stimulatory properties of IL-17A. elevation of proliferation of vascular and coronary endothelial Supporting that psoriasis also functions as a model for other cells (Sakurai et al.,2008;Tianet al., 2008; Gabunia et al., immune-mediated disorders, in which the IL-23/IL-17 axis 2011; Jain et al., 2011; Gabunia et al., 2012). On the other often plays a pathogenetic role, we hope that our study will hand, the expression signature of IL-19-treated epidermis pave the way for investigating IL-19 functions in further models showed differences from the signature of untreated immune-mediated diseases.

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MATERIALS AND METHODS To investigate IL-19 production and the cooperative action of IL-19 Patients with other cytokines, keratinocytes were stimulated or not stimulated To compare mRNA expressions of different cytokines, punch (control) with IL-19 (100 ng ml 1), 10 ng ml 1 of IL-6, IL-17A, IL-22, biopsies were obtained from diseased skin of 16 adult patients with IL-23, TNF-a, GM-CSF, and IFN-g, or combinations of cytokines as chronic plaque psoriasis and from 10 healthy volunteers. To analyze indicated (all from R&D Systems) for 24 hours–48 hours. therapy-induced changes in cutaneous IL-19 expression, biopsies EpiDerm-201 underdeveloped human epidermis models were were obtained from each of 3 patients with chronic plaque psoriasis cultured in inserts at the air–liquid interface as described in before and at defined time points after initiation of UV therapy Wolk et al. (2009a) and stimulated by either supplementing the (psoralen þ UVA for two patients and narrowband UVB irradiation culture medium or not supplementing (control) with 20 ng ml 1 for the third patient) or etanercept therapy as indicated, whereas the IL-22, 10 ng ml 1 IL-17A, 10 ng ml 1 IFN-g, 2 ng ml 1 TNF-a, same donor lesion per patient was used. To assess IL-19 protein levels 100 ng ml 1 IL-19, or combinations thereof as indicated (all from in human skin, biopsies were taken from 5 patients with chronic R&D Systems). After 72 hours, biopsies were taken, and the culture plaque psoriasis and from four healthy volunteers. medium was recovered. To quantify IL-19 protein in human skin, skin For assessment of systemic cytokine levels, serum and plasma were biopsies were cultured for 20 hours in keratinocyte medium to allow taken from venous blood obtained from 25 patients with chronic IL-19 diffusion into the culture medium. plaque psoriasis, from 13 (Figure 1c) and 26 (Figure 1e) healthy volunteers, from 17 patients with asthma, from 20 patients with Mice Crohn’s disease, and from 20 patients who had undergone renal Male mice were purchased from Charles River (Wilmington, MA) and transplantation (being treated by anti-IL-2R antibody as immunosup- were used at the ages of 96–110d. To study DNFB-induced cutaneous pressive induction therapy followed by a triple maintenance therapy inflammation, C57BL/6 mice were sensitized with 25 mlof0.5% with tacrolimus, mycophenolat mofetil, and prednisone). In an (v/v) DNFB in acetone/olive oil (80/20) (Sigma-Aldrich, Taufkirchen, additional approach to analyze therapy-related changes in blood IL- Germany) and repeatedly challenged on the skin of the ears with 19 levels, serum was obtained from venous blood of six psoriasis 25 ml of 0.3% (v/v) DNFB after 5d, 6d, and 7d. One hundred mgof patients before and 4 weeks after the first application of ustekinumab polyclonal anti-human IL-20R1 goat IgG or the respective isotype and from six patients before and 2–5 weeks after the start of UV-based control were given intraperitoneally 1 hours before the first challenge. combination therapy (two patients with psoralen þ UVA and 4 Ear thickness was measured before (0 hours) and 24 hours, 48 hours, patients with narrowband UVB irradiation; the UV-accompanying and 72 hours after the first challenge. Cytokine levels were analyzed therapy components included dithranol (all six patients), tar-contain- in ear homogenates 72 hours after the first challenge. To study ing ointments (all six patients), local corticosteroids (three patients), lipopolysaccharide-induced systemic inflammation, BALB/c mice calcipotriol (three patients), fumaric acid esters (three patients), and were intraperitoneally injected with 5 mg/g mouse lipopolysaccharide cyclosporin A (one patient). All skin and blood samples were from Escherichia coli 0111:B4 (Sigma-Aldrich). One hundred mgof approved by the clinical institutional review board of the Charite´ polyclonal anti-human IL-20R1 goat IgG or the respective isotype university hospital, Berlin, and written informed consent was control (both from R&D Systems) were given 16 hours before the obtained from all participants. The study was conducted according challenge, and blood plasma was collected 6 hours post lipopoly- to the Declaration of Helsinki Principles. saccharide challenge. All these studies have been approved by the regional authorities for provisions on labor, health, and technical Cell and tissue cultures safety in Berlin, Germany. Primary human epidermal keratinocytes, dermal fibroblasts, dermal microvascular endothelial cells, and melanocytes were obtained and qRT-PCR cultured as described previously (Wolk et al., 2009a). For assessment Tissue homogenization, isolation of total cellular RNA, reverse of IL-19 receptor expression, these cells were directly prepared for transcription of messenger RNA, and quantitative quantitative reverse transcriptase–PCR (qRT-PCR) analysis. To assess analysis by TaqMan real-time PCR either using the ABI Prism 7700 keratinocyte migration, confluent cultures of these cells were starved Sequence Detection System or the Stepone plus (both from Applied for 4 hours by replacing the culture medium with keratinocyte basal Biosystems, Darmstadt, Germany) from human samples were carried medium gold (Lonza, Verviers, Belgium) without supplements and the out as described previously using –exon spanning fluorescent cells were wounded by scratching the surface of the cell monolayers probes (Wolk et al., 2002, 2009b). The expression of human IL-10, IL- with a pipette tip (Sarstedt, Nu¨mbrecht, Germany), followed by two 19, IL-20, IL-22, IL-24, IL-26, IL-20R1, IL-20R2, BD2, and BD3 was consecutive washes. Thereafter, cells were stimulated with 40 ng ml 1 detected using of previously established systems (Wolk et al.,2002, IL-19 (R&D Systems, Wiesbaden-Nordenstadt, Germany) or 2004, 2011). All other detection systems were purchased from 10 ng ml 1 EGF (Peprotech, Hamburg, Germany) for 24 hours. Mon- Applied Biosystems. All expression levels were analyzed in itoring of scratch wound closure was performed at 0 and 24 hours after triplicate and in parallel with those of the housekeeping gene cytokine stimulation by means of AxioVision Release 4.6.3 image hypoxanthine phosphoribosyl-transferase 1 to allow calculation software (Zeiss, Jena, Germany). relative to hypoxanthine phosphoribosyl-transferase 1 (Wolk et al., To assess cellular proliferation, keratinocytes were either stimulated 2011). or not stimulated (control) with 40 ng ml 1 IL-19, 10 ng ml 1 IL-17A, or 10 ng ml 1 IFN-b (all from R&D Systems) for 5d in keratinocyte Histology and immunohistochemistry basal medium gold (Lonza) supplemented according to the manu- Frozen EpiDerm-201 tissues were processed as previously described facturer´s instructions, with the exception of EGF. (Wolk et al., 2009a, 2011). Sections were either stained with a mouse

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antibody to human S100A7 or the corresponding isotype control Capon F, Di Meglio P, Szaub J et al. (2007) Sequence variants in the genes for antibody or left unstained and were finally counterstained with the -23 receptor (IL23R) and its ligand (IL12B) confer protection against psoriasis. Hum Genet 122:201–6 Mayer’s hemalaun and covered as described in Wolk et al. (2011). Living cell layer thickness was measured by means of AxioVision Coccia M, Harrison OJ, Schiering C et al. (2012) IL-1beta mediates chronic intestinal inflammation by promoting the accumulation of IL-17A Release 4.6.3 image software (Zeiss). secreting innate lymphoid cells and CD4( þ ) Th17 cells. J Exp Med 209: 1595–609 ELISA Donnelly RP, Sheikh F, Dickensheets H et al. (2010) Interleukin-26: an Quantification of human IL-19, IL-20, IL-22, and CCL20 in IL-10-related cytokine produced by Th17 cells. Cytokine patient samples and/or culture medium was performed using the Rev 21:393–401 respective ELISA kits according to the manufacturer´s instructions Dumoutier L, Leemans C, Lejeune D et al. (2001) Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes (R&D Systems). of two types. J Immunol 167:3545–9 Gabunia K, Ellison SP, Singh H et al. (2012) Interleukin-19 (IL-19) induces Flow cytometry for cell counting heme oxygenase-1 (HO-1) expression and decreases reactive oxygen Cells were stained with propidium iodide staining solution (BD species in human vascular smooth muscle cells. JBiolChem287: Biosciences, Heidelberg, Germany), and live cell counts (propidium 2477–84 iodide-negative) were analyzed by means of a FACS Calibur instru- Gabunia K, Jain S, England RN et al. (2011) Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of ment and Cellquest software (BD Biosciences, Germany). A defined cytoskeletal regulators of VSMC motility. Am J Physiol Cell Physiol volume of CaliBRITE FITC-beads (BD Biosciences) added to kerati- 300:C896–906 nocyte suspensions served as the internal control. Gallagher G, Dickensheets H, Eskdale J et al. (2000) Cloning, expression and initial characterization of interleukin-19 (IL-19), a novel homologue of Statistical analyses human interleukin-10 (IL-10). Genes Immun 1:442–50 Samples of patients and healthy control participants were compared Griffiths CE, Barker JN (2007) Pathogenesis and clinical features of psoriasis. using the Mann–Whitney U-test (2-tailed) (*Pp0.05, **Po0.01, Lancet 370:263–71 ***Po0.001). Significance of differences between treatment groups He M, Liang P (2010) IL-24 transgenic mice: in vivo evidence of overlapping functions for IL-20, IL-22, and IL-24 in the epidermis. JImmunol of in vitro cultures and of patient samples before, during, and after 184:1793–8 therapy was tested by the Wilcoxon matched-pairs signed-rank test Hsing CH, Chiu CJ, Chang LY et al. (2008) IL-19 is involved in the pathogenesis (*Pp0.05, **Po0.01, ***Po0.001). For testing correlations between of endotoxic shock. Shock 29:7–15 parameters, Spearman’s rank correlation test was used (2-tailed) Huang F, Wachi S, Thai P et al. (2008) Potentiation of IL-19 expression in (*Pp0.05, **Po0.01, ***Po0.001). For all analyses, SPSS 19 soft- airway epithelia by IL-17A and IL-4/IL-13: important implications in ware (IBM, Ehningen, Germany) was used. asthma. J Allergy Clin Immunol 121:1415–21 Jain S, Gabunia K, Kelemen SE et al. (2011) The anti-inflammatory cytokine interleukin 19 is expressed by and angiogenic for human endothelial cells. CONFLICT OF INTEREST Arterioscler Thromb Vasc Biol 31:167–75 K. Asadullah is an employee and stockholder of Bayer AG. The remaining Kingo K, Mossner R, Koks S et al. (2007) Association analysis of IL19, IL20 and authors state no conflict of interest. IL24 genes in palmoplantar pustulosis. Br J Dermatol 156:646–52 Kingo K, Mossner R, Traks T et al. (2010) Further association analysis of ACKNOWLEDGMENTS chr 6q22-24 suggests a role of IL-20RA polymorphisms in psoriasis. The authors acknowledge Annette Buss, Brigitte Ketel, and Beate Pust for their J Dermatol Sci 57:71–3 steady excellent technical assistance. This study was supported by research Koks S, Kingo K, Vabrit K et al. (2005) Possible relations between the grant SA 1868/2–1 (to Robert Sabat) from the Deutsche Forschungsge- polymorphisms of the cytokines IL-19, IL-20 and IL-24 and plaque-type meinschaft (German Research Foundation). psoriasis. Genes Immun 6:407–15 Kumari S, Bonnet MC, Ulvmar MH et al. 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