Tnfa signaling through Tnfr2 protects skin against oxidative stress–induced inflammation

Tnfa Signaling Through Tnfr2 Protects Skin Against Oxidative Stress–Induced Inflammation Sergio Candel 1,2. , Sofı ´a de Oliveira 1,2,3. , Azucena Lo ´pez-Mun ˜oz 1,2 , Diana Garcı ´a-Moreno 1,2 , Raquel Espı ´n-Palazo ´n 1,2 , Sylwia D. Tyrkalska 1,2,4 ,Marı ´a L. Cayuela 2,5 , Stephen A. Renshaw 6 , Rau ´l Corbala ´n-Ve ´lez 7 , Inmaculada Vidal-Abarca 8 , Huai-Jen Tsai 9 , Jose ´Meseguer 1,2 ,Marı ´a P. Sepulcre 1,2 , Victoriano Mulero 1,2 * 1Departamento de Biologı ´a Celular e Histologı ´a, Facultad de Biologı ´a, Universidad de Murcia, Murcia, Spain, 2Instituto Murciano de Investigacio ´n Biosanitaria (IMIB), Murcia, Spain, 3Carlota Saldanha Lab, Instituto de Medicina Molecular, Instituto de Bioquı ´mica, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal, 4Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientı ´ficas (CSIC), Vigo, Spain, 5Grupo de Telo ´meros, Envejecimiento y Ca ´ncer, Unidad de Investigacio ´n, Departamento de Cirugı ´a, CIBERehd. Hospital Universitario ‘‘Virgen de la Arrixaca,’’ Murcia, Spain, 6MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom, 7Servicio de Dermatologı ´a, Hospital Universitario ‘‘Virgen de la Arrixaca,’’ Murcia, Spain, 8Servicio de Anatomı ´a Patolo ´gica, Hospital Universitario ‘‘Virgen de la Arrixaca,’’ Murcia, Spain, 9Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan Abstract TNFaoverexpression has been associated with several chronic inflammatory diseases, including psoriasis, lichen planus, rheumatoid arthritis, and inflammatory bowel disease. Paradoxically, numerous studies have reported new-onset psoriasis and lichen planus following TNFaantagonist therapy. Here, we show that genetic inhibition of Tnfa and Tnfr2 in zebrafish results in the mobilization of neutrophils to the skin. Using combinations of fluorescent reporter transgenes, fluorescence microscopy, and flow cytometry, we identified the local production of dual oxidase 1 (Duox1)-derived H 2 O 2 by Tnfaand Tnfr2-deficient keratinocytes as a trigger for the activation of the master inflammation transcription factor NF-kB, which then promotes the induction of genes encoding pro-inflammatory molecules. In addition, pharmacological inhibition of Duox1 completely abrogated skin inflammation, placing Duox1-derived H 2 O 2 upstream of this positive feedback inflammatory loop. Strikingly, DUOX1 was drastically induced in the skin lesions of psoriasis and lichen planus patients. These results reveal a crucial role for TNFa/TNFR2 axis in the protection of the skin against DUOX1-mediated oxidative stress and could establish new therapeutic targets for skin inflammatory disorders. Citation: Candel S, de Oliveira S, Lo ´pez-Mun ˜oz A, Garcı ´a-Moreno D, Espı ´n-Palazo ´n R, et al. (2014) Tnfa Signaling Through Tnfr2 Protects Skin Against Oxidative Stress–Induced Inflammation. PLoS Biol 12(5): e1001855. doi:10.1371/journal.pbio.1001855 Academic Editor: Douglas R. Green, St. Jude Children’s Research Hospital, United States of America Received November 8, 2013; Accepted March 28, 2014; Published May 6, 2014 Copyright: ß2014 Candel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Spanish Ministry of Science and Innovation (grants BIO2011-23400 and CSD2007-00002 to VM, and PhD fellowship to SC, all co-funded with Fondos Europeos de Desarrollo Regional/European Regional Development Funds), the Fundacio ´nSe ´neca-Murcia (grant 04538/GERM/06 to VM and PhD fellowship to RE-P), Fundac¸a ˜o para a Cie ˆncia e Tecnologia (PhD fellowship to SdO, SFRH/BD/62674/2009), a Medical Research Council Senior Clinical fellowship to SAR (G0701932), and the European 7th Framework Initial Training Network FishForPharma (PhD fellowship to SDT, PITG-GA-2011-289209). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] .These authors contributed equally to this work. Abbreviations: CHT, caudal hematopoietic tissue; DN, dominant negative; Duox, dual oxidase; HIF, hypoxia-inducible factor; H&E, haematoxylin and eosin; IBD, inflammatory bowel disease; Il1b, interleukin-1b; IP, incontinentia pigmenti; MO, morpholino; PFA, paraformaldehyde; Ptgs2, prostaglandin-endoperoxide synthase 2; ROS, reactive oxygen species; Tnf, tumor necrosis factor, Tnfr, Tnf receptor. Introduction Tumor necrosis factor a(TNFa) is a multifunctional cytokine that mediates key roles in acute and chronic inflammation, antitumor responses, and infection. TNFabinds TNF receptor 1 (TNFR1, also known as TNFRSF1A or P55) and TNFR2 (also known as TNFRSF1B or P75) for stimulation of two opposing signaling events [1]. In general, TNFR1 signaling results in the trigger of a cascade that can result in apoptosis [2]. This is dependent upon the cell type, the state of activation of the cell, and the cell cycle. In contrast, a TNFR2 signal induces cell survival pathways that can result in cell proliferation [2]. Enhanced TNFasynthesis is associated with the development of autoimmune/chronic inflammatory diseases, including psoriasis, lichen planus, rheumatoid arthritis, and inflammatory bowel disease (IBD). The inhibition of TNFaactivities in these diseases has been remarkably successful [3,4]. Paradoxically, however, numerous studies have reported new-onset psoriasis and lichen planus, or worsening of existing psoriasis, following TNFa antagonist therapy in adult patients [5–10]. Despite these clinical data pointing to an ambiguous function of TNFain psoriasis and lichen planus, the role of TNFa, and in particular the contribution of each TNFR, in the regulation of skin inflammation has been scarcely studied. An earlier study using gene-targeted mutant mice lacking either TNFR1 or TNFR2 showed that skin inflammation induced indirectly by irritant chemicals or directly by intradermal administration of TNFawas greatly attenuated in TNFR1deficient mice, whereas TNFR2-deficient siblings responded normally [11]. In addition, mice with an arrested canonical NF-kB activation pathway in the keratinocytes develop a severe PLOS Biology | www.plosbiology.org 1 May 2014 | Volume 12 | Issue 5 | e1001855 inflammatory skin disease shortly after birth, which is caused by TNFaand macrophage-mediated, but T-cell–independent, mechanisms [12–16]. The characteristics of this complex disorder are strikingly similar to those associated with the human X-linked genodermatosis incontinentia pigmenti (IP) [17]. To the best of our knowledge, however, the role played by TNFain the homeostasis of healthy skin has never been studied. TNFaand TNFRs are conserved in all vertebrates. Recent studies have shown that in the zebrafish (Danio rerio) Tnfa functions as a pro-inflammatory cytokine [18] and Tnfr signaling plays an important role in the homeostasis of endothelial cells [19]. In the present study, we have taken advantage of the strengths of the zebrafish embryo model to study the impact of Tnfa, Tnfr1, and Tnfr2 deficiencies in a whole vertebrate organism. We found that Tnfa and Tnfr2 are both crucial, whereas Tnfr1 is dispensable, for the homeostasis of the skin. Genetic inhibition of Tnfa and Tnfr2 promotes H 2 O 2 -mediated skin infiltration by neutrophils, increased keratinocyte proliferation, and the local activation of the master inflammation transcription factor NF-kB, which then promotes the induction of genes encoding pro-inflammatory molecules. In addition, DUOX1 was strongly induced in keratinocytes of human psoriasis and lichen planus patients. Results Tnfa or Tnfr2 Deficiency Results in Neutrophil Mobilization to the Skin In wild-type larvae, most neutrophils (approximately 90%) were located in the caudal hematopoietic tissue (CHT) [20] by 3 d postfertilization (dpf) (Figure 1A–C), which is consistent with neutrophil localization patterns described previously [21,22]. However, in Tnfaand Tnfr2-deficient larvae, approximately 40% of neutrophils were located outside the CHT (Figure 1A–C). In addition, Tnfr1-deficient animals showed a normal neutrophil distribution, whereas their double deficient siblings for both Tnfr1 and Tnfr2 showed also a distribution pattern more similar to single Tnfr2-deficient fish (Figure 1A–C). The specificity of this phenotype was confirmed with a dominant negative (DN) Tnfr2, which is lacking the entire intracellular signaling domain, but is identical to full-length Tnfr2 in its transmembrane and extracellular domains, and therefore, its trimerization with endogenous Tnfr2 extinguishes Tnfr2 signaling [19]. The results showed that the altered neutrophil distribution of Tnfr2 morphants was phenocopied by overexpression of DN-Tnfr2 (Figure S1A). In addition, the scattered distribution of Tnfaand Tnfr2-deficient larvae was partially rescued by overexpression of wild-type Tnfa and Tnfr2 RNAs, respectively (Figure S1B). These results prompted us to examine the distribution of macrophages in TNFaand TNFR2-deficient fish, and surprisingly, macrophage distribution was apparently normal in all cases (Figure S2). To ascertain the precise localization of neutrophils in Tnfa/ Tnfr2-deficient larvae, we knocked down Tnfr2 in transgenic mpx:eGFP animals followed by whole mount immunohistochemistry (WIHC) against p63 (basal keratinocyte marker) to visualize neutrophils (GFP + ) and skin keratinocytes (p63 + ) at the same time in whole larvae. The results revealed that although neutrophils from wild-type animals were mainly located in the CHT, a high proportion of neutrophils were seen in close contact with keratinocytes in Tnfr2-deficient animals (Figure 1D and Videos S1 and S2). Collectively, these results indicate that deficiency of either Tnfa or Tnfr2 specifically promotes neutrophil infiltration into the skin of zebrafish during early development. Tnfa or Tnfr2 Deficiency Triggers the Induction of Genes Encoding Pro-Inflammatory Mediators in Keratinocytes The phenotype of Tnfaand Tnfr2-deficient fish is reminiscent of that of spint1a and clint1 mutant fish, which show chronic skin inflammation characterized by increased interleukin-1b(IL-1b) production and neutrophil infiltration [23–25]. This led us to examine the expression of three genes encoding major proinflammatory molecules, namely Tnfa itself, IL-1b, and prostaglandin-endoperoxide synthase 2b (PTGS2b, also known as COX2b), in whole wild-type and Tnfr2-deficient larvae at 3 dpf as well as in sorted mpx:eGFP + cells—that is, neutrophils. It was found that Tnfr2 deficiency triggered the expression of tnfa,il1b, and ptgs2b genes (Figure 2A). Although neutrophils highly expressed the genes encoding Tnfa and Il1b as well as both Tnfrs, they did not mediate the induction of il1b observed in Tnfr2-deficient fish (Figures 2B, S3A, and S4A). Nevertheless, the transcript levels of tnfa were higher in neutrophils from Tnfr2deficient fish than in neutrophils from their wild-type siblings (Figure 2B), but this might reflect a positive feedback loop in response to Tnfr2 deficiency [19]. In addition, Tnfr2-deficient embryos showed higher transcript levels of il1b at 24 hpf (Figure S5), soon after the development of the first neutrophils in the zebrafish embryo [21,22,26] and before hatching. We then sorted krt18 + cells from Tnfaand Tnfr2-deficient animals at 3 dpf and found that they show much higher transcript levels of il1b and ptgs2b than krt18 + cells from wild-type animals (Figures 2C and S3B). Notably, krt18 + cells expressed both Tnf receptors (Figure S4B) and the specific marker of basal keratinocytes p63 (Figure S3B). We next wondered whether knockdown of Il1b using a specific morpholino (MO) [27] might rescue the neutrophil dispersion of Tnfr2-deficient animals. As shown in Figure 2D, genetic inhibition of Il1b failed to rescue the neutrophil dispersion observed in Tnfr2 morphants. These results taken together indicate that the Tnfa/ Tnfr2 axis is required for skin homeostasis in zebrafish and that the deficiency of either ligand or receptor triggers an inflammatory response characterized by the induction of pro-inflammatory mediators and neutrophil infiltration. Author Summary Psoriasis and lichen planus are chronic, debilitating skin diseases that affect millions of people worldwide. TNFais a multifunctional cytokine that mediates acute and chronic inflammation. While TNFaantagonist therapy is used for autoimmune or chronic inflammatory diseases, such as inflammatory bowel disease (IBD), numerous studies have reported new-onset psoriasis and lichen planus following such therapy. We have used the unique advantages of the zebrafish embryo to identify a novel phenotype that mirrors this unexplained and paradoxical onset of psoriasis and lichen planus. We found that depletion of Tnfa or its receptor Tnfr2 caused skin inflammation and hyperproliferation of keratinocytes through the activation of a Duox1/H 2 O 2 /NF-kB positive feedback inflammatory loop. Strikingly, DUOX1 was drastically induced in the skin lesions of psoriasis and lichen planus patients, and pharmacological inhibition of Duox1 abrogated skin inflammation, placing Duox1-derived H 2 O 2 upstream of this inflammatory loop. Our results suggest that therapies targeting DUOX1 and H 2 O 2 could provide innovative approaches to the management of skin inflammatory disorders. Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 2 May 2014 | Volume 12 | Issue 5 | e1001855 Tnfa and Tnfr2 Deficiencies Induce NF-kB Activation in the Skin The master regulator of inflammation NF-kB plays an essential role in the homeostasis of skin. Thus, genetic inhibition of the NFkB pathway in keratinocytes triggers a severe inflammatory skin disease in newborn mice, which is completely rescued by Tnfa and Tnfr1 depletion [12–16]. We therefore use a NF-kB reporter line [28] to visualize the dynamics of NF-kB in whole Tnfr2-deficient larvae. Injection of bacterial DNA, which activated TLR9, resulted in a drastic activation of NF-kB in the whole larvae Figure 1. TNFaand Tnfr2 deficiencies result in neutrophil mobilization to the skin. Zebrafish one-cell mpx:eGFP and/or krt18:RFP embryos were injected with standard control (Std), Tnfr1, Tnfr2, Tnfa, or Tnfr1+Tnfr2 MOs. (A) Representative images, bright field and green channels, of the morphants at 3 dpf showing the differences in the neutrophils distribution. (B) Fluorescence intensity was measured for all the groups in the area indicated (A), which includes the CHT, where most neutrophils are located in wild-type larvae at 3 dpf. The images were converted to a fluorescence value matrix where the value obtained for each pixel transversally was the mean 6S.E.M. for all the pixels for each row (15 larvae per treatment from 3 different experiments). The area corresponding to the CHT has been labeled and highlighted. The notochord (nt) location has been indicated to facilitate the larval orientation. auf, arbitrary units of fluorescence. (C) The neutrophil mobilization from the CHT in Tnfaand Tnfr2-deficient larvae was quantified as the percentage of neutrophils outside the CHT in 20 larvae per group from 3 different experiments. The mean 6S.E.M. for each group is shown. (D) Representative frontal (xy) and lateral (yz) views of tridimensional reconstructions from confocal microscopy images of WIHC of mpx:eGFP larvae stained at 3 dpf with anti-p63 antibodies (basal keratinocyte marker, red) showing the neutrophils’ distribution in the CHT area of control and Tnfr2-deficient larvae. Note that most neutrophils (eGFP, green) are located in the CHT in control larvae (white arrowheads), while many of them infiltrate the skin (blue arrowheads) of Tnfr2-deficient larvae, whereas they are mainly located in the CHT in their wild-type siblings. Scale bars, 100 mm. ns, not significant. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pbio.1001855.g001 Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 3 May 2014 | Volume 12 | Issue 5 | e1001855 (Figure 3A–B), as expected from previous results [29,30]. Interestingly, Tnfr2 deficiency promoted a restricted activation of NF-kB in the skin (Figure 3A–E and Videos S3 and S4). Furthermore, although skin integrity was unaffected up to 5 dpf in Tnfr2-deficient larvae, as assayed by histology (Figure S6), they showed increased keratinocyte proliferation, as assayed in double transgenic NF-kB:eGFP; krt18:RFP animals and double WIHC with anti-RFP and anti-phosphorylated H3 (Figure 4). Tnfa and Tnfr2 Deficiencies Trigger H 2 O 2 Production in the Skin Hydrogen peroxide gradients were recently shown to contribute to the early influx of neutrophils in wound [31] and tumor [32]. Interestingly, however, H 2 O 2 is not required for neutrophil detection of localized infection [33]. These gradients are created by the dual oxidase 1 (Duox1) [31] and sensed by neutrophils through the tyrosine kinase Lyn [34]. Although identified and best studied in the zebrafish, H 2 O 2 is likely to play the same function in human neutrophils [34]. We first analyzed the expression of the gene encoding Duox1 and found that Tnfr2-deficient keratinocytes showed higher transcript levels of duox1 than wild-type animals (Figure 5A). Next, using an H 2 O 2 -detecting fluorescence probe, we observed that Tnfr2-deficient larvae also produced H 2 O 2 in the skin (Figure 5B,C). We observed similar levels of labeling with the H 2 O 2 probe in Tnfr2-deficient keratinocytes and in local keratinocytes after wounding (Figure S7). Notably, H 2 O 2 production by Tnfr2-deficient keratinocytes preceded the activation of NF-kB (Figure S8). Consistent with these observations, genetic inhibition of Duox1 with a specific MO [31] was able to partially prevent the infiltration of neutrophils into the skin of Tnfr2-deficient larvae (Figure 5D,E). To further confirm this result, we designed a DN form of Duox1 [35], and notably, overexpression of DN-Duox1 was also able to partially prevent neutrophil infiltration in Tnfr2-deficient larvae (Figure S9A,B). Furthermore, we knocked down the H 2 O 2 sensor of neutrophils, Lyn [34], and found full prevention of neutrophil infiltration in both Tnfr2and Tnfa-deficient animals (Figure 5F,G). Pharmacological Inhibition of Duox1 Restores Skin Homeostasis in Tnfaand Tnfr2-Deficient Animals The above results prompted us to evaluate whether pharmacological inhibition of Duox1 using the NADPH oxidase inhibitor dibenziodolium chloride (DPI), which has been shown to inhibit Duox1 and H 2 O 2 gradient formation in zebrafish [31,34], may Figure 2. TNFa and Tnfr2 deficiencies trigger skin inflammation. Zebrafish one-cell mpx:eGFP or krt18:RFP embryos were injected with standard control (Std), Tnfr2, Il1b, and/or Tnfa MOs. The expression of tnfa,il1b, and ptgs2b genes was measured by RT-qPCR in whole body (A), FACSsorted neutrophils (B), and FACS-sorted keratinocytes (C) from Std and Tnfr2 morphants at 3 dpf. (D) The phenotype of 3 dpf morphant larvae was classified as neutrophils grouped in the CHT or scattered, as described in Figure 1. Note that IL-1bknockdown failed to rescue the neutrophil mobilization in Tnfr2-deficient larvae. ns, not significant. *p,0.05; **p,0.01; ***p,0.001. doi:10.1371/journal.pbio.1001855.g002 Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 4 May 2014 | Volume 12 | Issue 5 | e1001855 restore skin homeostasis in Tnfaand Tnfr2-deficient larvae. The results showed that DPI treatment completely inhibited the generation of H 2 O 2 in the skin (Figure 5B,C), the infiltration of neutrophils (Figure 6A–C) into this tissue, and more importantly, skin NF-kB activation (Figure 6D–F) in both Tnfaand Tnfr2deficient animals. Collectively, these results demonstrate that the Tnfa/Tnfr2 axis is indispensable for skin homeostasis and its inhibition results in the release of Duox1-derived H 2 O 2 , local activation of NF-kB, induction of genes encoding Duox1 and proinflammatory mediators, and neutrophil infiltration. DUOX1 Is Induced in Human Psoriasis and Lichen Planus Lesions The crucial role of Duox1-generated H 2 O 2 in the infiltration of neutrophils into the skin and the induction of NF-kB prompted us to investigate if this inflammatory signal may also play a role in human psoriasis and lichen planus. We analyzed by immunohistochemistry 10 healthy skins and 8 lichen planus and 15 psoriasis lesions using an antibody to human DUOX1 (Figure 7). The results showed that although DUOX1 was expressed at low levels in healthy epidermis, mainly in the granular layer, a drastic Figure 3. Tnfa and Tnfr2 deficiencies result in skin NF-kB activation. Zebrafish one-cell NF-kB:eGFP (A, B, D, E) or NF-kB:eGFP;krt18:RFP (C) embryos were injected with standard control (Std) or Tnfr2 MOs alone or in the presence of 2.3 ng/egg of V. anguillarum genomic DNA (VaDNA), as a positive control for NF-kB activation. (A) Representative pictures showing the induction of NF-kB activation in the skin (red arrowheads) of Tnfr2deficient larvae at 72 hpf and the ubiquitous, strong induction in their VaDNA-injected siblings. Note the strong expression of NF-kB in neuromasts of control larvae (white arrowheads). (B) The mean GFP fluorescence was quantified in whole larvae, and no significant differences between Tnfr2morphants and control larvae were observed. Each dot represents the mean GFP fluorescence per single larva. The mean 6S.E.M. of the whole GFP fluorescence for each group of larvae is also shown. (C) Representative frontal (xy) and lateral (yz) views of tridimensional reconstructions from confocal microscopy images of WIHC of NF-kB:eGFP;krt18:RFP larvae stained at 3 dpf with anti-RFP antibodies (keratinocytes, blue) showing the induction of NF-kB in the skin (eGFP, green) of Tnfr2-deficient larvae. (D, E) Quantification of NF-kB activation in the skin of Tnfr2-deficient larvae at 72 hpf. (D) Fluorescence intensity was measured in the area indicated of wild-type and Tnfr2-deficient larvae, as explained in the legend to Figure 1 (15 larvae per treatment from 3 different experiments). The skin and the neuromasts have been labeled to facilitate the larval orientation. Note the activation of NF-kB in the skin of Tnfr2-deficient larvae. (E) The skin NF-kB activation index was defined as the fluorescence in the skin (a+b) relative to the total fluorescence of the whole larvae (c). Each dot represents the skin NF-kB activation index per single larva. The mean 6S.E.M. of the skin NFkB activation index for each group of larvae is also shown. Scale bars, 100 mm. ns, not significant; auf, arbitrary units of fluorescence. *p,0.05; **p,0.01; ***p,0.001. doi:10.1371/journal.pbio.1001855.g003 Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 5 May 2014 | Volume 12 | Issue 5 | e1001855 induction of this enzyme was obvious in the keratinocytes of the spinous layer of the epidermis from both psoriasis and lichen planus lesions. In some patients, the induction was obvious in all keratinocytes of the spinous layer, whereas in others it was observed only in the upper layers of this stratum. It was noticeable the localization of DUOX1 in the plasma membrane of psoriasis and lichen planus keratinocytes and also in their cytoplasm, where it was accumulated in the upper side of these cells—that is, facing the cornified layer. Although this particular distribution deserves further investigation, these results strongly suggest a role for DUOX1 in psoriasis and lichen planus. Discussion Increased production of TNFais associated with the development of autoimmune/chronic inflammatory diseases, including psoriasis, lichen planus, rheumatoid arthritis, and IBD. We have used the unique advantages of the zebrafish embryo for in vivo imaging and cell tracking to demonstrate that the genetic depletion of Tnfa or Tnfr2, but not Tnfr1, caused the infiltration of neutrophils into the skin and hyperproliferation of keratinocytes through the activation of an H 2 O 2 /NF-kB/Duox1 positive feedback inflammatory loop (Figure 8). Strikingly, neutrophils, but not macrophages, are rapidly attracted to the skin. However, the activation of NF-kB and the induction of the gene encoding Il1b in the skin occurred before the appearance of the first neutrophils in the developing embryo. More importantly, DUOX1 was also strongly induced in the skin lesions of psoriasis and lichen planus patients. Collectively, these results (i) indicate a critical role of TNFa/TNFR2 signaling in the protection of the skin against oxidative stress, (ii) might explain the appearance of psoriasis and lichen planus in patients treated with anti-TNFa therapies [5–10], and (iii) support the idea that specific inhibition of the TNFa/TNFR1 signaling axis while leaving TNFa/TNFR2 signaling unaffected would inhibit the pathological effects of TNFa and reduce the side effects associated with this therapy [19,36]. This apparent discrepancy with TNFa-deficient mice, which do not show skin inflammation, may be due to developmental and/or physiological compensations, which probably do not exist in humans [37–39]. One of the most intriguing observations from this study is that impaired Tnfr2 signaling led to the induction of duox1 and the production of H 2 O 2 by keratinocytes. H 2 O 2 gradient was recently shown to contribute to the early influx of neutrophils in wound [31] and tumor [32], although it seems to be dispensable for neutrophil detection of localized infection [33]. To the best of our knowledge, this is the first study showing a role for Duox1-derived H 2 O 2 in the induction of NF-kB in the skin in vivo, suggesting that H 2 O 2 might play a critical role in the initiation and maintenance of chronic inflammatory diseases in both zebrafish and human. These observations suggest that antioxidants or inhibition of Duox1 might be therapeutic for the treatment of patients suffering from psoriasis, lichen planus, and other inflammatory diseases. Supporting this notion, several studies using psoriasis and IBD mouse models have shown that transgenic overexpression of endogenous antioxidant genes promotes protection, while antioxidant gene knockout promotes sensitization (reviewed by [40,41]). Even more importantly, the antioxidant levels and the oxidative stress biomarkers are usually correlated with the disease severity and the extent of inflammation in the psoriasis and IBD patients [40–42]. Therefore, all these results taken together suggest that antioxidants should be considered as part of a more specific and effective therapy for the treatment of inflammatory skin diseases, including psoriasis and lichen planus. The ability of Duox1 Figure 4. Tnfr2 deficiency results in increased proliferation of skin keratinocytes. Zebrafish one-cell krt18:RFP embryos were injected with standard control (Std) or Tnfr2 MOs. (A) Representative frontal (xy) and lateral (yz) tridimensional reconstructions from confocal microscopy images of WIHC of krt18:RFP larvae stained at 3 dpf with anti-RFP (keratinocytes, blue) and anti-phosphorylated H3 (pH 3, proliferation marker) antibodies. (B) Quantification of the number of pH3/RFP + (i.e., proliferating keratinocytes) cells in the CHT area. Each dot represents one single larva, and the mean 6S.E.M. for each group of larvae is also shown. Scale bars, 100 mm. *p,0.05. doi:10.1371/journal.pbio.1001855.g004 Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 6 May 2014 | Volume 12 | Issue 5 | e1001855 inhibition by pharmacological approaches, but not of IL-1b,to restore skin homeostasis in Tnfaand Tnfr2-deficient zebrafish embryos further supports this conclusion. It is known that different reactive oxygen species (ROS) act as second messengers, influencing various cellular signal transduction pathways, including NF-kB. However, there are still many inconsistencies concerning the influence of oxidative stress on NF-kB activity [43], and unfortunately, most studies have been performed in vitro using H 2 O 2 and cultured cells [44,45]. Such studies have shown that H 2 O 2 can act as an activator of IkB kinases (IKKs) [46] or can inactivate these proteins [47], probably depending on the cell type. More recently, it has been found that the same prolyl hydroxylases that confer oxygen sensitivity to the hypoxia-inducible factor (HIF) pathway, namely PHD1 and PHD2, seem to act as repressors of the canonical NF-kB pathway through mechanisms that could include direct hydroxylation of IKKb[48]. Our epistasis study in zebrafish demonstrates for the first time that the absence of Tnfa/Tnfr2 signaling led to the production of H 2 O 2 by keratinocytes, which, in turn, resulted in NF-kB activation and the induction of genes encoding proinflammatory mediators. This self-perpetuating cycle may be of clinical importance in view of the presumably key role played by oxidative stress [40–42], HIF [49,50], and NF-kB in psoriasis and IBD. It is tempting to speculate that the Tnfa/Tnfr2 axis would be required to prevent skin oxidative stress through the regulation of ROS-detoxifying enzymes, as it has been reported for oligodendrocyte progenitor cells in vitro [51]. The model reported here might contribute to clarify the mechanisms involved in the regulation of oxidative stress by TNFa, the regulation of NF-kB activity by ROS, and the crosstalk between oxidative stress and inflammation in vivo. The essential role played by NF-kB in the homeostasis of the skin is evidenced by the human X-linked genodermatosis IP, which affects the regulatory subunit of IKK (IKKc, NEMO) [17]. Humans suffering from this genetic disease exhibit severe skin inflammation, paradoxically due to impaired NF-kB activation Figure 5. Tnfa and Tnfr2 deficiencies result in the Duox1-derived H 2 O 2 production by keratinocytes. Zebrafish one-cell krt18:RFP (A), wild-type (B, C), or mpx:eGFP (D–G) embryos were injected with standard control (Std), Tnfr2, Tnfa, Duox1/p53, and/or Lyn MOs. (A) The expression of the duox1 gene was measured by RT-qPCR in FACS-sorted keratinocytes from 72 hpf wild-type and Tnfr2-deficient larvae. (B, C) Wild-type and Tnfr2deficient larvae were dechorionated at 24 hpf and treated by immersion in 100 mM DPI or vehicle alone (DMSO) for 24 h and then labeled with 50 mM acetyl-pentafluorobenzene sulphonyl fluorescein. Representative images of green channels of Std and Tnfr2 morphants are shown. Note that single keratinocytes are labeled with the H 2 O 2 probe in Tnfr2-deficient larvae (inset). (D–G) Rescues with Duox1 (D, E) and Lyn (F, G) MOs at 72 hpf. The differences in the neutrophil distribution (D, F) and quantification of neutrophil mobilization from the CHT to the skin in the indicated number of larvae per group from three different experiments (E, G) are shown. The mean 6S.E.M. for each group is shown. Scale bars, 100 mm. ns, not significant. ***p,0.001. doi:10.1371/journal.pbio.1001855.g005 Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 7 May 2014 | Volume 12 | Issue 5 | e1001855 and reduced resistance to TNFa/TNFR1-mediated apoptosis [52,53]. Similarly, although NF-kB actively participates in the excessive inflammatory response observed in IBD patients [54,55], recent studies with mice defective in NF-kB activation have revealed that epithelial NF-kB activation is essential to preserve intestinal homeostasis [56,57]. Therefore, a critical NF-kB signaling balance is required for skin and gut homeostasis, as both excessive and defective epithelial NF-kB activation can result in inflammation. Similarly, although the TNFa/TNFR1 axis was earlier appreciated to be involved in the apoptosis of both keratinocytes and enterocytes in the absence of NF-kB signaling [52,53,56,57], our results show that TNFasignaling through TNFR2 is also critically required for skin homeostasis. Whether the TNFa/TNFR2 axis is also required for gut homeostasis will require further investigation using germ-free and gnotobiotic zebrafish larvae, as host–microbe interactions have a profound impact in gut physiology and are usually involved in IBD. In conclusion, we have found that Tnfa signaling through Tnfr2 is indispensably required for the protection of the skin against oxidative stress-induced inflammation in the zebrafish. Thus, the absence of this signal triggers the local production of H 2 O 2 by Duox1, which, in turn, activates NF-kB and results in the upregulation of genes encoding pro-inflammatory mediators and neutrophil infiltration. These results, together with the induction of DUOX1 in the skin lesions of psoriasis and lichen planus patients, reveal a crucial role of H 2 O 2 and DUOX1 in skin inflammation and suggest that pharmacologic and genetic therapies that target these two key factors could provide innovative approaches to the management of psoriasis, lichen planus, and other chronic inflammatory diseases. Materials and Methods Ethics Statement The experiments performed comply with the Guidelines of the European Union Council (86/609/EU). Experiments and procedures were performed as approved by the Bioethical Committee of the University of Murcia (approval no. 537/2011) and the Ethical Clinical Research Committee of the University Hospital Virgen de la Arrixaca (approval no. 8/13). Animals Zebrafish (Danio rerio H.) were obtained from the Zebrafish International Resource Center and mated, staged, raised, and processed as described [58]. The lines Tg(mpx:eGFP) i114 [59], Tg(lyz:dsRED) nz50 [60], Tg(mpeg1:eGFP) gl22 [61], and Tg(krt18:RFP) Figure 6. Pharmacological inhibition of Duox1 prevents skin inflammation in Tnfaand Tnfr2-deficient zebrafish. Zebrafish one-cell mpx:eGFP (B, C) and NF-kB:eGFP (D–F) embryos were injected with standard control (Std), Tnfr2, or Tnfa MOs. (A) Scheme showing the experimental procedure: embryos were dechorionated at 24 hpf and treated by immersion in 100 mM DPI or vehicle alone (DMSO) for 24 h. (B, C) Representative images of bright field and green channels of the morphants at 48 hpf showing the differences in the neutrophils distribution (B) and quantification of neutrophil mobilization from the CHT to the skin in the indicated number of larvae per group from three different experiments (C). (D–F) Quantification of NF-kB activation in the skin of Tnfr2and Tnfa-deficient larvae at 72 hpf. (E) Fluorescence intensity was measured for all the groups in the area indicated, as explained in the legend to Figure 1 (15 larvae per treatment from 3 different experiments). The skin and the notochord (nt) have been labeled to facilitate the larval orientation. Note the activation of NF-kB in the skin (red arrowheads) of Tnfr2-deficient larvae. Note the strong expression of NF-kB in neuromasts (white arrowheads). (F) Skin NF-kB activation index was defined as the fluorescence in the skin (a+b) relative to the total fluorescence of the whole larvae (c). Each dot represents the skin NF-kB activation index per single larva. The mean 6S.E.M. of the skin NF-kB activation index for each group of larvae is also shown. Scale bars, 100 mm. ns, not significant. ***p,0.001. doi:10.1371/journal.pbio.1001855.g006 Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 8 May 2014 | Volume 12 | Issue 5 | e1001855 Figure 7. DUOX1 is induced in human psoriasis and lichen planus lesions. Representative images of sections from two healthy, two psoriatic, and two lichen planus skin biopsies that have been immunostained with an anti-DUOX1 goat polyclonal antibody and then slightly counterstained with hematoxilin. Note that DUOX1 is weakly expressed in healthy epidermis, mainly in the granular layer (GL), whereas it is strongly expressed (red arrowheads) in the spinous layer (SL) of both psoriasis and lichen planus lesions. CL, cornified layer; D, dermis. Scale bars, 100 mm (left panel) and 30 mm (right panel). doi:10.1371/journal.pbio.1001855.g007 Tnfa/Tnfr2 Axis Regulates Skin Homeostasis PLOS Biology | www.plosbiology.org 9 May 2014 | Volume 12 | Issue 5 | e1001855