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      Tnfa Signaling Through Tnfr2 Protects Skin Against Oxidative Stress–Induced Inflammation


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          A new zebrafish model of skin inflammatory disease explains new-onset and worsening psoriasis and lichen planus in patients receiving anti-TNFα therapy.


          TNFα overexpression 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 TNFα antagonist 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 2O 2 by Tnfa- and Tnfr2-deficient keratinocytes as a trigger for the activation of the master inflammation transcription factor NF-κB, 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 2O 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 TNFα/TNFR2 axis in the protection of the skin against DUOX1-mediated oxidative stress and could establish new therapeutic targets for skin inflammatory disorders.

          Author Summary

          Psoriasis and lichen planus are chronic, debilitating skin diseases that affect millions of people worldwide. TNFα is a multifunctional cytokine that mediates acute and chronic inflammation. While TNFα antagonist 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 2O 2/NF-κB 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 2O 2 upstream of this inflammatory loop. Our results suggest that therapies targeting DUOX1 and H 2O 2 could provide innovative approaches to the management of skin inflammatory disorders.

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          Most cited references 55

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          A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish

          Barrier structures (e.g. epithelia around tissues, plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometers within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Due to their fast diffusion and versatile biological activities, reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are interesting candidates for wound-to-leukocyte signalling. We probed the role of H2O2 during the early events of wound responses in zebrafish larvae expressing a genetically encoded H2O2 sensor1. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting ∼3 min after wounding and peaking at ∼20 min, which extended ∼100−200 μm into the tail fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by Dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role.
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            mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.

            Macrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology.
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              Prolyl hydroxylase-1 negatively regulates IkappaB kinase-beta, giving insight into hypoxia-induced NFkappaB activity.

              Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFkappaB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFkappaB through a pathway involving activation of IkappaB kinase-beta (IKKbeta) leading to phosphorylation-dependent degradation of IkappaBalpha and liberation of NFkappaB. Furthermore, through increasing the pool and/or activation potential of IKKbeta, hypoxia amplifies cellular sensitivity to stimulation with TNFalpha. Within its activation loop, IKKbeta contains an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases (PHDs). Mimicking hypoxia by treatment of cells with siRNA against PHD-1 or PHD-2 or the pan-prolyl hydroxylase inhibitor DMOG results in NFkappaB activation. Conversely, overexpression of PHD-1 decreases cytokine-stimulated NFkappaB reporter activity, further suggesting a repressive role for PHD-1 in controlling the activity of NFkappaB. Hypoxia increases both the expression and activity of IKKbeta, and site-directed mutagenesis of the proline residue (P191A) of the putative IKKbeta hydroxylation site results in a loss of hypoxic inducibility. Thus, we hypothesize that hypoxia releases repression of NFkappaB activity through decreased PHD-dependent hydroxylation of IKKbeta, an event that may contribute to tumor development and progression through amplification of tumorigenic signaling pathways.

                Author and article information

                Role: Academic Editor
                PLoS Biol
                PLoS Biol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                May 2014
                6 May 2014
                : 12
                : 5
                [1 ]Departamento de Biología Celular e Histología, Facultad de Biología, Universidad de Murcia, Murcia, Spain
                [2 ]Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain
                [3 ]Carlota Saldanha Lab, Instituto de Medicina Molecular, Instituto de Bioquímica, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
                [4 ]Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Vigo, Spain
                [5 ]Grupo de Telómeros, Envejecimiento y Cáncer, Unidad de Investigación, Departamento de Cirugía, CIBERehd. Hospital Universitario “Virgen de la Arrixaca,” Murcia, Spain
                [6 ]MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom
                [7 ]Servicio de Dermatología, Hospital Universitario “Virgen de la Arrixaca,” Murcia, Spain
                [8 ]Servicio de Anatomía Patológica, Hospital Universitario “Virgen de la Arrixaca,” Murcia, Spain
                [9 ]Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
                St. Jude Children's Research Hospital, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: SC SdO DG-M RE-P VM. Performed the experiments: SC SdO AL-M DG-M RE-P SDT MLC VM. Analyzed the data: SC SdO AL-M DG-M RE-P SDT MLC SAR RC-V IV-A JM MPS VM. Contributed reagents/materials/analysis tools: RC-V IV-A H-JT. Wrote the paper: VM.


                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.

                Page count
                Pages: 14
                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 Fundación Séneca-Murcia (grant 04538/GERM/06 to VM and PhD fellowship to RE-P), Fundação para a Ciência 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.
                Research Article
                Biology and Life Sciences
                Biological Tissue
                Epithelial Cells
                Cell Biology
                Cell Processes
                Cellular Stress Responses
                Cellular Types
                Animal Cells
                Immune Cells
                Signal Transduction
                Cell Signaling
                Signaling Cascades
                Molecular Cell Biology
                Developmental Biology
                Molecular Development
                Immune Response
                Immune System
                Innate Immune System
                Medicine and Health Sciences
                Inflammatory Diseases
                Research and Analysis Methods
                Model Organisms
                Animal Models

                Life sciences


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