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Keratinocyte Expression of A20/TNFAIP3 Controls Skin Inflammation Associated with Atopic Dermatitis and Psoriasis

  • Author Footnotes
    6 These authors contributed equally to this work.
    Michael Devos
    Footnotes
    6 These authors contributed equally to this work.
    Affiliations
    Molecular Signaling and Cell Death Unit, VIB-UGent Center for Inflammation Research, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Author Footnotes
    6 These authors contributed equally to this work.
    Denis A. Mogilenko
    Footnotes
    6 These authors contributed equally to this work.
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France
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  • Sébastien Fleury
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France
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  • Barbara Gilbert
    Affiliations
    Molecular Signaling and Cell Death Unit, VIB-UGent Center for Inflammation Research, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Coralie Becquart
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France

    Upper Airways Research Laboratory, Department of Otorhinolaryngology, Ghent University, Ghent, Belgium
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  • Sandrine Quemener
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France
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  • Hélène Dehondt
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France
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  • Peter Tougaard
    Affiliations
    Molecular Signaling and Cell Death Unit, VIB-UGent Center for Inflammation Research, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Bart Staels
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France
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  • Claus Bachert
    Affiliations
    Upper Airways Research Laboratory, Department of Otorhinolaryngology, Ghent University, Ghent, Belgium
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  • Peter Vandenabeele
    Affiliations
    Molecular Signaling and Cell Death Unit, VIB-UGent Center for Inflammation Research, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Geert Van Loo
    Affiliations
    Molecular Signaling and Cell Death Unit, VIB-UGent Center for Inflammation Research, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Delphine Staumont-Salle
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France

    Department of Dermatology, Centre Hospitalier Universitaire de Lille, Lille, France
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  • Author Footnotes
    7 These authors contributed equally to this work as senior authors.
    Wim Declercq
    Correspondence
    Wim Declercq, VIB-UGent Center for Inflammation Research, Technologiepark 927, 9052 Gent, Belgium.
    Footnotes
    7 These authors contributed equally to this work as senior authors.
    Affiliations
    Molecular Signaling and Cell Death Unit, VIB-UGent Center for Inflammation Research, Ghent, Belgium

    Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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  • Author Footnotes
    7 These authors contributed equally to this work as senior authors.
    David Dombrowicz
    Correspondence
    Correspondence: David Dombrowicz, INSERM U1011, Institut Pasteur de Lille, 1 rue du Prof. Camette BP245, 59019 Lille Cedex, France.
    Footnotes
    7 These authors contributed equally to this work as senior authors.
    Affiliations
    Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France
    Search for articles by this author
  • Author Footnotes
    6 These authors contributed equally to this work.
    7 These authors contributed equally to this work as senior authors.
Open ArchivePublished:August 14, 2018DOI:https://doi.org/10.1016/j.jid.2018.06.191
      Keratinocytes are key players in chronic inflammatory skin diseases. A20 regulates NF-κB–dependent expression of proinflammatory genes and cell death, but the impact of its expression in keratinocytes on systemic inflammation and skin disorders has not been determined. Comparative transcriptomic analysis of microdissected epidermis showed that A20 is down-regulated in involved epidermis, but not in dermis, of psoriasis and atopic dermatitis patients, suggesting that loss of A20 expression in keratinocytes increases the vulnerability for psoriasis/atopic dermatitis induction. We have previously shown that epidermis-specific A20 knockout mice (A20EKO) develop mild epidermal hyperplasia but no macroscopic skin inflammation. We now show that various cytokines and chemokines are up-regulated in A20EKO mouse skin. A20EKO mice also display systemic proinflammatory changes, even in the absence of skin immune cell infiltration, and an exacerbated disease severity upon induction of experimental psoriasis, atopic dermatitis, or skin barrier disruption. Keratinocytes showed increased proinflammatory gene expression in the absence of A20 in unstimulated and IL-17A–stimulated conditions, in part resulting from uncontrolled MyD88-dependent signaling. Our findings indicate that absence of A20 in keratinocytes leads to systemic inflammation at homeostatic conditions and is sufficient to exacerbate inflammatory skin disorders associated with different immune profiles by increasing cytokine and chemokine expression.

      Abbreviations:

      AD (atopic dermatitis), KO (knockout), IMQ (imiquimod), Th (T helper)

      Introduction

      Psoriasis and atopic dermatitis (AD) are the two most common inflammatory skin disorders, with different etiologies. However, certain specific subtypes of AD tend to have overlapping cytokine profiles and histopathology with psoriasis (
      • Guttman-Yassky E.
      • Krueger J.G.
      Atopic dermatitis and psoriasis: two different immune diseases or one spectrum?.
      ). Psoriasis, affecting 2%–3% of the global population, is mostly characterized by keratinocyte hyperproliferation, erythema, and ichthyosis (
      • Boehncke W.H.
      • Schon M.P.
      ,
      • Greb J.E.
      • Goldminz A.M.
      • Elder J.T.
      • Lebwohl M.G.
      • Gladman D.D.
      • Wu J.J.
      • et al.
      ). Initiation and maintenance of psoriasis depend on IL-12– and IL-23–producing dendritic cells (
      • Ganguly D.
      • Chamilos G.
      • Lande R.
      • Gregorio J.
      • Meller S.
      • Facchinetti V.
      • et al.
      Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8.
      ,
      • Lande R.
      • Gregorio J.
      • Facchinetti V.
      • Chatterjee B.
      • Wang Y.H.
      • Homey B.
      • et al.
      Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.
      ) and T helper (Th) lymphocytes, especially Th17 cells (
      • McGeachy M.J.
      • Chen Y.
      • Tato C.M.
      • Laurence A.
      • Joyce-Shaikh B.
      • Blumenschein W.M.
      • et al.
      The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo.
      ). AD, or atopic eczema, is a more prevalent chronic skin disease (up to 7% in adults and 25% in children) characterized by impaired skin barrier function, dry pruritic lesions, eosinophilia, and elevated IgE production (
      • Brunner P.M.
      • Guttman-Yassky E.
      • Leung D.Y.
      The immunology of atopic dermatitis and its reversibility with broad-spectrum and targeted therapies.
      ,
      • Weidinger S.
      • Novak N.
      Atopic dermatitis.
      ). Although often considered as distinct entities, based on genome-wide comparative analysis (
      • Baurecht H.
      • Hotze M.
      • Brand S.
      • Buning C.
      • Cormican P.
      • Corvin A.
      • et al.
      Genome-wide comparative analysis of atopic dermatitis and psoriasis gives insight into opposing genetic mechanisms.
      ,
      • Quaranta M.
      • Knapp B.
      • Garzorz N.
      • Mattii M.
      • Pullabhatla V.
      • Pennino D.
      • et al.
      Intraindividual genome expression analysis reveals a specific molecular signature of psoriasis and eczema.
      ) and on specific features displayed in pathogenesis, psoriasis and AD share some important features. Indeed, in addition to keratinocyte hyperproliferation and epidermal thickening, in chronic phases Th17 and Th22 responses are present in both diseases (
      • Eyerich K.
      • Dimartino V.
      • Cavani A.
      IL-17 and IL-22 in immunity: driving protection and pathology.
      ). Moreover, some patients might simultaneously develop psoriasis and AD (
      • Eyerich S.
      • Onken A.T.
      • Weidinger S.
      • Franke A.
      • Nasorri F.
      • Pennino D.
      • et al.
      Mutual antagonism of T cells causing psoriasis and atopic eczema.
      ). IFNγ, by decreasing skin long-chain fatty acid ceramides, seems to be involved in the development of both diseases (
      • Tawada C.
      • Kanoh H.
      • Nakamura M.
      • Mizutani Y.
      • Fujisawa T.
      • Banno Y.
      • et al.
      Interferon-γ decreases ceramides with long-chain fatty acids: possible involvement in atopic dermatitis and psoriasis.
      ). A neutrophilic signature is also associated with both diseases (
      • Choy D.F.
      • Hsu D.K.
      • Seshasayee D.
      • Fung M.A.
      • Modrusan Z.
      • Martin F.
      • et al.
      Comparative transcriptomic analyses of atopic dermatitis and psoriasis reveal shared neutrophilic inflammation.
      ). Finally, deficiency of the TNF superfamily molecule TWEAK (TNFSF12) in mice results in reduced skin inflammation in mouse models of house dust mite-induced AD and imiquimod (IMQ)-induced psoriasis, respectively due to defective maintenance of AD-specific Th2 and psoriasis-specific Th17 cells in the skin (
      • Sidler D.
      • Wu P.
      • Herro R.
      • Claus M.
      • Wolf D.
      • Kawakami Y.
      • et al.
      TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis.
      ). Therefore, one may hypothesize that the pathogenesis of both psoriasis and AD can be driven by common defects in controlling mechanisms in the skin that provide anti-inflammatory feedback to prevent chronic skin inflammation.
      The transcription factor NF-κB plays a central role in maintaining homeostasis and innate immune regulation in the skin. Numerous studies in mice have shown that both impairment and overactivation of NF-κB signaling in keratinocytes triggers chronic inflammation (
      • Kumari S.
      • Pasparakis M.
      Epithelial cell death and inflammation in skin.
      ,
      • Pasparakis M.
      Role of NF-κB in epithelial biology.
      ). To ensure control on NF-κB signaling, several of its target genes act as negative regulators, such as TNFAIP3, also named A20. Indeed, A20-deficient mice die prematurely of multiorgan inflammation and cachexia, as a result of excessive TLR signaling (
      • Lee E.G.
      • Boone D.L.
      • Chai S.
      • Libby S.L.
      • Chien M.
      • Lodolce J.P.
      • et al.
      Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice.
      ,
      • Turer E.E.
      • Tavares R.M.
      • Mortier E.
      • Hitotsumatsu O.
      • Advincula R.
      • Lee B.
      • et al.
      Homeostatic MyD88-dependent signals cause lethal inflammation in the absence of A20.
      ). A20 contains an N-terminal ovarian tumor (OTU) domain and seven zinc-finger (ZF) domains at the C-terminal part (
      • Wertz I.E.
      • O’Rourke K.M.
      • Zhou H.
      • Eby M.
      • Aravind L.
      • Seshagiri S.
      • et al.
      De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling.
      ). It holds both deubiquitinase and ubiquitin ligase activity; however, the physiological relevance of its catalytic activities is currently not clear (
      • De A.
      • Dainichi T.
      • Rathinam C.V.
      • Ghosh S.
      The deubiquitinase activity of A20 is dispensable for NF-κB signaling.
      ,
      • Draber P.
      • Kupka S.
      • Reichert M.
      • Draberova H.
      • Lafont E.
      • de Miguel D.
      • et al.
      LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing effects on linear ubiquitin in signaling complexes.
      ,
      • Lu T.T.
      • Onizawa M.
      • Hammer G.E.
      • Turer E.E.
      • Yin Q.
      • Damko E.
      • et al.
      Dimerization and ubiquitin mediated recruitment of A20, a complex deubiquitinating enzyme.
      ,
      • Skaug B.
      • Chen J.
      • Du F.
      • He J.
      • Ma A.
      • Chen Z.J.
      Direct, noncatalytic mechanism of IKK inhibition by A20.
      ,
      • Tokunaga F.
      • Nishimasu H.
      • Ishitani R.
      • Goto E.
      • Noguchi T.
      • Mio K.
      • et al.
      Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation.
      ,
      • Verhelst K.
      • Carpentier I.
      • Kreike M.
      • Meloni L.
      • Verstrepen L.
      • Kensche T.
      • et al.
      A20 inhibits LUBAC-mediated NF-κB activation by binding linear polyubiquitin chains via its zinc finger 7.
      ). Loss of A20 has also been shown to either enhance pro-survival signaling via up-regulation of anti-apoptotic proteins or sensitize to cell death, possibly depending on the cellular context (
      • Lee E.G.
      • Boone D.L.
      • Chai S.
      • Libby S.L.
      • Chien M.
      • Lodolce J.P.
      • et al.
      Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice.
      ,
      • Onizawa M.
      • Oshima S.
      • Schulze-Topphoff U.
      • Oses-Prieto J.A.
      • Lu T.
      • Tavares R.
      • et al.
      The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis.
      ,
      • Opipari Jr., A.W.
      • Hu H.M.
      • Yabkowitz R.
      • Dixit V.M.
      The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity.
      ,
      • Tavares R.M.
      • Turer E.E.
      • Liu C.L.
      • Advincula R.
      • Scapini P.
      • Rhee L.
      • et al.
      The ubiquitin modifying enzyme A20 restricts B cell survival and prevents autoimmunity.
      ,
      • Vereecke L.
      • Sze M.
      • Mc Guire C.
      • Rogiers B.
      • Chu Y.
      • Schmidt-Supprian M.
      • et al.
      Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis.
      ). Keratinocyte-specific deletion of A20 results in epidermal hyperproliferation and ectodermal defects, but skin infiltration of immune cells has not been observed (
      • Lippens S.
      • Lefebvre S.
      • Gilbert B.
      • Sze M.
      • Devos M.
      • Verhelst K.
      • et al.
      Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.
      ). Genome-wide association studies identified TNFAIP3/A20 as a susceptibility locus for psoriasis (
      • Haase O.
      • Mosaad H.
      • Eldarouti M.A.
      • Elramly A.Z.
      • Samir N.
      • Abdelhady M.M.
      • et al.
      TNFAIP3 and IL12B gene polymorphisms associated with psoriasis vulgaris in an Egyptian cohort.
      ,
      • Indhumathi S.
      • Rajappa M.
      • Chandrashekar L.
      • Ananthanarayanan P.H.
      • Thappa D.M.
      • Negi V.S.
      TNFAIP3 and TNIP1 polymorphisms confer psoriasis risk in South Indian Tamils.
      ,
      • Li X.L.
      • Yu H.
      • Wu G.S.
      Investigating the genetic association of HCP5, SPATA2, TNIP1, TNFAIP3 and COG6 with psoriasis in Chinese population.
      ,
      • Nair R.P.
      • Duffin K.C.
      • Helms C.
      • Ding J.
      • Stuart P.E.
      • Goldgar D.
      • et al.
      Genome-wide scan reveals association of psoriasis with IL-23 and NF-κB pathways.
      ; and
      • Zhang C.
      • Zhu K.J.
      • Liu H.
      • Quan C.
      • Liu Z.
      • Li S.J.
      • et al.
      The TNFAIP3 polymorphism rs610604 both associates with the risk of psoriasis vulgaris and affects the clinical severity.
      ), and its role as a negative regulator in Th2-associated lung inflammation, in particular through its expression by dendritic cells, has been well characterized (
      • Schuijs M.J.
      • Willart M.A.
      • Vergote K.
      • Gras D.
      • Deswarte K.
      • Ege M.J.
      • et al.
      Farm dust and endotoxin protect against allergy through A20 induction in lung epithelial cells.
      ,
      • Vroman H.
      • Bergen I.M.
      • van Hulst J.A.C.
      • van Nimwegen M.
      • van Uden D.
      • Schuijs M.J.
      • et al.
      TNF-α–induced protein 3 levels in lung dendritic cells instruct TH2 or TH17 cell differentiation in eosinophilic or neutrophilic asthma.
      ). On the other hand, some cases of A20 haploinsufficiency yield distinct autoinflammatory and/or autoimmune skin pathology, possibly with skin ulcerations and pustular abscesses (
      • Kadowaki T.
      • Ohnishi H.
      • Kawamoto N.
      • Hori T.
      • Nishimura K.
      • Kobayashi C.
      • et al.
      Haploinsufficiency of A20 causes autoinflammatory and autoimmune disorders.
      ,
      • Takagi M.
      • Ogata S.
      • Ueno H.
      • Yoshida K.
      • Yeh T.
      • Hoshino A.
      • et al.
      Haploinsufficiency of TNFAIP3 (A20) by germline mutation is involved in autoimmune lymphoproliferative syndrome.
      ,
      • Zhou Q.
      • Wang H.
      • Schwartz D.M.
      • Stoffels M.
      • Park Y.H.
      • Zhang Y.
      • et al.
      Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease.
      ). In addition, TNFAIP3/A20 single nucleotide polymorphisms correlate with response to TNF blockade in psoriasis patients (
      • Tejasvi T.
      • Stuart P.E.
      • Chandran V.
      • Voorhees J.J.
      • Gladman D.D.
      • Rahman P.
      • et al.
      TNFAIP3 gene polymorphisms are associated with response to TNF blockade in psoriasis.
      ). All these data show a clinically relevant association between mutations in the A20 gene and skin disease.
      Seeking for additional commonalities between psoriasis and AD, we found that in humans, A20 epidermal expression is strongly decreased in both diseases. Therefore, we studied the effect of keratinocyte-specific A20 deletion in experimental mouse models relevant to the human pathology (
      • Kabashima K.
      • Nomura T.
      Revisiting murine models for atopic dermatitis and psoriasis with multipolar cytokine axes.
      ). Our findings show that A20 expression in keratinocytes is required to establish a threshold to prevent systemic inflammation under homeostatic conditions and to limit inflammatory responses in skin pathologies with distinct immunological programs.

      Results

      A20/TNFAIP3 is down-regulated in human AD and psoriasis

      To determine common pathways leading to the two most frequent inflammatory skin diseases, psoriasis and AD, we performed comparative transcriptomic analysis of laser microdissected affected epidermis from patients with psoriasis and AD, as well as healthy epidermis from individuals with no skin diseases. Among transcripts significantly up-regulated in epidermis (at least 2-fold), 369 were common to both diseases, and 359 were common among the significantly down-regulated transcripts (Figure 1a), suggesting that both skin pathologies share common underlying molecular mechanisms in keratinocytes. The common up-regulated transcripts included known genes with affected expression in psoriasis and/or AD, such as S100A family members, KRT16, KRT6A, IL36G, LCE3 family members, CNFN, TNC, and others. The up-regulated genes were enriched for pathways related to the immune system and defense processes and pathways controlling keratinocyte differentiation and keratinization. The most down-regulated pathways among common transcripts in psoriasis and AD were diverse and included epithelial and epidermal development, intracellular signal transduction, negative regulation of response to stimulus, negative regulation of cell communication, and regulation of cell death (Figure 1b). We found A20/TNFAIP3 among the down-regulated genes in involved epidermis of both psoriasis and AD patients (Figure 1a). A20/TNFAIP3 expression was significantly decreased in uninvolved epidermis in psoriasis patients compared with epidermis from healthy skin and was further decreased in involved epidermis versus uninvolved epidermis in psoriasis patients. In AD, A20/TNFAIP3 expression was significantly lower in involved epidermis compared with epidermis from healthy skin. By contrast, no significant difference in A20/TNFAIP3 expression was observed between healthy dermis and both uninvolved and involved dermis in psoriasis and AD (Figure 1c). These results suggest that down-regulation of A20/TNFAIP3 in involved epidermis in psoriasis and AD is likely due to a decrease of its expression by keratinocytes, associated with pathogenesis of these diseases.
      Figure thumbnail gr1
      Figure 1A20/TNFAIP3 is down-regulated in epidermis of psoriasis and atopic dermatitis patients. (a) Significantly deregulated transcripts (Benjamini-Hochberg adjusted P-values < 0.05) in human epidermis isolated by laser capture microdissection from healthy skin (n = 5) or affected skin from patients with psoriasis (n = 6) or atopic dermatitis (n = 5). Some common up- and down-regulated genes are shown. (b) Gene set enrichment analysis of common genes significantly dysregulated in psoriasis and atopic dermatitis samples shown in a. (c) TNFAIP3 is decreased in human epidermis but not in dermis in psoriasis and atopic dermatitis. Relative expression of TNFAIP3 from healthy skin (n = 5) or involved and uninvolved skin from patients with psoriasis (n = 9) or atopic dermatitis (n = 5). P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by paired and unpaired Mann-Whitney U test. Graphs present mean ± standard error of the mean. AD, atopic dermatitis; FC, fold change; GO, gene ontology; Pso, psoriasis.

      Keratinocyte-specific A20/TNFAIP3 deficiency in mice leads to basal systemic inflammation

      Mice with keratinocyte-specific A20 deletion (A20EKO) do not spontaneously develop macroscopic or histological signs of skin inflammation, as reported previously (
      • Lippens S.
      • Lefebvre S.
      • Gilbert B.
      • Sze M.
      • Devos M.
      • Verhelst K.
      • et al.
      Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.
      ). Indeed, no infiltrating immune cells were observed in hematoxylin and eosin-stained sections of skin of A20EKO mice (see Supplementary Figure S1a online). However, analysis of inflammation-associated gene expression in the skin of these mice by quantitative real-time reverse transcriptase–PCR showed significant increases in TNF, Ccl20, Cxcl1, Lcn2, IL-22, IL-23a, and TSLP expression compared with A20Fl/Fl mice (see Supplementary Figure S1b). In addition, keratinocyte-specific A20 deficiency led to systemic inflammatory effects. Indeed, A20EKO mice displayed splenomegaly and a lower body weight than their A20Fl/Fl littermates, even when housed in specific pathogen-free conditions (see Supplementary Figure S1c). In line with this, the percentage of splenic neutrophils and Ly6Chi monocytes was increased in A20EKO animals (see Supplementary Figure S1d). Although the percentages of splenic CD4+ and CD8+ T lymphocytes were similar in both genotypes, the percentage of naïve T cells was decreased, whereas the frequency of effector T cells was increased in A20EKO mice (see Supplementary Figure S1e). Although a slight increase in frequency of splenic classical dendritic cells was observed in A20EKO animals, frequency of monocytes and plasmacytoid dendritic cells, natural killer, natural killer T, and γδ T cells was not altered (see Supplementary Figure S1f). Because K14-driven A20 deletion in the thymic epithelium cannot be excluded (
      • Kuraguchi M.
      • Wang X.P.
      • Bronson R.T.
      • Rothenberg R.
      • Ohene-Baah N.Y.
      • Lund J.J.
      • et al.
      Adenomatous polyposis coli (APC) is required for normal development of skin and thymus.
      ), we analyzed thymic cell populations in A20EKO and A20Fl/Fl mice and found that thymic composition was comparable for both genotypes, with only a marginal difference in the proportion of CD25+CD5hi CD4+T cells (see Supplementary Figure S2 online). These results indicate that A20 deficiency in keratinocytes results in an inflammatory gene signature in the skin and systemic proinflammatory changes in immune cell populations.

      Keratinocyte-specific A20/TNFAIP3 deficiency exacerbates experimental psoriasis

      We induced psoriasis-like inflammation by epicutaneous application of IMQ (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ) in A20EKO and A20Fl/Fl mice. Upon IMQ treatment, A20EKO mice displayed an increased disease severity score at the macroscopic level (Figure 2a and b ) but no sign of psoriatic arthritis (not shown). Despite increased erythema and scaling, epidermal thickening was similar in A20EKO and A20Fl/Fl mice (Figure 2c). In contrast, A20EKO mice showed significantly increased skin infiltration of neutrophils, including their epidermal accumulation, similar to Munro microabscesses in human psoriatic epidermis (Figure 2d). In keeping with morphological signs of inflammation, skin expression of psoriasis-associated genes involved in skin barrier and stress (Krt16, Lce3c, Lcn2, S100a8) as well as in inflammatory responses (IL-23a, IL-22, Cxcl1) was significantly increased in A20EKO mice upon treatment with IMQ as compared to A20Fl/Fl mice (Figure 2e). Furthermore, the frequency of classical dendritic cells and macrophages in skin draining lymph nodes harboring activated cells was increased in A20EKO mice, whereas the percentage of Langerhans, IL-17–producing CD4+ T, and γδ T cells was unchanged, αβ CD4+ T cells were decreased (Figure 2f and g). In contrast, A20TKO mice, generated by crossing A20FL/FL and CD4-Cre mice, reacted to IMQ in a similar way as A20FL/FL control mice (see Supplementary Figure S3 online). Because IL-23a expression was increased in skin from A20EKO compared with A20Fl/Fl mice and IL-23 injection also represents a relevant experimental model for human psoriasis (
      • Kabashima K.
      • Nomura T.
      Revisiting murine models for atopic dermatitis and psoriasis with multipolar cytokine axes.
      ), we intradermally injected IL-23 and observed that A20EKO mice developed exacerbated skin pathology compared with A20Fl/Fl mice (see Supplementary Figure S4 online). Taken together, these results indicate that A20 deficiency in keratinocytes sensitizes to psoriasis-like inflammation in mice.
      Figure thumbnail gr2
      Figure 2A20/TNFAIP3 deficiency in keratinocytes exacerbates IMQ-induced experimental psoriasis. (a) Abdominal skin morphology and histology. May-Grünwald Giemsa staining, scale bar = 50 μm. (b) Disease severity score. (c) Average epidermal thickness. (d) Infiltration of neutrophils (brown) in dermis and epidermis detected by Ly6G immunostaining. (e) Quantitative real-time reverse transcriptase–PCR analysis of inflammatory genes in whole skin. (f) Flow cytometry of classical dendritic cells (cDC), Langerhans cell (LCs), and macrophages in inguinal lymph nodes and (g) flow cytometry of γδT cells, γδT17, CD4 T cells and Th17 in inguinal lymph nodes (iLNs), during/after 6 days in the IMQ-induced model of psoriasis on A20 FL/FL and A20EKO mice. n = 5–6 mice per group. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by two-way analysis of variance with Sidak post hoc test or two-sided t test. Graphs present mean ± standard error of the mean. IMQ, imiquimod.

      Keratinocyte-specific A20/TNFAIP3 deficiency exacerbates experimental AD

      We next evaluated the contribution of A20/TNFAIP3 to the regulation of AD induced upon repeated epicutaneous sensitization with ovalbumin in the absence of adjuvant (
      • Spergel J.M.
      • Mizoguchi E.
      • Brewer J.P.
      • Martin T.R.
      • Bhan A.K.
      • Geha R.S.
      Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice.
      ). Antigen-sensitized A20EKO mice displayed increased epidermal thickness compared with A20Fl/Fl animals (Figure 3a and b ). Phosphate buffered saline-treated A20EKO mice also showed, to some extent, epidermal thickening, suggesting that in addition to the classical allergic response, an increased response to the mechanical stress caused by the repeated sensitization procedure was taking place (Figure 3a and b). Surprisingly, the strong eosinophilia associated to Th2 responses induced upon sensitization of A20Fl/Fl animals was significantly abolished in A20EKO mice (Figure 3c). The humoral response, a characteristic of AD, was exacerbated in A20EKO mice, with increased concentrations of antigen-specific Ig (Figure 3d), associated with both Th2 (IgE and IgG1) and Th1 immune responses (IgG2a and IgG2c). Accordingly, although the percentage of total CD4+ T cells in draining lymph nodes remained unchanged, the percentage of CD4+ T cells expressing IL-5, IL-13, IL-10, or Foxp3 was increased in both antigen- and vehicle-treated A20EKO mice compared with A20Fl/Fl animals (Figure 3e). IL-17A+ CD4+ cells were significantly increased in A20EKO animals compared with A20Fl/Fl mice only upon ovalbumin sensitization, and an increase in IL-4+ CD4+ cells occurred only in phosphate buffered saline-treated A20EKO animals (Figure 3e). No significant alteration was observed for IFNγ-producing CD4+ cells (Figure 3e). Taken together, these results show that keratinocyte-specific A20/TNFAIP3 deficiency exacerbates not only the antigen-specific immune response associated with AD but also the response associated with mechanical skin damage that takes place in vehicle-treated animals.
      Figure thumbnail gr3
      Figure 3A20/TNFAIP3 deficiency in keratinocytes exacerbates OVA-induced experimental atopic dermatitis. (a) Abdominal skin morphology and histology (May-Grünwald Giemsa staining, scale bar = 20 μm); (b) average epidermal thickness; (c) quantification of eosinophils in dermis; (d) total and OVA-specific IgE, IgG1, IgG2a, and IgG2c in serum; and (e) flow cytometry of T cells in inguinal lymph nodes after the epicutaneous OVA sensitization model of atopic dermatitis on A20 FL/FL and A20EKO mice. n = 6–8. P-value of interaction analyzed by two-way analysius of variance is depicted on graph. Graphs present mean ± standard error of the mean. iLN, inguinal lymph node; OVA, ovalbumin; PBS, phosphate buffered saline.

      Skin barrier disruption in A20EKO mice results in inflammation resembling interface dermatitis

      Given the observed sensitivity to mechanical stress in the phosphate buffered saline-treated animals in the described ovalbumin-based AD model, we next applied an established model of skin barrier disruption by challenging the skin of A20EKO and A20Fl/Fl mice by repetitive acetone wiping twice a day for 5 consecutive days. We used both nontreated and saline-treated mice as controls. Histological sections of back skin showed that A20EKO mice, in contrast to A20Fl/Fl mice, developed epidermal hyperplasia and infiltration of immune cells at the dermoepidermal junction upon topical treatment with acetone (Figure 4a and b ). These histological findings are characteristic of interface dermatitis, which is also observed in keratin5-IKK2 transgenic mice, a model for NF-κB overactivation (
      • Page A.
      • Navarro M.
      • Garin M.
      • Perez P.
      • Casanova M.L.
      • Moreno R.
      • et al.
      IKKβ leads to an inflammatory skin disease resembling interface dermatitis.
      ). In addition, we observed increased chemokine (Ccl20, Cxcl1), allergy-related TSLP, and stress molecule S100a8 mRNA levels in the skin of acetone-treated A20EKO versus A20Fl/Fl mice, but TNF was not further increased upon acetone treatment (Figure 4c). Taken together, these results suggest that physical abrasion is sufficient to induce skin inflammation and innate responses when A20 is deleted in keratinocytes, demonstrating its general anti-inflammatory role in the skin.
      Figure thumbnail gr4
      Figure 4Keratinocyte-specific A20/TNFAIP3 deficiency in combination with skin barrier disruption results in inflammation resembling interface dermatitis. (a) Back skin histology (hematoxylin and eosin, scale bar = 50 μm), (b) epidermal thickness, and (c) quantitatitve real-time reverse transcriptase–PCR analysis of inflammatory genes in whole back skin of A20Fl/Fl and A20EKO mice that were left untreated or subjected to repetitive treatment two times daily with saline or acetone wipes for 5 days. The inset magnifying the dermoepidermal junction in a shows the presence of inflammatory cells. n = 3–7. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by two-way analysis of variance with Bonferroni posttest graphs present mean ± standard error of the mean.

      A20/TNFAIP3 deficiency increases proinflammatory gene expression in mouse keratinocytes

      To further study the intrinsic regulation of the keratinocyte inflammatory responses by A20, we isolated keratinocytes from the epidermis of A20EKO mice and A20Fl/Fl littermates. Despite the fact that A20 deficiency sensitizes to TNF-induced cell death in other cell types (
      • Lee E.G.
      • Boone D.L.
      • Chai S.
      • Libby S.L.
      • Chien M.
      • Lodolce J.P.
      • et al.
      Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice.
      ,
      • Vereecke L.
      • Sze M.
      • Mc Guire C.
      • Rogiers B.
      • Chu Y.
      • Schmidt-Supprian M.
      • et al.
      Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis.
      ), no difference in proliferation and survival was observed in keratinocytes in the absence of A20 whether at resting state or upon TNF stimulation (see Supplementary Figure S5 online). However, quantitative real-time reverse transcriptase–PCR analysis showed that basal expression of inflammation-associated genes is higher in the absence of A20, including target genes of IL-17A (Ccl20, Cxcl1, Csf3) (Figure 5a). Although we confirmed that A20 restricted IL-17A–dependent NF-κB and MAPK activation in mouse embryonic fibroblast (MEF) cells (see Supplementary Figure S6a online) (
      • Garg A.V.
      • Ahmed M.
      • Vallejo A.N.
      • Ma A.
      • Gaffen S.L.
      The deubiquitinase A20 mediates feedback inhibition of interleukin-17 receptor signaling.
      ), we were not able to observe differences in IκB-α degradation and JNK-, Erk- and p38-phosphorylation after stimulation with IL-17A of keratinocytes isolated from A20EKO mice and A20Fl/Fl littermates (see Supplementary Figure S6b). In line with this, two-way analysis of variance of IL-17A–induced chemokine expression (Ccl20, Cxcl1, Csf3) in keratinocytes showed a nonsignificant interaction between the effects of A20 expression and IL-17A stimulation (Figure 5b). In addition, loss of A20 did not influence IL-17A–induced expression of the transcriptional co-activator IκB-ζ (Nfkbiz) (Figure 5c), another IL-17A–responsive gene important in psoriasis (
      • Johansen C.
      IκBζ: a key protein in the pathogenesis of psoriasis.
      ). Taken together, these observations show that A20 is not a negative regulator of IL-17A signaling in keratinocytes and raises the possibility that a different signaling pathway being sensitized in the absence of A20 might cross talk with IL-17A–induced gene expression. As MyD88-dependent signals underlie the lethal phenotype in full A20-knockout (KO) mice (
      • Turer E.E.
      • Tavares R.M.
      • Mortier E.
      • Hitotsumatsu O.
      • Advincula R.
      • Lee B.
      • et al.
      Homeostatic MyD88-dependent signals cause lethal inflammation in the absence of A20.
      ), we knocked down MyD88 in A20EKO and A20Fl/Fl keratinocytes and observed a reduced expression of chemokines (Ccl20, Cxcl1, Csf3) both in unstimulated and IL-17A–stimulated conditions (Figure 5d), showing that increased sensitivity upon loss of A20 in keratinocytes is, at least in part, dependent on MyD88 signaling. In contrast to MyD88 knockdown, knockdown of TNFR1 did not result in a similar reduction of Cxcl1 and Csf3 expression while Ccl20 levels were increased (see Supplementary Figure S7 online). We were unable to explain the latter observation. Inactivating Nfkbiz almost completely abrogated Ccl20, Cxc11, and Csf3 expression both in unstimulated and IL-17A–stimulated conditions (Figure 5e), giving evidence that IL-17A–induced chemokine expression is mostly dependent on the induction of the transcription co-activator IκB-ζ. Taken together, these data indicate that loss of A20 in keratinocytes leads to enhanced cytokine and chemokine production partly as a result of uncontrolled MyD88 signaling, which is further augmented through IL-17A–mediated IκB-ζ expression in an A20-independent manner.
      Figure thumbnail gr5
      Figure 5A20/TNFAIP3 deficiency increases proinflammatory gene expression in keratinocytes. (a) Quantitative real-time reverse transcriptase–PCR analysis of inflammatory genes of primary keratinocytes derived from A20Fl/Fl and A20EKO mice. n = 5–6. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by two sided t test. (b, c) Quantitative real-time reverse transcriptase–PCR analysis of indicated genes of unstimulated and in vitro IL-17A–stimulated (6 hours, 50 ng/ml) primary keratinocytes derived from A20FL/FL and A20EKO mice. n = 6. P-value of interaction analyzed by two-way analysis of variance is depicted on graph. (d, e) Quantitative real-time reverse transcriptase–PCR analysis of indicated genes of primary keratinocytes derived from A20FL/FL and A20EKO mice transfected with indicated siRNAs, unstimulated or in vitro stimulated with IL-17A (6 hours, 50 ng/ml). n = 3. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by two-way analysis of variance with Bonferroni posttest. Graphs present mean ± standard error of the mean or matched individual measurements. Ctrl, control; siRNA, small interfering RNA.

      Discussion

      Based on comparative transcriptomics on human biopsy samples and the use of in vivo mouse models, we found that specific down-regulation of A20 in keratinocytes, but not in T cells, determines sensitivity to both AD and/or psoriasis. Because keratinocytes show increased proinflammatory gene expression upon loss of A20, probably as a result of cross talk between different signaling pathways, we provide a mechanism by which A20 expression in keratinocytes is able to control inflammation in various skin immune disorders.
      First, transcriptome analysis of laser microdissected epidermis, but not dermis, from psoriasis and AD patients showed down-regulation of A20/TNFAIP3 in affected skin compared with healthy control samples. In addition, down-regulation of A20 mRNA expression could also be observed in the noninvolved skin of psoriatic patients. Second, using the best available and most relevant mouse models (
      • Kabashima K.
      • Nomura T.
      Revisiting murine models for atopic dermatitis and psoriasis with multipolar cytokine axes.
      ), we show that AD and psoriasis severity was increased in A20EKO mice upon repeated ovalbumin sensitization, IMQ application, or IL-23–injection. Although epidermal thickening was not significantly different in wild-type and A20EKO mice upon IMQ-induced psoriasis-like skin inflammation, we found significant increases in several important parameters relevant for the human disease: disease severity score; neutrophilia with neutrophilic abscesses (the latter having never been reported in mice to the best of our knowledge); expression of key immune genes, such as IL-22 and IL-23; expression of stress molecule S100A8; and expression of structural proteins Krt16, Lce3c, and Lcn2. In the AD model, A20EKO mice, compared with wild-type animals, displayed exacerbation of the key disease features except eosinophilia, which was barely induced by sensitization. This is possibly due to the higher number of IL-17A–positive CD4+ T cells as an inverse correlation between Th17 and eosinophilia that has been reported in AD (
      • Hayashida S.
      • Uchi H.
      • Moroi Y.
      • Furue M.
      Decrease in circulating Th17 cells correlates with increased levels of CCL17, IgE and eosinophils in atopic dermatitis.
      ). Furthermore, we also found that keratinocytes show increased proinflammatory gene expression upon loss of A20, probably as a result of cross talk between different signaling pathways, providing a mechanism by which A20 expression in keratinocytes is able to control inflammation in various skin immune disorders.
      Unlike K5-IKK2 transgenic mice or keratinocyte-specific deletion of the NF-κB inhibitor IκBα, a model for NF-κB over-activation in keratinocytes, A20EKO mice do not show spontaneous dermal and/or epidermal immune cell infiltration (
      • Lippens S.
      • Lefebvre S.
      • Gilbert B.
      • Sze M.
      • Devos M.
      • Verhelst K.
      • et al.
      Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.
      ,
      • Page A.
      • Navarro M.
      • Garin M.
      • Perez P.
      • Casanova M.L.
      • Moreno R.
      • et al.
      IKKβ leads to an inflammatory skin disease resembling interface dermatitis.
      ,
      • Rebholz B.
      • Haase I.
      • Eckelt B.
      • Paxian S.
      • Flaig M.J.
      • Ghoreschi K.
      • et al.
      Crosstalk between keratinocytes and adaptive immune cells in an IκBα protein-mediated inflammatory disease of the skin.
      ). Indeed, A20 is an NF-κB responsive gene that needs to be induced to prevent persistent trigger-induced NF-κB activation, so A20 might be redundant for skin immune homeostasis in the absence of pathogen- or stress-inducing stimuli. In contrast, keratinocyte-specific deletion of IκBα or constitutive IKK2 activation in K5-IKK2 transgenic epidermis results in continuous NF-κB overactivation and skin inflammation, without the need for an inflammatory trigger (
      • Page A.
      • Navarro M.
      • Garin M.
      • Perez P.
      • Casanova M.L.
      • Moreno R.
      • et al.
      IKKβ leads to an inflammatory skin disease resembling interface dermatitis.
      ). In support for this hypothesis, we found that disrupting the stratum corneum barrier in A20EKO mice by acetone treatment, which enables the penetration of skin commensals that can be recognized by pattern recognition receptors, results in an inflammatory skin condition resembling the interface dermatitis observed in K5-IKK2 mice (
      • Page A.
      • Navarro M.
      • Garin M.
      • Perez P.
      • Casanova M.L.
      • Moreno R.
      • et al.
      IKKβ leads to an inflammatory skin disease resembling interface dermatitis.
      ). This argues that in patients expressing lower A20 levels in the epidermis, physical abrasion could lie at the basis of skin inflammation. Remarkably, absence of IKK2 in keratinocytes (IKK2EKO) also results in TNF-dependent spontaneous severe skin inflammation (
      • Pasparakis M.
      • Courtois G.
      • Hafner M.
      • Schmidt-Supprian M.
      • Nenci A.
      • Toksoy A.
      • et al.
      TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2.
      ). Although unclear, this might result either from the fact that IKK2 can act as a negative regulator of RIPK1 (
      • Dondelinger Y.
      • Jouan-Lanhouet S.
      • Divert T.
      • Theatre E.
      • Bertin J.
      • Gough P.J.
      • et al.
      NF-κB-independent role of IKKα/IKKβ in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.
      ), thereby leading to a hypersensitivity toward TNF-induced RIPK1-dependent cell death, and/or from the fact that NF-κB is no longer activated in keratinocytes, causing RIPK1-dependent TNF-induced cell death (
      • Grinberg-Bleyer Y.
      • Dainichi T.
      • Oh H.
      • Heise N.
      • Klein U.
      • Schmid R.M.
      • et al.
      Cutting edge: NF-κB p65 and c-Rel control epidermal development and immune homeostasis in the skin.
      ,
      • Xu C.
      • Wu X.
      • Zhang X.
      • Xie Q.
      • Fan C.
      • Zhang H.
      Embryonic lethality and host immunity of RelA-deficient mice are mediated by both apoptosis and necroptosis.
      ).
      Even though skin from unchallenged A20EKO mice is devoid of infiltrating immune cells, we found an increase in proinflammatory gene expression in the skin of these mice, suggesting that A20 represses the skin inflammatory program even under homeostatic conditions. Furthermore, A20EKO mice show signs of basal systemic inflammation, indicating an impact of keratinocyte-specific A20 deficiency extending beyond skin. Such findings might in part explain (distal) articular manifestations found in psoriatic arthritis (
      • Ritchlin C.T.
      • Colbert R.A.
      • Gladman D.D.
      Psoriatic arthritis.
      ), which is also associated with A20/TNFAIP3 gene polymorphisms but is not present in our mouse model, probably because of the very short duration of the experimental protocol or cell-specific A20 deletion.
      Given the strong down-regulation of A20 transcripts in epidermis, but not dermis from psoriasis and AD patients compared with healthy control individuals, we suggest that the decrease in A20 expression occurred in keratinocytes. Although the mechanism controlling a decrease of A20 expression in epidermis in psoriasis and AD is not clear, the reduced homeostatic capacity of keratinocytes to counterbalance inflammatory signals and/or their increased production of proinflammatory factors, in the absence of A20, appears to control skin inflammatory susceptibility to a wide range of stimuli. Because the development of AD and psoriasis depends on cross talk between immune cells and keratinocytes (
      • Lowes M.A.
      • Suarez-Farinas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ), A20 expression in keratinocytes appears to play an important role in limiting skin immunoinflammatory responses in these pathologies, as also suggested by the link between hypomorphic A20 expression and psoriasis susceptibility (
      • Aki A.
      • Nagasaki M.
      • Malynn B.A.
      • Ma A.
      • Kagari T.
      Hypomorphic A20 expression confers susceptibility to psoriasis.
      ). Because keratinocyte responses to T cell-derived cytokines are paramount to both pathologies (
      • Guttman-Yassky E.
      • Krueger J.G.
      Atopic dermatitis and psoriasis: two different immune diseases or one spectrum?.
      ), a decrease of A20 expression in keratinocytes, by not yet established factor(s), seems to be a key trigger of pathogenic circuits in both diseases. The A20-binding partner TNIP1 is also linked to psoriasis by genome-wide association studies (
      • Heyninck K.
      • De Valck D.
      • Vanden Berghe W.
      • Van Criekinge W.
      • Contreras R.
      • Fiers W.
      • et al.
      The zinc finger protein A20 inhibits TNF-induced NF-κB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-κB-inhibiting protein ABIN.
      ,
      • Nair R.P.
      • Duffin K.C.
      • Helms C.
      • Ding J.
      • Stuart P.E.
      • Goldgar D.
      • et al.
      Genome-wide scan reveals association of psoriasis with IL-23 and NF-κB pathways.
      ), and epidermis-specific deletion of TNIP1 sensitizes mice to experimental psoriasis (
      • Ippagunta S.K.
      • Gangwar R.
      • Finkelstein D.
      • Vogel P.
      • Pelletier S.
      • Gingras S.
      • et al.
      Keratinocytes contribute intrinsically to psoriasis upon loss of Tnip1 function.
      ). Like A20, TNIP1 also provides negative feedback regulation in TLR pathways (
      • Zhou J.
      • Wu R.
      • High A.A.
      • Slaughter C.A.
      • Finkelstein D.
      • Rehg J.E.
      • et al.
      A20-binding inhibitor of NF-κB (ABIN1) controls Toll-like receptor-mediated CCAAT/enhancer-binding protein beta activation and protects from inflammatory disease.
      ), so regarding inflammatory skin diseases, it is tempting to speculate that A20 and TNIP act together to repress excessive inflammatory signaling.
      Paradoxically, upon anti-TNF treatment of inflammatory bowel disease and rheumatoid arthritis, psoriasis-like syndrome can occur. A recent study suggested this was due to reduced levels of TNF-induced A20 allowing p38-MAPK and PKC-driven IL-17A production in CD4+ T cells (
      • Urbano P.C.M.
      • Aguirre-Gamboa R.
      • Ashikov A.
      • van Heeswijk B.
      • Krippner-Heidenreich A.
      • Tijssen H.
      • et al.
      TNF-α-induced protein 3 (TNFAIP3)/A20 acts as a master switch in TNF-α blockade-driven IL-17A expression.
      ). Because A20 was previously reported to negatively regulate IL-17 receptor signaling in the ST2 stromal cell line and in embryonic fibroblasts (
      • Garg A.V.
      • Ahmed M.
      • Vallejo A.N.
      • Ma A.
      • Gaffen S.L.
      The deubiquitinase A20 mediates feedback inhibition of interleukin-17 receptor signaling.
      ), a similar mechanism involving IL-17R could in part account for the inverse correlation between A20 expression in keratinocytes and psoriasis/AD severity. We found that A20-deficient cultured keratinocytes showed up-regulation of proinflammatory genes already in unstimulated conditions, including the IL-17A target chemokines Ccl20, Cxcl1, and Csf3. Inactivating MyD88, a crucial adaptor protein for all TLRs except TLR3, and IL-1R in A20-deficient keratinocytes significantly decreased chemokine expression. However, A20 had no significant impact on IL-17A–mediated induction of Ccl20, Cxc1, and Csf3 in keratinocytes. These data suggest that A20 has no direct influence on IL-17 receptor signaling in keratinocytes, in contrast to embryonic fibroblasts (
      • Garg A.V.
      • Ahmed M.
      • Vallejo A.N.
      • Ma A.
      • Gaffen S.L.
      The deubiquitinase A20 mediates feedback inhibition of interleukin-17 receptor signaling.
      ), suggesting some cell specificity in this regulatory process. It also indicates that other signaling pathway(s) are activated in the absence of A20 and cross talk with IL-17A–induced gene expression. Cross talk between IL-17 signaling and other signaling pathways, such as TNF and IL-1, are mediated by communication between NF-κB and its co-activator IκB-ζ, which acts as a transcription enhancer primarily through chromatin remodeling, resulting in increased mRNA expression from their target promoters (
      • Johansen C.
      IκBζ: a key protein in the pathogenesis of psoriasis.
      ). A20 had no impact on basal and IL-17A–induced IκB-ζ expression, again showing that, as such, A20 does not influence IL-17R signaling in keratinocytes. However, we found that knocking down IκB-ζ abolished chemokine expression both in unstimulated and IL-17A–stimulated conditions. IκB-ζ has been shown to be a key driver in the development of psoriasis in different mouse models (
      • Johansen C.
      IκBζ: a key protein in the pathogenesis of psoriasis.
      ,
      • Johansen C.
      • Mose M.
      • Ommen P.
      • Bertelsen T.
      • Vinter H.
      • Hailfinger S.
      • et al.
      IκBζ is a key driver in the development of psoriasis.
      ). Taken together, we propose that, at homeostasis, loss of A20 sensitizes keratinocytes to signaling events in part induced by MyD88-dependent environmental triggers that amplify IL-17A–induced cytokine production, resulting in exacerbation of skin inflammation in psoriasis and AD.
      In summary, our findings show that subclinical skin inflammation caused by loss of A20 in keratinocytes is able to drive systemic inflammation under homeostatic conditions. In turn, this sensitivity primes or potentiates disease severity of distinct skin pathologies. How A20 expression could be restored/increased in keratinocytes will be an interesting challenge for better therapies for psoriasis, AD, and potentially other inflammatory conditions of the skin.

      Materials and Methods

      Patients

      Skin biopsy samples from involved and noninvolved areas were obtained from nine patients with psoriasis, five patients with AD, and five healthy volunteers (see Supplementary Materials online) after written informed consent was obtained as part of the FRADAPSO study, approved by the Committee for Protection of Persons Nord-Ouest IV under the number 2010-A00856-33.

      Mice

      All experiments on mice were conducted after approval by the local Ethics Committee of Ghent University (EC2017-015) and the Ethics Committee for Animal Experimentation from Nord-Pas de Calais Region (CEEA75-#01-2002R and APAFIS#7160-2017040313471173).
      Epidermis-specific (A20EKO) and CD4+ T cell-specific (A20TKO) A20-deficient mice were generated on a C57BL/6J background as previously described (
      • Drennan M.B.
      • Govindarajan S.
      • Verheugen E.
      • Coquet J.M.
      • Staal J.
      • McGuire C.
      • et al.
      NKT sublineage specification and survival requires the ubiquitin-modifying enzyme TNFAIP3/A20.
      ,
      • Lippens S.
      • Lefebvre S.
      • Gilbert B.
      • Sze M.
      • Devos M.
      • Verhelst K.
      • et al.
      Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.
      ) (see Supplementary Materials).

      In vivo skin inflammation models

      Experimental models for psoriasis (IMQ, IL-23 injection), AD (OVA), and barrier disruption (acetone) were induced as previously described (
      • Desmet J.
      • Verstraete K.
      • Bloch Y.
      • Lorent E.
      • Wen Y.
      • Devreese B.
      • et al.
      Structural basis of IL-23 antagonism by an Alphabody protein scaffold.
      ,
      • Hoste E.
      • Denecker G.
      • Gilbert B.
      • Van Nieuwerburgh F.
      • van der Fits L.
      • Asselbergh B.
      • et al.
      Caspase-14-deficient mice are more prone to the development of parakeratosis.
      ,
      • Staumont-Salle D.
      • Abboud G.
      • Brenuchon C.
      • Kanda A.
      • Roumier T.
      • Lavogiez C.
      • et al.
      Peroxisome proliferator-activated receptor α regulates skin inflammation and humoral response in atopic dermatitis.
      ,
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ) (see Supplementary Materials).

      Microarray analysis

      Epidermis and dermis were obtained from human skin biopsy samples by laser capture microdissection using Arcturus XT (MDS Analytical Technologies, Sunnyvale, CA) system (
      • Chinetti-Gbaguidi G.
      • Baron M.
      • Bouhlel M.A.
      • Vanhoutte J.
      • Copin C.
      • Sebti Y.
      • et al.
      Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways.
      ), followed by RNA isolation with Arcturus Picopure RNA Isolation Kit (MDS Analytical Technologies).

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported in part by grants from Agence Nationale de la Recherche (ANR) and European Union: EGID ANR-10-LABX-46 (to BS and DD) and Fondation pour la Recherche Médicale (to DD). BS is a professor at Institut Inter Universitaire de France. In addition, the research has been supported by the Flanders Institute for Biotechnology (VIB); Interuniversity Attraction Poles, IAP7/32; a Methusalem grant (BOF09/01M00709) from the Flemish Government to PV; and a UGent grant (GOA-01G01914). We also thank the VIB Microscopy core facility for excellent assistance.

      Supplementary Material

      References

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