AP1S3 Mutations Cause Skin Autoinflammation by Disrupting Keratinocyte Autophagy and Up-Regulating IL-36 Production
Identifieur interne : 002688 ( Pmc/Corpus ); précédent : 002687; suivant : 002689AP1S3 Mutations Cause Skin Autoinflammation by Disrupting Keratinocyte Autophagy and Up-Regulating IL-36 Production
Auteurs : Satveer K. Mahil ; Sophie Twelves ; Katalin Farkas ; Niovi Setta-Kaffetzi ; A. David Burden ; Joanna E. Gach ; Alan D. Irvine ; Lászl Képír ; Maja Mockenhaupt ; Hazel H. Oon ; Jason Pinner ; Annamari Ranki ; Marieke M. B. Seyger ; Pere Soler-Palacin ; Eoin R. Storan ; Eugene S. Tan ; Laurence Valeyrie-Allanore ; Helen S. Young ; Richard C. Trembath ; Siew-Eng Choon ; Marta Szell ; Zsuzsanna Bata-Csorgo ; Catherine H. Smith ; Paola Di Meglio ; Jonathan N. Barker ; Francesca CaponSource :
- The Journal of Investigative Dermatology [ 0022-202X ] ; 2016.
Abstract
Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting
Url:
DOI: 10.1016/j.jid.2016.06.618
PubMed: 27388993
PubMed Central: 5070969
Links to Exploration step
PMC:5070969Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en"><italic>AP1S3</italic>
Mutations Cause Skin Autoinflammation by Disrupting Keratinocyte Autophagy and Up-Regulating IL-36 Production</title>
<author><name sortKey="Mahil, Satveer K" sort="Mahil, Satveer K" uniqKey="Mahil S" first="Satveer K." last="Mahil">Satveer K. Mahil</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Twelves, Sophie" sort="Twelves, Sophie" uniqKey="Twelves S" first="Sophie" last="Twelves">Sophie Twelves</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Farkas, Katalin" sort="Farkas, Katalin" uniqKey="Farkas K" first="Katalin" last="Farkas">Katalin Farkas</name>
<affiliation><nlm:aff id="aff2">MTA-SZTE Dermatological Research Group, Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Setta Kaffetzi, Niovi" sort="Setta Kaffetzi, Niovi" uniqKey="Setta Kaffetzi N" first="Niovi" last="Setta-Kaffetzi">Niovi Setta-Kaffetzi</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Burden, A David" sort="Burden, A David" uniqKey="Burden A" first="A. David" last="Burden">A. David Burden</name>
<affiliation><nlm:aff id="aff3">Department of Dermatology, University of Glasgow, Glasgow, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Gach, Joanna E" sort="Gach, Joanna E" uniqKey="Gach J" first="Joanna E." last="Gach">Joanna E. Gach</name>
<affiliation><nlm:aff id="aff4">Department of Dermatology, Birmingham Children’s Hospital, Birmingham, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Irvine, Alan D" sort="Irvine, Alan D" uniqKey="Irvine A" first="Alan D." last="Irvine">Alan D. Irvine</name>
<affiliation><nlm:aff id="aff5">Paediatric Dermatology, Our Lady’s Children’s Hospital, Dublin, Ireland</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kepir, Laszl" sort="Kepir, Laszl" uniqKey="Kepir L" first="Lászl" last="Képír">Lászl Képír</name>
<affiliation><nlm:aff id="aff6">Department of Dermatology and Allergology, University of Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Mockenhaupt, Maja" sort="Mockenhaupt, Maja" uniqKey="Mockenhaupt M" first="Maja" last="Mockenhaupt">Maja Mockenhaupt</name>
<affiliation><nlm:aff id="aff7">Dokumentationszentrum schwerer Hautreaktionen (dZh) and RegiSCAR-study, Department of Dermatology, Medical Center–University of Freiburg, Freiburg, Germany</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Oon, Hazel H" sort="Oon, Hazel H" uniqKey="Oon H" first="Hazel H." last="Oon">Hazel H. Oon</name>
<affiliation><nlm:aff id="aff8">National Skin Centre, Singapore</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Pinner, Jason" sort="Pinner, Jason" uniqKey="Pinner J" first="Jason" last="Pinner">Jason Pinner</name>
<affiliation><nlm:aff id="aff9">Department of Medical Genomics, Royal Prince Alfred Hospital, Camperdown, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ranki, Annamari" sort="Ranki, Annamari" uniqKey="Ranki A" first="Annamari" last="Ranki">Annamari Ranki</name>
<affiliation><nlm:aff id="aff10">Department of Skin and Allergic Diseases, Helsinki University Central Hospital, Helsinki, Finland</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Seyger, Marieke M B" sort="Seyger, Marieke M B" uniqKey="Seyger M" first="Marieke M. B." last="Seyger">Marieke M. B. Seyger</name>
<affiliation><nlm:aff id="aff11">Department of Dermatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Soler Palacin, Pere" sort="Soler Palacin, Pere" uniqKey="Soler Palacin P" first="Pere" last="Soler-Palacin">Pere Soler-Palacin</name>
<affiliation><nlm:aff id="aff12">Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d'Hebron, Barcelona, Spain</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Storan, Eoin R" sort="Storan, Eoin R" uniqKey="Storan E" first="Eoin R." last="Storan">Eoin R. Storan</name>
<affiliation><nlm:aff id="aff13">Department of Dermatology, University Hospital, Galway, Ireland</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tan, Eugene S" sort="Tan, Eugene S" uniqKey="Tan E" first="Eugene S." last="Tan">Eugene S. Tan</name>
<affiliation><nlm:aff id="aff8">National Skin Centre, Singapore</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Valeyrie Allanore, Laurence" sort="Valeyrie Allanore, Laurence" uniqKey="Valeyrie Allanore L" first="Laurence" last="Valeyrie-Allanore">Laurence Valeyrie-Allanore</name>
<affiliation><nlm:aff id="aff14">Department of Dermatology, Henri Mondor Hospital, Paris, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Young, Helen S" sort="Young, Helen S" uniqKey="Young H" first="Helen S." last="Young">Helen S. Young</name>
<affiliation><nlm:aff id="aff15">Department of Dermatology, University of Manchester</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Trembath, Richard C" sort="Trembath, Richard C" uniqKey="Trembath R" first="Richard C." last="Trembath">Richard C. Trembath</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Choon, Siew Eng" sort="Choon, Siew Eng" uniqKey="Choon S" first="Siew-Eng" last="Choon">Siew-Eng Choon</name>
<affiliation><nlm:aff id="aff16">Department of Dermatology, Hospital Sultanah Aminah, Johor Bahru, Malaysia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Szell, Marta" sort="Szell, Marta" uniqKey="Szell M" first="Marta" last="Szell">Marta Szell</name>
<affiliation><nlm:aff id="aff2">MTA-SZTE Dermatological Research Group, Szeged, Hungary</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff17">Institute of Medical Genetics, University of Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Bata Csorgo, Zsuzsanna" sort="Bata Csorgo, Zsuzsanna" uniqKey="Bata Csorgo Z" first="Zsuzsanna" last="Bata-Csorgo">Zsuzsanna Bata-Csorgo</name>
<affiliation><nlm:aff id="aff2">MTA-SZTE Dermatological Research Group, Szeged, Hungary</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff6">Department of Dermatology and Allergology, University of Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Smith, Catherine H" sort="Smith, Catherine H" uniqKey="Smith C" first="Catherine H." last="Smith">Catherine H. Smith</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Di Meglio, Paola" sort="Di Meglio, Paola" uniqKey="Di Meglio P" first="Paola" last="Di Meglio">Paola Di Meglio</name>
<affiliation><nlm:aff id="aff18">Mill Hill Laboratory, The Francis Crick Institute, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Barker, Jonathan N" sort="Barker, Jonathan N" uniqKey="Barker J" first="Jonathan N." last="Barker">Jonathan N. Barker</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Capon, Francesca" sort="Capon, Francesca" uniqKey="Capon F" first="Francesca" last="Capon">Francesca Capon</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main"><italic>AP1S3</italic>
Mutations Cause Skin Autoinflammation by Disrupting Keratinocyte Autophagy and Up-Regulating IL-36 Production</title>
<author><name sortKey="Mahil, Satveer K" sort="Mahil, Satveer K" uniqKey="Mahil S" first="Satveer K." last="Mahil">Satveer K. Mahil</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Twelves, Sophie" sort="Twelves, Sophie" uniqKey="Twelves S" first="Sophie" last="Twelves">Sophie Twelves</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Farkas, Katalin" sort="Farkas, Katalin" uniqKey="Farkas K" first="Katalin" last="Farkas">Katalin Farkas</name>
<affiliation><nlm:aff id="aff2">MTA-SZTE Dermatological Research Group, Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Setta Kaffetzi, Niovi" sort="Setta Kaffetzi, Niovi" uniqKey="Setta Kaffetzi N" first="Niovi" last="Setta-Kaffetzi">Niovi Setta-Kaffetzi</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Burden, A David" sort="Burden, A David" uniqKey="Burden A" first="A. David" last="Burden">A. David Burden</name>
<affiliation><nlm:aff id="aff3">Department of Dermatology, University of Glasgow, Glasgow, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Gach, Joanna E" sort="Gach, Joanna E" uniqKey="Gach J" first="Joanna E." last="Gach">Joanna E. Gach</name>
<affiliation><nlm:aff id="aff4">Department of Dermatology, Birmingham Children’s Hospital, Birmingham, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Irvine, Alan D" sort="Irvine, Alan D" uniqKey="Irvine A" first="Alan D." last="Irvine">Alan D. Irvine</name>
<affiliation><nlm:aff id="aff5">Paediatric Dermatology, Our Lady’s Children’s Hospital, Dublin, Ireland</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kepir, Laszl" sort="Kepir, Laszl" uniqKey="Kepir L" first="Lászl" last="Képír">Lászl Képír</name>
<affiliation><nlm:aff id="aff6">Department of Dermatology and Allergology, University of Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Mockenhaupt, Maja" sort="Mockenhaupt, Maja" uniqKey="Mockenhaupt M" first="Maja" last="Mockenhaupt">Maja Mockenhaupt</name>
<affiliation><nlm:aff id="aff7">Dokumentationszentrum schwerer Hautreaktionen (dZh) and RegiSCAR-study, Department of Dermatology, Medical Center–University of Freiburg, Freiburg, Germany</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Oon, Hazel H" sort="Oon, Hazel H" uniqKey="Oon H" first="Hazel H." last="Oon">Hazel H. Oon</name>
<affiliation><nlm:aff id="aff8">National Skin Centre, Singapore</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Pinner, Jason" sort="Pinner, Jason" uniqKey="Pinner J" first="Jason" last="Pinner">Jason Pinner</name>
<affiliation><nlm:aff id="aff9">Department of Medical Genomics, Royal Prince Alfred Hospital, Camperdown, Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ranki, Annamari" sort="Ranki, Annamari" uniqKey="Ranki A" first="Annamari" last="Ranki">Annamari Ranki</name>
<affiliation><nlm:aff id="aff10">Department of Skin and Allergic Diseases, Helsinki University Central Hospital, Helsinki, Finland</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Seyger, Marieke M B" sort="Seyger, Marieke M B" uniqKey="Seyger M" first="Marieke M. B." last="Seyger">Marieke M. B. Seyger</name>
<affiliation><nlm:aff id="aff11">Department of Dermatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Soler Palacin, Pere" sort="Soler Palacin, Pere" uniqKey="Soler Palacin P" first="Pere" last="Soler-Palacin">Pere Soler-Palacin</name>
<affiliation><nlm:aff id="aff12">Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d'Hebron, Barcelona, Spain</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Storan, Eoin R" sort="Storan, Eoin R" uniqKey="Storan E" first="Eoin R." last="Storan">Eoin R. Storan</name>
<affiliation><nlm:aff id="aff13">Department of Dermatology, University Hospital, Galway, Ireland</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tan, Eugene S" sort="Tan, Eugene S" uniqKey="Tan E" first="Eugene S." last="Tan">Eugene S. Tan</name>
<affiliation><nlm:aff id="aff8">National Skin Centre, Singapore</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Valeyrie Allanore, Laurence" sort="Valeyrie Allanore, Laurence" uniqKey="Valeyrie Allanore L" first="Laurence" last="Valeyrie-Allanore">Laurence Valeyrie-Allanore</name>
<affiliation><nlm:aff id="aff14">Department of Dermatology, Henri Mondor Hospital, Paris, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Young, Helen S" sort="Young, Helen S" uniqKey="Young H" first="Helen S." last="Young">Helen S. Young</name>
<affiliation><nlm:aff id="aff15">Department of Dermatology, University of Manchester</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Trembath, Richard C" sort="Trembath, Richard C" uniqKey="Trembath R" first="Richard C." last="Trembath">Richard C. Trembath</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Choon, Siew Eng" sort="Choon, Siew Eng" uniqKey="Choon S" first="Siew-Eng" last="Choon">Siew-Eng Choon</name>
<affiliation><nlm:aff id="aff16">Department of Dermatology, Hospital Sultanah Aminah, Johor Bahru, Malaysia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Szell, Marta" sort="Szell, Marta" uniqKey="Szell M" first="Marta" last="Szell">Marta Szell</name>
<affiliation><nlm:aff id="aff2">MTA-SZTE Dermatological Research Group, Szeged, Hungary</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff17">Institute of Medical Genetics, University of Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Bata Csorgo, Zsuzsanna" sort="Bata Csorgo, Zsuzsanna" uniqKey="Bata Csorgo Z" first="Zsuzsanna" last="Bata-Csorgo">Zsuzsanna Bata-Csorgo</name>
<affiliation><nlm:aff id="aff2">MTA-SZTE Dermatological Research Group, Szeged, Hungary</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff6">Department of Dermatology and Allergology, University of Szeged, Hungary</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Smith, Catherine H" sort="Smith, Catherine H" uniqKey="Smith C" first="Catherine H." last="Smith">Catherine H. Smith</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Di Meglio, Paola" sort="Di Meglio, Paola" uniqKey="Di Meglio P" first="Paola" last="Di Meglio">Paola Di Meglio</name>
<affiliation><nlm:aff id="aff18">Mill Hill Laboratory, The Francis Crick Institute, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Barker, Jonathan N" sort="Barker, Jonathan N" uniqKey="Barker J" first="Jonathan N." last="Barker">Jonathan N. Barker</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Capon, Francesca" sort="Capon, Francesca" uniqKey="Capon F" first="Francesca" last="Capon">Francesca Capon</name>
<affiliation><nlm:aff id="aff1">Division of Genetics and Molecular Medicine, King’s College London, London, UK</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">The Journal of Investigative Dermatology</title>
<idno type="ISSN">0022-202X</idno>
<idno type="eISSN">1523-1747</idno>
<imprint><date when="2016">2016</date>
</imprint>
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<front><div type="abstract" xml:lang="en"><p>Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting <italic>AP1S3</italic>
, a disease gene previously identified by our group and validated here in a newly ascertained patient resource. We first showed that <italic>AP1S3</italic>
expression is distinctively elevated in keratinocytes. Because <italic>AP1S3</italic>
encodes a protein implicated in autophagosome formation, we next investigated the effects of gene silencing on this pathway. We found that <italic>AP1S3</italic>
knockout disrupts keratinocyte autophagy, causing abnormal accumulation of p62, an adaptor protein mediating NF-κB activation. We showed that as a consequence, <italic>AP1S3</italic>
-deficient cells up-regulate IL-1 signaling and overexpress IL-36α, a cytokine that is emerging as an important mediator of skin inflammation. These abnormal immune profiles were recapitulated by pharmacological inhibition of autophagy and verified in patient keratinocytes, where they were reversed by IL-36 blockade. These findings show that keratinocytes play a key role in skin autoinflammation and identify autophagy modulation of IL-36 signaling as a therapeutic target.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Invest Dermatol</journal-id>
<journal-id journal-id-type="iso-abbrev">J. Invest. Dermatol</journal-id>
<journal-title-group><journal-title>The Journal of Investigative Dermatology</journal-title>
</journal-title-group>
<issn pub-type="ppub">0022-202X</issn>
<issn pub-type="epub">1523-1747</issn>
<publisher><publisher-name>Elsevier</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">27388993</article-id>
<article-id pub-id-type="pmc">5070969</article-id>
<article-id pub-id-type="publisher-id">S0022-202X(16)32094-2</article-id>
<article-id pub-id-type="doi">10.1016/j.jid.2016.06.618</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject>
<subj-group><subject>Keratinocytes/Epidermis</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group><article-title><italic>AP1S3</italic>
Mutations Cause Skin Autoinflammation by Disrupting Keratinocyte Autophagy and Up-Regulating IL-36 Production</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Mahil</surname>
<given-names>Satveer K.</given-names>
</name>
<xref rid="aff1" ref-type="aff">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Twelves</surname>
<given-names>Sophie</given-names>
</name>
<xref rid="aff1" ref-type="aff">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Farkas</surname>
<given-names>Katalin</given-names>
</name>
<xref rid="aff2" ref-type="aff">2</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Setta-Kaffetzi</surname>
<given-names>Niovi</given-names>
</name>
<xref rid="aff1" ref-type="aff">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Burden</surname>
<given-names>A. David</given-names>
</name>
<xref rid="aff3" ref-type="aff">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Gach</surname>
<given-names>Joanna E.</given-names>
</name>
<xref rid="aff4" ref-type="aff">4</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Irvine</surname>
<given-names>Alan D.</given-names>
</name>
<xref rid="aff5" ref-type="aff">5</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Képíró</surname>
<given-names>László</given-names>
</name>
<xref rid="aff6" ref-type="aff">6</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Mockenhaupt</surname>
<given-names>Maja</given-names>
</name>
<xref rid="aff7" ref-type="aff">7</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Oon</surname>
<given-names>Hazel H.</given-names>
</name>
<xref rid="aff8" ref-type="aff">8</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Pinner</surname>
<given-names>Jason</given-names>
</name>
<xref rid="aff9" ref-type="aff">9</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ranki</surname>
<given-names>Annamari</given-names>
</name>
<xref rid="aff10" ref-type="aff">10</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Seyger</surname>
<given-names>Marieke M.B.</given-names>
</name>
<xref rid="aff11" ref-type="aff">11</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Soler-Palacin</surname>
<given-names>Pere</given-names>
</name>
<xref rid="aff12" ref-type="aff">12</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Storan</surname>
<given-names>Eoin R.</given-names>
</name>
<xref rid="aff13" ref-type="aff">13</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Tan</surname>
<given-names>Eugene S.</given-names>
</name>
<xref rid="aff8" ref-type="aff">8</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Valeyrie-Allanore</surname>
<given-names>Laurence</given-names>
</name>
<xref rid="aff14" ref-type="aff">14</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Young</surname>
<given-names>Helen S.</given-names>
</name>
<xref rid="aff15" ref-type="aff">15</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Trembath</surname>
<given-names>Richard C.</given-names>
</name>
<xref rid="aff1" ref-type="aff">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Choon</surname>
<given-names>Siew-Eng</given-names>
</name>
<xref rid="aff16" ref-type="aff">16</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Szell</surname>
<given-names>Marta</given-names>
</name>
<xref rid="aff2" ref-type="aff">2</xref>
<xref rid="aff17" ref-type="aff">17</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Bata-Csorgo</surname>
<given-names>Zsuzsanna</given-names>
</name>
<xref rid="aff2" ref-type="aff">2</xref>
<xref rid="aff6" ref-type="aff">6</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Smith</surname>
<given-names>Catherine H.</given-names>
</name>
<xref rid="aff1" ref-type="aff">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Di Meglio</surname>
<given-names>Paola</given-names>
</name>
<xref rid="aff18" ref-type="aff">18</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Barker</surname>
<given-names>Jonathan N.</given-names>
</name>
<xref rid="aff1" ref-type="aff">1</xref>
<xref rid="fn1" ref-type="fn">19</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Capon</surname>
<given-names>Francesca</given-names>
</name>
<email>francesca.capon@kcl.ac.uk</email>
<xref rid="aff1" ref-type="aff">1</xref>
<xref rid="fn1" ref-type="fn">19</xref>
<xref rid="cor1" ref-type="corresp">∗</xref>
</contrib>
</contrib-group>
<aff id="aff1"><label>1</label>
Division of Genetics and Molecular Medicine, King’s College London, London, UK</aff>
<aff id="aff2"><label>2</label>
MTA-SZTE Dermatological Research Group, Szeged, Hungary</aff>
<aff id="aff3"><label>3</label>
Department of Dermatology, University of Glasgow, Glasgow, UK</aff>
<aff id="aff4"><label>4</label>
Department of Dermatology, Birmingham Children’s Hospital, Birmingham, UK</aff>
<aff id="aff5"><label>5</label>
Paediatric Dermatology, Our Lady’s Children’s Hospital, Dublin, Ireland</aff>
<aff id="aff6"><label>6</label>
Department of Dermatology and Allergology, University of Szeged, Hungary</aff>
<aff id="aff7"><label>7</label>
Dokumentationszentrum schwerer Hautreaktionen (dZh) and RegiSCAR-study, Department of Dermatology, Medical Center–University of Freiburg, Freiburg, Germany</aff>
<aff id="aff8"><label>8</label>
National Skin Centre, Singapore</aff>
<aff id="aff9"><label>9</label>
Department of Medical Genomics, Royal Prince Alfred Hospital, Camperdown, Australia</aff>
<aff id="aff10"><label>10</label>
Department of Skin and Allergic Diseases, Helsinki University Central Hospital, Helsinki, Finland</aff>
<aff id="aff11"><label>11</label>
Department of Dermatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands</aff>
<aff id="aff12"><label>12</label>
Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d'Hebron, Barcelona, Spain</aff>
<aff id="aff13"><label>13</label>
Department of Dermatology, University Hospital, Galway, Ireland</aff>
<aff id="aff14"><label>14</label>
Department of Dermatology, Henri Mondor Hospital, Paris, France</aff>
<aff id="aff15"><label>15</label>
Department of Dermatology, University of Manchester</aff>
<aff id="aff16"><label>16</label>
Department of Dermatology, Hospital Sultanah Aminah, Johor Bahru, Malaysia</aff>
<aff id="aff17"><label>17</label>
Institute of Medical Genetics, University of Szeged, Hungary</aff>
<aff id="aff18"><label>18</label>
Mill Hill Laboratory, The Francis Crick Institute, London, UK</aff>
<author-notes><corresp id="cor1"><label>∗</label>
Correspondence: Francesca Capon, Division of Genetics and Molecular Medicine, 9th floor Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, UK.Division of Genetics and Molecular Medicine9th floor Tower WingGuy’s HospitalGreat Maze PondLondon SE1 9RTUK <email>francesca.capon@kcl.ac.uk</email>
</corresp>
<fn id="fn1"><label>19</label>
<p id="ntpara0010">These authors contributed equally to this work.</p>
</fn>
</author-notes>
<pub-date pub-type="pmc-release"><day>1</day>
<month>11</month>
<year>2016</year>
</pub-date>
<pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>11</month>
<year>2016</year>
</pub-date>
<volume>136</volume>
<issue>11</issue>
<fpage>2251</fpage>
<lpage>2259</lpage>
<history><date date-type="received"><day>23</day>
<month>3</month>
<year>2016</year>
</date>
<date date-type="rev-recd"><day>15</day>
<month>6</month>
<year>2016</year>
</date>
<date date-type="accepted"><day>25</day>
<month>6</month>
<year>2016</year>
</date>
</history>
<permissions><copyright-statement>© 2016 The Authors</copyright-statement>
<copyright-year>2016</copyright-year>
<license license-type="CC BY" xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</license-p>
</license>
</permissions>
<abstract id="abs0010"><p>Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting <italic>AP1S3</italic>
, a disease gene previously identified by our group and validated here in a newly ascertained patient resource. We first showed that <italic>AP1S3</italic>
expression is distinctively elevated in keratinocytes. Because <italic>AP1S3</italic>
encodes a protein implicated in autophagosome formation, we next investigated the effects of gene silencing on this pathway. We found that <italic>AP1S3</italic>
knockout disrupts keratinocyte autophagy, causing abnormal accumulation of p62, an adaptor protein mediating NF-κB activation. We showed that as a consequence, <italic>AP1S3</italic>
-deficient cells up-regulate IL-1 signaling and overexpress IL-36α, a cytokine that is emerging as an important mediator of skin inflammation. These abnormal immune profiles were recapitulated by pharmacological inhibition of autophagy and verified in patient keratinocytes, where they were reversed by IL-36 blockade. These findings show that keratinocytes play a key role in skin autoinflammation and identify autophagy modulation of IL-36 signaling as a therapeutic target.</p>
</abstract>
<kwd-group id="kwrds0010"><title>Abbreviations</title>
<kwd>3-MA, 3-methyladenine</kwd>
<kwd>AID, autoinflammatory disorder</kwd>
<kwd>CRISPR, clustered regularly-interspaced short palindromic repeats</kwd>
<kwd>Cas9, CRISPR-associated endonuclease 9</kwd>
<kwd>GFP, green fluorescent protein</kwd>
<kwd>MALP-2, macrophage-activating lipopeptide 2</kwd>
<kwd>siRNA, small interfering RNA</kwd>
<kwd>TLR-2/6, Toll-like receptor 2/6</kwd>
</kwd-group>
</article-meta>
<notes><p id="misc0010">accepted manuscript published online 5 July 2016; corrected proof published online 12 August 2016</p>
</notes>
</front>
<body><sec id="sec1"><title>Introduction</title>
<p>Autoinflammatory disorders (AIDs) are a group of inherited conditions caused by abnormal activation of the innate immune system. AIDs typically present with recurrent and seemingly unprovoked episodes of systemic upset, which are almost invariably accompanied by joint and skin inflammation (<xref rid="bib2" ref-type="bibr">Aksentijevich and Kastner, 2011</xref>
). The latter can manifest with urticarial, pustular, or ulcerative eruptions, which are considered important markers of disease activity (<xref rid="bib3" ref-type="bibr">Beer et al., 2014</xref>
).</p>
<p>In the last 15 years, genetic studies have identified more than 30 AID genes, illuminating fundamental innate immune pathways and highlighting pathogenic mechanisms (most notably, abnormal IL-1 production) that have been successfully targeted by therapeutic interventions (<xref rid="bib5" ref-type="bibr">de Jesus et al., 2015</xref>
).</p>
<p>Despite these successes, the basis of organ-specific disease manifestations is still unclear. This is particularly true of skin pathology, because the nature of the cells and molecular mechanisms that mediate cutaneous inflammation in AID remain poorly defined (<xref rid="bib3" ref-type="bibr">Beer et al., 2014</xref>
).</p>
<p>We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a severe AID manifesting with repeated eruptions of painful skin pustules. These can be localized to the palms and soles (palmar plantar pustulosis), toes and fingertips (acrodermatitis continua of Hallopeau) or affect most of the body surface (generalized pustular psoriasis). Although the lesions can be accompanied by arthritis and systemic upset, cutaneous involvement is the most prominent clinical feature of the disease (<xref rid="bib8" ref-type="bibr">Griffiths and Barker, 2010</xref>
). This makes pustular psoriasis an ideal model for investigating the molecular mechanisms that drive skin inflammation in AID.</p>
<p>We specifically investigated the pathogenic role of <italic>AP1S3</italic>
, a gene that we found to be mutated in all forms of pustular psoriasis (<xref rid="bib22" ref-type="bibr">Setta-Kaffetzi et al., 2014</xref>
). <italic>AP1S3</italic>
encodes a subunit of AP-1, a heterotetramer that mediates membrane trafficking between the post-Golgi network and the endosome (<xref rid="bib20" ref-type="bibr">Robinson, 2004</xref>
). The complex is composed of two large (AP-1γ1 and AP-1β1), one medium (AP-1μ1) and one small subunit (AP-1σ1). The latter exists in three alternative forms (AP-1σ1A, AP-1σ1B and AP-1σ1C), encoded by paralogous genes (<italic>AP1S1</italic>
, <italic>AP1S2</italic>
, <italic>AP1S3</italic>
), so that the <italic>AP1S3</italic>
product is AP-1σ1C (<xref rid="fig1" ref-type="fig">Figure 1</xref>
a). The σ1 subunit confers stability to AP-1 tetramers, so that mutations in <italic>AP1S</italic>
genes are expected to disrupt the entire complex (<xref rid="bib20" ref-type="bibr">Robinson, 2004</xref>
).</p>
<p>The AP-1 complex has also been implicated in the formation of autophagosomes (<xref rid="bib9" ref-type="bibr">Guo et al., 2012</xref>
). These are specialized vesicles that mediate the degradation of cellular components by autophagy, a catabolic process that can be activated by nutrient stress (e.g., starvation). Given that autophagy modulates cytokine production downstream of various pattern recognition receptors (<xref rid="bib17" ref-type="bibr">Netea-Maier et al., 2016</xref>
), we hypothesized that <italic>AP1S3</italic>
mutations would disturb autophagic activity, causing innate immune dysregulation. We then validated our pathogenic model in a variety of in vitro experimental systems and in patient cells.</p>
</sec>
<sec id="sec2"><title>Results</title>
<sec id="sec2.1"><title>Validation of <italic>AP1S3</italic>
as a pustular psoriasis gene</title>
<p>Although we previously reported that two <italic>AP1S3</italic>
mutations (p.Phe4Cys and p.Arg33Trp) account for approximately 10% of European pustular psoriasis patients (<xref rid="bib22" ref-type="bibr">Setta-Kaffetzi et al., 2014</xref>
), the rarity of the disease has hindered the replication of this finding. To address this issue, we screened the <italic>AP1S3</italic>
coding region in 85 newly ascertained patients (53 European and 32 non-European subjects) (see <xref rid="appsec1" ref-type="sec">Supplementary Table S1</xref>
online), recruited across Europe and East Asia. This uncovered p.Phe4Cys and p.Arg33Trp alleles in five unrelated individuals (n = 3 generalized pustular psoriasis and n = 2 palmar plantar pustulosis patients) (<xref rid="tbl1" ref-type="table">Table 1</xref>
). All were of European descent, confirming the limited geographic distribution of the two mutations. Two of the three generalized pustular psoriasis patients carried the <italic>AP1S3</italic>
mutation in conjunction with a deleterious change in <italic>IL36RN</italic>
, a pustular psoriasis gene encoding the IL-36 receptor antagonist (<xref rid="bib15" ref-type="bibr">Marrakchi et al., 2011</xref>
, <xref rid="bib18" ref-type="bibr">Onoufriadis et al., 2011</xref>
). One of these individuals exhibited a particularly severe, recalcitrant phenotype and had a sister with a milder form of the disease, who only carried the <italic>IL36RN</italic>
mutation (see <xref rid="appsec1" ref-type="sec">Supplementary Table S2</xref>
online).</p>
<p>Taken together, these observations validate the involvement of <italic>AP1S3</italic>
in pustular psoriasis and suggest the possibility of epistasis between <italic>IL36RN</italic>
and <italic>AP1S3</italic>
alleles.</p>
</sec>
<sec id="sec2.2"><title><italic>AP1S3</italic>
mutations disrupt protein function in keratinocytes</title>
<p>Structural homology modeling indicates that the p.Phe4Cys change maps to a β-sheet required for protein folding, whereas the p.Arg33Trp substitution is expected to disrupt the interaction between AP-1σ1C and AP-1μ1A (<xref rid="bib22" ref-type="bibr">Setta-Kaffetzi et al., 2014</xref>
). This strongly suggests that both mutations are loss-of-function alleles.</p>
<p>To validate these predictions, we first examined the effect of p.Phe4Cys on the thermal stability of AP-1σ1C. After transfection of wild-type and mutant <italic>AP1S3</italic>
constructs into HEK293 cultures, we subjected cell lysates to a temperature gradient and monitored AP-1σ1C levels by western blotting. We found that p.Phe4Cys proteins were denatured significantly more quickly than their wild-type counterparts (<xref rid="fig1" ref-type="fig">Figure 1</xref>
b), confirming that the mutation disrupts AP-1σ1C stability.</p>
<p>To investigate the impact of the p.Arg33Trp allele, we carried out co-immunoprecipitation experiments, using FLAG-<italic>AP1M1</italic>
and myc-<italic>AP1S3</italic>
constructs transfected into HEK293 cells. As expected, we found that wild-type myc-AP1σ1C co-precipitated with FLAG-AP1μ1A. This interaction, however, was disrupted when FLAG-<italic>AP1M1</italic>
was co-transfected with a p.Arg33Trp myc-<italic>AP1S3</italic>
cDNA (<xref rid="fig1" ref-type="fig">Figure 1</xref>
c). Similar results were obtained in immunofluorescence experiments, showing that wild-type myc-AP1σ1C co-localized with FLAG-AP1μ1A, whereas the mutant p.Arg33Trp protein did not (see <xref rid="appsec1" ref-type="sec">Supplementary Figure S1</xref>
online). Thus, we concluded that the p.Arg33Trp mutation disturbs the interaction between AP-1σ1C and AP-1μ1A, as predicted in-silico.</p>
<p>Having validated the loss-of-function nature of disease alleles, we sought to establish which cell types are most likely to be affected by <italic>AP1S3</italic>
deficiency. We therefore measured gene expression in biologically relevant cell populations. Although transcript levels were low in neutrophils and virtually undetectable in CD4<sup>+</sup>
T lymphocytes, we observed abundant gene expression in keratinocytes (<xref rid="fig1" ref-type="fig">Figure 1</xref>
d). The impact of disease alleles was therefore modeled in this cell type.</p>
</sec>
<sec id="sec2.3"><title><italic>AP1S3</italic>
deficiency causes impaired keratinocyte autophagy</title>
<p>Because autophagosome formation requires a functional AP-1 complex (<xref rid="bib9" ref-type="bibr">Guo et al., 2012</xref>
), we hypothesized that <italic>AP1S3</italic>
loss-of-function mutations may disrupt keratinocyte autophagy.</p>
<p>We first examined this possibility in a HaCaT keratinocyte cell line stably transduced with a silencing <italic>AP1S3</italic>
small hairpin RNA (<xref rid="bib22" ref-type="bibr">Setta-Kaffetzi et al., 2014</xref>
) (<xref rid="fig2" ref-type="fig">Figure 2</xref>
a). After inducing autophagy by starvation, we monitored the conversion of the LC3-I protein into its modified form (LC3-II), which is a well-recognized autophagosome marker (<xref rid="bib11" ref-type="bibr">Klionsky et al., 2012</xref>
). We found that LC3-II levels were significantly reduced in <italic>AP1S3</italic>
knockdown versus control cell lines (<xref rid="fig2" ref-type="fig">Figure 2</xref>
b).</p>
<p>We then repeated the experiment in a HEK293 <italic>AP1S3</italic>
knockout cell line, generated by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated endonuclease-9 (Cas9) genome editing (<xref rid="fig2" ref-type="fig">Figure 2</xref>
c). This confirmed that <italic>AP1S3</italic>
silencing causes a very significant decrease in starvation-induced LC3-II accumulation (<xref rid="fig2" ref-type="fig">Figure 2</xref>
d).</p>
<p>To further validate our findings, we used fluorescence microscopy to visualize the expression of LC3-green fluorescent protein (GFP) constructs transfected into the HEK293 <italic>AP1S3</italic>
knockout cell line. We found that the number of autophagosomes that had incorporated LC3-GFP was significantly reduced in knockout versus control cells. This phenotype was rescued by the overexpression of wild-type but not mutant (p.Arg33Trp) <italic>AP1S3</italic>
constructs (<xref rid="fig2" ref-type="fig">Figure 2</xref>
e).</p>
<p>Thus, <italic>AP1S3</italic>
deficiency disrupts autophagy induction in multiple experimental systems.</p>
</sec>
<sec id="sec2.4"><title><italic>AP1S3</italic>
deficiency results in abnormal p62 accumulation and enhanced Toll-like receptor (TLR) 2/6 signaling</title>
<p>It has been shown that keratinocyte autophagy modulates NF-κB activation downstream of TLR-2/6 by regulating the degradation of the p62 adaptor protein (<xref rid="bib12" ref-type="bibr">Lee et al., 2011</xref>
). This led us to hypothesize that <italic>AP1S3</italic>
deficiency would cause an abnormal accumulation of p62, resulting in enhanced NF-κB signaling. We therefore measured p62 protein levels in keratinocytes cultured from the hair plucks of one affected individual (carrying the <italic>AP1S3</italic>
p.Arg33Trp mutation) and two healthy control subjects. We found that p62 expression was markedly increased in the patient’s cells (<xref rid="fig3" ref-type="fig">Figure 3</xref>
a). A similar up-regulation was observed in normal primary keratinocytes transfected with <italic>AP1S3</italic>
small interfering RNAs (siRNAs) (<xref rid="fig3" ref-type="fig">Figure 3</xref>
b and 3c) and in a HaCaT <italic>AP1S3</italic>
knockout cell line (see <xref rid="appsec1" ref-type="sec">Supplementary Figure S2</xref>
a and b online). We therefore concluded that the abnormal p62 accumulation observed in the patient was a result of <italic>AP1S3</italic>
deficiency.</p>
<p>To further explore these findings, we measured macrophage-activating lipopeptide 2 (MALP-2)–induced cytokine expression in primary keratinocytes transiently transfected with <italic>AP1S3</italic>
siRNAs (<xref rid="fig3" ref-type="fig">Figure 3</xref>
d). Although there was no <italic>IL1B</italic>
, <italic>IL6,</italic>
or <italic>IL8</italic>
induction at the examined time point, we detected a marked increase in <italic>TNFA</italic>
levels. We also observed a significant induction of <italic>IL36A</italic>
(but not <italic>IL36B</italic>
or <italic>IL36G</italic>
), a cytokine that drives abnormal immune signaling in patients with <italic>IL36RN</italic>
mutations (<xref rid="bib18" ref-type="bibr">Onoufriadis et al., 2011</xref>
). Importantly, the induction of <italic>TNFA</italic>
and <italic>IL36A</italic>
was significantly enhanced in <italic>AP1S3</italic>
-deficient cells compared with control (<xref rid="fig3" ref-type="fig">Figure 3</xref>
d).</p>
<p>We then repeated the MALP-2 stimulations in the HaCaT <italic>AP1S3</italic>
knockout cell line. This confirmed the abnormal induction of <italic>TNFA</italic>
and <italic>IL36A</italic>
in knockout cells (see <xref rid="appsec1" ref-type="sec">Supplementary Figure S2</xref>
c).</p>
</sec>
<sec id="sec2.5"><title><italic>AP1S3</italic>
deficiency causes abnormal IL-1 signaling and up-regulates baseline IL-36 expression</title>
<p>Autophagy-mediated degradation of p62 also regulates IL-1 signaling (<xref rid="bib13" ref-type="bibr">Lee et al., 2012</xref>
), a response that plays a major role in autoinflammation. To determine whether <italic>AP1S3</italic>
deficiency would also affect this pathway, we transfected primary keratinocytes with <italic>AP1S3</italic>
siRNA pools and measured cytokine levels after IL-1 stimulation. Although <italic>TNFA</italic>
expression was unchanged at the examined time point, we observed a clear up-regulation of <italic>IL1B</italic>
, <italic>IL8,</italic>
and <italic>IL36A</italic>
transcripts. The induction of all cytokines was markedly up-regulated in <italic>AP1S3</italic>
-deficient cells compared with control (<xref rid="fig4" ref-type="fig">Figure 4</xref>
a). These observations were replicated in HaCaT <italic>AP1S3</italic>
-knockout cells (see <xref rid="appsec1" ref-type="sec">Supplementary Figure S3</xref>
a online), thus validating the effects of gene silencing on IL-1 signaling.</p>
<p>Surprisingly, our experiments showed that baseline <italic>IL36A</italic>
expression was markedly increased in <italic>AP1S3-</italic>
deficient cells, both at the RNA and protein levels (<xref rid="fig4" ref-type="fig">Figures 4</xref>
a and b, and see <xref rid="appsec1" ref-type="sec">Supplementary Figure S2</xref>
c). A similar, although less pronounced, effect was also observed for <italic>IL36B</italic>
and <italic>IL36G</italic>
mRNA expression (see <xref rid="appsec1" ref-type="sec">Supplementary Figures S4a and S4</xref>
b online) and IL-8 protein secretion (<xref rid="fig4" ref-type="fig">Figure 4</xref>
b).</p>
<p>To determine whether this up-regulation was also a consequence of impaired autophagy, we cultured normal primary keratinocytes in medium supplemented with 3-methyladenine (3-MA), an agent that blocks the formation of autophagosomes (<xref rid="bib11" ref-type="bibr">Klionsky et al., 2012</xref>
). As expected, we found that 3-MA treatment caused an increase in IL-1–dependent cytokine expression. <italic>IL36A</italic>
baseline expression was also up-regulated by 3-MA (<xref rid="fig4" ref-type="fig">Figure 4</xref>
c). These observations, which were replicated in HaCaT keratinocytes (see <xref rid="appsec1" ref-type="sec">Supplementary Figure S3</xref>
b), show that the proinflammatory effects of <italic>AP1S3</italic>
deficiency are mediated by a disruption of keratinocyte autophagy.</p>
</sec>
<sec id="sec2.6"><title>Patients harboring <italic>AP1S3</italic>
mutations up-regulate IL-36 expression and IL-1 signaling</title>
<p>To validate the pathophysiological relevance of our findings, we cultured keratinocytes from the hair plucks of two affected individuals (each carrying an <italic>AP1S3</italic>
mutation and a wild-type <italic>IL36RN</italic>
sequence) and two healthy control subjects. Although we observed only a weak response to MALP-2 stimulation, we found that cytokine levels were robustly up-regulated after IL-1 treatment. Importantly, the induction of <italic>IL1B, IL8,</italic>
and <italic>IL36A</italic>
transcripts was increased in the keratinocytes of patients compared with control (<xref rid="fig5" ref-type="fig">Figure 5</xref>
a), replicating the results generated in <italic>AP1S3</italic>
-knockdown cells.</p>
<p>The basal expression of IL-36 cytokines was also up-regulated in patient keratinocytes (<xref rid="fig5" ref-type="fig">Figure 5</xref>
a and b and see <xref rid="appsec1" ref-type="sec">Supplementary Figure S4</xref>
c), further validating the data obtained in <italic>AP1S3</italic>
-deficient cells. <italic>IL1B</italic>
and <italic>IL8</italic>
baseline transcripts were also significantly overexpressed in the examined individuals (<xref rid="fig5" ref-type="fig">Figure 5</xref>
a).</p>
<p>To further investigate the mechanisms underlying these observations, we measured transcript levels after autophagy induction by starvation, or blockade, of the IL-36 receptor with a recombinant antagonist (IL-36Ra). We found that both treatments could lower patient cytokine expression to the levels observed in healthy control subjects (<xref rid="fig5" ref-type="fig">Figure 5</xref>
c, and see <xref rid="appsec1" ref-type="sec">Supplementary Figure S4</xref>
d). Although the experiment was carried out in a single patient, the results were also replicated in <italic>AP1S3</italic>
-knockout cells (see <xref rid="appsec1" ref-type="sec">Supplementary Figure S5</xref>
online), suggesting that impaired autophagy and enhanced IL-36 signaling both contribute to the abnormal immune profile associated with <italic>AP1S3</italic>
mutations.</p>
</sec>
</sec>
<sec id="sec3"><title>Discussion</title>
<p>The aim of our study was to characterize the molecular mechanisms underlying the cutaneous features of AIDs. We therefore investigated the pathogenesis of pustular psoriasis, focusing our attention on <italic>AP1S3,</italic>
a gene that is specifically mutated in this disease. We first validated the pathogenic involvement of this locus by demonstrating the presence of disease alleles in five of the 53 European patients (9.4%) who were included in our screening. We observed that <italic>AP1S3</italic>
mutations can be inherited in conjunction with <italic>IL36RN</italic>
changes, modifying the phenotypic effect of the latter. This suggests that <italic>AP1S3</italic>
alleles may exacerbate the effects of <italic>IL36RN</italic>
deficiency by disturbing IL-36 homeostasis, a notion that is borne out by the results of our functional studies.</p>
<p>First, our experiments showed that <italic>AP1S3</italic>
expression was low or undetectable in cells that do not respond to IL-36 stimulation (neutrophils and CD4<sup>+</sup>
T cells), whereas transcript levels were abundant in keratinocytes, where IL-36 signaling can be activated by TLR agonists (<xref rid="bib7" ref-type="bibr">Gabay and Towne, 2015</xref>
). The only other known gene for pustular psoriasis (<italic>CARD14</italic>
) is also abundantly expressed in keratinocytes (<xref rid="bib4" ref-type="bibr">Berki et al., 2015</xref>
), suggesting that these cells play an important role in cutaneous autoinflammation. This is in keeping with the emerging view of keratinocytes as immune sentinels contributing to host defense through the engagement of innate receptors and the production of proinflammatory mediators (<xref rid="bib6" ref-type="bibr">Di Meglio et al., 2011</xref>
, <xref rid="bib14" ref-type="bibr">Lowes et al., 2013</xref>
).</p>
<p>The involvement of <italic>AP1S3</italic>
in IL-36 regulation is also supported by repeated observations of increased <italic>IL36A</italic>
expression in <italic>AP1S3</italic>
-deficient cells and in nonlesional keratinocytes, derived from patient hair plucks. Of note, stable <italic>AP1S3</italic>
knockout also led to constitutive up-regulation of <italic>IL1B</italic>
and <italic>IL8</italic>
(see <xref rid="appsec1" ref-type="sec">Supplementary Figure S3</xref>
). Although this phenotype mirrored the expression profile observed in patients, it was not replicated in the transient gene-silencing experiments, where mRNA levels were measured shortly after knockdown initiation. Although <italic>IL36A</italic>
was up-regulated at this early time point, the other two cytokines were not, suggesting that the overexpression of <italic>IL1B</italic>
and <italic>IL8</italic>
may be secondary to IL-36 accumulation. Indeed, our experiments showed that IL-36 receptor blockade is sufficient to normalize <italic>IL1B</italic>
and <italic>IL8</italic>
levels in patient keratinocytes.</p>
<p>Thus, our observations place IL-36 at the center of a proinflammatory loop that drives enhanced cytokine production in skin autoinflammation (see <xref rid="appsec1" ref-type="sec">Supplementary Figure S6</xref>
online). This is in keeping with the results of recent studies, indicating that <italic>IL36A</italic>
is markedly up-regulated in psoriatic skin and that this is unlikely to be a secondary consequence of inflammation (<xref rid="bib24" ref-type="bibr">Swindell et al., 2016</xref>
). Given that therapeutics blocking IL-36 are now under development (<xref rid="bib25" ref-type="bibr">Wolf and Ferris, 2014</xref>
), these discoveries have important translational implications.</p>
<p>Our experiments show that the effects of <italic>AP1S3</italic>
mutations on cytokine production are mediated by disruption of keratinocyte autophagy, causing abnormal p62 accumulation and enhanced NF-κB activation downstream of TLR-2/6 and IL-1R. Of note, p62 transcripts are up-regulated in psoriatic lesions, whereas the expression of molecules that contribute to skin inflammation is reduced in p62-deficient keratinocytes (<xref rid="bib12" ref-type="bibr">Lee et al., 2011</xref>
).</p>
<p>Here, IL-1 treatment of patient cells (which overexpress p62) caused a moderate (∼2-fold) induction of <italic>IL1B</italic>
transcripts (<xref rid="fig5" ref-type="fig">Figure 5</xref>
) but a substantial up-regulation of <italic>IL8</italic>
(>20-fold). Given that the latter cytokine plays a fundamental role in driving neutrophilic skin infiltration, this finding has a clear pathological relevance.</p>
<p>Autophagy can also modulate cytokine production at the posttranslational level, by degrading components of the inflammasome, the molecular complex that cleaves pro-IL1β into a bioactive molecule (<xref rid="bib23" ref-type="bibr">Shi et al., 2012</xref>
). Although this process has been chiefly investigated in mouse macrophages, it might also be active in human keratinocytes, where it could amplify the effects of <italic>AP1S3</italic>
mutations.</p>
<p>It is now widely accepted that perturbations of protein degradation play a pathogenic role in various AIDs with prominent dermatological features (<xref rid="bib16" ref-type="bibr">Martinon and Aksentijevich, 2015</xref>
). Evidence recently generated in animal models also indicates that therapeutic effects of anakinra (an IL-1 blocker widely used for the treatment of AIDs) are partly mediated by the rescue of defective autophagy (<xref rid="bib10" ref-type="bibr">Iannitti et al., 2016</xref>
). In the light of this evidence, our work warrants further studies of impaired keratinocyte autophagy as a pathogenic mechanism and therapeutic target in skin autoinflammation.</p>
</sec>
<sec id="sec4"><title>Methods</title>
<sec id="sec4.1"><title>Participants</title>
<p>This study was performed in accordance with the declaration of Helsinki and was approved by the ethics committees of participating institutions. Written informed consent was obtained from all participants. Patients were ascertained by trained dermatologists (see <xref rid="appsec1" ref-type="sec">Supplementary Table S3</xref>
online) on the basis of established diagnostic criteria (<xref rid="bib8" ref-type="bibr">Griffiths and Barker, 2010</xref>
). Patients 1 and 2 were described elsewhere as T002206 and T001882, respectively (<xref rid="bib22" ref-type="bibr">Setta-Kaffetzi et al., 2014</xref>
). Healthy volunteers were recruited within King’s College London. All affected individuals were screened for <italic>IL36RN</italic>
and <italic>AP1S3</italic>
mutations as described (<xref rid="bib18" ref-type="bibr">Onoufriadis et al., 2011</xref>
, <xref rid="bib22" ref-type="bibr">Setta-Kaffetzi et al., 2014</xref>
).</p>
</sec>
<sec id="sec4.2"><title>Plasmids and constructs</title>
<p>The wild-type and mutant myc-tagged <italic>AP1S3</italic>
constructs are described elsewhere (<xref rid="bib22" ref-type="bibr">Setta-Kaffetzi et al., 2014</xref>
). The FLAG-<italic>AP1M1</italic>
construct was generated by cloning the gene coding sequence into a c-Flag pcDNA3 vector (Addgene #20011). CRISPR/Cas9 guide RNAs (see <xref rid="appsec1" ref-type="sec">Supplementary Table S4</xref>
online) were designed with the CRISPR design tool (<ext-link ext-link-type="uri" xlink:href="http://crispr.mit.edu/" id="intref0010">http://crispr.mit.edu/</ext-link>
) and cloned into a pSpCas9BB-2A-GFP vector (Addgene #48138), as described elsewhere (<xref rid="bib19" ref-type="bibr">Ran et al., 2013</xref>
). All constructs were validated by Sanger sequencing.</p>
</sec>
<sec id="sec4.3"><title>Primary cell culture</title>
<p>Primary keratinocytes and dermal fibroblasts were isolated from healthy skin discarded after plastic surgery. The keratinocytes were maintained in Epilife keratinocyte medium supplemented with Supplement 7 and 1% penicillin-streptomycin, and the fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (all reagents from Gibco, Waltham, MA).</p>
<p>Keratinocytes were derived from hair plucks as described elsewhere (<xref rid="bib1" ref-type="bibr">Aasen and Izpisua Belmonte, 2010</xref>
). Briefly, 12 hairs were plucked from the temporal scalp and placed in mTeSR1 medium (Stem Cell Technologies, Vancouver, Canada) containing 1% penicillin-streptomycin and 250 ng/ml amphotericin B (Sigma, St. Louis, MO). Once outgrowths were visible, mTeSR1 was replaced with Epilife keratinocyte medium containing Supplement 7 and 1% penicillin-streptomycin. After 14 days, cells were stimulated.</p>
</sec>
<sec id="sec4.4"><title>Thermal stability assay</title>
<p>HEK293 cells were transfected with the indicated constructs, using Lipofectamine 2000 (Life Technologies, Waltham, MA). Cell lysates were then incubated for 5 minutes across a 37–57 °C temperature gradient. Samples were centrifuged for 30 minutes at 13,000 rpm at 4 °C, and the soluble fraction (supernatant) was analyzed by Western blotting.</p>
</sec>
<sec id="sec4.5"><title>CRISPR/Cas9 genome editing</title>
<p>The protocol described by <xref rid="bib19" ref-type="bibr">Ran et al. (2013)</xref>
was used to edit HaCaT and HEK293 cells maintained in complete DMEM. Briefly, the guide RNA construct was transfected into the cells, using Lipofectamine 2000. After 48 hours, GFP-positive cells were isolated by flow cytometry and seeded for clonal expansion. The resulting cell lines were validated by Sanger sequencing of the target region, paralogous loci, and off-target sites predicted by the CRISPR design tool. The expression of <italic>AP1S1</italic>
, <italic>AP1S2,</italic>
and <italic>AP1S3</italic>
was also measured by real-time PCR. Control cells were transfected with an empty pSpCas9BB-2A-GFP vector.</p>
</sec>
<sec id="sec4.6"><title>Cell stimulation</title>
<p>For autophagy induction, cells were starved for 18 hours in Hank’s Balanced Salt solution (Gibco), and protein extracts were analyzed by Western blotting. For autophagy inhibition, cells were pretreated with 10mmol/L of 3-MA (Sigma) for 5 hours and then stimulated with 20ng/ml of IL-1β (Sigma) for 2 hours, in the presence 3-MA.</p>
<p>Alternatively, primary or immortalized keratinocytes were treated with 100ng/ml of MALP2 (Bio-techne, Minneapolis, MN) for 42 hours, 20ng/ml of IL-1β for 2 hours, 100ng/ml of IL-36Ra (Bio-techne) for 5 hours or were starved as described.</p>
<p>For transient gene-silencing experiments, cells were transfected for 48 hours with 33 nmol/L of <italic>AP1S3</italic>
ON-TARGET plus SMARTpool siRNA or ON-TARGETplus nontargeting siRNA (GE Dharmacon, Lafayette, CO) using Lipofectamine 2000 and stimulated as described above.</p>
</sec>
<sec id="sec4.7"><title>Real-time PCR and ELISA</title>
<p>RNAs isolated from skin, lymphocytes, in vitro derived macrophages/dendritic cells, and neutrophils were provided by Frank Nestle, Susan John, Leonie Taams (King’s College London), and Benjamin Fairfax (Wellcome Trust Centre for Human Genetics, Oxford), respectively. All remaining RNAs were isolated using the RNeasy Mini Plus kit (Qiagen, Hilden, Germany). Gene expression was assessed by real-time PCR by using the primers listed in <xref rid="appsec1" ref-type="sec">Supplementary Table S4</xref>
online. Transcript levels were normalized to <italic>PPIA</italic>
or <italic>B2M</italic>
expression, measured with Applied Biosystems (Foster City, CA) TaqMan probes. IL-36α and IL-8 production was measured with the Human IL36A ELISA Kit (Sigma) and Human IL-8 ELISA Kit (Sigma).</p>
</sec>
<sec id="sec4.8"><title>Co-immunoprecipitation and Western blotting</title>
<p>A rabbit monoclonal anti-FLAG (1:50, Cell Signaling Technology, Danvers, MA) was used in co-immunoprecipitation experiments, whereas Western blots were probed with rabbit polyclonal anti-LC3 (Cell Signaling Technology), rabbit polyclonal anti-β actin (Cell Signaling Technology), rabbit polyclonal anti-p62 (Sigma), or mouse monoclonal anti-myc (Thermo Scientific, Waltham, MA) (all 1:1,000). Densitometry was undertaken with Image J software (<xref rid="bib21" ref-type="bibr">Schneider et al., 2012</xref>
).</p>
</sec>
<sec id="sec4.9"><title>Immunofluorescence microscopy</title>
<p>In the co-localization experiments, HEK293 cells were co-transfected with the indicated constructs, using Lipofectamine 2000. After 24 hours, cells were fixed and incubated with 1:500 mouse monoclonal anti-myc (Cell Signaling Technology) and 1:600 rabbit monoclonal anti-FLAG. Slides were imaged by using a C2 confocal microscope (Nikon, Tokyo, Japan), and z-stack images of at least 15 cells per slide were taken.</p>
<p>In autophagy induction experiments, HEK293 cells were transfected with a pEGFP-LC3 plasmid (Addgene #24920) and the indicated construct, using Lipofectamine 2000. After 24 hours, cells were starved for 18 hours in Hank’s Balanced Salt solution supplemented with 0.1 μmol/L of Bafilomycin A1 (Sigma). Cells were imaged as described above and autophagosomes were counted by using NIS-Elements Advanced Research software (Nikon).</p>
</sec>
<sec id="sec4.10"><title>Statistics</title>
<p>Means were compared with unpaired Student <italic>t</italic>
tests. Error bars represent standard error of the mean.</p>
</sec>
</sec>
<sec id="sec5"><title>ORCID</title>
<p>Francesca Capon: <ext-link ext-link-type="uri" xlink:href="http://orcid.org/0000-0003-2432-5793" id="interref0010">http://orcid.org/0000-0003-2432-5793</ext-link>
</p>
<p>Alan Irvine: <ext-link ext-link-type="uri" xlink:href="http://orcid.org/0000-0002-9048-2044" id="intref0015">http://orcid.org/0000-0002-9048-2044</ext-link>
</p>
</sec>
<sec id="sec6"><title>Conflict of Interest</title>
<p>Maja Mockenhaupt is the coordinator of the international RegiSCAR-project, which was/is funded (among others) by a consortium of pharmaceutical companies (Bayer Vital, Boehringer-lngelheim, Cephalon, GlaxoSmithKline, MSD Sharp and Dohme, Merck, Novartis, Pfizer, Roche, Sanofi-Aventis, Servier, Tibotec, Grünenthal, Falk Pharma, UCB Biopharma, AB-Science). Maja Mockenhaupt is also a member of expert panels/advisory boards in the field of severe cutaneous adverse reaction coordinated by pharmaceutical companies (Boehringer Ingelheim, Merck, Sanofi). She has also been an expert in litigations concerning Stevens Johnson syndrome/toxic epidermal necrolysis. Helen Young is/has been a consultant or speaker to Abbott/Abbvie, Amgen, Janssen-Cilag, Leo-Pharma, Novartis, Lily, Stiefel, Teva Pharmaceuticals, and Wyeth/Pfizer.</p>
</sec>
</body>
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</p>
</sec>
<ack id="ack0010"><title>Acknowledgments</title>
<p>This work was supported by the Department of Health via the National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s & St. Thomas’ National Health Service Foundation Trust in partnership with King’s College London, King’s College Hospital National Health Service Foundation Trust. FC, RCT, and JNB are supported by the Medical Research Council (award MR/L011808/1). ZB and MS were supported by Orszagos Tudomanyos Kutatasi Alap (National Scientific Research Fund) grants OTKA K105985 and OTKA K111885. MM and LV are part of The International Registry of Severe Cutaneous Adverse Reaction (RegiSCAR) Consortium, funded by the European Commission (QLRT-2002-01738), GIS-Institut des Maladies Rares, and Institut National de la Santé et de la Reserche Médicale (INSERM) (4CH09G) in France and by a consortium of pharmaceutical companies. SKM is funded by a Medical Research Council Clinical Training Fellowship (MR/L001543/1). ST is supported by the King’s Bioscience Institute and the Guy’s and St Thomas’ Charity Prize PhD Programme in Biomedical and Translational Science. NSK received a British Skin Foundation PhD studentship (grant 3007s).</p>
</ack>
<fn-group><fn id="appsec2" fn-type="supplementary-material"><p>Supplementary material is linked to the online version of the paper at <ext-link ext-link-type="uri" xlink:href="http://www.jidonline.org" id="intref0020">www.jidonline.org</ext-link>
, and at <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.jid.2016.06.618" id="intref0025">http://dx.doi.org/10.1016/j.jid.2016.06.618</ext-link>
.</p>
</fn>
</fn-group>
</back>
<floats-group><fig id="fig1"><label>Figure 1</label>
<caption><p><bold><italic>AP1S3</italic>
loss-of-function mutations are most likely to affect skin keratinocytes.</bold>
(<bold>a</bold>
) Schematic representation of AP-1 structure. (<bold>b</bold>
) HEK293 cells were transfected with wild-type and mutant <italic>AP1S3</italic>
constructs. Lysates were subjected to the indicated temperature gradient, and soluble (nondenatured) proteins were analyzed by Western blotting. The densitometry shows the fraction of nondenatured protein (mean ± standard error of the mean of the results obtained in two experiments). (<bold>c</bold>
) HEK293 cells were transfected with myc-tagged <italic>AP1S3</italic>
and FLAG-tagged <italic>AP1M1</italic>
constructs. Lysates were subjected to immune precipitation (IP) and immune blotting (IB) as indicated. The image is representative of results obtained in two experiments. (<bold>d</bold>
) Real-time PCR analysis showing abundant <italic>AP1S3</italic>
expression in keratinocytes. The data show the mean ± standard error of the mean of measurements obtained in two donors. <sup>∗</sup>
<italic>P</italic>
≤ 0.05. IB, immune blotting; IP, immune precipitation; WCE, whole cell extracts; wt, wild type.</p>
</caption>
<graphic xlink:href="gr1"></graphic>
</fig>
<fig id="fig2"><label>Figure 2</label>
<caption><p><bold><italic>AP1S3</italic>
deficiency results in impaired autophagy.</bold>
(<bold>a</bold>
) After the generation of a HaCaT <italic>AP1S3</italic>
knockdown cell line, gene silencing was measured by real-time PCR, because of cross-reactivity of the anti-AP1σ1c antibody with the proteins encoded by <italic>AP1S1</italic>
and <italic>AP1S2</italic>
. (<bold>b</bold>
) Starvation-induced LC3-II accumulation was measured by Western blotting and densitometry. The data are presented as mean ± standard error of the mean of measurements obtained in four independent experiments. (<bold>c</bold>
) HEK293 <italic>AP1S3</italic>
knockout cell lines harboring a c.124delC change (highlighted by a red asterisk in the chromatogram) were generated by CRISPR/Cas-9 editing. (<bold>d</bold>
) Cells were starved to induce autophagy, and LC3-II accumulation was measured by Western blotting. The data are presented as described. (<bold>e</bold>
) Control and <italic>AP1S3</italic>
KO HEK293 cells were transfected with GFP-LC3 and either an empty vector (control and KO panels) or a rescue construct (wild-type <italic>AP1S3</italic>
in KO/wt panel and p.Arg33Trp <italic>AP1S3</italic>
in KO/mut). Starvation-induced LC3 punctae were visualized by confocal fluorescence microscopy. The data are presented as mean ± standard error of the mean of measurements obtained in at least 15 cells per experiment. Scale bar = 5 μm. <sup>∗</sup>
<italic>P</italic>
≤ 0.05, <sup>∗∗∗∗</sup>
<italic>P</italic>
≤ 0.0001. Cas9, CRISPR-associated protein-9; CRISPR, clustered regularly interspaced short palindromic repeats; GFP, green fluorescent protein; KD, knockdown; KO, knockout; mut, mutated; ns, not significant; wt, wild-type.</p>
</caption>
<graphic xlink:href="gr2"></graphic>
</fig>
<fig id="fig3"><label>Figure 3</label>
<caption><p><bold>Abnormal p62 accumulation and enhanced TLR-2/6 signaling in <italic>AP1S3</italic>
-deficient keratinocytes.</bold>
(<bold>a</bold>
) p62 levels were measured in patient and control subject keratinocytes by Western blotting and densitometry. (<bold>b</bold>
) After the transfection of silencing (<italic>AP1S3</italic>
siRNA) and nonsilencing (control) siRNA pools into primary keratinocytes, (<bold>c</bold>
) baseline p62 levels were measured by Western blotting and densitometry. (<bold>d</bold>
) Alternatively, cells were stimulated with MALP-2 in triplicate, and the induction of TLR2/6-dependent genes was measured by real-time PCR. The data are representative of results obtained in at least two independent experiments and are presented as mean ± standard error of the mean of duplicate stimulations. <sup>∗</sup>
<italic>P</italic>
≤ 0.05, <sup>∗∗</sup>
<italic>P</italic>
≤ 0.01, <sup>∗∗∗</sup>
<italic>P</italic>
≤ 0.001. c, control; ns, not significant; siRNA, small interfering RNA; TLR, Toll-like receptor.</p>
</caption>
<graphic xlink:href="gr3"></graphic>
</fig>
<fig id="fig4"><label>Figure 4</label>
<caption><p><bold><italic>AP1S3</italic>
-deficient primary keratinocytes exhibit an abnormal immune profile, which can be recapitulated by autophagy inhibition.</bold>
(<bold>a</bold>
) After siRNA-mediated <italic>AP1S3</italic>
silencing, primary keratinocytes were stimulated with IL-1β, and gene expression was determined by real-time PCR. (<bold>b</bold>
) Alternatively, cells were cultured for a further 48 hours in the absence of stimuli, and cytokine production was measured by ELISA. (<bold>c</bold>
) Normal primary keratinocytes were cultured in the presence or absence of 3-MA and subsequently stimulated with IL-1β. Gene expression was determined by real-time PCR. All data are representative of results obtained in two independent experiments and are presented as mean ± standard error of the mean of (<bold>a</bold>
) duplicate or (<bold>b, c</bold>
) triplicate measurements. <sup>∗</sup>
<italic>P</italic>
≤ 0.05, <sup>∗∗</sup>
<italic>P</italic>
≤ 0.01. 3-MA, 3-methyladenine; siRNA, small interfering RNA.</p>
</caption>
<graphic xlink:href="gr4"></graphic>
</fig>
<fig id="fig5"><label>Figure 5</label>
<caption><p><bold>Abnormal cytokine expression in the keratinocytes of patients harboring <italic>AP1S3</italic>
mutations.</bold>
(<bold>a</bold>
) Primary keratinocytes were stimulated with IL-1β, and cytokine induction was measured by real-time PCR. The data are presented as mean ± standard error of the mean of duplicate stimulations carried out in the cells of two unrelated patients and two healthy control subjects. (<bold>b</bold>
) IL-36α and IL-8 production was measured in culture supernatants by ELISA. Data are presented as mean ± standard error of the mean of triplicate measurements. (<bold>c</bold>
) Primary keratinocytes were starved to induce autophagy or cultured in the presence of IL-36Ra. Gene expression was measured by real-time PCR. The data are presented as mean ± standard error of the mean of triplicate measurements, obtained in one patient and two healthy control subjects. <sup>∗</sup>
<italic>P</italic>
< 0.05, <sup>∗∗</sup>
<italic>P</italic>
< 0.01, <sup>∗∗∗</sup>
<italic>P</italic>
< 0.001. ns, not significant.</p>
</caption>
<graphic xlink:href="gr5"></graphic>
</fig>
<table-wrap id="tbl1" position="float"><label>Table 1</label>
<caption><p>Clinical phenotype of affected individuals bearing <italic>AP1S3</italic>
disease alleles</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>Patient ID</th>
<th>Sex</th>
<th>Ethnicity</th>
<th>Diagnosis</th>
<th>Concurrent PV</th>
<th>Age of Onset, years</th>
<th><italic>IL36RN</italic>
Genotype</th>
<th><italic>AP1S3</italic>
Genotype</th>
</tr>
</thead>
<tbody><tr><td>T010091</td>
<td>F</td>
<td>European</td>
<td>GPP</td>
<td>U</td>
<td align="char">68</td>
<td>p.Ser113Leu/–</td>
<td>p.Phe4Cys/–</td>
</tr>
<tr><td>T030865</td>
<td>F</td>
<td>European</td>
<td>GPP</td>
<td>N</td>
<td align="char"><1</td>
<td>p.Ser113Leu/–</td>
<td>p.Phe4Cys/–</td>
</tr>
<tr><td>T016713</td>
<td>F</td>
<td>European</td>
<td>PPP</td>
<td>N</td>
<td align="char">55</td>
<td>–/–</td>
<td>p.Arg33Trp/–</td>
</tr>
<tr><td>T026517</td>
<td>F</td>
<td>European</td>
<td>PPP</td>
<td>N</td>
<td align="char">50</td>
<td>–/–</td>
<td>p.Arg33Trp/–</td>
</tr>
<tr><td>T028754</td>
<td>F</td>
<td>European</td>
<td>PPP</td>
<td>N</td>
<td align="char">49</td>
<td>–/–</td>
<td>p.Arg33Trp/–</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn><p>Abbreviations: F, female; GPP, generalized pustular psoriasis; ID, identifier; N, no; PPP, palmar plantar pustulosis; PV, psoriasis vulgaris; U, unknown.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</floats-group>
</pmc>
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