Journal of Cell Science nlmaoyntoki kntruhinterleukin-18 Roth through Wera skin an in in network participates and inflammatory integrity skin maintains 1 Article Research A)(rw n cen 02 j ta. 00.E is 2006). EI Segre, 2001; 2010). al., al., et Schmuth et 2004; Oji McLean, defects in barrier and eczema 2012; and mutations (Lane hyperkeratosis atopic erosions, McLean, skin whereas disorder by and characterized 2006), inflammatory (Brown the Segre, 2004; (AE) cause McLean, 2001; and can Lane al., 2011; et al., et and Schmuth (Arin KRT1 hyperkeratosis defects erosions, barrier fact, epidermolytic skin and by In congenital characterized 113800) function. to MIM associated (EI, lead barrier the mutations for and the KRT10 crucial to led of are This subset 2011). filaggrin al., a et that Simpson 2005; hypothesis envelope, epidermal al., the cornified/lipid et constitutes (Candi complex the cells, barrier a Langerhans forms to with addition ultimately together of in which, lipids, , these of expression to suprabasal Krt10 series crosslinking not in Covalent but precedes Krt1 proteins. of envelope upregulation cornified major and filaggrin Their the I type heterodimer form its . and (Schweizer KRT1 KRT10 keratin epithelia II partner type all The 2006). in al., the et constitute cytoskeleton its Keratins filament 2007). of (Kim al., intermediate et keratins Many Magin including 2007; 2011). proteins Coulombe, and structural al., on et depend of functions (Simpson virtue injury by keratinocytes mechanical homeostasis immune epidermal against regulates organism and dehydration, an and protects The Introduction words: Key barrier a by keratinocytes. accompanied murine proteins, in S100A9 network and inflammatory S100A8 the an and in rescued (IL-18) partially participates IL-18 we interleukin-18 and of control, in integrity Depletion inflammation increase lethality. skin and perinatal prenatal formation of and a in barrier defect maintenance in caused cytoskeleton for keratins Krt1 filament crucial recurrent of of intermediate and role is Absence integrity the the Krt1 barrier and of that of pathomechanisms constituents loss unknown here by major largely show characterized the are humans, of (KRT10) in search ichthyosis 10 In epidermolytic erythema. keratin cause partner mutations KRT1 heterodimer epidermis. its suprabasal and (KRT1) 1 Keratin Summary 10.1242/jcs.116574 doi: 5269–5279 125, ß Science Cell of Journal 2012 October 8 Accepted ( correspondence for *Author 7 6 5 4 3 2 1 u aasgetafntoa ikbtenKT n ua nlmaoysi diseases. skin simplex. inflammatory bullosa human epidermolysis and and deficiency KRT1 Krt1-mediated Krt5 between a from revealed link different profiling functional but transcriptome a psoriasis, Finally, and suggest pathology. eczema data the atopic Our in to KRT1 similar of signature role expression upstream an supporting dependent, caspase-1 So hmsVogl Thomas nttt fClua eiieadNrhEs nln tmCl nttt,NwateUiest,Nwateuo yeN24H UK 4HH, NE2 Tyne upon Newcastle University, Newcastle Institute, Cell Stem England East Germany North Germany Bonn, and Bonn, Universita 53115 Medicine 53115 Bonn, Cellular Bonn, Mu of of of of University Institute University University Institute, Biochemistry, Immunology, LIMES Germany Cell of Immunoregulation, Leipzig, of Institute Switzerland and 04103 Division Zurich, Genomics Leipzig, Biology, 8006 of of Molecular Zurich, Department University and of Biology, Biochemistry University of of Dermatology, Institute Institute of and Department (TRM) Hospital, Medicine University Regenerative for Centre Translational 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. e Thiering ren ¨ t-atlnk Ruprecht-Karls-Universita ¨ts-Hautklinik, eai yokltn pdra are,Int muiy nelui-8 tpceczema Atopic Interleukin-18, immunity, Innate barrier, Epidermal cytoskeleton, io Kumar Vinod , 5 oansRoth Johannes , 1 nraStaratschek-Jox Andrea , [email protected] 1 asDemrBeer Hans-Dietmar , ntr 84 Mu 48149 ¨nster, 5 ui Reichelt Julia , tHiebr,610Hiebr,Germany Heidelberg, 69120 Heidelberg, ¨t ) 4 nraHofmann Andrea , ntr Germany ¨nster, 6 2 nrdHausser Ingrid , iimRichter Miriam , Krt1 2 ciaino aps- stgtyrgltdb h inflammasome, the by regulated secretion 2009). tightly is (Dinarello, unconventional caspase-1 keratinocytes of including Activation before cells, of proteases variety a from other by cleavage or is requiring IL-18 caspase-1 propeptide 2012). N-terminal al., an et are with (Kou levels AE synthesized from IL-18 suffering and humans 2002), in AE-like al., elevated in et results (Konishi mice lesions 2009). in inflammatory IL-18 (Dinarello, of keratinocytes IL-1 overexpression suprabasal the Skin-specific of by cytokines Interleukins produced pro-inflammatory 2009). are family al., IL-33 et and IL-18 (Nestle al., linked (IL) et diseases are Vogl therefore skin 2009; and inflammatory al., skin psoriatic to et in Nestle elevated 2009; are al., They (Eckert 2007). et keratinocytes Ehrchen autocrine on 2004; as 4 al., (TLR) et act receptor they Toll-like of released, by activators defects Once barrier upon antimicrobial secretion. heterodimers as unconventional as act released S100 can are proteins the and that to peptides proteins MHCII belong proteins calcium-binding of A9 of and family S100A8 expression 2009). al., by cytokines, et (Nestle of and poorly secretion peptides by antimicrobial responses remain immune and inflammatory pathomechanisms al., et underlying Quigley 2009; al., understood. the of et Nestle role 2012; 2009), diseases primary skin McLean, chronic a and and (Brown established acute in have proteins -resident disorders these Although / 2 eie muecls eaioye euaeskin regulate keratinocytes cells, immune Besides ie L1 ees a eaioyeatnmu,KT and KRT1 keratinocyte-autonomous, was release IL-18 mice. 4 aiaKreusch Fatima , 7 1 lui Wohlenberg Claudia , n hmsM Magin M. Thomas and 4 oci .Schultze L. Joachim , 3 ruaReuter Ursula , 1, * 4 , 3 , 5269 Journal of Cell Science hntp Rihl ta. 01,ls fKt asdperinatal a in revealed caused signature profiling Krt1 expression Transcriptional gene of defect. barrier loss a 2001), mild and a lethality al., with et mice viable (Reichelt to led phenotype which deficiency, Krt10 to contrast nlmaoysi iessA n srai.Dpeino IL-18 of Depletion psoriasis. rescued partially and AE diseases skin inflammatory eei akrud nieKt0dpein(ecete al., et (Reichelt neonatal 129/Ola depletion of mixed Krt10 Histology a unlike 2001). in background, of lethality absence genetic neonatal the caused Surprisingly, 1B,D; S1B). Krt1 (Fig. Fig. mice material Krt1-deficient generated supplementary 2007; we 2007) Coulombe, al., and epidermal et (Kim Magin during formation functions barrier and keratin differentiation isotype-specific address barrier To skin the controls Krt1 control. keratinocyte damage the intracellular whether in is involved question RIG is intriguing (Davis cytoskeleton An including beta and 2011). amyloid and al., signals, NOD crystals et cholesterol danger ATP, or acids, self-derived nucleic (PAMPs) recognizing patterns receptors, pathogen- either molecular sensing TLRs, associated include a and al., are understood activation et partially inflammasome containing (Davis controlling mechanisms procaspase-1/-5 protein The protein and 2011). adaptor domain) the speck-like recruitment keratinocytes, caspase (apoptosis-associated in NLRP3 scaffold ASC as of consisting such complex proteins multiprotein cytoplasmic large a natsrtfe pdri,wtotlclifamto and inflammation handling-induced from local possibly without lesions, keratinocyte epidermis, localized stratified intact 5270 sgtkeeso muersossi knadln cytokine Results link and KRT1. skin of loss in the responses to immune release of gatekeepers as ots hshptei,w aegenerated have we hypothesis, this test To ora fCl cec 2 (22) 125 Science Cell of Journal Krt1 2 / 2 ie u aaietf eai proteins keratin identify data Our mice. 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Krt5/14 desmosome presence maintained both of epidermis that organization suggested This intracellular 2C–E). (Fig. unaffected lcrnmcocp eeldsas eai grgtsin aggregates keratin 1C,D). (Fig. sparse unaltered revealed Krt1 remained microscopy distribution Electron intracellular its sebe ihtp Ikrtn r5o r6in Krt6 or Krt5 keratins II Krt10 type whether intermediate address contributed with To and 2001). atypical al., present assembled et were (Reichelt but integrity Krt14 skin and to detectable, Krt1 between were filament aggregates Krt1 2 / 2 kndet oso r1 niaiglc of lack indicating Krt1, of loss to due skin Krt1 hcns aksi etosfrom sections skin back thickness Krt1 nlsso r1()adKt0()epeso nttlskin total in expression (C) Krt10 from and extracts (B) Krt1 of analysis 20 top, bars: Scale P0. etosfrom sections gis r6adKt6 oi ieidctsbasement 10 indicates bars: line Scale Solid membrane. 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Fig. 2007) Coulombe, material of and defects supplementary (Kim healing 1E; wound (Fig. and defects barrier os(EL a nrae wfl nneonatal in twofold water trans-epidermal increased contrast, was In (TEWL) 3A). loss (Fig. skin the penetrated pdra are nert mlyn netbihddye- intact established in an established barrier This an examine 1999). epidermal al., to employing outside-in et (Segre us integrity assay prompted penetration barrier S2A) Fig. epidermal material (supplementary to utemr,tenme fitc onfe neoe was envelopes cornified intact 2012). of al., to et reduced number significantly (Wallace mice the these of Furthermore, state barrier the not the Possibly, may reveal mice 3D). fully Krt1/10-doubly-deficient (Fig. in performed barrier assay inside-out biotin defective a revealing h nw atcpto fKT,btntKT0t cornified to KRT10 not but KRT1, of participation known The Krt10 hsi loi gemn ihtercnl described recently the with agreement in also is This . +/+ 2 and / 2 Krt1 ie r o ibe(ecete l,20;Reichelt 2001; al., et (Reichelt viable not are mice, , 2 / 5 in 55% 2 ieadtento that notion the and mice Krt1 , 7 in 17% 2 Krt10 / 2 ieta aietdpostnatally manifested that mice +/+ Krt1 Krt1 os kn Fg 3B,C,E; (Fig. skins mouse 2 2 / 2 / 2 oprdto compared ebrs sn dye no as newborns, Krt1 2 Krt1 / 2 ncontrast in , 2 , / 2 4 in 84% mice, in r1cnrl nifamtr ewr nsi 5271 skin in network inflammatory an controls Krt1 O1adocui dt o hw)in shown) not of (data and 3F) occludin between (Fig. and Cldn5 and ZO-1 junction Cldn4 barrier spaces (Cldn), tight unaltered claudin-1 The to of 2012), paracellular examined. expression were al., contribute distribution et and the expression (Kubo which protein keratinocytes sealing junctions layer the granular address tight by To integrity. of barrier functionality a in substantiated role Krt1 data II type known our the 2012), of al., role envelopes major et cornified (Wallace of 60% intact mice remained deficient doubly Krt1/10 in that upre rmr eetdrcl eae oKrt1. to related directly defect primary a supported eoye dt o hw) tE85 efud13genes 163 two found the we in in downregulated E18.5, expressed At two only differentially shown). and upregulated gene not when E15.5, Krt1 (data only day At development, genotypes the 0. 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S5 al., Table et material (Merico supplementary details) (see supplementary S4 and in Table numbered listed material are are clusters IDs the pathway of corresponding are sets clusters gene functional Depicted gene to shown. connected according fully grouped of (cliques) network sets pathway resulting the and rsn nboth in present analyses. comparison present respective ( identifiers of gene intersection identical the of in numbers the are set Shown data the and patients the EBS from and sets psoriasis data AE, profiling from transcriptional derived genome-wide and set data ( h o 0 nihdptwy iha with pathways enriched database. 100 MSigDB top the The using (GSEA) analyses enrichment he ieprgntp eeaaye.Rdidctsgenes indicates Red in analyzed. upregulated were from genotype Samples per (B,D). mice and pathways three (A,C) response formation immune CE to with linked associated (C,D) P0 and (A,B) ifrnilyepesdgnsin genes expressed differentially skin inflammatory human diseases. to linked differentially genes with expressed overlap and prenatally establish fint muersos ee in genes response activation immune with innate coincide of defects barrier Functional 4. 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IL-18 human primary differentiated terminally keratinocytes. in IL-18 of secretion of Knockdown 7. Fig. 5276 uiekrtncts cl as 10 primary bars: keratin-cytoskeleton-free Scale and keratinocytes. wild-type murine in NLRP3 ( for control. staining siRNA scrambled of Scr, knockdown (siCasp1). siRNA casapse-1 tissue after and keratinocytes differentiated extracts human from cell precipitation primary in acetone by KRT10 concentrated and supernatants KRT1 culture (CASP-1), caspase-1 IL-18, of ni eety eaiswr rdmnnl eaddas regarded predominantly were keratins recently, Until m )o h a-aps niio A (10 VAD inhibitor pan-caspase the or M) ora fCl cec 2 (22) 125 Science Cell of Journal ( A V raito fpiayhmnkrtnctsmediates keratinocytes human primary of irradiation UVB ) KRT1 xrsinidcscaspase-1-dependent induces expression m m. m ) ( M). C B Immunofluorescence ) muoltanalysis Immunoblot ) Krt1 sKt)and (siKrt1) 2 nesoeuiu r1fntos(e loTbe1). covalently Table data is mouse also our Krt10, (see with three together functions not 1998) all Krt1 al., but unique et in underscore Krt1, (Candi CE fragility that the cell to notion cross-linked the of and extent the models limited in aggregates The Krt10 cytoplasmic latter. of presence the to connected h lgtyeeae rglt fKrt1/IL-18 Given of 2012). fragility al., elevated et slightly (Tam reported the in was Krt6A al., extracts of fragments keratinocyte et of corneal (Depianto activity antimicrobial response recently, inflammatory More was 2010). Th2-dominated Krt17 and a Th1- of a 2012; to from cytokines absence Th17- al., inflammatory in this, et change a of (Kou inflammati by characterized support profile attenuate Th2 In and to a 2009). reported cytoskeleton to al., Th1 et epithelial a Wittmann the from shifted whi of responses and activation components pharmacological between inflammasome partial suggest data link the these Collectively, by phenotype. a Krt1 underscored the of is rescue genetic IL-18 setting of importance this The IL-18. in of upstream Krt1 cultured place from keratinocytes data release IL-18 with caspase-1-dependent and together KRT1- showing levels, posttranscriptional and transcriptional ieaayi fbt iew pclt htteaprnl more apparently of the single that phenotype speculate with side-by we a mice severe than of both absence of severe the In analysis 2012). side more al., one et fragility of (Wallace As knockouts gene skin 2001). deletion by al., lacking combined et accompanied mice Krt1, (Reichelt predict, of partner phenotype of might mild heterodimer is very loss Krt1 its the of Krt10, upon specificity by The supported of prenatally involvement. further primary release manifest its and underlining cornified cytokines upregulation importantly, transcriptional integrity distinct Most cell defects, stress. preserving in envelope mechanical in role elevated its role from to upon major release addition IL-18 a in restricting plays in keratinocytes, and the proteins, formation of member barrier cytoskeleton a epidermal Krt1, al., of that et time family Magin first 2011; the keratin 2007; al., for in Coulombe, show et and we Here, observed Kim Arin 2007). 2011; 2010; phenotypes al., al., et with et Chamcheu (Arin agreement keratinopathies apparent epidermal in insults, su fwehrcniin nwihKT smttd(e.g. (Arin infections) mutated HPV is and cell healing KRT1 squamous wound (e.g. cutaneous which the downregulated carcinoma, raise in or data conditions ichthyosis) our epidermolytic context, phenotype whether about wider questions of a mild to In issue leads KRT1. unexpectedly filaggrin of hypersensitivity involvement The of the loss contact 2012). complete in from al., arising responses loss, et enhanced water not trans-epidermal (Kawasaki does in elevated mice results an in cause but filaggrin system 2012). or of al., survival deletion immune et affect prediction, Kou responding 2012; to al., the contrast et In Kezic and 2012; McLean, barrier and resulting (Brown defective possibly event, a late a from represents AE cytokines filaggrin-related pro-inflammatory through in of of elevation acts material absence Furthermore, (supplementary Krt1 S2B). the processing Fig. filaggrin whether that affect of not find does issue Krt1 We the mechanisms. raise filaggrin-dependent patients AE and rflsado omnyrgltdptwy in protein pathways keratin-associated regulated cytokine commonly the of of similarity in The and 2012). profiles mutations McLean, and to (Brown filaggrin linked AE, to ciiy fcnevdi os e animals. surviving in mouse inflammation strong of in absence the to conserved contribute if activity, h rntlurglto fI-8ta curdat occurred that IL-18 of upregulation prenatal The h nlmaoysgauein signature inflammatory The Krt1 / 10 hmgtcnrbt oimmune to contribute might ch 2 ni oeso ct dermatitis, acute of models in on / 2 Krt1 iemlkrtncts might keratinocytes, pidermal oprdwith compared 2 / 2 Krt1 2 iebassimilarity bears mice / 2 pdri,sc an such epidermis, and Krt1 Krt10 Krt1 2 / 2 2 / mice 2 was is Journal of Cell Science ti aucit;Kt0(ecete l,20) r1Kt0(alc ta. 02,Kt1 MGwne l,20;Tn n olme 2006). 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Krieg, H., Traupe, I., Hausser, S., Emmert, V., Oji, J., M. Arin, OFR etceFrcuggmishf DG11/ to Reichelt]; 1316/9 J. Thyssen [DFG the and from T.M.M. Forschungsgemeinschaft grants to Deutsche by 10.02.1.103 supported BONFOR, [Thyssen partially was Foundation work This Funding the of generation the for Bornheim Roland thank We at set Acknowledgements S10. was Table level material alpha supplementary in The shown groups. are two data than statistical more for unpaired tests , variance by of determined analysis was significance Statistical analysis Statistical days. 6 for was USA) differentiation NJ, mJ/cm Rutherford, 2007), 50 East with al., (Cambrex, UV-irradiated et were KBM-2 Keratinocytes in (Feldmeyer culture keratinocytes by induced primary human In transfection and culture Keratinocyte rn .J,Gibr,G,Shmn,H,D led,H,J,Cag .R,Tadini, R., Y. Chang, Jr, H., Almeida, De H., Schumann, G., Grimberg, J., M. Arin, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.116574/-/DC1 online available material Supplementary Medicine Regenerative T.M.M.]. for to Centre 0315883 [PtJ-Bio, Translational Leipzig the and T.M.M.]; fsRA SgaAdih t oi,M,UA sdi rvddin provided is used USA) MO, Louis, St. S9. days; Table (Sigma-Aldrich, 6 information material Sequence after supplementary precipitated. harvested siRNAs acetone For transfection, were was supernatants supernatant h. (Polyplus of the 4 and Interferin from Cells after protein and used. total harvested were siRNAs and France) nM (vehicle) Illkirch, 10 DMSO experiments, or knockdown Germany) Lorrach, Sciences, oh,Buh,Gray ntepeec ftesnhtcttaetd YVAD tetrapeptide synthetic the of presence 20 the in (Tyr-Val-Ala-Asp; Germany) Bruehl, Boehm, .5 aawr nlzdwt im lt1. n lte ihEcl All Excel. with plotted and 11.0 Plot Sigma with analyzed were Data 0.05. h nlmaoemdae V-nue ciainadsceino interleukin- of secretion and keratinocytes. activation by UVB-induced 1beta mediates innate inflammasome as The (calprotectin) cancer. 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