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A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells

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A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells
  LETTERS A key role for autophagy and the autophagy gene  Atg16l1  in mouse and human intestinal Paneth cells Ken Cadwell 1 , John Y. Liu 1 , Sarah L. Brown 1 , Hiroyuki Miyoshi 1 , Joy Loh 1 , Jochen K. Lennerz 1 , Chieko Kishi 5 ,Wumesh Kc 1 , Javier A. Carrero 1 , Steven Hunt 2 , Christian D. Stone 3 , Elizabeth M. Brunt 1 , Ramnik J. Xavier 6 ,Barry P. Sleckman 1 , Ellen Li 3 , Noboru Mizushima 5 , Thaddeus S. Stappenbeck 1 *  & Herbert W. Virgin IV 1,4 * Susceptibility to Crohn’s disease, a complex inflammatory diseaseinvolving the small intestine, is controlled by over 30 loci 1 . OneCrohn’s disease risk allele is in  ATG16L1 , a gene homologous tothe essential yeast autophagy gene  ATG16   (ref. 2). It is not knownhow ATG16L1 or autophagy contributes to intestinal biology orCrohn’sdiseasepathogenesis.To addressthesequestions, we gener-ated and characterized mice that are hypomorphic for ATG16L1proteinexpression,andvalidatedconclusionsonthebasisofstudiesin these mice by analysing intestinal tissues that we collected fromCrohn’s disease patients carrying the Crohn’s disease risk allele of  ATG16L1 . Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a secondessential autophagy protein ATG5 are selectively important for thebiology of the Paneth cell, a specialized epithelial cell that functionsin part by secretion of granule contents containing antimicrobialpeptides and other proteins that alter the intestinal environment 3 .ATG16L1-andATG5-deficientPanethcellsexhibitednotableabnor-malities in the granule exocytosis pathway. In addition, transcrip-tional analysis revealed an unexpected gain of function specific toATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor(PPAR) signalling and lipid metabolism, of acute phase reactantsandoftwoadipocytokines,leptinandadiponectin,knowntodirectly influence intestinal injury responses. Importantly, Crohn’s diseasepatients homozygous for the  ATG16L1  Crohn’s disease risk alleledisplayed Paneth cell granule abnormalities similar to thoseobserved in autophagy-protein-deficient mice and expressedincreased levels of leptin protein. Thus, ATG16L1, and probably theprocessofautophagy,havearolewithintheintestinalepitheliumof mice and Crohn’s disease patients by selective effects on the cellbiology and specialized regulatory properties of Paneth cells. Crohn’s disease typically involves the distal ileum and in its mostsevere form is characterized by pathological changes including trans-mural acute and chronic inflammation. Crohn’s disease is associatedwith increased expression of immunoregulatory cytokines includingleptin 4 and adiponectin 5 . Multiple genetic factors predispose toCrohn’s disease, but the specific relationship between the functionofthesegenesandthediversepathologiesobservedinCrohn’sdiseaseis not well understood. One Crohn’s disease susceptibility allele is inthe predicted autophagy gene  ATG16L1  (refs 6–9). Autophagy is anevolutionarilyconservedprocessthatrecyclescellularcomponentsby means of delivery of double-membrane-bound vesicles containingcytoplasm and cytoplasmic organelles to the lysosome 10 . Autophagy has an important role in cell and tissue homeostasis, and has beenimplicatedina rangeofhumandiseases 10 .ThemammalianATG16L1protein contains an amino-terminal domain that is homologous to yeastAtg16(ref.2),whichfunctionsinautophagyaspartofacomplex withautophagyproteins Atg5 and Atg12 (refs 2,11).Atg16 isrespon-sible,inyeastandmammaliancells,forpropersubcellularlocalizationof the autophagy machinery  8,11,12 .Todetermine the role of ATG16L1 and autophagy in the intestine,we generated two mouse lines with gene-trap-mediated disruptionsof   Atg16l1  and a third line lacking  Atg5   in intestinal epithelial cells.Gene-trap vectors introduce a false splice acceptor into an intron,and can inhibit expression of intact messenger RNA 13 (Fig. 1a). Thiscanresultindecreasedexpression ofaprotein,potentiallygeneratingviable mice when full disruption of a gene is lethal. This approach isattractive because ATG5 is part of the ATG16L1 complex and fulldisruptionof  Atg5  islethal 2,14 .Mouselineshomozygousforgene-trapmutations were generated from commercially available embryonicstemcellscarryinggene-trapmutationsintheintron3 9 toeitherexon6 or exon 10 of   Atg16l1  (ref. 13).  Atg16l1  mutant mouse embryonicfibroblasts(MEFs)expressedlowlevelsofATG16L1protein(Fig.1b)indicating that both mouse lines, ATG16L1 HM1 and ATG16L1 HM2 ,are hypomorphic (HM) for expression of the ATG16L1 protein.ATG16L1 HM mice were born at Mendelian ratios (Supplementary Fig. 1a) and survive to adulthood; the characteristics of the twoATG16L1 HM lines  in vivo   were similar across all experiments.To determine whether ATG16L1 is an autophagy protein, westudied low-passage and transformed MEFs from ATG16L1 HM micecompared to MEFs lacking the essential autophagy protein ATG5(ref. 14). Rapamycin-induced and autophagy-dependent 15 degrada-tion of the adaptor protein sequestosome (SQSTM1, also known asP62) was diminished in ATG16L1 HM and  Atg5  2 / 2 cells (Fig. 1c–e).Decreased degradation of P62 in ATG16L1 HM cells was restored by expressing ATG16L1 (Supplementary Fig. 1b, c). ATG16L1 HM MEFsalso showed diminished rapamycin-induced production of LC3-II,the phosphatidylethanolamine-conjugated form of microtubule-associated protein 1 light chain 3 (LC3-I) generated during autop-hagy  10 (Fig. 1e,f).The induction ofautophagosomes, asmeasuredby LC3-positive dot formation after rapamycin treatment or starvation,was also decreased in ATG16L1 HM MEFs (Supplementary Fig. 2),although the defect of ATG16L1 HM2 cells was more subtle in starvedcells; this was confirmed in cells transfected with green fluorescentprotein (GFP)–LC3 (Supplementary Fig. 3). Mammalian ATG16L1is therefore an autophagy protein.We next determined whether markers of autophagy were abnor-mal in the distal small intestine (ileum, a common site of Crohn’sdisease) of ATG16L1 HM mice by measuring the expression of ATG16L1, LC3 and P62 proteins in ileal lysates (Fig. 1g–i).  Atg16l1 * These authors contributed equally to this work. 1 DepartmentofPathologyandImmunology, 2 DepartmentofSurgery, 3 DepartmentofMedicine, 4 DepartmentofMolecularMicrobiology,WashingtonUniversitySchool ofMedicine,St Louis, Missouri 63110, USA.  5 Department of Physiology and Cell Biology, Tokyo Medical and Dental University Graduate School and Faculty of Medicine, Tokyo 113-8519, Japan. 6 Center for Computational and Integrative Biology and Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA. doi:10.1038/nature07416 1  ©2008 Macmillan Publishers Limited. All rights reserved  mRNAisexpressedthroughout thecrypt–villusaxis(Supplementary Fig. 4). ATG16L1 HM mice expressed 23–37% of the expected level of ATG16L1 protein (Fig. 1g, h). Consistent with a role for ATG16L1 inileal autophagy, both the ratio of LC3-I to LC3-II and the totalamount of LC3-I and P62 were increased in lysates fromATG16L1 HM mice (Fig. 1g, i). To validate these results, we studiedileal lysates from mice generated by breeding  Atg5   flox/flox  mice 16 withmice expressing the Cre recombinase under the control of the intest-inal-epithelium-specific villin promoter ( Atg5   flox/flox  villin -Cre  mice) 17 . ATG5 expression was diminished in ileal lysates of  Atg5   flox/flox  villin -Cre   mice, and these mice exhibited changes inexpression of LC3 and P62 similar to those observed inATG16L1 HM mice (Supplementary Fig. 5). These data are consistentwith a role for ATG16L1 and ATG5 in ileal autophagy.Deficiency in ATG16L1 had no effect on the overall morphology of theileumorthecolon,asmeasuredbyanalysisofcryptheightorvilluslength (data not shown). However, there were obvious abnormalitiesin Paneth cells, leading us to focus our studies on these critically important intestinal innate immune cells. Paneth cells are ileal epithe-lial cells thought to have a role in control of intestinal microbiota by means of secretion of granule contents including antimicrobial pep-tides and lysozyme 18 . Staining of whole mounts of ileum revealed theexpected co-localization of lysozyme and mucus (Fig. 2a). However,there was a notable lack of lysozyme staining in the mucus of ATG16L1 HM mice, suggesting an abnormality of Paneth cell secretion(Fig. 2b). We examined periodic acid-Shiff (PAS)/alcian-blue-stainedsectionsandfoundextraordinaryabnormalitiesinPanethcellsinclud-ing aberrant, disorganized granules as well as decreased granule num-bers (Fig. 2c, d and data not shown). Blinded analysis of thesesectionsfrom 7 control and 12 mutant mice revealed a 100% concordancebetween ATG16L1 HM genotype and abnormal Paneth cell morpho-logy. We quantified these Paneth granule abnormalities by stainingsections for lysozyme, which is normally packaged efficiently in thegranules 18 (Fig. 2e–h). In these sections, we observed a notable popu-lation of ATG16L1 HM cells with diffuse lysozyme staining (Fig. 2f, h).We also observed the presence of intact granules in the crypt lumen of   ATG50 5 10 15050100150  Atg5 +/+  Atg5  –/– Time (h)    P  e  r  c  e  n   t  a  g  e  o   f   P   6   2  r  e  m  a   i  n   i  n  g  ATG16L10 5 10 15WTHM1HM2 WT0 4 8 12 0 4 8 12 0 4 8 12HM1 HM2  Atg5 +/+ 0 4 8 12 0 4 8 12  Atg5 –/–  ActinP62 ** * *** ******* a bc β geo HM1HM2WT β geo β geo β geo  ATG16L1 β  ATG16L1 α  ActinWT HM2HM1  ATG16L1Short exposure ActinWT HM2HM1 ATG16L1Long exposureLC3-ILC3-IIP62020406080100WT HM1 HM20123WT HM1 HM2 ATG16L1P62 P  < 0.004 P  < 0.004 P  < 0.02 P  < 0.03 g hi                                                                                                                                                                                                                                                                      F                                                                                                                                                                                                 o                                                                                                                                                                                                                                                                         l                                                                                                                                                                                                                                                                    d                                                                                                                                                                                                                                                                         i                                                                                                                                                                                                 n                                                                                                                                                                                                 c                                                                                                                                                                                                      r                                                                                                                                                                                                 e                                                                                                                                                                                                      a                                                                                                                                                                                                       s                                                                                                                                                                                                     e                                                                                                                                                                                                                                                                           W                                                                                                                                                                                                                                                                      T                                                                                                                                                                                                                                                                            (                                                                                                                                                                                                                                                                                                                                               %                                                                                                                                                                                                                                                                                  )                                                                         Rap+CHX(h): def LC3-ILC3-II WT HM1 HM2DMSO + + +Rapamycin + + +  ActinWT HM1 HM2Induced LC3-II P  = 0.0008 P  = 0.0036    W   T   L   C   3  -   I   I   (   %   ) 080100204060050100150200 Figure 1  |  Role of ATG16L1 in autophagy in cells and intestine fromATG16L1 HM mice. a , A gene-trap vector containing a splice acceptorfollowed by the  b geo ( b -galactosidase/neomycin resistance fusion cassette)exon disrupts the  Atg16l1  gene locus by insertions within the intronicregions after exons 6 and 10 for the ATG16L1 HM1 and ATG16L1 HM2 lines,respectively. WT, wild type.  b , MEFs were generated from day 12.5 embryosof   Atg16l1  mutant mice, and western blot analysis was used to detect the  a and  b  isoforms of ATG16L1 from whole-cell lysates.  c ,  d ,  Atg5 2 / 2 andATG16L1 HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 m gml 2 1 ) and cyclohexamide (CHX,5 m gml 2 1 ) for 0, 4, 8 and 12h. Cell lysates were analysed by western blot forloss of P62 expression ( c ). P62 levels were quantified by densitometry normalized to actin ( n 5 3;  d ).  * P  , 0.04;  ** P  , 0.008;  *** P  5 0.0003. e ,  f , Western blot analysis of LC3 expression in MEFs grown in the presenceof rapamycin (50 m gml 2 1 ) or dimethylsulphoxide (DMSO) control for 4h( e ). LC3-II expression in the presence of rapamycin was quantified andnormalized to actin ( n 5 6;  f ).  g , Detection of ATG16L1, LC3 and P62 by  western blot of ileal lysates from ATG16L1 HM mice. ATG16L1 can bedetected in both mutant lines on longer exposures.  h ,  i , Quantification of ATG16L1( h )andP62( i )normalizedtoactindetectedinileallysates( n 5 3). P   values were calculated using two-tailed Student’s  t  -test. Error bars, s.e.m. D 0  D 1  D 2  D 3   ** ******** ****** *** D 0  D 1  D 2  D 3 c de fgha bi 100806040200100806040200    P  e  r  c  e  n   t  a  g  e  o   f   t  o   t  a   l   P  a  n  e   t   h  c  e   l   l  s   P  e  r  c  e  n   t  a  g  e  o   f   t  o   t  a   l   P  a  n  e   t   h  c  e   l   l  s WT ATG16L1 HM  Atg5 flox/flox   Atg5 flox/flox  Villin- Cre D 0  D 1  D 2  D 3 Figure 2  |  Mutation of ATG16L1 or ATG5 leads to disruption of the Panethcell granule exocytosis pathway. a ,  b , Whole-mount images takenimmediately above the ileal mucosal surface from WT ( a ) and ATG16L1 HM ( b ) littermate mice stained with  Helix pomatia  lectin that labels mucus(green) and antisera directed against lysozyme (red). Images arerepresentativeof3WTand3ATG16L1 HM mice. c , d ,IlealsectionsfromWT( c ) and ATG16L1 HM ( d ) mice were stained with PAS/alcian blue to detectPanethgranulesbylight microscopy(dashedlinedenotesthecrypt unitandarrowheads indicate Paneth cells). Images are representative of 7 WT, 7ATG16L1 HM1 and5ATG16L1 HM2 mice;morethan100cryptswereanalysedfor each.  e ,  f , Representative images of indirect immunofluorescence of sections stained for lysozyme (red) in WT ( e ) and ATG16L1 HM ( f ) mouseileal crypts.  g , Paneth cells display one of four patterns of lysozymeexpression (represented in red; white represents areas that excludelysozyme): normal (D 0 ), disordered (D 1 ), depleted (D 2 ) and diffuse (D 3 ). h ,  i , Number of Paneth cells from ATG16L1 HM ( h ) and  Atg5  flox/flox   villin -Cre  ( i )micedisplayingeachpatternoflysozymeexpression( n 5 6,660cellsfrom5 ATG16L1 HM mice and 5,634 cells from 5 WT mice;  n 5 1,756 cells from 2  Atg5  flox/flox   villin -Cre   mice and 1,649 from 2  Atg5  flox/flox  mice). Scale bars: a , b , 200 m m; c – f , 10 m m. * P  , 0.05, ** P  , 0.01, *** P  , 0.001. P   valueswerecalculated using two-tailed Student’s  t  -test. Error bars, s.e.m. LETTERS  NATURE 2  ©2008 Macmillan Publishers Limited. All rights reserved  whole mounts of ileum from ATG16L1 HM mice (Supplementary Fig.6a–c), a finding confirmed by electron microscopy (Supplementary Fig. 6d, e), potentially explaining the absence of lysozyme staining inthe mucus layer of the ileum (Fig. 3a, b). These observations indicatethat ATG16L1 is required for maintaining the integrity of the Panethcell granule exocytosis pathway. On the basis of these data, and thepublished role for NOD2 in resistance to infection 19 , we orally chal-lenged ATG16L1 HM mice with  Listeria monocytogenes  . We found nochangein L. monocytogenes  titresinspleen,liverandmesentericlymphnodes (Supplementary Fig. 7), indicating that the marked changes inreleaseofgranulesinATG16L1 HM micedidnotaffect L.monocytogenes  resistance.Thisarguesthatthephenotypesofmutationsanalysedsofarin ATG16L1 and NOD2 are distinct.Despite these profound alterations in the granule exocytosis path-way, we found no evidence for increased epithelial cell death or pro-liferation, as determined by quantification of apoptotic bodies and WT     1    0    0    %    5    0  –    7    5    %    0  –    5    0    %    0    % begac d PPARsignallingStatin pathway Adipocytokine signallingPolyunsaturated fatty acid biosythesisSt Wnt Ca 2+  cyclic GMP pathway† γ  -Hexachlorocyclohexane degradation‡Dorsal–ventral axis formationNitrogen metabolismKrebs TCA cycleFolate biosynthesis γ  -Hexachlorocyclohexane degradation‡‡ P  < 0.05–Log(  P -value)01234567 f 21NSCathepsin E19.6NSDeath-associated protein kinase 17.9NS Ramp1 4.63.4Haemoglobin α  adult chain 1 or 2NS4.4Complement component factor 1NS5.1Complement factor D (adipsin)NS5.7Serum amyloid A1NS5.8HaptoglobinNS4.8Resistin-like α NS5.5LeptinNS6.4 AdiponectinNS4.4 Apolipoprotein A-IVNS8.2Lipoprotein lipaseNS10Stearoyl-coenzyme A desaturase 1ThymocytePanethcellFold increase in ATG16L1-deficient cellGene name Figure 3  |  Critical role of ATG16L1 in the structure and transcriptionalprofileofPanethcells. a – d ,ElectronmicrographoflittermateWT( a , c )andATG16L1 HM ( b , d )Panethcells(for a , b ,dashedlinesdenoteindividualcells,asterisk indicates normal progenitor cell; for  c ,  d , blue stars indicate normalmitochondria in WT Paneth cells and ATG16L1 HM progenitor cells, reddiamonds indicate degenerating mitochondria, the dashed box showsnormal Golgi compartments, and yellow asterisks indicate granules;  n 5 3micepergenotype). e ,Mitochondrialintegritywasquantifiedonthebasisof the percentage of the visible mitochondrial section displaying intactcisternae ( n 5 49 WT and 71 ATG16L1 HM cells).  f , Enrichment of pathwaysassociated with human orthologues mapped from genes differentially expressed in ATG16L1-deficient Paneth cells relative to WT cells, using thecanonical pathway collection from MSigDB 24 . The bar chart displays thenegative log of the enrichment  P   values for each pathway using thehypergeometric distribution (see Methods). Pathways with only one geneassignedwereexcludedfromthechart. { referstotheWnt/Frizzledreceptor-mediated cyclic GMP pathway obtained from the Signal TransductionKnowledge database (STKE,;CMP_12420).  {  and  {{  denote similar pathways associated with c -hexachlorocyclohexane degradation as curated by KEGG and GenMAPP,respectively. g ,ChartofselectedgenesforwhichmRNAisenrichedineitherATG16L1-deficient Paneth cells (baseline is WT Paneth cells) or ATG16L1-deficient thymocytes (baseline is WT thymocytes). Only haemoglobin is incommon in these two sets of analyses. NS, not significant. Scale bars: a ,  b , 10 m m;  c ,  d , 500nm. NATURE  LETTERS 3  ©2008 Macmillan Publishers Limited. All rights reserved  M-phasecells(notshown).Importantly,deletionof  Atg5  intheintest-inal epithelium in  Atg5   flox/flox  villin -Cre   mice led to Paneth cell andgranule abnormalities similar to those observed in ATG16L1 HM mice(Fig. 2i and Supplementary Fig. 8), whereas other epithelial cellsappeared normal. This indicates that, within the ideal epithelium,Paneth cells have a unique sensitivity to autophagy gene disruption.To characterize better the effects of ATG16L1 deficiency in Panethcells,we used transmission electron microscopy. We observed degen-erating mitochondria, loss of granules and the frequent absence of apical microvilli in ATG16L1 HM Paneth cells (Fig. 3a–e andSupplementary Fig. 6d, e). Electron microscopy also showedATG16L1 HM Paneth cells with marked increases in cytoplasmic vesi-cles (Fig. 3b, d); a similar abnormality has been reported in Panethcellsfroma Crohn’sdiseasepatient 20 .Importantly,suchnotablefind-ings were notpresent in epithelial progenitors or enterocytes (Fig. 3b,d and Supplementary Fig. 9), confirming that ATG16L1 deficiency selectively affects Paneth cells within the intestinal epithelium.We next performed transcriptional profiling of Paneth cell RNAprocuredby lasercapture microdissection (LCM) 21 (seemethodsandanalysisinSupplementaryFig.10).Consistentwithalackofcelldeathor degeneration, as detected by electron microscopy, microarray ana-lysis revealed that less than 1.5% of probe sets detected changes inRNA levels using low-stringency criteria ( $ 1.3-fold difference;Supplementary Table 1 and Supplementary Fig. 11). Cluster analysisof significantly enriched transcripts in ATG16L1-deficient Panethcells revealed a notable signature of genes involved in peroxisomeproliferator-activated receptor (PPAR) pathways, adipocytokine sig-nalling and aspects of lipid metabolism (Fig. 3f and Supplementary Fig. 12). Additionally, transcripts for several acute-phase reactantsincluding serum amyloid A1, haptoglobin and complement factorsD and I were increased (Fig. 3g). Of particular interest was the obser-vation that the adipocytokines leptin and adiponectin, previously reported to be increased in Crohn’s disease patients 4,5 , were amongstthe most highly enriched transcripts (Fig. 3g).To determine whether these transcriptional changes are unique toATG16L1-deficient Paneth cells, we examined a second primary celltypefromATG16L1 HM mice.Autophagyisimportantinthefunctionof T cells 22 . Thymocytes from ATG16L1 HM mice were normal innumberbutexpressedlowlevelsofATG16L1andexhibiteddecreasedconversion of LC3I to LC3II (Supplementary Fig. 13), demonstratingthat ATG16L1 is important for autophagy in thymocytes. Microarray analysisofthesecellsrevealedthat,inmarkedcontrasttoPanethcells,expression of only 27 genes was altered more than 1.3-fold(Supplementary Fig. 14). There was no notable change in expressionof the most significant clusters of transcripts altered in ATG16L1 HM Paneth cells,and only one gene altered in thymocytes was also alteredin Paneth cells (Fig. 3g). Therefore, the transcriptional signature of ATG16L1 deficiency is specific to Paneth cells, again emphasizing theunique effects of ATG16L1 deficiency on these cells. Taken togetherwiththeextensivemorphologicabnormalitiesdocumentedabove,weconcludethatATG16L1deficiencyisassociatedwithprofoundaltera-tions in the specialized properties of Paneth cells including defectivegranule exocytosis and unexpected increases in expression of genesinvolved in regulating injury responses.Given the importance of ATG16L1 and ATG5 in Paneth cells inmice, we examined the role of the human  ATG16L1  Crohn’s diseaserisk allele by means of a retrospective analysis of ileocolic resectionspecimens from patients with Crohn’s disease. We studied tissue sec-tions from the uninvolved proximal margins, containing little or noinflammation, from 10 Crohn’s disease patients homozygous for the ATG16L1  risk allele compared to 7 Crohn’s disease controls withoutthe risk allele. All 17 patients lackedthe three major  NOD2   risk allelesand the protective  IL23R   allele for Crohn’s disease 7,23 . IndependentblindedexaminationbyT.S.S.andE.M.B.revealedthat100%ofatrisk patients and 0% of controls contained abnormal Paneth cells, similarto the results in ATG16L1 HM mice (Fig. 4a, b). We quantified lyso-zyme staining in Crohn’s disease patient specimens, and, likeATG16L1 HM mice, found that patients carrying the  ATG16L1  risk allele contained an increased proportion of Paneth cells with disorga-nized or diminished granules or exhibiting diffuse cytoplasmic lyso-zyme staining (Fig. 4c–g). Moreover, consistent with transcriptionalanalysis in mice, an increased proportion of Paneth cells from at risk patients that exhibited diffuse lysozyme staining D 3  cells in Fig. 4g)also stained positive for leptin protein compared to similar cells incontrols( P  , 0.05;Fig.4e–g).Thesefindingsdemonstrateaconcord-ancebetween thepathologyandtranscriptionalprofileofPanethcellsfrom ATG16L1 HM mice and Paneth cell abnormalities observed inCrohn’s disease patients with the risk allele of   ATG16L1 . D 0  D 1  D 2  D 3 ********* a bc dge f    P  e  r  c  e  n   t  a  g  e  o   f   t  o   t  a   l   P  a  n  e   t   h  c  e   l   l  s 100806040200Unmutated  ATG16L1  T300A  Figure 4  |  Crohn’s disease patients homozygous for the disease risk alleleof ATG16L1 display Paneth cell abnormalities similar to ATG16L1 HM mice.a ,  b , Haematoxylin-and-eosin-stained sections of uninvolved areas fromileo–colicresectionsfrompatientswith Crohn’sdiseasehomozygousforthesafe ( a ) or risk ( b ) allele of   ATG16L1  (blue dashed line denotes crypt unit). c – f , Immunofluorescence images of Paneth cells from control patients( c , e )andpatientswiththeriskallele( d , f )stainedforlysozyme(red; c , d )anddouble-labelled additionally for leptin (green;  e ,  f ; yellow dashed linedenotes the crypt unit).  g , Aberrant lysozyme expression was quantifiedusing the same criteria as that used for mouse sections in Fig. 3 ( n 5 6,829cells for at risk patients and 8,182 for control,  n 5 5 patients for eachgenotype). Leptin-positive D 3  cells were quantified in patient samples ( n 5 4 patients per genotype) homozygous for the risk allele (76 out of 322 D 3 cellswerepositivefromatotal of580cryptsexamined)andhomozygousforthe safe allele (11 out of 93 D 3  cells were positive from a total 749 cryptsexamined). Scale bars:  a – f , 10 m m.  * P  , 0.05,  ** P  , 0.01,  *** P  , 0.001. P   values were calculated using two-tailed Student’s  t  -test. Error bars, s.e.m. LETTERS  NATURE 4  ©2008 Macmillan Publishers Limited. All rights reserved  ThesedataindicatethatATG16L1hasaspecificroleinhumansandmice in regulating the specialized properties of Paneth cells, and pro-vide a new and relevant mouse model that emulates one of the many diverse pathological hallmarks of human Crohn’s disease. We show that ATG16L1 and a second autophagy protein ATG5 are critically important for the known role of Paneth cells in secretion of granulecontents that may alter the intestinal microbiota. In addition, wedemonstrateapreviouslyunknownroleofATG16L1intheregulationofPanethcellexpressionofadipocytokines,previouslyassociatedwithCrohn’sdisease.Withintheintestinalepithelium,themarkedeffectof autophagy-proteindeficiencyonPanethcells, butnotonenterocytes,whichshareacommonprogenitor,indicatesthatautophagycancon-tribute to disease pathogenesis by means of a highly specific rolewithin a single cell lineage. Indeed, the effects of hypomorphicexpression of ATG16L1 were specific to Paneth cells and not seen inATG16L1-deficient thymocytes. An important implication of thistype of ‘within-lineage’ specificity is that future studies will need tofocus on how ATG16L1 polymorphisms affect the function of differ-entiated Paneth cells. Because both environment and genotypehave arole in human Crohn’s disease pathogenesis 7 , it will be interesting todetermine whether environmental triggers including agents thatdamage the intestine or pathogens alter the pathological featuresassociated with compromised ATG16L1 function. METHODSSUMMARY The embryonic stem cell lines BC0122 (ATG16L1 HM1 ) and XR0164(ATG16L1 Hm2 ) containing insertions of the gene-trap vector were purchasedfrom Bay Genomics. Founder chimaeric mice were mated with C57BL/6 mice,and heterozygous progenies were mated to each other to generate experimentalmice. Littermate mice that are homozygous for the undisrupted allele were usedas controls in all experiments.For immunohistochemistry, paraffin-embedded tissue was washed for 5minthreetimeswithxylene,twicewith100%EtOH,oncewith95%EtOH,oncewith70%EtOH,andoncewithPBS.SectionswereboiledinTrilogy(CellMarque)forantigen retrieval, rinsed in deionized water for 15min, and washed with PBS.Sections were blocked in 1% BSA, 0.3% Triton X-100 PBS, and incubated with a1:100 dilution of goat polyclonal anti-lysozyme C antibody (C-19), Santa CruzBiotechnology. The specificity of the antibody was confirmed with a mousemonoclonal anti-lysozyme antibody (BGN/06/961), Abcam. Identical stainingwasobservedwhenvisualizedwithappropriatesecondaryantibodiesconjugatedto Alexa594 (1:500).For whole-mount immunofluorescence, dissected ileal samples were carefully opened with fine scissors and the intestine was pinned on black wax in 10%buffered formalin for 1h. The tissue was rinsed three times for 5min in PBS andincubated with a 1:100 dilution of anti-lysozyme (both mouse and goat anti-bodies were equivalent) for one hour at 24 u C. After PBS washes, the tissue wasincubated with a 1:500 dilution of Cy3-labelled anti-goat or anti-mouse anti-bodyaswellasa1:100dilutionofFITC-labelled H.pomatia  lectinforonehourat24 u C.TissuewasmountedonglassslidesafterPBSwashesandwasviewedbyanepifluorescence microscope. Full Methods  and any associated references are available in the online version ofthe paper at Received 4 August; accepted 15 September 2008.Published online 5 October 2008. 1. Barrett, J. C.  et al.  Genome-wide association defines more than 30 distinctsusceptibility loci for Crohn’s disease.  Nature Genet.  40,  955 – 962 (2008).2. Mizushima, N.  et al.  Mouse Apg16L, a novel WD-repeat protein, targets to theautophagic isolation membrane with the Apg12 – Apg5 conjugate.  J. Cell Sci.  116, 1679 – 1688 (2003).3. Ouellette,A. J.Paneth cell a -defensinsynthesisandfunction.  Curr. Top. Microbiol.Immunol.  306,  1 – 25 (2006).4. Barbier, M.  et al.  Overexpression of leptin mRNA in mesenteric adipose tissue ininflammatory bowel diseases.  Gastroenterol. Clin. Biol.  27,  987 – 991 (2003).5. Yamamoto, K.  et al.  Production of adiponectin, an anti-inflammatory protein, inmesenteric adipose tissue in Crohn’s disease.  Gut  54,  789 – 796 (2005).6. The Wellcome Trust Case Control Consortium.. Genome-wide association studyof 14,000 cases of seven common diseases and 3,000 shared controls.  Nature 447,  661 – 678 (2007).7. Xavier,R.J.&Podolsky,D.K.Unravellingthepathogenesisofinflammatoryboweldisease.  Nature  448,  427 – 434 (2007).8. Rioux,J.D. et al. Genome-wideassociationstudyidentifiesnewsusceptibility locifor Crohn disease and implicates autophagy in disease pathogenesis.  NatureGenet.  39,  596 – 604 (2007).9. Hampe, J.  et al.  A genome-wide association scan of nonsynonymous SNPsidentifies a susceptibility variant for Crohn disease in ATG16L1.  Nature Genet.  39, 207 – 211 (2007).10. Levine, B. & Kroemer, G. Autophagy in the Pathogenesis of Disease.  Cell  132, 27 – 42 (2008).11. Fujita, N., Itoh, T., Fukuda, M., Noda, T. & Yoshimori, T. The Atg16L complexspecifies the site of LC3 lipidation for membrane biogenesis in autophagy.  Mol.Biol. Cell  19,  2092 – 2100 (2008).12. Kuma, A., Mizushima, N., Ishihara, N. & Ohsumi, Y. Formation of theapproximately 350-kDa Apg12 – Apg5.Apg16 multimeric complex, mediated byApg16 oligomerization, is essential for autophagy in yeast.  J. Biol. Chem.  277, 18619 – 18625 (2002).13. Stryke, D.  et al.  BayGenomics: a resource of insertional mutations in mouseembryonic stem cells.  Nucleic Acids Res.  31,  278 – 281 (2003).14. Kuma, A.  et al.  The role of autophagy during the early neonatal starvation period. Nature  432,  1032 – 1036 (2004).15. Komatsu, M.  et al.  Homeostatic levels of p62 control cytoplasmic inclusion bodyformation in autophagy-deficient mice.  Cell  131,  1149 – 1163 (2007).16. Hara, T.  et al.  Suppression of basal autophagy in neural cells causesneurodegenerative disease in mice.  Nature  441,  885 – 889 (2006).17. Madison,B.B. et al. Ciselementsofthevillingenecontrolexpressioninrestricteddomains of the vertical (crypt) and horizontal (duodenum, cecum) axes of theintestine.  J. Biol. Chem.  277,  33275 – 33283 (2002).18. Porter,E.M.,Bevins,C.L.,Ghosh,D.&Ganz,T.ThemultifacetedPanethcell. Cell.Mol. Life Sci.  59,  156 – 170 (2002).19. Kobayashi, K. S.  et al.  Nod2-dependent regulation of innate and adaptiveimmunity in the intestinal tract.  Science  307,  731 – 734 (2005).20. Dvorak, A. M. & Dickersin, G. R. Crohn’s disease: transmission electronmicroscopic studies I. Barrier function. Possible changes related to alterations ofcell coat, mucous coat, epithelial cells, and Paneth cells.  Hum. Pathol.  11,  561 – 571(1980).21. Stappenbeck, T. S., Mills, J. C. & Gordon, J. I. Molecular features of adult mousesmall intestinal epithelial progenitors.  Proc. Natl Acad. Sci. USA  100,  1004 – 1009(2003).22. Pua, H. H., Dzhagalov, I., Chuck, M., Mizushima, N. & He, Y. W. A critical role forthe autophagy gene Atg5 in T cell survival and proliferation.  J. Exp. Med.  204, 25 – 31 (2007).23. Duerr, R. H.  et al.  A genome-wide association study identifies IL23R as aninflammatory bowel disease gene.  Science  314,  1461 – 1463 (2006).24. Subramanian, A.  et al.  Gene set enrichment analysis: a knowledge-basedapproach for interpreting genome-wide expression profiles.  Proc. Natl Acad. Sci.USA  102,  15545 – 15550 (2005). Supplementary Information  is linked to the online version of the paper Acknowledgements  We thank N. Abe for technical assistance with fluorescencemicroscopy, V. Cavalli for microscope use, J. Eisenberg and A. Ng for help withbioinformatics analysis, A. J. Ouellette for technical advice, and I. Mysorekar andJ. Mills for help with antibodies. This research was supported by grant U54AI057160 Project 6 and the Broad Foundation (K.C., J. Loh and H.W.V.), traininggrant NIH T32 AR07279 (K.C.), the Lallage Feazel Wall Fellowship DRG-1972-08(K.C.) from the Damon Runyon Cancer Research Foundation; the Pew Foundation(J.Liu,S.L.B.,H.M.,W.K.andT.S.S.);theWashingtonUniversityDigestiveDiseasesResearch Core Center P30 DK52574, Barnes Jewish Foundation, Johnson andJohnson Translational Seed Award, and the Crohn’s Colitis Foundation of America(E.L., S.H. and C.S.); grants-in-aid for Scientific Research from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan, and the TorayScience Foundation (C.K. and N.M.); NIH R01 AI062832 (J.A.C.); and NIHAI062773 and DK43351 (R.J.X.). AuthorContributions ThesrcinalhypothesisofthearticlewasformulatedbyK.C.,H.W.V.andT.S.S.AutophagyexperimentswereperformedbyK.C.andC.K.Mouselines were established by K.C., J. Loh and B.P.S. Mouse analysis experiments wereperformed by K.C., J. Liu, S.L.B., H.M., W.K., T.S.S. and J. Lennerz. Histologicalexamination was performed by T.S.S, E.M.B. and J. Lennerz.  L. monocytogenes experiments were performed by J.C. and K.C. Microarray analysis was performedby R.J.X. and T.S.S. Human tissue collection, genotyping and preparation wereperformed by E.L., S.H. and C.S. N.M. provided advice, the antibody to ATG16L1,andexperimentsbyC.K.wereperformedinN.M.’slaboratory.Themanuscriptwaswritten by K.C., H.W.V. and T.S.S., and all authors commented on the manuscript,data and conclusions before submission. Author Information  CEL files have been submitted to GEOfor both LCM procuredcrypt base samples (accession numbers GSM318315 (ATG16 HM1-1),GSM318588 (ATG16 HM1-2), GSM318589 (WT1) and GSM318590 (WT2)) andthe thymocyte samples (accession number GSE12707). Reprints and permissionsinformation is available at Correspondence andrequests for materials should be addressed to H.W.V. ( andT.S.S. ( NATURE  LETTERS 5  ©2008 Macmillan Publishers Limited. All rights reserved
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