Crohn’s disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn’s disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296–299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn’s disease.
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Manolio, T. A. Bringing genome-wide association findings into clinical use. Nature Rev. Genet. 14, 549–558 (2013)
Adolph, T. E. et al. Paneth cells as a site of origin for intestinal inflammation. Nature 503, 272–276 (2013)
Abraham, C. & Cho, J. H. Inflammatory bowel disease. N. Engl. J. Med. 361, 2066–2078 (2009)
Gardet, A. & Xavier, R. J. Common alleles that influence autophagy and the risk for inflammatory bowel disease. Curr. Opin. Immunol. 24, 522–529 (2012)
Kaser, A. et al. XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell 134, 743–756 (2008)
He, C. & Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009)
Baxt, L. A., Garza-Mayers, A. C. & Goldberg, M. B. Bacterial subversion of host innate immune pathways. Science 340, 697–701 (2013)
Hampe, J. et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nature Genet. 39, 207–211 (2007)
Saitoh, T. et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production. Nature 456, 264–268 (2008)
Cadwell, K. et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 456, 259–263 (2008)
Cadwell, K. et al. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell 141, 1135–1145 (2010)
Cooney, R. et al. NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation. Nature Med. 16, 90–97 (2010)
Fujita, N. et al. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy: analysis of the organization of the Atg16L1 complex in fibroblasts. J. Biol. Chem. 284, 32602–32609 (2009)
Messer, J. S. et al. The Crohn's disease: associated ATG16L1 variant and Salmonella invasion. BMJ Open 3, e002790 (2013)
Sorbara, M. T. et al. The protein ATG16L1 suppresses inflammatory cytokines induced by the intracellular sensors Nod1 and Nod2 in an autophagy-independent manner. Immunity 39, 858–873 (2013)
Conway, K. L. et al. Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection. Gastroenterology 145, 1347–1357 (2013)
Stennicke, H. R., Renatus, M., Meldal, M. & Salvesen, G. S. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. Biochem. J. 350, 563–568 (2000)
Rawlings, N. D., Barrett, A. J. & Bateman, A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 40, D343–D350 (2012)
McStay, G. P., Salvesen, G. S. & Green, D. R. Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 15, 322–331 (2008)
Jost, P. J. et al. XIAP discriminates between type I and type II FAS-induced apoptosis. Nature 460, 1035–1039 (2009)
McAlpine, F., Williamson, L. E., Tooze, S. A. & Chan, E. Y. Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2. Autophagy 9, 361–373 (2013)
Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010)
Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012)
Bialik, S. et al. The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes. Circ. Res. 85, 403–414 (1999)
Caro-Maldonado, A. et al. Glucose deprivation induces an atypical form of apoptosis mediated by caspase-8 in Bax-, Bak-deficient cells. Cell Death Differ. 17, 1335–1344 (2010)
Altman, B. J. & Rathmell, J. C. Metabolic stress in autophagy and cell death pathways. Cold Spring Harb. Perspect. Biol. 4, a008763 (2012)
Kuballa, P., Huett, A., Rioux, J. D., Daly, M. J. & Xavier, R. J. Impaired autophagy of an intracellular pathogen induced by a Crohn’s disease associated ATG16L1 variant. PLoS ONE 3, e3391 (2008)
Altshuler, D. M. et al. Integrating common and rare genetic variation in diverse human populations. Nature 467, 52–58 (2010)
Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075 (2008)
Choi, A. M., Ryter, S. W. & Levine, B. Autophagy in human health and disease. N. Engl. J. Med. 368, 651–662 (2013)
Jostins, L. et al. Host–microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 491, 119–124 (2012)
Brown, E. M., Sadarangani, M. & Finlay, B. B. The role of the immune system in governing host-microbe interactions in the intestine. Nature Immunol. 14, 660–667 (2013)
Grassl, G. A., Valdez, Y., Bergstrom, K. S., Vallance, B. A. & Finlay, B. B. Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis. Gastroenterology 134, 768–780 (2008)
Dube, P. H., Revell, P. A., Chaplin, D. D., Lorenz, R. G. & Miller, V. L. A role for IL-1α in inducing pathologic inflammation during bacterial infection. Proc. Natl Acad. Sci. USA 98, 10880–10885 (2001)
Prescott, N. J. et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn’s disease and is independent of CARD15 and IBD5. Gastroenterology 132, 1665–1671 (2007)
Mills, S. D. et al. Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein. Proc. Natl Acad. Sci. USA 94, 12638–12643 (1997)
Erfurth, S. E. et al. Yersinia enterocolitica induces apoptosis and inhibits surface molecule expression and cytokine production in murine dendritic cells. Infect. Immun. 72, 7045–7054 (2004)
Denecker, G. et al. Yersinia enterocolitica YopP-induced apoptosis of macrophages involves the apoptotic signaling cascade upstream of bid. J. Biol. Chem. 276, 19706–19714 (2001)
Nakahira, K. et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nature Immunol. 12, 222–230 (2011)
Shi, C. S. et al. Activation of autophagy by inflammatory signals limits IL-1β production by targeting ubiquitinated inflammasomes for destruction. Nature Immunol. 13, 255–263 (2012)
Marchiando, A. M. et al. A deficiency in the autophagy gene Atg16L1 enhances resistance to enteric bacterial infection. Cell Host Microbe 14, 216–224 (2013)
Clausen, B. E., Burkhardt, C., Reith, W., Renkawitz, R. & Forster, I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 8, 265–277 (1999)
Kuida, K. et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384, 368–372 (1996)
Barrett, J. C. et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nature Genet. 40, 955–962 (2008)
Wang, G. G. et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nature Methods 3, 287–293 (2006)
de la Calle, C., Joubert, P.-E., Law, H. K. W., Hasan, M. & Albert, M. Simultaneous assessment of autophagy and apoptosis using multispectral imaging cytometry. Autophagy 7, 1045–1051 (2011)
The authors would like to thank M. Zepeda for coordinating human donors, R. A. Flavell for providing Casp3-knockout mice, J. E. Cupp, W. Ortmann, J. Borneo, J. Ruan, J. Ting and L. Rangell for technical assistance, D. Holmes, N. Kayagaki, C. J. Spooner, M. E. Keir, A. Ashkenazi and T. W. Behrens for critical evaluation of the manuscript.
All authors are employees of Genentech, Inc., a for-profit institution.
Extended data figures and tables
Extended Data Figure 1 T300A enhances whereas D299E abolishes caspase-3-mediated ATG16L1 processing.
a, In vitro translated variants of V5-tagged ATG16L1 (wild-type, T300A, D299E) were incubated with recombinant active caspase 1, 3, 6 or 7 for 1 h followed by assessment of ATG16L1 cleavage by V5 immunoblot. b, HeLa cells were transfected with indicated C-terminal V5-tagged ATG16L1 construct, followed by treatment with 20 ng ml−1 TNF + 10 μg ml−1 cycloheximide (CHX) for indicated times. Immunoblot was performed for V5. *non-specific band. c, LysMCre+Atg16l1loxp/loxp bone marrow progenitors were immortalized with ER–HoxB8 and reconstituted with the indicated murine ATG16L1 retroviral constructs. Macrophage differentiation was induced by removal of β-oestradiol for 10 days. Immunoblotting with an ATG16L1 antibody indicates complete deletion of endogenous protein in vector-transfected cells and successful reconstitution with the listed ATG16L1 variants. Expression of the truncated splice variant ATG16L1β′ (52 kDa) is observed along with full-length ATG16L1β (68 kDa). d, ER–HoxB8 immortalized progenitors transduced with above constructs were differentiated into macrophages, and cells were treated with indicated doses of TNF + 10 μg ml−1 CHX. C-terminal fragment of cleaved ATG16L1 was detected by immunoblotting with a V5 antibody. Data in a are representative of 3 independent experiments; data in b–d are representative of 2 independent experiments.
Extended Data Figure 2 ATG16L1 cleavage is enhanced in primary human macrophages with T300A variant during TNF-α-mediated apoptosis whereas caspase 3 and PARP cleavage remain unaltered in both groups.
Representative immunoblots depicting cleavage of ATG16L1, caspase 3 and PARP in non-risk (WT) and T300A cells following TNF-α + CHX treatment. Scatter plots are gated on donor macrophages demonstrating TNF-α-mediated caspase 3 cleavage.
Extended Data Figure 3 Generation of T316A knock-in mutant mice.
a, Atg16l1 T316A knock-in construct design. b, Genomic PCR analysis of embryonic stem cell clone used for microinjection to generate colony (clone H4-1-B2-1), wild-type and knock-in mice. WT = 470bp; knock-in = 436bp. c, Atg16l1loxp/loxp construct design. d, Atg16l conditional knockout mice were generated by crossing Atg16l1loxp/loxp mice with LysMCre+ mice. Western blots demonstrate protein levels of ATG16L1β/α and α-tubulin in lysates from thioglycollate-elicited macrophages from LysMCre+Atg16l1loxp/loxp and Atg16l1wt/wt mice.
Extended Data Figure 4 Relative abundance of myeloid and lymphoid cell subsets in ATG16L1 T316A knock-in mice and WT controls under basal conditions.
a, Flow cytometry analysis of indicated myeloid cell populations in bone marrow, spleen, mesenteric and peripheral (inguinal and brachial) lymph nodes. n = 5 female mice aged 6–10 weeks per genotype were analysed. Scatterplot bars represent mean. b, Flow cytometry analysis of indicated lymphoid cell populations in spleen, mesenteric and peripheral lymph nodes. T-cell activation was measured by CD69 staining. Five female mice aged 6–10 weeks per genotype were analysed. Scatterplot bars represent mean.
Extended Data Figure 5 Histological analysis and quantification of small intestine Paneth cell granules in ATG16L1 T316A knock-in and WT control mice under basal conditions.
a, Haematoxylin and eosin staining depicting normal villus and crypt architecture, with comparable Paneth cell abundance in WT and T316A female mice aged 6–10 weeks. b–d, Similar size and morphology of Paneth cell granules identified by lysozyme staining in WT and T316A mice. Graph in c represents quantification of lysozyme-positive granule allocation patterns as described10. Data are mean ± S.D., n = 5 female mice per genotype, aged 6–10 weeks. A total of 701 (WT) and 891 (T316A) Paneth cells were quantified. Arrows indicate specific Paneth cell morphologies as described in reference 10. Error bars depict standard deviation from mean.
Extended Data Figure 6 Enhanced cleavage of murine ATG16L1 α and β isoforms in macrophages harbouring the T316A genotype; comparable ATG16L1 stability under baseline conditions.
a, Multiple exposures of ATG16L1 immunoblots reveal increased processing of ATG16L1β/α harbouring Τ316Α variant. Graphs quantify cleavage of ATG16L1α and β isoforms. Data are pooled from 5 independent experiments. b, 35S pulse-chase illustrating comparable turnover of wild-type and T316A variants of ATG16L1. Densitometry analysis of ATG16L1 levels is depicted in the graph using data from 3 independent experiments.
Extended Data Figure 7 ATG16L1 processing requires caspase 3.
a, HeLa cells were pre-treated with vehicle (DMSO) or pan-caspase inhibitor (zVAD-fmk), followed by stimulation with 20 ng ml−1 TNF + 10 μg ml−1 CHX, 0.5 μg ml−1 Fas-agonist antibody + 10 μg ml−1 CHX or 2.0 μM staurosporine for indicated times. b, RAW 264.7 cells were pre-treated with DMSO or zVAD-fmk followed by treatment with 20 ng ml−1 TNF-α + 10 μg ml−1 CHX for indicated times. c, Bax+/+ or Bax−/− colon carcinoma epithelial cells (HCT116) were pre-treated with DMSO or 20 μM zVAD-fmk for 3 h, followed by treatment with 20 ng ml−1 TNF + 10 μg ml−1 CHX for indicated times. d, HeLa cells transfected with control, caspase-3- or caspase 7-specific siRNAs were stimulated with 2.0 μM staurosporine for indicated times. e, MCF-7 cells were pre-treated with DMSO or zVAD-fmk followed by stimulation with 20 ng ml−1 TNF + 10 μg ml−1 CHX, 0.5 μg ml−1 Fas-agonist antibody + 10 μg ml−1 CHX or 2.0 μM staurosporine for indicated times. Data in a–e are representative of 2 independent experiments. Atg16l1 genotypes of cell lines used are listed in Supplementary Table 4. f, Bone-marrow-derived macrophages from WT mice were treated as indicated to induce canonical NLRP3 inflammasome activation. ATG16L1, caspase 1 and IL-1β processing were assessed by immunoblotting. upLPS = ultrapure LPS. Data are representative of 2 independent experiments.
Extended Data Figure 8 Reduced autophagic flux induced by nutrient starvation of bone-marrow-derived macrophages from T316A mice or mice with a macrophage-specific deletion of Atg16l1 (LysMCre+Atg16l1loxp/loxp).
a, Immunoblot analysis of LC3-lipidation following amino acid and serum starvation for 4 h. 400 nM bafilomycin A1 was added where indicated to promote accumulation of lipidated LC3 (LC3-II). b, Cells were nutrient starved by culture in glucose-free medium without serum for indicated time points. 400 nM bafilomycin A1 was added where indicated to promote accumulation of lipidated LC3 (LC3-II). LC3-II spots enumerated by counting the number of punctae identified by LC3 bright detail intensity analysis in Fig. 3. 10,000 cells per sample were analysed, n = 4 mice. Scatterplot bars represent mean. c, Cells were nutrient starved by culture in amino-acid depleted medium without serum for indicated time points. 400 nM bafilomycin A1 was added where indicated to promote accumulation of lipidated LC3 (LC3-II). LC3-II area was quantified using image-based flow cytometry. d, Cells were cultured as in c (2 × 104 cells per well), followed by caspase 3 activity analysis using a luciferase-based caspase 3 substrate cleavage assay. e, Cells were treated with 2.5 μg ml−1 rapamycin for 4 h. 400 nM bafilomycin A1 was added where indicated. LC3-II area was quantified as in c. f, Cells were cultured as in e (2 × 104 cells per well), followed by caspase 3 activity analysis as in d. g, h, Bone-marrow-derived macrophages from control (LysMCre+Atg16l1wt/wt) or LysMCre+Atg16l1loxp/loxp mice were starved of glucose in the presence or absence of bafilomycin A1 for 4 h, and image-based flow cytometry used to measure the area occupied by punctate LC3 (LC3-II area, g) and LC3-II spots (h) for 10,000 cells per sample, n = 3 mice. Scatter plot bars represent mean. Data in a–h are representative of 2 independent experiments.
Extended Data Figure 9 Defective Y. enterocolitica clearance in T316A mutant or ATG16L1 deficient macrophages; comparable Y. enterocolitica invasion and caspase 3/7 activation in wild type and T316A macrophages.
a, Thioglycollate-elicited peritoneal macrophages (2 × 104 cells per well) were infected with 20 m.o.i. Y. enterocolitica for 30 min followed by 6 h of culture in gentamycin sulphate. Cells were lysed and caspase 3 activity measured using a luciferase-based caspase 3 activity assay. 2.0 μM staurosporine treatment for 3 h was used as a positive control for caspase 3 activation. zVAD-fmk was added where indicated to abolish caspase 3 activity. b, Thioglycollate-elicited peritoneal macrophages from LysMCre+Atg16l1+/+ or Atg16l1loxp/loxp mice (1.25 × 105 cells per well) were infected with Y. enterocolitica for 30 min followed by 6 h of culture in gentamycin sulphate. Bacterial c.f.u. were calculated following cell lysis. c, Thioglycollate-elicited peritoneal macrophages from WT and T316A mutant mice (1.25 × 105 cells per well) were infected with indicated m.o.i. of Y. enterocolitica for 30 min followed by 6 h of culture in gentamycin sulphate to measure pathogen clearance. n = 4 mice, scatterplot bars represent mean. d, Macrophages were infected as in c and immediately washed with gentamycin sulphate followed by cell lysis and plating to measure bacterial invasion. n = 4 mice, scatterplot bars represent mean. Data in a–d are representative of 2 independent experiments.
Extended Data Figure 10 Tnfa, Il1b and Il6 transcription and secretion by murine macrophages following stimulation with heat-killed Y. enterocolitica, TLR ligands and muramyl dipeptide (MDP, NOD2 ligand); ileal inflammation and systemic IL-1β release induced by oral gavage of Y. enterocolitica.
a–c, Thioglycollate-elicited peritoneal macrophages from WT or T316A mice were stimulated with heat-killed Y. enterocolitica (bacteria heated to 90 °C for 30 min) for indicated time points. a, Tnfa transcript levels were analysed by quantitative PCR, and TNF-α protein release was measured by ELISA. Cytokine transcript levels are normalized to Gapdh (2−ΔCt). b, Il1b transcript levels and IL-1β protein release measured as in a. c, Il6 transcript levels and IL-6 protein release measured as in a. d, e, TNF-α (d) and IL-6 (e) release from thioglycollate-elicited macrophages measured by ELISA following stimulation with indicated ligands for 24 h. f. IL-1β release from thioglycollate-elicited macrophages measured by ELISA following stimulation with LPS, MDP or both for 24 h. Data in a–f are representative of 2 independent experiments. g, Histological analysis (haematoxylin and eosin) depicting ileal but not colonic inflammation in WT and T316A mice 48 h following oral gavage of Y. enterocolitica. Images are representative of 3 PBS-treated and 7–8 Y. enterocolitica-infected mice. All mice were males aged 7–10 weeks. h, Serum ELISA of murine IL-1β 48 h following oral gavage of Y. enterocolitica. Data are pooled from 2 independent experiments of 3 PBS-treated and 7–8 Y. enterocolitica infected mice, all aged 7–10 weeks. Male mice were used in the first experiment; female mice were used in the second experiment. Dotted line depicts lower detection limit of the ELISA. Scatterplot bars depict means.
This file contains Supplementary Tables 1-5. The following is extra information for Supplementary Table 1: Comparison of the reference (A) and risk (G) allele frequency indicates that the Crohn’s disease risk associated G allele is the major allele in northern and western Europeans (CEU, frequency of 0.567), Tuscan Italians (TSI, frequency of 0.540) and near dominant in Gujarati Indians (GIH, frequency of 0.480). (PDF 161 kb)
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Murthy, A., Li, Y., Peng, I. et al. A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3. Nature 506, 456–462 (2014). https://doi.org/10.1038/nature13044
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