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

Abstract

Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn'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 second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.

Figure 1. Role of Atg16L1 in autophagy in cells and intestine

a, Gene trap vector containing a splice acceptor followed by the βgeo exon disrupts the Atg16L1 gene locus by insertions within the intronic regions following exon 6 and 10 for the Atg16L1^HM1 and Atg16L1^HM2 lines respectively. b, Detection of the α and β isoforms of Atg16L1 by Western blot analysis in whole cell lysates from Murine embryo fibroblasts (MEFs). c-d, Atg5^−/− and Atg16L1^HM MEFs were grown in the presence of rapamycin (50μg/ml) and cyclohexamide (5μg/ml) and analyzed by Western blot for loss of p62 expression (c) and quantified (n = 3) (d). e-f, Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin (50μg/ml) or DMSO control for 4 h (e). Quantification of LC3-II expression in the presence of rapamycin (n = 6) (f). g, Detection of Atg16L1, LC3, and p62 by Western blot of ileal lysates from Atg16L1^HM mice. Atg16L1 can be detected in both mutant lines upon longer exposures. h-i, Quantification of Atg16L1 (h) and p62 in ileal lysates (n = 3). Protein levels were quantified by densitometry and normalized to actin. P values were calculated using two-tailed student's t test. Error bars represent SEM.

Figure 2. Mutation of Atg16L1 or Atg5 leads to disruption of the Paneth cell granule exocytosis pathway

a-b, Whole-mount images taken immediately above the ileal mucosal surface from WT (a) and Atg16L1^HM (b) mice stained with Helix pomatia lectin that labels mucus (green) and antisera directed against lysozyme (red). Images are representative of 3 WT and 3 Atg16L1^HM mice. c-d, Ileal sections from WT (c) and Atg16L1^HM (d) mice stained with PAS-alcian blue (dotted line denotes crypt unit and arrowheads indicate Paneth cells). Images are representative of 16 WT, 16 Atg16L1^HM1, and 14 Atg16L1^HM2, greater than 100 crypts analyzed for each. e-f, Representative images of indirect immunofluorescence of sections stained for lysozyme (red) in WT (e) and Atg16L1^HM (f) mouse ileal crypts. g, Paneth cells display one of four patterns of lysozyme expression (represented in red, white represents areas that exclude lysozyme): normal (D₀), disordered (D₁), depleted (D₂), and diffuse (D₃). h-i, Number of Paneth cells from Atg16L1^HM (h) and Atg5^flox/floxvillin-Cre (i) mice displaying each pattern of lysozyme expression (n = 6660 cells from 5 Atg16L1^HM mice and 5634 cells from 5 WT mice; n = 1756 cells from 2 Atg5^flox/floxvillin-Cre mice and 1649 from 2 Atg5^flox/flox mice). Scale bars: a-b, 200 μm; c-f, 10 μm. *p<0.05, **p<0.01, ***p<0.001. P values were calculated using two-tailed student's t test. Error bars represent SEM.

Figure 3. Critical role of Atg16L1 in the structure and transcriptional profile of Paneth cells

a-d, EM of littermate WT (a, c) and Atg16L1^HM (b, d) Paneth cells (a, b: dotted lines denote individual cells, asterisk indicates normal progenitor cell; c, d: blue stars indicate normal mitochondria in WT Paneth cells and Atg16L1^HM progenitor cells, red diamonds indicate degenerating mitochondria, dotted Box shows normal Golgi compartments, and yellow asterisks indicate granules) (n = 3 mice/genotype). e, Mitochondrial integrity was quantified based on the percentage of the visible mitochondrial section displaying intact cisternae (n = 49 WT and 71 Atg16L1^HM cells). f, Enrichment of pathways associated with human orthologs mapped from genes differentially expressed in Atg16L1 deficient Paneth cells relative to WT cells, using the canonical pathway collection from MSigDB. The bar chart displays the negative log of the enrichment p-values for each pathway using the hypergeometric distribution (see methods). *denotes pathways found to be significantly enriched (p<0.05). Pathways with only one gene assigned were excluded from the chart. g, Chart of selected genes whose mRNA are enriched in either Atg16L1 deficient Paneth cells (baseline = WT Paneth cells) or Atg16L1 deficient thymocytes (baseline = WT thymocytes). N.S. = not significant. Scale bars: a-b, 10 μm; c-d, 500nm.

Figure 4. Crohn's Disease patients homozygous for the disease risk allele of ATG16L1 display Paneth cell abnormalities similar to Atg16L1^HM mice

a-b, H&E stained sections of uninvolved areas from ileo-colic resections from patients with Crohn's disease homozygous for the safe (a) or risk allele (b) of ATG16L1 (blue dotted line denotes crypt unit). c-f, Immunofluorescence images of Paneth cells from control (c, e) and patients with the risk allele (d, f) stained for lysozyme (red) (c, d) and double labeled additionally for leptin (green) (e, f) (yellow dotted line denotes crypt unit). g, Aberrant lysozyme expression was quantified by the same criteria used for mouse sections in Fig. 3 (n = 6829 cells for at risk patients and 8182 for control in c and e, n = 5 patients for both genotypes in d and e). Leptin positive D3 cells were quantified in patient samples homozygous for the risk allele (76/322 D3 cells were positive from a total of 580 crypts examined) and homozygous for the safe allele (11/93 D3 cells were positive from a total 749 crypts examined). Scale bars: a-f, 10 μm. *p<0.05, **p<0.01, ***p<0.001. P values were calculated using two-tailed student's t test. Error bars represent SEM.