Volume 142, Issue 5, Pages 1097-1099.e4 (May 2012) Abnormal Activation of Autophagy-Induced Crinophagy in Paneth Cells From Patients With Crohn's Disease  Élodie Thachil, Jean–Pierre Hugot, Brigitte Arbeille, Régine Paris, Alain Grodet, Michel Peuchmaur, Patrice Codogno, Frédérick Barreau, Éric Ogier–Denis, Dominique Berrebi, Jérôme Viala  Gastroenterology  Volume 142, Issue 5, Pages 1097-1099.e4 (May 2012) DOI: 10.1053/j.gastro.2012.01.031 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 (A) CD Paneth cells showed strong LC3 staining compared with controls (CD inset: arrowhead points to a Paneth cell stained by H&E). (B) Quantifications of LC3-positive Paneth cells in duodenum and ileum. Each point indicates the value for 1 patient. (C) LC3-positive Paneth cells in noninflamed and inflamed CD duodenum compared with inflamed celiac duodenum (left inset: LC3 staining of Paneth cells was cytoplasmic with a heterogeneous and granular pattern. Central inset: H&E staining of an active and focal CD duodenitis). (D) In CD, transmission electron microscopy of CD Paneth cells showed numerous autophagic vacuoles (Av), primary (Ly I), and secondary (Ly II) lysosomes. Lamp1 accumulation (arrowhead) within CD Paneth cells compared with control Paneth cells (arrowhead) (inset: H&E staining of Paneth cells). **P < .01; ***P < .001. Gastroenterology 2012 142, 1097-1099.e4DOI: (10.1053/j.gastro.2012.01.031) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 (A) Transmission electron microscopy showed a decrease of secretory granules in CD Paneth cells associated with autophagic vacuoles engulfing granules (arrowheads). The number of secretory granules per Paneth cell is decreased in CD (B). High magnification of CD Paneth cells showed a high increase of different steps of crinophagy, including the fusion of secretory granules together (upper left) or with autophagolysome structures (upper inset), the early and late (lower inset) degradation of content stored in CD Paneth cells (right), leading to the complete elimination of a secretory granule in CD Paneth cells (arrowheads in the lower picture). The chart compares the quantification of crinophagic structures per Paneth cell (mean ± SEM). **P < .01; ***P < .001. Gastroenterology 2012 142, 1097-1099.e4DOI: (10.1053/j.gastro.2012.01.031) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 LC3 antibody was specific. To confirm the specificity of the LC3 antibody, LC3 staining was studied in granular cells tumor (A).2 In the intestinal mucosa, the nerve fibers within the lamina propria were LC3-positive and served as an internal positive control for immunostaining (B).3 The absence of mucosal staining was verified on serial sections in case of primary antibody omission (C) or in case of irrelevant antibody (data not shown). Gastroenterology 2012 142, 1097-1099.e4DOI: (10.1053/j.gastro.2012.01.031) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Mucosa LC3-positive cells are Paneth cells. In controls or CD intestinal samples, no LC3 staining was observed in enterocytes or mononuclear cells. The LC3-positive cells were identified as Paneth cells because of their localization at the bottom of the crypts (A), their progressive disappearance from the right to left colon, and their expression of the CD15 marker in double-labeling experiments using anti-CD15 (red staining) and anti-LC3 antibodies (dark-brown staining) (B). Although the number of LC3-positive Paneth cells was increased in CD patients, the total number of Paneth cells per crypt was not changed (C). In the left colon, some crypts expressed a high level of LC3 corresponding to the presence of metaplastic Paneth cells, which frequently occurs during CD, with a haphazard distribution (D). Gastroenterology 2012 142, 1097-1099.e4DOI: (10.1053/j.gastro.2012.01.031) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 The LC3 expression was not correlated to inflammation in ileum. In ileum biopsies, CD Paneth cells expressed LC3 in noninflamed (A) and inflamed samples (B), whereas the LC3 staining was negative in ulcerative colitis sections (C). Gastroenterology 2012 142, 1097-1099.e4DOI: (10.1053/j.gastro.2012.01.031) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 LC3 staining is not correlated to CD risk alleles of ATG16L1 or IRGM. (A, B) Quantitative analyses of LC3 expression in CD patients carrying ATG16L1 300A or 300T variants, and rs10065172 polymorphism localized in IRGM in duodenum and in terminal ileum. (C, D) IRGM duodenal staining within Paneth cells is similar from controls and CD patients, respectively. (E) Quantitative analyses of IRGM-positive Paneth cells per crypt in duodenum from CD patients and controls. (F) Quantitative analyse of IRGM-positive Paneth cells of CD patients carrying rs10065172 polymorphism of IRGM (n = 7). C allele and T allele are respectively the frequent and CD-associated alleles of IRGM. Values are mean ± SEM. Gastroenterology 2012 142, 1097-1099.e4DOI: (10.1053/j.gastro.2012.01.031) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Intermediary degradation steps of secretory granules during crinophagy in CD Paneth cells. High magnification visualization show partially degraded secretory granules occurring during crinophagy in different CD Paneth cells (A−D). Gastroenterology 2012 142, 1097-1099.e4DOI: (10.1053/j.gastro.2012.01.031) Copyright © 2012 AGA Institute Terms and Conditions