Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense

Abstract

A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1β production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1β production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.

Fig. 1.

Generation and characterization of T300A mice. (A) Schematic of targeted genomic region (gray), exon 9 (black), neomycin resistance cassette (green), and amino acids at position 300. Southern blot probes are indicated (dashed lines). (B) Genomic DNA of ES cells were detected via Southern blot. (C) Ileal sections were stained with lysozyme (red) and DAPI (blue). White dotted lines outline a single crypt. (Scale bar, 10 µm.) (D) Paneth cell phenotypes were classified as normal or abnormal (with disordered, diminished, diffuse, or excluded granule phenotypes) based on lysozyme-positive secretory granule morphology. Details of these categories are provided in SI Materials and Methods. (E) PAS/Alcian blue-stained colonic sections. Black dotted lines outline surface epithelium. Arrows indicate surface goblet cells. (Scale bar, 100 µm.) (F) Percentage of surface goblet cells (black arrows in E) greater than 320 μm². Data shown as mean ± SEM; n = 6–7 mice per group, n = 200–300 goblet cells per mouse (**P < 0.01). (G) Quantification of average goblet cell size (average area of cytoplasmic mucin/goblet cell) in WT and T300A ascending colon (n = 6 mice per group; ***P < 0.005, Student t test). (H) Organoid formation per WT Lgr5⁺ intestinal stem cells cocultured with sorted Paneth cells from indicated mice (data shown as mean ± SD; n ≥ 4).

Fig. 2.

ATG16L1 T300A is more susceptible to caspase 3- and caspase 7-mediated cleavage. (A) Immunoblot analysis of LC3-I/II and p62 in MEFs untreated or stimulated for 4 h with 10 µg/mL E64d plus pepstatin or 100 nM Torin 1 and 10 μg/mL E64d plus pepstatin. Blots were probed for LC3-I/II and p62. Actin served as a loading control. (B) Schematic of ATG16L1 domains with caspase cleavage consensus sequence and location of the T300A polymorphism. CC, coiled-coil domain. WD, WD40 domain. (C–E) ATG16L1 WT (isoform 1) or ATG16L1 T300A, with or without an additional D299E mutation, was in vitro-translated with [³⁵S]methionine and incubated with recombinant caspases for 1 h at 37 °C. ATG16L1 cleavage was observed by autoradiography. (F and G) Western blots showing ATG16L1 cleavage from HeLa cells transfected with indicated constructs and treated for 4 h with 1 μM staurosporine or 20 μM zVAD. FL, full-length.

Fig. 3.

T300A polymorphism enhances IL-1β secretion and is associated with increased susceptibility to bacteria-induced inflammation. (A) CD11c⁺ cells were isolated from the mesenteric lymph node and stimulated with LPS. IL-1β levels in culture supernatants were assessed after 24 h (data shown as mean ± SD; n = 2; *P = 0.047). Cell lysates were analyzed by Western blot for pro–IL-1β levels. Actin served as a loading control. (B) CD11b⁺ cells were isolated from the spleen and colonic lamina propria (LP) and stimulated with LPS and MDP. IL-1β levels in culture supernatants were assessed after 24 h (data shown as mean ± SD; n ≥ 5; *P = 0.04, WT vs. T300A splenic; *P < 0.05, WT vs. T300A lamina propria). (C) CD11b⁺ cells were isolated from the spleen and stimulated with LPS and MDP. IL-1β levels in culture supernatants were assessed after 24 h (data shown as mean ± SD; n = 10; *P = 0.04, WT vs. T300A; **P = 0.01, ATG16L1 KO/WT vs. ATG16L1 KO/T300A). (D) CD11b⁺ cells were isolated from the spleen and infected with S. flexneri. IL-1β levels in culture supernatants were assessed after 16 h. (Data shown as mean ± SD and are representative of n = 3; *P = 0.018). (E) S. flexneri or S. flexneri ΔicsB survival as assessed by an intracellular bacterial protection assay. Relative bacterial luciferase units were measured in live MEFs at the indicated times postinfection. Fold replication was calculated for each well as raw luciferase units at the indicated time point divided by raw luciferase units at t 1.5 h (n = 4; data shown as mean ± SD; ***P < 0.0004, Atg16L1 WT vs. T300A with Shigella WT; ***P < 10⁻⁵, Atg16L1 WT vs. T300A with Shigella ΔicsB; *P = 0.02, Shigella WT vs. Shigella ΔicsB in Atg16L1 WT MEFs). (F) S. flexneri ΔicsB intracellular replication in small intestinal epithelial cells. Fold replication was calculated for each well as cfu counts per 100,000 cells at the indicated time point/raw luciferase units (n > 3; *P = 0.0315). (G) Serum IL-1β levels 6 d after S. Typhimurium infection (n ≥ 4; **P < 0.01, WT vs. T300A; **P < 0.01, Rag1 KO vs. T300A Rag1 KO). (H) Histological score for inflammation in cecal tissues 6 d after S. Typhimurium infection (data shown as mean ± SD; n ≥ 4; *P = 0.013).

Fig. 4.

Quantitative proteomics identifies ATG16L1-dependent proteins involved in IL-1β secretion and antibacterial autophagy. (A) Schematic of quantitative iTRAQ proteomic experimental design. Atg16L1^−/− MEFs were transfected with empty vector, FLAG-WT hATG16L1 isoform 1, FLAG-hATG16L1 isoform 2, or FLAG-hATG16L1 T300A. Immunoprecipitated complexes were labeled, combined, and analyzed by liquid chromatography/MS. (B) Summary of proteomic interactors selected for follow-up analysis. Proteins in bold were previously identified as members of the autophagy interactome (23); proteins also in italics were previously identified as ATG16L1 interactors. (C) Interactome analysis shows network after extension. Proteins identified by proteomics are shown in purple and blue, with high-confidence autophagy-associated genes shown in purple. Intervening nodes are shown in green. High-confidence interactions are shown with red lines, and lower-confidence interactions are visualized with blue lines. (D) Immortalized BMDMs were infected with the indicated shRNA lentiviruses, and transduced cells were selected with puromycin. Cells were stimulated with IFN-γ (100 ng/mL), LPS (100 ng/mL), and MDP (10 μg/mL) for 24 h, and harvested supernatants were analyzed by ELISA for IL-1β secretion (data shown as mean ± SD; n = 4). (E) HeLa cells stably transduced with GFP-LC3 were transfected for 48 h with control siRNA or siRNAs against the indicated genes and then infected with DsRed-labeled Salmonella for 1 h. GFP-LC3/bacteria colocalization was measured at six sites per well, and data shown are mean ± SD of three wells per condition. Data are representative of three independent experiments. (F) Salmonella survival as assessed by an intracellular bacterial protection assay in HeLa cells treated with the indicated siRNA for 48 h. Fold replication was calculated for each well as raw luciferase units at the indicated time point divided by raw luciferase units at t 1.5 h (data shown as mean ± SD; n = 3). (G) Representative confocal fluorescence microscopy images of HeLa cells treated as described in E. (H) siRNA-treated cells were infected as described in E and stained with anti-NDP52. NDP52/bacteria colocalization was measured at six sites per well, and data shown are mean ± SD of three wells per condition. Data are representative of three independent experiments (*P = 0.045, siCtrl vs. siNOLC1). (I) Representative confocal fluorescence microscopy images of HeLa cells treated as in G.