Faculty Bibliography

It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFalpha and IFNgamma and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.

Autophagy is implicated in many functions of mammalian cells such as organelle recycling, survival and differentiation, and is essential for the maintenance of T and B lymphocytes. Here, we demonstrate that autophagy is a constitutive process during T cell development. Deletion of the essential autophagy genes Atg5 or Atg7 in T cells resulted in decreased thymocyte and peripheral T cell numbers, and Atg5-deficient T cells had a decrease in cell survival. We employed functional-genetic and integrative computational analyses to elucidate specific functions of the autophagic process in developing T-lineage lymphocytes. Our whole-genome transcriptional profiling identified a set of 699 genes differentially expressed in Atg5-deficient and Atg5-sufficient thymocytes (Atg5-dependent gene set). Strikingly, the Atg5-dependent gene set was dramatically enriched in genes encoding proteins associated with the mitochondrion. In support of a role for autophagy in mitochondrial maintenance in T lineage cells, the deletion of Atg5 led to increased mitochondrial mass in peripheral T cells. We also observed a correlation between mitochondrial mass and Annexin-V staining in peripheral T cells. We propose that autophagy is critical for mitochondrial maintenance and T cell survival. We speculate that, similar to its role in yeast or mammalian liver cells, autophagy is required in T cells for the removal of damaged or aging mitochondria and that this contributes to the cell death of autophagy-deficient T cells.

The analysis of autophagy in cells and tissue has principally been performed via qualitative measures. These assays identify autophagosomes or measure the conversion of LC3I to LC3II. However, qualitative assays fail to quantitate the degradation of an autophagic substrate and therefore only indirectly measure an intact autophagic system. "Autophagic flux" can be measured using long-lived proteins that are degraded via autophagy. We developed a quantifiable luciferase reporter assay that measures the degradation of a long-lived polyglutamine protein aggregate, polyQ80-luciferase. Using this reporter, the induction of autophagy via starvation or rapamycin in cells preferentially decreases polyQ80-luciferase when compared with a nonaggregating polyQ19-luciferase after four hours of treatment. This response was both time- and concentration-dependent, prevented by autophagy inhibitors and absent in ATG5 knockout cells. We adapted this assay to living animals by electroporating polyQ19-luciferase and polyQ80-luciferase expression constructs into the right and left tibialis anterior (TA) muscles of mice, respectively. The change in the ratio of polyQ80-luciferase to polyQ19-luciferase signal before and after autophagic stimulation or inhibition was quantified via in vivo bioluminescent imaging. Following two days of starvation or treatment with intraperitoneal rapamycin, there was a approximately 35% reduction in the ratio of polyQ80:polyQ19-luciferase activity, consistent with the selective autophagic degradation of polyQ80 protein. This autophagic response in skeletal muscle in vivo was abrogated by co-treatment with chloroquine and in ATG16L1 hypomorphic mice. Our study demonstrates a method to quantify the autophagic flux of an expanded polyglutamine via luciferase reporters in vitro and in vivo.

Recently identified genetic determinants for enhanced susceptibility to Crohn disease (CD) included polymorphisms in the ATG16L1 and IRGM1 loci suggesting that the autophagy pathway plays a role in the pathogenesis of this disease. We have generated and analyzed three mouse models with diminished expression of autophagy proteins and show how the loss of function of various autophagy components contributes to CD pathogenesis. In the mouse small intestine, one common cellular target of Atg16L1, Atg5 and Atg7 is the Paneth cell, a specialized epithelial cell whose main function is the delivery of antimicrobial factors into the intestinal lumen by production and secretion of its characteristic cytoplasmic granules. Autophagy-deficient Paneth cells exhibited a striking loss of function in this granule exocytosis pathway. Transcriptional analysis revealed a gain of function whereby the gene expression associated with inflammatory responses was increased in autophagy-deficient Paneth cells. Importantly, we validated these findings by analyzing intestinal tissues from CD patients. Similar Paneth cell abnormalities were observed in CD patients homozygous for the ATG16L1 risk allele. Thus, one role for the autophagy pathway in CD pathogenesis is through selective effects on the biology and specialized properties of Paneth cells.

Polymorphisms associated with two genes in the autophagy pathway, ATG16L1 and IRGM1, have been implicated in susceptibility to Crohn's disease, an idiopathic inflammatory disease typically involving the gastrointestinal tract. The intestinal mucosa is a site of careful immune regulation where the epithelium and immune cells encounter pathogens as well as a robust and diverse population of indigenous microbes that are predominately bacteria. Since the role of autophagy in immunity is broad and expanding, it is unclear which downstream functions of autophagy and which cell types are the key factors in Crohn's disease susceptibility. This chapter reviews the recent literature on the roles of ATG16L1 and IRGM1 in the autophagy pathway, inflammation, antimicrobial immunity, and the biology of the intestine, and discusses how these genes may contribute to Crohn's disease pathogenesis.

The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. Using mice with granulocyte- and macrophage-specific deletion of the essential autophagy protein Atg5, we show that Atg5 is required for in vivo resistance to the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii. In primary macrophages, Atg5 was required for interferongamma (IFN-gamma)/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect classical hallmarks of autophagy, such as autophagosomes enveloping T. gondii, Atg5 was required for recruitment of IFN-gamma-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane, an event that mediates IFN-gamma-mediated clearance of T. gondii. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo, and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.

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.

Kaposi's sarcoma-associated herpesvirus encodes two homologous E3 ligases, MIR1 and MIR2, that mediate the ubiquitination and subsequent downregulation of several cell surface proteins, and in particular major histocompatibility complex class I (MHC-I) molecules. We have previously shown that, in addition to lysine ubiquitination, MIR1 has the unique ability of transferring ubiquitin onto MHC-I molecules lacking available lysine residues, in a cysteine-dependent manner. Here we report that MIR1 activity is maximal when either a lysine or cysteine residue is placed approximately 15 amino acids away from the transmembrane domain, whereas MIR2 preferentially targets residues, including cysteines, that are closer to the transmembrane domain. Thus MIR1 and -2 can distinguish their substrates based on the position of the lysine or cysteine residues, suggesting that these proteins have evolved to target different sets of surface molecules. These results indicate that the position of target residues within a substrate is an essential determinant of E3 ubiquitin ligase specificity.

Macroautophagy (herein autophagy) is an evolutionarily conserved process, requiring the gene ATG5, by which cells degrade cytoplasmic constituents and organelles. Here we show that ATG5 is required for efficient B cell development and for the maintenance of B-1a B cell numbers. Deletion of ATG5 in B lymphocytes using Cre-LoxP technology or repopulation of irradiated mice with ATG5-/- fetal liver progenitors resulted in a dramatic reduction in B-1 B cells in the peritoneum. ATG5-/- progenitors exhibited a significant defect in B cell development at the pro- to pre-B cell transition, although a proportion of pre-B cells survived to populate the periphery. Inefficient B cell development in the bone marrow was associated with increased cell death, indicating that ATG5 is important for B cell survival during development. In addition, B-1a B cells require ATG5 for their maintenance in the periphery. We conclude that ATG5 is differentially required at discrete stages of development in distinct, but closely related, cell lineages.

Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to beta2-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.