In one of the first genome-wide association studies (GWAS) we and our colleagues of the NIDDK IBD Genetics Consortium tested approximately 300,000 SNPs in one thousand patients with Crohn’s disease (CD) and in one thousand healthy individuals, and identified association with variants in the autophagy related 16-like 1 (S. cerevisiae) or ATG16L1 gene ⁵. The ATG16 gene product is part of a multimeric protein complex that is essential for autophagy, a biological process that mediates the bulk degradation of cytoplasmic components in lysosomes and vacuoles ⁶. This recent GWAS specifically identified an associated SNP that encodes a nonsynonymous amino acid change – an alanine to threonine substitution in exon 8 (a.k.a. Ala197Thr) – in the human equivalent of the ATG16 gene. In all of the populations examined, the threonine allele is the minor allele and has a protective effect. The same causal variant in ATG16L1 had also been identified in an independent screen of ~7000 common non-synonymous coding variants in a German CD study ⁷. Alone or combined, these two studies provide incontrovertible evidence that a protein involved in the autophagic machinery is also involved with a chronic inflammatory disease of the digestive tract.

The basic mechanisms of the autophagic process seem to be highly conserved amongst eukaryotes. Upon induction of autophagy, a membrane cisterna, known as the isolation membrane, appears and, by the addition of new membrane of unknown origin, curves around part of the cytoplasm (elongation). Sealing of the structure leads to the formation of an autophagosome, which differs from the conventional phagosome by the presence of a double delimiting membrane and intralumenal cytosolic content, and from other membranes in having few intramembrane proteins. Both of these features allow easy detection of autophagosomes by electron microscopy. The subsequent fusion between an autophagosome and lysosomes (maturation) results in a degradative compartment termed the autolysosome ^12,23,24. Experiments in yeast demonstrated that multimeric complexes formed by three autophagy gene (ATG) products, specifically ATG5, ATG12, and ATG16, were essential for the formation of the autophagosome ²⁵. Despite the high level of conservation of the autophagy apparatus from yeast to human there are significant differences. One of these differences is that mammalian ATG16L1 proteins possess seven WD repeats at their C-terminus (Figure 1A), which are completely absent in the yeast ATG16 gene product. Thus once ATG16L1 was identified as a CD susceptibility gene it was important to begin establishing its role in human autophagy and potential role in CD pathogenesis. Since WD repeats are most often associated with protein-protein or protein-membrane interactions, it is thought that those of ATG16L1 potentially reflect (i) a more complex regulation of mammalian versus yeast autophagy and (ii) a key role played by ATG16L1 in the autophagic clearance of pathogens, fulfilling a function absent in yeast. In an experimental system we knocked down the expression of the human ATG16L1 gene and demonstrated that it is essential for the formation of autophagosomes in response to serum starvation or bacterial infection confirming the role of the human ATG16L1 in autophagy in addition to implicating this process for the first time in the pathogenesis of CD ⁵.

The relative contribution of autophagy to this host defense balance may be tissue or organ dependant. Indeed, there are certain features of the gut mucosa and its resident immune cells that may increase the reliance upon autophagy for both homeostasis and immune defense. Chief amongst these is the relatively refractory nature of the gut immune compartment, despite constant exposure to high bacterial and antigen load. Many innate immune cells from the intestinal mucosal compartment lack high-level expression of TLRs and other pathogen-detecting molecules, or are otherwise refractory to antigen exposure. To compensate for such lack, it is plausible that innate immune cells rely mainly upon detection/destruction of pathogens following phagocytosis and internalization. Thus these cells must be able to induce a variety of programs to destroy internalized pathogens before they, themselves are incapacitated. The use of the autophagic apparatus might therefore be favored under these circumstances. Induction of autophagy in response to pathogens is thought to be specific, but the sensors driving the process are yet to be identified. This predisposition to autophagic control of pathogenic microbes may be regulated by compartment-specific modulators of the autophagic apparatus, as have been observed in recent studies³⁶. TLR signaling is already known to be required for intestinal homeostasis, recovery from injury and response to pathogenic challenge, and most recently a direct link between TLR signaling, autophagy and phagocytosis has been demonstrated ^37-39.

The lack of hair-trigger pattern detection in the gut is also likely to affect adaptive immunity. Antigen presentation of pathogen-derived components is likely to be dependant upon autophagosome-HLA crosstalk, rather than the conventional lysosomal pathways. A number of studies have demonstrated that autophagy pathways efficiently transfer cytosolic antigens to late endosomal or lysosomal compartments, where they can be loaded onto HLA class II molecules. This cross-presentation process will promote HLA class II presentation of cytosolic antigen in cells with high levels of autophagy. Thus one can envision that, via autophagy, the antigen portfolio presented upon HLA class II may differ in intestinal epithelial cells, lamina propria APCs and B cells in the intestinal mucosa when compared to mucosal surfaces not exposed to a dense microbial load. This potential bias towards HLA class II presentation of cytosolic antigen may also contribute to the enhanced effectiveness of mucosal vaccination against enteric pathogens versus parenteral antigen exposure ^40,41.