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 typeand pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease. H uman genetic studies offer an unbiased approach to identify genes and DNA variants underlying susceptibility to complex diseases. Although this approach has been successful at identifying more than 160 loci associated with Crohn disease (CD), a chronic inflammatory condition affecting the gastrointestinal tract (1, 2), ascribing function to specific risk variants has been difficult. Individuals who harbor a common threonine to alanine coding variant at position 300 in autophagy related 16-like 1 (ATG16L1) (T300A) are at increased risk of developing CD compared with individuals who possess a threonine at this position (T300T) (3, 4). The T300A variant lies within a structurally unclassified region of ATG16L1, making it challenging to identify the effect of this polymorphism.ATG16L1 is a component of the core autophagy machinery that plays a critical role in immunity and inflammation. Initial studies investigating ATG16L1 used hypomorphic Atg16L1 mouse models, which show Paneth cell abnormalities relevant to CD such as abnormal mitochondria, irregular patterns of granule morphology and lysozyme distribution, and increased expression of genes implicated in inflammation (5, 6). Although these studies have been useful in highlighting the important role of autophagy proteins in intestinal cells such as Paneth cells and goblet cells, the precise mechanisms by which ATG16L1 T300A influences pathogenesis remain unclear (7)(8)(9)(10).Previous studies have demonstrated that Atg16L1-deficient macrophages produce elevated levels of active caspase 1 and secrete higher levels of the cytokines IL-1β and IL-18 u...

Small-molecule enhancers of autophagy modulate cellular disease phenotypes suggested by human genetics

“…More specifically, diseaseassociated alleles in genes that regulate bacterial recognition (NOD2, IRGM) and autophagy (ATG16L1) are associated with reduced clearance of intracellular bacteria (4). Furthermore, expression of a coding variant in ATG16L1 (T300A) correlates with compromised bacterial defense, increased release of inflammatory cytokines (IL-1β), and reduced formation of antimicrobial granules in gut Paneth cells (5,6). Similar organassociated phenotypes have been observed for variants linked to other diseases.…”

ATG16L1: A multifunctional susceptibility factor in Crohn disease

“…Furthermore, the presence of the T300A mutation in Atg16l1 leads to aberrant functionality of Paneth cells. Coculturing LGR5 C stem cells 78 with Paneth cells from Atg16l1 T300A mice causes a reduced organoid formation, while coculturing with Paneth cells from wild-type mice conversely enhances organoid formation, 73 demonstrating a crucial role of ATG16L1 not only in the control of inflammatory immune responses but also for epithelial stem cell maintenance and function in the intestine. Furthermore, elevated secretion of the pro-inflammatory cytokines IL1B (interleukin 1 b) and IL18 can be observed after stimulation with lipopolysaccharide in ATG16L1-deficient myeloid cells.…”

“…72 A recent paper has shown that ATG16L1 T300A might also be susceptible to CASP7 degradation. 73,73 However, cells lacking CASP3 are completely incapable of cleaving ATG16L1 even in the presence of activated CASP7. 72 Thus, physiologically CASP7 is not capable of cleaving ATG16L1, possibly due to its lower abundance or potency compared to CASP3.…”

“…77 These mice are highly susceptible to dextran sulfate sodium-induced acute experimental colitis. 77 Recently, Lassen et al 73 generated a knock-in mouse model expressing ATG16L1 T300A . Such mice do not develop spontaneous inflammation, although they exhibit morphological defects in both Paneth cells and goblet cells.…”

“…85 Thus, a strong association between defective autophagy and impaired properties counteracting bacterial infections has been reported by numerous studies. 73,[86][87][88][89] However, a recent study has revealed a protective role of Atg16l1 deficiency against intestinal disease induced by the bacterial pathogen model, Citrobacter rodentium. This immunosuppressive role of ATG16L1 deficiency is dependent on the presence of NOD2 90 and adds to the complexity of the role of ATG16L1 in bacterial clearance.…”