Defects in Autophagy Induce Alterations in the Secretory Pathway and Proinflammatory Signaling of Paneth Cells

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Cadwell K, Liu JY, Brown SL, et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 2008;456:259–263.

An exciting and unforeseen finding from the genome-wide association studies is the potential role of autophagy in Crohn's disease (CD) pathogenesis. However, information on the mechanisms linking alterations in autophagy functions and CD pathogenesis was lacking. In that regard, the paper published in Nature by Cadwell et al (Nature 2008;456:259–263) demonstrates a role of the autophagy-related 16-like 1 (ATG16L1) protein in intestinal Paneth cell biology and provides a potential mechanism for the association of ATG16L1 polymorphism with an increased risk of developing CD.

ATG16L1-deficient animals die shortly after birth and cells derived from such animals show autophagy defects in vitro (Nature 2008;456:264–268), indicating that ATG16L1 is essential for surviving the period of neonatal starvation. To bypass the lethality of ATG16L1 gene deletion, Cadwell et al generated mice that are hypomorphic for ATG16L1 expression (ATGL16^HM) by means of intronic insertion of a gene-trap vector, and they used these mice to assess the effects of lower ATG16L1 expression on autophagy in the intestine.

Embryonic fibroblasts from ATGL16^HM mice show defects in autophagy-dependent pathways, comparable with those observed in fibroblasts from animals deficient in the key autophagy-related protein autophagy-related 5 (ATG5), demonstrating that mammalian ATG16L1 protein is indeed involved in autophagy. ATGL16^HM mice show decreased ATG16L1 mRNA throughout the crypt–villus axis. Interestingly, low ATG16L1 protein expression does not affect the overall morphology of the ileal and colonic crypts and villi, but does result in abnormal levels of other ATG proteins (p62 and LC3-I) and in the dysfunction of Paneth cell secretory activity. This important observation prompted the authors to further characterize granule formation by staining for lysozyme in ileal sections from wild-type and ATGL16^HM mice. By using a variety of morphologic measurements, prominent histologic abnormalities in Paneth cells of ATG16L1^HM mice were detected. Importantly, these changes were also obvious in resected ileal tissue from patients with CD who are homozygous for the risk ATG16L1 allele. In both settings, Paneth cells show morphologic defects in antibacterial lysosyme-containing secretory granules and granule exocytosis. Further supporting the functional relevance of autophagy in Paneth cells biology, targeted deletion of ATG5 in the intestinal epithelium in ATG5^flox/floxvillin–Cre mice led to comparable Paneth cell granule abnormalities.

Moreover, transcriptional profiling of Paneth cells in ATGL16^HM mice showed increased expression of genes involved in peroxisome proliferator-activated receptor signaling as well as leptin and adiponectin, adipocytokines that have been linked to intestinal inflammation. Most interestingly, in patients homozygous for the disease-associated ATG16L1 variant (T300A), the authors also demonstrated increased leptin expression reminiscent of the observation in their ATGL16^HM mouse model.

In summary, this key study provides the basis for understanding how the presence of the risk allele for ATG16L1 can influence CD pathophysiology. Most important, it brings new evidence on the role of the epithelial barrier, and in particular of the granule exocytosis pathway of Paneth cells, in maintaining intestinal homeostasis.

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Autophagy is an ancient, highly conserved cellular process used by all eukaryotic cells. Classical macroautophagy (referred as ‘autophagy’ here) involves the sequestration of cytoplasmic contents in a characteristic double-membrane vacuole, the autophagosome. Fusion of the outer autophagosomal membrane with the lysosome and the subsequent breakdown of the inner membrane results in the exposure of the sequestered cytoplasmic material to lysosomal hydrolases (Cell 2008;132:27–42; Nature 2008;451:1069–1075). Two key protein–protein and protein–lipid complexes are associated with the autophagosome controlled elongation and stabilization: ATG12 is covalently conjugated to ATG5 and associates with ATG16L1 to form an 800-kDa ATG12–ATG5–ATG16L1 complex, which localizes to the cytoplasmic face of the isolation membrane. This complex participates in the lipidation process of LC3, an essential step in the formation and elongation of the isolation membrane.

The autophagy pathway has 2 main physiological functions: It rids the cell of unwanted constituents and it recycles cytoplasmic material so that cells can maintain macromolecular synthesis and energy homeostasis during stressful conditions. Furthermore, studies by multiple groups from diverse disciplines have converged to reveal the role of autophagy in innate and adaptive immunity. The first link between autophagy and the innate immune system was shown by the discovery that intracellular pathogens (primarily bacteria and viruses) can be eliminated from cells via the autophagy pathway (Cell Death Differ 2009;16:57–69). One of the signals to initiate pathogen-induced autophagy is the activation of the Toll-like receptors (EMBO J 2008;27:1110–1121). The involvement of autophagy in adaptive immunity has been established by studies demonstrating that cells expressing major histocompatibility complex class II proteins use the autophagy pathway in processing peptide antigens for presentation to CD4⁺ T cells (Immunity 2007;26:79–92). Experiments in mice using targeted deletions of key autophagy genes have shown that lymphocyte homeostasis, T-cell development, and central tolerance are also dependent on this cellular pathway.

Two genes involved in this process, ATG16L1 and immunity-related GTPase family, M (IRGM), were found to be significantly associated with the risk to develop CD. A third autophagy-associated gene, leucine-rich repeat kinase 2, is 1 of 2 CD candidate genes on chromosome 12q12 (Trends Genet 2009;25:137–146). The CD-associated ATG16L1 risk allele encodes a protein with a threonine-to-alanine substitution (T300A) in the carboxyterminal domain. However, it remained to be defined how the ATG16L1 polymorphism confers the phenotypic development of CD.

The data provided in the paper by Cadwell et al (Nature 2008;456:259–263) demonstrate a role for autophagy in regulating the specialized functions of Paneth cells. In particular, low expression of ATG16L1 protein in mice or the presence of the ATG16L1 risk allele in CD patients, specifically target Paneth cells secretory pathway and transcriptional profile. Interestingly, no such dramatic changes are present in other cell types (eg, splenocytes) despite significantly low ATG16L1 protein expression. The model may be relevant to humans carrying the allele encoding ATG16L1 T300A, because it has been suggested that the T300A substitution may lower ATG16L1 function because of altered stability (Nat Immunol 2009;10:461–470).

Because Paneth cells function as a barrier to bacterial invasion, in part by secreting granule contents that contain antimicrobial peptides, and as regulators of intestinal inflammation, the findings reported suggest that defects in ATG16L1 function in Paneth cells may contribute to the pathogenesis of CD. Studies in mice with intestinal epithelial cell–specific deletion of other autophagy genes, including ATG5 or ATG7, have demonstrated similar abnormalities in Paneth cells (Autophagy 2009;5:250–252; Nature 2008;456:259–263). Thus, several components of the autophagic machinery that act at the membrane expansion–completion stage (such as ATG16L1, ATG5, and ATG7) are involved in the maintenance of Paneth cell function and can potentially play a role in maintain epithelial cell homeostasis.

Paneth cells represent the major source for antimicrobial peptide in the small intestine and it is reasonable to assume that defects in Paneth cell biology could affect the composition and response to the intestinal flora. Previous studies had demonstrated that CD patients have decreased antibacterial activity, specifically α-defensins, in ileal mucosal extracts (Science 2005;307:731–734). Remarkably, another CD susceptibility gene, NOD2 is also predominantly expressed in Paneth cells. NOD2-deficient mice show a decreased expression of antibacterial peptides and an increased susceptibility to oral Listeria monocytogenes (Science 2005;307:731–734). Cadwell et al show that the ATGL16^HM mice are, in contrast, not compromised in their ability to clear the same Gram-positive bacteria, suggesting that the phenotypes derived from ATG16L1 and NOD2 polymorphisms are distinct.

Interestingly, neither ATGL16^HM mice (Nature 2008;456:259–263), ATG16L1-deficient, fetal liver chimeric mice (Nature 2008;456:264–268), NOD2-deficient, or mutant mice (Science 2005;307:731–734) develop spontaneous intestinal inflammation. These observations are consistent with an inability of Paneth cell abnormalities alone to induce intestinal inflammation. Specifically, Paneth cell depletion (J Biol Chem 1997;272:23729–23740) or an inability to activate α-defensins (Science 1999;286:113–117) are not associated with spontaneous intestinal inflammation. This strongly argues against a dominant inflammation-inducing effect owing to alterations in the microflora alone. Rather, a growing body of evidence supports a “2-hit hypothesis,” wherein host (or potentially environmentally)-mediated alterations in the intestinal microbiota may only induce dysregulated intestinal inflammation characteristic of CD when present together with a tendency to hyper-respond to microbial stimuli. It remains to be seen if the ATGL16^HM mice exhibit higher susceptibility to colitis and, if so, what are the main mechanisms involved in such a response. An accompanying study in the same issue of Nature shows that chimeric mice with ATG16L1-deficient hematopoietic cells are indeed highly susceptible to colitis induced by dextran sodium sulfate, further supporting a link between a defect in ATG16L1 function and intestinal inflammation (Nature 2008;456:264–268).

Compared with ATG16L1, less information is available on other ATG genes associated with CD, such as IRGM. IRGM was initially found to have an important role in protection against Mycobacterium tuberculosis infection by activation of the autophagy pathway (Science 2006;313:1438–1441). However, IRGM-related autophagy genes are not highly conserved, making it more difficult to compare function between yeast and murine model systems and humans. In humans, only 2 IRG genes have been found, whereas in mice there is a large family comprising >20 genes. Recently, a copy number variation, a 20-kb deletion polymorphism immediately upstream of the gene, has been identified, which is in perfect linkage disequilibrium with the most strongly associated SNP (Nat Genet 2008;40:1107–1112). When tested on a panel of heterozygote cell lines, the 2 IRGM haplotypes give different patterns of expression, indicating that the copy number variation influences the regulation of IRGM. Further, reducing the level of IRGM mRNA in HeLa cells by treatment with siRNA substantially compromised the efficiency of antibacterial autophagy (Nat Genet 2008;40:1107–1112).

Overall, these genetic and functional studies, following the implication of ATG16LI and IRGM in CD, point to autophagy as a key pathway in the pathogenesis of CD. Common genetic variations in these and other autophagy genes are likely to have a major impact on the response of the innate immune system to intestinal microbiota and susceptibility to inflammatory bowel disease. Converging evidence suggests that autophagy proteins, although critical for normal immune responses, may also function to prevent immunologic hyperresponsiveness in certain cell types, such as macrophages, fibroblasts, and Paneth cells; this may at least in part underlie the associations between polymorphisms in autophagy genes and CD.

The many functions of autophagy and autophagy genes in immunity provide both opportunities and risks for manipulating autophagy therapeutically. Enhancing autophagy might ameliorate CD if agents that overcome deficiencies in the function of ATG16L1 or IRGM can be identified. It is noteworthy that rapamycin, the most commonly used laboratory agent to induce autophagy, has been in clinical use in a number of different clinical conditions for several years. One recent case report has described the clinical use of rapamycin in a patient with severe CD inducing a marked and sustained improvement in symptoms and endoscopic lesions (Gut 2008;57:1294–1296). Additional understanding of the molecular basis of the diverse functions of autophagy genes in immunity may open new doors for rational intervention in a variety of diseases without untoward immunologic consequences.