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Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
An alteration in ATG16L1 stability in Crohn disease ab
Kara G Lassen a
& Ramnik J Xavier
abc
Broad Institute; Cambridge, MA USA
b
Center for Computational and Integrative Biology; Massachusetts General Hospital; Boston, MA USA c
Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease; Massachusetts General Hospital; Harvard Medical School; Boston, MA USA Published online: 12 Aug 2014.
Click for updates To cite this article: Kara G Lassen & Ramnik J Xavier (2014) An alteration in ATG16L1 stability in Crohn disease, Autophagy, 10:10, 1858-1860, DOI: 10.4161/auto.29963 To link to this article: http://dx.doi.org/10.4161/auto.29963
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Autophagic Punctum Autophagic Punctum
Autophagy 10:10, 1858–1860; October 2014; © 2014 Landes Bioscience
An alteration in ATG16L1 stability in Crohn disease Kara G Lassen1,2,* and Ramnik J Xavier1,2,3 Broad Institute; Cambridge, MA USA; 2Center for Computational and Integrative Biology; Massachusetts General Hospital; Boston, MA USA; Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease; Massachusetts General Hospital; Harvard Medical School; Boston, MA USA
Downloaded by [UT Southwestern Medical Center] at 19:47 23 January 2015
3
I
ndividuals who harbor a common coding polymorphism (Thr300Ala) within a structurally unclassified region of ATG16L1 are at increased risk for the development of Crohn disease. Recently, we reported on the generation and characterization of knockin mice carrying the ATG16L1 T300A variant. We demonstrate that multiple cell types from T300A knock-in mice exhibit reduced selective autophagy, and we mechanistically link this phenotype with an increased susceptibility of ATG16L1 T300A to CASP3- and CASP7-mediated cleavage. These findings demonstrate how a single polymorphism can result in cell type- and pathway-specific disruptions of selective autophagy and alterations in the inflammatory milieu that can contribute to disease.
Keywords: Crohn disease, ATG16L1, IL1B, antibacterial autophagy, caspase cleavage Submitted: 07/05/2014 Revised: 07/15/2014 Accepted: 07/16/2014 Published Online: 08/12/2014 http://dx.doi.org/10.4161/auto.29963 *Correspondence to: Kara G Lassen; Email: [email protected] Punctum to: Lassen KG, Kuballa P, Conway KL, Patel KK, Becker CE, Peloquin JM, Villablanca EJ, Norman JM, Liu TC, Heath RJ, et al. Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc Natl Acad Sci U S A 2014; 111:7741–6; PMID:24821797; http://dx.doi. org/10.1073/pnas.1407001111
Autophagy is a prosurvival degradation pathway in which cytoplasmic contents are engulfed and targeted for degradation by the lysosome. Recent work has focused on the diverse roles of autophagy in both innate and adaptive immunity, including its function as an intracellular defense mechanism against pathogens. Polymorphisms in genes functioning in the autophagy pathway have been linked to inflammatory disorders including Crohn disease, a chronic condition affecting the gastrointestinal tract. ATG16L1 T300A is a common coding polymorphism that predisposes individuals to Crohn disease. While studies in human cells have suggested that ATG16L1 T300A decreases antibacterial autophagy, increases IL1B secretion, and alters Paneth cell morphology, the precise mechanism by which
1858 Autophagy
ATG16L1 T300A alters the autophagy pathway and contributes to disease has been more challenging to elucidate. To investigate the influence of the ATG16L1 T300A polymorphism in vivo, we generated a knock-in mouse model with a Thr to Ala substitution at position 300 in ATG16L1 (position 300 in isoform β, position 281 in isoform α, and position 316 in isoform γ). Expanding on the human findings, the T300A mouse model recapitulates each of these previously reported human phenotypes and provides a model to study multiple discrete cellular phenotypes both in vivo and ex vivo. Using immunofluorescence to precisely observe and quantify changes in lysozyme distribution within the epithelium of T300A mice, we showed disruptions not only in Paneth cell morphology, but goblet cell morphology as well. This alteration in goblet cell morphology was previously unknown given the technical limitations in staining human tissue. An organoid-forming assay involving the coculture of intestinal stem cells and Paneth cells demonstrates reduced organoid formation in co-cultures containing Paneth cells with T300A. Studies have suggested a critical role for secretion of soluble factors from Paneth cells to increase organoid formation in this assay. It is likely that reduced secretion from both Paneth and goblet cells in the intestinal epithelium of patients homozygous for ATG16L1 T300A is a key factor leading to altered gut homeostasis in Crohn disease (Fig. 1). Higher levels of IL1B have been reported in peripheral blood mononuclear cells from patients homozygous for ATG16L1 T300A after stimulation with
Volume 10 Issue 10
©2014 Landes Bioscience. Do not distribute.
1
lipopolysaccharide and muramyl dipeptide. Recapitulating these human findings, we found that ATG16L1 T300A macrophages and dendritic cells isolated from the gut and gut-associated lymph nodes produce higher levels of IL1B upon stimulation with lipopolysaccharide/muramyl dipeptide or after infection with intracellular pathogens. Both ATG16L1 T300A mouse embryonic fibroblasts and primary cultures of small intestinal epithelial cells exhibit increased susceptibility to intracellular bacterial infection. Interestingly, the
most common region of the gastrointestinal tract affected by Crohn disease, the terminal ileum, contains the highest commensal bacterial burden in the body. Taken together, these data suggest a model in which pathogenic bacteria or opportunistic pathobionts could have diverse effects on epithelial damage, inflammation, and cellular homeostasis stemming from small alterations in the autophagy pathway (Fig. 1). In this model, higher levels of epithelial damage and cellular stress resulting from increased
susceptibility of cells to bacterial infection could be sufficient to induce a hyperinflammatory state. Increased intestinal inflammation could feed back and alter epithelial integrity, resulting in a self-perpetuating feedback loop altering intestinal homeostasis. To further understand how ATG16L1 regulates IL1B secretion and antibacterial autophagy, we used quantitative proteomic analysis of ATG16L1 to identify a highly interconnected network of novel ATG16L1-interacting proteins. Follow-up
www.landesbioscience.com Autophagy 1859
©2014 Landes Bioscience. Do not distribute.
Downloaded by [UT Southwestern Medical Center] at 19:47 23 January 2015
Figure 1. The T300A polymorphism in ATG16L1 alters numerous pathways in diverse cell types. Reduced secretion from Paneth and goblet cells in the intestinal epithelium could alter susceptibility to infection. A feedback loop altering intestinal homeostasis can be generated by bacterial infection or other forms of cellular stress that decrease epithelial integrity and increase caspase activity. Since ATG16L1 T300A is more susceptible to CASP3- and CASP7-mediated cleavage compared with wild-type ATG16L1, an increase in the level of caspase activity results in lower levels of full-length, functional ATG16L1. This results in increases in bacterial infection, intracellular replication, and production of pro-inflammatory cytokines. Dendritic cells and macrophages bearing ATG16L1 T300A then produce higher levels of IL1B in response to bacterial ligands or infection. This cycle perpetuates a hyperinflammatory milieu in the intestine.
immediately preceded by a caspase consensus motif. We employed a highly sensitive assay using untagged 35S-methioninelabeled ATG16L1 proteins incubated with recombinant caspases to demonstrate that ATG16L1 T300A is more susceptible to CASP3- and CASP7-mediated cleavage than is wild-type ATG16L1. CASP3 more potently cleaves ATG16L1 T300A compared with similar levels of CASP7. Importantly, inhibitors of CASP3 or CASP3 and CASP7 rescue autophagic flux in ATG16L1 T300A mouse embryonic fibroblasts. These results indicate that both CASP3 and CASP7 are likely physiological enzymes for ATG16L1; however, cleavage of ATG16L1 in vivo is likely dependent on the relative expression and activation of CASP3 and CASP7 in different cell types. These data support a model in which cell types expressing ATG16L1 T300A
1860 Autophagy
with increased levels of active caspases would have an accumulation of cleaved ATG16L1 and less full-length functional ATG16L1 protein, helping to explain some of the observed cell type restriction of ATG16L1 T300Aassociated phenotypes (Fig. 1). Additionally, various types of cellular stress that are known to modulate caspase function could be exacerbated by environmental factors, increasing the chances of disease manifestations. These findings create a context for understanding how a single amino acid change in a core autophagy protein can alter the autophagy pathway at the molecular level, thereby altering cellular and tissue homeostasis. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Volume 10 Issue 10
©2014 Landes Bioscience. Do not distribute.
Downloaded by [UT Southwestern Medical Center] at 19:47 23 January 2015
assays investigated the role of each of the identified interactors in IL1B secretion and antibacterial autophagy. Knockdowns of 4 (Suclg1, Mccc2, Slc25a11, and Vps28) of the genes encoding identified interactors were found to increase IL1B secretion upon knockdown similar to ATG16L1, and 6 (BTD, AVIL, GARS, CALU, NOLC1, and SLC25A11) gene knockdowns were found to alter antibacterial autophagy. Interestingly, only SLC25A11 played a role in both pathways. The nonoverlapping functions of these ATG16L1 interactors suggest that discrete steps in the autophagy pathway involve the association of required cofactors to confer functional specificity to core autophagy proteins. The T300A polymorphism lies in a structurally unclassified region of ATG16L1, making it challenging to determine its effect on ATG16L1 function. Interestingly, the T300A polymorphism is
Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
An alteration in ATG16L1 stability in Crohn disease ab
Kara G Lassen a
& Ramnik J Xavier
abc
Broad Institute; Cambridge, MA USA
b
Center for Computational and Integrative Biology; Massachusetts General Hospital; Boston, MA USA c
Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease; Massachusetts General Hospital; Harvard Medical School; Boston, MA USA Published online: 12 Aug 2014.
Click for updates To cite this article: Kara G Lassen & Ramnik J Xavier (2014) An alteration in ATG16L1 stability in Crohn disease, Autophagy, 10:10, 1858-1860, DOI: 10.4161/auto.29963 To link to this article: http://dx.doi.org/10.4161/auto.29963
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Autophagic Punctum Autophagic Punctum
Autophagy 10:10, 1858–1860; October 2014; © 2014 Landes Bioscience
An alteration in ATG16L1 stability in Crohn disease Kara G Lassen1,2,* and Ramnik J Xavier1,2,3 Broad Institute; Cambridge, MA USA; 2Center for Computational and Integrative Biology; Massachusetts General Hospital; Boston, MA USA; Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease; Massachusetts General Hospital; Harvard Medical School; Boston, MA USA
Downloaded by [UT Southwestern Medical Center] at 19:47 23 January 2015
3
I
ndividuals who harbor a common coding polymorphism (Thr300Ala) within a structurally unclassified region of ATG16L1 are at increased risk for the development of Crohn disease. Recently, we reported on the generation and characterization of knockin mice carrying the ATG16L1 T300A variant. We demonstrate that multiple cell types from T300A knock-in mice exhibit reduced selective autophagy, and we mechanistically link this phenotype with an increased susceptibility of ATG16L1 T300A to CASP3- and CASP7-mediated cleavage. These findings demonstrate how a single polymorphism can result in cell type- and pathway-specific disruptions of selective autophagy and alterations in the inflammatory milieu that can contribute to disease.
Keywords: Crohn disease, ATG16L1, IL1B, antibacterial autophagy, caspase cleavage Submitted: 07/05/2014 Revised: 07/15/2014 Accepted: 07/16/2014 Published Online: 08/12/2014 http://dx.doi.org/10.4161/auto.29963 *Correspondence to: Kara G Lassen; Email: [email protected] Punctum to: Lassen KG, Kuballa P, Conway KL, Patel KK, Becker CE, Peloquin JM, Villablanca EJ, Norman JM, Liu TC, Heath RJ, et al. Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc Natl Acad Sci U S A 2014; 111:7741–6; PMID:24821797; http://dx.doi. org/10.1073/pnas.1407001111
Autophagy is a prosurvival degradation pathway in which cytoplasmic contents are engulfed and targeted for degradation by the lysosome. Recent work has focused on the diverse roles of autophagy in both innate and adaptive immunity, including its function as an intracellular defense mechanism against pathogens. Polymorphisms in genes functioning in the autophagy pathway have been linked to inflammatory disorders including Crohn disease, a chronic condition affecting the gastrointestinal tract. ATG16L1 T300A is a common coding polymorphism that predisposes individuals to Crohn disease. While studies in human cells have suggested that ATG16L1 T300A decreases antibacterial autophagy, increases IL1B secretion, and alters Paneth cell morphology, the precise mechanism by which
1858 Autophagy
ATG16L1 T300A alters the autophagy pathway and contributes to disease has been more challenging to elucidate. To investigate the influence of the ATG16L1 T300A polymorphism in vivo, we generated a knock-in mouse model with a Thr to Ala substitution at position 300 in ATG16L1 (position 300 in isoform β, position 281 in isoform α, and position 316 in isoform γ). Expanding on the human findings, the T300A mouse model recapitulates each of these previously reported human phenotypes and provides a model to study multiple discrete cellular phenotypes both in vivo and ex vivo. Using immunofluorescence to precisely observe and quantify changes in lysozyme distribution within the epithelium of T300A mice, we showed disruptions not only in Paneth cell morphology, but goblet cell morphology as well. This alteration in goblet cell morphology was previously unknown given the technical limitations in staining human tissue. An organoid-forming assay involving the coculture of intestinal stem cells and Paneth cells demonstrates reduced organoid formation in co-cultures containing Paneth cells with T300A. Studies have suggested a critical role for secretion of soluble factors from Paneth cells to increase organoid formation in this assay. It is likely that reduced secretion from both Paneth and goblet cells in the intestinal epithelium of patients homozygous for ATG16L1 T300A is a key factor leading to altered gut homeostasis in Crohn disease (Fig. 1). Higher levels of IL1B have been reported in peripheral blood mononuclear cells from patients homozygous for ATG16L1 T300A after stimulation with
Volume 10 Issue 10
©2014 Landes Bioscience. Do not distribute.
1
lipopolysaccharide and muramyl dipeptide. Recapitulating these human findings, we found that ATG16L1 T300A macrophages and dendritic cells isolated from the gut and gut-associated lymph nodes produce higher levels of IL1B upon stimulation with lipopolysaccharide/muramyl dipeptide or after infection with intracellular pathogens. Both ATG16L1 T300A mouse embryonic fibroblasts and primary cultures of small intestinal epithelial cells exhibit increased susceptibility to intracellular bacterial infection. Interestingly, the
most common region of the gastrointestinal tract affected by Crohn disease, the terminal ileum, contains the highest commensal bacterial burden in the body. Taken together, these data suggest a model in which pathogenic bacteria or opportunistic pathobionts could have diverse effects on epithelial damage, inflammation, and cellular homeostasis stemming from small alterations in the autophagy pathway (Fig. 1). In this model, higher levels of epithelial damage and cellular stress resulting from increased
susceptibility of cells to bacterial infection could be sufficient to induce a hyperinflammatory state. Increased intestinal inflammation could feed back and alter epithelial integrity, resulting in a self-perpetuating feedback loop altering intestinal homeostasis. To further understand how ATG16L1 regulates IL1B secretion and antibacterial autophagy, we used quantitative proteomic analysis of ATG16L1 to identify a highly interconnected network of novel ATG16L1-interacting proteins. Follow-up
www.landesbioscience.com Autophagy 1859
©2014 Landes Bioscience. Do not distribute.
Downloaded by [UT Southwestern Medical Center] at 19:47 23 January 2015
Figure 1. The T300A polymorphism in ATG16L1 alters numerous pathways in diverse cell types. Reduced secretion from Paneth and goblet cells in the intestinal epithelium could alter susceptibility to infection. A feedback loop altering intestinal homeostasis can be generated by bacterial infection or other forms of cellular stress that decrease epithelial integrity and increase caspase activity. Since ATG16L1 T300A is more susceptible to CASP3- and CASP7-mediated cleavage compared with wild-type ATG16L1, an increase in the level of caspase activity results in lower levels of full-length, functional ATG16L1. This results in increases in bacterial infection, intracellular replication, and production of pro-inflammatory cytokines. Dendritic cells and macrophages bearing ATG16L1 T300A then produce higher levels of IL1B in response to bacterial ligands or infection. This cycle perpetuates a hyperinflammatory milieu in the intestine.
immediately preceded by a caspase consensus motif. We employed a highly sensitive assay using untagged 35S-methioninelabeled ATG16L1 proteins incubated with recombinant caspases to demonstrate that ATG16L1 T300A is more susceptible to CASP3- and CASP7-mediated cleavage than is wild-type ATG16L1. CASP3 more potently cleaves ATG16L1 T300A compared with similar levels of CASP7. Importantly, inhibitors of CASP3 or CASP3 and CASP7 rescue autophagic flux in ATG16L1 T300A mouse embryonic fibroblasts. These results indicate that both CASP3 and CASP7 are likely physiological enzymes for ATG16L1; however, cleavage of ATG16L1 in vivo is likely dependent on the relative expression and activation of CASP3 and CASP7 in different cell types. These data support a model in which cell types expressing ATG16L1 T300A
1860 Autophagy
with increased levels of active caspases would have an accumulation of cleaved ATG16L1 and less full-length functional ATG16L1 protein, helping to explain some of the observed cell type restriction of ATG16L1 T300Aassociated phenotypes (Fig. 1). Additionally, various types of cellular stress that are known to modulate caspase function could be exacerbated by environmental factors, increasing the chances of disease manifestations. These findings create a context for understanding how a single amino acid change in a core autophagy protein can alter the autophagy pathway at the molecular level, thereby altering cellular and tissue homeostasis. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Volume 10 Issue 10
©2014 Landes Bioscience. Do not distribute.
Downloaded by [UT Southwestern Medical Center] at 19:47 23 January 2015
assays investigated the role of each of the identified interactors in IL1B secretion and antibacterial autophagy. Knockdowns of 4 (Suclg1, Mccc2, Slc25a11, and Vps28) of the genes encoding identified interactors were found to increase IL1B secretion upon knockdown similar to ATG16L1, and 6 (BTD, AVIL, GARS, CALU, NOLC1, and SLC25A11) gene knockdowns were found to alter antibacterial autophagy. Interestingly, only SLC25A11 played a role in both pathways. The nonoverlapping functions of these ATG16L1 interactors suggest that discrete steps in the autophagy pathway involve the association of required cofactors to confer functional specificity to core autophagy proteins. The T300A polymorphism lies in a structurally unclassified region of ATG16L1, making it challenging to determine its effect on ATG16L1 function. Interestingly, the T300A polymorphism is
