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      Integrative analysis of Paneth cell proteomic and transcriptomic data from intestinal organoids reveals functional processes dependent on autophagy

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          ABSTRACT

          Paneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.

          This article has an associated First Person interview with the joint first authors of the paper .

          Abstract

          Editor's choice: Using an integrative approach encompassing intestinal organoid culture, proteomics, transcriptomics and protein-protein interaction networks, we list Paneth cell functions dependent on autophagy.

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          Most cited references58

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production.

            Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.
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              Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis.

              Building and maintaining a homeostatic relationship between a host and its colonizing microbiota entails ongoing complex interactions between the host and the microorganisms. The mucosal immune system, including epithelial cells, plays an essential part in negotiating this equilibrium. Paneth cells (specialized cells in the epithelium of the small intestine) are an important source of antimicrobial peptides in the intestine. These cells have become the focus of investigations that explore the mechanisms of host-microorganism homeostasis in the small intestine and its collapse in the processes of infection and chronic inflammation. In this Review, we provide an overview of the intestinal microbiota and describe the cell biology of Paneth cells, emphasizing the composition of their secretions and the roles of these cells in intestinal host defence and homeostasis. We also highlight the implications of Paneth cell dysfunction in susceptibility to chronic inflammatory bowel disease.
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                Author and article information

                Journal
                Dis Model Mech
                Dis Model Mech
                DMM
                dmm
                Disease Models & Mechanisms
                The Company of Biologists Ltd
                1754-8403
                1754-8411
                1 March 2019
                18 March 2019
                18 March 2019
                : 12
                : 3
                : dmm037069
                Affiliations
                [1 ]Earlham Institute, Norwich Research Park , Norwich NR4 7UZ, UK
                [2 ]Quadram Institute, Norwich Research Park , Norwich NR4 7UA, UK
                [3 ]Norwich Medical School, University of East Anglia , Norwich NR4 7TJ, UK
                [4 ]National Institute of Health Research, University of Liverpool , Liverpool L3 5RF, UK
                [5 ]School of Biological Sciences, University of East Anglia , Norwich NR4 7TJ, UK
                Author notes
                [*]

                These authors contributed equally to this work

                []Author for correspondence ( Tamas.Korcsmaros@ 123456earlham.ac.uk )
                Author information
                http://orcid.org/0000-0002-5600-8189
                http://orcid.org/0000-0003-1717-996X
                Article
                DMM037069
                10.1242/dmm.037069
                6451430
                30814064
                103df035-719d-47a3-bc32-50b159dd1e03
                © 2019. Published by The Company of Biologists Ltd

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

                History
                : 5 September 2018
                : 1 February 2019
                Funding
                Funded by: Earlham Institute;
                Award ID: BB/CSP17270/1
                Funded by: Quadram Institute;
                Award ID: BB/P016774/1
                Funded by: BBSRC, http://dx.doi.org/10.13039/501100000268;
                Award ID: BB/J004529/1
                Award ID: BB/CCG1720/1
                Award ID: BB/M011216/1
                Award ID: BB/J01754X/1
                Award ID: BB/J004529/1
                Funded by: Norwich Medical School;
                Funded by: Wellcome Trust, http://dx.doi.org/10.13039/100010269;
                Award ID: 100
                Award ID: 974/C/13/Z
                Funded by: Medical Research Council, http://dx.doi.org/10.13039/501100000265;
                Award ID: MR/P026028/1
                Categories
                Research Article

                Molecular medicine
                paneth cells,atg16l1,intestinal organoids,quantitative proteomics,selective autophagy

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