Abstract
ABSTRACTPaneth 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.
Highlights
Paneth cells, located at the bottom of the crypts of Lieberkühn in the small intestine, secrete various types of antimicrobial compounds to regulate the microbial composition of the intestine, as well as growth factors that maintain the cryptassociated stem cell population (Bevins and Salzman, 2011)
Paneth-cell-enriched Atg16l1ΔIEC organoids are a valid model to study the role of autophagy in intestinal epithelial homeostasis We have generated a mouse model that lacks Atg16l1 in intestinal epithelial cells (Atg16l1ΔIEC) and have used selforganising in vitro organoid cultures generated from small-intestinal crypts (Sato et al, 2009) to assess the impact of autophagy on Paneth cell functions
Detection of mRNA transcripts by linear reverse transcription PCR (RT-PCR) for Lgr5, ChgA, Muc2 and Cd24 cDNAs along with the housekeeping β-actin gene revealed that Atg16l1ΔIEC organoids expressed markers for stem cells, enteroendocrine cells, goblet cells and Paneth cells at similar levels as WT organoids (Fig. 2B), confirming similar differentiation expression levels in both genotypes of important cell types found in the in vivo small-intestinal epithelium
Summary
Paneth cells, located at the bottom of the crypts of Lieberkühn in the small intestine, secrete various types of antimicrobial compounds (e.g. lysozyme, defensins) to regulate the microbial composition of the intestine, as well as growth factors that maintain the cryptassociated stem cell population (Bevins and Salzman, 2011). Conventional protein secretion involves trafficking through the endoplasmic reticulum (ER) and Golgi (Farquhar and Palade, 1981; Viotti, 2016) Paneth cell defects such as altered granule morphology and increased susceptibility to ER stress are seen in mouse models in which autophagy is lost from intestinal epithelial cells (Liu et al, 2013; Wileman, 2013). Autophagy is a pivotal recycling process that sequesters cytoplasmic misfolded proteins or damaged organelles as well as clearing the cytosol from invading pathogens These targets are captured in double-membrane vesicles called autophagosomes that are subsequently delivered for degradation to lysosomal compartments (Deretic et al, 2013; Glick et al, 2010; Todde et al, 2009; Wileman, 2013). These events eventually result in cargo engulfment by the autophagosome, which
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