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

Abstract

Using an integrative approach encompassing intestinal organoid culture, proteomics and proteinprotein interaction networks, we link Paneth cell biological functions often found affected in Crohn's disease to autophagy impairment. AbstractCrohn's disease (CD) is associated with Paneth cell dysfunctions such as altered antimicrobial secretion that is dependent on autophagy which recycles cellular components. Patients carrying the CD risk allele in ATG16L1 -an important component of the autophagy machinery -have Paneth cell abnormalities, as reproduced in Atg16l1-deficient mouse models. However, the direct effect of Atg16l1-deficiency and autophagy-impairment in Paneth cells has not been analyzed.To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells (Atg16l1 △ IEC ) making these cells impaired in autophagy. Using a 3D intestinal organoid culture model that we enriched for Paneth cells, we compared the proteomic profiles of organoids derived from the wild-type (WT) and Atg16l1 △ IEC mice. We used an integrated computational approach combining protein-protein interaction networks, autophagy targeted proteins and functional information to identify the mechanistic link between autophagyimpairment and disrupted cellular processes. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids compared to WT organoids indicating a reduced protein degradation. Interestingly, the 284 differentially abundant proteins comprised 116 proteins (41%), which are potentially targeted by selective autophagy proteins such as p62, LC3 and ATG16l1. Our integrative analysis revealed autophagy-mediated mechanisms which degrade essential proteins belonging to key Paneth cell functions such as exocytosis, apoptosis and DNA damage repair. We performed validation experiments focusing on Paneth cell-derived lysozyme to confirm our inferred observation of down-regulated exocytosis. Our observations could explain how protein level alterations in CD as a result of autophagy impairment could affect Paneth cell functions.

Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches

“…In addition, signatures of enteroendocrine cells were increased in both differentiation protocols, albeit at a lower amount than the target cell type. This finding correlates with previous investigations at both the transcriptomic and proteomic levels (25,(27)(28)(29) and is likely due to the shared differentiation pathways of these secretory cells. Nevertheless, as enteroids contain a mixed population of cell types by nature and because intercellular communication is key to a functioning epithelium (19,71), the increased proportion of non-targeted secretory lineages should not be an issue for the application of these models to research.…”

“…A recent study by Mead et al found that Paneth cells from enriched enteroids more closely represent their in vivo counterparts than those isolated from conventionally differentiated enteroids, based on transcriptomics, proteomics and morphologic data (27). Furthermore, we have shown that enteroids enriched for Paneth cells and goblet cells recapitulate in vivo characteristics on the proteomics level (28,29) and that they are a useful tool for the investigation of health and disease related processes in specific intestinal cell types (29).…”

“…Single cell sequencing, however, comes at a greater financial cost and provides lower coverage which can be problematic for rare cell types and lowly expressed RNAs. Furthermore, a number of previous studies have shown that these cell type enriched enteroid models, which offer a simplified and manipulatable version of the intestinal environment, are useful for the investigation of health and disease related processes (26,28,29). It must be considered, however, that through applying chemical inhibitors to enteroids to enrich particular cell types, we may observe changes in gene expression which are related to the direct effects of the inhibitor but not to differentiation.…”

“…Murine enteroids were generated as described previously (18,20,29). Briefly, the entire small intestine was opened longitudinally, washed in cold PBS then cut into ~5mm pieces.…”

The roles and functions of Paneth cells in Crohn’s disease: A critical review

“…Based on a recent study, autophagy impairment resulting from ATG16L1 deficiency alters the proteomic abundance profiles in PCs 39 . In the experiment conducted, the levels of 283 proteins (corresponding to 284 human orthologue proteins) were detected in PC‐rich organoids derived from both WT and ATG16L1 deficiency mice.…”