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

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. .

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.…”

“…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. Due to the nature of the inhibitors, it would be challenging to separate these effects.…”

“…26 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 Nevertheless, the effect of Paneth cell and goblet cell enrichment of enteroids on key regulatory landscapes has not been extensively characterised.…”

“…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 Nevertheless, the effect of Paneth cell and goblet cell enrichment of enteroids on key regulatory landscapes has not been extensively characterised. In this study, we comprehensively profiled mRNAs, miRNAs and lncRNAs from mouse derived, 3D conventionally differentiated enteroids (control), Paneth cell enriched enteroids (PCeE) and goblet cell enriched enteroids (GCeE) to determine the extent to which these enteroids display increased Paneth cell and goblet cell signatures.…”

Assessment of Ecosystem Services and Capabilities of Communities from different Scales and Niches - Implications on Sustainability Goals

“…Microbial communities comprising a mixture of bacteria, viruses, fungi etc and differing in composition and abundance reside in the ecosystems of both the gut and the soil. While gut microbial communities have an important influence on human health/disease (Sánchez et al, 2017), previous research has shown how molecular components of the mammalian gut are involved in mediating homeostatic functions (Jones et al, 2018) as well as interacting with microbial proteins (Sudhakar et al, 2019). In a similar manner, the microbial communities in the soil are central to several key services which are not only key to agriculture and food production (Jacoby et al, 2017) but also in several other critical processes.…”