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Small RNA transcriptome in distinct cell types of the jejunal intestinal epithelium of germfree and conventionalized mice

ABSTRACT: We evaluated miRNA expression profiles by next-generation high-throughput small RNA-sequencing in distinct IEC subtypes of germ-free (GF) and conventionalized (CV) mice. We used the highly characterized Sox9-EGFP transgenic mouse model, which permits the isolation and analysis of four distinct IEC populations using fluorescence-activated cell sorting (FACS) based on differing levels of cellular EGFP intensity. These are Sox9-EGFP Low (actively cycling IESCs), Sox9-EGFP Sublow (progenitor cells), Sox9-EGFP Neg (mostly differentiated enterocytes as well as goblet cells and Paneth cells), and Sox9-EGFP High (primarily EECs).

ORGANISM(S): Mus musculus

PROVIDER: GSE76179 | GEO | 2017/01/04

SECONDARY ACCESSION(S): PRJNA306526

REPOSITORIES: GEO

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Project description:Crohn’s disease (CD) is associated with multiple Paneth cell dysfunctions such as altered antimicrobial secretion that relies on autophagy - an essential process controlling the turnover of cellular components. Genome-wide association studies have linked CD to mutations in the ATG16l1 gene, encoding an important component of the autophagy machinery. Patients carrying the ATG16l1 CD risk allele have Paneth cell abnormalities, as reproduced in Atg16l1-deficient mouse models. However, the direct effect of the ATG16L1-deficiency and autophagy-impairment in Paneth cells has not been analysed in detail. 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 small intestinal organoid culture model, which we enriched for Paneth cells and compared the proteomic profiles of organoids derived from the wild-type (WT) and the Atg16l1△IEC mice. We used an integrated computational approach combining protein-protein interaction networks, list of proteins potentially targeted by autophagy and functional information on the proteins with altered protein levels to identify the mechanistic link between autophagy-impairment and disrupted cellular process. 198 (70%) of the 284 altered proteins were more abundant in autophagy-impaired organoids than WT organoids that could be due to a reduced protein turnover. Combining the proteomic dataset with the potential target proteins of selective autophagy proteins revealed that 116 (41%) of the differentially abundant proteins could rely on autophagy-mediated turnover. Taken together, our results suggest that proteins with increased levels in autophagy-impaired background require an autophagy-mediated turnover mechanism, and that their altered levels in CD could affect key cellular processes. Consequently, we pointed out the importance of autophagy in controlling the turnover of proteins relevant in key Paneth cell functions such as exocytosis, apoptosis and DNA damage repair. Our study unravels autophagy-dependent cellular processes of Paneth cells that not directly relies on the autophagy process, rather than a selective protein turnover mechanism. The identified proteins and processes open new avenues for targeted, cell type-specific therapeutic interventions in CD.

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Small RNA transcriptome in distinct cell types of the jejunal intestinal epithelium of germfree and conventionalized mice

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