Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse Peyers patch M cells, villous M-like cells, and intestinal epithelial cells

ABSTRACT: Separate populations of M cells have been detected in the follicle-associated epithelium of Peyerâs patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that M cells are rare but M-like cells positive for lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM16-2-4 are induced by oral administration of cholera toxin in the duodenal villous epithelium. Here, we determined the gene expression of PP M cells, villous M-like cells, and intestinal epithelial cells (IECs) isolated by a novel approach using FACS. Specific expression of glycoprotein 2 (GP2) and MARCKS-like protein (MLP) by PP M cells and not villous M-like cells was confirmed by additional mRNA and protein analyses. Comprehensive gene profiling also suggested that villous M-like cells share traits of both PP M cells and IECs, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology. Experiment Overall Design: Epithelial cells in duodenal Peyer's patches or villi of BALB/c mice treated with or without cholera toxin were dissociated and stained with NKM16-2-4 monoclonal antibody (mAb)-FITC, lectin UEA-1-PE, and anti-CD45 mAb-APC-Cy7. Naive PP M cells (NKM16-2-4+, UEA-1+, and CD45- cells), CT-induced villous M-like cells (NKM16-2-4+, UEA-1+, and CD45- cells), and naive intestinal epithelial cells (NKM16-2-4-, UEA-1-, and CD45- cells) were isolated by FACS. Affymetrix GeneChip Mouse Genome 430 2.0 Array was used in triplicate for each cell fraction.

ORGANISM(S): Mus musculus

SUBMITTER: Kazutaka Terahara

PROVIDER: E-GEOD-7838 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:M cells in the follicle-associated epithelium (FAE) of Peyer’s patches (PPs) serve as a main portal for external antigens. Limitation in the number of M cells has hampered identification of their specific molecules until recently. Efforts by us as well as others has gradually unraveled the molecular mechanisms for the development and differentiation of M cells. However, molecular mechanisms of antigen transcytosis and M cell differentiation were not fully elucidated. Recent studies reported that small non-coding RNAs including microRNA (miRNA) regulate gene expression that controls various biological processes such as cellular differentiation and functions. In fact, intestinal epithelial miRNAs play a critical role in goblet cell differentiation and maturation. However, the expression and function of miRNAs in FAE including M cell that function as the sentinels in the mucosal immune system are largely unknown. To address this notion, we performed microarray analysis to characterize expression profiles of miRNA in intestinal villous epithelium (VE) and FAE of PP. We also generated mice with intestinal epithelial cell-specific deletion of Dicer1 (DicerΔIEC), in which intestinal phenotypes including M-cell differentiation and epithelial morphology were examined. The microarray analysis identified that 72 miRNAs were up-regulated whereas 11 miRNAs were down-regulated, in FAE compared to VE. DicerΔIEC mice displayed a prominent decrease in mature M cells, suggesting an essential role of miRNAs in maturation of the cell type. Furthermore, electron micrographs clearly showed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake in M cells was impaired in DicerΔIEC mice compared to control floxed Dicer mice. These results raised the possibility that miRNAs play a significant role in the regulation of M cells maturation, and consequently secure mucosal immune homeostasis. Follicle associated epithelium or villous epithelium was isolated from Peyer's patch(PP). PP was soaked Hank's balanced salt solution (HBSS;GIBCO) containing 30mM EDTA. After incubation at 4℃ for 20 min, FAE or VE was isolated by manipulation with fine needle under stereomicroscopic monitoring. The isolated epithelial cell sheets were kept in ice-cold HBSS until RNA extraction(n=2).

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Transcription profiling of mouse Peyers patch M cells, villous M-like cells, and intestinal epithelial cells

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