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M cell, FAE and VAE expresssion profiles

ABSTRACT: M cells are specialized epithelial cells occurring in the follicle-associated epithelium (FAE) overlying the Peyer’s patch. M cells transcytose macromolecules and microorganisms from the gut lumen to the underlying organized mucosal lymphoid follicles. Thus, M cells are a portal to the mucosal immune system and are crucial for the study of pathogenesis and mucosal vaccine design. Although characterization has been primarily performed to date using immunohistochemistry and in vitro cell culture techniques, little is known about the genetic factors in vivo distinguishing M cells from other intestinal epithelial cells. Our aim was to develop a method to isolate viable murine M cells for the purpose of generating an expression profile of murine M cells compared to FAE enterocytes and villus associated enterocytes (VAE). Peyer’s patches were microdissected from murine small intestine and the FAE isolated by enzymatic digestion. M cells were isolated using magnetic bead cell sorting of FITC-UEAI lectin stained FAE. Analysis of M cell, FAE and VAE gene expression was performed using cDNA arrays and Real-time RT-PCR. Altered expression of several genes involved with cell structure, differentiation and cell-interaction was observed. In murine M cells; E-cadherin, Ccl20, Ccl25 and Lamr1 were increased in expression, compared to the neighboring FAE enterocytes. Cxcr4 and PIN were found increased, with Il-18 and Bad decreased in expression, in both M cell and FAE enterocytes compared to VAE.This gene expression profile of in vivo murine M cells, FAE and VAE provides a portal into M cell biology previously undiscovered. This expression profile of M cells will aid in understanding pathogen-host immune interactions and contribute to the understanding of intestinal M cell biology. Keywords: Cell type comparison

ORGANISM(S): Mus musculus

PROVIDER: GSE3434 | GEO | 2006/09/27

SECONDARY ACCESSION(S): PRJNA93469

REPOSITORIES: GEO

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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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M cell, FAE and VAE expresssion profiles

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