12 research outputs found
Epithelium-Intrinsic MicroRNAs Contribute to Mucosal Immune Homeostasis by Promoting M-Cell Maturation.
M cells in the follicle-associated epithelium (FAE) of Peyer's patches (PPs) serve as a main portal for external antigens and function as a sentinel in mucosal immune responses. The scarcity of these cells has hampered identification of M cell-specific molecules. Recent efforts have begun to provide insight into antigen transcytosis and differentiation of M cells; however, the molecular mechanisms underlying these processes are not fully elucidated. Small non-coding RNAs including microRNA (miRNA) have been reported to regulate gene expression and control various biological processes such as cellular differentiation and function. To evaluate the expression of miRNAs in FAE, including M cells, we previously performed microarray analysis comparing intestinal villous epithelium (VE) and PP FAE. Here we confirmed FAE specific miRNA expression levels by quantitative PCR. To gain insight into miRNA function, we generated mice with intestinal epithelial cell-specific deletion of Dicer1 (DicerΔIEC) and analyzed intestinal phenotypes, including M-cell differentiation, morphology and function. DicerΔIEC mice had a marked decrease in M cells compared to control floxed Dicer mice, suggesting an essential role of miRNAs in maturation of these cells. Furthermore, transmission electron microscopic analysis revealed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake by M cells was impaired in DicerΔIEC mice. These results suggest that miRNAs play a significant role in M cell differentiation and help secure mucosal immune homeostasis
Cutting edge : Brucella abortus exploits a cellular prion protein on intestinal m cells as an invasive receptor
miRNA expression profiles in intestinal epithelium.
<p>Q-PCR analysis was performed for miRNA expression in FAE and VE. The relative levels of each miRNA relative to the small nucleolar RNA <i>Sno202</i> are shown. Values are mean ± SE of three samples from different mice. *P<0.05.</p
FAE miRNAs involved in M cell maturation.
<p>(A) Flowchart of M cell maturation. (B) Q-PCR analysis was performed for <i>Marcksl1</i>, <i>SpiB</i>, <i>Ccl9 and Gp2</i> mRNA expression in Dicer<sup>ΔIEC</sup> FAE and Dicer<sup>F/F</sup> FAE. The relative expression levels of each gene to <i>Gapdh</i> are shown. Values represent the mean ± SD of three samples from different mice. *<i>P</i> < 0.05 **<i>P</i> < 0.01.</p
Cutting edge : Brucella abortus exploits a cellular prion protein on intestinal m cells as an invasive receptor
Morphology of M cells in Dicer<sup>ΔIEC</sup>.
<p>Electron micrographs of Dicer<sup>F/F</sup> and Dicer<sup>ΔIEC</sup> PP. (A) Surface of FAE by scanning electron microscopy. Arrowheads indicate M cells. Scale bars: 10 μm (B) Transmission electron micrographs of an M cell in FAE and enterocyte in VE. Scale bars: 1.8 μm (C) High magnification image of (B). Arrow indicate the endosomes in M cell. Scale bars: 500 nm.</p
Total number of M cells in Peyer’s patches is decreased in Dicer<sup>ΔIEC</sup> mice.
<p>(A) H&E staining of small intestines VE region of Dicer<sup>ΔIEC</sup> and Dicer<sup>F/F</sup>. (B) H&E staining in PPs of Dicer<sup>ΔIEC</sup> and Dicer<sup>F/F</sup>. (C) The total number of follicles and surface area in Dicer<sup>ΔIEC</sup> and Dicer<sup>F/F</sup>. Data are means± SE (n = 3). *<i>P</i> < 0.05. (D) Whole mount immunostaining of PPs with anti-GP2 (red) and F-actin (green) analyzed using a confocal microscope. Scale bars: 100 μm (E) M cell number/mm<sup>2</sup> in FAE of each mouse strain. Data are means and SE. *<i>P</i> < 0.05.</p
Impaired antigen uptake by Dicer<sup>ΔIEC</sup> M cells.
<p>(A) Dicer<sup>ΔIEC</sup> and Dicer<sup>F/F</sup> mice were inoculated by gavage with 1 x 10<sup>11</sup> FluoSpheres. After 4 hours, frozen sections were prepared to examine translocated beads in PPs. Scale bars: 100 μm (B) Count data of beads taken up in PP each mouse strain. Data are expressed as the mean ± SE of four different samples for each group. **<i>P</i> < 0.01. (C) Dicer<sup>ΔIEC</sup> and Dicer<sup>F/F</sup> mice were inoculated intragastrically by gavage with 1 x 10<sup>8</sup> CFU of <i>Yersinia enterocolitica</i>. After 24 hours, the bacterial translocation to Peyer’s patches was examined by plating PP homogenates. Data are expressed as the mean ± SE of five different mice/each group. *<i>P</i> < 0.05.</p