Allograft inflammatory factor 1 is a regulator of transcytosis in M cells - PubMed (original) (raw)
Allograft inflammatory factor 1 is a regulator of transcytosis in M cells
Sari Kishikawa et al. Nat Commun. 2017.
Abstract
M cells in follicle-associated epithelium (FAE) are specialized antigen-sampling cells that take up intestinal luminal antigens. Transcription factor Spi-B regulates M-cell maturation, but the molecules that promote transcytosis within M cells are not fully identified. Here we show that mouse allograft inflammatory factor 1 (Aif1) is expressed by M cells and contributes to M-cell transcytosis. FAE in Aif1-/- mice has suppressed uptake of particles and commensal bacteria, compared with wild-type mice. Translocation of Yersinia enterocolitica, but not of Salmonella enterica serovar Typhimurium, leading to the generation of antigen-specific IgA antibodies, is also diminished in Aif1-deficient mice. Although β1 integrin, which acts as a receptor for Y. enterocolitica via invasin protein, is expressed on the apical surface membranes of M cells, its active form is rarely found in Aif1-/- mice. These findings show that Aif1 is important for bacterial and particle transcytosis in M cells.
Conflict of interest statement
The authors declare no competing financial interests.
Figures
Figure 1. Specific expression of Aif1 in GP2-positive M cells among IECs.
(a) Relative Aif1 expression in indicated cell populations from Spib+/+ and Spib −/− mice. Each result was normalized against the expression of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and is representative of three independent experiments. (b,c) Whole-mount staining of GP2-positive M cells and Aif1. Peyer's patches (PPs) prepared from wild-type C57BL/6J (b) and Spi-B-deficient mice (c) were stained with anti-GP2 antibody (green), anti-Aif1 antibody (red) and phalloidin (blue). The yellow box region in (b, bottom left) was magnified to produce the image in (b, bottom right). Dotted lines indicate the periphery of the FAE region. Data are representative of three independent experiments. Duo, duodenal; ECs, epithelial cells; FAE, follicle-associated epithelium, haemato, haematopoetic cells; Ile, ileum; LI, large intestine.
Figure 2. Normal M-cell numbers in Aif1-deficient mice.
(a) Relative expression of Gp2 in FAE from Aif1+/+ and Aif1 −/− mice. Each result was normalized against the expression of Gapdh. Data are shown as means±s.e.m. (_n_=2 per genotype) from one experiment representative of two independent experiments. P value was determined with Student's _t_-test. (b) Whole-mount staining of GP2-positive M cells in the FAE of Aif1+/+ and Aif1 −/− mice. The number of GP2-positive cells found in each 0.01-mm2 field was counted to provide the data in c. Dotted lines indicate the periphery of the FAE region. Data are representative of three independent experiments. (c) M cells were counted as GP2-positive cells, and the average number of M cells found in each 0.01 mm2 field is shown as the mean±s.e.m. (_n_=7 per genotype). P value was determined with Student's _t_-test.
Figure 3. Ultrastructure of M cells in Aif1-deficient mice.
(a) Scanning electron microscopic images of FAE from control and Aif1 _−/−_mice. Right panels are magnified images of the boxed regions in the left panels. (b) M cells were counted as sunken cells, and the average number of M cells found in each 0.01 mm2 field is shown as the mean±s.e.m. of one experiment representative of two independent experiments (Aif1+/+, _n_=9; Aif1 −/−, _n_=10). P value was determined with Student's _t_-test. (c) Transmission electron microscopic images of FAE from control and Aif1 _−/−_mice. Arrows indicate M cells. Data are representative of three independent experiments. N, nucleus; L, lymphocyte. (d) Magnified transmission electron microscopic images of the apical side of M cells. Arrows show endosomes. Data are representative of three independent experiments.
Figure 4. Influence of Aif1 deficiency on transcytosis function of M cells.
Evaluation of particle (a) and Lactobacillus reuteri (b) sampling by M cells. Mice fasted for 24 h were given fluorescence-conjugated 200 nm particles (1 × 1011 beads) or FITC-labelled bacteria (5 × 109 CFUs) orally. Two hours later, PPs were collected from the duodenum, fixed in 4% paraformaldehyde, and cut into 10 or 15 consecutive sections. The numbers of particles or labelled bacteria that had entered each PP were then counted under a fluorescence microscope. Each dot represents an individual PP from three independent experiments (For particles: Aif1+/+, _n_=12; Aif1 −/−, _n_=11. For L. reuteri: Aif1+/+, _n_=10; Aif1 −/−, _n_=9). Horizontal bars indicate each mean. P values were determined with Student's _t_-test. Right panels are representative images of each section. Dotted lines indicate FAE regions and circles indicate each particle or bacterium. Scale bar, 20 μm.
Figure 5. Impaired translocation of pathogenic bacteria in Aif1 −/− mice.
Evaluation of transcytosis of Y. enterocolitica (a; 1 × 109 CFUs given orally) and indicated strains of S. Typhimurium (b–d; 1 × 108 CFUs given orally). Twenty-four hours after administration of the bacteria, PPs were collected from the ileum, homogenized and diluted. Each diluted sample was cultured on the appropriate plates. Colonies were counted and the values normalized against the weight of the PPs. Data are shown as means±s.e.m. from two independent experiments (_n_=18 per genotype for Y. enterocolitica; _n_=10 to 20 per genotype for S. Typhimurium). P values were determined with Student's _t_-test.
Figure 6. Importance of M-cell-intrinsic Aif1 for uptake of particles and Y. enterocolitica.
Evaluation of 200 nm bead uptake (a) and transcytosis of Y. enterocolitica (b) in bone-marrow (BM) chimeric mice to which we transferred Aif1 −/− BM cells (Aif1 −/− BM→Ly5.1) or vice versa (Ly5.1 BM→Aif1 −/−). Both experiments used the same protocols as described in Fig. 4a (particles) and Fig. 5a (Y. enterocolitica; for particles: _n_=12 per group. For Y. enterocolitica: Aif1 −/− BM→Ly5.1, n_=5; Ly5.1 BM→_Aif1 −/−, _n_=6). P values were determined with Student's _t_-test.
Figure 7. Importance of Aif1 for initiation of the mucosal IgA immune response.
(a) Quantification of total faecal IgA. Faeces were prepared from the chimeric mice indicated. Concentrations of total IgA were determined by ELISA. Data are means±s.e.m. from two independent experiments (_n_=8 per group). P value was determined with Student's _t_-test. (b) Representative plots of bacterial flow cytometry. Faeces were collected from the chimeric mice indicated and suspended in PBS. Bacteria recognized by IgA were detected and their percentages determined. (c) The percentages determined in b are shown as means±s.e.m. from two independent experiments (_n_=12 per group). P value was determined with Student's _t_-test. (d) Quantification of total IgA after long-term infection with Y. enterocolitica. Faeces were prepared from the indicated chimeric mice on day 28 after infection. Concentrations of total IgA were determined by ELISA. Data are means±s.e.m. from two independent experiments (_n_=6 per group). P value was determined with Student's _t_-test. (e) Comparison of anti-_Y. enterocolitica-_specific IgA production after long-term infection. Serial diluted faecal extracts prepared from _Y. enterocolitica-_infected mice were subjected to ELISA by using _Y. enterocolitica-_coated plates. Data are means±s.e.m. from one experiment representative of two independent experiments (_n_=4 per group). *P<0.05, determined with Student's _t_-test.
Figure 8. Aif1-dependent activation of β1 integrin.
(a) Relative gene expression of β1 integrin in FAE from wild-type and Aif1 −/− mice. Data are means±s.e.m. of one experiment representative of two independent experiments (_n_=4 per genotype). P value was determined with Student's _t_-test. (b,c) Whole-mount staining of PPs prepared from Aif1+/+ and Aif1 −/− mice with anti-total β1 integrin antibody (b, red) or anti-activated-β1 integrin antibody (c, red), together with anti-GP2 antibody (b,c, green). Each yellow box region in c (left panels) was magnified to produce the image in c (right panels). Dotted lines indicate the periphery of the FAE region. Data are representative of three independent experiments.
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