Efficient norovirus and reovirus replication in the mouse intestine requires microfold (M) cells - PubMed (original) (raw)

Efficient norovirus and reovirus replication in the mouse intestine requires microfold (M) cells

Mariam B Gonzalez-Hernandez et al. J Virol. 2014 Jun.

Abstract

Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. M cells have also been suggested as a portal for pathogen entry into the host. While virus particles have been observed in M cells, it is not known whether viruses use M cells to initiate a productive infection. Noroviruses (NoVs) are single-stranded RNA viruses that infect host organisms via the fecal-oral route. Murine NoV (MNV) infects intestinal macrophages and dendritic cells and provides a tractable experimental system for understanding how an enteric virus overcomes the intestinal epithelial barrier to infect underlying target cells. We found that replication of two divergent MNV strains was reduced in mice depleted of M cells. Reoviruses are double-stranded RNA viruses that infect hosts via respiratory or enteric routes. In contrast to MNV, reovirus infects enterocytes in the intestine. Despite differences in cell tropism, reovirus infection was also reduced in M cell-depleted mice. These data demonstrate that M cells are required for the pathogenesis of two unrelated enteric viruses that replicate in different cell types within the intestine.

Importance: To successfully infect their hosts, pathogens that infect via the gastrointestinal tract must overcome the multilayered system of host defenses. Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. Virus particles have been observed within M cells. However, it is not known whether viruses use M cells to initiate a productive infection. To address this question, we use MNV and reovirus, two enteric viruses that replicate in different cell types in the intestine, intestinal epithelial cells for reovirus and intestinal mononuclear phagocytes for MNV. Interestingly, MNV- and reovirus-infected mice depleted of M cells showed reduced viral loads in the intestine. Thus, our work demonstrates the importance of M cells in the pathogenesis of enteric viruses irrespective of the target cell type in which the virus replicates.

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Figures

FIG 1

Neutral red assay distinguishes between input and replicated MNV. (A) Distal ileum (tissue) homogenate from BALB/c mice (7 to 8 weeks old) were mixed with ∼104 PFU of neutral red (NR) containing MNV-1 or CR3 stock. Viral titers were determined by plaque assay in the dark (total) or light (replicated). (B) BALB/c mice (7 to 8 weeks old) were inoculated perorally with 105 PFU of a neutral red-containing MNV-1. Distal ileum and colon were harvested at the times shown. Viral titers were determined by plaque assay in the light (replicated) or dark (total). (C to E) MNV strains differ in replication kinetics. BALB/c mice were inoculated perorally with 105 PFU of either MNV-1 or CR3 containing neutral red. The GI tract was dissected 12, 24, or 72 h later, and viral titers were determined by plaque assay as described for panels A and B. Data are expressed as means ± SEM for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ND, not detectable; J/D, jejunum/duodenum; PI, proximal ileum; DI, distal ileum; CE, cecum; CO, colon; FE, feces.

FIG 2

MNV infection is reduced in the GI tract following M cell depletion. (A) Schematic of the experimental design for MNV infection of M cell-depleted mice. BALB/c mice (7 to 8 weeks old) were left untreated or were inoculated i.p. with anti-RANKL or isotype control antibody every other day for a total of four doses and then infected with either MNV-1 (NR) or CR3 (NR) 36 h later, and regions of the GI tract were harvested 12 hpi for MNV-1 and 18 hpi for CR3. (B to E) The number of mice analyzed in each group is indicated in parentheses. Viral titers in indicated tissues were quantified by plaque assay in the dark (total; B and D) or in the light (replicated; C and E). Data are expressed as means ± SEM for two to three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; ND, not detectable; ST, stomach; J/D, jejunum/duodenum; PI, proximal ileum; DI, distal ileum; CE, cecum; CO, colon; FE, feces.

FIG 3

Whole-mount staining of Peyer's patches shows decreased numbers of M cells in anti-RANKL-treated mice. BALB/c mice were left untreated or were inoculated i.p. with anti-RANKL or isotype control antibody every other day for a total of four doses and then infected with either MNV-1 (NR) or CR3 (NR) 36 h later, and Peyer's patches were harvested after infection and immunostained for two M cell markers. (A to I) Representative confocal microscopic images of Peyer's patches stained with M cell markers GP2 (green) and UEA-1 (red) from untreated (A to C), isotype control-treated (D to F), and anti-RANKL-treated (G to I) mice. DAPI was used to stain the nuclei. A 10-μm scale bar is shown in the upper right corner of each image. (J) Quantification of whole-mount staining with M cell markers GP2 and UEA-1 of one Peyer's patch per mouse from a total of 5 to 6 mice per group. Immunofluorescence staining was quantified using the scoring system of intensities by the Metamorph Premier v6.3 image analysis software. Data are expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 4

MNV does not replicate in M cells. BALB/c and STAT1−/− mice were infected with MNV, and Peyer's patches were harvested 24 hpi for cryosectioning and immunostaining. (A and C) The secondary antibody-only control (red) and an isotype control for GP2 (green) were used to identify background staining. (B and D) MNV replication was detected using an antibody against the nonstructural protein N-term (red), and M cells were detected using an anti-GP2 antibody (green). Arrowheads in the inset indicate GP2-positive M cells.

FIG 5

Reovirus infection is reduced in the GI tract following M cell depletion. BALB/c mice (3 to 4 weeks old) were depleted of M cells as described in the legend to Fig. 1 and inoculated perorally with T1L reovirus 24 h later, and regions of the GI tract were harvested 24 hpi. The number of mice analyzed in each group is indicated in parentheses. Viral titers were quantified by plaque assay. Data are expressed as means ± SEM from two independent experiments. *, P < 0.05; ***, P < 0.001; ND, not detectable; ST, stomach; J/D, jejunum/duodenum; PI, proximal ileum; DI, distal ileum; CE, cecum; CO, colon; FE, feces.

FIG 6

Reovirus replicates in enterocytes adjacent to and in M cells of control but not M cell-depleted mice. Isotype control-treated (A) or anti-RANKL-treated (B) BALB/c mice were infected with reovirus for 24 h. Peyer's patches were harvested for paraffin embedding and immunostaining. Reovirus replication was detected using a polyclonal antibody against the sigma nonstructural (σNS) protein (red), and M cells were detected using an anti-GP2 antibody (green). Arrowheads indicate uninfected GP2-positive M cells, while stars indicate reovirus-infected M cells.

FIG 7

Proposed mechanisms of MNV and reovirus entry into the intestinal mucosa. MNV (black circles) and reovirus (red circles) in the intestinal lumen initially interact with M cells within the follicle-associated epithelium (FAE) overlying Peyer's patches (PP) to establish a productive infection in untreated animals. In mice depleted of M cells, reovirus infection in the gastrointestinal tract is substantially diminished, while MNV infection is partially reduced, suggesting alternative route(s) of entry for MNV, for example, via transepithelial dendritic cells.

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Efficient norovirus and reovirus replication in the mouse intestine requires microfold (M) cells - PubMed (original) (raw)