IFN-λ derived from nonsusceptible enterocytes acts on tuft cells to limit persistent norovirus

Figures

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  Fig. 1.. IFN-λ is derived from radiation-insensitive cells for control of persistent MNoV. (A and B) MNoV genome copies in fecal pellets (A) and colon (B) at 7dpi with CR6 in bone marrow chimeras (n = 7 to 8) generated from WT and Ifnl2−/−Ifnl3−/− mice as indicated. Pie chart depicting percentage of chimerism in peripheral blood at 8 weeks after transplant before MNoV infection. Results were analyzed using Kruskal-Wallis analysis of variance (ANOVA) with Dunn’s post-test from two independent experiments. **P < 0.01; ns, not significant. Bars indicate mean of all data points.

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  Fig. 2.. Mavs/Mda5 axis is critical for controlling persistent MNoV infection. (A to D) MNoV genome copies in fecal pellets and colon tissues of WT (n = 5 to 7), Mavs−/− (n = 9), and Mda5−/− (n = 6) mice at 7 dpi. (E) Quantification of CR6-positive tuft cells per tissue section of the entire ileum, with portions of ileum sections depicted in (F). (F) Representative ileal sections of WT, Mavs−/−, and Mda5−/− mice at 7 dpi with CR6, hybridized with probes for MNoV (green) and stained with anti-DCLK1 (red). Cell nuclei were stained using DAPI (blue). Results were analyzed by Mann-Whitney test (A to D) from three (A and B) and two (C to E) independent experiments. **P < 0.01; *P < 0.05. Bars indicate mean of all data points. White boxes reflect the magnified inset images.

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  Fig. 3.. Mavs is required in epithelial cells but is dispensable in tuft cells for control of persistent MNoV. (A) Relative quantification of Mavs mRNA levels in IECs from small intestine of Mavsf/f (n = 5) and Mavsf/f-Vilcre (n = 5) mice. (B and C) MNoV genome copies in fecal pellets and colon tissues of Mavsf/f (n = 18) and Mavsf/f-Vilcre (n = 15) mice at 7 dpi with CR6. (D) Relative quantification of Mavs mRNA levels in tuft cells from small intestine of Mavsf/f (n = 6) and Mavsf/f-Dclk1cre (n = 6) mice. (E and F) MNoV genome copies in fecal pellets and colon tissues of Mavsf/f (n = 8) and Mavsf/f-Dclk1cre (n = 8) mice at 7 dpi with CR6. (G and H) Relative quantification of Mavs mRNA levels in (G) tuft cells and nontuft IECs, and (H) splenocytes from small intestine of Mavsf/f (n = 4) and Mavsf/f-Vavicre (n = 6) mice. (I and J) MNoV genome copies in fecal pellets and colon tissues of Mavsf/f (n = 6) and Mavsf/f-VaviCre (n = 6) mice at 7 dpi with CR6. Results were analyzed by Mann-Whitney test (A to F, I, and J) from three (A to E) and two (I and J) independent experiments. *P < 0.05; **P < 0.01. Bars indicate mean of all data points.

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  Fig. 4.. Mavs is dispensable in secretory cells but required in enterocytes for control of persistent MNoV. (A) Relative quantification of Mavs mRNA levels in goblet cells and nongoblet IECs from small intestine of Mavsf/f (n = 6) and Mavsf/f-Math1cre (n = 7) mice. (B and C) MNoV genome copies in fecal pellets and colon tissues of Mavsf/f (n = 10) and Mavsf/f-Math1cre (n = 7 to 10) mice at 7 dpi with CR6. (D) Relative quantification of Mavs mRNA levels in IECs from Mavsf/f and Mavsf/f-AlpicreERT2 at 3 days after tamoxifen treatment. (E and F) MNoV genome copies in fecal pellets and colon tissues of Mavsf/f (n = 9) and Mavsf/f-AlpicreERT2 (n = 10) mice at 7 dpi. (G) Quantification of CR6-positive tuft cells in the entire ileum, with portions of ileum sections depicted in (H). (H) Representative ileal sections of WT, Mavs−/−, and Mda5−/− mice at 7 dpi with CR6, hybridized with probes for MNoV (green) and stained with anti-DCLK1 (red). Cell nuclei were stained using DAPI (blue). Results were analyzed by Mann-Whitney test (A to G) from three (A to F) and two (G and H) independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05. Bars indicate mean of all data points. White boxes reflect the…

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  Fig. 5.. IFN-λ acts on tuft cells to regulate persistent MNoV infection. (A) MNoV genomes quantified in fecal pellets of WT (n = 5), Ifnlr1−/− treated with PBS (n = 5), and Ifnlr1−/− mice treated with succinate (n = 6) at 7 dpi. (B) Electron microscopy (EM) images of tuft cell from CR6-infected Ifnlr1−/− mice pretreated with succinate. Microvesicular bodies (MVBs) containing putative MNoV are denoted by arrows, and white box denotes corresponding magnified image in (C). (C) Magnified image of MVB depicting putative CR6 virion size. (D) EM images of CR6 virions isolated from BV2 cells. (E) Immunofluorescence images of ileal sections of Mx1gfp mice treated with mock (PBS) or IFN-λ for 24 hours, with tuft cell marker DCLK1 (red) and GFP (green). Cell nuclei were stained using DAPI (blue). (F) Relative quantification of Ifnlr1 mRNA levels in tuft cells and nontuft IECs from small intestine (SI) of Ifnlr1f/f (n = 3) and Ifnlr1f/f-Dclk1cre (n = 5) mice. (G and H) Longitudinal analysis of MNoV genome copies in fecal pellets of Ifnlr1f/f (n = 6) and Ifnlr1f/f-Dclk1cre (n = 7) mice infected with CR6 for 21 days before IFN-λ treatment, and colon tissues at 28 dpi. Results were analyzed using…