An enteric virus can replace the beneficial function of commensal bacteria

Abstract

Intestinal microbial communities have profound effects on host physiology1. Whereas the symbiotic contribution of commensal bacteria is well established, the role of eukaryotic viruses that are present in the gastrointestinal tract under homeostatic conditions is undefined2,3. Here we demonstrate that a common enteric RNA virus can replace the beneficial function of commensal bacteria in the intestine. Murine norovirus (MNV) infection of germ-free or antibiotic-treated mice restored intestinal morphology and lymphocyte function without inducing overt inflammation and disease. The presence of MNV also suppressed an expansion of group 2 innate lymphoid cells observed in the absence of bacteria, and induced transcriptional changes in the intestine associated with immune development and type I interferon (IFN) signalling. Consistent with this observation, the IFN-α receptor was essential for the ability of MNV to compensate for bacterial depletion. Importantly, MNV infection offset the deleterious effect of treatment with antibiotics in models of intestinal injury and pathogenic bacterial infection. These data indicate that eukaryotic viruses have the capacity to support intestinal homeostasis and shape mucosal immunity, similarly to commensal bacteria.

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Figure 1: MNV reverses intestinal abnormalities in GF mice.
Figure 2: MNV reverses intestinal abnormalities induced by antibiotics.
Figure 3: MNV-induced changes are dependent on a type I IFN response.
Figure 4: MNV protects antibiotic-treated mice from intestinal injury and C. rodentium superinfection.

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Primary accessions

Gene Expression Omnibus

Referenced accessions

NCBI Reference Sequence

Data deposits

RNA-seq data have been deposited in the Gene Expression Omnibus under accession number GSE60163. The MNV.SKI capsid sequence has been deposited in the NCBI Reference Sequence database under accession number KM463105.

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Acknowledgements

We would like to thank S. Koralov and P. Loke for advice on the manuscript, E. Venturini for assistance with deep sequencing, S. Brown and Z. Tang for data analysis, L. Ciriboga for CD3 staining, the flow cytometry and histopathology cores (Cancer Center Support Grant, P30CA016087) for assistance with sample preparation and analyses, M. Alva and D. Littman for assistance with breeding and maintaining GF mice, and H. Moura Silva for sample collection for MNV isolation. This research was supported by National Institutes of Health grant R01 DK093668 (K.C.) and a New York University Whitehead Fellowship (K.C.), Vilcek Fellowship (E.K.) and Erwin Schrödinger Fellowship from the Austrian Science Foundation (E.K.).

Author information

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Authors

Contributions

E.K. performed all the experiments, Y.D. analysed and scored histological sections, K.C. and E.K. designed the study and wrote the manuscript.

Corresponding author

Correspondence to Ken Cadwell.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Mono-association of GF mice with MNV does not lead to uncontrolled viral replication or disease.

a, Schematic of MNV mono-association procedure (see Methods for additional details). GF breeder pairs (F0) within a gnotobiotic isolator were infected with 3 × 106 p.f.u. of MNV.CR6, which was allowed to transmit naturally to offspring (F1). Weaned offspring were maintained in isolators until adulthood, and then either used for analysis or as breeders to generate additional experimental animals (F2). b, Successful transmission to offspring and the persistent presence of the virus in mono-associated GF mice (GF+MNV) was confirmed by performing a plaque assay using stool harvested from 8-week-old offspring (1 month after weaning). The amount of virus in stool from GF mice, antibiotic (ABX)-treated wild-type (WT) and Ifnar1−/− mice, and conventional (Conv) mice infected with 3 × 106 p.f.u. of the indicated strains of MNV for 10 days are also shown, n = 5 mice per group. cf, Mice receiving the indicated treatments did not display marked histopathology in the small intestine (c, e) and colon (d, f) based on blind quantification of H&E-stained sections using a previously described scoring system37. Mice receiving a pathology score of 1 displayed mild blunting of villi (additional details in Methods). No histopathology was detected in spleens, and no other signs of disease were noted. Note that a previous publication in which mice were reported to display pathologies after MNV infection used a different strain of MNV, an early time point (24 h after infection), and mice on a different background (129/Sv)38. The lack of pathology in C57BL/6 mice persistently infected with MNV.CR6 is consistent with our previous publication15. n = 5–7 mice per group. All graphs show means ± s.e.m.

Extended Data Figure 2 MNV improves several deficiencies related to intestinal immunity in GF mice.

a, b, Representative images of crypts from small intestinal tissue sections stained with H&E (a) and an anti-lysozyme antibody (b) harvested from GF, GF+MNV (mono-association) and conventional (Conv) mice. Scale bar, 1 µm. c, d, Quantification of the above images shows an increase in granules per Paneth cell (c) and lysozyme-positive cells per crypt (d) in GF+MNV mice, indicating that the presence of MNV partially reverses Paneth cell abnormalities due to the absence of bacteria. n = 5 mice per group. eh, MNV mono-association of GF mice increases the number of CD4+ (e, g) and CD8+ (f, h) T cells (TCR-β+) in small intestinal (SI) lamina propria cells and MLNs. in, Flow cytometry analysis indicates that MNV mono-association of GF mice also increases the number of IFN-γ-expressing CD4+ and CD8+ T cells in small intestine lamina propria (i, k) and MLNs (j, l). IL-17 expression by CD4+ T cells is also influenced by the presence of MNVs (m, n). n = 10. o, p, GF+MNV mice display increased IgA levels in small intestine tissue (o) and IgG2c levels in serum (p). n = 5 mice per group. q, Percentage of T-bet+ cells in the small intestine lamina propria after gating on live and Lin cells remain unchanged by MNV infection of GF mice. n = 10 mice per group. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ANOVA with Holm–Sidak correction. All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Figure 3 MNV infection of adult GF mice has similar effects to mono-association of GF mice from birth.

af, Six- to eight-week old adult GF mice were infected with MNV.CR6 for 10 days and examined for reversal of intestinal abnormalities. a, b, Quantification of villus width (a) and granules per Paneth cell (b) in H&E-stained small intestinal sections. n = 5 mice per group. c, Quantification of the number of CD3+ T cells in the small intestine lamina propria (LP) by flow cytometric analysis. n = 6 mice per group. d, e, Quantification of IFN-γ-producing CD3+CD4+ (d) and CD3+CD8+ (e) T cells by flow cytometry. f, Quantification of small intestinal IgA by enzyme-linked immunosorbent assay (ELISA). n = 6 mice per group. *P < 0.05. Unpaired two-tailed t-test. All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Figure 4 The effect of MNV is specific to mice depleted of bacteria.

ah, Conventional mice were infected with 3 × 106 p.f.u. of MNV.CR6 to determine the effect of MNV in the presence of commensal bacteria. a, b, Representative images of H&E-stained small intestinal sections of conventional (Conv and Conv mice infected with MNV (Conv+MNV) mice showing no aberrant changes after MNV infection. Scale bar, 100 µm (a); 1 µm (b). c, d, Villus width (c) and Paneth cell granules (d) were quantified from at least 50 villi and 30 crypts of 2–5 mice per group. eh, Cell numbers of CD4+TCR-β+ (e), CD8+TCR-β+ (f), IFN-γ producing CD4+ (g) and IFN-γ producing CD8+ T cells in small intestine lamina propria (h). n = 6 mice per group. NS, not significant. *P < 0.05. Unpaired two-tailed t-test. All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Figure 5 MNV mono-association of GF mice increases colonic lymphocyte populations.

a, Representative images of H&E-stained colonic small intestinal sections of GF, GF+MNV (mono-association with MNV.CR6) and conventional (Conv) mice. Scale bar, 100 µm. b, In these mice the crypt height was measured, showing a significant difference between GF and conventional mice. c, Percentages of NK T cells (CD1d+, TCR-β+) in colonic lamina propria of GF, GF+MNV and conventional mice. dg, Percentages of CD4+TCR-β+ (d) and CD8+TCR-β+ cells (f), and percentages of IFN-γ-producing CD4+ (e) and CD8+ T cells (g) in the colonic lamina propria of GF, GF+MNV and conventional mice. n = 5 mice per group. NS, not significant. *P < 0.05, **P < 0.01. ANOVA with Holm–Sidak correction. All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Figure 6 The effect of MNV on the small intestine of GF mice is not strain specific.

a, Phylogenetic tree of the capsid sequences of the indicated MNV strains. bd, Quantification of the villus width (b), granules (c) and CD3+ cells (d) in small intestinal sections prepared from conventional (Conv) mice, GF mice, and GF mice infected with the indicated strains of MNV for 10 days. eh, Percentages of CD4+TCR-β+ (e) and CD8+TCR-β+ cells (g), and percentages of IFN-γ-producing CD4+ (f) and CD8+ T cells (h) in the small intestine lamina propria of the indicated mice. n = 6 mice per group. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ANOVA with Holm–Sidak correction. All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Figure 7 Antibiotics treatment induces intestinal abnormalities that can be reversed by B. thetaiotaomicron, L. johnsonii or MNV.CR6.

ag, Percentages of CD4+TCR-β+ (a) and CD8+TCR-β+ cells (b), and percentages of IFN-γ-producing CD4+ (c) and CD8+ T cells (d) in small intestine lamina propria in conventional (Conv) mice with and without antibiotic (ABX) treatment. n = 5–10 mice per group. e, Schematic of antibiotic treatment for introducing bacteria. After 14 days, antibiotic-containing water was replaced by regular water. Mice were then inoculated with B. thetaiotaomicron (B. theta), L. johnsonii, or left untreated for 10 days before analyses. fh, Bacterial loads of colon (f), small intestine (g) and stool (h) of antibiotic-treated mice inoculated with B. thetaiotaomicron or L. johnsonii for 10 days. i, j, Small intestinal sections stained with H&E (i) or anti-CD3 antibody (j) indicating that inoculation with MNV.CR6, B. thetaiotaomicron or L. johnsonii have similar effects on intestinal morphology. kp, Mice that received antibiotics during the whole course of the experiment with or without MNV (ABX and ABX+MNV) were compared with mice treated as in e using the previously described measurements: quantification of villus width (k), CD3+ cells per villi (l), and percentages of CD4+ (m), CD8+ (o), IFN-γ+CD4+ (n) and IFN-γ+CD8+ (p) T cells in the small intestine lamina propria. n = 8 mice per group. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001. ad, kp, Unpaired two-tailed t-test (ad), ANOVA with Holm–Sidak correction (kp). All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Figure 8 Type I IFN induction by poly(I:C) changes small intestinal architecture without affecting the T-cell compartment.

a, qRT–PCR quantification of the IFN-I inducible gene Mx2 in small intestinal tissue of untreated GF mice, GF mice injected for 10 days with poly(I:C), MNV.CR6 mono-associated GF mice (GF+MNV), and conventional (Conv) mice indicate that the poly(I:C) injection procedure induces an IFN-I response. Values represent fold induction of Mx2 compared with untreated GF mice after normalizing to Gapdh. bd, Quantification of the villus width (b), granules (c) and CD3+ cells in the small intestine of the same mice as in a of H&E-stained small intestinal sections. eh, Percentages of CD4+TCR-β+ (e), CD8+TCR-β+ cells (g) and IFN-γ-producing CD4+ (f) and CD8+ (h) T cells from the small intenstine lamina propria. n = 6 mice per group. NS, not significant. ***P < 0.001, ****P < 0.0001. ah, ANOVA with Holm–Sidak correction (ad), unpaired two-tailed t-test (eh). All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Figure 9 MNV infection alters the host response to a super infection with C. rodentium.

a, Taxon-specific 16S qRT–PCR for Lactobacilli, Enterobacteriacea, Fusobacteria, Bacteroides and C. rodentium normalized to total 16S gene expression in stool of mice infected for 9 days with C. rodentium after antibiotic (ABX) pre-treatment with or without MNV.CR6 infection, showing similar colonization of mice throughout the groups. b, c, Fold induction of C. rodentium virulence factors tir (b) and ler (c) compared with the antibiotic group after normalization to recA in stool of indicated mice on day 9 after C. rodentium infection. df, MNV.CR6 infection of antibiotic-treated mice before C. rodentium infection increases IgA levels in colonic tissue (d) and stool (e), and IgG2c levels in serum (f) at day 9 after C. rodentium infection. gi, At day 9 after C. rodentium infection, antibiotics plus MNV mice display elevated expression of IFN-γ (g), Gbp2 (h) and IL-10 (i) in colonic tissue compared with antibiotics-only mice. n = 5 mice per group. ND, not detectable. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired two-tailed t-test. All graphs show means ± s.e.m. from at least two independent experiments.

Extended Data Table 1 Genes displaying increased expression after MNV infection or conventionalization of GF mice

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Researcher Ken Cadwell on the surprising benefits of norovirus for the mouse gut.

Supplementary information

Supplementary Information

This file contains the RNAseq table. Sheet 1 and sheet 2 show the mRNA counts for GF (uni N = 3), GF+MNV (MNV, N = 4) and GF+conv (conv, N = 4) which are above the threshold cutoff and which have been ranked according to their p-value after edgeR analysis. In sheet 1, ranked genes in the comparison GF to GF+MNV are shown; in sheet 2, GF was compared to GF+conv. For each gene the log fold change (logFC), log counts per million (log CPM) as well as the false discovery rate (FDR, calculated with the Benjamini’s and Hochberg algorithm) are shown. (XLS 5128 kb)

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Kernbauer, E., Ding, Y. & Cadwell, K. An enteric virus can replace the beneficial function of commensal bacteria. Nature 516, 94–98 (2014). https://doi.org/10.1038/nature13960

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