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Interferon-λ and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection

Nature Immunology volume 16, pages 698–707 (2015)Cite this article

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Abstract

The epithelium is the main entry point for many viruses, but the processes that protect barrier surfaces against viral infections are incompletely understood. Here we identified interleukin 22 (IL-22) produced by innate lymphoid cell group 3 (ILC3) as an amplifier of signaling via interferon-λ (IFN-λ), a synergism needed to curtail the replication of rotavirus, the leading cause of childhood gastroenteritis. Cooperation between the receptor for IL-22 and the receptor for IFN-λ, both of which were 'preferentially' expressed by intestinal epithelial cells (IECs), was required for optimal activation of the transcription factor STAT1 and expression of interferon-stimulated genes (ISGs). These data suggested that epithelial cells are protected against viral replication by co-option of two evolutionarily related cytokine networks. These data may inform the design of novel immunotherapy for viral infections that are sensitive to interferons.

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Figure 1: Control of rotavirus infection requires IL-22.
Figure 2: Rotavirus infection induces production of IFN-λ by IECs and of IL-22 by ILC3 cells.
Figure 3: IL-1α produced by IECs controls rotavirus-induced production of IL-22 by ILC3 cells.
Figure 4: Control of rotavirus replication by IL-22 depends on signaling via IFN-λR.
Figure 5: Cooperation between IL-22 and IFN-λ is required for induction of an efficient antiviral state in IECs.
Figure 6: IL-22 and IFN-λ act synergistically for the phosphorylation of STAT1.
Figure 7: Enhancement of IFN-λ-dependent ISG expression by IL-22 requires STAT1 signaling.

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Acknowledgements

We thank V. Sexl (University of Veterinary Medicine Vienna) for mice with a Stat3fl allele (provided with permission from V. Poli, Universita di Torino); M. Stemmler (Max-Planck-Institute of Immunobiology & Epigenetics, Freiburg) for Vil1-Cre mice (provided with permission from S. Robine. Institute Curie, Paris); C. Johner (Max-Planck-Institute of Immunobiology & Epigenetics, Freiburg) for Rag2−/− and Rag2−/−Il2rg−/− mice; D. Littman (Skirball Institute of Biomolecular Medicine) for Rorc-CreTG mice; N. Ghilardi (Genentech) for Il23a−/− mice; M. Hornef (Hannover Medical School) for the rat IEC line IEC6 and the mouse rotavirus strain EDIM and for discussions; S. Koike (Tokyo Metropolitan Institute of Medical Science) for poliovirus type I Mahoney strain; G. Häcker for support; members of the Diefenbach laboratory for discussions; A. Triantafyllopoulou for critical reading of the manuscript; K. Oberle, S. Woltemate and Z. Fiebig for technical assistance; and M. Follo, K. Geiger and J. Bodinek-Wersing for cell sorting. Supported by ERC (311377 to A.D.), Deutsche Forschungsgemeinschaft (Priority Program 1656 “Intestinal Microbiota” DI764/3 to A.D. and SU133/9 to S.S.; GRK1104 to A.D. and F.G.; and SFB900/Z1 to S.S.) and the Excellence Initiative of the Deutsche Forschungsgemeinschaft and the German Science Council (Spemann Graduate School of Biology and Medicine (GSC-4 to T.M.); and the Cluster of Excellence “Inflammation at Interfaces” (Borstel-Kiel-Lübeck-Plön) EXC306 to C.H.).

Author information

Author notes

  1. Pedro P Hernández and Tanel Mahlakõiv: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Centre Immunology and Institute of Medical Microbiology and Hygiene, University of Mainz Medical Centre, Mainz, Germany

    Pedro P Hernández, Vera Schwierzeck, Fabian Guendel, Konrad Gronke & Andreas Diefenbach

  2. Department of Medical Microbiology and Hygiene, Institute for Medical Microbiology and Hygiene, Freiburg University Medical Centre, Freiburg, Germany

    Pedro P Hernández, Vera Schwierzeck, Nam Nguyen, Fabian Guendel, Konrad Gronke & Andreas Diefenbach

  3. Max-Planck-Institute for Immunobiology and Epigenetics, Freiburg, Germany

    Pedro P Hernández & Konrad Gronke

  4. Department of Medical Microbiology and Hygiene, Institute for Virology, Freiburg University Medical Centre, Freiburg, Germany

    Tanel Mahlakõiv & Peter Staeheli

  5. Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany

    Tanel Mahlakõiv

  6. Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany

    Ines Yang & Sebastian Suerbaum

  7. German Center for Infection Research, Hannover, Germany

    Ines Yang & Sebastian Suerbaum

  8. Research Training Group (GRK1104) of Organogenesis, Freiburg, Germany

    Fabian Guendel, Konrad Gronke & Andreas Diefenbach

  9. INEM–UMR7355, Molecular Immunology, University of Orleans, Orleans, France

    Bernhard Ryffel

  10. CNRS, Orleans, France

    Bernhard Ryffel

  11. Institute of Infectious Disease, University of Cape Town, Cape Town, South Africa

    Bernhard Ryffel

  12. Infection Immunology Research, Research Center Borstel, Borstel, Germany

    Christoph Hölscher

  13. Cluster of Excellence Inflammation at Interfaces, Borstel-Kiel-Lübeck-Plön, Germany

    Christoph Hölscher

  14. Ludwig Institute for Cancer Research, Université Catholique de Louvain, Brussels, Belgium

    Laure Dumoutier & Jean-Christophe Renauld

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  1. Pedro P Hernández
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Contributions

P.P.H. and T.M. performed most of the experiments with the help of V.S., N.N., F.G. and K.G.; I.Y. and S.S. performed the microbiota analysis; B.R., C.H., L.D. and J.-C.R. provided mice, reagents and input on the manuscript; P.S. and A.D. designed the study; P.P.H., T.M., P.S. and A.D. analyzed the data; and A.D. directed the study and wrote the manuscript (with contributions from P.P.H., T.M. and P.S.).

Corresponding author

Correspondence to Andreas Diefenbach.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gene-expression analysis during the first days of rotavirus infection.

a-i, Groups of wildtype suckling mice were infected with 7x102 ID50 rotavirus by oral inoculation. Transcript levels of the indicated genes were quantified by RT-qPCR from small intestinal tissue obtained at the specified time-points after infection. All RT-qPCR data are shown as fold (2-∆Ct) relative to Hprt. n ≥ 3. Mean ± SEM.

Supplementary Figure 2 Presence of a functional Mx1 gene has no effect on the control of rotavirus infection in suckling mice.

(a) Groups of suckling wildtype mice with a functional (Mx1+) or a non-functional (Mx1−) Mx1 allele were infected with rotavirus. Virus titers and transcript levels of the indicated genes were quantified by RT-qPCR from small intestinal tissue obtained at the indicated time-points after oral infection. n ≥ 3. Mean ± SEM. One-way ANOVA. NS, not significant (P > 0.05). (b) H&E stain of small intestinal tissue of suckling mice at day 4 after rotavirus infection to illustrate virus-mediated tissue damage. Circled areas: vacuolizations; arrowheads: epithelial erosions; arrows: villus disruptions. Scale bar, 100μm. (c, d) Adult (6-8 week old) mice of the indicated strains were orally infected with rotavirus. Rotavirus transcripts in small intestine at day 4 after infection were determined by RT-qPCR (c). Virus shedding via feces was quantified by ELISA on day 4 (d). Mean ± SEM. n ≥ 9, One-way ANOVA. NS, not significant (P > 0.05). (e-h) Groups of suckling mice of the indicated genotypes were infected with rotavirus and were injected with BrdU at day 3 following infection. BrdU incorporation by IECs was assessed. (e, f) Numbers of BrdU-positive IECs per crypt where quantified 2 hours after BrdU injection. (g, h) BrdU incorporation by IECs at 24 hours after BrdU pulse starting at day three after infection. Relative distance of BrdU-positive IECs from crypt bottom to villus tip as percentage of villus length (h). Data are representative of two independent experiments. All RT-qPCR data are shown as fold (2-∆Ct) relative to Hprt. (e-h) n ≥ 3. Mean ± SEM; Student’s t test. One-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant (P > 0.05). Scale bars, 50 μm.

Supplementary Figure 3 Composition of microbial communities in Il22−/− and Ifnlr1−/− mice.

(a-f) Rarefaction curves of the 16S rDNA library and PCoA plots of microbiota compositions. Rarefaction curves of the 16S rDNA library OTU dataset. Adult mice (a), individual samples. Colour coded by genotype and litter. Suckling mice (d): Average values of litters of one genotype (solid lines), with standard deviations (dotted lines). PCoA plot of the microbiota composition of adult mice (b, c) of two different genotypes from two different litters, based on Jaccard index distances. PC 1 explains 23.1% of the variation; PC 2, 18.2%, and PC 3, 17.7%. One litter marked by squares (n = 4), the other by triangles (n = 6); Il22−/− mice are marked in blue (n = 5), WT mice in violet (n = 5). PCoA plot of the microbiota composition of suckling mice (e, f) of different genotypes, based on Jaccard index distances. One litter per genotype. PC 1 explains 20.9 % of the variation; PC 2, 18.7 %, and PC 3, 12.1%. Suckling Il22−/− mice are marked in green (n = 4), B6.A2G-Mx1-Il22−/− (MX_IL22_KO) sucklings in dark blue (n = 5), B6.A2G-Mx1-Ifnlr1−/− (MX_lamdaR_KO) sucklings in blue-green (n = 5), and B6.A2G-Mx1 (MX_WT) sucklings in pink (n = 5).

Supplementary Figure 4 IFN-λ is produced by intestinal epithelial cells.

Groups of suckling and adult wildtype mice were infected with rotavirus. Control groups received PBS. (a) Ifnl2/3 expression in small intestinal epithelial cells (IEC, black columns) and lamina propria leukocytes (LPL, white columns) isolated from adult mice at day 4 following infection was determined by RT-qPCR. (b) Rotavirus load in purified cells of the small intestine from suckling mice at day 4 after infection was determined by RT-qPCR. Purity of isolated cell fractions confirmed by expression of leukocyte marker Ptprc (encoding CD45) (c) or epithelial marker Cdh1 (encoding E-cadherin) (d). (e-h) Cells isolated from small intestinal tissues of rotavirus infected suckling mice at day 4 following rotavirus infection were stained for CD45 (hematopoietic cells) and EpCAM (IECs). The dot plot represents the gating strategy to isolate highly purified hematopoietic cells (CD45+ EpCAM-) or IECs (CD45- EpCAM+) (see Fig. 2c). (f-h) ISG expression in highly purified IECs (CD45− EpCAM+) and hematopoietic cells (CD45+ EpCAM−) was analyzed by RT-qPCR using RNA obtained from day 1 infected suckling mice. All RT-qPCR data are shown as fold (2-∆Ct) relative to Hprt. (a-h) Mean ± SEM. (a-d) n ≥ 6. (f-h) n ≥ 3. Data are representative of three (a-d) and two (f-h) independent experiments. (a, c, d, f-h) Two-way ANOVA. (b) Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary Figure 5 Upregulation of IL-22 expression by CCR6+ RORγt+ ILC3 cells following rotavirus infection does not require IFN-λ signaling.

(a-e) Groups of suckling or adult mice of the indicated genotypes were orally infected with rotavirus and samples were collected at the indicated days following infection. Uninfected mice received PBS (mock) injections. (a) Il22 expression (RT-qPCR) in IECs and LPLs from suckling mice at day 1 following infection. (b-d) LPLs from the small intestine of suckling mice at day 1 following infection were stained with the indicated antibodies. Histograms (c) represent analysis of RORγt expression of gated IL-22− cells (grey) or IL-22+ (open) cells of all CD19− CD11b− cells (b). Contour plots (d) represent CCR6 and RORγt expression by IL-22− or IL-22+ cells of all CD19− CD11b− cells. (e) Rotavirus titers measured by ELISA on day 4 post infection in adult mice of the indicated genotypes. (f-i) Suckling mice were infected with rotavirus and expression of the indicated genes in small intestinal tissue was determined by RT-qPCR at day 1 following infection. Small intestinal sections were stained as indicated (i). Scale bars, 50 μm. All RT-qPCR data are shown as fold (2-∆Ct) relative to Hprt. (a-h) Mean ± SEM; n ≥ 5. Data are representative of three (a-d, f-i) and two (e) independent experiments (a, f-h) Two-way ANOVA. (e) One-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (P > 0.05). ND, not detectable (or not able to be displayed on the scale here).

Supplementary Figure 6 IL-1α is required for rotavirus-induced expression of IL-22 by RORγt+ ILC3 cells.

Groups of suckling mice were orally infected with rotavirus. (a, b) Expression of Il1b and Il23a in IECs and LPLs from wildtype mice at day 1 following infection was analyzed by RT-qPCR. (c) Ifnl2/3 expression at day 1 following infection of the indicated mouse strains was analyzed by RT-qPCR using RNA from small intestinal tissue. (d-i) Twelve hours prior to infection, wildtype mice received intraperitoneal injections with 100 μg of the indicated neutralizing antibody. Il22 expression at day 1 following infection was analyzed by RT-qPCR using RNA from small intestinal tissue (d). Percentage (e) and absolute numbers (f) of IL-22+ cells among CD11b− CD19− CD3− RORγt+ lamina propria leukocytes at day 1 after infection. Rotavirus titers in small intestinal tissue determined by RT-qPCR (g). (h-i) Expression of IL-1-inducible genes by RT-qPCR. All RT-qPCR data are shown as fold (2-∆Ct) relative to Hprt. (a-i) Mean ± SEM. (a, b) n = 5. (c) n ≥ 3. (d-i) n ≥ 4. Data are representative of three (a-c) and two (e-i) independent experiments. (a-c, e-g) Two-way ANOVA. (d, h, i) One-way ANOVA. *P < 0.05; **P < 0.01; NS, not significant (P > 0.05). UI, uninfected mice. ND, not detectable (or not able to be displayed on the scale here).

Supplementary Figure 7 IL-22 and IFN-λ act synergisitcally for ISG induction.

Gene expression analysis by RT-qPCR of isolated small intestinal epithelial cells from rotavirus infected animals sacrificed at day 1 (a) or day 4 (b) after infection. The expression of 114 genes (ISGs, IL-22-regulated genes, STAT1 and/or STAT3 targets and genes involved in tissue repair) was analysed. Genes are divided as ISG and non-ISGs based on the Interferome online database (http://interferome.its.monash.edu.au) and organized according to the fold induction upon rotavirus infection in WT mice. Heat maps depict the ratio of gene expression as indicated. Thresholds of the color code are indicated at the bottom. (a, b) Left row indicates the ratio of gene expression between infected (I) vs. uninfected (UI) wildtype (WT) mice, middle row indicates the ratio of gene expression ratio between infected Il22−/− vs. infected WT mice and the right column indicates gene expression ratio between infected Ifnlr1−/− vs. infected WT mice. (c, d) Gene expression analysis by RT-qPCR in isolated IECs from rotavirus infected or mock infected animals. (e-l) The intestinal epithelial cell lines IEC6 (e-g) and Caco2 (i-l) were stimulated for 16 hours with the indicated amounts of mouse (for IEC6) or human (for Caco2) IFN-λ2 alone or with 100 ng/ml of mouse or human IL-22. Expression of the indicated ISGs was evaluated by RT-qPCR. All RT-qPCR data are shown as fold (2-∆Ct) relative to Hprt. (c-k) Mean ± SEM. (a, b) n ≥ 5. (c, d) n ≥ 4. (e-k) n = 4. Data are representative of two (a, b) or three (c-l) independent experiments. (e-l) Student’s t test. (c, d) Two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (P > 0.05). ND, not detectable (or not able to be displayed on the scale here).

Supplementary Figure 8 IL-22 and IFN-λ act together for the phosphorylation of STAT1.

(a-f) Specific deletion of STAT3 in intestinal epithelial cells. Analysis of the expression of Stat3 (a, b) and the indicated Stat1 (c) and Stat3 (d-f) target genes by RT-qPCR in IECs and LPLs isolated from adult wildtype and Stat3∆IEC mice. Stat3 transcripts were determined by using primer pairs outside (a) and inside (b) the deleted region. (g) Wildtype mice were subcutaneously injected with 1 μg of IFN-λ2 and/or 1 μg of IL-22 and sacrificed 1 hour post injection when STAT phosphorylation was assessed in small intestinal tissue. (h-i) Phospho-STAT analysis in intestinal epithelial cell lines IEC6 (h) and Caco2 (i) stimulated as indicated with 20 ng/ml of murine or human IFN-λ2 and 100 ng/ml of murine or human IL-22 for 30 min. For quantification, Image Studio Software for Odyssey® Fc System was used to image the blots and quantify the signal. Phospho-STAT values were normalized to the respective non-phosphorylated STATs. IFN-λ-stimulated sample signal was set to 100% for pSTAT1/STAT1 signal analysis and IL-22-stimulated sample signal was set to 100% in pSTAT3/STAT3 signal analysis. Data from three different experiments were pooled. (j) Groups of suckling mice from the indicated mouse strains were orally infected with rotavirus. Expression of the indicated genes in small intestinal tissue from suckling mice was determined by RT-qPCR at day 4 following infection. (k) Gene knockdown for the indicated STAT proteins was performed in IEC6 cells using siRNAs. Cells were stimulated with 100 ng/ml of IL-22 and/or IFN-λ2 for 16 hours. Gene expression was evaluated by RT-qPCR (upper graph) or Western blotting (below). All RT-qPCR data are shown as fold (2-∆Ct) relative to Hprt. Mean ± SEM. (a-f) n ≥ 3. (g-i) n = 4. (j) n ≥ 3. (k) n = 4. Data are representative of three (a-j) or four (k) independent experiments. (a-j) One-way ANOVA. (k) Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant (P > 0.05). ND, not detectable (or not able to be displayed on the scale here).

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Hernández, P., Mahlakõiv, T., Yang, I. et al. Interferon-λ and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection. Nat Immunol 16, 698–707 (2015). https://doi.org/10.1038/ni.3180

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