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An innate antiviral pathway acting before interferons at epithelial surfaces

Nature Immunology volume 17, pages 150–158 (2016)Cite this article

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Abstract

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.

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Figure 1: Vaginal HSV-2 infection induces early production of CXCR3 chemokines.
Figure 2: CXCR3 is essential for early antiviral defense before the requirement for the IFN-α/β system.
Figure 3: Early CXCL10 production is derived from vaginal epithelial cells.
Figure 4: Early CXCL10 production is independent of known innate sensors recognizing HSV.
Figure 5: Viral entry and early replication events are not required for induction of CXCL10 expression.
Figure 6: Disturbance of mucosal integrity induces IFN-independent CXCL10 expression.
Figure 7: HSV-2 infection stimulates early recruitment of neutrophils in a CXCR3-dependent manner.

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Acknowledgements

We thank K.S. Petersen and I.M. Poulsen for technical assistance and the AU FACS Core facility for technical help. Supported by The Danish Medical Research Council (12-124330 to S.R.P.; 1331-00133B to H.H.W.), the Novo Nordisk Foundation (S.R.P.), the Lundbeck Foundation (R34-3855 to S.R.P.), Aarhus University Research Foundation (S.R.P.), the EU FP7 Mobilex program (1333-00090A to M.K.T.), the US National Institutes of Health (AI065309 to B.C.H.), Faculty of Health Sciences, AU (M.B.I.), the Danish National Research Foundation (DNRF107 to H.H.W.), the Excellence Programme for Interdisciplinary Research (CDO2016 to H.H.W.), the German Research Foundation (DFG, SFB 1054 to J.R.) and the European Research Council (FP7, grant agreement 322865 to J.R.).

Author information

Author notes

  1. Line S Reinert and Martin K Thomsen: These authors contributed equally to this work.

  2. Christian K Holm and Søren R Paludan: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Biomedicine, University of Aarhus, Aarhus, Denmark

    Marie B Iversen, Line S Reinert, Martin K Thomsen, Ramya Nandakumar, Thaneas Prabakaran, Sine K Kratholm, Steen V Petersen, Sebastian Frische, Christian K Holm & Søren R Paludan

  2. Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark

    Marie B Iversen, Line S Reinert, Martin K Thomsen, Ramya Nandakumar, Thaneas Prabakaran, Sine K Kratholm, Christian K Holm & Søren R Paludan

  3. Department of Cellular and Molecular Medicine, Centre for Glycomics, University of Copenhagen, Copenhagen, Denmark

    Ieva Bagdonaite, Sergey Y Vakhrushev & Hans H Wandall

  4. Department of Pediatrics and Microbiology, Albert Einstein College of Medicine, New York, USA

    Natalia Cheshenko & Betsy C Herold

  5. Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology, Warsaw, Poland

    Malgosha Krzyzowska

  6. Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia, USA

    Fernando Ruiz-Perez

  7. Institute for Medical Immunology, Université Libre de Bruxelles, Gosselies, Belgium

    Stanislas Goriely

  8. Department of Biostatistics, University of Aarhus, Aarhus, Denmark

    Bo Martin Bibby

  9. Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden

    Kristina Eriksson

  10. Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany

    Jürgen Ruland

  11. Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark

    Allan R Thomsen

Authors
  1. Marie B Iversen
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  2. Line S Reinert
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  8. Sergey Y Vakhrushev
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Contributions

M.B.I., C.K.H. and S.R.P. conceived of the study; M.B.I., L.S.R., M.K.T., I.B., R.N., N.C., T.P., S.Y.V., M.K. and S.K.K. performed experiments; M.B.I., B.M.B., H.H.W., S.F., C.K.H. and S.R.P. analyzed data; F.R.-P., S.G., K.E., J.R., A.R.T., B.C.H., S.V.P. and H.H.W. provided reagents and intellectual guidance; and M.B.I. and S.R.P. wrote the paper.

Corresponding author

Correspondence to Søren R Paludan.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Production of cytokines and chemokines after vaginal HSV-2 infection.

C57BL/6 mice were (a) treated intravaginally with 20 µL of PBS or infected with 6.7x104 pfu of HSV-2 in the same volume, or were (b) infected intravaginally with 6.7x104 pfu of HSV-2 produced in murine neurons. Vaginal washes were isolated 24 h p.i. and levels of CXCL10 were determined by ELISA. (c-k) C57BL/6 mice were infected intravaginally with 6.7x104 pfu of HSV-2. Vaginal washes were isolated at the indicated time points p.i., and analyzed for levels of (c) CCL2, (d) CCL5, (e) CXCL1, (f) IL-1β, (g) IL-10, (h) IL-12 p40, (i) IL-15, (j) IL-33, (k) TNF-α. Data are presented as means +/- st.dev. n = 6-8 per group. ** 0.001<p<0.01; *** p<0.001; ns, not statistically significant.

Supplementary Figure 2 An essential role for CXCR3 in early control of corneal HSV-1 infection.

C57BL/6 and Cxcr3-/- mice were infected in the cornea with 2x106 pfu of HSV-1 (McKrae). (a). Eye balls were isolated 24 h p.i. and analyzed for the levels of CXCL10 and bioactive type I IFN. n=8 per group. (b) Total RNA isolated from homogenized eyes of mice infected for 24 h was analyzed for gB and Gapdh mRNA by RT-qPCR. Data are presented as means +/- standard deviation n=5-8 per group. * 0.01<p<0.05, ** 0.001<p<0.01, *** p<0.001.

Supplementary Figure 3 Type II and III IFNs are redundant for innate control of genital HSV-2 infection.

C57BL/6, Il28ra-/-, and Ifngr-/- mice were infected intravaginally with 6.7x104 pfu of HSV-2. (a, c) Vaginal washes were isolated on day 1 and 2 points p.i., and analyzed for viral load by plaque assay on Vero cells. Data points represent individual mice (n= 5-10 per group). (b, d) The mice were scored daily for clinical signs of disease. Data are presented as mean disease score +/- standard deviation. ns, not statistically significant.

Supplementary Figure 4 Re-establishment of a mucosal layer by vaginal epithelial cells in vitro.

Vaginal tissue was excised from female mice and treated with Collagenase and Dispase as described in the method section. The subepithelium was removed, and the epithelial cell layer was sat in culture for 24 h. (a) Total RNA was isolated and analyzed for expression of Muc1, 2, 5b, and Gapdh. For comparison, RNA from murine bone-marrow-derived dendritic cells (BMDCs) was analyzed in parallel. Data are presented as means +/- st.dev. n=3 per group. (b) Periodic acid–Schiff staining of vaginal epithelial cells in culture after the treatment described above. As control is included a staining of a section from vaginal tissue treated with medium not supplemented with Collagenase/Dispase. The periodic acid–Schiff stains glycans, which are abundantly present in the mucus layer (red).

Supplementary Figure 5 Expression of PRRs in the vagina of mice.

(a) RNA was isolated from vaginas and bone-marrow-derived dendritic cells from C57BL/6 mice. Levels of Tlr2, Tlr3, Tlr9, Rig-I, Mda5, Mb21d1 (cGAS) and β-actin mRNA were measured by RT-qPCR. For each PRR, data was normalized to β-actin and expression levels are presented as mean of the ratio between vaginal epithelial cells and dendritic cells +/- standard deviation. n = 3. (b) The Ct values from the PCR data set on dendritic cells used for calculation of expression ratios.

Supplementary Figure 6 Development of disease after infection with infectious or attenuated HSV-2.

C57BL/6 mice were infected intravaginally with 6.7x104 pfu of infectious or UV-inactivated HSV-2 or HSV-2 ΔgD-R (revertant) or HSV-2 ΔgD. (a) On subsequent days, the animals were scored for clinical signs of disease (n = 8). Data are presented as mean disease score +/- st. dev. (b) Tissue sections of the vagina from mice left untreated or infected for 24 h with HSV-2 or HSV-2 ΔgD were stained with anti-HSV-2. Scale bar, 100 μm.

Supplementary Figure 7 Characterization of HSV-2 devoid of O-linked sugars.

(a-d) HSV-2 stocks were treated as indicated for 30 min at 37oC and subjected to (a) SDS-PAGE and Western blotting using antibodies against gB and gD, (b, d) infection on MEFs followed by confocal microscopy (stainings: anti-VP5, anti-STING, and DAPI) and (c) viral plaque titration on Vero cells. (e) EdC-labeled HSV-2 grown in parental or Cosmc KO cells were incubated at 37 °C for 30 min. DNA and capsid were visualized with CLICK chemistry and anti-VP5 respectively. Data are presented as mean % CLICK+VP5- foci of the total number of counted VP5+ foci +/- st.dev. (f-g) C57BL/6 mice were infected intravaginally with 6.7x104 pfu of (f) HSV-2, which has been treated with buffer or an enzyme mix cleaving O-linked sugars before infection, or (g) HSV-2 grown in either parental or Cosmc KO HaCaT cells. Vaginal washes were harvested 48 hours p.i. for and levels of CXCL10 were measured (n= 8). (h) Virus titer for preparations of HSV-2 grown in parental or Cosmc KO HaCaT cells. Data are presented as means +/- st.dev. * 0.01<p<0.05, ** 0.001<p<0.01, *** p<0.001. ns, not statistically significant.

Supplementary Figure 8 Identification of NK cells and neutrophils by flow cytometry.

C57BL/6 mice were infected intravaginally with 6.7x104 pfu of HSV-2 or left untreated. 24 hours p.i., single-cell leukocytes were isolated from the vaginal tissue and analyzed by flow cytometry (a) Ly6G+CD11b+ cells, and (b) CD45+ NK1.1+ cells. The plots represent one infected C57BL/6 mouse and one naive C57BL/6 mouse. (c) Vaginal washes were isolated 24 hours p.i. and analyzed for levels of CXCL10. Data are presented as means +/- standard deviation. n = 8 per group. ns, not statistically significant.

Supplementary information

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Supplementary Figures 1–8 and Supplementary Table 2 (PDF 1784 kb)

Supplementary Table 1

Supplementary information on mass spectrometry analysis of HSV-2 O-glycoproteome (XLSX 122 kb)

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Iversen, M., Reinert, L., Thomsen, M. et al. An innate antiviral pathway acting before interferons at epithelial surfaces. Nat Immunol 17, 150–158 (2016). https://doi.org/10.1038/ni.3319

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