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GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses

Nature Immunology volume 15, pages 63–71 (2014)Cite this article

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Abstract

Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I–like receptors. Activation of GEF-H1 controls RIG-I–dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-β expression in macrophages. Generation of Arhgef2−/− mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.

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Figure 1: GEF-H1 is essential for RLR-mediated production of IFN-β.
Figure 2: GEF-H1 enhances TBK1-dependent IRF3 activation.
Figure 3: GEF-H1 controls microtubule-dependent induction of IFN-β expression.
Figure 4: Polarized microtubules are required for the activation of GEF-H1 and interaction with TBK1.
Figure 5: GEF-H1 mediates host defenses against ssRNA viruses.
Figure 6: GEF-H1 is required for the control of influenza A infection.

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Acknowledgements

This work was supported by grants AI-093588 (H.-C.R.), DK-068181 (H.-C.R.), DK-033506 (H.-C.R.), DK-043351 (H.-C.R. and C.T.) and DK-52510 (C.T.) from the US National Institutes of Health.

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  1. Hao-Sen Chiang and Yun Zhao: These authors contributed equally to this work.

Authors and Affiliations

  1. Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Hao-Sen Chiang, Yun Zhao, Joo-Hye Song, Song Liu, Megha Basavappa, Kate L Jeffrey & Hans-Christian Reinecker

  2. Division of Immunology, Beth Israel Deaconess Medical Center and Center for the Study of Inflammatory Bowel Disease, Harvard Medical School, Boston, Massachusetts, USA

    Ninghai Wang & Cox Terhorst

  3. Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA

    Arlene H Sharpe

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Contributions

H.-S.C., Y.Z., J.-H.S., S.L., N.W. and M.B. carried out experiments; K.J., A.H.S. and C.T. supported the development of research tools and mice; K.L.J. provided virus and advised on virus infection experiments; H.-C.R. conceived of and directed all research, and along with H.-S.C. prepared the manuscript.

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Correspondence to Hans-Christian Reinecker.

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Integrated supplementary information

Supplementary Figure 1 Normal distribution of T cells, B cells and mononuclear phagocytes in mesenteric lymph nodes and spleens of Arhgef2−/− mice.

Flow cytometry analysis of T cells, B cells, and mononuclear phagocytes in mesenteric lymph nodes (MLN) and spleens of C57BL/6 wild-type, Arhgef2+/–, and Arhgef2−/− mice (n=3 per group).

Supplementary Figure 2 TLR4-mediated IFN-β and proinflammatory cytokine production in wild-type and GEF-H1–deficient macrophages.

(a) IFN-β in supernatants of WT or Arhgef2−/− macrophages incubated with LPS for 24 h. (b) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT, Arhgef2+/– and Arhgef2−/− macrophages in the presence of LPS (0.2 μg/ml) for 24 h. (c) Quantitative RT-PCR analysis of Il6 and Tnf mRNA expression in WT, Arhgef2+/– and Arhgef2−/− macrophages incubated for 24 h with LPS (0.2 μg/ml). Results are presented relative to the expression of GAPDH. UT, untreated. ND, not detectable. Data are from one experiment representative of three independent experiments and reflect mean ± SD.

Supplementary Figure 3 Poly(I:C) uptake, microtubule and actin networks in C57BL/6 wild-type and Arhgef2−/− macrophages.

(a) Confocal microscopic images of wild-type and Arhgef2−/− macrophages in the presence of 0.5 μg/ml poly(I:C)-Rhodamine. (b) Flow cytometry analysis of wild-type (blue line) and Arhgef2−/− (red line) macrophages following an incubation with Rhodamine-labeled poly(I:C) (0.5 μg/ml). Data are from one experiment representative of three independent experiments. (c, d) Confocal images of α-tubulin (c) and F-actin (d) in macrophages form Arhgef2−/− mice and their wild-type littermate control in the presence of 0.5 μg/ml poly(I:C) for 3 h. (e) Confocal images of nocodazole-treated (10μM, 40 minutes) wild-type and Arhgef2−/− macrophages in the presence of 0.5 μg/ml poly(I:C)-Rhodamine for 3 h. UT, untreated. Confocal images are from one experiment representative of three independent experiments.

Supplementary Figure 4 GEF-H1–deficient macrophages are susceptible to Influenza A and VSV infection.

(a) Viral replication was visualized by confocoal microscopy of WT or Arhgef2-/- macrophages 12 h after infection (MOI=1) with a recombinant influenza A/PR/8/34 carrying RNA for an NS1-GFP fusion protein. At least 200 cells of WT or Arhgef2−/− macrophages were imaged. Mean fluorescence intensity of each cell is shown in the scatter plot. (b) Plaque assays of the supernatants of VSV-infected C56BL/6 WT and GEF-H1-deficient macrophages. The bar graph represents the viral titers in the supernatants of macrophages after VSV infection (MOI=1). (c) ELISA of IFN-β in the supernatants of VSV-infected C56BL/6 WT and GEF-H1-deficient macrophages (MOI=1). (d) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in VSV-infected C56BL/6 WT and MAVS-deficient macrophages (MOI=1). Results are presented relative to the expression of GAPDH. ND, not detectable. Data reflect mean ± SD. **, P < 0.01, *, P < 0.05 (Student's t-test). (e) Immunoblot analysis of STAT1 phosphorylation (tyrosine 701) in C57BL/6 wild-type or Arhgef2-/- bone marrow-derived macrophages treated with IFN-β (500 U/ml) for 10 minutes, 30 minutes, 4 hours or 16 hours. β-actin serves as a loading control. Data are from one experiment representative of three independent experiments

Supplementary Figure 5 GEF-H1 is dispensable for Ifnb1 and Tnf mRNA induction during S. typhimurium infection.

(a, b) Quantitative RT-PCR analysis of (a) Ifnb1 mRNA and (b) Tnf expression in S. typhimurim-infected (MOI=10) C56BL/6 WT and GEF-H1-deficient macrophages. (c) Intracellular CFU was measured by a gentamicin protection assay in WT and GEF-H1 deficient macrophages infected with S. typhimurim at MOI=10. Results are presented relative to the expression of GAPDH. ND, not detectable (Student's t-test). Data reflect mean ± SD. Data are from one experiment representative of three independent experiments.

Supplementary Figure 6 Graphical abstract.

GEF-H1 emerges as a critical signaling intermediate for cytoplasmic recognition of nucleic acid through the MAVS and STING pathway. GEF-H1 is required for sensing of 5'-triphosphate dsRNA by RIG-I and poly(I:C) by MDA5, and mediates IFN-β expression upon DDX41 activation. GEF-H1 elicits this selective function through its nucleotide exchange activity after release from a microtubule bound reservoir by dephosphorylation. GEF-H1 subsequently becomes part of TBK1 containing signaling complexes that mediate phosphorylation of IRF3 and subsequent Ifnb1 promoter induction. Disruption of the microtubule network prevents activation of GEF-H1 and MAVS signaling. In contrast, deletion of GEF-H1 or disruption of microtubule function in macrophages does not prevent NF-κB activation by surface and endosomal TLRs involved in the detection of viral RNA and viral glycoproteins. GEF-H1 is required for IFN-β secretion by macrophages in response to infection by EMCV and influenza A and controls influenza A virus infection in mice.

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Chiang, HS., Zhao, Y., Song, JH. et al. GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses. Nat Immunol 15, 63–71 (2014). https://doi.org/10.1038/ni.2766

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