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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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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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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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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DOI: https://doi.org/10.1038/ni.2766