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

Chiang HS, Zhao Y, Song JH, Liu S, Wang N, Terhorst C, Sharpe AH, Basavappa M, Jeffrey KL, Reinecker HC - Nat. Immunol. (2013)

Bottom Line: 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.Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.

View Article: PubMed Central - PubMed

Affiliation: 1] Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. [2].

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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GEF-H1 is essential for RLR-mediated IFN-β production. (a) Schematic diagram of the gene-trap vector and its site of insertion into Arhgef2 gene. GEF-H1 mRNA expression in wild-type (WT), Arhgef2 heterozygous (+/-), and Arhgef2 homozygous (−/−) mice was determined by RT-PCR. (b) Immunoblot analysis of GEF-H1 expression in WT and Arhgef2−/− macrophages. (c, d) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT and Arhgef2−/− macrophages incubated for 16 h with transfected (c) 5′ppp-dsRNA (0.5 μg/ml), HMW poly(I:C) (0.5 μg/ml) or c-di-GMP (10 μg/ml) and (d) Pam3CSK4 (1 μg/ml), HKLM (108 cells/ml), LPS (0.2 μg/ml), Flagellin (1 μg/ml), FSL-1 (1 μg/ml), ssRNA (1 μg/ml) complexed with Lipofectamine (lipofect), and CpG ODN1826 (5 μM) complexed with Lipofectamine. (e-g) IFN-β secretion by WT and Arhgef2−/− macrophages in the presence of 5′ppp-dsRNA, LMW poly(I:C) or HMW poly(I:C) (0.5 μg/ml) complexed with or without Lipofectamine for 24 h. (h) Immunoblot analysis of GEF-H1 expression in WT macrophages treated with transfected 5′ppp-dsRNA (0.5 μg/ml) for 1 or 3 h. (i) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT, Arhgef2+/- or Arhgef2−/− macrophages in the presence of HMW poly(I:C) (0.5 μg/ml) for 24 h. (j) Quantitative RT-PCR analysis of Il6 and Tnf mRNA expression in WT and Arhgef2−/− macrophages incubated for 24 h with transfected 5′ppp-dsRNA (0.5 μg/ml). Results are presented relative to the expression of GAPDH. UT, untreated. ND, not detectable. **, P < 0.01 (Student’s t-test). Data are from one experiment representative of three independent experiments. Error bars indicate mean ± SD.
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Figure 1: GEF-H1 is essential for RLR-mediated IFN-β production. (a) Schematic diagram of the gene-trap vector and its site of insertion into Arhgef2 gene. GEF-H1 mRNA expression in wild-type (WT), Arhgef2 heterozygous (+/-), and Arhgef2 homozygous (−/−) mice was determined by RT-PCR. (b) Immunoblot analysis of GEF-H1 expression in WT and Arhgef2−/− macrophages. (c, d) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT and Arhgef2−/− macrophages incubated for 16 h with transfected (c) 5′ppp-dsRNA (0.5 μg/ml), HMW poly(I:C) (0.5 μg/ml) or c-di-GMP (10 μg/ml) and (d) Pam3CSK4 (1 μg/ml), HKLM (108 cells/ml), LPS (0.2 μg/ml), Flagellin (1 μg/ml), FSL-1 (1 μg/ml), ssRNA (1 μg/ml) complexed with Lipofectamine (lipofect), and CpG ODN1826 (5 μM) complexed with Lipofectamine. (e-g) IFN-β secretion by WT and Arhgef2−/− macrophages in the presence of 5′ppp-dsRNA, LMW poly(I:C) or HMW poly(I:C) (0.5 μg/ml) complexed with or without Lipofectamine for 24 h. (h) Immunoblot analysis of GEF-H1 expression in WT macrophages treated with transfected 5′ppp-dsRNA (0.5 μg/ml) for 1 or 3 h. (i) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT, Arhgef2+/- or Arhgef2−/− macrophages in the presence of HMW poly(I:C) (0.5 μg/ml) for 24 h. (j) Quantitative RT-PCR analysis of Il6 and Tnf mRNA expression in WT and Arhgef2−/− macrophages incubated for 24 h with transfected 5′ppp-dsRNA (0.5 μg/ml). Results are presented relative to the expression of GAPDH. UT, untreated. ND, not detectable. **, P < 0.01 (Student’s t-test). Data are from one experiment representative of three independent experiments. Error bars indicate mean ± SD.

Mentions: To define the role of GEF-H1 in innate immune activation by foreign nucleic acids, we generated GEF-H1-deficient mice using C57BL/6 embryonic stem cells with a gene-trap insertion between exons 4 and 5 of Arhgef2 on mouse chromosome 3 that prevents GFH-H1 mRNA (Fig. 1a) and protein expression (Fig. 1b). These mice had normal T cell, B cell and mononuclear phagocyte numbers in spleen and lymph nodes (Supplementary Fig. 1).


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

Chiang HS, Zhao Y, Song JH, Liu S, Wang N, Terhorst C, Sharpe AH, Basavappa M, Jeffrey KL, Reinecker HC - Nat. Immunol. (2013)

GEF-H1 is essential for RLR-mediated IFN-β production. (a) Schematic diagram of the gene-trap vector and its site of insertion into Arhgef2 gene. GEF-H1 mRNA expression in wild-type (WT), Arhgef2 heterozygous (+/-), and Arhgef2 homozygous (−/−) mice was determined by RT-PCR. (b) Immunoblot analysis of GEF-H1 expression in WT and Arhgef2−/− macrophages. (c, d) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT and Arhgef2−/− macrophages incubated for 16 h with transfected (c) 5′ppp-dsRNA (0.5 μg/ml), HMW poly(I:C) (0.5 μg/ml) or c-di-GMP (10 μg/ml) and (d) Pam3CSK4 (1 μg/ml), HKLM (108 cells/ml), LPS (0.2 μg/ml), Flagellin (1 μg/ml), FSL-1 (1 μg/ml), ssRNA (1 μg/ml) complexed with Lipofectamine (lipofect), and CpG ODN1826 (5 μM) complexed with Lipofectamine. (e-g) IFN-β secretion by WT and Arhgef2−/− macrophages in the presence of 5′ppp-dsRNA, LMW poly(I:C) or HMW poly(I:C) (0.5 μg/ml) complexed with or without Lipofectamine for 24 h. (h) Immunoblot analysis of GEF-H1 expression in WT macrophages treated with transfected 5′ppp-dsRNA (0.5 μg/ml) for 1 or 3 h. (i) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT, Arhgef2+/- or Arhgef2−/− macrophages in the presence of HMW poly(I:C) (0.5 μg/ml) for 24 h. (j) Quantitative RT-PCR analysis of Il6 and Tnf mRNA expression in WT and Arhgef2−/− macrophages incubated for 24 h with transfected 5′ppp-dsRNA (0.5 μg/ml). Results are presented relative to the expression of GAPDH. UT, untreated. ND, not detectable. **, P < 0.01 (Student’s t-test). Data are from one experiment representative of three independent experiments. Error bars indicate mean ± SD.
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Related In: Results  -  Collection

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Figure 1: GEF-H1 is essential for RLR-mediated IFN-β production. (a) Schematic diagram of the gene-trap vector and its site of insertion into Arhgef2 gene. GEF-H1 mRNA expression in wild-type (WT), Arhgef2 heterozygous (+/-), and Arhgef2 homozygous (−/−) mice was determined by RT-PCR. (b) Immunoblot analysis of GEF-H1 expression in WT and Arhgef2−/− macrophages. (c, d) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT and Arhgef2−/− macrophages incubated for 16 h with transfected (c) 5′ppp-dsRNA (0.5 μg/ml), HMW poly(I:C) (0.5 μg/ml) or c-di-GMP (10 μg/ml) and (d) Pam3CSK4 (1 μg/ml), HKLM (108 cells/ml), LPS (0.2 μg/ml), Flagellin (1 μg/ml), FSL-1 (1 μg/ml), ssRNA (1 μg/ml) complexed with Lipofectamine (lipofect), and CpG ODN1826 (5 μM) complexed with Lipofectamine. (e-g) IFN-β secretion by WT and Arhgef2−/− macrophages in the presence of 5′ppp-dsRNA, LMW poly(I:C) or HMW poly(I:C) (0.5 μg/ml) complexed with or without Lipofectamine for 24 h. (h) Immunoblot analysis of GEF-H1 expression in WT macrophages treated with transfected 5′ppp-dsRNA (0.5 μg/ml) for 1 or 3 h. (i) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in WT, Arhgef2+/- or Arhgef2−/− macrophages in the presence of HMW poly(I:C) (0.5 μg/ml) for 24 h. (j) Quantitative RT-PCR analysis of Il6 and Tnf mRNA expression in WT and Arhgef2−/− macrophages incubated for 24 h with transfected 5′ppp-dsRNA (0.5 μg/ml). Results are presented relative to the expression of GAPDH. UT, untreated. ND, not detectable. **, P < 0.01 (Student’s t-test). Data are from one experiment representative of three independent experiments. Error bars indicate mean ± SD.
Mentions: To define the role of GEF-H1 in innate immune activation by foreign nucleic acids, we generated GEF-H1-deficient mice using C57BL/6 embryonic stem cells with a gene-trap insertion between exons 4 and 5 of Arhgef2 on mouse chromosome 3 that prevents GFH-H1 mRNA (Fig. 1a) and protein expression (Fig. 1b). These mice had normal T cell, B cell and mononuclear phagocyte numbers in spleen and lymph nodes (Supplementary Fig. 1).

Bottom Line: 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.Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.

View Article: PubMed Central - PubMed

Affiliation: 1] Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. [2].

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.

Show MeSH
Related in: MedlinePlus