Combating herpesvirus encephalitis by potentiating a TLR3–mTORC2 axis (original) (raw)

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Further details of the experimental procedures are provided in the Life Sciences Reporting Summary. The data that support the findings of the study are available from the corresponding author upon request.

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Acknowledgements

We thank P.W. Kincade for critical reading of the manuscript; K. Kontani (Meiji Pharmaceutical University) for rabbit polyclonal antibody to mouse Arl8; T. Kitamura (The University of Tokyo) for retroviral pMXpuro vectors; S. Akira (Osaka University) for C57BL/6 _Tlr3_–/– mice; and N. Tanimura, C. Sato and Y. Motoi for advice on the experiments. Supported by Grant-in-Aid for Scientific Research (S) (16H06388 to K.M.); Grant-in-Aid for Scientific Research (B) (17H04080 to Y.K.); Grant-in-Aid for Scientific Research (C) (16K08827 and 17K09369 to T.I.; 17K08851 to J.A.; and 18K07169 to R.F.); Grant for Joint Research Project of the Institute of Medical Science; the US National Institutes of Health (NIAID/AI079336 to G.N.B.); the University of Tokyo; a Grant-in-Aid for Young Scientists (A) (17H05069 to A.K.; and 18K15133 to Y.M.); Grant-in-Aid for Challenging Exploratory Research (17K19548 to K.M.; and 17K19549 to A.K.); Grant-in-Aid for Scientific Research on Innovative Areas (16H06433, 16H06429 and 16K21723 to Y.K.; 18H04666 to K.M.; 18H04968 to A.K.; and 18H04856 to S.-I.S.); and a contract research fund from the Japan Initiative for Global Research Network on Infectious Diseases (JP18fm0108006 to Y.K.).

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Author notes

1. These authors contributed equally: Ryota Sato, Akihisa Kato.

Authors and Affiliations

1. Division of Innate Immunity, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
   Ryota Sato, Shin-Ichiroh Saitoh, Takuma Shibata, Yusuke Murakami, Ryutaro Fukui, Kaiwen Liu, Yun Zhang & Kensuke Miyake
2. Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
   Akihisa Kato, Jun Arii & Yasushi Kawaguchi
3. Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
   Akihisa Kato, Jun Arii & Yasushi Kawaguchi
4. Division of Neuronal Network, Department of Basic Medical Sciences, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
   Takahiko Chimura & Toshiya Manabe
5. Department of Biochemistry, Faculty of Pharmaceutical Sciences, Doshisha Women’s College, Kyoto, Japan
   Ge-Hong Sun-Wada
6. Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan
   Yoh Wada
7. Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
   Tsuneo Ikenoue
8. UM/Sylvester Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA
   Glen N. Barber

Authors

1. Ryota Sato
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2. Akihisa Kato
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3. Takahiko Chimura
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4. Shin-Ichiroh Saitoh
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5. Takuma Shibata
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6. Yusuke Murakami
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7. Ryutaro Fukui
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8. Kaiwen Liu
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9. Yun Zhang
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10. Jun Arii
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11. Ge-Hong Sun-Wada
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12. Yoh Wada
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13. Tsuneo Ikenoue
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14. Glen N. Barber
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15. Toshiya Manabe
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16. Yasushi Kawaguchi
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17. Kensuke Miyake
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Contributions

R.S., A.K., T.C., S.-I.S., T.S., Y.M, R.F., K.L, Y.Z. and J.A. conducted the experiments; G.-H.S.-W., Y.W., T.I., and G.N.B. provided reagents; T.M. and Y.K. discussed the results; and R.S. and K. M. wrote the manuscript.

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Correspondence toYasushi Kawaguchi or Kensuke Miyake.

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Supplementary Figure 1 TLR3 responses in primary neurons and fibroblasts.

(a) WT mice (n = 8 per group) were intracranially infected with indicated doses of HSV-1 and mice survival was evaluated. (b) Primary neurons, astrocytes, and microglia were stained with antibodies to a neuron marker NeuN, an astrocyte marker GFAP, and a microglia marker CD11b (red histograms). Black histograms show staining with the second reagent alone. (c) Primary neurons were left unstimulated or stimulated with poly(I:C) (PIC) at 25 μg/ml, cGAMP at 7 μM with Lipofectoamine or ISD 1 μg/ml with Lipofectoamine for 24 h. Real-time PCR was conducted to measure Ccl5 mRNA. These results are normalized by HPRT mRNA and represented by the mean value ± s. d. of triplicate wells. Statistical analysis was performed by the two-tailed Student’s t test. ***P < 0.001. (d) WT and Tlr3 –/– primary neurons were left uninfected (U) or infected (HSV-1) with HSV-1 at an MOI of 10. Real-time PCR was conducted to measure Ifnb1 mRNA. The results were analyzed as in (c). (e) Membrane permeabilized staining of TLR3 and Unc93B1-Flag in NIH3T3 cells or those expressing the indicated molecules (red histograms). Black histograms indicate labeling with the secondary antibody alone. (f) NIH3T3 cells or those expressing the indicated molecules were left unstimulated (U) or stimulated with poly(I:C) at 5 (PIC 5) or 25 (PIC 25) μg/ml for 24 h. The concentration of CCL5 in the supernatant was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. (g, h) NIH3T3-TLR3-Unc93B1 cells were treated with vehicle, Rapamycin at 50 or 250 nM or Torin 1 at 50 or 250 nM for 2 h. The treated cells were left unstimulated (U) or stimulated with poly(I:C) at 5 (P5) or 25 μg/ml (P25) for 24 h (g) or at 25 μg/ml for the indicated periods of time (h). Immunoblotting of the indicated molecules was conducted (h). Grb2 is shown as a loading control. The concentration of CCL5 in the supernatant was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. Statistical analysis was performed by the two-tailed Student’s t test. ***P < 0.001. These experiments were repeated more than three times. (i) The deletion of RICTOR is shown by immunoblot of the whole cell lysate. Grb2, a loading control. Shown on the right is membrane permeabilized staining of TLR3 in WT and Rictor Δ/Δ fibroblasts (red histograms). Black histograms indicate labeling with the secondary antibody alone

Supplementary Figure 2 Innate immune responses to HSV-1 in fibroblasts and macrophages.

(a) WT MEF cells or those expressing the indicated molecules were stained with the anti-TLR3 mAb (PaT3) or -flag mAb for Unc93B1. The results are shown in red histograms, and the black histograms show the staining with the secondary reagent alone. (b, c) MEF-TLR3-Unc93B1 cells were left unstimulated (U), stimulated with poly(I:C) at 25 μg/ml, or with HSV-1 at an MOI of 10 for indicated periods of times. Real-time PCR was conducted to evaluate Ifnb1 mRNA expression. The results are normalized by Hprt mRNA and represented by the mean value ± s. d. from triplicates. (d) MEF cells expressing indicated molecules were left uninfected (U) or infected with HSV-1 at an MOI of 10 for 24 h. IFN-β production was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. (e) MEF-TLR3-Unc93B1 cells were infected with HSV-1 at an MOI of 10. After the indicated periods of times, cell lysate was prepared, and immunoblotting of the indicated molecules was conducted. ICP4, an HSV-1 regulatory protein, is shown as a marker indicating HSV-1 infection. Grb2, a loading control. (f) MEF-TLR3-Unc93B1 cells were infected with HSV-1 at an MOI of 10 for indicated periods of times. Immunoblotting of indicated molecules in the whole lysates was conducted. (g) MEF cells expressing hCas9 with or without TRAF3 gRNA were left uninfected (U) or infected with HSV-1 at an MOI of 10. After 24 h, IFN-β production was measured by ELISA. The deletion of TRAF3 is shown by immunostaining of the whole cell lysate. Grb2, a loading control. (h) Bone marrow derived macrophages were stimulated with poly(I:C) at 25 μg/ml for indicated periods of times. Immunostaining of the indicated molecules in the whole lysate was conducted. *P < 0.05, ***P < 0.001

Supplementary Figure 3 Cytoskeletal changes and TLR3 trafficking are mediated by PKC and mTORC2.

(a, b) NIH3T3-TLR3-Unc93B1 cells were left untreated or treated with Rapamycin at 400 nM (R), Torin 1 at 250 nM (T), or Go 6976 at 250 nM (G) for 2 h. Cells were left unstimulated or stimulated (PIC) with poly(I:C) at 25 μg/ml for 18 h. Polymerized α-tubulin and actin were stained. The mean fluorescence intensity of α-tubulin and actin was statistically analyzed (b). Statistical analysis was performed by one-way ANOVA and Tukey’s test. ***P < 0.001. Scale bar, 20 μm. These experiments were repeated more than three times. (c) The wildtype and Rictor Δ/Δ fibroblasts were transduced to express TLR3 and Unc93B1. These cells were left unstimulated (U), stimulated (PIC) with poly(I:C) at 25 μg/ml for 16 h or infected (HSV-1) with HSV-1 at an MOI of 10 for 6 h. TLR3 (green), LAMP-1 (red) and nuclei (blue) were stained and analyzed by confocal microscopy. The statistical analyses of the peripheral TLR3 is also shown. Statistical analysis was performed by one-way ANOVA and Tukey’s test. ***P < 0.001, Scale bar, 20 μm. These experiments were repeated more than three times

Supplementary Figure 4 TLR3 trafficking to the cell periphery depends on microtubule polymerization.

MEF/TLR3/Unc93B1 cells were untreated (UT) or treated for 2 h with a Nocodazole at 10 µM. Cells were left unstimulated (U), stimulated (PIC) with poly(I:C) at 25 μg/ml for 16 h or infected (HSV-1) with HSV-1 at an MOI of 10 for 6 h. TLR3 (green), LAMP-1 (red) and nuclei (blue) were stained and analyzed by confocal microscopy. The statistical analyses of the peripheral TLR3 and LAMP-1 are also shown. Statistical analysis was performed by one-way ANOVA and Tukey’s test. ***P < 0.001, Scale bar, 20 μm. These experiments were repeated more than three times

Supplementary Figure 5 Cytoskeletal changes and TLR3 trafficking in HSV-1-infected fibroblasts depend on mTOR, PKC and Rab7a.

(a, b) MEF-TLR3-Unc93B1 cells were left untreated (-) or treated with Go 6976 at 250 nM (G) or Torin 1 at 250 nM for 2 h (T). Cells were left uninfected (-) or infected with HSV-1 at an MOI of 10. After 6 h, polymerized α-tubulin and actin were stained (a). The statistical analyses of the mean fluorescence intensity of α-tubulin and actin are shown (b). ***P < 0.001. These experiments were repeated more than three times. (c) MEF-TLR3-Unc93B1 cells were untreated (U) or treated for 2 h with Go 6976 at 250 nM or Torin 1 at 1 μM. The treated cells were left uninfected (U) or infected with HSV-1 at an MOI of 10 for 6 h. TLR3 (green) and nuclei (blue) were stained. The percentages of peripheral TLR3 are also shown. (d) WT and _Rab7a_Δ/Δ MEF-TLR3-Unc93B1 cells were left uninfected (U) or infected with HSV-1 at an MOI of 10 for 6 h. TLR3 (green) and nuclei (blue) were stained, and the percentages of peripheral TLR3 were statistically analyzed. Statistical analyses were performed by one-way ANOVA and Tukey’s test. ***P < 0.001, Scale bar, 20 μm. These experiments were repeated more than three times

Figure Supplementary 6 The effect of inhibitors, molecular deficiency, and anti-TLR3 mAb on HSV-1 infection.

(a) Progeny virus yields (PFU/ml) in supernatants from MEF-TLR3-Unc93B1 cells untreated (-) or treated for 2 h with Go 6976 (G) at 250 nM or Torin 1 (T) at 1 μM before the HSV-1 infection for 24 h at an MOI of 10. The progeny virus yields were determined with Vero cells. (b) WT and Rab7a Δ/Δ MEF-TLR3-Unc93B1 cells were infected with the HSV-1 gB-RFP (red fluorescent protein) at an MOI of 10. RFP expression enables detection of HSV-1 in infected cells by confocal microscopy. After 6 h post-infection, the cells were immunostained with anti-TLR3 mAb, and the subcellular distribution of HSV-1 gB-RFP and TLR3 was analyzed. Statistical analyses of TLR3 colocalization with gB-RFP are shown. These experiments were repeated more than three times. (c) MEF-TLR3-Unc93B1 cells were treated with DMSO (Vehicle) or Torin 1 at 1 μM for 24 h and stained with Annexin V to determine cell viability. (d) WT mice (n = 10 per group) were intracranially administered with DMSO (Vehicle) or 6 μg of Torin1. Mice survival is shown. (e) WT mice were intracranially treated with 6 μg of Torin1. After 2 h, cell lysate was prepared, and immunoblotting of the indicated molecules was conducted. Grb2, a loading control. These experiments were repeated more than three times. (f) MEF-TLR3-Unc93B1 cells were left untreated or treated with control IgG or anti-TLR3 mAb (PaT3) at 4 or 20 μg/ml for 2 h. The treated cells were left unstimulated (U), stimulated (PIC) with poly(I:C) at 25 μg/ml for 24 h, or infected (HSV) with HSV-1 at an MOI of 10 for 24 h. The concentration of CCL5 and IFN-β in the supernatants was determined by ELISA. The results are represented by the mean value ± s.d. of triplicate wells. (g) WT mice (n = 8 per group) were intracranially infected with indicated doses of HSV-1 and mice survival was evaluated. (h, i) WT and _Ifnar1_−/− mice were intracranially infected with 103 PFU of HSV-1 (h) Mice survival is shown. n = 8 per group. (i) Virus yields in the brain at 2 days post infection. n > 3 per group. Statistical analysis was performed by the log-rank test (h) or the two-tailed Student’s t test (i). ***P < 0.001

Supplementary Figure 7 TLR3-mTORC2 axis is required for protection against HSV-1 infection.

Prior to its activation, TLR3 is associated with the GTPase Rab7a and is localized in late endosomes/lysosomes around the nucleus. Upon Herpes Simplex Virus-1 (HSV-1) infection, TLR3 is activated and recruits mTORC2, leading to TRAF6-dependent NF-κB activation to produce proinflammatory cytokines such as the chemokine CCL5. TLR3-dependent mTORC2 activation also induces microtubule elongation to the cell periphery by activating PKCs. Rab7a links TLR3-containing lysosomes with elongated microtubules for TLR3 trafficking to the cell periphery. This trafficking is required for the interaction between TLR3 and type I interferon (type I IFN) signaling molecules, such as TRAF3 and mTORC1. A monoclonal antibody to TLR3 shows the rescuing effect against Herpes Simplex Encephalitis by enhancing TLR3 responses. N, nucleus

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Sato, R., Kato, A., Chimura, T. et al. Combating herpesvirus encephalitis by potentiating a TLR3–mTORC2 axis.Nat Immunol 19, 1071–1082 (2018). https://doi.org/10.1038/s41590-018-0203-2

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• Received: 08 September 2017
• Accepted: 31 July 2018
• Published: 10 September 2018
• Issue Date: October 2018
• DOI: https://doi.org/10.1038/s41590-018-0203-2