Research ArticleImmunology

Guanabenz inhibits TLR9 signaling through a pathway that is independent of eIF2α dephosphorylation by the GADD34/PP1c complex

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Science Signaling  23 Jan 2018:
Vol. 11, Issue 514, eaam8104
DOI: 10.1126/scisignal.aam8104
  • Fig. 1 GBZ mimics GADD34 inactivation and impairs TLR3 and TLR9 signaling.

    (A to F) Bone marrow–derived Flt3L-DCs (dendritic cells) (A and B), bone marrow–derived granulocyte-macrophage colony-stimulating factor (GM-CSF) DCs (C and D), and splenic DCs (E and F) from wild-type (WT) or GADD34ΔC/ΔC (ΔC/ΔC) mice were stimulated for 6 hours with lipopolysaccharide (LPS) (100 ng/ml; from Escherichia coli), low–molecular weight (LMW) poly(I:C) (pI:C; 10 μg/ml), or 1 μM ODN 1585. NT, nontreated. Guanabenz (GBZ) was added at 50 μM. The concentrations of secreted interferon-β (IFN-β) (A, C, and E) or interleukin-6 (IL-6) (B, D, and F) were measured by enzyme-linked immunosorbent assay (ELISA). Statistical significance was assigned using two-tailed t test on at least three independent experiments (*P < 0.05; **P < 0.005; ***P < 0.0005). N.D., not detected; N.S., not significant.

  • Fig. 2 GBZ inhibits TLR9 activation in mouse Flt3L-DCs.

    (A) Sorting strategy for bone marrow–derived Flt3L-DCs. (B) IFN-β and IL-6 secretion in sorted DC1 and plasmacytoid DCs (pDCs) activated for 6 hours with LMW poly(I:C) (10 μg/ml) or 1 μM ODN 1585, respectively. GBZ was added at 50 μM. (C) Sorted pDCs were also analyzed for the transcriptional induction of genes encoding ISGs (IRF7 and ISG15) and cytokines (IFNA4, IL12B, IL6, and TNF) at different time points. Data are represented as fold increase compared to untreated pDCs. Statistical significance was assigned using two-tailed t test on at least n = 3 independent experiments (*P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001).

  • Fig. 3 GBZ impairs TLR9 signaling in human cells.

    (A) Human Toll-like receptor 4 (hTLR4), hTLR3, hTLR9, and hTLR7 reporters expressed in HEK-Blue cells were activated with LPS (100 ng/ml), LMW poly(I:C) (10 μg/ml), 2.5 μM ODN 2006, or imiquimod (5 μg/ml), respectively. HEK-293, human embryonic kidney 293 cells; OD 405 nm, optical density at 405 nm. GBZ was added at 50 μM. TLR activity was measured indirectly through secreted alkaline phosphatase (SEAP) activity as absorbance at 405 nm. (B) Human primary pDCs were isolated from blood and activated either with 3 μM ODN 2216 or 2% serum from lupus patients. GBZ was added at 50 μM. Secreted IFN-α and tumor necrosis factor–α (TNF-α) were measured at the indicated time points or at 16 hours. (C) CAL-1 cells were stimulated with 3 μM ODN 2216. GBZ was added at 50 μM. Secreted TNF-α was measured 3 hours after activation. Gene expression of TNF and IFNA2 were measured by quantitative polymerase chain reaction (qPCR) and normalized on untreated cells. (D) Mean fluorescence intensity (MFI) for phospho-ribosomal protein S6 (P-S6) detected by intracellular phospho-flow cytometry in CpG-A–activated CAL-1 cells treated or not with GBZ. Data of three experiments were normalized to those from nontreated cells. Statistical significance was assigned using two-tailed t test on at least three independent experiments (*P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001).

  • Fig. 4 GBZ rescues mice from TLR9-dependent cytokines shock.

    (A and B) WT (A) and GADD34ΔC/ΔC (B) mice were injected with d-GalN (20 mg per mouse), ODN 1826 or phosphate-buffered saline (PBS) for control mice (50 μg/20 g), and GBZ or clonidine (Clo; 2 mg/kg). Survival is represented as total percentage of survival after 48 hours (left) or in a Kaplan-Meier plot (right). (C) The concentrations of circulating TNF-α, IL-10, and IL-6 were measured at 1 and/or 3 hours. (D) Liver tissue damage (histological score) was determined in a blinded fashion by a pathologist. Statistical significance was assigned on sample comparison by a one-way analysis of variance (ANOVA), followed by Tukey range test, and on the survival curve using log-rank test, followed by Benjamini-Yekutieli correction (*P < 0.05; **P < 0.005; ***P < 0.0005.). n values in the figure indicate the number of mice.

  • Fig. 5 GBZ decreases the severity lupus-like symptoms in the pristane injection model.

    (A) The experimental protocol: Mice were injected at day 0 (D0) with PBS or 500 μl of tetramethylpentadecane (TMPD) and then injected every 2 days for 2 weeks with PBS/dimethyl sulfoxide (DMSO) or GBZ (2 mg/kg). At day 7, mice were injected with ODN 1585 (50 μg/20 g). Blood was collected monthly to determine the circulating autoantibody level. Mice were sacrificed at week 24 (W24) after disease induction. (B and C) Flow cytometry analysis of peritoneal exudate cell (PEC) populations at day 14. (D) Mice were sacrificed at week 24, and gene expression of IFN-stimulated genes (ISGs) and cytokines in PECs was determined by qPCR. Statistical significance was assigned using two-tailed Student’s t test (*P < 0.05). (E) Level of circulating anti-RNA antibodies (normalized to control mice), antinuclear antibodies (arbitrary units), the presence of lipogranulomas in the peritoneal cavity (arbitrary score), and glomerulopathy (histological score was determined in a blinded fashion by a pathologist) at week 24. Statistical significance was assigned by a one-way ANOVA, followed by Tukey range test (*P < 0.05; **P < 0.005; ***P < 0.0005). n values in the figure indicate the number of mice.

  • Fig. 6 GBZ reduces TLR-dependent Ch25h induction.

    (A) Principal components (Comp) analysis generated from microarray gene expression studies comparing GM-CSF DCs activated for 8 hours with high–molecular weight (HMW) poly(I:C) or P. mirabilis, with or without GBZ. (B) Graphical representation of the cholesterol biosynthesis pathway and its regulation with regard to CH25H, SREBP (sterol regulatory element–binding protein), GBZ, and TLRs. (C) List of genes involved in the cholesterol biosynthesis and their expression fold change (Fc) compared to untreated control cells. na, not applicable. (D) TLR9, GADD34, and ATF3 mRNA expression in mouse spleen pDCs activated with 1 μM ODN 1585. CH25H mRNA level in GM-CSF DCs activated for 8h with LPS (100 ng/ml), HMW poly(I:C) (10 μg/ml), or 1 μM ODN 1585, with or without GBZ (50 μM) (bottom). (E) CH25H mRNA (left) and IL-6 secretion (right) levels in WT and GADD34ΔC/ΔC Flt3L-DCs treated with CpG and GBZ, exposed or not to exogenously added type I IFN. Statistical significance was assigned using two-tailed t test on at least three independent experiments (*P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001).

  • Fig. 7 25-HC antagonizes GBZ inhibitory activity on TLR9.

    (A) CAL-1 cells were stimulated with 3 μM ODN 2216. GBZ was added at 50 μM. Immunofluorescence proximity ligation assays (iPLAs) were performed at the indicated time points on the following pairs of proteins: TLR9/MyD88, TLR9/VAMP3, and TLR9/IRF7. Scale bars, 10 μM. (B) CAL-1 cells were stimulated with 3 μM ODN 2216 with or without GBZ (50 μM) and/or 25-hydroxycholesterol (25-HC) (100 nM). iPLAs were performed at the indicated time points on the following pairs of proteins: TLR9/MyD88, TLR9/VAMP3, TLR9/MyD88 (3 hours) or TLR9/IRF7 (3 hours). Scale bars, 10 μM. (C) Representative immunoblot for ribosomal protein S6 (S6) and associated TLR-dependent phosphorylation in CAL-1 cells stimulated for 1 hour. Quantification of the blots is shown on the right (n = 3 independent experiments). (D) TNF-α secretion by CAL-1 cells stimulated with 3 μM ODN 2216 for 3 hours with or without GBZ (50 μM) and/or 25-HC (100 nM) was measured by ELISA. Statistical significance was assigned using two-tailed t test on at least three independent experiments (*P < 0.05; **P < 0.005; ***P < 0.0005).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/514/eaam8104/DC1

    Fig. S1. GBZ inhibits TLR9 signaling in B cells.

    Fig. S2. TMPD and CpG synergize to promote inflammation.

    Fig. S3. GBZ does not inhibit endosomal acidification or protein synthesis.

    Fig. S4. GBZ and 25-HC do not reduce TLR9 abundance in pDCs.

    Table S1. List of genes in DCs with increased expression in response to poly(I:C) or P. mirabilis that is inhibited by GBZ.

    Table S2. List of the primers used for gene expression analysis by qPCR.

    Table S3. List of the antibodies used for sorting DC1, DC2, and pDCs from Flt3L-DC culture.

    Table S4. List of the antibodies used for flow cytometry.

  • Supplementary Materials for:

    Guanabenz inhibits TLR9 signaling through a pathway that is independent of eIF2α dephosphorylation by the GADD34/PP1c complex

    Jessica Perego, Andreia Mendes, Clarisse Bourbon, Voahirana Camosseto, Alexis Combes, Hong Liu, Thien-Phong Vu Manh, Alexandre Dalet, Lionel Chasson, Lionel Spinelli, Nathalie Bardin, Laurent Chiche, Manuel A. S. Santos, Evelina Gatti,* Philippe Pierre*

    *Corresponding author. Email: gatti{at}ciml.univ-mrs.fr (E.G.); pierre{at}ciml.univ-mrs.fr (P.P.)

    This PDF file includes:

    • Fig. S1. GBZ inhibits TLR9 signaling in B cells.
    • Fig. S2. TMPD and CpG synergize to promote inflammation.
    • Fig. S3. GBZ does not inhibit endosomal acidification or protein synthesis.
    • Fig. S4. GBZ and 25-HC do not reduce TLR9 abundance in pDCs.
    • Table S1. List of genes in DCs with increased expression in response to poly(I:C) or P. mirabilis that is inhibited by GBZ.
    • Table S2. List of the primers used for gene expression analysis by qPCR.
    • Table S3. List of the antibodies used for sorting DC1, DC2, and pDCs from Flt3L-DC culture.
    • Table S4. List of the antibodies used for flow cytometry.

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    Citation: J. Perego, A. Mendes, C. Bourbon, V. Camosseto, A. Combes, H. Liu, T.-P. V. Manh, A. Dalet, L. Chasson, L. Spinelli, N. Bardin, L. Chiche,M. A. S. Santos, E. Gatti, P. Pierre, Guanabenz inhibits TLR9 signaling through a pathway that is independent of eIF2α dephosphorylation by the GADD34/PP1c complex. Sci. Signal. 11, eaam8104 (2018).

    © 2018 American Association for the Advancement of Science

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