Research ArticleIMMUNOLOGY

TRIM8 is required for virus-induced IFN response in human plasmacytoid dendritic cells

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Science Advances  20 Nov 2019:
Vol. 5, no. 11, eaax3511
DOI: 10.1126/sciadv.aax3511
  • Fig. 1 Constitutively expressed and virus-induced TRIM genes in human primary pDCs.

    (A) Systematic analysis of constitutive expression of all human TRIM genes in pDCs by RT-qPCR analysis. Data represent the mean ± SD of independent experiments performed in duplicate on purified pDCs from three different blood donors. Red arrows point to TRIM genes that are constitutively expressed at detectable levels above the geometric mean of the six different housekeeping genes (2−ΔCt > 1). (B) Purified pDCs were stimulated overnight with HIV-1 MN AT-2 or influenza A virus (IAV). Cytokine secretion was blocked for 12 hours using BFA at 1 μg/ml. Intracellular IFN-α was measured by fluorescence-activated cell sorting (FACS) after fixation and saponin permeabilization. (C and D) IFN secreted in the culture medium by virus-activated pDCs was titrated using two different techniques: The concentration of IFN-α2, IFN-β, and IFN-γ was quantified by the multiplex bead-based immunoassay LEGENDplex (BioLegend) (C), or global (type I and type II) IFN activity was quantified using the STING-37 reporter cell line, which expresses luciferase under the control of five IFN-stimulated response elements (ISRE) (D). (E) Purified pDCs from three donors were activated with HIV-1 MN AT-2 or IAV for 16 hours, and the transcriptional profiles of 114 genes [6 housekeeping genes, 17 IFN-induced genes (ISGs), 16 virus-induced cytokines and chemokines, and the 75 human TRIM genes] were analyzed simultaneously by RT-qPCR. The expression of each transcript was normalized to the geometric mean of a set of stably expressed reference genes (SDHA, PPIB, TBP, POLR2A, PPIA, and B2M). Data represent the mean of three independent experiments performed in duplicate on purified pDCs from different blood donors. Values were converted to log2 and represented as heat maps. Raw data are shown in fig. S1B.

  • Fig. 2 Effect of TRIM gene silencing on virus-induced IFN response in human primary pDCs.

    (A to C) Purified pDCs from three donors were transfected with nontargeting siRNA (C) or with siRNA targeting selected TRIM transcripts (TRIM8, TRIM20, TRIM21, TRIM22, TRIM28, TRIM36, TRIM38, TRIM46, or TRIM49) or IRF7 as a control. After 24 hours, pDCs were activated with HIV-1 MN AT-2 or IAV for 16 hours before the expression of type I IFN was determined both at the transcript level by RT-qPCR (A and B) and at the protein level in the culture medium (C). The relative expression of IFN-α2 and IFN-β transcripts in activated pDCs is represented as mean values ± SD, with each bar representing a single donor (A) and as mean fold change of all donors relative to control siRNA (B). (C) For the quantification of IFN in the culture medium, STING-37 cells were incubated for 24 hours with the supernatant of activated pDCs or with known concentrations of recombinant IFN-α2. Statistical significance (P value) was calculated by one-way analysis of variance (ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. (D) Schematic model of TRIM proteins identified as positive (in green) and negative (in red) regulators of TLR7-dependent IFN response in human pDCs. The putative compartment (cytoplasm or nucleus) where TRIM proteins act is based on their known subcellular localization (8).

  • Fig. 3 TRIM8 knockdown inhibits IFN secretion through a decrease of pIRF7 in human primary pDCs.

    (A) Subcellular localization of TRIM8 and IRF7 or pIRF7 was visualized by immunofluorescent confocal microscopy on naive pDCs (NS) or pDCs stimulated for 6 hours with IAV. (B) Raw intensity density per cell of TRIM8 and pIRF7 staining was measured using ImageJ software. Data represented by box and whiskers with median ± min to max represent the analysis of 30 cells from nine different fields. *P < 0.05, by Mann-Whitney test. (C) Expression of pIRF7 and IRF7 in IAV-stimulated pDCs was assessed by Western blot. (D) Mander’s coefficients were determined using the ImageJ JACoP plug-in to evaluate the colocalization between IRF7 and TRIM8 or between pIRF7 and TRIM8 in unstimulated or IAV-stimulated cells, respectively. The analysis was performed on 30 cells from nine different fields. **P < 0.01, by Mann-Whitney test. (E to G) Human pDCs were transfected with nontargeting siRNA (CTR) or with a TRIM8-specific siRNA and activated 24 hours later with IAV for 16 hours. (E) The concentration of IFN-α2 and IFN-β secreted in the culture medium by IAV-activated pDCs was quantified with the multiplex bead-based immunoassay LEGENDplex (BioLegend). (F) TRIM8 expression was verified by RT-qPCR. To quantify the amount of pIRF7 by flow cytometry, cells were fixed, permeabilized, and labeled with mouse Alexa Fluor 488 anti-IRF7 (pS477/pS479) antibody. (G) Representative flow cytometry histogram of pIRF7 expression in nonactivated control (dotted line), IAV-stimulated control (gray), or TRIM8 knockdown pDCs (green). (H) pDCs were transfected with nontargeting siRNA (siCTR) or with a TRIM8-specific (siTRIM8) siRNA and activated for 16 hours with R848 (5 μg/ml). Activated pDCs were then cocultured with autologous PBMCs at a ratio of 1:10. After 24 hours of coculture, PBMCs were mock-infected (/) or infected with 2.5 × 104 PFU/well of the IAV nanoluciferase-expressing reporter virus for 24 hours before nanoluciferase activity was measured on an Infinite F200 Pro (Tecan) plate reader. As controls, PBMCs were cultivated without pDCs and were either left untreated (/) or treated with 1000 IU of IFN-α2 before challenged with the same dose of IAV reporter virus. Data represent the mean ± SD of one experiment performed in triplicate, representative of two independent experiments performed on cells from two blood donors. (I and J) Human pDCs were activated with HIV-1 MN or IAV, and TRIM8 expression was determined by RT-qPCR (I) and Western blot (J) 6 or 24 hours after stimulation, respectively.

  • Fig. 4 TRIM8 regulates pIRF7 stability independently of its E3 ubiquitin ligase activity.

    (A) HEK293T cells were transfected with nontargeting control (CTR) or TRIM8-specific (T8) siRNA. Twenty-four hours after transfection, expression of endogenous IRF7 was induced by treating the cells with 1000 IU of IFN-α2 for 16 hours and cells were infected with SeV to trigger IRF7 phosphorylation. Equal amounts of cell extracts were analyzed by Western blot for the expression of TRIM8, IRF7, and pIRF7. (B) HEK293T cells expressing Flag-tagged phosphomimetic IRF7 (S477D/S479D) construct (Flag-pmIRF7) were transfected with nontargeting control (CTR) or TRIM8-specific siRNA. After 48 hours, whole-cell lysates were analyzed by Western blot with anti-Flag or anti-TRIM8 antibodies (Abs). (C) HEK293T cells were treated with 1000 IU of IFN-α2 for 16 hours and transfected or not with a plasmid expressing V5-TRIM8. The expression of endogenous IRF7, pIRF7, and pIRF3 following SeV infection was evaluated by Western blot. (D) Whole-cell lysates obtained from HEK293T cells expressing Flag-pmIRF7 and transfected with increasing amounts of V5-TRIM8 for 48 hours were subjected to Western blot analysis using anti-Flag or anti-V5 antibodies. (E) Flag-pmIRF7–expressing HEK293T cells were transfected with V5-TRIM8. After IP using anti-Flag antibodies, TRIM8 was detected using anti-V5 antibodies. (F) HEK293T cells expressing Flag-pmIRF7 were transfected with either a nontargeting siRNA (CTR) or an siRNA targeting TRIM8 for 48 hours. Six hours before cell extraction, cells were treated with MG132 to block proteasomal degradation. Expression of Flag-pmIRF7 and TRIM8 was assessed by Western blot using anti-Flag and anti-TRIM8 antibodies, respectively. (G) Lysates from HEK293T cells cotransfected with Flag-pmIRF7, V5-TRIM8, and empty vector (EV), HA-Ub (wt), HA-Ub (K48), or HA-Ub (K63) plasmids were subjected to IP with anti-Flag antibody followed by Western blot analysis with anti-Flag, anti-V5, or anti-HA antibodies. (H) HEK293T were cotransfected with Flag-pmIRF7 and HA-Ub (K63) or HA-Ub (K48) plasmids and with nontargeting control (CTR) or TRIM8-specific siRNA. After 48 hours, whole-cell lysates were subjected to IP with anti-Flag antibody followed by Western blot analysis with anti-Flag, anti-TRIM8, and anti-HA antibodies. (I) Flag-pmIRF7–expressing HEK293T cells were transfected with V5-TRIM8 wt, V5-TRIM8 mutant C15S/C18S/C35S/C38S (V5-TRIM8 C4S), or empty vector. After 48 hours, cell extracts were subjected to Western blot analysis using anti-Flag or anti-V5 antibodies. All panels show typical results representative of at least two independent experiments.

  • Fig. 5 pIRF7 is a Pin1 substrate.

    (A) HEK293T cells were transfected with HA-Pin1 together with or without Flag-tagged phosphomimetic IRF7 (Flag-pmIRF7). Following IP with anti-HA antibodies, pmIRF7 was revealed using an anti-Flag antibody. (B) HEK293T cells were transfected with Flag-pmIRF7 and with nontargeting (CTR) or Pin1-specific siRNA, as indicated. Pin1 and pIRF7 expression was assessed by Western blot using anti-Pin1 or anti-Flag antibodies, respectively. To follow the phosphorylation of endogenous IRF3 as an internal control, cells were also infected with SeV for 6 hours before preparing the extracts, and the expression of IRF3 and pIRF3 was assessed by Western blot. (B) and (C) show typical results representative of at least two independent experiments. (C) Pin1 expression was silenced in HEK293T cells using siRNA, while endogenous IRF7 expression was induced by 1000 IU of IFN-α2. Cells were then infected with SeV for 6 hours, and the amount of pIRF7 was evaluated by flow cytometry. Graph shows the mean of three independent experiments ± SD. Statistical significance (P value) was determined by Student’s t test. *P < 0.05. (D) HEK293T cells were transfected with a nontargeting (CTR) or a Pin1-targeting siRNA. IRF7 expression was induced by a 16-hour treatment with 1000 IU of IFN-α2, and activation of IRF7 was triggered by SeV infection. The expression levels of pIRF7, IRF7, and Pin1 were assessed by Western blot at different time points after infection, as indicated. (E) Human pDCs from two different donors were transfected with nontargeting siRNA (CTR) or with a Pin1-specific siRNA. Twenty-four hours after transfection, cells were stimulated with HIV-1 for 16 hours, and IFN-α2 and IFN-β mRNA was quantified by RT-qPCR. The results shown are mean ± SD. Statistical significance (P value) was determined by Student’s t test. ***P < 0.001. Data are representative of three independent experiments.

  • Fig. 6 TRIM8 interferes with the recognition of pIRF7 by Pin1.

    (A) HEK293T cells expressing Flag-tagged phosphomimetic IRF7 (Flag-pmIRF7) were transfected with increasing quantity of the V5-TRIM8 plasmid. Following IP with anti-Flag antibodies, the presence of Pin1, V5-TRIM8, and Flag-pmIRF7 was assessed by Western blot. (B) Flag-pmIRF7–expressing HEK293T cells were transfected with a nontargeting (CTR) or a TRIM8-specific siRNA. Flag-pmIRF7 was pulled down using anti-Flag antibodies, and Pin1 was detected by Western blot. (C) Flag-pmIRF7–expressing HEK293T cells were transfected with empty vector, Myc-tagged full-length TRIM8 (wt), or TRIM8 deleted from one domain [RING (R), B box (B), coiled coil (CC), or C-terminal domain (Ct)] in the presence of MG132 to prevent pmIRF7 degradation. Following pull-down with anti-Myc antibodies, the presence of pmIRF7 was revealed by Western blot using anti-Flag antibodies. (D) HEK293T cells expressing Flag-pmIRF7 were transfected with wt or ΔRING (R) Myc-tagged TRIM8. TRIM8 and pmIRF7 expression was assessed by Western blot using anti-Myc or anti-Flag antibodies, respectively. (E) Primary human monocyte-derived dendritic cells (MoDCs) were treated for 16 hours with 1000 IU of IFN-α2 to induce endogenous IRF7 expression and infected or not with SeV for 3 hours to induce its phosphorylation. Following IP with anti-Pin1 or anti-IRF7 antibodies, the presence of pIRF7, IRF7, Pin1, and TRIM8 was assessed by Western blot, as indicated. (F) Primary MoDCs were transfected with a nontargeting (CTR) or a TRIM8-specific siRNA, treated for 16 hours with 1000 IU of IFN-α2, and infected or not with SeV for 3 hours. Forty-eight hours after transfection, expression levels of TRIM8, pIRF7, IRF7, and Pin1 were assessed by Western blot. Following IP with anti-Pin1 antibodies, the presence of Pin1, pIRF7, and TRIM8 was assessed by Western blot.

  • Fig. 7 trim8b positively regulates type I IFN responses in zebrafish.

    (A) MXA reporter zebrafish embryos were injected at the one-cell stage with control, trim8a-targeting, or trim8b-targeting morpholinos (MOs) and, at 3 dpf, inoculated or not with CHIKV. After 48 hours, larvae were imaged with a fluorescence microscope. Images are of the lateral view, dorsal to top, anterior to left, and merge of transmitted light image (gray) with red fluorescence (magenta). The red signal in the lens is due to the secondary cryaa:DsRed reporter to identify transgene carriers. Scale bar, 1 mm. (B) Corresponding quantification of MXA reporter signal. Mean red fluorescence intensity (MFI) was measured posterior to the eyes; average background fluorescence from control larvae was subtracted before plotting the data. (C) Disease score assessed 3 days after CHIKV inoculation. (D) RT-qPCR measurement of type I IFN gene expression in MO-injected, CHIKV-infected wt larvae 40 hours after infection, displayed as fold induction over uninfected control larvae. (E to H) Outcome of CHIKV-GFP infection in MO-injected ifnphi3ip7/ip7 larvae: (E) disease score 3 days after infection; (F) infection level 3 days after infection, quantified by GFP fluorescence, with two representative images shown in (G) as a merge of transmitted light and green fluorescence; (H) RT-qPCR assessment of CHIKV-E1 transcripts 40 hours after infection. Means ± SD are provided over individual data points. A.U., arbitrary units. (B and C) ANOVA with Sidak’s multiple comparisons test was performed. (D to F and J) Unpaired t tests were performed. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Table 1 List of the 114 transcripts simultaneously quantified by RT-qPCR profiling, including 6 housekeeping genes (1 to 6), 75 TRIM genes (7 to 81), 17 ISGs (82 to 98), 9 IFNs (99 to 107), and 7 inflammatory cytokines or chemokines (108 to 114).

    For each transcript, the official name, the alias name, if any, and the RefSeq accession number are indicated.


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Supplementary Materials

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. TRIM expression and silencing in human primary pDCs.
    • Fig. S2. Verification of knockdown efficiency in zebrafish.

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