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Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity

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Science Translational Medicine  09 Sep 2015:
Vol. 7, Issue 304, pp. 304ra142
DOI: 10.1126/scitranslmed.aaa3863
  • Fig. 1. Dengue NS1 activates human and mouse innate immune responses, and is not inhibited by LPS-binding antibiotic polymyxin B.

    (A) NS1 induces IL-6 secretion by human PBMCs. Cells were treated for 24 hours with LPS (100 ng/ml), NS1 (40 μg/ml), or an analogous NS1 sample immunodepleted with anti-NS1 or anti-E antibody. IL-6 was measured by enzyme-linked immunosorbent assay (ELISA). Data are means ± SD of three donors. The inset immunoblot is of the supernatant fraction after immunodepletion using anti-NS1 antibody or isotype-matched control antibody. (B) Polymyxin B does not block NS1-induced IL-6 in human PBMCs. NS1 and LPS were preincubated for 30 min with or without polymyxin B, then added to PBMCs, and incubated for 24 hours. The final concentration of polymyxin B was 25 μg/ml, with varying concentrations of LPS (0.1, 1, and 10 ng/ml) and NS1 (5, 10, and 20 μg/ml). The experiment was run in parallel with (A) and separated for clarity (mean ± SD of three donors). (C) Polymyxin B does not block NS1-induced loss of surface CSF1R on BMMs. NS1 and LPS were preincubated for 30 min with or without polymyxin B and then added to BMMs that had been starved of CSF1 overnight. The final concentration of polymyxin B was 25 μg/ml (19.2 μM), with LPS (10-fold dilutions from 100 ng/ml, or 10 nM assuming average Mr = 10 kD) and NS1 (2-fold dilutions from 10 μg/ml, or 33.3 nM). After 1 hour of treatment, CSF1R levels were determined by flow cytometry, with the % of cells with high levels of receptor plotted. Data are means ± SD from three independent experiments.

  • Fig. 2. NS1 from several sources has immunostimulatory activity.

    (A) NS1 immunodepletion prevents the down-modulation of surface CSF1R by S2 cell–expressed NS1. BMMs starved of CSF1 overnight were treated with either phosphate-buffered saline (PBS), CSF1 (104 U/ml), LPS (100 ng/ml), NS1 (10 μg/ml), or NS1 immunodepleted with anti-NS1 antibody or control anti-E antibody. Data are means ± SD from three independent experiments. (B) Minimally processed CHO cell–expressed NS1 has similar activity to S2 cell–derived NS1. CHO cells were transfected with either empty expression vector or a plasmid encoding NS1 under the control of the cytomegalovirus promoter. Culture medium, concentrated with 100-kD cutoff filters from untransfected cells (control SN), empty vector (pcDNA SN), or NS1 expression vector–transfected cells (NS1 SN), was applied to CSF1-starved BMMs. The final concentration of NS1 (5 μg/ml) (as quantified by capture ELISA) represents about fourfold higher levels than produced in the CHO cell medium. Cells were also exposed to NS1 SN after immunodepletion with anti-NS1 antibody or control anti-E antibody and to CSF1. Data are means ± range from two independent experiments. Immunoblot analysis for NS1 confirms immunodepletion. (C) CHO cell–derived NS1 induces IL-6 in human PBMCs. Culture medium from transient expression concentrated as above was applied to human PBMCs. Treatments are as per (B), with the inclusion of medium from CHO cells exposed to PEI transfection reagent (PEI SN) as an extra control. Released IL-6 was measured by ELISA, and data are means ± SD from four donors, except for immunodepleted samples, which represent three donors.

  • Fig. 3. Induction of TNF-α and IL-6 mRNAs in mouse macrophages and human PBMCs in response to S2-derived NS1 protein.

    (A) Dose-dependent production of cytokine mRNAs from treated murine BMMs. BMMs were incubated with purified NS1 (1.25, 2.5, 5, 10, 20, and 40 μg/ml) or LPS (0.1, 1, 10, and 100 ng/ml) and harvested after 3 hours. Expression of individual mRNAs was measured by real-time polymerase chain reaction (PCR) and expressed relative to Hprt mRNA. (B) Time course of BMM response to LPS and NS1. BMMs were incubated with either no additions (control), NS1 (40 μg/ml), or LPS (1 ng/ml) for the indicated times. Cytokine mRNAs were measured as described in (A). A control sample is included for each time point, but generally cannot be seen on this scale. (C) Time course of PBMC response to LPS and NS1. PBMCs were treated with no additions (control), NS1 (10 μg/ml), or LPS (10 ng/ml) for the indicated times. Cytokine mRNAs were measured by real-time PCR and expressed relative to Hprt mRNA. Data are means ± range from two independent experiments (A and B) or means ± SD from four donors (C).

  • Fig. 4. NS1 activates cells via TLR4.

    (A) Down-modulation of BMM cell surface CSF1R by NS1 requires TLR4. CSF1R on wild-type (WT) C57BL/6 BMMs, Tlr4−/−, and MyD88−/−/Trif−/− BMMs was measured after 1 hour of treatment with CSF1 (104 U/ml), LPS (100 ng/ml), and NS1 (10 μg/ml). (B) Down-modulation of BMM cell surface CSF1R by NS1 does not require TLR2. BALB/c WT BMMs and Tlr2−/− BMMs were treated as in (A) or with Pam3CSK4 (100 ng/ml) as a TLR2 stimulus. (C and D) NS1 induction of TNF-α and IL-1β mRNAs requires TLR4. WT (C57BL/6) and Tlr4−/− BMMs were incubated with NS1 (10 μg/ml), LPS (1 ng/ml), or Pam3CSK4 (100 ng/ml) for 3 hours, and mRNA expression was determined using real-time PCR relative to Hprt mRNA. (E and F) NS1 induction of cytokine mRNAs does not require TLR2. WT BALB/c and Tlr2−/− BMMs were treated as in (C). (G and H) Human embryonic kidney (HEK) 293 cells expressing human TLR4 and MD-2 (G), but not TLR2 (H), respond to 3 hours of treatment with NS1 (10 μg/ml), with induction of IL-8 mRNA, measured by real-time PCR relative to Hprt mRNA, and normalized to the control sample. LPS (10 ng/ml) and Pam3CSK4 (100 ng/ml) provide control stimuli for TLR4 and TLR2, respectively. Data are means ± SD of three independent experiments (A) or means ± range from two independent experiments (B to H).

  • Fig. 5. Colocalization of NS1 and TLR4 on adherent PBMCs.

    PBMCs were allowed to adhere to coverslips for 2 hours and were then treated with NS1 (10 μg/ml) for 45 min before fixation. Cells were stained for cell surface TLR4 (red) and NS1 (green) without permeabilization. (A) Orthographic and planar projections were obtained by confocal microscopy. In the right hand panel, regions of colocalization appear as yellow (selected regions arrowed). Single-color controls showed no bleed between fluorescence channels. There was no background staining with secondary antibodies alone, and the secondary antibodies were demonstrated to be specific for the species of primary antibody (fig. S5). (B) NS1 binding correlates with TLR4 expression in the mixed PBMC population. Cells were stained as per (A).

  • Fig. 6. TLR4 antagonism blocks NS1 activity in vitro and reduces vascular leak in a murine DENV infection model.

    (A) NS1-induced IL-6 production by PBMCs was inhibited by LPS-RS. Cells were preincubated with LPS-RS (10 μg/ml) for 30 min and subsequently stimulated with LPS (100 ng/ml), NS1 (10 μg/ml), or Pam3CSK4 (500 ng/ml) for 24 hours. IL-6 was quantified by ELISA. (B) Anti-TLR4 antibody reduces the response to NS1. PBMCs were preincubated with anti-TLR4 (2 and 10 μg/ml) for 1 hour and then stimulated with NS1, LPS, or Pam3CSK4 for 24 hours as above. IL-6 was quantified by ELISA. Data are means ± range for two donors (A and B). (C) NS1 induces permeability of HMEC-1 monolayers, and its action is inhibited by LPS-RS and anti-TLR4 antibody. Confluent cells in Transwells were pretreated with or without LPS-RS (100 μg/ml) or anti-TLR4 antibody (TLR4 Ab) (20 μg/ml) for 1 hour, and then treated with LPS (100 ng/ml) or NS1 (10 μg/ml). Monolayer integrity was assessed by measurement of transendothelial electrical resistance (TEER) at the indicated times. Data are means ± SD over four replicate wells. (D and E) AG129 mice were injected intraperitoneally with 50 μg of LPS-RS or saline, 1 hour before infection with DENV-2 strain D220 [104 plaque-forming units (PFU), intraperitoneally]. Mice were treated daily with the same doses of LPS-RS or saline. Evans blue was administered intravenously on day 4 after infection and allowed to circulate for 2 hours before mice were anesthetized and perfused, and organs were harvested. Dye content from intestines (D) and liver (E) was assessed colorimetrically after extraction into dimethylformamide to assess relative vascular integrity. Means ± SD are indicated for n = 5 to 7. Statistical significance was assessed by one-way analysis of variance (ANOVA) (Sidak multiple comparisons test; *P < 0.05, **P < 0.01, ****P < 0.0001 for comparisons indicated on the graphs).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/304/304ra142/DC1

    Materials and Methods

    Fig. S1. Purification and characterization of DENV-2 NS1 from S2 cells.

    Fig. S2. Dengue NS1 activates a mouse macrophage NF-κB reporter cell line.

    Fig. S3. Polymyxin B can efficiently inhibit LPS in the presence of NS1.

    Fig. S4. NS1 protein causes the loss of surface CSF1R on murine BMMs.

    Fig. S5. Recombinant NS1 from other flaviviruses has immunostimulatory activity.

    Fig. S6. Induction of cytokine mRNAs in C57BL/6 BMMs and human PBMCs in response to S2-derived NS1 protein.

    Fig. S7. NS1 activates cells via TLR4.

    Fig. S8. Controls for colocalization of NS1 and TLR4 on adherent PBMCs.

    Fig. S9. Inhibition of PBMC production of IL-8 in response to NS1 by anti-TLR4 antibody and TLR4 antagonist.

    Table S1. List of mouse primers for RT-PCR.

    Table S2. List of human primers for RT-PCR.

    Movie S1. TLR4 and NS1 colocalization on PBMCs; video of immunofluorescence confocal microscopy Z stack.

    Primary data tables (Excel file)

    References (57, 58)

  • Supplementary Material for:

    Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity

    Naphak Modhiran, Daniel Watterson, David A. Muller, Adele K. Panetta, David P. Sester, Lidong Liu, David A. Hume, Katryn J. Stacey,* Paul R. Young*

    *Corresponding author. E-mail: p.young{at}uq.edu.au (P.R.Y.); katryn.stacey{at}uq.edu.au (K.J.S.)

    Published 9 September 2015, Sci. Transl. Med. 7, 304ra142 (2015)
    DOI: 10.1126/scitranslmed.aaa3863

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Purification and characterization of DENV-2 NS1 from S2 cells.
    • Fig. S2. Dengue NS1 activates a mouse macrophage NF-κB reporter cell line.
    • Fig. S3. Polymyxin B can efficiently inhibit LPS in the presence of NS1.
    • Fig. S4. NS1 protein causes the loss of surface CSF1R on murine BMMs.
    • Fig. S5. Recombinant NS1 from other flaviviruses has immunostimulatory activity.
    • Fig. S6. Induction of cytokine mRNAs in C57BL/6 BMMs and human PBMCs in response to S2-derived NS1 protein.
    • Fig. S7. NS1 activates cells via TLR4.
    • Fig. S8. Controls for colocalization of NS1 and TLR4 on adherent PBMCs.
    • Fig. S9. Inhibition of PBMC production of IL-8 in response to NS1 by anti-TLR4 antibody and TLR4 antagonist.
    • Table S1. List of mouse primers for RT-PCR.
    • Table S2. List of human primers for RT-PCR.
    • Legend for movie S1
    • References (57, 58)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). TLR4 and NS1 colocalization on PBMCs; video of immunofluorescence confocal microscopy Z stack.
    • Primary data tables (Excel file)

    [Download Movie S1]

    [Download Primary data tables]

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