DHX9 pairs with IPS-1 to sense double-stranded RNA in myeloid dendritic cells

Abstract

The innate immune system is equipped with many molecular sensors for microbial DNA/RNA to quickly mount antimicrobial host immune responses. In this paper, we identified DHX9, a DExDc helicase family member, as an important viral dsRNA sensor in myeloid dendritic cells (mDCs). Knockdown of DHX9 expression by small heteroduplex RNA dramatically blocked the ability of mDCs to produce IFN-α/β and proinflammatory cytokines in response to polyinosine-polycytidylic acid, influenza A, and reovirus. DHX9 could specifically bind polyinosine-polycytidylic acid via its double-strand RNA binding motifs. DHX9 interacted with IPS-1 via the HelicC-HA2-DUF and CARD domains of DHX9 and IPS-1, respectively. Knockdown of DHX9 expression in mDCs blocked the activation of NF-κB and IFN regulatory factor 3 by dsRNA. Collectively, these results suggest that DHX9 is an important RNA sensor that is dependent on IPS-1 to sense pathogenic RNA.

Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1

DHX9 senses poly I:C and RNA virus in mDCs. A, Immunoblot (IB) showing the knockdown efficiency of shRNAs targeting the indicated proteins in D2SC cells, a mouse mDC cell line. Nontargeting shRNA served as a control (first left lane). Actin blots are shown as loading controls (lower panel). ELISA of IFN-α/β, TNF-α, and IL-6 production from D2SC cells with the indicated shRNA after a 16-h stimulation with 2.5 µg/ml of short poly I:C (B), 2.5 µg/ml long poly I:C (C), 1 µg/ml poly dA-dT (D), 2 mM CpG A (E), reovirus (F), or Flu A virus (G). Nucleic acids were delivered to the cells by Lipofectamine 2000. Viruses were added to the cells at a multiplicity of infection (MOI) = 10 for 2 h. Cells were collected, washed with PBS, and cultured for an additional 16 h. N-STM, scrambled shRNA-treated D2SC cells without stimulation.

FIGURE 2

DHX9 knockdown in BMDCs abolishes their cytokine responses to poly I:C and reovirus. A, Immunoblot (IB) showing the knockdown efficiency of shRNA targeting the indicated genes in BMDCs. Nontargeting shRNA served as a control (first left lane). β-Actin blots are shown as loading controls (lower panel). ELISA of IFN-α/β production by BMDCs with the indicated shRNA after 16 h stimulation with 2.5 µg/ml short poly I:C (B), 2.5 µg/ml of long poly I:C (C), or reovirus (D) at a multiplicity of infection (MOI) = 10. Nucleic acids were delivered to the cells by Lipofectamine 2000. E, qPCR of reovirus copies from BMDCs with shRNA knockdown of indicated protein. BMDCs were incubated with reovirus at MOI of 10 for 2 h. Cells were collected, washed with PBS, and cultured for an additional 16 h, followed by RNA extract and RT-PCR. F, qPCR of TNF-α and IFN-β mRNA extract from BMDCs treated with scrambled shRNA (control) or shRNA targeting DHX9. N-STM, scrambled shRNA-treated D2SC cells without stimulation.

FIGURE 3

DHX9 requires DSRM domain to bind poly I:C. A, Immunoblot using anti-HA Ab of pulldown assays in which purified HA-DHX9 or HA-DHX36 recombinant proteins were incubated with biotinylated poly I:C, poly dA-dT, poly dG-dC, poly dI-dC, CpG A, or CpG B, followed by addition of NA beads. B, Schematic representations of full-length and serial truncations of DHX9. HA-DHX9, DHX9 full size; HA-9ΔDSRM, DHX9-deleted DSRM; HA-9ΔDEXDc, DHX9-deleted DSRM and DEXDc; HA-9ΔHelicC, DHX9-deleted DSRM, DEADc and HelicC; HA-9CΔDUF, DHX9-deleted DUF; HA-9CΔHD, DHX9-deleted HA2 and DUF. Numbers denote amino acid residues. C, Immunoblot using anti-HA Ab of pulldown assays in which purified HA-tagged serial truncations of DHX9 were individually incubated with biotinylated poly I:C, followed by addition of NA beads. D, Immunoblot using anti-HA Ab of pulldown competition assays in which 0.5, 5, or 50 µg/ml CpG B, poly A, poly A:U, poly I:C, or CpG A was added to a mixture of HA-DHX9 protein plus biotinylated poly I:C, followed by addition of NA beads. DEAD, Asp-Glu-Ala-Asp box motif.

FIGURE 4

Overexpression of DHX9 leads to enhanced Ifnb promoter activity in response to poly I:C. Fold activation of the Ifnb promoter in HEK293T cells transfected with IFN-β luciferase reporter (100 ng) plus MyD88, IPS-1, DHX9, or DHX9-deleted DSRM expression vectors at 20, 100, or 200 ng concentrations. The Renilla-luciferase reporter gene (2 ng) was transfected simultaneously for the internal control. At 30 h posttransfection, cells were without stimulation or stimulated for 6 h with 2.5 µg/ml poly I:C delivered by Lipofectamine 2000 and then harvested.

FIGURE 5

HelicC-HA2-DUF domain of DHX9 and CARD domain of IPS-1 required for DHX9:IPS-1 interaction. A, Immunoblot (IB) of indicated proteins precipitated with anti-DHX9 Ab from whole-cell lysates of unstimulated (−), short poly I:C-, or long poly I:C-stimulated (+) D2SC cells. IgG Ab served as a control; input, D2SC lysates used in immunoprecipitation assays; B, IB using anti-HA Ab of pulldown assays in which purified MyC-IPS-1 was incubated with full-length or truncated HA-DHX9 protein, followed by addition of anti-MyC beads; C, schematic representations of full-length and serial truncations of IPS-1. MyC-IPS-1: IPS-1 full size; Myc-ΔCARD: IPS-1–deleted CARD. D, IB using anti-Myc Ab of pulldown assays in which purified HA-DHX9 was incubated with Myc-IPS-1 or Myc-ΔCARD protein, followed by addition of anti-HA beads. E, IB showing the knockdown efficiency of shRNA targeting DHX9 in RIG-I deficiency D2SC. Non-targeting shRNA served as a control (first left lane). β-Actin blots are shown as loading controls (lower panel). ELISA of IFN-α production by D2SC after 16 h stimulation with 2.5 µg/ml short poly I:C. CARD, N-terminal caspase recruitment domain; N-STM, scrambled shRNA-treated RIGI deficiency D2SC cells without stimulation.

FIGURE 6

DHX9 and IPS-1 are required for activating the NF-κB and IFN-response signal transduction pathways upon dsRNA stimulation. Immunoblot of indicated proteins from lysates of D2SC with indicated shRNA after stimulation with 2.5 µg/ml poly I:C delivered by Lipofectamine 2000. β-Actin served as loading control.