Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells

Abstract

Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I IFN responses in plasmacytoid dendritic cells (pDCs). Previous studies suggest the presence of myeloid differentiation primary response gene 88 (MyD88)-dependent DNA sensors other than TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)-binding proteins from human pDCs, pDC-cell lines, and interferon regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively bound the aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36), whereas CpG-B selectively bound DExD/H-box helicase 9 (DHX9). Although the aspartate-glutamate-alanine-histidine box motif (DEAH) domain of DHX36 was essential for CpG-A binding, the domain of unknown function 1605 (DUF1605 domain) of DHX9 was required for CpG-B binding. DHX36 is associated with IFN-alpha production and IRF7 nuclear translocation in response to CpG-A, but DHX9 is important for TNF-alpha and IL-6 production and NF-kappaB activation in response to CpG-B. Knocking down DHX9 or DHX36 significantly reduced the cytokine responses of pDCs to a DNA virus but had no effect on the cytokine responses to an RNA virus. We further showed that both DHX9 and DHX36 are localized within the cytosol and are directly bound to the Toll-interleukin receptor domain of MyD88 via their helicase-associated domain 2 and DUF domains. This study demonstrates that DHX9/DHX36 represent the MyD88-dependent DNA sensors in the cytosol of pDCs and suggests a much broader role for DHX helicases in viral sensing.

Fig. 1.

DHX36 and DHX9 associate with CpG-A and CpG-B, respectively. (A) Coomassie staining of CpG-A–associated proteins and CpG-B–associated proteins purified with NA beads from primary pDCs (Left), from a pDC cell line (Gen2.2) (Center), and from a B-cell line (Namalwa) (Right) treated with both CpG-A and CpG-B (CpG-A?B), with biotin-CpG-A, or with biotin-CpG-B. Proteins identified by LC-MS are as indicated. The sample treated with CpG-A?B served as control. (B) Purified CpG-A–bound proteins and CpG-B–-bound proteins from pDCs (Left), from Gen2.2 cells (Center), and from Namalwa cells (Right) were analyzed by immunoblotting with anti-DHX36 and anti-DHX9 antibodies, as indicated. (C) Phylogeny of 59 human DExD/H helicases was analyzed by ClustalW analysis.

Fig. 2.

Domains responsible for specific interactions of DHX36 with CpG-A and of DHX9 with CpG-B. (A) Pulldown assays were performed by incubating whole-cell lysates from HA-DHX36– and HA-DHX9–expressing 293T cells with biotin-CpG-A or biotin-CpG-B with NA beads. Bound proteins were analyzed by immunoblotting with anti-HA antibody. (B) Pulldown and immunoblotting assays were performed as described in A but with whole-cell lysates from HA-Mock or HA-TLR9–expressing 293T cells. (C) Competitive pulldown assays. (Left) Whole-cell lysates from HA-DHX36–expressing 293T cells were preincubated in the absence or presence of nonconjugated CpG-A (10× and 50×; 1×=1 μM) or CpG-B (50×) and then were incubated with 1× of biotin-CpG-A or biotin-CpG-B along with NA beads. (Right) Whole-cell lysates from HA-DHX9–expressing 293T cells were preincubated in the absence or presence of nonconjugated CpG-B (10× and 50×) or CpG-A (50×) and then were incubated with 1× of biotin-CpG-B or biotin-CpG-A along with NA beads. Bound proteins were analyzed by immunoblotting with anti-HA antibody. (D) Schematic representations of DHX36 and DHX9 and their serial-deletion mutants. Numbers denote amino acid residues. DEAH, aspartate-glutamate-alanine-histidine box motif; DSRM, double-stranded RNA binding motif; DUF 1605, domain of unknown function 1605; HA2, helicase-associated domain 2; HelicC, helicase C-terminal domain. HA2, helicase-associated domain 2. (E and F) Pulldown assays were performed by incubating lysates from 293T cells expressing HA-tagged full-length and deletion mutants of DHX36 with biotin-CpG-A (E) or by incubating HA-tagged full-length and deletion mutants of DHX9 with biotin-CpG-B (F) along with NA beads. Bound proteins were analyzed by immunoblotting with anti-HA. NS, nonspecific band.

Fig. 3.

DHX36 and DHX9 are critical for microbial DNA-mediated cytokine responses. (A) Gen2.2 cells were transfected with nonspecific siRNA (siControl), two siRNAs targeting DHX36 (siDHX36-1 and siDHX36-2), two siRNAs targeting DHX9 (siDHX9-1 and siDHX9-2), or a MyD88-targeting siRNA (siMyD88). Endogenous DHX36, DHX9, and MyD88 were monitored by immunoblotting with anti-DHX36, anti-DHX9, and anti-MyD88 antibodies, as indicated at the right. NS, nonspecific bands. (B and C) ELISAs to monitor IFN-α production from Gen2.2 cells transfected with siRNA, as indicated, upon treatment with 2 μM of CpG-A for 12 h or 0.5 μM of CpG-B for 12 h (B) or to monitor TNF-α and IL-6 production upon treatment with 0.5 μM of CpG-B for 4 h (C). (D) ELISAs to monitor IFN-α and TNF-α production from Gen2.2 cells transfected with siRNA, as indicated, upon treatment with HSV or Flu A (multiplicity of infection, 10) for 12 h. Data are mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 versus sample transfected with siControl and treated with CpG or virus.

Fig. 4.

DHX36 and DHX9 interact with MyD88 in the cytosol of pDCs. (A) Confocal images of pDCs untreated or treated with CpG-A or CpG-B. The three columns on the left show staining of DHX9 and TfR (an early endosome marker) or LAMP1 (a late endosome marker). The column on the right shows staining of DHX36 and TfR or LAMP1. (B) Endosomal fractionation. Fractionated early endosome and late endosome by ultracentrifugation based on step-gradient sucrose was examined by immunoblotting with anti-DHX9, anti-DHX36, anti-TfR, anti-LAMP1, anti-histone deacetylase 1 (anti-HDAC1), and anti-β-Actin antibodies. (C) Schematic representations of MyD88 and two deletion mutants. Numbers denote amino acid residues. DD, death domain; TIR, Toll-IL-1R homologous domain. (D) Lysates from 293T cells expressing Myc-tagged full-length or deletion mutants of MyD88 with HA-DHX36 or HA-DHX9 were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-Myc antibody, or vice versa. (E) Lysates from 293T cells expressing HA-tagged full-length or deletion mutants of DHX36 or DHX9 with Myc-MyD88 were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-HA antibody.

Fig. 5.

DHX36 and DHX9 trigger downstream signaling cascades. (A and B) Nuclear fractions from Gen2.2 cells stimulated with 2 μM of CpG-A (A) or 0.5 μM of CpG-B (B) in a time-dependent manner were immunoblotted with anti-IRF7 or anti-p50 antibodies. HDAC1 and β-Actin were used as nuclear and cytosolic markers, respectively. (C) Nuclear fractions from Gen2.2 cells transfected with siRNA, as indicated, upon treatment with 2 μM of CpG-A for 3 h (Upper) or 0.5 μM of CpG-B for 1 h (Lower) were immunoblotted with anti-IRF7 or anti-p50 antibodies. HDAC1 was used as a loading control. (D) Endogenous DHX36, DHX9, TLR9, and MyD88 were monitored by immunoblotting with anti-DHX36, anti-DHX9, anti-TLR9, and anti-MyD88 antibodies, as indicated at the right.