Zhiqiang Zhang

The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells

The recognition of pathogenic DNA is important to the initiation of antiviral responses. Here we report the identification of DDX41, a member of the DEXDc family of helicases, as an intracellular DNA sensor in myeloid dendritic cells (mDCs). Knockdown of DDX41 expression by short hairpin RNA blocked the ability of mDCs to mount type I interferon and cytokine responses to DNA and DNA viruses. Overexpression of both DDX41 and the membrane-associated adaptor STING together had a synergistic effect in promoting Ifnb promoter activity. DDX41 bound both DNA and STING and localized together with STING in the cytosol. Knockdown of DDX41 expression blocked activation of the mitogen-activated protein kinase TBK1 and the transcription factors NF-κB and IRF3 by B-form DNA. Our results suggest that DDX41 is an additional DNA sensor that depends on STING to sense pathogenic DNA.

The E3 ubiquitin ligase TRIM21 negatively regulates the innate immune response to intracellular double-stranded DNA

DDX41 is a sensor of intracellular double-stranded DNA (dsDNA) in myeloid dendritic cells (mDCs) that triggers a type I interferon response via the signaling adaptor STING. We identified the E3 ligase TRIM21 as a DDX41-interacting protein and found that knockdown of or deficiency in TRIM21 resulted in enhanced type I interferon responses to intracellular dsDNA and DNA viruses. Overexpression of TRIM21 resulted in more degradation of DDX41 and less production of interferon-β (IFN-β) in response to intracellular dsDNA. The SPRY-PRY domain of TRIM21 interacted with the DEADc domain of DDX41. Lys9 and Lys115 of DDX41 were the targets of TRIM21-mediated ubiquitination. TRIM21 is therefore an interferon-inducible E3 ligase that induces the Lys48 (K48)-linked ubiquitination and degradation of DDX41 and negatively regulates the innate immune response to intracellular dsDNA.

The DHX33 RNA Helicase Senses Cytosolic RNA and Activates the NLRP3 Inflammasome

The NLRP3 inflammasome plays a major role in innate immune responses by activating caspase-1, resulting in secretion of interleukin-18 (IL-18) and IL-1β. Although cytosolic double-stranded RNA (dsRNA) and bacterial RNA are known to activate the NLRP3 inflammasome, the upstream sensor is unknown. We investigated the potential function of DExD/H-box RNA helicase family members (previously shown to sense cytosolic DNA and RNA to induce type 1 interferon responses) in RNA-induced NLRP3 inflammasome activation. Among the helicase family members tested, we found that targeting of DHX33 expression by short hairpin RNA efficiently blocked the activation of caspase-1 and secretion of IL-18 and IL-1β in human macrophages that were activated by cytosolic poly I:C, reoviral RNA, or bacterial RNA. DHX33 bound dsRNA via the helicasexa0Cxa0domain. DHX33 interacted with NLRP3 and formed the inflammasome complex following stimulation with RNA. We therefore identified DHX33 as axa0cytosolic RNA sensor that activates the NLRP3 inflammasome.

Human NLRP3 inflammasome senses multiple types of bacterial RNAs

Significance The innate immune system has evolved to protect the host from potential pathogens. The nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome is one of the platforms that can sense pathogenic bacteria, which can cause fatal bacterial infections. Here we show that all three types of bacteria-derived RNA—mRNA, tRNA, and rRNAs—as well as synthetic 20-guanosine ssRNA, are capable of activating the NLRP3 inflammasome and inducing human macrophages to secrete inflammatory cytokines. Interestingly, only bacterial mRNA is able to activate the murine Nlrp3 inflammasome. Therefore human macrophages may have evolved in a unique fashion, adapting to the bacterial environment. This study could provide important clues for developing efficient medicines for bacterial infections and various immunotherapies such as anticancer vaccines. Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.

Identification of a role for TRIM29 in the control of innate immunity in the respiratory tract

The respiratory tract is heavily populated with innate immune cells, but the mechanisms that control such cells are poorly defined. Here we found that the E3 ubiquitin ligase TRIM29 was a selective regulator of the activation of alveolar macrophages, the expression of type I interferons and the production of proinflammatory cytokines in the lungs. We found that deletion of TRIM29 enhanced macrophage production of type I interferons and protected mice from infection with influenza virus, while challenge of Trim29−/− mice with Haemophilus influenzae resulted in lethal lung inflammation due to massive production of proinflammatory cytokines by macrophages. Mechanistically, we demonstrated that TRIM29 inhibited interferon-regulatory factors and signaling via the transcription factor NF-κB by degrading the adaptor NEMO and that TRIM29 directly bound NEMO and subsequently induced its ubiquitination and proteolytic degradation. These data identify TRIM29 as a key negative regulator of alveolar macrophages and might have important clinical implications for local immunity and immunopathology.

The E3 Ubiquitin Ligase Tripartite Motif 33 Is Essential for Cytosolic RNA–Induced NLRP3 Inflammasome Activation

NLRP3 is a key component of caspase-activating macromolecular protein complexes called inflammasomes. It has been found that DHX33 is a cytosolic dsRNA sensor for the NLRP3 inflammasome, which induces caspase-1–dependent production of IL-1β and IL-18 upon activation. However, how the cytosolic dsRNAs induce the interaction between DHX33 and the NLRP3 inflammasome remains unknown. In this study, we report that TRIM33, a member of the tripartite motif (TRIM) family, can bind DHX33 directly and induce DHX33 ubiquitination via the lysine 218 upon dsRNA stimulation. Knocking down of TRIM33 abolished the dsRNA-induced NLRP3 inflammasome activation in both THP-1–derived macrophages and human monocyte-derived macrophages. The ubiquitination of DHX33 by TRIM33 is lysine 63 specific and is required for the formation of the DHX33–NLRP3 inflammasome complex.

The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells.

In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA (dsRNA) sensor in myeloid dendritic cells (mDCs). The knockdown of DHX33 using small heteroduplex RNA (shRNA) blocked the ability of mDCs to produce type I interferon (IFN) in response to poly I:C and reovirus. The HELICc domain of DHX33 was shown to bind poly I:C. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 (also referred to MAVS and VISA). The inhibition of DHX33 expression by RNA interference blocked the poly I:C-induced activation of MAP kinases, NF-κB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I:C and RNA viruses in mDCs.

DHX15 Senses Double-Stranded RNA in Myeloid Dendritic Cells

Many members of the DEXD/H box helicase family play important roles in the innate immune system against viral infection. Therefore, we isolated dsRNA complex in myeloid dendritic cells. We found that DHx15, a DEXDc helicase family member, is one of the components of this complex. Knockdown of DHX15 expression by short hairpin RNA efficiently reduced the ability of myeloid dendritic cells to produce IFN-β, IL-6, and TNF-α in response to dsRNA and RNA virus. DHX15 specifically bound polyinosine-polycytidylic acid via its helicase C-terminal domain. DHX15 interacted with MAVS and formed a complex following stimulation with polyinosine-polycytidylic acid. The N-terminal domain containing a DEXDc motif in DHX15 bound the C terminus of MAVS. DHX15 is required to activate IRF3 phosphorylation as well as NF-κB and MAPK signaling during RNA virus infection. We, therefore, identified DHX15 as a new RNA virus sensor mediated by MAVS to activate the immune responses to RNA.

Corrigendum: The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells

Corrigendum: The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells

Corrigendum: Identification of a role for TRIM29 in the control of innate immunity in the respiratory tract

Nat. Immunol.; doi:10.1038/ni.3580; corrected online 10 October 2016 In the version of this article initially published online, the immunoblots in Figures 1a, 5a, 5b, 5c, 5d, 5f, 6a, 6b, 7a, 7b, 8a and 8b were in color. These have been replaced with black and white immunoblots. The error has been corrected for the print, PDF and HTML versions of this article.