Yong-Jun Liu

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN− dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.

The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

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.

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.

Opposing Roles of Dectin-1 Expressed on Human Plasmacytoid Dendritic Cells and Myeloid Dendritic Cells in Th2 Polarization

Dendritic cells (DCs) can induce and control host immune responses. DC subset-dependent functional specialties and their ability to display functional plasticity, which is mainly driven by signals via pattern recognition receptors, identify DCs as immune orchestrators. A pattern recognition receptor, Dectin-1, is expressed on myeloid DCs and known to play important roles in Th17 induction and activation during fungal and certain bacterial infections. In this study, we first demonstrate that human plasmacytoid DCs express Dectin-1 in both mRNA and protein levels. More interestingly, Dectin-1–activated plasmacytoid DCs promote Th2-type T cell responses, whereas Dectin-1–activated myeloid DCs decrease Th2-type T cell responses. Such contrasting outcomes of Th2-type T cell responses by the two DC subsets are mainly due to their distinct abilities to control surface OX40L expression in response to β-glucan. This study provides new insights for the regulation of host immune responses by Dectin-1 expressed on DCs.

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.

Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendritic Cells

Type I IFNs are crucial mediators of human innate and adaptive immunity and are massively produced from plasmacytoid dendritic cells (pDCs). IFN regulatory factor (IRF)7 is a critical regulator of type I IFN production when pathogens are detected by TLR 7/9 in pDC. However, hyperactivation of pDC can cause life-threatening autoimmune diseases. To avoid the deleterious effects of aberrant pDC activation, tight regulation of IRF7 is required. Nonetheless, the detailed mechanisms of how IRF7 transcription is regulated in pDC are still elusive. MYC is a well-known highly pleiotropic transcription factor; however, the role of MYC in pDC function is not well defined yet. To identify the role of transcription factor MYC in human pDC, we employed a knockdown technique using human pDC cell line, GEN2.2. When we knocked down MYC in the pDC cell line, production of IFN-stimulated genes was dramatically increased and was further enhanced by the TLR9 agonist CpGB. Interestingly, MYC is shown to be recruited to the IRF7 promoter region through interaction with nuclear receptor corepressor 2/histone deacetylase 3 for its repression. In addition, activation of TLR9-mediated NF-κB and MAPK and nuclear translocation of IRF7 were greatly enhanced by MYC depletion. Pharmaceutical inhibition of MYC recovered IRF7 expression, further confirming the negative role of MYC in the antiviral response by pDC. Therefore, our results identify the novel immunomodulatory role of MYC in human pDC and may add to our understanding of aberrant pDC function in cancer and autoimmune disease.

Structural and functional analyses of human DDX41 DEAD domain

DEAD-box proteins, which are named after the strictly conserved amino acid sequence Asp-Glu-Ala-Asp, were first identified as a distinct family in the late 1980s when alignments based on eight homologues of the yeast eIF4A highlighted the presence of several conserved motifs (Linder et al., 1989). DEAD-box proteins are widely distributed in different life forms, ranging from bacteria to human and constitute the largest RNA helicase family (Jiang et al., 2016). They are involved in many aspects of RNA metabolism, such as splicing, mRNA export, transcriptional and translational regulation, ribosome biogenesis and RNA decay (Rocak and Linder, 2004). The core of DEAD-box proteins is organized into two major domains. Domain 1 (DEAD domain) consists of motifs Q, I (Walker A, P-loop), II (Walker B, DEAD-box), Ia, GG, Ib and III, whereas domain 2 (Helicase domain) consists of motifs IV, V and VI. Different motifs are involved in nucleotide binding (Q, I and II), RNA binding (Ia, Ib, IV and V) and ATP hydrolysis (III and possibly VI). Compared with the two conserved domains, the Nand C-terminal regions are variable and divergent. Their functions are not fully characterized, but they are thought to confer their own specificity on different proteins (Hogbom et al., 2007). The recognition of pathogen-associated molecular patterns (PAMPs) of pathogens by pattern recognition receptors (PRRs) is important for the induction of type I interferons (IFN) (Medzhitov and Janeway, 2000). DDX41, a member of the DEAD-box proteins, containing a disordered N-terminal region, a DEAD domain and a Helicase domain (Fig. 1A), was identified as an intracellular DNA sensor in myeloid dendritic cells (mDCs) by Yong-Jun Liu’s group. They showed that DDX41 directly binds DNA and STING via its DEAD domain and triggers activation of signaling mediated by mitogen-activated protein kinases TBK1 and transcription factor IRF3, resulting IFN production (Zhang et al., 2011). DDX41 can also detect bacterial secondary messengers like cyclic di-GMP (c-di-GMP) and cyclic di-AMP (c-di-AMP), leading to formation of a complex with STING. This complex transmits the signal of bacterial intrusion to TBK1-IRF3 and activates the interferon response (Parvatiyar et al., 2012). Phosphorylation of Tyr414 of DDX41 is a pre-requisite for foreign dsDNA recognition and recruitment of STING. Besides, BTK’s kinase domain can bind the DEAD domain of DDX41 (Lee et al., 2015). After immune response, DDX41 will be ubiquitinated by TRIM21 through K48-mediated linkage for degradation. The ubiquitination sites are Lys9 and Lys115 (Zhang et al., 2013). Somatic DDX41 mutations have been reported in the study of sporadic acute myeloid leukemia (AML) syndrome (Ding et al., 2012). A familial MDS/ AML syndrome characterized by long latency and germline mutations in DDX41 gene is also identified (Polprasert et al., 2015). DDX41 can associate with spliceosomal proteins, and its defects lead to loss of tumor suppressor function due to altered pre-mRNA splicing and RNA processing (Polprasert et al., 2015). Although DDX41 plays important roles in innate immunity and diseases, the precise mechanism as well as the extent of involvement the protein in these processes is poorly understood. Here, we report the crystal structure of human DDX41 (hDDX41) DEAD domain complexed with an SO4 2− and an Mg to 2.26 Å resolution. There are strong interactions between different motifs to stabilize the whole structure. The P-loop presents in a half-open conformation. The DEAD domain protein can bind ADP and AMP but not ATP in vitro because of the steric hindrance. Most mutated amino acids related with familial MDS/AML are conserved. In addition, the N-terminal disordered region (amino acid 1–152) is shown targeting hDDX41 protein to the nucleus. Our study provides basic structural information for further researches on hDDX41 biological function and valuable insights for the treatment of DDX41-related diseases in the future. The crystal structure of hDDX41 DEAD domain was solved by molecular replacement using the structure of DDX5 domain I (PDB code: 3FE2) as the search model and refined to 2.26 Å resolution with an R factor of 0.19 (Rfree = 0.23). Details of data collection and refinement statistics are listed in Table S1. The crystal used for data collection belonged to space group P21. One asymmetric unit consists of two molecules of the protein based on the calculated solvent content of 44.15%. The hDDX41 DEAD domain consists of an α/β fold, which is similar to those observed for other members of the DEAD-box proteins for which structures are available. The overall structure consists of ten α-helices (α1–α10) and a β-sheet formed by eight β-

Erratum to: Structural and functional analyses of human DDX41 DEAD domain

Yan Jiang, Yanping Zhu, Weicheng Qiu, Yong-Jun Liu, Genhong Cheng, Zhi-Jie Liu, Songying Ouyang 1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2 Baylor Research Institute, Baylor Scott and White Health, Dallas, TX 75246, USA 3 Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA 4 Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China & Correspondence: zjliu@ibp.ac.cn (Z.-J. Liu), ouyangsy@ibp.ac.cn (Songying Ouyang)