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The DNA damage response (DDR) induces the expression of type I interferons (IFNs), but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Double-stranded DNA (dsDNA) derived from pathogen- or host-damaged cells triggers innate immune responses when exposed to cytoplasm. However, the machinery underlying the primary recognition of intracellular dsDNA is obscure. Here we show that the DNA damage sensor, meiotic recombination 11 homolog A (MRE11), serves as a cytosolic sensor for dsDNA. Cells with a mutation of MRE11 gene derived from a patient with ataxia-telangiectasia-like disorder, and cells in which Mre11 was knocked down, had defects in dsDNA-induced type I IFN production. MRE11 physically interacted with dsDNA in the cytoplasm and was required for activation of stimulator of IFN genes (STING) and IRF3. RAD50, a binding protein to MRE11, was also required for dsDNA responses, whereas NBS1, another binding protein to MRE11, was dispensable. Collectively, our results suggest that the MRE11-RAD50 complex plays important roles in recognition of dsDNA and initiation of STING-dependent signaling, in addition to its role in DNA-damage responses.

Aluminum-based adjuvants (aluminum salts or alum) are widely used in human vaccination, although their mechanisms of action are poorly understood. Here we report that, in mice, alum causes cell death and the subsequent release of host cell DNA, which acts as a potent endogenous immunostimulatory signal mediating alum adjuvant activity. Furthermore, we propose that host DNA signaling differentially regulates IgE and IgG1 production after alum-adjuvanted immunization. We suggest that, on the one hand, host DNA induces primary B cell responses, including IgG1 production, through interferon response factor 3 (Irf3)-independent mechanisms. On the other hand, we suggest that host DNA also stimulates 'canonical' T helper type 2 (T(H)2) responses, associated with IgE isotype switching and peripheral effector responses, through Irf3-dependent mechanisms. The finding that host DNA released from dying cells acts as a damage-associated molecular pattern that mediates alum adjuvant activity may increase our understanding of the mechanisms of action of current vaccines and help in the design of new adjuvants.

Fragments of double-stranded DNA (dsDNA) forming a right-handed helical structure (B-DNA) stimulate cells to produce type I interferons (IFNs). While an adaptor molecule, IFN-beta promoter stimulator 1 (IPS-1), mediates dsDNA-induced cellular signaling in human cells, the underlying molecular mechanism is not fully understood. Here, we demonstrate that the extrachromosomal histone H2B mediates innate antiviral immune responses in human cells. H2B physically interacts with IPS-1 through the association with a newly identified adaptor, CIAO (COOH-terminal importin 9-related adaptor organizing histone H2B and IPS-1), to transmit the cellular signaling for dsDNA but not immunostimulatory RNA. Extrachromosomal histone H2B was biologically crucial for cell-autonomous responses to protect against multiplication of DNA viruses but not an RNA virus. Thus, the present findings provide evidence indicating that the extrachromosomal histone H2B is engaged in the signaling pathway initiated by dsDNA to trigger antiviral innate immune responses.

Microbial nucleic acids are critical for the induction of innate immune responses, a host defense mechanism against infection by microbes. Recent studies have indicated that double-stranded DNA (dsDNA) induces potent innate immune responses via the induction of type I IFN (IFN) and IFN-inducible genes. However, the regulatory mechanisms underlying dsDNA-triggered signaling are not fully understood. Here we show that the translocation and assembly of the essential signal transducers, stimulator of IFN genes (STING) and TANK-binding kinase 1 (TBK1), are required for dsDNA-triggered innate immune responses. After sensing dsDNA, STING moves from the endoplasmic reticulum (ER) to the Golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with TBK1. The addition of an ER-retention signal to the C terminus of STING dampens its ability to induce antiviral responses. We also show that STING co-localizes with the autophagy proteins, microtubule-associated protein 1 light chain 3 (LC3) and autophagy-related gene 9a (Atg9a), after dsDNA stimulation. The loss of Atg9a, but not that of another autophagy-related gene (Atg7), greatly enhances the assembly of STING and TBK1 by dsDNA, leading to aberrant activation of the innate immune response. Hence Atg9a functions as a regulator of innate immunity following dsDNA stimulation as well as an essential autophagy protein. These results demonstrate that dynamic membrane traffic mediates the sequential translocation and assembly of STING, both of which are essential processes required for maximal activation of the innate immune response triggered by dsDNA.

Mammalian innate immunity possesses a distinct system to recognize aberrant DNA inside the cell. One class of DNA sensors is the Toll-like receptor 9, which is expressed in the specialized immune cells, binds to single-stranded DNA in the endosome to transmit cellular signaling through myeloid differentiation primary response protein 88 (MyD88). Another class of DNA sensors exists in the cytoplasm of most type of cells in the tissue, detecting double-stranded DNA to signal through TANK-binding kinase-1 (TBK1)-mediated type-I interferon production and apoptosis-associated speck-like protein containing a CARD (ASC)-mediated IL-1beta secretion. Since DNA sensors have potential to recognize aberrant DNA of both self and nonself origin, their physiological roles in microbial infection, tissue damage, autoimmune diseases, and DNA-based therapeutic applications are being intensively investigated.

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.

DNA in microbes or host cells is normally sequestered from the immune system, and therefore inert, but becomes an active immunostimulatory molecule during infection or tissue damage. Recent evidence suggests that Toll-like receptor (TLR)9, currently the only known immune sensor for DNA, recognizes more diverse elements in its ligand than initially thought, and must cooperate with additional host factors to provoke an optimal innate immune response in the physiological environment. Moreover, the innate immune system possesses a TLR9-independent, as-yet-undefined intracellular recognition machinery of double-stranded DNA that induces type I interferons through distinct signaling pathways. TLR9-dependent and TLR9-independent immune recognition of DNA might play crucial roles in DNA-associated protective immunity and in pathological autoimmunity.

Introducing double-stranded DNA (dsDNA) into the cytoplasm of macrophages and dendritic cells triggers the activation of these professional antigen-presenting cells (APCs). This process is characterized by the up-regulation of costimulatory molecules and the production of various cytokines, chemokines, and antibacterial/viral factors. Current findings indicate that interferon-beta (IFN-beta) plays a key role in the stimulatory cascade triggered by dsDNA. Both immune and non-immune cells respond to intracytoplasmic dsDNA by up-regulating IFN-beta) expression, a process that reduces host susceptibility to infection. The immune activation induced by dsDNA is independent of MyD88, TRIF and DNA-PKcs, indicating that a Toll-like receptor-independent mechanism underlies the cellular activation mediated by intracytoplasmic dsDNA.

The innate immune system recognizes nucleic acids during infection or tissue damage; however, the mechanisms of intracellular recognition of DNA have not been fully elucidated. Here we show that intracellular administration of double-stranded B-form DNA (B-DNA) triggered antiviral responses including production of type I interferons and chemokines independently of Toll-like receptors or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding interferon-beta through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-kappaB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of interferon-beta and NF-kappaB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5'-->3' direction, and that a 5' motif can block recognition of immediately 3' sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs.

The immunoreceptor NKG2D originally identified in natural killer (NK) cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DL) is induced by the DNA damage response (DDR), which is often activated constitutively in cancer cells, revealing them to NK cells as a mechanism of immunosurveillance. Here, we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines that express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. Irf3(+/-);Eμ-Myc mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared with Irf3(+/+);Eμ-Myc mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance.

Successful vaccines contain not only protective antigen(s) but also an adjuvant component that triggers innate immune activation and is necessary for their optimal immunogenicity. In the case of DNA vaccines, this consists of plasmid DNA; however, the adjuvant element(s) as well as its intra- and inter-cellular innate immune signalling pathway(s) leading to the encoded antigen-specific T- and B-cell responses remain unclear. Here we demonstrate in vivo that TANK-binding kinase 1 (TBK1), a non-canonical IκB kinase, mediates the adjuvant effect of DNA vaccines and is essential for its immunogenicity in mice. Plasmid-DNA-activated, TBK1-dependent signalling and the resultant type-I interferon receptor-mediated signalling was required for induction of antigen-specific B and T cells, which occurred even in the absence of innate immune signalling through a well known CpG DNA sensor—Toll-like receptor 9 (TLR9) or Z-DNA binding protein 1 (ZBP1, also known as DAI, which was recently reported as a potential B-form DNA sensor). Moreover, bone-marrow-transfer experiments revealed that TBK1-mediated signalling in haematopoietic cells was critical for the induction of antigen-specific B and CD4+ T cells, whereas in non-haematopoietic cells TBK1 was required for CD8+ T-cell induction. These data suggest that TBK1 is a key signalling molecule for DNA-vaccine-induced immunogenicity, by differentially controlling DNA-activated innate immune signalling through haematopoietic and non-haematopoietic cells. [ABSTRACT FROM AUTHOR]

Introducing double-stranded DNA (dsDNA) into the cytoplasm of macrophages and dendritic cells triggers the activation of these professional antigen-presenting cells (APCs). This process is characterized by the up-regulation of costimulatory molecules and the production of various cytokines, chemokines, and antibacterial/viral factors. Current findings indicate that interferon-β (IFN-β) plays a key role in the stimulatory cascade triggered by dsDNA. Both immune and non-immune cells respond to intracytoplasmic dsDNA by up-regulating IFN-β) expression, a process that reduces host susceptibility to infection. The immune activation induced by dsDNA is independent of MyD88, TRIF and DNA-PKcs, indicating that a Toll-like receptor-independent mechanism underlies the cellular activation mediated by intracytoplasmic dsDNA. [ABSTRACT FROM AUTHOR]

Objective: To examine the effect of suppressive oligodeoxynucleotides (ODNs) on the pulmonary inflammation induced by immunostimulatory CpG DNA.Design: Prospective, randomized, controlled study.Setting: Research laboratories.Subjects: RAW 264.7 murine macrophage-like cell line and BALB/c mice.Interventions: RAW 264.7 cells were incubated with bacterial DNA or CpG ODN, alone or combined with suppressive ODN. The in vivo effect of suppressive ODN was determined using an acute lung injury model. CpG ODN alone or combined with suppressive ODN was instilled into the mouse lung.Measurements and Main Results: Production of tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2 by RAW 264.7 cells were measured by enzyme-linked immunosorbent assay (ELISA), whereas their messenger RNA levels were determined by reverse transcriptase-polymerase chain reaction. Synthetic ODN containing CpG motifs (CpG ODN) mimicked the ability of bacterial DNA to stimulate the production of TNF-alpha and MIP-2. Suppressive ODN significantly inhibited the activation of RAW 264.7 cells by both bacterial DNA and CpG ODN. In the lung injury model, production of proinflammatory cytokines (TNF-alpha and IL-6) and chemokines (MIP-2 and KC) in bronchoalveolar lavage (BAL) fluids was measured by ELISA. Neutrophil accumulation in the alveolar spaces was also evaluated. Instillation of CpG ODN into the lungs of normal mice triggered the synthesis of TNF-alpha, IL-6, MIP-2, and KC. Suppressive ODN significantly blocked the production of these proinflammatory cytokines and chemokines and also reduced neutrophil mobilization into the alveolar spaces by CpG DNA.Conclusions: Proinflammatory cytokines and chemokines are up-regulated by CpG motifs in bacterial DNA. Suppressive ODN significantly inhibits the inflammatory response induced by CpG DNA in murine macrophages and the lung. This study supports the use of suppressive ODN to reduce the deleterious inflammatory responses induced by bacterial DNA. [ABSTRACT FROM AUTHOR]

Synthetic oligodeoxynucleotides (ODN) expressing non-methylated “CpG motifs” patterned after those present in bacterial DNA have characteristic immunomodulatory effects. CpG DNA is recognized as a pathogen-associated molecular pattern, and triggers a rapid innate immune response. CpG ODN are being harnessed for a variety of therapeutic uses, including as immune adjuvants, for cancer therapy, as anti-allergens, and as immunoprotective agents. The signal transduction pathway mediated by the engagement of CpG DNA with Toll-like receptor 9 (TLR9) is shared with other members of the TLR family. Recent studies demonstrate that formation and maturation of CpG DNA-containing endosomes are regulated by phosphatidylinositol 3 kinases and the Ras-associated GTP-binding protein, Rab5, which are essential for the initiation of TLR9-mediated signaling. [Copyright &y& Elsevier]