Biological DNA Sensor: The Impact of Nucleic Acids on Diseases and Vaccinology

USA

Preface

Deoxyribonucleic acid (DNA), the fundamental molecule that packs genetic instructions for the development of living organisms, has been discovered to hold yet another vital function for our biology – inflammation. The fact that DNA is an abundant commodity in our body and causes its immunogenic property is one that cannot be taken lightly. Now, we know that DNA induced inflammation is responsible for the pathogenicity of autoimmune and infectious diseases and metabolic disorders. Furthermore, we have evidence that DNA induced inflammation is involved in the development of cancer. Therefore the awareness of the immunogenic nature of dsDNA has provided us with the opportunity to gain further insights into the workings of human diseases. DNA seemed to be an ironic choice as a trigger molecule for inflammation. After all, who would expect that self-products containing our genetic blueprint could turn against us and induce adverse inflammatory reactions upon detection by the immune system? However, on deeper reflection, it does make practical sense to have nucleic acids as signaling entities that sound off the alarm to indicate impending danger to the body. Pathogens including viruses, bacteria and parasites, like human beings, have their genetic information stored in nucleic acids. Their invasion into the host system is likely to be associated with the introduction of their genetic materials and therefore it would be the most appropriate indication of pathogenic invasion. On the other hand, the release of our own DNA into the surrounding physiological environment could also indicate cell death as a result of trauma, which may invite infection to occur and require immune defenses to be put in place. Therefore in more ways than one, DNA is an aptly chosen alarm molecule for the immune system to react to danger. Regarding the potential impact it has on various aspects of our health, we have seen an explosion of research on DNA sensing in the past eight years. This surge in publication was due in part to the discovery that DNA could be sensed not only by TLR9 in the endosomes, but also by sensors present in the cytosol. More importantly, DNA from pathogens as well as from mammalian derived sources is equally potent in inducing inflammation.

It is the purpose of this book to bring together the research on the signaling mechanism of DNA induced inflammation as well as its impact on diseases and vaccinology. The book has three sections: In Section I, Ishii, Fitzgerald, Barber and Saitoh discuss the signaling pathway leading to DNA induced inflammation, in Section II, Kawai, Marshak-Rothstein, Opitz, Bowie, Gasser and Arditi discuss the impact of DNA inflammation on human diseases and lastly, in Section III, Coban, Desmet and Lenz comment on how the inflammatory response of DNA could influence the outcome of vaccination in DNA vaccination strategies and adjuvants targeting the DNA sensing pathway.

We are greatly indebted to the contributors whose participation and cooperation made this book possible. We thank them for their patience with our persistent requests for completion of their manuscript. We are appreciative for the editorial and technical assistance provided by Elizabeth Gibson and Mary Preap. To the publisher we are grateful for this timely opportunity to consolidate the wealth of knowledge we have acquired on DNA sensing which could facilitate future investigations. Lastly, we would like to pay tribute to Alick Issacs and his colleagues who were way ahead of their time in that they had the audacity to identify a molecule that is so fundamental to our existence, namely, DNA, as a danger signaling molecule.

Section A

The Discovery of dsDNA Immunogenicity and the Definition of DNA Sensing Pathways

Outline

Chapter 1 Route to Discovering the Immunogenic Properties of DNA from TLR9 to Cytosolic DNA Sensors

Chapter 2 The PYHIN Family of Molecules and their Functions Sensing dsDNA

Chapter 3 Cytosolic DNA-Sensing and the STING Pathway

Chapter 4 Regulation of Intracellular dsDNA-Induced Innate Immune Responses by Autophagy-Related Proteins

Chapter 1

Route to Discovering the Immunogenic Properties of DNA from TLR9 to Cytosolic DNA Sensors

Choon Kit Tang¹,²,³,⁴, Cevayir Coban², Shizuo Akira³ and Ken J. Ishii¹,⁴,    ¹Vaccine Science Laboratory, Immunology Frontier Research Centre (IFReC), Osaka University, Japan,    ²Malaria Immunology Laboratory, IFReC, Osaka University, Japan,    ³Host Defense Laboratory, IFReC, Osaka University, Japan,    ⁴Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation (NIBIO), Saito-Asagi, Osaka, Japan

In this chapter, we reflect on our early understanding of the immunogenic properties of dsDNA and give a chronological account of the journey we have taken to discover the individual cellular DNA sensors which have played important roles in mediating DNA induced inflammation.

Keywords

innate immunity; inflammation; cytosolic DNA sensors; TLR9; PAMPs; DAMPs

Introduction

The innate immune system relies on cues in the form of products released during injury or pathogenic invasion for its activation. Immunologists have come to appreciate deoxyribonucleic acid (DNA) as one of such products that signal danger to the innate immune system and induce inflammation. For example, uncleared DNA released during mechanical injury to tissues or the introduction of viral and bacterial DNA during infection was found to elicit inflammatory responses. This immune response induced by DNA is not a random process but rather it is detected by specific receptors that trigger distinct cell signaling pathways to produce inflammatory products. Our journey in understanding the mechanism of DNA sensing and inflammation began with the discovery of Toll-like receptor 9 (TLR9) in endosomal compartments and its ability to detect CpG motifs present specifically in the DNA of microbes. The immune response generated by TLR9 detection of CpG DNA is potent and capable of protection against pathogenic infection and it was also studied for applications in vaccine adjuvants. It was realized however that TLR9 could not be the sole DNA sensor present in the cell as inflammation induced by DNA persisted despite the absence of TLR9. This was not clearly understood until it was realized that DNA when directly introduced into the cytosol of cells could elicit potent type-I-interferon (type-I-IFN) responses that were independent of TLR9 but completely relied on the TBK1–IRF3 signaling axis. This discovery led to the conclusion that there are other sensors of DNA present in the cytosol and not in the endosomal compartments which could induce inflammation. Since then, several cytosolic DNA sensors have been published and more continue to be reported (Figure 1.1). In this chapter, we will reflect on our early understanding of the immunogenic properties of dsDNA and give a chronological account of the journey we have taken to discover the individual cellular DNA sensors that have played important roles in mediating DNA induced inflammation.

Figure 1.1 The current understanding of the DNA sensing pathways.

negative regulators of the pathways.

The Importance of the Innate Immune System

PAMPS and DAMPs

The immune responses of higher animals can be broadly categorized into those of the innate immune system and the adaptive immune system. They differ in the cell types and cytokines involved as well as the level of specificity and most importantly the responding time. Adaptive immunity provides protection with high degree of specificity and efficiency by targeting antigens present on the invading pathogen. This is put in place through a series of immunological processes including degradation of pathogens, antigen presentation and clonal expansion of T and B cells. As suggested by the multi-step process, time is required for the adaptive immune responses to be mounted. In contrast, the innate immune system is an evolutionarily older form of immunity that offers less specific but faster protection compared to adaptive immunity. Instead of reacting to specific epitopes, the innate immunity senses the small molecular motifs that are uniquely associated with pathogens such as bacterial flagellin or viral glycoproteins, which are collectively known as pathogen-associated molecular patterns (PAMPs). No further degradation or presentation to specialized cells is required for the triggering of the innate immune system by PAMPS. It is directly sensed by a class of germline-encoded receptors named pattern-recognition receptors (PRRs). So far, there are five families of PRRs identified: Toll-like receptors (TLRs), cytosolic RIG-I-like receptors (RLRs), Nod-like receptors, C-type lectins and absence inmelanoma-2 (AIM2)-like receptors. These PRRs differ in their cellular locations, type of PAMPs they recognize and the induction of downstream immune effector responses.

More recently, it has been proposed that the innate immune system should encompass the detection and reaction to another category of danger signals termed damage-associated molecular patterns (DAMPs) [1]. As the name suggests, DAMPs are non-pathogen derived self-products that are sequestered and restricted from interacting with the immune system during unprovoked physiological conditions but are exposed during cellular stress or injury. This may include intracellular proteins such as histones and heat-shock proteins that are released during necrotic or apoptotic cell death. It could also include extracellular products released after mechanical tissue injury such as heparin sulfate and hyaluronan. Irritant particles such as silica and crystals which are known to induce inflammatory responses are also considered as DAMPs. In essence, DAMPs are the agonists of sterile inflammation. Although DAMPs are categorically different from PAMPs, the patterns of inflammatory responses they induce are the same, such as infiltration of macrophages and neutrophils and the release of TNF-α and IL-1 families of cytokines. Therefore they are considered to be sensed by the same set of PRRs.

The ‘shoot first ask later’ nature of the innate immune system provides an effective first line of defense against pathogen invasion, but also remains as one of the greatest flaws in our immune system. In the state of chronic inflammation, when the source of the inflammatory agonists is not clear, potent cytokines and factors continually released by the innate immune system can cause widespread indiscriminate injury to the surrounding tissue, and outcomes of these processes are manifested as autoimmune diseases. On the other hand, it is also these characteristics of the innate immune response which allow adjuvants to provide the right conditions for efficacious vaccine response. In fact, the activation of the innate immune system has been viewed to be the essential first step that could pre-determine the type and magnitude of the adaptive immune response that is subsequently generated. Inflammatory cytokines induced by the innate immune system are potent immune-modulators that are capable of initiating and steering the immune response induced by vaccine preparations. For a long time, Alum has been thought to provide vaccines with adjuvant help by slowly releasing the antigen of interest which has been incorporated into its content by emulsification, a process termed depot effect. Now, we know that Alum is able to activate different arms of the innate immune system. Reports have shown that Alum can cause cell death which leads to DNA release that acts as DAMP. The release of proinflammatory cytokines by these pathways could be involved in the adjuvant activity of Alum.

Therefore the induction of the innate immunity triggered by PAMPs and DAMPs is an important process that affects many different processes in our biology including host defense, autoimmune disease and vaccine adjuvants.

Pathogen Derived Nucleic Acids are Potent Activators of the Innate Immune System

Nucleic acids have been known to be immunogenic for a long time. One of the earliest demonstrations of its immunogenic property was presented by Alick Isaacs and colleagues in 1963 [2]. At that time, interferon was recognized as an antiviral substance released by mammalian cells during viral infection, however it was not clear which component of the virus was responsible for this response [3]. Alick Isaacs and colleagues showed that nucleic acids derived from viruses including encephalomyocarditis virus and turnip yellow mosaic virus were able to induce interferon production and inhibit Chikungunya virus plaque formations when added directly to mouse and chick embryonic fibroblasts. The addition of homologous (self) DNA did not however induce strong production of interferon or give protective antiviral responses [2,4]. This was the first direct demonstration of the immunogenic properties of nucleic acids from pathogens.

Bacterial derived DNA was similarly noted to be immunogenic. In the 19th century, an innovative physician, William B. Coley, formulated a mixture composed of bacterial cell lysates (termed Coley’s Toxin) for the treatment of carcinoma based on the observation that in certain cases, infection could actually be therapeutic in tumor malignancy. This form of treatment still exists in the present day, for example Mycobacterium bovis derived Bacillus Calmette–Guérin (BCG) is used for the therapy of bladder cancer [5]. It was not understood how this crude preparation of bacteria could have a therapeutic effect on cancer progression until the investigations made by Tokunaga and colleagues were published in 1984 [6]. In their study, they fractionated the BCG preparation and tested each of the fractions for its anti-tumor activity in guinea pigs. The anti-tumor component of BCG was narrowed to a fraction which contained mainly bacterial DNA and had no detectable cell wall components. In addition to anti-tumor activity, this fraction of BCG was found to have immunogenic properties. It was able to activate NK cell lytic activity and induce type-I-IFN response from human peripheral blood mononuclear cells (PBMCs) [7]. Subsequently, Messina et al. extended this observation and demonstrated that the immunogenic nature of DNA was not restricted to mycobacteria, but was also present in other bacterial derived DNA [8]. They showed that bacterial DNA could induce proliferation in mouse splenocytes and also directly activate B cells to proliferate and produce IgM antibodies in the absence of T cell help. This phenomenon however did not extend to mammalian derived DNA.

Another clue from early studies which suggested bacteria derived DNA is immunogenic comes from a report that investigated the generation of anti-DNA antibodies in the context of systemic lupus erythematosus (SLE). It was shown that Escherichia coli and Clostridium perfringens DNA were able to induce anti-double stranded DNA antibodies when injected into mice [9,10]. This effect was less prominent when DNA from vertebrate origin such as calf thymus, chicken blood, human placenta and salmon testes were used. These observations brought to light the immunogenic property of bacterial DNA and identified it as a source of a danger signal which forces the innate immune system to act and prepare for host defense during pathogen invasion.

Endosomal-Membrane Bound DNA Sensor – TLR9

Toll-Like Receptors in Innate Immunity

Toll-like receptors belong to a family of transmembrane proteins that are phylogenetically conserved and play important roles in the innate immunity. Toll-protein was first discovered in Drosophila (fruit fly). In addition to its role in controlling dorsal–ventral patterning during embryogenesis [11], Drosophila-Toll was found to be essential in host defense against microbial invasion as exemplified by the anti-fungal response against Aspergillus fumigatus[12]. Flies expressing a mutant non-functional form of Toll-protein were unable to secrete the anti-fungal factor drosomycin and succumbed to the fungal infection. A human homolog of Drosophila-Toll was later identified by Medzhitov et al. in 1997 [13]. At the same time, Rock et al. reported that they had cloned five different forms of human equivalent Toll-proteins and labeled them toll-like receptors (TLR) 1–5 [14]. Coincidentally, the first TLR identified was TLR4 in the report prepared by Rock et al.

Both Drosophila- and human-Toll are type-I transmembrane proteins. They are composed of a leucine rich repeat (LRR) extracellular N-terminal domain, a transmembrane region and a cytoplasmic domain that is homologous to the human IL-1 receptor (IL-1R) cytoplasmic domain (termed Toll/interleukin-1 receptor (TIR) motif in human/mammalian TLR) [15].The signaling events triggered by human IL-1R and Drosophila-Toll bear many similarities. In humans, the detection of IL-1β by IL-1R triggers activation of NF-κB through signal transduction mediated by its cytoplasmic domain. The activation of NF-κB requires it to be released from IκB proteins, which inhibits NF-κB phosphorylation and translocation into the nucleus. In Drosophila, upon engagement and activation of the Toll protein, a signaling cascade is initiated which eventually leads to activation of the Dorsal protein by disengaging the inhibiting Cactus protein; these proteins are human equivalents of NF-κB and IκB respectively. Hence, the presence of TIR domain in human TLR and the similarities in signaling events triggered by human IL-1R and Drosophila-Toll indicate that human TLR might have functions in mediating the inflammatory response. Indeed, the transfection of a constitutively active form of human-Toll into THP-1 cells (human monocytic cell line) can trigger the activation of NF-κB and induce the expression of co-stimulatory molecule B7.1 and proinflammatory cytokines IL-1, IL-6 and IL-8. These responses are indicative of innate immune activity.

Although TLR4 was identified, its exact role in innate immunity was unknown until it was found to be the receptor responsible for the detection of lipopolysaccharides (LPS) from Gram-negative bacteria and the induction of inflammatory cytokines that cause sepsis. This discovery was made by studying C3H/HeJ and C57BL/10ScCr mice, which have macrophages that are strongly resistant to LPS stimulation but are highly susceptible to Gram-negative infection [16,17]. It was determined that C3H/HeJ mice have a single missense in the TIR domain of TLR4, and C57BL/10ScCr mice do not express the TLR4 gene, which rendered these mice unresponsive to LPS stimulation. Since then, 11 mouse TLRs and 13 human TLRs based on the different ligands recognized have been identified. Four out of these identified human and mouse TLRs sense microbial nucleic acids. These TLRs have distinct specificities for the type of nucleic acids they can detect and react to. TLR3 recognizes double stranded RNA (dsRNA) while TLR7 and TLR8 recognize single stranded RNA (ssRNA). A newly identified TLR13, which exists only in mice, was found to recognize a conserved region of the bacterial 23S ribosomal RNA on to which certain antibiotics such as erythromycin bind [18]. TLR9 is the only TLR that recognizes DNA in humans and mice.

The identification of TLR and its role in the innate immune system is a significant discovery that is fundamental to our understanding of host defense mechanisms against pathogens. For their role in helping to define this area of research, Bruce A. Beutler, Jules A. Hoffmann and Ralph M. Steinman were awarded the Nobel Prize in Physiology or Medicine in 2011 [19].

TLR9 and CpG

Ten years after Tokunaga and colleagues reported on the immunogenic nature of mycobacteria derived DNA, Krieg et al. made a connection between the CpG motifs present in microbial DNA sequences and their immunogenicity [20]. CpG motifs are DNA sequences that contain a cytosine that is immediately followed by a guanine. The ‘p’ in the middle refers to the phosphodiester backbone of DNA. Two major differences between human and microbial DNA were reported [21,22]. Firstly, microbial DNA has four times as much CpG motif as vertebrate DNA. Secondly, the cytosines of CpG motifs found in microbes are hypomethylated compared to vertebrate DNA. True to their suspicion, Krieg et al. demonstrated that the methylation of bacterial DNA with CpG methylase completely inhibits the proliferation and the upregulation of MHC class II expression in mouse splenic derived B cells. Synthetic DNA that contains CpG motifs was also able to mimic bacterial DNA in inducing B cell proliferation and IgM production. Conversely, the removal of CpG motifs or the replacement of cytosine with 5-methylcytosine eliminated the stimulatory effect of synthetic DNA. CpG was shown to activate several immune cell types such as T cells, natural killer cells, monocytic cells and B cells. The immunogenic effect was most profound on B cells. B cells stimulated with CpG DNA were shown to induce strong IL-6, IL-12 and IFNγ secretion in-vitrowithin 1 to 2 hours [23]. An early attempt to determine the signaling pathway induced by bacterial DNA was made by Stacey et al. [24]. The authors demonstrated on macrophages that unmethylated CpG containing plasmid DNA was similar to LPS in the inflammatory signaling pathways it activates. They showed that both LPS and plasmid DNA could activate NF-κB to induce TNF-α and IL-1β responses as well as the LPS-responsive gene plasminogen activator inhibitor type-2 (PAI-2) and the integrated HIV-1 LTR. iNOS was also found to be induced by plasmid DNA, but only when the macrophages were primed with IFNγ. The awareness of the potent stimulatory effect of CpG DNA resulted in its application in various therapies including as vaccine adjuvants. Due to the cytokine milieu induced by CpG activation, it was found to trigger a strong Th1-type response when used as an adjuvant [25].

The discovery of the cellular receptor for CpG DNA came as Hemmi et al. uncovered another mammalian TLR, named TLR9, which contained a TIR motif that associates with MyD88 and TRAF6 [26]. Prior to this publication, the authors had reported that CpG responses were dependent on MyD88 and TRAF6, but TLR2 and TLR4 which signal through these molecules were not responsible for detection. This prompted Hemmi et al. to investigate whether TLR9 could be the receptor for CpG DNA. Indeed, by performing studies on TLR9 knockout mice, they confirmed that TLR9 was the cellular receptor for CpG which mediates its immunogenic effects. In the absence of TLR9, splenocytes were unable to proliferate in the presence of CpG or induce increased expression of MHC class II molecules. Similarly, peritoneal macrophages from TLR9−/− mice were unable to secrete detectable levels of proinflammatory cytokines such as TNF-α, IL-6 and IL-12 in response to CpG stimulation. On the contrary, TLR9−/− macrophages responded with a response similar to WT cells when stimulated with ligands of other TLR such as lipoprotein (TLR2/1 and TLR2/6), peptidoglycan (TLR2/2), zymosan (TLR2/6) and LPS (TLR4). TLR9 was also investigated for the role in mediating Th1 response by CpG DNA on DCs. Bone marrow derived DCs generated from TLR9−/− mice were unable to respond with IL-12 production or upregulate co-stimulatory molecules CD40, CD80 and CD86 when stimulated with CpG DNA. The adjuvant activity of CpG DNA was also abolished in TLR9−/− mice as exemplified by the lack of OVA specific IFNγ response from the splenocytes of OVA+CpG DNA immunized TLR9−/− mice. At the signaling level, CpG DNA failed to activate NF-κB, c-JUN N-terminal kinase (JNK) and IRAK in TLR9−/− macrophages. Finally, TLR9−/− mice were demonstrated to be resistant to the septic shock resulting from the cytokine storm induced by CpG after D-galactosamine sensitization. Latz et al. subsequently showed that CpG could indeed directly associate with TLR9 in ligand-binding studies [27]. They further demonstrated that, unlike TLR2 and TLR4, TLR9 was found to reside at the endoplasmic reticulum during the resting state. Instead of being detected on the cell surface, CpG DNA was internalized through endocytosis into the same endosomal compartments to which TLR9 were shown to migrate in conjunction with the accumulation of MyD88. All this evidence clearly demonstrates the role and specificity of TLR9 for the detection of CpG DNA in the endosomes.

The identification of TLR9 as the cellular receptor for the detection of CpG DNA remains a cornerstone in our understanding of DNA sensing. It shows that inflammation induced by DNA is not a random process, but rather it is detected by a specific receptor that induces inflammation via a distinct signaling pathway.

Cytosolic DNA Sensors

Signs that TLR9 may not be the only DNA Intracellular Sensor

The discovery of TLR9 ability to detect hypomethylated CpG-rich DNA has provided an explanation for the immuno-stimulatory property of DNA, although it is not a complete one. Numerous items of evidence have pointed to a redundancy in the system and that the detection of CpG by TLR9 could not completely account for all the inflammatory response induced by DNA. For example, it was initially thought that non-adjuvanted naked DNA vaccines were effective in mice due to the un-methylated CpG motifs present in the DNA plasmids triggering TLR9 activation. Although it was demonstrated that TLR9−/− DCs are defective in the production of IL-12p40 when transfected with DNA, opposing reports soon arose demonstrating that TLR9−/− mice responded with similar immune responses to WT mice after DNA vaccination [28–30]. This suggests that there are other danger signals present in DNA vaccines that could be detected by other innate immune sensors.

There were also reports which dispute the observation made in early studies that mammalian DNA is not immunogenic. During an investigation on the abnormal increase of MHC molecule expression in autoimmune diseases, it was found that nucleic acids released by dying cells were immunogenic and the effect could be duplicated by directly introducing dsDNA and dsRNA into the cytoplasm of cells including non-immune cells by lipofection [31]. This implied that homologous (self) and mammalian derived nucleic acids could induce inflammatory responses if delivered into the cytoplasm instead of plain addition to the culture media of in-vitro systems. In fact, the cytoplasmic delivery via transfection of DNA from different vertebrate sources including calf thymus, salmon sperm and genomic DNA were stimulatory. Cytosol-delivered mammalian DNA was able to upregulate MHC class I, MHC class II expression and other genes that are required for antigen processing and presentation such as LMP2 and TAP1, in mammalian non-immune cell types. Further characterization of this response revealed that the cytosolic DNA has to be double stranded and larger than 25 base pairs (bp) for it to be immunogenic; it is however not dependent on specific sequences or motifs being present. At the signaling level, cytosolic dsDNA was demonstrated to activate Stat1, Stat3, MAPK, NF-κB and IRF1. Furthermore, our group found that the immuno-stimulatory effect of mammalian derived DNA was functional and could activate the functions of antigen presenting cells (APCs), such as macrophages and DCs. As a result, C57BL/6 mice immunized with OVA mixed with dsDNA had higher levels of IgG1, IgG2a and antigen specific cytotoxic T cell activity compared to mice that were immunized with OVA alone. This adjuvant effect of DNA remains despite the treatment with methylase, therefore indicating that CpG is not involved in the stimulatory effect [32].

The immunogenic property of cytosolic dsDNA and the presence of DNA sensors other than TLR9 were also implicated in a study which investigated the lethality of mice lacking DNase II [33]. In macrophages, the primary function of DNase II is to clear redundant DNA that is taken into the cytosol during engulfment of apoptotic cells. Okabe et al. found that macrophages derived from the fetal liver of DNase II−/− embryos constitutively expressed a set of genes that was exclusive and not upregulated in heterozygote macrophages; these genes include IFNβ, TNF-α and CXCL10. Other interferon inducible genes including IFNγ, 2′5′-oligo(A) synthetase (OAS), IRF7 and ISG15 were also exclusively expressed in DNase II−/− cells, but were not restricted to macrophages and applied to other non-macrophage cell types in the fetal liver. Due to this potent induction of pro-inflammatory cytokines from the constant stimulation of uncleared DNA, DNase II deficient mice were found to die in utero. This lethality could be however be reversed if DNase II−/− mice were crossed with type-I-IFN receptor deficient mice, which confirmed that type-I-IFN production was responsible for the lethal effect. However, when DNase II−/− mice were crossed with TLR3−/−, TLR9−/− and mice deficient for other adaptor molecules required for TLR signaling including MyD88 and TRIF, the lethal effect of DNase II persisted. Therefore this indicated the presence of a TLR independent sensing mechanism which detects DNA and induces potent type-I-IFN responses.

This evidence challenged our conventional view on how DNA is sensed at the cellular level after the discovery of TLR9, and a few possibilities are suggested: (1) All sources of DNA and not just CpG motif containing microbial DNA maybe immunogenic; (2) DNA may not only be sensed in the endosomal compartments but also in the cytoplasm; (3) Most importantly, TLR9 may not be the only receptor that detects DNA triggering the innate immune response.

DsDNA Utilizes the TBK1-IRF3 Signaling Pathway

The next leap forward in our understanding of the immuno-stimulatory effects of DNA began with realizing that DNA of non-pathogen origins could also be immuno-stimulatory when delivered directly into the cytoplasm. Investigations in our laboratory have revealed that dsDNA, when transfected using lipofection methods, is a potent inducer of type-I-IFN and chemokinegenes such as Cxcl10, Ccl5 and Ccl2 in mouse and human dendritic cells (DCs) and stromal cells [34]. The criteria for immunogenicity were different between added CpG DNA and transfected dsDNA. Cytosolic dsDNA remained stimulatory, irrespective of its sequence, and was able to induce type-I-IFN even when it was methylated. It was also not restricted to pathogen derived DNA because calf thymus DNA and synthetic DNA which lacked the CpG motif were equally stimulatory to E. coli and HSV-1, when transfected. Furthermore, MEF cells which were previously demonstrated to be unresponsive to CpG because of the lack of TLR9 expression were shown to induce type-I-IFN response when transfected with DNA. The type-I-IFN stimulatory effect of dsDNA was dependent on its size. Longer dsDNA induced a stronger type-I-IFN response. Only dsDNA was stimulatory; ssDNA such as complementary DNA (cDNA) was found to be immunologically inert. The stimulatory effect was also restricted to dsDNA in the B-form (B-DNA) but not the Z-form (Z-DNA) conformation. B-DNA is a commonly occurring low energy state of dsDNA structure that adopts a right-handed helix conformation. Conversely, Z-DNA adopts a high energy left-handed helix structure that occurs less frequently in physiological states. Synthetic dsDNA made up of AT repeats poly(dA-dT)•poly(dT-dA) (poly(dA:dT) hereafter) tends to adopt the B-conformation and was able to induce potent type-I-IFN response when transfected into MEFs. On the other hand, synthetic dsDNA made up of GC repeats, poly(dG-dC)•poly(dC-dG) (poly(dG:dC) hereafter), which adopts the Z-conformation, was not stimulatory when transfected into MEFs. These secondary structures of DNA remained intact even when complexed with liposomal-based transfecting reagents