Inhibitory Oligodeoxynucleotides Research Articles

Advances in the mechanisms and applications of inhibitory oligodeoxynucleotides against immune-mediated inflammatory diseases.

Inhibitory oligodeoxynucleotides (ODNs) are short single-stranded DNA, which capable of folding into complex structures, enabling them to bind to a large variety of targets. With appropriate modifications, the inhibitory oligodeoxynucleotides exhibited many features of long half-life time, simple production, low toxicity and immunogenicity. In recent years, inhibitory oligodeoxynucleotides have received considerable attention for their potential therapeutic applications in immune-mediated inflammatory diseases (IMIDs). Inhibitory oligodeoxynucleotides could be divided into three categories according to its mechanisms and targets, including antisense ODNs (AS-ODNs), DNA aptamers and immunosuppressive ODNs (iSup ODNs). As a synthetic tool with immunomodulatory activity, it can target RNAs or proteins in a specific way, resulting in the reduction, increase or recovery of protein expression, and then regulate the state of immune activation. More importantly, inhibitory oligodeoxynucleotides have been used to treat immune-mediated inflammatory diseases, including inflammatory disorders and autoimmune diseases. Several inhibitory oligodeoxynucleotide drugs have been developed and approved on the market already. These drugs vary in their chemical structures, action mechanisms and cellular targets, but all of them could be capable of inhibiting excessive inflammatory responses. This review summarized their chemical modifications, action mechanisms and applications of the three kinds of inhibitory oligodeoxynucleotidesin the precise treatment of immune-mediated inflammatory diseases.

Incorporation of a TGF-β2-inhibiting oligodeoxynucleotide molecular adjuvant into a tumor cell lysate vaccine to enhance antiglioma immunity in mice.

Transforming growth factor β2 (TGF-β2), also known as glioma-derived T-cell suppressor factor, is associated with the impairment of tumor immune surveillance. Therefore, blocking TGF-β2 signaling probably be a feasible strategy to develop a novel type of adjuvant for glioma vaccines to enhance antitumor immunity. A TGF-β2 inhibitory oligodeoxynucleotide, TIO3, was designed with sequences complementary to the 3' untranslated region of TGF-β2 mRNA. The expression of TGF-β2 and MHC-I was detected by qPCR, western and flow cytometry in vitro. All the percentage and activation of immune cells were detected by flow cytometry. Subsequently, TIO3 was formulated with Glioma cell lysate (TCL) and investigated for its antitumor effects in GL261 murine glioma prophylactic and therapeutic models. TIO3 could efficiently downregulate the expression of TGF-β2 while increase the MHC-I's expression in GL261 and U251 glioma cells in vitro. Meanwhile, TIO3 was detected in mice CD4+ T, CD8+ T, B and Ly6G+ cells from lymph nodes after 24 hours incubation. Moreover, TCL+TIO3 vaccination significantly prolonged the survival of primary glioma-bearing mice and protected these mice from glioma re-challenge in vivo. Mechanistically, TCL+TIO3 formulation strongly evoke the antitumor immune responses. 1) TCL+TIO3 significantly increased the composition of CD4+ and CD8+ T cells from draining lymph nodes while promoted their IFN-γ production and reduced the expression of TGF-β2 and PD1. 2) TCL+TIO3 activated the NK cells with the elevation of CD69 or NKG2D expression and PD1 reduction. 3) TCL+TIO3 increased the glioma-specific lysis CTLs from spleen. 4) TCL+TIO3 downregulated PD-L1 expression in glioma tissues and in Ly6G+ cells among glioma-infiltrating immune cells. TIO3 is a promising adjuvant for enhancing TCL-based vaccines to produce a more vigorous and long-lasting antitumor response by interfering with TGF-β2 expression.

Potential of HMGB-inhibitory oligodeoxynucleotide ISM ODN to neutrophil recruitment in mouse model of hepatitis.

High-mobility group box 1 (HMGB1) is a nucleotide-binding chromatin protein that has also been characterized as a prototypical damage-associate molecular pattern. It triggers inflammatory responses upon release from damaged or dying cells. In fact, HMGB1 has been linked to the induction of many inflammatory diseases through immune cell activation including neutrophil recruitment. In this study, we examined the impact of HMGB1-binding inhibitory oligodeoxynucleotide (ISM ODN) on the development of hepatitis using a murine model of the disease. Our results indicate that ISM ODN effectively suppresses pathological features of hepatitis, including neutrophil accumulation. This study therefore may offer clinical insight into the treatment of hepatitis and possibly other inflammatory diseases.

DNA Nanoflowers' Amelioration of Lupus Symptoms in Mice via Blockade of TLR7/9's Signal.

Inhibitory oligodeoxynucleotides (INH-ODN) can exert an immunomodulatory effect to specifically block TLR7 and TLR9 signaling in systemic lupus erythematosus (SLE). To extend the half-life of INH-ODN in vivo, the phosphorothioate backbone, instead of the native phosphodiester, is preferred due to its strong resistance against nuclease degradation. However, its incomplete degradation in vivo may lead to potential risk. To solve these problems and enhance the blockage of TLR7 and TLR9, we prepared highly compressed DNA nanoflowers with prolonged native DNA backbones and repeated INH-ODN motifs. Three therapeutic types of nanoflower, incorporating INH-ODN sequences, including IRS 661, IRS 869, and IRS 954, were prepared by rolling circle amplification and were subcutaneously injected into MRL/lpr mice. The TLR7 blocker of the IRS 661 nanoflower and the TLR9 antagonist of the IRS 869 nanoflower could decrease autoantibodies, reduce cytokine secretion, and alleviate lupus nephritis in mice. However, the IRS 954 nanoflower, the TLR7 and TLR9 dual antagonist, did not have additive or opposing effects on lupus nephritis but only showed a decrease in serum IFNα, suggesting that the TLR7 and TLR9 antagonist may have a competition mechanism or signal-dependent switching relationship. INH-ODN nanoflowers were proposed as a novel and potential therapeutic nucleic acids for SLE.

Anti-DNA antibodies cross-reactive with β2-glycoprotein I induce monocyte tissue factor through the TLR9 pathway

Antibodies specific for cardiolipin (CL)-β2-glycoprotein I (β2GPI) are known to induce tissue factor (TF) expression by monocytes and endothelial cells which leads to a prothrombotic state in antiphospholipid syndrome (APS), but the mechanism is not fully elucidated. Previously, we reported that the mouse monoclonal anti-CL-β2GPI antibody WB-6 cross-reacts with DNA, enters monocytes via binding to cell surface DNA, and induces TF expression. The current study aimed to identify the intracellular signaling pathways involved in this process. The binding of WB-6 to CL-β2GPI or DNA, and endocytosis was not prevented by chloroquine, but pre-treatment of the cells with chloroquine significantly suppressed TF expression. TLR9 inhibitory oligodeoxynucleotide also suppressed the WB-6-induced TF expression, suggesting a pivotal role of the TLR9 pathway in TF production. Serum antibodies obtained from a patient with APS accompanying systemic lupus erythematosus (SLE) bound to both CL-β2GPI and DNA, and induced TF in normal monocytes. This effect was suppressed by chloroquine, and abolished by removal of the DNA-binding activity. These results suggest that induction of TF expression results from TLR9 activation by DNA which was internalized together with cross-reactive antibodies produced in secondary APS accompanying SLE.

Endotracheal intubation results in acute tracheal damage induced by mtDNA/TLR9/NF-κB activity.

Tracheitis secondary to placement of an endotracheal tube (ETT) is characterized by neutrophil accumulation in the tracheal lumen, which is generally associated with epithelial damage. Mitochondrial DNA (mtDNA), has been implicated in systemic inflammation and organ dysfunction following trauma; however, less is known about the effects of a foreign body on local trauma and tissue damage. We hypothesized that tracheal damage secondary to the ETT will result in local release of mtDNA at sufficient levels to induce TLR9 and NF‐κB activation. In a swine model we compared the differences between uncoated, and chloroquine (CQ) and N‐acetylcysteine (NAC) coated ETTs as measured by tracheal lavage fluids (TLF) over a period of 6 h. The swine model allowed us to recreate human conditions. ETT presence was characterized by neutrophil activation, necrosis, and release of proinflammatory cytokines mediated by TLR9/NF‐κB induction. Amelioration of the tracheal damage was observed in the CQ and NAC coated ETT group as shown in tracheal tissue specimens and TLF. The role of TLR9/NF‐κB dependent activity was confirmed by HEK‐Blue hTLR9 reporter cell line analysis after coincubation with TLF specimens with predetermined concentrations of NAC or CQ alone or TLR9 inhibitory oligodeoxynucleotide (iODN). These findings indicate that therapeutic interventions aimed at preventing mtDNA/TLR9/NF‐κB activity may have benefits in prevention of acute tracheal damage.

The TLR9 Antagonist iCpG-ODN at Different Dosages Inhibits Cerebral Ischemia/Reperfusion Injury in Mice.

Inflammatory responses are important mechanisms that are involved in cerebral ischemia/reperfusion(I/R) injury. Whether toll-like receptor 9(TLR9), which belongs to the innate immune system, takes part in the inflammatory responses following cerebral I/R remains unclear. This study examined the effect of different dosages of the TLR9 antagonist inhibitory oligodeoxynucleotide (iCpG-ODN) on cerebral I/R injury by using a mouse model of transient middle cerebral artery occlusion. Neurological function, infarct size, splenocytes and the expression of TLR9 and the downstream products of the TLR9 pathways were determined after cerebral I/R for up to 72 hours. The Clark's focal symptom scoring showed iCpG-ODN improved neurological deficits following focal cerebral I/R. The iCpG-ODN administration significantly decreased the infarct size in a dose-dependent manner. RT-PCR showed that iCpG-ODN attenuated the I/R-induced RNA expression of TLR9. Immunoblot showed that iCpG-ODN prevented I/R-induced increases in NFκB and IRF7 levels and that it further downregulated the levels of IL-1β, TNF-α, and INF-β in the brain. iCpG-ODN did not alter the levels of TNF-α or INF-β in the peripheral blood or affect stroke-induced changes in the number of splenocytes. These findings suggest that iCpG-ODN induced protection against cerebral I/R via inhibiting inflammatory responses in a dose-dependent manner and may be useful in therapy for stroke patients.

Oligodeoxynucleotide inhibition of Toll‐like receptors 3, 7, 8, and 9 suppresses cytokine production in a human rheumatoid arthritis model

Toll-like receptors (TLRs) are innate immune receptors that respond to both exogenous and endogenous stimuli and are suggested to contribute to the perpetuation of chronic inflammation associated with rheumatoid arthritis (RA). In particular, the endosomal TLRs 3, 7, 8, and 9 have more recently been postulated to be of importance in RA pathogenesis. In this study, pan inhibition of the endosomal TLRs by a phosphorothioate-modified inhibitory oligodeoxynucleotide (ODN) is demonstrated in primary human B cells, macrophages, and RA fibroblasts. Inhibition of TLR8 was of particular interest as TLR8 has been associated with RA pathogenesis in both human and murine arthritis models. ODN1411 competitively inhibited TLR8 signaling and was observed to directly bind to a purified TLR8 ectodomain, suggesting inhibition was through a direct interaction with the receptor. Addition of ODN1411 to human RA synovial membrane cultures significantly inhibited spontaneous cytokine production from these cultures, suggesting a potential role for one or more of the endosomal TLRs in inflammatory cytokine production in RA and the potential for inhibitory ODNs as novel therapies.

Self-assembling DNA hydrogel-based delivery of immunoinhibitory nucleic acids to immune cells

Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical EditorToll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.

Therapeutic Implications of Orally Delivered Immunomodulatory Oligonucleotides

DNA has multiple and complex effects on the immune system. Bacterial DNA contains immunostimulatory CpG motifs that trigger cells expressing Toll-like receptor 9 (TLR9) to proliferate, mature, and support T helper type 1 (Th1)-biased immune responses (reviewed in ref. 1). This response confers a selective advantage to the host by improving resistance to infection. In contrast, mammalian DNA contains sequence motifs that downregulate the immune system. Synthetic oligodeoxynucleotides that mimic this inhibitory activity (iODNs) slow or prevent the development of inflammatory and autoimmune diseases, consistent with the release of DNA from dying host cells inhibiting autoaggressive immune responses (reviewed in ref. 2). As reported in this issue, Wang et al. break new ground in the study of immunomodulatory ODNs by showing that the severity of chemically induced atopic dermatitis can be reduced by oral delivery of iODNs and worsened by CpG ODNs.3

Non-CpG Oligonucleotides Exert Adjuvant Effects by Enhancing Cognate B Cell-T Cell Interactions, Leading to B Cell Activation, Differentiation, and Isotype Switching.

Natural and synthetic nucleic acids are known to exert immunomodulatory properties. Notably, nucleic acids are known to modulate immune function via several different pathways and various cell types, necessitating a complex interpretation of their effects. In this study we set out to compare the effects of a CpG motif containing oligodeoxynucleotide (ODN) with those of a control and an inhibitory non-CpG ODN during cognate B cell-T cell interactions. We employed an antigen presentation system using splenocytes from TCR transgenic DO11.10 mice and the ovalbumin peptide recognized by the TCR as model antigen. We followed early activation events by measuring CD69 expression, late activation by MHC class II expression, cell division and antibody production of switched, and nonswitched isotypes. We found that both of the tested non-CpG ODN exerted significant immunomodulatory effects on early T cell and on late B cell activation events. Importantly, a synergism between non-CpG effects and T cell help acting on B cells was observed, resulting in enhanced IgG production following cognate T cell-B cell interactions. We propose that non-CpG ODN may perform as better adjuvants when a strong antigen-independent immune activation, elicited by CpG ODNs, is undesirable.

Pathogen-Triggered Activation of Plasmacytoid Dendritic Cells Induces IL-10–Producing B Cells in Response to Staphylococcus aureus

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9+/+), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.

Effects of Toll-like receptor 7 and Toll-like receptor 9 signaling stimulators and inhibitors on chronic lymphocytic leukemia cells ex vivo and their interactions with cladribine

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant B lymphocytes in the peripheral blood that are regarded as poorly antigenic to the host immune system. Nonetheless, they are thought to be able to undergo stimulation and become antigen-presenting cells possibly through Toll-like receptors (TLRs). Several studies have examined the effect of TLR7 and TLR9 stimulation on the biology of CLL cells, revealing contradictory results in terms of cell proliferation and apoptosis. On the other hand, suppression of TLRs has not been studied in CLL so far, and the rationale for this may be increasing evidence of the supportive role of TLR signaling in CLL. In our study we assessed the effect of a synthetic oligodeoxynucleotide with immunoregulatory sequences (IRS 954) on peripheral blood cells from patients with untreated CLL, in terms of expression of costimulatory molecules, production of cytokines and cell viability ex vivo. Agonists of TLR7 (imiquimod, IMI) and TLR9 (oligodeoxynucleotide ODN 2006) acted as positive internal controls. ODN 2006 most markedly induced CD86 expression compared to IMI and IRS 954. Both oligodeoxynucleotides – IRS 954 and ODN 2006 – caused 1.5- and 5-fold increases of CD40 on CLL cells, respectively. Immunostimulatory ODN 2006 induced CD95 expression 1.5-fold. Changes in costimulatory molecule expression were accompanied by a moderate response from CD4 + and CD8 + T lymphocytes. TLR7 and TLR9 agonists led to significantly higher production of interleukin 6 (IL-6) and IL-10. IRS 954 and ODN 2006 markedly increased the concentration of tumor necrosis factor α (TNFα). IL-17A was significantly decreased by 50% after IMI. IRS 954 and IMI induced significant necrosis at all concentrations, and the effect was augmented by the addition of cladribine (2CdA). ODN 2006 presented a dual effect on cell viability, which was related to disease stage and baseline IL-17A concentration. The addition of 2CdA had little effect in a group where ODN 2006 supported cell survival, and further enhanced cytotoxicity of ODN 2006 in the second group. Inhibitory oligodeoxynucleotides seem to exert promising antileukemic effects regardless of sample background, and thus may become a new modality in CLL. The response of leukemic cells to ODN 2006 varies between samples and cannot yet be predicted.

Immune Tolerance Induction to Factor IX Through B Cell Gene Transfer – Delineating Between Tolerogenic and Immunogenic B Cells.

Abstract 3156At present, the most serious complication in hemophilia therapy is the development of neutralizing antibodies (inhibitors) to intravenous administrated recombinant protein, which compromises therapy, creates immune-toxicity, and increases costs. Although inhibitor formation is less frequent in hemophilia B, it is more prevalent in severe hemophilia B patients, often with additional consequences - up to 50% patients with inhibitors to factor IX (F.IX) develop anaphylactic reactions. These further increase risks of morbidity and mortality. Available bypass therapy is expensive and at risk for thrombosis. Clinical immune tolerance induction (ITI) protocols are lengthy, expensive, and are often terminated in hemophilia B due to anaphylactic reactions or nephrotic syndrome. Therefore, effective protocols to induce immune tolerance to F.IX are urgently needed. B cells have been identified as antigen presenting cells with potentially immune suppressive/regulatory roles. Upon gene transfer, primary B cells were found to induce tolerance to the expressed transgene product. Hence, we use autologous gene-modified primary B cells expressing F.IX antigen fused with immunoglobulin-G heavy chain in a murine model of hemophilia B. Our murine hemophilia B model is unique in both developing high-titer inhibitors and fatal anaphylactic reactions to protein replacement therapy. Retroviral transduced B cells, expressing either full-length or shorter version of F.IX, markedly reduced inhibitor titers up to 30-fold and completely prevented fatal anaphylactic reactions. After 7 weeks of treatment with recombinant human F.IX (IV, 1 IU/mouse, once per week), mice receiving control B cells (n=6) had developed inhibitor titers of 23±8 BU, and 50% died after the last injection. Mice tolerized to F.IX by B cell transplant (n=7) had formed <1 BU, essentially undetectable by this assay, and all survived without anaphylactic reactions. We also tested the B cell-based therapy in already primed mice. Animals receiving B cells expressing the F.IX-IgG fusion successfully reversed the inhibitor and total anti-F.IX IgG titers markedly, whereas animals receiving B cells expressing IgG control had insignificant changes of inhibitor/antibody levels. Our data suggested that B cell-based gene therapy is a promising strategy in not only prevention but also treatment of inhibitors against F.IX. Besides retroviral gene transfer, we tested alternative methods such as DNA nucleofection. Interestingly, although achieving higher gene transfer efficiency, nucleofection of the plasmid encoding the retroviral expression cassette increased rather than decreased immune responses to F.IX. This was likely caused by activation of innate immune mediators and inflammatory cytokine expression as indicated by expression array analysis. Among the 29 genes tested, IL-6 and type I IFN were significantly upregulated in nucleofected B cells compared with retroviral infected B cells, which was further confirmed by ELISA. IL-6 and type I IFN are known to abrogate tolerance such as in transplant rejection and anti-tumor immunity. We suspected that the endosomal DNA sensor TLR9 may induce these cytokines in response to nucleofection. Consistent with this hypothesis, using a TLR9 inhibitory oligodeoxynucleotide (ODN 2088), we significantly reduced nucleofection-associated IL-6 and type I IFN production compared to passive ODN control. These data provide insights into the mechanisms that control the immune phenotype of gene-modified primary B cells, which become tolerogenic under conditions of limited innate responses and immunogenic upon activation of inflammatory and IFN I gene expression. Disclosures:Herzog:Genzyme Corp.: Royalties, AAV-FIX technology, Royalties, AAV-FIX technology Patents & Royalties.

Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression

We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′-TTAGGG-3′, which has a six-base loop head structure, and a 3′-oligo (dG)3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)-immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)-6, IL-12p35, IL-12p40, and interferon (IFN) γ mRNA expression, but also IL-4 and IL-13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen-immunized mice, suggesting usefulness in the treatment of diseases induced by over-active immune activation.

Abstract 80: Accumulation of Mitochondrial DNA in Autolysosome Causes Inflammation and Heart Failure Through Toll-Like Receptor 9 (TLR9) Signaling

Backgrounds Nuclear DNA in apoptotic cell is digested by lysosomal deoxyribonuclease II (DNase II) in macrophages. Improper DNA digestion can lead to inflammation. We previously reported that cardiac-specific DNase II-deficient mice (CKO) exhibited heart failure after transverse aortic constriction (TAC). We observed inflammatory response and DNA accumulation in autolysosome in TAC-operated CKO heart. They were considered to be mitochondrial DNA (mtDNA). In present study, we elucidated the mechanism of inflammation integrated by DNA accumulation in TAC-operated CKO hearts. Furthermore we investigated the pathogenesis of inflammation and heart failure in wild-typeTAC-operated mice. Methods &amp; Results First, we identified the origin of accumulated DNA in lysosome. To label cardiac mtDNA, EdU (5-ethynyl 2’ deoxyuridine) were injected into mice before TAC. In TAC-operated CKO mice, EdU- and LAMP2a (lysosomal marker) or LC3 (autophagosome marker) positive deposits were observed, indicating that mtDNA accumulated in autolysosome. Then, we examined the mechanism how the mtDNA accumulation leads to inflammation. mtDNA has similarities to bacterial DNA, which contains inflammatogenic unmethylated CpG motif. TLR9, localized in the endolysosome, senses DNA with unmethylated CpG motifs. Therefore, we hypothesized that undigested mtDNA is sensed by TLR9. We administrated the inhibitory oligodeoxynucleotides against TLR9 to TAC-operated CKO mice. They attenuated the development of cardiomyopathy in CKO mice. Ablation of Tlr9 also canceled the cardiac phenotype of CKO mice. Next, we examined the involvement of DNA accumulation and TLR9 signaling in wild-type TAC-operated mice. DNase II activity was up-regulated in hypertrophied hearts, but not in failing hearts. LAMP2a- or LC3- positive DNA accumulation was observed in failing hearts. To determine the significance of TLR9 signaling pathway in the pathogenesis of heart failure, we subjected TLR9-deficient mice to TAC. They showed significant resistance to pressure-overload. TLR9-inhibitory oligodeoxynucleotides also improved the mortality in wild-type TAC-operated mice. Conclusion mtDNA-TLR9 axis is involved in inflammation in failing hearts in response to pressure overload.

Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure

Heart failure is a leading cause of morbidity and mortality in industrialized countries. Although infection with microorganisms is not involved in the development of heart failure in most cases, inflammation has been implicated in the pathogenesis of heart failure1. However, the mechanisms responsible for initiating and integrating inflammatory responses within the heart remain poorly defined. Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA2,3,4. Mitochondria damaged by external hemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes5. Here, we show that mitochondrial DNA that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated inflammatory responses in cardiomyocytes and is capable of inducing myocarditis, and dilated cardiomyopathy. Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions, but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload. Early in the pathogenesis, DNase II-deficient hearts exhibited infiltration of inflammatory cells and increased mRNA expression of inflammatory cytokines, with accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium. Administration of the inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA6, or ablation of Tlr9 attenuated the development of cardiomyopathy in DNase II-deficient mice. Furthermore, Tlr9-ablation improved pressure overload-induced cardiac dysfunction and inflammation even in mice with wild-type Dnase2a alleles. These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.

B-cell receptor for antigen modulates B-cell responses to complex TLR9 agonists and antagonists: implications for systemic lupus erythematosus

The capacity to make secondary structures significantly affects the ability of Toll-like receptor 9 (TLR9) agonists and antagonists to either induce or block TLR9-dependent activation in B cells. However, it has a minor impact on TLR9-induced activation in interferon alpha (IFNα)-producing dendritic cells. Based on the ability of inhibitory oligodeoxynucleotides to form predictable secondary structures, we have classified TLR9-antagonists into Class R ('restricted', palindromic) and Class B ('broadly reactive', linear) oligodeoxynucleotides. In non-autoreactive B cells, Class R oligodeoxynucleotides are at least 10-fold less potent TLR9-inhibitors. We wanted to determine whether engagement of the B-cell receptor for antigen could overcome this restriction. Here we show that in non-autoreactive mouse B cells, B-cell receptor for antigen engagement increased the potency of Class R oligodeoxynucleotides for TLR9 activation at least 10-fold, making it equal in potency to linear oligodeoxynucleotides. However, this enhanced potency was selective for TLR9-induced B-cell cycling and apoptosis protection while TLR9-induced IL-6, an event that strongly depends on signaling via late endosomes, still required 10 times more Class R oligodeoxynucleotides. Thus, pathway-specific effects of Class R oligodeoxynucleotides for TLR9/B-cell receptor for antigen co-stimulated B cells may have therapeutic advantages over non-selective targeting of B cells, a strategy that may be seen as a potential therapy for human systemic lupus erythematosus.

Bacterial DNA delays human eosinophil apoptosis

Oligodeoxynucleotide (ODN) sequences containing unmethylated cytidine phosphate guanosine (CpG) motifs prevalent in bacterial DNA attenuate allergic lung inflammation in experimental models of asthma but failed to inhibit eosinophilia and improve lung function in patients with asthma. Bacterial respiratory tract infections exacerbate asthma in humans. Increased eosinophil survival is a critical factor leading to persistent eosinophilic airway inflammation. Apoptosis is regarded as a key mechanism in the resolution of eosinophilic inflammation. The aim of this study was to investigate the effects of bacterial DNA and CpG ODNs on human eosinophil apoptosis in vitro and to elucidate the signalling pathway. Eosinophils were isolated from human peripheral blood by CD16- or CD16-, CD19- and CD304-negative selection. Apoptosis was determined by flow cytometric analysis of relative DNA content, Annexin-V staining and/or morphological analysis. Toll-like receptor 9 (TLR9) expression was studied by using western blotting and intracellular flow cytometry. Bacterial DNA and phosphorothioate-modified CpG ODNs, but not vertebrate DNA, were found to delay spontaneous eosinophil apoptosis. The effect of CpG ODNs was dependent on endosomal acidification and reversed by inhibitory ODN, which suggests involvement of TLR9 pathway. Furthermore, we demonstrated TLR9 expression in eosinophils derived from both atopic and healthy donors. Non-CpG ODNs had occasionally parallel but less profound effect on eosinophil apoptosis, which was not dependent on endosomal acidification. The anti-apoptotic effect of CpG ODNs was dependent on phosphatidylinositol 3-kinase (PI3K) and nuclear factor-κB (NF-κB) but not mitogen-activated protein kinases (MAPKs) as determined by inhibitor studies. Although our results suggest CpG-dependent involvement of TLR9 in the action of phosphorothioate-modified ODNs, we interestingly found that the anti-apoptotic action of native bacterial DNA in eosinophils is not dependent on unmethylated CpG motifs. This suggests that bacterial DNA contains a currently unknown recognition structure lacking from vertebrate DNA. Bacterial DNA-mediated suppression of eosinophil apoptosis is a novel mechanism for exacerbation of eosinophilic lung inflammation associated with bacterial respiratory tract infection.