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Background: Cytosine-guanosine dinucleotide (CpG) motifs are immunostimulatory components of bacterial DNA and activators of innate immunity through Toll-like receptor 9 (TLR9). Administration of CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. It was investigated whether physiologic CpG/TLR9 interactions are critical for the homeostasis of the intestinal immune system., Methods: Mesenteric lymph node cell and lamina propria mononuclear cell (LPMC) populations from BALB/c wild-type (wt) or TLR9 mice were assessed by flow cytometry and proteome profiling. Cytokine secretion was determined and nuclear extracts were analyzed for nuclear factor kappa B (NF-κB) and cAMP response-element binding protein activity. To assess the colitogenic potential of intestinal T cells, CD4-enriched cells from LPMC of wt or TLR9 donor mice were injected intraperitoneally in recipient CB-17 SCID mice., Results: TLR9 deficiency was accompanied by slight changes in cellular composition and phosphorylation of signaling proteins of mesenteric lymph node cell and LPMC. LPMC from TLR9 mice displayed an increased proinflammatory phenotype compared with wt LPMC. NF-κB activity in cells from TLR9 mice was enhanced, whereas cAMP response-element binding activity was reduced compared with wt. Transfer of lamina propria CD4-enriched T cells from TLR9 mice induced severe colitis, whereas wt lamina propria CD4-enriched T cells displayed an attenuated phenotype., Conclusions: Lack of physiologic CpG/TLR9 interaction impairs the function of the intestinal immune system indicated by enhanced proinflammatory properties. Thus, physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counterregulating anti-inflammatory mechanisms.

Nucleic acid-reactive B cells frequently arise in the bone marrow but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the precise nature of its apparently contradictory roles in autoimmunity remained unclear. In this study, using the 3H9 anti-DNA BCR transgene in the autoimmune-prone MRL.Fas(lpr) mouse model of systemic lupus erythematosus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for L chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell life span in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer life spans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell-surface BCR expression. In contrast, whereas TLR9-intact anergic B cells accumulate near the T/B border, TLR9-deficient anti-DNA B cells are somewhat more dispersed throughout the follicle. Nonetheless, in older autoimmune-prone animals, TLR9 expression specifically within the B cell compartment is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into Ab-forming cells via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells, yet is also required for their activation.

Preterm birth, the major cause of neonatal mortality in developed countries, is associated with intrauterine infections and inflammation, although the exact mechanisms underlying this event are unclear. In this study, we show that circulating fetal DNA, which is elevated in pregnancies complicated by preterm labor or preeclampsia, triggers an inflammatory reaction that results in spontaneous preterm birth. Fetal DNA activates NF-κB, shown by IκBα degradation in human PBMCs resulting in production of proinflammatory IL-6. We show that fetal resorption and preterm birth are rapidly induced in mice after i.p. injection of CpG or fetal DNA (300 μg/dam) on gestational day 10-14. In contrast, TLR9(-/-) mice were protected from these effects. Furthermore, this effect was blocked by oral administration of the TLR9 inhibitor chloroquine. Our data therefore provide a novel mechanism for preterm birth and preeclampsia, highlighting TLR9 as a potential therapeutic target for these common disorders of pregnancy.

Background: It has been shown that dietary nucleotides modulate immune response. Due to their unique properties in immune responses, nucleotides are used as immunonutrition in the field of clinical nutrition., Aim of the Study: In this study, we examined the effect of dietary deoxynucleic acid (DNA) on antigen (Ag)-specific immune response in ovalbumin (OVA)-immunized BALB/c mice and determined the mechanism using toll-like receptor 9 (TLR9) knock-out (KO) mice., Methods: BALB/c or TLR9 KO mice were fed control and 1% DNA diets and immunized with OVA. Spleen cells from OVA-immunized mice were stimulated with OVA in vitro, and the contents of IFN-γ and IL-4 in supernatants were measured by an ex vivo system. CD11c(+) dendritic cells were purified, and ability of cytokine induction to CD4(+) cells was examined., Results: The level of OVA-specific IL-4 production in the DNA group was significantly higher than that in the control group. In contrast, the level of OVA-specific IFN-γ production in the DNA group was lower than that in the control group. The DNA diet decreased Ag-specific IL-4 production and enhanced Ag-specific IFN-γ production in TLR9 KO mice. CD11c(+) DCs from mice fed the DNA diet had a greater ability than CD11c(+) DCs from mice fed the control diet to induce the production of IL-4 from DO11.10 CD4(+) T cells., Conclusions: Dietary DNA increases Ag-specific IL-4 production and decreases IFN-γ production through a TLR9-dependent pathway. CD11c(+) dendritic cells are target cells in dietary DNA-induced immune regulation.

Bicyclol, a novel synthetic anti-hepatitis drug, has a potent hepatocyte-protective effect and a mild anti-hepatitis virus function. However, its pharmaceutical effects and mechanism are still unclear. In the present study, we found that bicyclol pre-treatment could attenuate the production of the inflammatory cytokines (TNF-alpha and IL-18) and chemokines (MCP-1, MIP-1 alpha and Rantes) and inhibit the activation of the p65-NF-kappaB and MAPK signaling pathways in CpG-ODN 2006-stimulated L02 hepatocytes in a dose-dependent manner. Collectively, these results suggest that bicyclol could exert its hepatocyte-protective effect through attenuating CpG-ODN 2006-stimulated inflammatory responses in L02 hepatocytes, which might be associated with the activations of NF-kappaB and MAPK pathways.

Although TLR9 was originally thought to specifically recognize microbial DNA, it is now evident that mammalian DNA can be an effective TLR9 ligand. However, the DNA sequence required for TLR9 activation is controversial, as studies have shown conflicting results depending on the nature of the DNA backbone, the route of DNA uptake, and the cell type being studied. In systemic lupus erythematosus, a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DCs), is through uptake as a DNA-containing immune complex. In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated CpG dinucleotides within dsDNA are required for murine DC TLR9 activation induced by a DNA-containing immune complex. The strongest activation is seen with dsDNA fragments containing optimal CpG motifs (purine-purine-CpG-pyrimidine-pyrimidine) that are common in microbial DNA but rare in mammalian DNA. Importantly, however, activation can also be induced by CpG-rich DNA fragments that lack these optimal CpG motifs and that we show are plentiful in CpG islands within mammalian DNA. No activation is induced by DNA fragments lacking CpG dinucleotides, although this CpG-free DNA can induce DC activation if internalized by liposomal transfection instead of as an immune complex. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis in which activation of TLR9 in DCs by self DNA has been implicated in disease pathogenesis.

To develop novel immunoregulatory oligodeoxynucleotides (ODNs), we have designed a series of ODNs based on the sequences in human microsatellite (MS) DNA. The ODNs, designated as human MS DNA mimicking ODNs (MS ODNs), have been studied for their inhibitory effects on human immune cells activated by TLR9-dependent and -independent stimulations. We find for the first time that MS08, a MS ODN composed entirely of TC dinucleotide (TC) repeats, inhibits CpG ODN (TLR9 ligand)-induced human PBMCs proliferation, CD80 and CD86 expression and production of interferon. In addition, MS08 also inhibits the proliferation of human PBMCs stimulated by PHA, PMA and alloantigens in a TLR9-independent manner. The inhibition correlates with competition of binding and uptake between MS08 and CpG ODN in human PBMCs. Structurally, TC, CT or CCT are revealed as essential suppressive motifs required for the inhibition. These findings suggest that TC repeat containing MS ODN could be of therapeutic use in pathologic situations due to excessive activation of immune cells.

Microbial sensing mediated by pattern recognition receptors (PRRs) is the first key step to trigger innate immune responses, represented by the induction of type I interferons (IFNs), proinflammatory cytokines and chemokines. This innate signaling elicits an efficient activation of more specific responses in adaptive immunity. Such coordinated responses in the two systems are essential for the optimal elimination of invading microbes. Despite a major advance in our understanding of RNA sensors, TLR9 remained the only known sensor of DNA. On the other hand, there has been accumulating evidence supporting the existence of TLR9-independent DNA recognition mechanism. In this regard, DAI (also termed as DLM-1/ZBP1), the first sensor of cytosolic DNA, has recently been identified with its activation of IFN-regulatory factors(IRFs) and NF-kappaB transcriptional factors. Several recent papers suggest the involvement of an additional cytosolic DNA sensor(s). There is also a recent report that cytosolic microbial and host DNA can trigger pro-inflammatory responses via the TLR- and IRF-indepnedent pathway mediated by the inflammasome, which is consisted of NLR family members together with the adaptor protein ASC and caspase-1. In addition, evidence has been provided that host- and virus-derived proteins, which contain DNA-binding motifs (Zalpha and/or Zbeta) similar to those of DAI(DLM-1/ZBP1), negatively regulates the immune response that is activated by cytosolic DNA. Thus, these recent findings reveal the complex DNA-sensing mechanism for triggering the activation of innate immunity, and the breakdown of this sensing mechanism may lead to autoimmune abnormalities.

Unmethylated DNA with CpG-motifs is recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Macrophages and conventional dendritic cells (cDCs) produce proinflammatory cytokines to B/K-type CpG-DNA, whereas plasmacytoid DCs induce type I interferons to A/D-type CpG-DNA and DNA viruses. The TLR9 mediated signaling pathway is not only responsible for activation of innate immune cells, but also for mounting acquired responses. Thus, it has been attempted to exploit TLR9 ligands as a vaccine adjuvant for anti-cancer immunotherapy. Further, TLR9 mediated signaling is implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus. Nevertheless, recent studies revealed that double-stranded DNA can be recognized by intracellular receptor(s) in a TLR9-independent manner. This review will focus on the roles of TLR9 in immune responses, and its signaling pathways.

The mammalian immune system senses pathogens through pattern recognition receptors (PRRs) and responds with activation. The Toll-like receptor (TLR) family that consists of 13 receptors plays a critical role in this process. TLRmediated signaling activates immune cells and leads to an innate immune response with subsequent initiation of an adaptive immune response. Toll-like receptor 9 (TLR9) recognizes deoxyribonucleic acid (DNA) leading to cellular activation and cytokine production influencing the immune response against viruses and bacteria. The stimulation of TLR9 will be exploited for adjuvant therapy and treatment of cancer or allergy. In this review we will discuss TLR9 ligands, TLR9 expression, signaling, and the therapeutic potential of TLR9 ligands in treatment of infectious or allergic diseases and cancer.

The presence of autoantibodies specific for nucleic acid-associated antigens is the hallmark of systemic lupus erythematosus (SLE). We have recently developed a specific inhibitor of TLR7 and TLR9, called immunoregulatory sequence (IRS) 954, and showed that it inhibits the induction of IFN-alpha by human plasmacytoid dendritic cells in response to DNA and RNA viruses and isolated immune complexes from lupus patients. In this study, we show that IRS 954 can prevent progression of disease when injected in the lupus prone (NZBxNZW)F1 mice. Following treatment, we observed a significant reduction of serum levels of nucleic acid-specific autoantibodies as well as decreased proteinuria, reduced glomerulonephritis, end-organ damage and increased survival. These data demonstrate that in addition to its ability to block IFN-alpha, IRS 954 can reduce symptoms in a lupus model and thus represents a promising therapeutic agent for the treatment of SLE.

Systemic lupus erythematosus is characterized by the production of autoantibodies directed against nuclear Ags, including nucleosome and DNA. TLR9 is thought to play a role in the production of these autoantibodies through the capacity of nuclear immunogenic particles to interact both with BCR and TLR9. To determine the role of TLR9 in SLE, C57BL/6-lpr/lpr-TLR9(-/-) and TLR9(+/+) mice were analyzed. The abrogation of TLR9 totally impaired the production of anti-nucleosome Abs, whereas no difference was observed in the frequency of anti-dsDNA autoantibodies whose titer was strikingly higher in TLR9(-/-) mice. In addition a higher rate of mesangial proliferation was observed in the kidney of TLR9-deficient animals. These results indicate that in C57BL/6-lpr/lpr mice, TLR9 is absolutely required for the anti-nucleosome Ab response but not for anti-dsDNA Ab production which is involved in mesangial proliferation.

Mouse follicular B cells express TLR9 and respond vigorously to stimulation with single-stranded CpG-oligodeoxynucleotides (ODN). Surprisingly, follicular B cells do not respond to direct stimulation with other TLR9 ligands, such as bacterial DNA or class A(D) CpG-ODN capable of forming higher-order structures, unless other cell types are present. Here, we show that priming with interferons or with B cell-activating factor, or simultaneous co-engagement of the B cell receptor for antigen (BCR), can overcome this unresponsiveness. The effect of interferons occurs at the transcriptional level and is mediated through an autocrine/paracrine loop, which is dependent on IRF-1, IL-6 and IL-12 p40. We hypothesize that the lack of bystander activation of follicular B cells with more complex CpG ligands may be an important safety mechanism for avoiding autoimmunity. This will prevent resting B cells from responding to foreign or self-derived hypomethylated double-stranded CpG ligands unless these ligands are either delivered through the B cell receptor or under conditions where B cells are simultaneously co-engaged by activated plasmacytoid dendritic cells or TH1 cells. A corollary is that the heightened responsiveness of lupus B cells to TLR9-induced stimulation cannot be ascribed to unprimed follicular B cells, but is rather mediated by hypersensitive marginal zone B cells.

Objective: Exogenous nucleic acids, including bacterial unmethylated DNA and viral single-stranded RNA, are potent activators of innate immunity through interaction with the Toll-like receptors (TLRs). In contrast, mammalian DNA has been generally thought to have a limited activation effect, or even a suppressive effect, on innate immunity. Since DNA is a major component of dying cells and recent studies indicate that mammalian nucleic acids may be stimulatory under certain conditions, we undertook this study to examine the effect of intracellular mammalian DNA on myeloid dendritic cell (DC) activation., Methods: Mammalian DNA was introduced into murine bone marrow-derived DCs (BMDCs) by transfection. BMDC activation was determined by flow cytometry (CD40, CD86). Production of tumor necrosis factor alpha and interleukin-6 was measured by enzyme-linked immunosorbent assay, and production of type I interferons (IFNs) by bioassay. Parallel studies were conducted using BMDCs from mice deficient in myeloid differentiation 88 (MyD88), TLR-9, and IFNalpha/beta receptor., Results: Intracellular mammalian DNA activated immature BMDCs, as determined by the up-regulation of CD40 and CD86 as well as by the production of significant quantities of type I IFN. The interferogenic response was shown to be relatively independent of TLR-9, and the TLR adaptor MyD88. The IFN response to intracellular DNA was reduced in BMDCs lacking IFNalpha/beta receptor but was intact in embryonic fibroblasts lacking protein kinase R., Conclusion: These results indicate that intracellular DNA stimulates BMDC maturation and IFN production predominantly through a TLR-independent pathway, and support a model whereby inefficient clearance and/or degradation of endogenous DNA may stimulate innate immune responses similar to the TLR-independent response to exogenous (i.e., viral) double-stranded RNA.

AM14 B cells are a prototype for those low affinity autoreactive B cells that routinely mature as naïve cells in peripheral lymphoid tissues. These cells express a transgene-encoded receptor specific for IgG2a and can be effectively activated by immune complexes that incorporate either mammalian DNA or mammalian RNA that has been released from dead or dying cells. Activation depends on the ability of the B-cell receptor to deliver antigen to an internal vesicular compartment containing either Toll-like receptor-9 (TLR9) or TLR7. Since TLR9 and TLR7 are thought to recognize microbial DNA and RNA preferentially, it is important to determine under what conditions mammalian DNA and RNA become effective TLR ligands, and whether the determining factor is delivery or structure. This issue has been addressed by using IgG2a mAbs to deliver immune complexes preloaded with defined fragments of DNA or RNA, or by using modified ODNs/ORNs. The data demonstrate that only certain nucleic acid sequences or structures can induce autoreactive B-cell proliferation, even when delivery to the appropriate TLR compartment is facilitated by uptake through the B-cell receptor (BCR).

One of the key issues of DNA-based therapies is the immunostimulatory effect caused by DNA, which it has a significant impact on the consequences of these therapies. This is a result of the recognition by Toll-like receptor-9 (TLR9); a pattern recognition receptor for unmethylated CpG motifs within DNA sequences. However, recent studies show that TLR9 can recognize non-CpG motifs and induce cellular activation when DNA is efficiently delivered to TLR9, for example, by cationic lipids. Moreover, recent research shows that TLR9-independent immunoactivation could take place. Here, we have attempted to present an overview of immunoactivation by DNA to optimize DNA-based therapies.