Intracisternally localized bacterial DNA containing CpG motifs induces meningitis

Unmethylated CpG motifs are frequently found in bacterial DNA, and have recently been shown to exert immunostimulatory effects on leukocytes. Since bacterial infections in the CNS will lead to local release of prokaryotic DNA, we wanted to investigate whether such an event might trigger meningitis. To that end, we have intracisternally injected mice and rats with bacterial DNA and oligonucleotides containing CpG motifs. Histopathological signs of meningitis were evident within 12 h and lasted for at least 14 days, and were characterized by an influx of monocytic, Mac-3(+) cells and by a lack of T lymphocytes. To study the mechanisms whereby unmethylated CpG DNA gives rise to meningitis, we deleted the monocyte/macrophage population leading to abrogation of brain inflammation. Also, interaction with NF-kappaB using antisense technology led to down-regulation of proinflammatory cytokine production and frequency of meningitis. Furthermore, specific interactions with vascular selectin expression and inhibition of NO synthase led to a significant amelioration of meningitis, altogether indicating that this condition is dependent on macrophages and their products. In contrast, neutrophils, NK cells, T/B lymphocytes, IL-12, and complement system were not instrumental in meningitis triggered by bacterial DNA containing CpG motifs. This study proves that bacterial DNA containing unmethylated CpG motifs induces meningitis, and indicates that this condition is mediated in vivo by activated macrophages.     

CpG DNA: recognition by and activation of monocytes

Unmethylated CpG motifs present in bacterial DNA rapidly trigger an innate immune response characterized by the activation of Ig- and cytokine-secreting cells. Synthetic oligonucleotides (ODNs) containing CpG motifs mimic this activity, triggering monocytes to proliferate, secrete and/or differentiate. Analysis of hundreds of novel ODNs led to the identification of two structurally distinct classes of CpG motif that differentially activate human monocytes. ODNs of the "K"-type interact with Toll-like receptor 9 and induce monocytes to proliferate and secrete IL-6. In contrast, "D"-type ODNs trigger monocytes to differentiate into mature dendritic cells.     

CXCL16 influences the nature and specificity of CpG-induced immune activation

Unmethylated CpG motifs are present at high frequency in bacterial DNA. They provide a danger signal to the mammalian immune system that triggers a protective immune response characterized by the production of Th1 and proinflammatory cytokines and chemokines. Although the recognition of CpG DNA by B cells and plasmacytoid dendritic cells is mediated by TLR 9, these cell types differ in their ability to bind and respond to structurally distinct classes of CpG oligonucleotides. This work establishes that CXCL16, a membrane-bound scavenger receptor, influences the uptake, subcellular localization, and cytokine profile induced by D oligonucleotides. This is the first example of a surface receptor modifying the cellular specificity and nature of the immune response mediated by an intracellular TLR.     

Bacterial DNA or oligonucleotides containing unmethylated CpG motifs can minimize lipopolysaccharide-induced inflammation in the lower respiratory tract through an IL-12-dependent pathway.

To determine whether the systemic immune activation by CpG DNA could alter airway inflammation, we pretreated mice with either i.v. bacterial DNA (bDNA) or oligonucleotides with or without CpG motifs, exposed these mice to LPS by inhalation, and measured the inflammatory response systemically and in the lung immediately following LPS inhalation. Compared with non-CpG oligonucleotides, i. v. treatment with CpG oligonucleotides resulted in higher systemic concentrations of polymorphonuclear leukocytes, IL-10, and IL-12, but significantly reduced the concentration of total cells, polymorphonuclear leukocytes, TNF-alpha, and macrophage inflammatory protein-2 in the lavage fluid following LPS inhalation. The immunoprotective effect of CpG-containing oligonucleotides was dose-dependent and was most pronounced in mice pretreated between 2 and 4 h before the inhalation challenge, corresponding to the peak levels of serum cytokines. bDNA resulted in a similar immunoprotective effect, and methylation of the CpG motifs abolished the protective effect of CpG oligonucleotides. The protective effect of CpG oligonucleotides was observed in mice with either a disrupted IL-10 or IFN-gamma gene, but release of cytokines in the lung was increased, especially in the mice lacking IFN-gamma. In contrast, CpG DNA did not protect mice with a disrupted IL-12 gene against the LPS-induced cellular influx, even though CpG DNA reduced the release of TNF-alpha and macrophage inflammatory protein-2 in the lung. These findings indicate that CpG-containing oligonucleotides or bDNA are protected against LPS-induced cellular airway inflammation through an IL-12-dependent pathway, and that the pulmonary cytokine and cellular changes appear to be regulated independently.     

Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes

Plasmid DNA (pDNA) is very important in non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits gene expression while improving immunological consequences. In this report, we investigated the cytokine secretion induced by pDNA/cationic liposome complexes using murine macrophages. Naked CpG DNA induced tumor necrosis factor-alpha (TNF-alpha) secretion from the macrophages, but DNA without CpG motif did not, demonstrating that the cytokine induction was mediated by CpG motifs. pDNA complexed with cationic liposomes, but not the cationic liposomes alone, produced a significant amount of TNF-alpha from the macrophages. Surprisingly, methylated pDNA and calf thymus DNA complexed with the cationic liposomes were also able to induce TNF-alpha production, indicating that these responses were not dependent on CpG motifs. Taken together, the present study demonstrated that for the first time DNA can stimulate murine macrophages in a CpG motif-independent manner when it is complexed with the cationic liposomes.     

Immunostimulatory DNA sequences influence the course of adjuvant arthritis.

Bacterial DNA is enriched in unmethylated CpG motifs that have been shown to activate the innate immune system. These immunostimulatory DNA sequences (ISS) induce inflammation when injected directly into joints. However, the role of bacterial DNA in systemic arthritis is not known. The purpose of the present experiments was to determine whether ISS contributes to the development of adjuvant arthritis in Lewis rats after intradermal injection of heat-killed Mycobacterium tuberculosis (Mtb). The results showed that Mtb DNA was necessary for maximal joint inflammation in adjuvant arthritis but could be replaced by synthetic ISS oligodeoxynucleotides. The arthritis-promoting effect of the Mtb DNA or of the ISS oligodeoxynucleotides correlated with an increased Th1 response to Mtb Ags, as measured by the production of IFN-gamma and increased production of the osteoclast differentiation factor, receptor activator of NF-kappaB ligand (RANKL). The Mtb DNA did not enter the joints but dispersed to the bone marrow and spleen before the onset of systemic joint inflammation. Thus, adjuvant arthritis is a microbial DNA-dependent disease. In this model, we postulate that massive and prolonged activation of macrophages, dendritic cells, and osteoclast precursors in the bone marrow may prime the joints for the induction of inflammatory Th1 immune responses to Mtb Ags.     

Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.     

In vivo role of p38 mitogen-activated protein kinase in mediating the anti-inflammatory effects of CpG oligodeoxynucleotide in murine asthma

DNA containing unmethylated CpG motifs is intrinsically immunostimulatory, inducing the production of a variety of cytokines and chemokines by immune cells. The strong Th1 response triggered by CpG oligodeoxynucleotide (ODN) inhibits the development of Th2-mediated allergic asthma in mice. This work documents that CpG ODN-induced IL-12 production plays a critical role in this process, because intrapulmonary CpG ODN inhibits allergic inflammation in wild-type but not IL-12(-/-) mice. CpG ODN rapidly localized to alveolar macrophages (AM), thereby triggering the phosphorylation of p38 mitogen-activated protein kinase (MAP kinase). AM cultured with CpG but not control ODN up-regulated IL-12 p40 expression and release, and these effects were blocked by the highly specific p38 MAP kinase inhibitor SB202190. Intrapulmonary administration of this inhibitor blocked the ability of CpG ODN to produce IL-12 in the lungs and reversed the anti-inflammatory effects of CpG ODN on allergic lung inflammation. These findings indicate that IL-12 production by AM is stimulated by intrapulmonary CpG ODN administration through a p38 MAP kinase-dependent process, and IL-12 is a key cytokine that mediates CpG ODN-induced protection against allergic lung inflammation.     

Stimulation of thymocyte proliferation by phosphorothioate DNA oligonucleotides

DNA is a complex macromolecule the immunological properties of which depend on short sequence motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences are mitogenic for B cells and can stimulate macrophage cytokine production. While these sequences do not directly activate T cells, they can augment effects of stimulation via the TCR. Furthermore, ISS can affect T cells because of macrophage production of IL-12 and IFN-alpha/beta. In these studies, we further evaluated the immune effects of DNA on T cells, testing the possibility that certain T cell populations can respond directly to this stimulus. We therefore tested the in vitro responses of thymocytes to a series of phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) varying in sequence. In in vitro cultures, phosphorothioate ODNs (sODNs) containing CpG motifs induced significant proliferation of murine thymocytes, although phosphodiester compounds lacked activity. The magnitude of stimulation varied with sequences flanking the CpG motifs, as both dA and dT sequences enhanced the stimulatory capacity of the CpG motif. Furthermore, CpG sODNs were strong costimulators of anti-CD3-mediated thymocyte activation, increasing proliferation compared to anti-CD3 in the absence of DNA. This activation was only partially inhibited by cyclosporine A and was not dependent on a calcium influx. Together, these results indicate that phosphorothioate oligonucleotides containing CpG motifs can directly induce thymocyte proliferation as well as augment TCR activation. These observations thus extend the range of actions of CpG DNA and suggest additional mechanisms for its function as an immunomodulatory agent or adjuvant.     

Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells

Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge-charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection.     

CpG DNA-mediated induction of acute liver injury in D-galactosamine-sensitized mice: the mitochondrial apoptotic pathway-dependent death of hepatocytes

Unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce innate inflammatory responses, including rapid induction of proinflammatory cytokines. Although innate inflammatory responses induced by CpG DNA and other pathogen-associated molecular patterns are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is detrimental to the host. In this study, we demonstrate that CpG DNA, but not control non-CpG DNA, induces a fulminant liver failure with subsequent shock-mediated death by promoting massive apoptotic death of hepatocytes in D-galactosamine (D-GalN)-sensitized mice. Inhibition of mitochondrial membrane permeability transition pore opening or caspase 9 activity in vivo protects D-GalN-sensitized mice from the CpG DNA-mediated liver injury and death. CpG DNA enhanced production of proinflammatory cytokines in D-GalN-sensitized mice via a TLR9/MyD88-dependent pathway. In addition, CpG DNA failed to induce massive hepatocyte apoptosis and subsequent fulminant liver failure and death in D-GalN-sensitized mice that lack TLR9, MyD88, tumor necrosis factor (TNF)-alpha, or TNF receptor I but not interleukin-6 or -12p40. Taken together, our results provide direct evidence that CpG DNA induces a severe acute liver injury and shock-mediated death through the mitochondrial apoptotic pathway-dependent death of hepatocytes caused by an enhanced production of TNF-alpha through a TLR9/MyD88 signaling pathway in D-GalN-sensitized mice.     

Complex roles of CpG in liposomal delivery of DNA and oligonucleotides

Unmethylated CpG in bacterial DNA has recently been recognized as a danger signal to the mammalian immune system. This CpG signal can be greatly amplified when DNA is delivered via a lipidic vector. The CpG effects are affected by the administration route, and can be either beneficial or harmful. In this review, we will summarize our current understanding about the mechanism of action of the immunostimulatory motifs. Emphasis will be placed on the discussion of the complicated roles of CpG when CpG DNA or oligonucleotides are administered in vivo using liposomes as a delivery vehicle.     

Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c- type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN

Two classes of nucleic acids, bacterial DNA containing unmethylated CpG motifs and dsRNA in viruses, induce the production of type I IFN that contributes to the immunostimulatory effects of these microbial molecules. Thus, it is important to determine which cells produce type I IFN in response to CpG DNA and dsRNA. CD4(+)CD11c(-) type 2 dendritic cell precursors (pre-DC2) were identified as the main producers of type I IFN in human blood in response to viruses. Here we asked whether pre-DC2 also produce type I IFN in response to CpG DNA and dsRNA. Oligodeoxynucleotides containing particular palindromic CpG motifs induced pre-DC2, but not CD11c(+) blood DC or monocytes, to produce IFN-alpha. In contrast, a synthetic dsRNA, polyinosinic polycytidylic-acid, induced CD11c(+) DC, but not pre-DC2 or monocytes, to produce IFN-alphabeta. These data indicate that CpG DNA and polyinosinic-polycytidylic acid stimulate different types of cells to produce type I IFN and that it is important to select oligodeoxynucleotides containing particular CpG motifs to induce pre-DC2 to produce type I IFN, which may play a key role in the strong adjuvant effects of CpG DNA.     

Comparative proteomics study reveals that bacterial CpG motifs induce tumor cell autophagy in vitro and in vivo

Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.     

Microbial DNA induces a host defense reaction of human respiratory epithelial cells

Epithelial cells represent the initial site of bacterial colonization in the respiratory tract. TLR9 has been identified in B cells and CD 123(+) dendritic cells and found to be involved in the recognition of microbial DNA. It was the aim of the study to investigate the role of TLR9 in the host defense reactions of the respiratory epithelium. Respiratory epithelial cell lines (IHAEo(-), Calu-3) or fully differentiated primary human cells as air-liquid interface cultures were stimulated with bacterial DNA or synthetic oligonucleotides containing CpG motifs (CpG oligodeoxynucleotides). Expression of TLR9, cytokines, and human beta-defensin 2 was determined by quantitative RT-PCR or by ELISA. We found that TLR9 is expressed by respiratory epithelial cell lines and fully differentiated primary epithelial cells at low levels. Stimulation of the above-mentioned cells with bacterial DNA or CpG oligodeoxynucleotide resulted in an inflammatory reaction characterized by a dose-dependent up-regulation of cytokines (IL-6, IL-8) and human beta-defensin 2. Up-regulation of NF-kappaB in epithelial cells in response to the CpG motif containing DNA was inhibited by overexpression of a dominant negative form of MyD88. These results provide clear evidence that the human respiratory epithelium is capable of detecting microbial DNA by TLR9. The respiratory epithelium has an important function in triggering innate immune responses and therefore represents an interesting target for anti-inflammatory therapy.     

CpG-A-induced monocyte IFN-gamma-inducible protein-10 production is regulated by plasmacytoid dendritic cell-derived IFN-alpha

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) are known for inducing a Th1 cytokine/chemokine environment, but the mechanisms regulating this have been unclear. Recent studies have defined two classes of CpG ODN, CpG-A ODN that induce plasmacytoid dendritic cells (pDC) to secrete very high levels of IFN-alpha, and CpG-B ODN that induce only low levels of IFN-alpha production, but strongly activate B cells. We now demonstrate that a CpG-A ODN directly activates pDC secretion of IFN-alpha and other soluble factors that secondarily induce purified monocytes to secrete high levels of the Th1-promoting chemokine IFN-gamma-inducible protein-10 (IP-10). Cell contact between the monocytes and pDC is not required for this interaction. IFN-alpha is necessary, but only partially sufficient, for this indirect CpG-induced monocyte IP-10 production. Although CpG ODN induce human PBMC to make only very slight amounts of IFN-gamma, we find that these low concentrations synergize with IFN-alpha for inducing monocyte production of IP-10. These studies provide a better understanding of the mechanisms through which CpG ODN create a Th1-like environment.     

CpG-ODN increases resistance of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection

Unmethylated cytosine-phosphate-guanine (CpG) dinucleotides flanked by specific bases in bacterial DNA induce a favorable immune response by acting as danger signals to the host. Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) also act like the unmethylated CpG oligonucleotides in bacterial DNA. In the present study, we investigated the effects of synthetic CpG-ODN on the protection of olive flounder (Paralichthys olivaceus) against infection by Philasterides dicentrarchi, a pathogen of scuticociliatosis, through two consecutive experiments (trial I and II). Fish were intraperitoneally (i.p.) injected with CpG-ODN 1668 or GpC-ODN 1720 at different doses (3 microg in trial I and 10 microg in trial II), and after one week the fish were i.p. challenged with P. dicentrarchi. In both trial I and II, fish injected with CpG-ODN 1668 showed significantly higher serum scuticocidal activity than fish injected with PBS alone, while the scuticocidal activity disappeared by heat-inactivation. This result suggests that CpG-ODN might activate an alternative pathway of complement of olive flounder, and complement-mediated killing might be an important innate immune factor in the resistance against P. dicentrarchi infection. Although the cumulative mortality was largely different between trials I and II, the relative survival rate of fish injected with a high dose of CpG-ODN 1668 was considerably higher than that of fish injected with a low dose of this ODN, while the relative survival rate was not different between fish injected with the high dose and low dose of GpC-ODN 1720. The results of the present study suggest that CpG-ODNs may be used as potential immunostimulants to lessen cultured fish loss caused by scuticociliates.     

Cytokine induction by a bacterial DNA-specific modified base

Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N(6)-methyladenine (N(6)-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N(6)-MeA induced cytokines when injected into mice. Co-injection of N(6)-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N(6)-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N(6)-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine.