Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “nature's adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS^®-fractionated MHC class II^low (termed immature DC) or MHC class II^high populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-α. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.     

TLR9-/- and TLR9+/+ mice display similar immune responses to a DNA vaccine

Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9-/-) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-gamma (IFN-gamma)-secreting cells in TLR9-/- mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.     

CpG-ODN enhances ingestion of apoptotic neutrophils by macrophages

The clearance of apoptotic neutrophils by macrophages plays an important role in the process of inflammatory response. In the present study, we examined the ability of macrophages to ingest apoptotic neutrophils after activated by synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) in vitro. The results showed that, while CpG-ODN at the experimental concentration had no cytotoxic effect on the viability of macrophages, the percentage of macrophages with ingested apoptotic neutrophils was increased from 23.6 to 42.30% by CpG-ODN stimulation. This effect was silenced when macrophages were treated with the mutation of CpG-ODN motifs. Both the total and cell surface protein of Toll-like receptor 9 (TLR9) expression in macrophages was up-regulated after CpG-ODN stimulation. While chloroquine (CHQ) had no effect on TLR9 expression in macrophages, it abolished the enhanced uptake of apoptotic neutrophils by macrophages. Although CpG-ODN had no significant effect on the IL-6 production, it was able to induce the increase of TNF-α protein expression and this effect was inhibited by CHQ pretreatment. Increased TNF-α production from macrophages induced by CpG-ODN stimulation was down-regulated after phagocytosis of apoptotic neutrophils. In conclusion, CpG-ODN could enhance the ingestion of apoptotic neutrophils by macrophages via TLR9 accompanied with an increasing in the level of TNF-α. After phagocytosis of apoptotic neutrophils, the increased TNF-α production from macrophages induced by CpG-ODN stimulation was down-regulated which the implications in the immune response remains for the further study.     

Endocytosis‐free DNA sensing by cell surface TLR9 in neutrophils: Rapid defense with autoimmune risks

TLR9 senses microbial DNA, but may also respond to self‐DNA. To prevent the initiation of innate immune responses to self‐DNA, TLR9 is thought to sense microbial DNA in endolysosomes, and not at the cell surface. A report by Lindau et al. in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43: 2101–2113] shows that TLR9 is expressed on the surface of human and mouse neutrophils and, furthermore, shows that cell surface TLR9, instead of endosomal TLR9, senses DNA in neutrophils. These findings demonstrate that DNA sensing by TLR9 in neutrophils is quite distinct from that in DCs or macrophages. The unique DNA sensing by cell surface TLR9 in neutrophils may reflect their role in inducing rapid inflammation by degranulation with a minimal role in engulfing microbial products for antigen presentation.     

DNA and its cationic lipid complexes induce CpG motif-dependent activation of murine dendritic cells

Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response of antigen-presenting cells (APC). In this study, we investigated the cytokine release from murine dendritic cells (DC) by the addition of various types of DNA in the free or complexed form with cationic lipids. Naked plasmid DNA and Escherichia coli DNA with immunostimulatory unmethylated CpG motifs induced pro-inflammatory cytokine secretion from granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow-derived DC and the DC cell-line, DC2.4 cells, though vertebrate calf thymus DNA (CT DNA) with less CpG motifs did not. These characteristics differed from mouse peritoneal resident macrophages that do not respond to any naked DNA. The amount of cytokines released from the DC was significantly increased by complex formation with cationic lipids when CpG-motif positive DNAs were used. Unlike murine macrophages or Flt-3 L cultured DC, GM-CSF DC did not release inflammatory cytokines in response to the addition of CT DNA/cationic lipid complex, suggesting that the activation is completely dependent on CpG motifs. Taken together, the results of the present study demonstrate that murine DC produce pro-inflammatory cytokines upon stimulation with CpG-containing DNAs and the responses are enhanced by cationic lipids. These results also suggest that DC are the major cells that respond to naked CpG DNA in vivo, although both DC and macrophages will release inflammatory cytokines after the administration of a DNA/cationic lipid complex.     

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) activates the MAPK pathway in human neutrophils

Infection by Helicobacter pylori causes an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by the infiltration of neutrophils andmononuclear inflammatory cells. The neutrophil-activating protein of Helicobacter pylori (HP-NAP) is a virulence factor that activates neutrophils, monocytes, and mast cells. However, the mechanism by which HP-NAP activates these cells is not fully understood. Here, we show that HP-NAP induces extracellular regulated kinase (ERK) and p38-mitogen-activated protein kinase (MAPK) activation in human neutrophils; c-Jun N-terminal kinase is not activated by HP-NAP. A MAPK/ERK kinase inhibitor and a p38-MAPK inhibitor suppress HP-NAP-mediated neutrophil oxidative burst, adhesion, andchemotaxis, but not actin polymerization. Pertussis toxin (PTX) inhibits all these neutrophil functions and the MAPK activation caused by HP-NAP. These results demonstrate that HP-NAP activates neutrophils through a PTX-sensitive pathway and that ERK and p38-MAPK are involved in many neutrophil functions stimulated by HP-NAP.     

Specific siRNA downregulated TLR9 and altered cytokine expression pattern in macrophage after CpG DNA stimulation

Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)candirectly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptorTLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial tothe subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are notfully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokineprofiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid uponCpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased whileTLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6,IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alterationof cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role ofCpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology.2005;2(2):130-135.     

Immunostimulatory effects of plasmid DNA and synthetic oligodeoxynucleotides

Nucleic acid immunization is a new vaccination technology. DNA vaccines do not only carry the genetic information for the antigen of interest but also deliver an adjuvant effect due to the presence of immunostimulatory sequences within the plasmid backbone. It is generally assumed that the adjuvant properties of plasmid DNA are equal to those described for oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. To challenge this hypothesis we have carried out a series of experiments comparing the ability of single- and double-stranded ODN containing CpG motifs to induce the activation of mouse spleen cells. Moreover, we compared the immunostimulatory properties of plasmids that were modified by the addition of two to four CpG motifs. Our results establish that plasmid DNA express their adjuvanticity as either double or single strands, and no differences were observed between modified and unmodified plasmids. On the other hand, the strongest stimulatory ODN sequences lost their adjuvant properties when administered as double-strand DNA. Furthermore, the profile of cytokines induced on spleen cells by plasmid DNA and ODN is different. Strikingly, plasmid DNA induces a moderate synthesis of IL-6 and a strong synthesis of IFN-gamma, whereas stimulation with ODN showed an inverse profile with a higher increase in the synthesis of IL-6 but a moderate increase in IFN-gamma. Finally, in vivo studies were consistent with the results obtained in vitro. Mice immunized with modified or unmodified plasmids encoding the glycoprotein D of HSV showed similar levels of cellular and humoral immune responses.     

Editorial: CpG Motifs to Modulate Innate and Adaptive Immune Responses

The vertebrate adaptive and innate immune systems have evolved to protect the host from pathogen infections. To achieve this mission, the innate immune system developed particular receptors, termed “pattern recognition receptors” (PRRs). These PRRs selectively bind certain types of structures expressed by pathogens but in principal absent in vertebrates. One of the best understood receptors is the Toll-like receptor (TLR) 9 that recognizes CpG sequence motifs in bacterial and viral DNA. Different classes of short synthetic phosphorothioate-stabilized CpG oligodeoxynucleotides were developed and are currently in human clinical trials in the fields of infectious disease, cancer, and asthma/allergy.     

Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor-α-mediated shock

Cell surface components of pathogens, such as lipopolysaccharide (LPS), are an important signal for receptor-mediated activation of immune cells. Here we demonstrate that DNA of gram-positive and gram-negative bacteria or certain synthetic oligonucleotides displaying unmethylated CpG-motifs can trigger macrophages in vitro to induce nuclear translocation of nuclear factor-xB, accumulate tumor necrosis factor (TNF)-α mRNA and release large amounts of TNF-α. In vivo these events culminate in acute cytokine-release syndrome which includes systemic but transient accumulation of TNF-α. D -Galactosamine (D -GalN)-sensitized mice succumb to lethal toxic shock due to macrophage-derived TNF-α resulting in fulminant apoptosis of liver cells. LPS and a specific oligonucleotide synergized in vivo as measured by TNF-α-release, suggesting that macrophages integrate the respective signals. The ability of macrophages to discriminate and to respond to bacterial DNA with acute release of pro-inflammatory cytokines may point out an important and as yet unappreciated sensing mechanism for foreign DNA.     

Multi-plasmid DNA vaccination avoids antigenic competition and enhances immunogenicity of a poorly immunogenic plasmid

DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi-plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single-plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin-related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co-injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single-plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single-and multi-plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4⁺ T helper cells. The sera from mice immunized with the four-plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.     

GM-CSF enhances a CpG-independent pathway of neutrophil activation triggered by bacterial DNA

We have previously demonstrated that bacterial DNA induces neutrophil activation through a CpG- and TLR9-independent but MyD88-dependent-pathway. In this study we determined that GM-CSF enhances the activation of neutrophils by bacterial DNA. Granulocyte-macrophage colony-stimulating factor increased IL-8 and IL-1beta secretion, and CD11b-upregulation induced by single-stranded bacterial DNA. It also enhanced neutrophil IL-8 production induced by double-stranded bacterial DNA, methylated single-stranded DNA, plasmid DNA, and phosphorothioated-CpG and non-CpG-oligodeoxynucleotides. Together these observations indicated that GM-CSF enhances neutrophil responses triggered by bacterial DNA in a CpG-independent fashion. We also found that GM-CSF enhanced the activation of the MAPKs p38 and ERK1/2 induced by bacterial DNA. Moreover, the pharmacological inhibition of these pathways significantly diminished GM-CSF ability to increase neutrophil activation by bacterial DNA. Finally, we observed that GM-CSF was unable to increase the activation of MyD88(-/-) neutrophils by bacterial DNA. Our findings suggest that GM-CSF modulates the CpG-independent, MyD88-dependent neutrophil response to bacterial DNA, by increasing the activation of the MAPKs p38 and ERK1/2.     

Fibrinogen-CD11b/CD18 interaction activates the NF-kappa B pathway and delays apoptosis in human neutrophils

The regulation of neutrophil half-life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2).Since NF-kappa B is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF-kappa B is involved in the control of PMN survival by sFbg. We show that sFbg triggers inhibitor protein kappa B (I kappa B-alpha) degradation and NF-kappa B activation. Furthermore, pharmacological inhibition of NF-kappa B abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF-kappa B translocation by sFbg, suggesting a relationship between ERK1/2 and NF-kappa B activation. Similar results are obtained when granulocytic-differentiated HL-60 cells are treated with sFbg, making this model highly attractive for integrin-induced gene expression studies. It can be concluded that NF-kappa B participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF-kappa B regulation may be of benefit for the resolution of the inflammatory response.     

TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism

Histamine is a bioactive amine that exerts immunomodulatory functions, including many allergic symptoms. It is preformed and stored in mast cells and basophils but recent evidence suggests that other cell types produce histamine in an inducible fashion. During infection, it has been suggested that neutrophils may produce histamine. We also observed that histamine is released in a neutrophil-mediated LPS-induced model of acute lung injury. Therefore, we sought to examine whether innate signals promote histamine production by neutrophils. Bone marrow-derived neutrophils stimulated with a range of TLR agonists secreted histamine in response to LPS or R837, suggesting TLR4 or TLR7 are important. LPS-driven histamine was enhanced by coculture with GM-CSF and led to a transient release of histamine that peaked at 8 h post stimulation. This was dependent upon de novo synthesis of histamine, since cells derived from histidine decarboxylase (HDC) deficient mice were unable to produce histamine but did generate reactive oxygen species upon stimulation. Using pharmacological inhibitors, we show that histamine production requires PI3 kinase, which has been shown to regulate other neutrophil functions, including activation and selective granule release. However, unlike mast cells, HDC deficiency did not alter the granule structure of neutrophils, suggesting that histamine does not participate in granule integrity in these cells. Consequently, our findings establish that neutrophils generate histamine in response to a select panel of innate immune triggers and that this might contribute to acute lung injury responses.     

Torquetenovirus DNA drives proinflammatory cytokines production and secretion by immune cells via toll-like receptor 9

Active infection with torquetenovirus (TTV) has been associated with an increased severity of diseases in which inflammation plays a particularly important pathogenetic role. Here, we report that cloned DNA of a genogroup 4 TTV (ViPiSAL) is an activator of proinflammatory cytokine production by murine spleen cells and that the effect is mediated via toll-like receptor (TLR)9. The same DNA also increased the levels of proinflammatory cytokines induced by two well-characterized TLR9 stimulants. Finally, in silico analyses of the genomes of ViPiSAL and other TTVs revealed marked differences in the representation of CpG motifs known to be most effective at activating immune cells via TLR9. These findings demonstrate for the first time that at least one TTV isolate has the potential to stimulate and co-stimulate inflammatory responses.     

The assembly of a short linear natural cytosine-phosphate-guanine DNA into dendritic structures and its effect on immunostimulatory activity

DNA containing unmethylated CpG dinucleotides, or CpG motifs, (CpG DNA) has been explored as a therapeutic agent, owing to its potent immunostimulatory activity. A previous study showing that Y-shaped (Y-) CpG DNA has a high immunostimulatory activity compared with single- or double stranded CpG DNA suggests the possibility that CpG DNA in a more complicated structure is a stronger activator of the immune system. In the present study, dendrimer-like DNA (DL-DNA) was prepared by ligating Y-DNA monomers. The DL-DNA of the second or third generation with 12 or 24 highly potent CpG motifs in one unit, respectively, were designed and successfully prepared for the first time. These DL-DNAs induced greater amounts of tumor necrosis factor-α and interleukin-6 from RAW264.7 macrophage-like cells than did a mixture of Y-DNA with the same sequences as the corresponding DL-DNA. DL-DNA was more efficiently taken up by RAW264.7 cells than Y-DNA, but the increase was lower than that exhibited by the levels of cytokine release. These results suggest that the dendritic structure formation is a potential approach to increasing the immunostimulatory activity of CpG DNA without any modifications of the chemical structure of the natural phosphodiester DNA.     

Stimulatory effect of fragments from transcribed region of ribosomal repeat on human peripheral blood lymphocytes

Fragments from the transcribed region of the ribosomal repeat include considerable amounts of unmethylated CpG DNA motifs. These motifs activate immune cells via the interaction with Toll receptors. In vitro experiments confirmed the stimulatory effect of transcribed region of ribosomal repeat on human lymphocytes. Culturing of lymphocytes in a medium containing 2–20,000 ng/ml fragments from transcribed region of ribosomal repeat was accompanied by structural changes in the nucleus in a considerable number of cells. These changes manifested in translocation of pericentromeric heterochromatin from the membrane to the center of the nucleus and activation of the nucleolus and were accompanied by a significant increase in interleukin-6 production and slight stimulation of tumor necrosis factor-α synthesis. The transcribed region of the ribosomal repeat and E. coli DNA had various effects on quantitative parameters of lymphocytes. Our results suggest the existence of mechanisms of stimulation not mediated by the interaction of CpG DNA motifs with Toll receptors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 10, pp. 409–413, October, 2006     

LL37 inhibits the inflammatory endothelial response induced by viral or endogenous DNA

In viral infection, morbidity and mortality often result from extrahepatic disease manifestations such as vasculitis. We hereby show that human microvascular endothelial cells express viral receptors of the innate immune system which are induced upon ligand engagement. Furthermore, stimulation of endothelial cells with the synthetic analog of viral DNA, poly (dA:dT), human DNA and hepatitis B virus-containing immunoprecipitates from a patient with polyarteritis nodosa induces an inflammatory response including the upregulation of adhesion molecules, which is mediated exclusively by TLR9 and involves an IRF3-dependent pathway. Thus, endothelial cells are able to actively participate in immune mediated vascular inflammation caused by viral infections. Furthermore, we provide evidence for the ability of LL37 to bind and internalize viral or endogenous DNA into non-immune cells. DNA nucleotides internalized by LL37 suppress the production of proinflammatory mediators suggesting a protective effect against direct responses to viral infection or circulating DNA-fragments of endogenous origin.     

Toll-like receptor 9-mediated induction of the immunosuppressive pathway of tryptophan catabolism

A series of recent studies, including an article in this issue of the European Journal of Immunology, have demonstrated that the administration of CpG-rich oligodeoxynucleotides (CpG-ODN) in experimental settings may lead to the activation of the immunosuppressive pathway of tryptophan catabolism, depending on several factors, including the route of CpG-ODN administration. These studies call attention to the need for a careful evaluation of the modalities of inclusion of CpG-ODN in vaccines for human use. At the same time, these studies may offer novel opportunities for use of CpG-ODN as immunosuppressive agents and may also lead to an improved understanding of the cellular events mediated by Toll-like receptor 9 signaling.     

Down-Regulation of TLR9 Expression Affects the Maturation and Function of Murine Bone Marrow-Derived Dendritic Cells Induced by CpG

Toll-like receptor 9 （TLR9） is expressed intracellularly by dendritic cells （DCs） and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequent immune responses. Here, RNA interference （RNAi） was used to identify the effect of CpG DNA signaling on DC function. The results showed that transfection of DCs with siRNA specific for TLR9 gene significantly down-regulated TLR9 expression. Immature DCs transfected with TLR9 siRNA did not differentiate into mature DCs with exposure to CpG. TLR9 siRNA-treated DCs expressed low levels of MHC II and CD40 without reducing endocytosis. Furthermore, TLR9 siRNA-transfected DCs exhibited a decreased allostimulatory capacity in a lymphocyte proliferation assay and attenuated Thl responses by decreasing IL-12p70 production. Our findings indicate that siRNA in silencing TLR9 gene in DCs may offer a potential tool to study the TLR9-CpG pathway.