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.     

Co-stimulation with TLR3 and TLR21 ligands synergistically up-regulates Th1-cytokine IFN-γ and regulatory cytokine IL-10 expression in chicken monocytes

Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune system for various conserved pathogen-associated molecular motifs. Chicken TLR3 and TLR21 (avian equivalent to mammalian TLR9) recognize poly I:C (double-stranded RNA) and CpG-ODN (a CpG-motif containing oligodeoxydinucleotide), respectively. Interaction between TLR3 and TLR21 agonists poly I:C and CpG-ODN has been reported to synergize in expression of proinflammatory cytokines and chemokines and the production of nitric oxide in chicken monocytes. However, the interaction between poly I:C and CpG-ODN on the expression of interferons (IFNs) and Th1/Th2 cytokines remains unknown. The objective of the present study was to investigate the effect of the interaction between poly I:C and CpG-ODN on the mRNA expression levels of IFN-α and IFN-β, Th1 cytokines IFN-γ and IL-12, Th2 cytokine IL-4, and regulatory IL-10 in chicken monocytes. When stimulated with either agonist alone, CpG-ODN significantly up-regulated the expression of INF-γ, IL-10, and IL-12p40, but not IFN-α and IFN-β; whereas poly I:C induced the expression of INF-γ, IFN-α, IFN-β, and IL-10; but not IL-12p40. However, stimulation with a combinatory CpG-ODN and poly I:C further synergistically increased the expression of IFN-γ and IL-10 mRNA. Our results provide strong evidence supporting the critical role of TLR3 and TLR21 in avian innate immunity against both viral and bacterial infections; and the synergistic interaction between the TLR3 and TLR21 pathways produces a stronger Th1-biased immune response in chicken monocytes. Our result also suggest a potential use of poly I:C and CpG-ODN together as a more efficient adjuvant for poultry vaccine development.     

DNA–polymer conjugates for immune stimulation through Toll-like receptor 9 mediated pathways

Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide motifs are agonists of Toll-like receptor 9 and are currently being investigated for use as vaccine adjuvants through the promotion of type I immunity. Several classes of ODN have been developed which differ in their propensity to aggregate, which in turn alters cytokine profiles and cellular subsets activated. Although aggregation state is correlated with the change in cytokine response, it is unknown if this results from a change in the number of ODNs available for binding and/or the possible engagement of multiple TLR9 molecules. Here, we examined the role of ligand valency on the activation of TLR9 through the synthesis of ODN–poly(acrylic acid) (PAA) conjugates. The compositions and size of the conjugates were characterized by UV-vis spectroscopy, proton nuclear magnetic resonance, gel permeation chromatography and dynamic light scattering. Enzyme-linked immunosorbent assays of cytokine secretion by murine-like macrophages indicate that these ODN–PAA polymer conjugates show enhanced immunostimulation at 100-fold lower concentrations than those required for ODN alone, for both TNF-α and IL-6 release, and are more potent than any other previously reported multivalent ODN constructs. Increasing valency was shown to significantly enhance cytokine expression, particularly for IL-6. Knockdown by siRNA demonstrates that these polymer conjugates are specific to TLR9. Our results define valency as a critical design parameter and polymer conjugation as an advantageous strategy for producing ODN immunomodulatory agents.     

Therapeutic Applications of Synthetic CpG Oligodeoxynucleotides as TLR9 Agonists for Immune Modulation

Vertebrate toll-like receptors (TLRs) sense invading pathogens by recognizing bacterial and viral structures and, as a result, activate innate and adaptive immune responses. Ten human functional TLRs have been reported so far; three of these (TLR7, 8, and 9) are expressed in intracellular compartments and respond to single-stranded nucleic acids as natural ligands. The pathogen structure selectively recognized by TLR9 in bacterial or viral DNA was identified to be CpG dinucleotides in specific sequence contexts (CpG motifs). Short phosphorothioate-stabilized oligodeoxynucleotides (ODNs) containing such motifs are used as synthetic TLR9 agonists, and different classes of ODN TLR9 agonists have been identified with distinct immune modulatory profiles. The TLR9-mediated activation of the vertebrate immune system suggests using such TLR9 agonists as effective vaccine adjuvants for infectious disease, and for the treatment of cancer and asthma/allergy. Immune activation by CpG ODNs has been demonstrated to be beneficial in animal models as a vaccine adjuvant and for the treatment of a variety of viral, bacterial, and parasitic diseases. Antitumor activity of CpG ODNs has also been established in numerous mouse models. In clinical vaccine trials in healthy human volunteers or in immunocompromised HIV-infected patients, CpG ODNs strongly enhanced vaccination efficiency. Most encouraging results in the treatment of cancers have come from human phase I and II clinical trials using CpG ODNs as a tumor vaccine adjuvant, monotherapy, or in combination with chemotherapy. Therefore, CpG ODNs represent targeted immune modulatory drugs with a broad range of potential applications.     

Investigation of the differential potentials of TLR agonists to elicit uveitis in mice

TLRs are critical for host defense and innate immunity. Emerging evidence also supports a role for TLRs in many chronic inflammatory diseases, including inflammatory eye disease, known as uveitis. The activation of TLR4 by endotoxin induces a standard model of murine uveitis. How activation of additional TLRs influences the onset and/or severity of anterior uveitis has not been examined. We sought to elucidate the potential of TLRs (TLR1/2, TLR2/6, TLR3, TLR4, TLR5, TLR7/8, and TLR9) to trigger uveitis in mice. Directly stimulated iris/ciliary body explants demonstrated a marked increase in production of inflammatory cytokines TNF-α, IL-6, IP-10/CXCL10, MCP-1, and KC with relatively little production of IFN-γ, IL-10, IL-12p40, IL-12p70, IL-17, IL-1β, IL-4, or RANTES. The cytokine-response profiles were comparable amongst the TLR agonists, albeit some differences were noted, such as greater IP-10 production following TLR3 activation. Intra-ocular injection of TLR agonists increased leukocyte interactions with the endothelium of the iris vasculature and resulted in chemotaxis into the iris tissue. Assessment of leukocytic responses by ivt videomicroscopy and histology revealed quantitative differences amongst responses to the TLR agonists with respect to the timing and numbers of rolling, adhering, iris-infiltrating, and aqueous humor-infiltrating cells, along with cytokine levels in vivo. Our data demonstrate the eye's responsiveness to a diverse array of microbial products, which activates TLRs, and reveal differences in relative cellular response among the various TLR agonists in vivo.     

A Point of View: HIV-1/AIDS is an Allergy but CpG ODN Treatments may Inhibit Virus Replication and Reactivate the Adaptive Immunity – Hypothesis and Implications

Reevaluation of the increase in the levels of IgE and IL-4 in sera of HIV-1 infected and AIDS patients led to the suggestion that AIDS resembles allergy. Studies on the properties of the viral shed gp120 revealed that it resemble environmental allergens in their ability to induce hematopoietic cells to release large amounts of Th2 cytokines, inhibitors of the patients adaptive immune response. Yet, induction of TLR9+ plasmacytoid DCs by CpG ODNs cause the release of type I interferons, inhibitors of HIV-1 replication and IL-4 release from hematopoietic cells. CpG ODN binding to TLR+ B cells inhibits IgE synthesis and reactivates the failing adaptive immunity. The possible use of CpG ODNs as treatment to patients is discussed.     

Nucleosome-induced neutrophil activation occurs independently of TLR9 and endosomal acidification: implications for systemic lupus erythematosus

The nucleosome is a major autoantigen known to activate PMN in systemic lupus erythematosus (SLE). TLR9 recognizes bacterial and even mammalian DNA under certain circumstances. Nevertheless, the role of TLR9 in SLE development is still unclear. Since nucleosomes are composed of DNA, we investigated whether TLR9 is required for nucleosome-induced PMN activation. Isolated neutrophils were cultured with nucleosomes, plasma from lupus patients and other stimuli in the presence/absence of various inhibitors. Cells were analyzed by flow cytometry, ELISA and confocal microscopy. We found that nucleosomes circulating in lupus plasma induce the secretion of pro-inflammatory cytokines by PMN. Nucleosomes activate human PMN independently of unmethylated CpG sequences in nucleosomal DNA, leading to IL-8/IL-6/TNF secretion and CD11b up-regulation. Nucleosomes accumulate in the cytoplasm of PMN upon endocytosis, induce TLR9 up-regulation and act synergistically with TLR9 ligands. Nucleosome-induced activation was not inhibited by polymyxin B (PB), chloroquine (CQ), ammonium chloride (AC) or a TLR9 antagonist. Moreover, both PMN isolated from WT and TLR9-KO mice were activated by nucleosomes, as detected by MIP-2 secretion and CD11b up-regulation. Activation occurred therefore independently of endotoxins, endosomal acidification, TLR9 and CpG motifs. TLR9 may thus be differently required in the triggering of nucleosome-induced innate immunity and anti-nucleosome B-cell autoimmunity.     

CC chemokine receptor 4 modulates Toll-like receptor 9-mediated innate immunity and signaling

The present study addressed the modulatory role of CC chemokine receptor 4 (CCR4) in Toll-like receptor (TLR) 9-mediated innate immunity and explored the underlying molecular mechanisms. Our results demonstrated that CCR4-deficient mice were resistant to both septic peritonitis induced by cecal ligation and puncture (CLP) and CpG DNA/D-galactosamine-induced shock. In bone marrow-derived macrophages (BMMPhi) from CLP-treated CCR4-deficient mice, TLR9-mediated pathways of MAPK/AP-1, PI3K/Akt, and IkappaB kinase (IKK)/NF-kappaB were impaired compared to wild-type (WT) cells. While TLR9 expression was not altered, the intensity of internalized CpG DNA was increased in CCR4-deficient macrophages when compared to WT macrophages. Pharmacological inhibitor studies revealed that impaired activation of JNK, PI3K/Akt, and/or IKK/NF-kappaB could be responsible for decreased proinflammatory cytokine expression in CCR4-deficient macrophages. Interestingly, the CCR4-deficient BMMPhi exhibited an alternatively activated (M2) phenotype and the impaired TLR9-mediated signal transduction responses in CCR4-deficient cells were similar to the signaling responses observed in WT BMMPhi skewed to an alternatively activated phenotype. These results indicate that macrophages deficient in CCR4 impart a regulatory influence on TLR9-mediated innate immunity.     

Innate immune responses in lupus-prone Palmerston North mice: differential responses to LPS and bacterial DNA/CpG oligonucleotides

Inadequate immune response to infectious danger may contribute to the pathogenesis of systemic autoimmune diseases, e.g., systemic lupus erythematosus. To test this hypothesis, we studied innate responses of prediseased lupus-prone Palmerston North (PN) mice to lipopolysaccharide (LPS), bacterial DNA, and synthetic CpG oligonucleotides. LPS and bacterial DNA/CpG oligodeoxyribonucleotides (ODNs) drove PN splenocytes into the cell cycle and protected B cells against spontaneous apoptosis, as in control lupus-free DBA-1 mice. LPS induced significantly higher IL-6 production in PN than in control splenocytes. In contrast, in PN splenocytes bacterial DNA and CpG ODNs induced approximately four- to sixfold lower IL-12p40 and approximately twofold lower IL-6 secretion than controls. This reduction in cytokine secretion in PN mice was not due to delayed kinetics but was related to significantly higher constitutive and CpG-inducible IL-10 secretion. Neutralizing anti-IL-10 antibodies almost completely restored PN IL-6 and IL-12p40 secretion to DBA-1 levels, whereas exogenous IL-10 inhibited in vitro IL-6 and IL-12p40 production in DBA-1 mice. Importantly, treatment with either IL-10 or anti-IL-10 antibody did not modulate CpG-induced cell cycle entry and apoptosis protection in either strain. In conclusion, lupus-prone PN mice show abnormal innate responses through their pattern-recognition TLR9 receptors, characterized by higher inducible IL-10 and lower IL-12p40 and IL-6 secretion, thus implying that response to infectious danger in PN mice is inappropriate and may be linked to lupus pathogenesis.     

Differential signaling by CpG DNA in DCs and B cells: not just TLR9

CpG-containing oligodeoxynucleotides (CpG ODNs) act on Toll-like receptor 9 (TLR9) that is expressed on B cells and plasmacytoid dendritic cells (pDCs) to stimulate the innate immune system, however, different types of CpG ODNs induce distinct responses. Recent papers suggest some CpG ODNs could require a second receptor or cofactor to signal. The different signaling complexes assembled might impact on the affinity with which CpG ODNs signal to TLR9 or activate additional pathways that lead to distinct immune responses.     

C7: A CpG oligodeoxynucleotide that induces protective immune response against megalocytivirus in Japanese flounder (Paralichthys olivaceus) via toll-like receptor 9-mediated signaling pathway

Megalocytivirus is the causative agent of severe disease outbreaks in farmed fish. Currently there is no effective control against megalocytivirus in aquaculture. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are known to possess marked immunostimulatory properties. In this study, we investigated the potentials of ten CpG ODNs as antiviral agents in a model of Japanese flounder (Paralichthys olivaceus). We found that, when administered into flounder, three of the ten CpG ODNs inhibited viral replication in kidney, spleen, and liver. ODN C7, which exhibited the strongest inhibitory activity, was able to promote proliferation of peripheral blood leukocytes and enhance activation of head kidney mononuclear adherent phagocytes. When the expression of toll-like receptor 9 (TLR9) was knocked down in vivo by small interfering RNA, C7-mediated immune response and antiviral activity were significantly blocked. Moreover, when C7 was co-administered with pCN86, a DNA vaccine against megalocytivirus, a significant increase in vaccine-induced protection was observed compared to administration with pCN86 alone. Further analysis showed that compared to fish immunized with pCN86, fish immunized with pCN86 plus C7 exhibited significantly upregulated expression of a wide range of genes involved in innate and adaptive immunity. Taken together, these results indicate that ODN C7 activates TLR9-mediated immune response and possesses antiviral and adjuvant potentials that may be exploited for the control of megalocytivirus infection in farmed flounder.     

Immunomodulatory effects of CpG oligodeoxynucleotides on established th2 responses

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-gamma) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-gamma, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8(+) T lymphocytes. Although IFN-gamma plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-gamma was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-gamma, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-gamma was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-gamma and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.     

Progesterone inhibits Toll-like receptor 4-mediated innate immune response in macrophages by suppressing NF-κB activation and enhancing SOCS1 expression

Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been implicated to have a functional role in immune response, mainly at concentrations commensurate with pregnancy. However, the underlying mechanisms remain to be fully understood. Here we present the evidences that progesterone inhibited immune response to lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (CpG ODNs) through modulating Toll-like receptor (TLR) signaling. Pretreatment with progesterone can significantly inhibit TLR4 and TLR9-triggered IL-6 and nitric oxide (NO) production in macrophages. Furthermore, we found that progesterone can significantly inhibit LPS-induced nitric oxide synthesis (iNOS), TLR4 expression and nuclear factor-κB (NF-κB) activation. Consistently, as a negative feedback inhibitor, the expression of suppressor of cytokine signaling (SOCS1) protein was up-regulated by progesterone in LPS-stimulated macrophages. These results support the concept that progesterone might inhibit innate immune response by suppressing NF-κB activation and enhancement of SOCS1 expression, providing a possible mechanistic explanation for the function of progesterone in regulating innate immune responses.     

Toll-like receptor 9-dependent immune activation by unmethylated CpG motifs in Aspergillus fumigatus DNA

Phagocytic defenses are critical for effective host defenses against the opportunistic fungal pathogen Aspergillus fumigatus. Previous studies found that following challenge with A. fumigatus, Toll-like receptor 9 (TLR9) knockout mice survived longer than wild-type mice. However, the mechanism responsible was not defined. Here we demonstrate that A. fumigatus contains unmethylated CpG sequences, the natural ligands for TLR9. A. fumigatus DNA and synthetic CpG-rich oligodeoxynucleotides (ODNs) containing sequences found in the A. fumigatus genome potently stimulated the production of proinflammatory cytokines in mouse bone marrow-derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells. The response was decreased when the fungal DNA was treated with a CpG methylase or with CpG-specific endonucleases. A role for TLR9 was demonstrated as cytokine production was abolished in BMDCs from TLR9-deficient mice. Moreover, transfection of HEK293 cells with human TLR9 conferred responsiveness to synthetic CpG-rich ODNs containing sequences found in A. fumigatus DNA. Taken together, these data demonstrate that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines, which may contribute to the immune response to the pathogen.     

Immunotherapeutic potential of CpG oligodeoxynucleotides in veterinary species

Abstract

Innate immunity plays a critical role in host defense against infectious diseases by discriminating between self and infectious non-self. The recognition of infectious non-self involves germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). The PAMPs are the components of pathogenic microbes which include not only the cell wall constituents but also the unmethylated 2′-deoxy-ribo-cytosine-phosphate-guanosine (CpG) motifs. These CpG motifs present within bacterial and viral DNA are recognized by toll-like receptor 9 (TLR9), and signaling by this receptor triggers a proinflammatory cytokine response which, in turn, influences both innate and adaptive immune responses. The activation of TLR9 with synthetic CpG oligodeoxynucleotides (ODNs) induces powerful Th1-like immune responses. It has been shown to provide protection against infectious diseases, allergy and cancer in laboratory animal models and some domestic animal species. With better understanding of the basic biology and immune mechanisms, it would be possible to exploit the potential of CpG motifs for animal welfare. The research developments in the area of CpG and TLR9 and the potential applications in animal health have been reviewed in this article.     

Plasmacytoid dendritic cells: the key to CpG

The vertebrate immune system has established TLR9 to detect microbial DNA based on unmethylated CG dinucleotides within certain sequence contexts (CpG motifs). In humans, the expression of toll-like receptor 9 (TLR9) is restricted to B cells and plasmacytoid dendritic cells (PDC). The PDC is characterized by the ability to rapidly synthesize large amounts of type I IFN (IFN- and IFN-β) in response to viral infection. In contrast to other dendritic cell subsets which express a broad profile of TLRs, the TLR profile in PDC is restricted to TLR7 and TLR9. So far, CpG DNA is the only defined microbial molecule recognized by PDC. An intriguing feature of PDC is its ability to simultaneously produce the two major Th1-inducing cytokines in humans, IFN- and IL-12, both at high levels. The ratio of IFN- versus IL-12 and the quantity of these cytokines are regulated by T helper cell-mediated costimulation via CD40 ligation. The ratio also depends on the differentiation stage of the PDC at the time of stimulation and the type of CpG ODN used. We propose a model in which the establishment of Th1 responses in vivo is improved by appropriately stimulated PDC that otherwise – in the absence of CpG DNA – support Th2 or Th0 responses and thus have been called DC2.     

Toll-like receptors and immune response in allergic disease

Allergic reactions are dominated by the preferential development of specific Th2 responses against innocuous antigens in atopic individuals. This can reflect alterations in innate immune mechanisms. Toll-like receptors (TLRs) have evolved as key molecules., in innate and adaptive immunity. Their activation by structurally distinct exogenous or endogenous, ligands present at the cell microenvironment plays a critical role in antimicrobial defense. The global view is that TLR activation induces antigen-presenting, cells to produce cytokines that favor Th1-type immune responses, suggesting that it might prevent the development of deleterious Th2 responses in allergy. On the basis of epidemiological studies and recent data, it has been established that TLRs play a role in the development of Th2 responses. However, more information is needed to fully understand the mechanism of TLR involvement and the implication of immune cells that express TLRs in the Th1/Th2 cytokine profiles. Several TLRs, such as TLR9, TLR7, and TLR8, can be considered as good target candidates. Some TLR ligands, such as CpG DNA, are effective adjuvants, strong inducers of both IL-5 and eosinophilia downregulation. They are also potential links to allergen epitopes that could provide new allergen-specific immunotherapy regimens for the treatment of allergic disorders.