Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct cytokine gene expression patterns in rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing rhesus monkey plasmacytoid dendritic cells.
ABSTRACT: To determine if deoxycytidyl-deoxyguanosine oligonucleotides (CpG ODN) can be used effectively as nonspecific inducers of innate immune defenses for preventative or therapeutic interventions in infectious disease models for nonhuman primates, the present study evaluated the response of rhesus monkey peripheral blood mononuclear cells to three different synthetic CpG ODN classes by defining the cytokine gene expression patterns and by characterizing IFN-alpha/beta responses. Depending on the type and dose of CpG ODN used for stimulation, distinct gene expression patterns were induced. CpG ODN class A (CpG-A ODN) and CpG-C ODN, but not CpG-B ODN, were potent inducers of alpha interferon (IFN-alpha), and this response was due to IFN-alpha production by TLR9-positive plasmacytoid dendritic cells. Importantly, there was a dose-dependent increase in IFN-alpha responses to CpG-A ODN but a dose-dependent decrease in IFN-alpha responses by CpG-B ODN. The most sustained IFN-alpha response was induced by CpG-A ODN and was associated with a stronger induction of interferon regulatory factor 7 and the induction of several interferon-stimulated genes. In contrast, and independent of the dose, CpG-B ODN were the weakest inducers of IFN-alpha but the most potent inducers of proinflammatory cytokines. CpG-C ODN induced cytokine gene expression patterns that were intermediate between those of CpG-A and CpG-B ODN. Thus, the different types of CpG ODN induce different post-TLR9 signaling pathways that result in distinct cytokine gene expression patterns. Based on these findings, A and C class CpG ODN, but not B class CpG ODN, may be particularly suited for use as therapeutic or prophylactic antiviral interventions.
Project description:The initial host response to viral infection occurs after Toll-like receptors (TLRs) on dendritic cells (DC) are stimulated by viral nucleic acids (double-stranded RNA, single-stranded RNA) and alpha interferon (IFN-alpha) and IFN-beta are produced. We hypothesized that pharmacologic induction of innate antiviral responses in the cervicovaginal mucosa by topical application of TLR agonists prior to viral exposure could prevent or blunt vaginal transmission of simian immunodeficiency virus (SIV). To test this hypothesis, we treated rhesus monkeys intravaginally with either the TLR9 agonist, CpG oligodeoxynucleotides (ODN), or the TLR7 agonist, imiquimod. Both immune modifiers rapidly induced IFN-alpha and other antiviral effector molecules in the cervicovaginal mucosa of treated animals. However, both CpG ODN and imiquimod also induced proinflammatory cytokine expression in the cervicovaginal mucosa. In the vaginal mucosa of imiquimod-treated monkeys, we documented a massive mononuclear cell infiltrate consisting of activated CD4(+) T cells, DC, and beta-chemokine-secreting cells. After vaginal SIV inoculation, all TLR agonist-treated animals became infected and had plasma vRNA levels that were higher than those of control monkeys. We conclude that induction of mucosal innate immunity including an IFN-alpha response is not sufficient to prevent sexual transmission of human immunodeficiency virus.
Project description:The interaction of cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) with Toll-like receptor 9 (TLR9) activates the immune system. Multimeric class A CpG ODNs induce interferon-? (IFN-?) and, to a lesser extent, interleukin-6. By contrast, monomeric class B CpG ODNs induce interleukin-6 but not IFN-?. This difference suggests that the multimerization of CpG ODN molecules is a key factor in IFN-? induction. We multimerized class B CpG ODN2006x3-PD molecules that consist entirely of a phosphodiester backbone onto quantum dot silicon nanoparticles with various binding modes. Herein, we present the binding mode-dependent bifurcation of cytokine induction and discuss its possible mechanism of CpG ODN and TLR9 interaction. Our discoveries also suggest that nanoparticles play roles in not only delivery of CpG ODNs but also control of CpG ODN activity.
Project description:Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells (pDCs) and triggers MyD88-dependent type I interferon (IFN) responses. To better understand TLR9 biology in pDCs, we established a yeast two-hybrid library for the identification of TLR9-interacting proteins. Here, we report that an IFN-inducible protein, phospholipid scramblase 1 (PLSCR1), interacts with TLR9 in pDCs. Knockdown of PLSCR1 expression by siRNA in human pDC cell line led to a 60-70% reduction of IFN-? responses following CpG-ODN (oligodeoxynucleotide) stimulation. Primary pDCs from PLSCR1-deficient mice produced lower amount of type 1 IFN than pDCs from the wild-type mice in response to CpG-ODN, herpes simplex virus and influenza A virus. Following CpG-A stimulation, there were much lower amounts of TLR9 in the early endosomes together with CpG-A in pDCs from PLSCR1-deficient mice. Our study demonstrates that PLSCR1 is a TLR9-interacting protein that plays an important role in pDC's type 1 IFN responses by regulating TLR9 trafficking to the endosomal compartment.
Project description:Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN-?) and pro-inflammatory (IL-6) cytokines by CpG-stimulated human pDCs and the human CAL-1 pDC cell line. Results show that interferon regulatory factor-5 (IRF-5) and NF-?B p50 are key co-regulators of IFN-? and IL-6 expression following TLR9-mediated activation of human pDCs. The nuclear accumulation of IRF-1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF-8 was identified as a novel negative regulator of gene activation in CpG-stimulated pDCs. As variants of IRF-5 and IRF-8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of "K" ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.
Project description:CpG oligodeoxynucleotides (ODNs) are proved to have strong immune stimulatory activity. Plasmids containing CpG ODNs could be conveniently and low-costly used as vaccine adjuvant. However, they are different among various plasmids, motif repeats, species, etc. In the present study, plasmid pcDNA3.1 (+) containing five repetitions of CpG ODN 1670A named pcDNA3.1-1670A*5 with strong immunostimulation was screened out from twelve recombinant plasmids and three empty vectors by cell proliferation activity, interferon promoter activities and immune related gene expressions in CIK cells. It works through TLR9-mediated signaling pathway, triggering the immune related genes expression. Furthermore, the potentiality of pcDNA3.1-1670A*5 as adjuvant was tested in vivo. pcDNA3.1-1670A*5 was co-inoculated with inactivated GCRV vaccine on grass carp fingerlings. Immunoglobulins (IgM, IgD, IgZ), TLR9, IFN?2, IFN1, TNF-?, Mx2 and VP4 were examined. Ultimately, pcDNA3.1-1670A*5 significantly enhanced the expressions of IgM in serum, head kidney and spleen, recognition receptor TLR9 as well as antiviral effector molecule Mx2, and inhibited GCRV proliferation in head kidney and spleen tissues. The present study explored the application and mechanism of plasmid containing CpG ODN as high-efficient adjuvant to promote efficiency of vaccine and control disease in grass carp, which will contribute to the development of new type CpG ODN adjuvant in aquaculture industry.
Project description:Bacterial DNA expressing unmethylated CpG motifs binds to TLR9, thereby stimulating a broadly protective, innate immune response. Although CpG-mediated signal transduction has been studied, the scope of TLR9-dependent gene expression is incompletely understood. To resolve these issues, mice were treated with immunostimulatory CpG oligonucleotides (ODN) and splenic mRNA levels monitored from 30 min through 3 days by microarray. Through the unique application of bioinformatic analysis to these experimental data, this study is the first to describe the complex regulatory networks responsible for TLR9-mediated gene expression. Current results are the first to establish that CpG-induced stimulation of the innate immune system proceeds in multiple waves over time, and gene up-regulation is mediated by a small number of temporally activated "major inducers" and "minor inducers". An additional study of TNF knockout mice supports the conclusion that the regulatory networks identified by our bioinformatic analysis accurately identified CpG ODN-driven gene-gene interactions in vivo. Equally important, this work identifies the counter-regulatory mechanisms embedded within the signaling cascade that suppresses the proinflammatory response triggered in vivo by CpG DNA stimulation. Identifying these network interactions provides novel and global insights into the regulation of TLR9-mediated gene activation, improves our understanding of TLR-mediated host defense, and facilitates the development of interventions designed to optimize the nature and duration of the ensuing response.
Project description:Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type-1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K-type) induces IFNs only weakly and STING ligands rather induce type-2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type-1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING-ligand cyclic GMP-AMP (cGAMP), culminating in NK cell IFN-? (type-II IFN) production, is due to the concurrent effects of IL-12 and type-I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type-1 adjuvant, capable of inducing strong Th 1-type responses, as demonstrated by enhanced antigen-specific IgG2c and IFN-? production, as well as cytotoxic CD8(+) T-cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen-free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type-II IFN inducer.
Project description:Background:C type CpG oligodeoxynucleotides (CpG-C ODNs), possessing the features of both A type and B type CpG ODNs, exert a variety of immunostimulatory activities and have been demonstrated as an effective antitumor immunotherapy. Based on the structural characteristics, we designed 20 potential ODNs with the aim of synthesizing an optimal, novel CpG-C ODN specific to human and murine Toll-like receptor 9 (TLR9). We also sought to investigate the in vitro immunostimulatory and in vivo antitumor effects of the novel CpG-C ODN. Methods:Twenty potential CpG-C ODNs were screened for their ability to secrete interferon (IFN)-?, and interleukin (IL)-6 and tumor necrosis factor (TNF)-? production for the three most promising sequences were assayed in human peripheral blood mononuclear cells (PBMCs) by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array assay. The functions of human and mouse B cells, and cytokine production in mice induced by the most promising sequence, HP06T07, were determined by flow cytometry and ELISA. Growth and morphology of tumor tissues in in vivo murine models inoculated with CT26 cells were analyzed by a growth inhibition assay and immunohistochemistry, respectively. Results:Among the 20 designed ODNs, HP06T07 significantly induced IFN-?, IL-6, and TNF-? secretion, and promoted B-cell activation and proliferation in a dose-dependent manner in human PBMCs and mouse splenocytes in vitro. Intratumoral injection of HP06T07 notably suppressed tumor growth and prolonged survival in the CT26 subcutaneous mouse model in a dose-dependent manner. HP06T07 administered nine times at 2-day intervals (I2) eradicated tumor growth at both primary and distant sites of CT26 tumors. HP06T07 restrained tumor growth by increasing the infiltration of T cells, NK cells, and plasmacytoid dendritic cells (pDCs). Conclusions:HP06T07, a novel CpG-C ODN, shows potent immunostimulatory activity in vitro and suppresses tumor growth in the CT26 subcutaneous mouse model.
Project description:Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-alpha and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-alpha production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-alpha production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1-positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-alpha from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1-positive endosomes accompanied by the loss of IFN-alpha production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-alpha and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-alpha response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs.
Project description:Due to their constant exposure to inhaled antigens, lungs represent a particularly immunosuppressive environment that limits excessive immune responses; however, cancer cells can exploit this unique environment for their growth. We previously described the ability of aerosolized CpG-ODN combined with Poly(I:C) (TLR9 and TLR3 agonists, respectively) to promote antitumor immunity in a B16 melanoma lung metastasis model. Here, we explored the possibility of improving the therapeutic efficacy of TLR9/TLR3 agonist combinations by including in the inhalant either an antibody directed to both Ly6G and Ly6C markers to locally deplete myeloid-derived suppressive cells (MDSCs) or IFN? to directly activate the natural killer (NK) and macrophage innate immune cells in the lung. Addition of nebulized anti-MDSC antibody RB6-8C5 to aerosolized CpG-ODN/Poly(I:C) resulted in reduced mRNA levels of immunsuppressive molecules (IL10, Arg-1, and Nos2), increased activation of resident NK cells and improved treatment outcome, with a significant reduction in established B16 melanoma lung metastases compared to treatment with CpG-ODN/Poly(I:C) alone. Likewise, addition of aerosolized IFN? led to increased mRNA levels of proinflammatory cytokines (IL15 and IFN?) in the lung and recruitment of highly activated NK cells, with no evident signs of toxicity and with a significantly improved antitumor effect as compared with aerosolized CpG-ODN/Poly(I:C). Combining both IFN? and RB6-8C5 with CpG-ODN/Poly(I:C) did not produce an additive effect compared to IFN? + CpG-ODN/Poly(I:C) or RB6-8C5 + CpG-ODN/Poly(I:C). Our results indicate that the inhalation therapy is a feasible and non-invasive strategy to deliver immunodulatory molecules, including antibodies and cytokines that reprogram the lung tumor microenvironment to foster immune destruction of tumors.