ABSTRACT
Recently, it has become clear that the tumour microenvironment (TME) is important in cancer immunotherapy. While immune checkpoint inhibitors are effective for some patients, the heterogeneous nature and status of the TME ('cold' tumours) play a critical role in suppressing antitumour immunity in non-responding patients. Converting 'cold' to 'hot' tumours through modulation of the TME may enable expansion of the therapeutic efficacy of immunotherapy to a broader patient population. This paper describes advances in intratumoural immunotherapy, specifically activation of nucleic acid sensing pattern recognition receptors to modulate the TME.
ABSTRACT
OBJECTIVES: Unstable atherosclerotic lesions in carotid arteries require surgical endarterectomy to reduce the risk of ischemic stroke. We aimed to identify microRNAs that exert a broad effect on atherosclerotic plaque formation and stability in the carotid artery. BACKGROUND: We made a selection of 164 genes involved in atherosclerosis. Using www.targetscan.org, we determined which microRNAs potentially regulate expression of these genes. We identified multiple microRNAs from the 14q32 microRNA cluster, which is highly involved in vascular remodeling. In human plaques, collected during carotid endarterectomy surgery, we found that 14q32 microRNA (miR-494) was abundantly expressed in unstable lesions. METHODS: We induced atherosclerotic plaque formation in hypercholesterolemic ApoE mice by placing semiconstrictive collars around both carotid arteries. We injected "Gene Silencing Oligonucleotides" against miR-494 (GSO-494) or negative control (GSO-control). Using fluorescently labeled GSOs, we confirmed uptake of GSOs in affected areas of the carotids, but not elsewhere in the vasculature. RESULTS: After injection of GSO-494, we observed significant downregulation of miR-494 expression in the carotid arteries, although miR-494 target genes were upregulated. Further analyses revealed a 65% decrease in plaque size after GSO-494 treatment. Plaque stability was increased in GSO-494-treated mice, determined by an 80% decrease in necrotic core size and a 50% increase in plaque collagen content. Inhibition of miR-494 also resulted in decreased cholesterol levels and decreased very low-density lipoprotein (VLDL) fractions. CONCLUSIONS: Treatment with GSO-494 results in smaller atherosclerotic lesions with increased plaque stability. Inhibition of miR-494 may decrease the risk of surgical complications or even avert endarterectomy surgery in some cases.
Subject(s)
Atherosclerosis/genetics , DNA/genetics , Gene Expression Regulation , MicroRNAs/genetics , Plaque, Atherosclerotic/genetics , Animals , Atherosclerosis/metabolism , Blotting, Western , Carotid Arteries , Disease Models, Animal , Humans , Male , Mice , MicroRNAs/biosynthesis , Plaque, Atherosclerotic/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.
Subject(s)
Adaptor Protein Complex 1/immunology , Adaptor Protein Complex 2/immunology , B-Lymphocytes/immunology , Membrane Transport Proteins/immunology , Monocytes/immunology , Toll-Like Receptors/immunology , Adaptor Protein Complex 1/genetics , Adaptor Protein Complex 2/genetics , Amino Acid Motifs , B-Lymphocytes/cytology , Cell Line, Tumor , HEK293 Cells , Humans , Membrane Transport Proteins/genetics , Monocytes/cytology , Protein Transport/genetics , Protein Transport/immunology , Toll-Like Receptors/geneticsABSTRACT
RATIONALE: Effective neovascularization is crucial for recovery after cardiovascular events. OBJECTIVE: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation. METHODS AND RESULTS: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation. CONCLUSIONS: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.
Subject(s)
Blood Vessels/metabolism , MicroRNAs/genetics , Animals , Blood Flow Velocity/genetics , Blood Flow Velocity/physiology , Blood Vessels/physiopathology , Cell Proliferation , Cells, Cultured , Chromosomes, Human, Pair 14/genetics , Endothelial Cells/metabolism , Gene Expression Profiling , Gene Silencing , HeLa Cells , Hindlimb/blood supply , Humans , Ischemia/physiopathology , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/blood supply , Myocytes, Smooth Muscle/metabolism , Oligonucleotide Array Sequence Analysis , Oligonucleotides/geneticsABSTRACT
Although the cause of Duchenne muscular dystrophy (DMD) is known, the specific factors that initiate and perpetuate disease progression are not well understood. We hypothesized that leaky dystrophin-deficient skeletal muscle releases endogenous danger signals (TLR ligands), which bind to Toll-like receptors (TLRs) on muscle and immune cells and activate downstream processes that facilitate degeneration and regeneration in dystrophic skeletal muscle. Here, we demonstrate that dystrophin-deficient mouse muscle cells show increased expression of several cell-surface and endosomal TLRs. In vitro screening identified ssRNA as a relevant endogenous TLR7 ligand. TLR7 activation led to myd88-dependent production of pro-inflammatory cytokines in dystrophin-deficient muscle cells, and cause significant degeneration/regeneration in vivo in mdx mouse muscle. Also, knockout of the central TLR adaptor protein, myd88 in mdx mice significantly improved skeletal and cardiac muscle function. Likewise, proof-of-concept experiments showed that treating young mdx mice with a TLR7/9 antagonist significantly reduced skeletal muscle inflammation and increased muscle force, suggesting that blocking this pathway may have therapeutic potential for DMD.
Subject(s)
Membrane Glycoproteins/physiology , Muscle, Skeletal/metabolism , Myeloid Differentiation Factor 88/metabolism , Myocardium/metabolism , Toll-Like Receptor 7/physiology , Toll-Like Receptor 9/metabolism , Animals , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Dystrophin/deficiency , Female , Humans , Male , Membrane Glycoproteins/agonists , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Knockout , Muscle, Skeletal/pathology , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/pathology , Myoblasts, Skeletal/immunology , Myoblasts, Skeletal/metabolism , Myocardium/pathology , Phenotype , Toll-Like Receptor 7/agonistsABSTRACT
Systemic Lupus Erythematosus is an autoimmune disease characterized by production of autoantibodies against nucleic acid-associated antigens. Endogenous DNA and RNA associated with these antigens stimulate inflammatory responses through Toll-like receptors (TLRs) and exacerbate lupus disease pathology. We have evaluated an antagonist of TLR7, 8 and 9 as a therapeutic agent in lupus-prone NZBW/F1 mice. NZBW/F1 mice treated with the antagonist had lower serum levels of autoantibodies targeting DNA, RNP, Smith antigen, SSA and SSB than did untreated mice. Reduction in blood urea nitrogen and proteinuria and improvements in kidney histopathology were observed in antagonist-treated mice. The antagonist treatment also reduced serum IL-12 and IL-1ß and increased IL-10 levels. Levels of mRNA for IL-6, iNOS and IL-1ß were lower in the kidneys and spleen of antagonist-treated mice than in those of untreated mice. Levels of mRNA for IP-10, TNFRSF9 and FASL were lower and IL-4 mRNA were higher in spleens of antagonist-treated mice than in spleens of untreated mice. mRNA for the inflammasome component NLRP3 was lower and mRNA for the antioxidant enzymes, catalase and glutathione peroxidase 1 was higher in the kidneys of antagonist-treated mice than in those of untreated mice. These results show that the antagonist of TLR7, 8 and 9 effectively inhibits inflammatory pathways involved in the development of lupus in NZBW/F1 mice and constitutes a potential therapeutic approach for the treatment of lupus and other autoimmune diseases.
Subject(s)
Deoxyribonucleotides/administration & dosage , Deoxyribonucleotides/antagonists & inhibitors , Down-Regulation/immunology , Lupus Erythematosus, Systemic/pathology , Lupus Erythematosus, Systemic/prevention & control , Membrane Glycoproteins/antagonists & inhibitors , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitors , Toll-Like Receptor 9/antagonists & inhibitors , Animals , Autoantibodies/biosynthesis , Autoantibodies/blood , Deoxyribonucleotides/pharmacology , Female , Inflammation Mediators/antagonists & inhibitors , Kidney/drug effects , Kidney/immunology , Kidney/pathology , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred NZBABSTRACT
Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.
Subject(s)
Autoimmune Diseases/metabolism , CD11b Antigen/biosynthesis , Gene Expression Regulation , Macrophage-1 Antigen/metabolism , Macrophages/metabolism , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Allosteric Site , Anti-Inflammatory Agents/pharmacology , Cell Adhesion , Down-Regulation , Heterozygote , Humans , Inflammation , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/metabolism , Macrophages/cytology , Models, Genetic , Myeloid Differentiation Factor 88/metabolism , RNA/metabolism , Signal TransductionABSTRACT
Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1ß. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases.
Subject(s)
Inflammasomes/metabolism , Membrane Glycoproteins/antagonists & inhibitors , Oligonucleotides/pharmacology , Psoriasis/pathology , Th1 Cells/pathology , Th17 Cells/pathology , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitors , Toll-Like Receptor 9/antagonists & inhibitors , Animals , Carrier Proteins/metabolism , Cytokines/metabolism , DNA-Binding Proteins , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Injections, Intradermal , Interleukin-1beta/metabolism , Interleukin-23/administration & dosage , Interleukin-23/adverse effects , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Nuclear Proteins/metabolism , Oligonucleotides/therapeutic use , Psoriasis/chemically induced , Psoriasis/metabolismABSTRACT
Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2'-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1ß and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.
Subject(s)
Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitors , Toll-Like Receptor 9/antagonists & inhibitors , Animals , Cells, Cultured , Cytokines/biosynthesis , Female , HEK293 Cells , Humans , Lupus Erythematosus, Systemic/immunology , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Oligoribonucleotides/chemistry , Oligoribonucleotides/pharmacology , Signal Transduction/drug effects , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 9/metabolism , Toll-Like Receptors/agonists , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Double-stranded RNA of viral origin and enzymatically synthesized poly I:C act as agonists of TLR3 and induce immune responses. We have designed and synthesized double-stranded synthetic oligoribonucleotides (dsORNs) which act as agonists of TLR3. Each strand of dsORN contains two distinct segments, namely an alignment segment composed of a heteronucleotide sequence and an oligo inosine (I) or an oligo cytidine (C) segment. We report here the results of studies of dsORNs containing varying lengths and compositions of alignment and oligo I/oligo C segments. dsORNs of 50-mer length with a 15-mer alignment segment and a 35-mer oligo I/oligo C segment form stable duplexes under physiological conditions and induce TLR3-mediated immune responses. dsORNs activated the IRF3 signaling pathway in J774 cells, induced production of cytokines, including IFN-ß, IFN-α, IP-10, IL-12 and IL-6, in murine and human cell-based assays and also induced multiple cytokines following systemic administration in mice and non-human primates.
Subject(s)
Drug Design , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/pharmacology , Toll-Like Receptor 3/agonists , Animals , Base Sequence , Blotting, Western , Cells, Cultured , Dose-Response Relationship, Drug , Female , Humans , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Oligoribonucleotides/chemistry , Sequence AlignmentABSTRACT
BACKGROUND & AIMS: The toll-like receptor 9 (TLR9) agonist IMO-2125 is currently evaluated in clinical trials for chronic hepatitis C therapy. The aim of this study was to investigate the in vivo mode of action of a closely related compound, referred to as immunomodulatory oligonucleotide (IMO). METHODS: We analyzed the Jak-STAT pathway activation and induction of interferon-stimulated genes in the liver of wild type, interferon-α/ß receptor-deficient and interferon-γ-deficient mice, after administration of IMO. RESULTS: IMO induced a prolonged activation of the Jak-STAT pathway and upregulation of interferon-stimulated genes in the mouse liver. Contrary to the response observed after interferon-α injection, the signalling induced by IMO was not abrogated following repeated administration. At early time points after IMO injection, STAT1 phosphorylation and interferon-stimulated gene induction required a functional interferon-α/ß receptor, whereas at the later time points, the activation was type I interferon-independent. Microarray analysis revealed that IMO induced a broad transcriptional response in the mouse liver. This included upregulation of cytokine and chemokine genes responsible for recruitment of IFN-γ producers, such as T cells and natural killer cells. Interferon-γ-deficient mice showed a transient response to IMO, demonstrating the central role of interferon-γ in sustained activation of Jak-STAT pathway by IMO. CONCLUSIONS: The bimodal kinetics of response to IMO in the mouse liver are driven by the sequential endogenous production of type I and II interferons. The lack of refractoriness to IMO, combined with the long-lasting induction of interferon-stimulated genes, reveals a favourable pharmacodynamics profile of this novel TLR9 agonist for the treatment of chronic viral hepatitis.
Subject(s)
Interferon Type I/biosynthesis , Interferon-gamma/biosynthesis , Liver/drug effects , Liver/immunology , Toll-Like Receptor 9/agonists , Animals , Chemokines/genetics , Cytokines/genetics , Immunologic Factors/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligodeoxyribonucleotides/pharmacology , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/genetics , STAT1 Transcription Factor/metabolism , Signal Transduction/drug effects , Toll-Like Receptor 9/deficiency , Toll-Like Receptor 9/genetics , Transcriptional Activation/drug effects , Up-Regulation/drug effectsABSTRACT
Oligodeoxynucleotides (ODNs) containing a CpG or certain synthetic dinucleotides, referred to as immune-stimulatory dinucleotides, induce Toll-like receptor 9 (TLR9)-mediated immune responses. Chemical modifications such as 2'-O-methylribonucleotides incorporated adjacent to the immune-stimulatory dinucleotide on the 5'-side abrogate TLR9-mediated immune responses. In this study, we evaluated the effect of the location of immune-stimulatory dinucleotides in ODNs on TLR9-mediated immune responses. We designed and synthesized ODNs with two immune-stimulatory dinucleotides, one placed toward the 5'-end region and the other toward the 3'-end region, incorporated 2'-O-methylribonucleotides selectively preceding the 5'- or 3'-immune-stimulatory dinucleotide or both, and studied TLR9-mediated immune responses of these compounds in cell-based assays and in vivo in mice. These studies showed that an immune-stimulatory dinucleotide located closer to the 5'-end is critical for and dictates TLR9-mediated immune responses. These studies provide insights for the use of ODNs when employed as TLR9 agonists and antagonists or antisense agents.
ABSTRACT
PURPOSE: Recent studies have shown that a new generation of synthetic agonist of Toll-like receptor (TLR) 9 consisting a 3'-3'-attached structure and a dCp7-deaza-dG dinucultodie shows more potent immunostimulatory effects in both mouse and human than conventional CpG oligonucleotides. Radiation therapy (RT) provides a source of tumor antigens that are released from dying, irradiated, tumor cells without causing systemic immunosuppression. We, therefore, examined effect of combining RT with a designer synthetic agonist of TLR9 on anti-tumoral immunity, primary tumor growth retardation and metastases in a murine model of lung cancer. METHODS: Grouped C57BL/6 and congenic B cell deficient mice (B(-/-)) bearing footpad 3LL tumors were treated with PBS, TLR9 agonist, control oligonucelotide, RT or the combination of RT and TLR9 agonist. Immune phenotype of splenocytes and serum IFN-γ and IL-10 levels were analyzed by FACS and ELISA, 24 h after treatment. Tumor growth, lung metastases and survival rate were monitored and tumor specific antibodies in serum and deposition in tumor tissue were measured by ELISA and immunofluorescence. RESULTS: TLR9 agonist expanded and activated B cells and plasmacytoid dendritic cells in wild-type mice and natural killer DCs (NKDCs) in B cell-deficient (B(-/-)) mice bearing ectopic Lewis lung adenocarcinoma (3LL). Combined RT with TLR9 agonist treatment inhibited 3LL tumor growth in both wild type and B(-/-) mice. A strong tumor-specific humoral immune response (titer: 1/3200) with deposition of mouse IgG auto-antibodies in tumor tissue were found in wildtype mice, whereas the number of tumor infiltrating NKDCs increased in B(-/-) mice following RT+ TLR9 agonist therapy. Furthermore, mice receiving combination therapy had fewer lung metastases and a higher survival than single treatment cohorts. CONCLUSIONS: Combination therapy with TLR9 agonist and RT induces systemic anti-tumoral humoral response, augments tumoral infiltration of NKDCs, reduces pulmonary metastases and improves survival in a murine model of 3LL cancer.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/radiotherapy , Antineoplastic Agents/pharmacology , Lung Neoplasms/drug therapy , Lung Neoplasms/radiotherapy , Toll-Like Receptor 9/agonists , Vaccination , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Animals , Antibodies, Neoplasm/immunology , Antigens, Neoplasm/immunology , Antineoplastic Agents/therapeutic use , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/radiation effects , Cell Line, Tumor , Combined Modality Therapy , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/radiation effects , Disease Progression , Humans , Immunomodulation/drug effects , Immunomodulation/radiation effects , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Male , Mice , Neoplasm Metastasis , Recurrence , Survival Analysis , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Tumor Microenvironment/radiation effectsABSTRACT
OBJECTIVE: The role of toll-like receptors (TLRs) in vascular remodeling is well established. However, the involvement of the endosomal TLRs is unknown. Here, we study the effect of combined blocking of TLR7 and TLR9 on postinterventional remodeling and accelerated atherosclerosis. METHODS AND RESULTS: In hypercholesterolemic apolipoprotein E*3-Leiden mice, femoral artery cuff placement led to strong increase of TLR7 and TLR9 presence demonstrated by immunohistochemistry. Blocking TLR7/9 with a dual antagonist in vivo reduced neointimal thickening and foam cell accumulation 14 days after surgery by 65.6% (P=0.0079). Intima/media ratio was reduced by 64.5% and luminal stenosis by 62.8%. The TLR7/9 antagonist reduced the arterial wall inflammation, with reduced macrophage infiltration, decreased cytoplasmic high-mobility group box 1 expression, and altered serum interleukin-10 levels. Stimulation of cultured macrophages with TLR7 and TLR9 ligands enhanced tumor necrosis factor-α expression, which is decreased by TLR7/9 antagonist coadministration. Additionally, the antagonist abolished the TLR7/9-enhanced low-density lipoprotein uptake. The antagonist also reduced oxidized low-density lipoprotein-induced foam cell formation, most likely not via decreased influx but via increased efflux, because CD36 expression was unchanged whereas interleukin-10 levels were higher (36.1 ± 22.3 pg/mL versus 128.9 ± 6.6 pg/mL; P=0.008). CONCLUSIONS: Blocking TLR7 and TLR9 reduced postinterventional vascular remodeling and foam cell accumulation indicating TLR7 and TLR9 as novel therapeutic targets.
Subject(s)
Atherosclerosis/etiology , Cell Movement , Coronary Vessels/pathology , Foam Cells/physiology , Macrophage Activation , Membrane Glycoproteins/physiology , Toll-Like Receptor 7/physiology , Toll-Like Receptor 9/physiology , Angioplasty, Balloon, Coronary , Animals , Cytokines/biosynthesis , HMGB1 Protein/analysis , Lipoproteins, LDL/physiology , Membrane Glycoproteins/antagonists & inhibitors , Mice , Neointima/prevention & control , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 9/antagonists & inhibitorsABSTRACT
PURPOSE: Radiation-induced gastrointestinal syndrome (RIGS) is due to the clonogenic loss of crypt cells and villi depopulation, resulting in disruption of mucosal barrier, bacterial invasion, inflammation and sepsis. Intestinal macrophages could recognize invading bacterial DNA via TLR9 receptors and transmit regenerative signals to the neighboring crypt. We therefore investigated whether systemic administration of designer TLR9 agonist could ameliorate RIGS by activating TLR9. METHODS AND MATERIALS: Male C57Bl6 mice were distributed in four experimental cohorts, whole body irradiation (WBI) (8.4-10.4 Gy), TLR9 agonist (1 mg/kg s.c.), 1 h pre- or post-WBI and TLR9 agonist+WBI+iMyd88 (pretreatment with inhibitory peptide against Myd88). Animals were observed for survival and intestine was harvested for histological analysis. BALB/c mice with CT26 colon tumors in abdominal wall were irradiated with 14 Gy single dose of whole abdominal irradiation (AIR) for tumor growth study. RESULTS: Mice receiving pre-WBI TLR9 agonist demonstrated improvement of survival after 10.4 Gy (p<0.03), 9.4 Gy (p<0.008) and 8.4 Gy (p<0.002) of WBI, compared to untreated or iMyd88-treated controls. Post-WBI TLR9 agonist mitigates up to 8.4 Gy WBI (p<0.01). Histological analysis and xylose absorption test demonstrated significant structural and functional restitution of the intestine in WBI+TLR9 agonist cohorts. Although, AIR reduced tumor growth, all animals died within 12 days from RIGS. TLR9 agonist improved the survival of mice beyond 28 days post-AIR (p<0.008) with significant reduction of tumor growth (p<0.0001). CONCLUSIONS: TLR9 agonist treatment could serve both as a prophylactic or mitigating agent against acute radiation syndrome and also as an adjuvant therapy to increase the therapeutic ratio of abdominal Radiation Therapy for Gastro Intestinal malignancies.
Subject(s)
Gastrointestinal Agents/therapeutic use , Gastrointestinal Diseases/etiology , Gastrointestinal Diseases/prevention & control , Radiation Injuries, Experimental/prevention & control , Toll-Like Receptor 9/agonists , Animals , Cell Line, Tumor , Cytoprotection/drug effects , Drug Evaluation, Preclinical , Gastrointestinal Agents/pharmacology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Radiation Tolerance/drug effects , Radiation-Protective Agents/pharmacology , Radiation-Protective Agents/therapeutic use , Rats , Syndrome , Toll-Like Receptor 9/metabolismABSTRACT
Viral single-stranded (ss) RNA is the natural ligand for TLR7 and TLR8. Synthetic ssRNA has been shown to act as a ligand for TLR7 and TLR8. We have previously reported a novel RNA structure, referred to as stabilized immune modulatory RNA (SIMRA), in which two short phosphorothioate oligoribonucleotides were linked through their 3'-ends via a linker. SIMRA compounds had greater stability in serum than unmodified ssRNA and induced immune responses via TLR7 and/or TLR8. SIMRA compounds were synthesized using phosphoramidite chemistry on controlled-pore glass solid support derivatized with a linker. After cleavage from the solid support and removal of protecting groups, SIMRA compounds were purified on an anion-exchange HPLC followed by desalting/dialysis, and lyophilization. SIMRA compounds were characterized for their purity and sequence integrity by anion-exchange HPLC, capillary gel electrophoresis, polyacrylamide gel electrophoresis, and MALDI-TOF mass spectrophotometric analysis. As SIMRA compounds induce TLR7- and/or TLR8-mediated Th1-type immune responses, they have potential utility as therapeutic agents for a broad range of diseases, including cancer, infectious diseases, asthma, and allergies, and as adjuvants with vaccines.
Subject(s)
Adjuvants, Immunologic/chemical synthesis , Chemistry, Pharmaceutical/methods , Immunologic Factors/chemical synthesis , Oligoribonucleotides/chemical synthesis , Phosphorothioate Oligonucleotides/chemical synthesis , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists , Adjuvants, Immunologic/isolation & purification , Adjuvants, Immunologic/pharmacology , Chromatography, High Pressure Liquid , Communicable Diseases/drug therapy , Communicable Diseases/immunology , Communicable Diseases/pathology , Drug Stability , Electrophoresis, Capillary , Humans , Hypersensitivity/drug therapy , Hypersensitivity/immunology , Hypersensitivity/pathology , Immunologic Factors/isolation & purification , Immunologic Factors/pharmacology , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/pathology , Oligoribonucleotides/isolation & purification , Oligoribonucleotides/pharmacology , Phosphorothioate Oligonucleotides/isolation & purification , Phosphorothioate Oligonucleotides/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Toll-Like Receptor 7/immunology , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/immunology , Toll-Like Receptor 8/metabolismABSTRACT
Methods and protocols for automated synthesis and purification of immune modulatory oligonucleotides (IMOs), a novel class of Toll-like receptor 9 (TLR9) agonists, are described. IMOs containing two short identical sequences of 11-mers with phosphorothioate linkages can be synthesized in parallel synthetic strategy. A C3-linker that mimics the natural inter-nucleotide distance was commonly used for joining the two segments of IMOs. NittoPhase solid support bearing a symmetrical C3-linker (glycerol) and nucleoside-ß-cyanoethyl-N,N-diisopropylphosphoramidites were used for IMO synthesis. The parallel synthesis was carried out in a 3'â 5' direction with removal of the final dimethoxytrityl (DMT) protecting group. After synthesis, the IMO was cleaved and deprotected by treating with aqueous ammonia. The product was purified on anion-exchange HPLC, desalted, lyophilized, and characterized by anion-exchange HPLC, capillary gel electrophoresis, polyacrylamide gel electrophoresis, and MALDI-TOF mass spectral analysis.
Subject(s)
Adjuvants, Immunologic/chemical synthesis , Chemistry, Pharmaceutical/methods , Immunologic Factors/chemical synthesis , Oligodeoxyribonucleotides/chemical synthesis , Phosphorothioate Oligonucleotides/chemical synthesis , Toll-Like Receptor 9/agonists , Adjuvants, Immunologic/isolation & purification , Adjuvants, Immunologic/pharmacology , Bacterial Infections/drug therapy , Bacterial Infections/immunology , Bacterial Infections/microbiology , Base Sequence , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Capillary , Glycerol/chemistry , Humans , Immunologic Factors/isolation & purification , Immunologic Factors/pharmacology , Molecular Sequence Data , Oligodeoxyribonucleotides/isolation & purification , Oligodeoxyribonucleotides/pharmacology , Phosphorothioate Oligonucleotides/isolation & purification , Phosphorothioate Oligonucleotides/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/metabolism , Virus Diseases/drug therapy , Virus Diseases/immunology , Virus Diseases/virologyABSTRACT
Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-κB in TLR7- and TLR8-transfected cell lines; induction of IFN-α, IL-6, TNF-α, IL-12, and IP-10 in human PBMCs; IFN-α induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-α, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-α, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-α in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-α acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.
Subject(s)
Oligoribonucleotides/pharmacology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists , Animals , Base Sequence , Cell Line , Cytokines/biosynthesis , Cytokines/blood , Dendritic Cells/drug effects , Dendritic Cells/immunology , Drug Design , Female , HEK293 Cells , Humans , Immunity, Innate , Interleukin-12/biosynthesis , Macaca mulatta , Mice , Mice, Inbred C57BL , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/genetics , Oligoribonucleotides/immunology , Recombinant Proteins/agonists , Recombinant Proteins/genetics , Toll-Like Receptor 7/genetics , Toll-Like Receptor 8/genetics , TransfectionABSTRACT
Oligonucleotides are being employed for gene-silencing activity by a variety of mechanisms, including antisense, ribozyme, and siRNA. In the present studies, we designed novel oligonucleotides complementary to targeted mRNAs and studied the effect of 3'-end exposure and oligonucleotide length on gene-silencing activity. We synthesized both oligoribonucleotides (RNAs) and oligodeoxynucleotides (DNAs) with phosphorothioate backbones, consisting of two identical segments complementary to the targeted mRNA attached through their 5'-ends, thereby containing two accessible 3'-ends; these compounds are referred to as gene-silencing oligonucleotides (GSOs). RNA and/or DNA GSOs targeted to MyD88, VEGF, and TLR9 mRNAs had more potent gene-silencing activity than did antisense phosphorothioate oligonucleotides (PS-oligos) in cell-based assays and in vivo. Of the different lengths of GSOs evaluated, 19-mer long RNA and DNA GSOs had the best gene-silencing activity both in vitro and in vivo. These results suggest that GSOs are novel agents for gene silencing that can be delivered systemically with broader applicability.
Subject(s)
Gene Silencing , Oligonucleotides/pharmacology , RNA, Messenger/chemistry , Animals , Base Sequence , Cell Line , DNA Primers , Humans , Mice , Myeloid Differentiation Factor 88/genetics , Oligonucleotides/chemistryABSTRACT
Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptors 7 and 8 (TLR7 and TLR8). We have reported a novel class of synthetic oligoribonucleotides, referred to as stabilized immune-modulatory RNA compounds, which act as agonists of TLR7, TLR8, or both TLR7 and TLR8 depending on the sequence composition and the presence of specific chemical modifications. In the present study, we evaluated the antitumor activity of a dual TLR7/8 agonist in tumor-bearing mice with peritoneal disseminated CT26.CL25 colon and 3LL-C75 lung carcinomas. Peritoneal administration of dual TLR7/8 agonist in mice bearing CT26.CL25 colon carcinomas had potent dose-dependent antitumor activity, which was associated with a marked decrease in CD4(+)CD25(+)Foxp3(+) T regulatory cells and a significant increase in tumor antigen-specific IFN-gamma-secreting effector cell responses in splenocytes and local tumor-infiltrating cells. In 3LL-C75 lung carcinoma, dual TLR7/8 agonist induced strong immune responses and antitumor effects in C57BL/6 and TLR9(-/-) mice, but not in TLR7(-/-) and MyD88(-/-) mice, indicating that the agonist induces immune responses via TLR7 and through the MyD88-dependent signaling pathway. TLR8 is not functional in mice. Additionally, s.c. administration of TLR7/8 agonist effectively prevented lung metastasis of tumors in the CT26.CL25 pulmonary metastasis model. These studies show that the dual TLR7/8 agonist induced Th1-type immune responses and potent antitumor activity in mice via TLR7 and through the MyD88-dependent pathway.