ABSTRACT
(1) Background: Aberrant activation of the hedgehog (HH)-GLI pathway in stem-like tumor-initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH-GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH-GLI-driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity, and monitored the oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO inhibitor-sensitive and -resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent TIC in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor-sensitive and -resistant tumors.
ABSTRACT
In hepatocellular carcinoma (HCC), blood platelets have been linked to tumor growth, epithelial-to-mesenchymal transition (EMT), extrahepatic metastasis and a limited therapeutic response to the multikinase inhibitor (MKi) sorafenib, the standard of care in advanced HCC for the last decade. Recent clinical data indicated an improved overall survival for sorafenib in combination with the HDAC inhibitor resminostat in a platelet count dependent manner. Here, the impact of platelets on the sorafenib and resminostat drug effects in HCC cells was explored. In contrast to sorafenib, resminostat triggered an anti-proliferative response in HCC cell lines independent of platelets. As previously described, platelets induced invasive capabilities of HCC cells, a prerequisite for extravasation and metastasis. Importantly, the resminostat/sorafenib drug combination, but not the individual drugs, effectively blocked platelet-induced HCC cell invasion. Exploration of the molecular mechanism revealed that the combined drug action led to a reduction of platelet-induced CD44 expression and to the deregulation of several other epithelial and mesenchymal genes, suggesting interference with cell invasion via EMT. In addition, the drug combination decreased phosphorylated ERK level, indicating inhibition of the mitogenic signaling pathway MEK/ERK. Taken together, the resminostat plus sorafenib combination counteracts platelet-mediated cancer promoting effects in HCC cells.
Subject(s)
Antineoplastic Agents/pharmacology , Blood Platelets/drug effects , Carcinoma, Hepatocellular/pathology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Liver Neoplasms/pathology , Sorafenib/pharmacology , Sulfonamides/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Therapy, Combination , Epithelial-Mesenchymal Transition/drug effects , Histone Deacetylase Inhibitors/therapeutic use , Humans , Hydroxamic Acids/therapeutic use , Liver Neoplasms/drug therapy , Mice , Signal Transduction/drug effects , Sorafenib/therapeutic use , Sulfonamides/therapeutic useABSTRACT
BACKGROUND: The efficacy of PD-(L)1 blockade depends on the composition of the tumor immune microenvironment (TIME) and is generally higher in tumors with pre-existing cytotoxic T cells (CTL) than in those with low CTL numbers. Nonetheless, a significant proportion of patients with pre-existing immunity fail to respond, indicating a therapeutic potential for combining PD-(L)1 blockade with additional immunomodulatory agents in both CTL-high and -low immune phenotypes. Here, we evaluated domatinostat (4SC-202), a class I-selective histone deacetylase (HDAC) inhibitor, for its effect on the TIME and its antitumoral efficacy using syngeneic mouse models with CTL-high or CTL-low tumors. METHODS: Domatinostat was evaluated in PD-1 blockade-insensitive CTL-low (CT26) and CTL-high (C38) syngeneic models alone and in combination with different immune-inhibitory and -stimulatory approaches. Effects on the immunophenotype were assessed via flow cytometry and RNA-seq analyses. The changes in RNA-seq-based immune signatures determined in a murine setting were investigated in patient samples from the first-dose cohort of the SENSITIZE trial (NCT03278665) evaluating domatinostat combined with pembrolizumab in advanced-stage melanoma patients refractory/nonresponding to PD-1 blockade. RESULTS: Domatinostat increased the expression of antigen-presenting machinery (APM) genes and MHC class I and II molecules, along with CTL infiltration, in tumors of both immune phenotypes. In combination with PD-(L)1 blockade, domatinostat augmented antitumor effects substantially above the effects of single-agent therapies, displaying greater benefit in tumors with pre-existing CTLs. In this setting, the combination of domatinostat with agonistic anti-4-1BB or both PD-1 and LAG3 blockade further increased the antitumor efficacy. In CTL-low tumors, domatinostat enhanced the expression of genes known to reinforce immune responses against tumors. Specifically, domatinostat increased the expression of Ifng and genes associated with responses to pembrolizumab and nivolumab. Clinically, these findings were confirmed in patients with advanced melanoma treated with domatinostat for 14 days, who demonstrated elevated expression of APM and MHC genes, the IFNG gene, and the IFN-γ and pembrolizumab response signatures in individual tumor samples. CONCLUSION: In summary, these data suggest a promising potential of domatinostat in combination with immunotherapy to improve the outcome of refractory cancer patients.
Subject(s)
Benzamides/pharmacology , Neoplasms/immunology , Neoplasms/pathology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Animals , Antigen Presentation/immunology , Antigens, Neoplasm/immunology , Antineoplastic Agents, Immunological/pharmacology , Biomarkers, Tumor , Biopsy , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Female , Histone Deacetylase Inhibitors/pharmacology , Humans , Immunophenotyping , Male , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Toll-like receptor 7 (TLR7) agonists are potent immune stimulants able to overcome cancer-associated immune suppression. Due to dose-limiting systemic toxicities, only the topically applied TLR7 agonist (imiquimod) has been approved for therapy of skin tumors. There is a need for TLR7-activating compounds with equivalent efficacy but less toxicity. SC1, a novel small molecule agonist for TLR7, is a potent type-1 interferon inducer, comparable to the reference TLR7 agonist resiquimod, yet with lower induction of proinflammatory cytokines. In vivo, SC1 activates NK cells in a TLR7-dependent manner. Mice bearing the NK cell-sensitive lymphoma RMA-S are cured by repeated s. c. administrations of SC1 as efficiently as by the administration of resiquimod. No relevant toxicities were observed. Mechanistically, SC1 reverses NK cell anergy and restores NK cell-mediated tumor cell killing in an IFN-α-dependent manner. TLR7 targeting by SC1-based compounds may form an attractive strategy to activate NK cell responses for cancer therapy.
ABSTRACT
A wide range of human malignancies displays aberrant activation of Hedgehog (HH)/GLI signaling, including cancers of the skin, brain, gastrointestinal tract and hematopoietic system. Targeting oncogenic HH/GLI signaling with small molecule inhibitors of the essential pathway effector Smoothened (SMO) has shown remarkable therapeutic effects in patients with advanced and metastatic basal cell carcinoma. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the use of SMO antagonists and urgently calls for the identification of novel targets and compounds.Here we report on the identification of the Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase 1B (DYRK1B) as critical positive regulator of HH/GLI signaling downstream of SMO. Genetic and chemical inhibition of DYRK1B in human and mouse cancer cells resulted in marked repression of HH signaling and GLI1 expression, respectively. Importantly, DYRK1B inhibition profoundly impaired GLI1 expression in both SMO-inhibitor sensitive and resistant settings. We further introduce a novel small molecule DYRK1B inhibitor, DYRKi, with suitable pharmacologic properties to impair SMO-dependent and SMO-independent oncogenic GLI activity. The results support the use of DYRK1B antagonists for the treatment of HH/GLI-associated cancers where SMO inhibitors fail to demonstrate therapeutic efficacy.
Subject(s)
Carcinoma, Basal Cell/pathology , Drug Resistance, Neoplasm , Hedgehog Proteins/metabolism , Pancreatic Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Smoothened Receptor/metabolism , Zinc Finger Protein GLI1/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blotting, Western , Carcinoma, Basal Cell/drug therapy , Carcinoma, Basal Cell/genetics , Carcinoma, Basal Cell/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Forkhead Transcription Factors/physiology , Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/genetics , Humans , Mice , Mice, Nude , NIH 3T3 Cells , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Smoothened Receptor/antagonists & inhibitors , Smoothened Receptor/genetics , Xenograft Model Antitumor Assays , Zinc Finger Protein GLI1/antagonists & inhibitors , Zinc Finger Protein GLI1/genetics , Dyrk KinasesABSTRACT
Microtubule inhibitors are invaluable tools in cancer chemotherapy: taxanes and vinca alkaloids have been successfully used in the clinic over the past thirty years against a broad range of tumors. However, two factors have limited the effectiveness of microtubule inhibitors: toxicity and resistance. In particular, the latter is highly unpredictable, variable from patient to patient and is believed to be the cause of treatment failure in most cases of metastatic cancers. For these reasons, there is an increasing demand for new microtubule inhibitors that can overcome resistance mechanisms and that, at the same time, have reduced side effects. Here we present a novel microtubule inhibitor, 4SC-207, which shows strong anti-proliferative activity in a large panel of tumor cell lines with an average GI50 of 11 nM. In particular, 4SC-207 is active in multi-drug resistant cell lines, such as HCT-15 and ACHN, suggesting that it is a poor substrate for drug efflux pumps. 4SC-207 inhibits microtubule growth in vitro and in vivo and promotes, in a dose dependent manner, a mitotic delay/arrest, followed by apoptosis or aberrant divisions due to chromosome alignment defects and formation of multi-polar spindles. Furthermore, preliminary data from preclinical studies suggest low propensity towards bone marrow toxicities at concentrations that inhibit tumor growth in paclitaxel-resistant xenograft models. In summary, our results suggest that 4SC-207 may be a potential anti-cancer agent.
Subject(s)
Microtubules/drug effects , Taxoids/pharmacology , Tubulin Modulators/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Flow Cytometry , Fluorescent Antibody Technique , HeLa Cells , Humans , Tubulin/metabolismABSTRACT
BACKGROUND: Dihydroorotate dehydrogenase (DHODH) is a key enzyme involved in pyrimidine biosynthesis. DHODH is a known target for the treatment of autoimmune diseases. 4SC-101 is a novel immunosuppressive drug that inhibits DHODH. A goal of our study was to examine the in vitro effects of 4SC-101 on IL-17 production by mononuclear cells. In addition, we evaluated the efficacy of 4SC-101 against acute TNBS (2,4,6-tritrobenzene sulfonic acid) and chronic dextran sodium sulfate (DSS)-induced colitis in mice. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy human donors were used to evaluate cellular proliferation and cytokine (IL-17, TNF-α) production. The oral effects of 4SC-101 (100 or 200 mg/kg) were examined following induction of chronic colitis by the administration of 3% DSS (4 cycles) to Balb/c mice. Morphometric and histological indices of colitis were evaluated as indicators of drug efficacy. 4SC-101 was also administered for 6 days after the intracolonic administration of TNBS (20 mg in 50% ethanol) to female Balb/c mice. The colons were analyzed for overall macroscopic damage, ulceration, total length, distal segment weight, MPO activity, and histological pathology as indicators for the effectiveness of 4SC-101. RESULTS: In vitro, 4SC-101 is a potent inhibitor of human DHODH, inhibits lymphocyte proliferation, and uniquely blocks phytohemagglutinin-stimulated IL-17 production by lymphocytes. In vivo, oral administration of 4SC-101 effectively improved both chronic DSS and acute TNBS colitis in mice. In these colitis models the overall efficacy profile of 4SC-101 was similar to that of dexamethasone. CONCLUSIONS: 4SC-101 is a novel immunosuppressive drug with excellent potential for the treatment of intestinal inflammation.
Subject(s)
Biphenyl Compounds/pharmacology , Colitis/drug therapy , Dicarboxylic Acids/pharmacology , Immunosuppressive Agents/pharmacology , Inflammatory Bowel Diseases/drug therapy , Interleukin-17/antagonists & inhibitors , Acute Disease , Animals , Blotting, Western , Cell Proliferation/drug effects , Chronic Disease , Colitis/chemically induced , Colitis/pathology , Dextran Sulfate/toxicity , Dihydroorotate Dehydrogenase , Disease Models, Animal , Female , Immunoenzyme Techniques , Inflammatory Bowel Diseases/pathology , Interleukin-17/metabolism , Mice , Mice, Inbred BALB C , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Trinitrobenzenesulfonic Acid/toxicityABSTRACT
Small interfering RNA (siRNA) is widely used to modulate gene expression, but its potential induction of cytokines via Toll-like receptors (TLR) strongly impairs its use. Selective 2'-O-ribose methylation of sense or antisense strand can abolish the immunostimulatory potential, however, no universal approach is available and the mechanism of action is unknown. Here, we demonstrate that alternating 2'-O-ribose methylation of the sense strand within a siRNA duplex specific for eGFP or beta(2)-microglobulin destroyed its immunostimulatory function in primary immune cells, while reduction in target gene expression was functional. Furthermore, addition of siRNA containing a 2'-O-ribose-methylated sense strand to immunostimulatory siRNA abolished its stimulatory activity and binding studies revealed that 2'-O-ribose-methylated RNA bound stronger to TLR7 than unmodified RNA. We conclude that 2'-O-ribose methylation acts as inhibitor for RNA-driven immune stimulation via TLR7 and recommend alternating 2'-O-ribose methylation of the sense strand as a universal approach for the generation of non-immunostimulatory siRNA.
Subject(s)
Dendritic Cells/metabolism , RNA, Small Interfering/immunology , Toll-Like Receptor 7/immunology , Animals , Cell Line, Transformed , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/pathology , Gene Expression Regulation , Humans , Immunization , Methylation , Mice , Mice, Knockout , Mice, Transgenic , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Ribose/analogs & derivatives , Ribose/chemistry , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolismABSTRACT
Toll-like receptor (TLR)-mediated signaling is proposed as an immunotherapeutic target against tumorigenesis. Natural killer (NK) cells play a critical role in host defense against tumors. Specifically, formation of tumor metastasis in various organs can be suppressed by the local activity of NK cells. In this study, we present a novel TLR7 agonist (termed SC-1) that induces pro-inflammatory cytokines in human blood cells, activates NK cell function, and is highly efficient in preventing lung metastases in a pulmonary metastatic Renca model. Furthermore, a second compound (termed SC-2), acting as dual-specific TLR7 and TLR8 agonist, was evaluated with respect to its immunostimulatory and NK cell-activating capacities. The release of pro-inflammatory cytokines was shown to be even more pronounced with this compound. Additional experiments showed a significant up-regulation of activation marker CD69 on NK cells and increased cytolytic activity of peripheral blood cells compared to the effect of a monospecific TLR7 agonist SC-1. Normally, TLR7 and TLR8 are expressed on different immune cell subpopulations. TLR7 expression on antigen-presenting cells is detected in plasmacytoid dendritic cells, CD34+-derived dendritic cells, and B-cells, whereas TLR8 is mainly expressed on cells of the myeloid lineage, such as monocytes, macrophages, and myeloid dendritic cells. Therefore, a compound that activates both TLR7 and TLR8 would result in a highly efficient immune system activation and may give rise to an enhanced anti-tumor activity in vivo compared to that elicited by a monospecific TLR7 agonist.
Subject(s)
Antibodies, Monoclonal/pharmacology , Lung Neoplasms/drug therapy , Lymphocyte Activation/drug effects , Membrane Glycoproteins/agonists , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists , Animals , Antibodies, Monoclonal, Humanized , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Cell Death/drug effects , Cell Line , Dendritic Cells/drug effects , Dendritic Cells/immunology , Drug Screening Assays, Antitumor , Female , Humans , Immunotherapy , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lectins, C-Type , Lung Neoplasms/immunology , Lung Neoplasms/secondary , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolismABSTRACT
The mammalian immune system senses pathogens through pattern recognition receptors and responds with activation. The Toll-like receptors (TLRs) that are expressed on antigen presenting cells such as macrophages and dendritic cells play a critical role in this process. Their signaling activates these cells and leads to an innate immune response with subsequent initiation of an adaptive immune response. Each TLR recognizes specific structures and induces common inflammatory cytokines. However, some TLRs have specific functions, such as induction of Type I interferons. The TLR dependent cytokine response is reflected in the induction of common and specific signaling pathways leading to adequate immune responses for different pathogens. Some TLRs are also activated by endogenous structures that are released during infection, but these structures may promote or sustain autoimmune diseases under certain circumstances. In addition, TLRs directly shape adaptive immune responses of T and B cells and play an important role in homeostasis of gut epithelium and lung repair after injury.
Subject(s)
Cytokines/immunology , Interferons/immunology , Protein Kinases/immunology , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism , Animals , Autoimmunity/immunology , Bacteria/immunology , Cytokines/metabolism , Fungi/immunology , Humans , Interferons/metabolism , Ligands , Parasites/immunology , Protein Kinases/metabolism , Signal Transduction/immunology , Toll-Like Receptors/chemistry , Viruses/immunologyABSTRACT
New structural classes of K(V)1.3 and IK-1 ion channel blockers have been identified based on a virtual high throughput screening approach using a homology model of KcsA. These compounds display inhibitory effects on T-cell and/or keratinocyte proliferation and immunosuppressant activity within a DTH animal model.
Subject(s)
Immunosuppressive Agents/chemical synthesis , Immunosuppressive Agents/pharmacology , Intermediate-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Kv1.3 Potassium Channel/antagonists & inhibitors , Potassium Channel Blockers/chemical synthesis , Potassium Channel Blockers/pharmacology , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Humans , Hypersensitivity, Delayed/drug therapy , Hypersensitivity, Delayed/metabolism , Immunosuppressive Agents/classification , Intermediate-Conductance Calcium-Activated Potassium Channels/chemistry , Intermediate-Conductance Calcium-Activated Potassium Channels/physiology , Keratinocytes/drug effects , Keratinocytes/physiology , Kv1.3 Potassium Channel/chemistry , Kv1.3 Potassium Channel/physiology , Patch-Clamp Techniques , Potassium Channel Blockers/classificationABSTRACT
The mammalian immune system senses pathogens through pattern recognition receptors (PRR) and responds with activation. Toll-like receptors (TLRs) that are expressed on immune and non-immune cells play a critical role in this process. As part of the innate immune response, TLRs lead to cellular activation and cytokine production with subsequent initiation of an adaptive immune response. TLR7-9 recognize single-stranded RNA, nucleoside analogs and single-stranded CpG-DNA, respectively, and their activation initiates the immune response against viruses and bacteria. Furthermore, the stimulation of these TLRs may be exploited for adjuvant therapy, vaccination and anti-tumor responses. However, a role in the generation or perpetuation of autoimmune diseases such as systemic lupus erythematosus (SLE) has also been suggested.
Subject(s)
Nucleic Acids/metabolism , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 9/metabolism , Amino Acid Sequence , Animals , CpG Islands , DNA, Single-Stranded/chemistry , Humans , Interleukin-6/biosynthesis , Mice , Models, Biological , Molecular Sequence Data , RNA/chemistry , Sequence Homology, Amino Acid , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 8/chemistry , Toll-Like Receptor 9/chemistryABSTRACT
The human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recognizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (dendritic cells) from TLR9-/- mice secreted reduced levels of IL-12, IFN-alpha, and IL-6, when compared with dendritic cells from wild-type mice. Intranasal infection of TLR9-/- and wild-type mice did not reveal any differences during lytic and latent infection. In contrast, when infected i.p., TLR9-/- mice showed markedly higher viral loads both during lytic and latent infection. Thus, we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific immunity.
Subject(s)
Gammaherpesvirinae , Herpesviridae Infections/immunology , Toll-Like Receptor 9/physiology , Animals , Dendritic Cells/immunology , Humans , Interferon-alpha/metabolism , Interleukin-12/metabolism , Interleukin-6/metabolism , Mice , Mice, Mutant Strains , Toll-Like Receptor 9/geneticsABSTRACT
The mammalian immune system senses pathogens through pattern recognition receptors (PRRs) and responds with activation. The Toll-like receptor (TLR) family that consists of 13 receptors plays a critical role in this process. TLRmediated signaling activates immune cells and leads to an innate immune response with subsequent initiation of an adaptive immune response. Toll-like receptor 9 (TLR9) recognizes deoxyribonucleic acid (DNA) leading to cellular activation and cytokine production influencing the immune response against viruses and bacteria. The stimulation of TLR9 will be exploited for adjuvant therapy and treatment of cancer or allergy. In this review we will discuss TLR9 ligands, TLR9 expression, signaling, and the therapeutic potential of TLR9 ligands in treatment of infectious or allergic diseases and cancer.
Subject(s)
DNA/physiology , Toll-Like Receptor 9/physiology , Animals , Anti-Infective Agents/pharmacology , Antineoplastic Agents/pharmacology , Bacterial Physiological Phenomena , Humans , Ligands , Signal Transduction/physiology , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/drug effects , Viruses/metabolismABSTRACT
Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.
Subject(s)
Immunity, Innate , Mononegavirales/immunology , Oligodeoxyribonucleotides/immunology , Oligoribonucleotides/immunology , RNA, Viral/immunology , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Animals , Cell Line , Conserved Sequence , Cytokines/biosynthesis , Cytokines/immunology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Female , Genome, Viral , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Mice , Mice, Inbred BALB C , Monocytes/drug effects , Monocytes/immunology , Mononegavirales/metabolism , Oligodeoxyribonucleotides/pharmacology , Oligoribonucleotides/pharmacology , Toll-Like Receptor 7/immunology , Toll-Like Receptor 8/immunologyABSTRACT
Single-stranded RNA stimulates immune cells and induces the secretion of pro-inflammatory cytokines and type I IFN. As adjuvant RNA can induce a T(h)2 type of humoral response, however, its potency in the induction of cytotoxic T cells in vivo has not been analyzed. Here we show that immunization with the antigen ovalbumin (OVA) and the adjuvant phosphodiester RNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) induced a Toll-like receptor-7-dependent cytotoxic T cell and humoral response. Staining with SIINFEKL-K(b) tetramers demonstrated the induction of antigen-specific T cells that were functional in in vivo cytotoxic T cell assays against SIINFEKL-loaded target cells. In infection experiments with OVA-secreting Listeria monocytogenes, the cytotoxic T cell response strongly reduced the bacterial load in liver and spleen. The RNA-driven humoral response was characterized by OVA-specific antibodies of the IgG1 isotype whereas CpG-DNA induced antigen-specific antibodies of the IgG2a (BALB/c) or IgG2c (C57BL/6) isotype. Furthermore, stimulation with RNA did not induce splenomegaly, a common feature of CpG-DNA-driven immune activation in mice. Taken together, our data confirm that RNA can be used as a safe adjuvant and induces a strong antibody response of the IgG1 isotype. Additionally, we demonstrate that RNA induces an antigen-specific immunity characterized by a potent cytotoxic T cell response to infection.
Subject(s)
Adjuvants, Immunologic , Bacterial Vaccines/immunology , Listeria monocytogenes/immunology , Membrane Glycoproteins/immunology , Oligonucleotides/immunology , RNA/immunology , T-Lymphocytes, Cytotoxic/immunology , Toll-Like Receptor 7/immunology , Adjuvants, Immunologic/toxicity , Animals , Antibody Formation , Antigens/immunology , Bacterial Vaccines/toxicity , Cells, Cultured , Disease Models, Animal , Fatty Acids, Monounsaturated/immunology , Female , Immunization , Immunoglobulin G/blood , Listeriosis/immunology , Listeriosis/prevention & control , Lymphocyte Activation , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Oligodeoxyribonucleotides/immunology , Oligonucleotides/toxicity , Ovalbumin/immunology , Quaternary Ammonium Compounds/immunology , RNA/toxicity , Th2 Cells/immunology , Toll-Like Receptor 7/deficiency , Toll-Like Receptor 7/geneticsABSTRACT
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor gammaRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8.
Subject(s)
Autoantigens/immunology , Immunity, Innate , Lupus Erythematosus, Systemic/immunology , RNA, Small Nuclear/immunology , Toll-Like Receptor 7/immunology , Toll-Like Receptor 8/immunology , Animals , Antibodies, Antinuclear/immunology , Cell Line , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Humans , Interferon Type I/immunology , Interferon Type I/metabolism , Male , Mice , Plasma Cells/immunology , Receptors, IgG/immunologyABSTRACT
Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which 'trim' polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-kappaB (nuclear factor kappaB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IkappaBalpha (inhibitor of kappaB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKgamma (IkappaB kinase gamma)/NEMO (NF-kappaB essential modifier) signal adaptors relies on their modification with 'nonclassical' forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-kappaB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.