RESUMEN
The aberrant secretion of proinflammatory cytokines by immune cells is the principal cause of inflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Toll-like receptor 7 (TLR7) and TLR9, sequestered to the endosomal compartment of dendritic cells and macrophages, are closely associated with the initiation and progression of these diseases. Therefore, the development of drugs targeting dysregulated endosomal TLRs is imperative to mitigate systemic inflammation. Here, we applied the principles of computer-aided drug discovery to identify a novel low-molecular-weight compound, TLR inhibitory compound 10 (TIC10), and its potent derivative (TIC10g), which demonstrated dual inhibition of TLR7 and TLR9 signaling pathways. Compared to TIC10, TIC10g exhibited a more pronounced inhibition of the TLR7- and TLR9-mediated secretion of the proinflammatory cytokine tumor necrosis factor-α in a mouse macrophage cell line and mouse bone marrow dendritic cells in a concentration-dependent manner. While TIC10g slightly prevented TLR3 and TLR8 activation, it had no impact on cell surface TLRs (TLR1/2, TLR2/6, TLR4, or TLR5), indicating its selectivity for TLR7 and TLR9. Additionally, mechanistic studies suggested that TIC10g interfered with TLR9 activation by CpG DNA and suppressed downstream pathways by directly binding to TLR9. Western blot analysis revealed that TIC10g downregulated the phosphorylation of the p65 subunit of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular-signal-regulated kinase, p38-MAPK, and c-Jun N-terminal kinase. These findings indicate that the novel ligand, TIC10g, is a specific dual inhibitor of endosomal TLRs (TLR7 and TLR9), disrupting MAPK- and NF-κB-mediated proinflammatory gene expression.
Asunto(s)
Bibliotecas de Moléculas Pequeñas , Receptor Toll-Like 7 , Receptor Toll-Like 9 , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 7/metabolismo , Animales , Ratones , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , HumanosRESUMEN
Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.
Asunto(s)
Nucleotidiltransferasas/antagonistas & inhibidores , Oligonucleótidos Antisentido/química , Receptor Toll-Like 9/antagonistas & inhibidores , Adulto , Animales , Secuencia de Bases , Células Cultivadas , ADN , Humanos , Ratones , Transducción de Señal , Receptor Toll-Like 3/antagonistas & inhibidores , Receptor Toll-Like 7/antagonistas & inhibidoresRESUMEN
Despite recent advances, non-Hodgkin's B cell lymphoma patients often relapse or remain refractory to therapy. Therapeutic resistance is often associated with survival signaling via nuclear factor κB (NF-κB) transcription factor, an attractive but undruggable molecular target. In this study, we describe a bipartite inhibitor comprising a NF-κB-specific decoy DNA tethered to a CpG oligodeoxynucleotide (ODN) targeting Toll-like receptor-9-expressing B cell lymphoma cells. The Bc-NFκBdODN showed efficient uptake by human diffuse large B cell (U2932, OCI-Ly3), Burkitt (RaJi), and mantle cell (Jeko1) lymphomas, respectively. We confirmed that Bc-NFκBdODN inhibited NF-κB nuclear translocation and DNA binding, resulting in CCND2 and MYC downregulation. Bc-NFκBdODN enhanced radiosensitivity of lymphoma cells in vitro. In xenotransplanted human lymphoma, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors. When combined with a local 3-Gy dose of radiation, Bc-NFκBdODN effectively arrested OCI-Ly3 lymphoma progression. In immunocompetent mice, intratumoral injections of Bc-NFκBdODN suppressed growth of directly treated and distant A20 lymphomas, as a result of systemic CD8 T cell-dependent immune responses. Finally, systemic administration of Bc-NFκBdODN to mice bearing disseminated A20 lymphoma induced complete regression and extended survival of most of the treated mice. Our results underscore clinical relevance of this strategy as monotherapy and in support of radiation therapy to benefit patients with resistant or relapsed B cell lymphoma.
Asunto(s)
Linfoma de Células B/terapia , FN-kappa B/antagonistas & inhibidores , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Apoptosis , Proliferación Celular , Humanos , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Oligodesoxirribonucleótidos/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Innate immunity serves as the first line of defence against invading pathogens such as bacteria and viruses. Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses. TLR9 recognizes bacterial and viral DNA containing the cytosine-phosphate-guanine (CpG) dideoxynucleotide motif. The molecular basis by which CpG-containing DNA (CpG-DNA) elicits immunostimulatory activity via TLR9 remains to be elucidated. Here we show the crystal structures of three forms of TLR9: unliganded, bound to agonistic CpG-DNA, and bound to inhibitory DNA (iDNA). Agonistic-CpG-DNA-bound TLR9 formed a symmetric TLR9-CpG-DNA complex with 2:2 stoichiometry, whereas iDNA-bound TLR9 was a monomer. CpG-DNA was recognized by both protomers in the dimer, in particular by the amino-terminal fragment (LRRNT-LRR10) from one protomer and the carboxy-terminal fragment (LRR20-LRR22) from the other. The iDNA, which formed a stem-loop structure suitable for binding by intramolecular base pairing, bound to the concave surface from LRR2-LRR10. This structure serves as an important basis for improving our understanding of the functional mechanisms of TLR9.
Asunto(s)
Islas de CpG/inmunología , ADN/química , ADN/inmunología , Receptor Toll-Like 9/química , Receptor Toll-Like 9/inmunología , Animales , Secuencia de Bases , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Humanos , Ligandos , Modelos Moleculares , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/antagonistas & inhibidoresRESUMEN
Toll-like receptor 9 (TLR9) plays a fundamental role in innate immune responses through pathogen-associated and danger-associated molecular pattern recognition. Ligand recognition by TLR9 results in activation of several signaling pathways, including those involving nuclear factor-kappa B, mitogen-activated protein kinases, and interfer-on-regulatory factors, which promote secretion of proinflammatory cytokines and type I interferons. TLR9 is expressed by immune-mediated cells and in clinical specimens and cell lines of various human cancers. TLR9 appears to act as a double-edged sword in cancer, with some studies indicating that it is associated with increased malignancy and others indicating that it contributes to immune response against cancer. At present, the mechanisms underlying the role of TLR9 in cancer pathophysiology are not completely clear, although various TLR9 agonists and antagonists are being tested in in vitro and in vivo cancer models as well as clinical trials. This review summarizes the current state of knowledge regarding TLR9 features, isoforms, structure, ligands, and signaling, and discusses the roles of TLR9 in cancer pathogenesis. Recent efforts to utilize TLR9 agonists and antagonists as potential anticancer immunotherapy agents are also highlighted.
Asunto(s)
Neoplasias/fisiopatología , Receptor Toll-Like 9/fisiología , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/antagonistas & inhibidoresRESUMEN
BACKGROUND: The recruitment of immune system cells into the central nervous system (CNS) has a profound effect on the outcomes of injury and disease. Glia-derived chemoattractants, including chemokines, play a pivotal role in this process. In addition, cytokines and chemokines influence the phenotype of infiltrating immune cells. Depending on the stimuli present in the local milieu, infiltrating macrophages acquire the classically activated M1 or alternatively activated M2 phenotypes. The polarization of macrophages into detrimental M1 versus beneficial M2 phenotypes significantly influences CNS pathophysiology. Earlier studies indicated that a toll-like receptor 9 (TLR9) antagonist modulates astrocyte-derived cytokine and chemokine release. However, it is not known whether these molecular changes affect astrocyte-induced chemotaxis and polarization of macrophages. The present studies were undertaken to address these issues. METHODS: The chemotaxis and polarization of mouse peritoneal macrophages by spinal cord astrocytes were evaluated in a Transwell co-culture system. Arrays and ELISA were utilized to quantify chemokines in the conditioned medium (CM) of pure astrocyte cultures. Immunostaining for M1- and M2-specific markers characterized the macrophage phenotype. The percentage of M2 macrophages at the glial scar was determined by stereological approaches in mice sustaining a mid-thoracic spinal cord contusion injury (SCI) and intrathecally treated with oligodeoxynucleotide 2088 (ODN 2088), the TLR9 antagonist. Statistical analyses used two-tailed independent-sample t-test and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p value < 0.05 was considered to be statistically significant. RESULTS: ODN 2088-treated astrocytes significantly increased the chemotaxis of peritoneal macrophages via release of chemokine (C-C motif) ligand 1 (CCL1). Vehicle-treated astrocytes polarized macrophages into the M2 phenotype and ODN 2088-treated astrocytes promoted further M2 polarization. Reduced CCL2 and CCL9 release by astrocytes in response to ODN 2088 facilitated the acquisition of the M2 phenotype, suggesting that CCL2 and CCL9 are negative regulators of M2 polarization. The percentage of M2 macrophages at the glial scar was higher in mice sustaining a SCI and receiving ODN 2088 treatment as compared to vehicle-treated injured controls. CONCLUSIONS: TLR9 antagonism could create a favorable environment during SCI by supporting M2 macrophage polarization and chemotaxis via modulation of astrocyte-to-macrophage signals.
Asunto(s)
Astrocitos/metabolismo , Quimiotaxis de Leucocito/fisiología , Activación de Macrófagos/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/metabolismo , Receptor Toll-Like 9/inmunologíaRESUMEN
Unmethylated CpG oligodeoxynucleotides (ODNs) activate plasmacytoid dendritic cells (pDCs) and B cells to induce humoral and cellular immunity, and are under development for the treatment of multiple cancers. However, the specific differences in antitumor effects among the three CpG ODN classes when administered as a monotherapy or in co-therapy with the anti-PD-1 antibody are unclear. We compared the immunostimulatory effects in vitro and antitumor effects in a CT26 subcutaneous mouse tumor model among the three CpG ODN classes. We found that CpG-A slightly suppressed tumor growth but possessed no synergistic antitumor effects with the anti-PD-1 antibody. CpG-B at low doses significantly inhibited tumor growth and possessed synergistic antitumor effects with the anti-PD-1 antibody. A high dose of CpG-C was required to achieve antitumor effects comparable to those of CpG-B, which was consistent with the immunostimulatory effects in B-cell proliferation and TLR9-NF-κB activation. Importantly, CpG-C in combination with anti-PD-1 antibody inhibited tumor growth more quickly and effectively than CpG-B because CpG-B significantly upregulated PD-L1 expression on multiple host immune cells to promote tumor immune escape. Moreover, co-therapy increased the infiltration of effector memory T cells. In summary, CpG-B and CpG-C with different optimal concentrations possessed strong antitumor effects, while CpG-C was more rapid and effective for co-therapy with the anti-PD-1 antibody.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias del Colon/tratamiento farmacológico , Islas de CpG , Inhibidores de Puntos de Control Inmunológico/farmacología , Oligodesoxirribonucleótidos/farmacología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Sinergismo Farmacológico , Femenino , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Transducción de Señal , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismo , Carga Tumoral/efectos de los fármacos , Escape del Tumor/efectos de los fármacos , Microambiente TumoralRESUMEN
Owing to the limited therapeutic efficacy of glioma vaccines, new strategies are required to improve cancer vaccines. Our study aimed to assess the therapeutic efficacy of a glioma vaccine called STDENVANT. This vaccine, comprising glioma stem-like cell (GSC) lysate, dendritic cells (DCs), and Toll-like receptor (TLR) 9 agonist CpG motif-containing oligodeoxynucleotides (CpG ODNs), was assessed using a GL261-C57BL/6 orthotopic mouse model of glioma. STDENVANT markedly improved survival and tumor regression by enhancing anti-tumor immune function. Moreover, STDENVANT upregulated programmed death 1 (PD-1) and its ligand PD-L1 on effector T cells, DCs, and glioma tissues, resulting in the accumulation of regulatory T (Treg) cells in the brain and lymph nodes. Combinatorial administration of anti-PD-L1 antibody and STDENVANT conferred a greater survival advantage and decreased the Treg cell population in the brain. The present results indicate that PD-L1 blockade can promote tumor regression via STDENVANT in a mouse model of glioma, and combinatorial administration of anti-PD-L1 antibody and STDENVANT increases the therapeutic anti-tumor efficacy of treatment.
Asunto(s)
Neoplasias Encefálicas/terapia , Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/inmunología , Glioma/terapia , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Encéfalo/citología , Encéfalo/inmunología , Encéfalo/patología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Femenino , Glioma/inmunología , Glioma/mortalidad , Glioma/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/farmacología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/inmunologíaRESUMEN
Increasing evidence indicates that innate immune receptors play important, yet controversial, roles in traumatic central nervous system (CNS) injury. Despite many advances, the contributions of toll-like receptors (TLRs) to spinal cord injury (SCI) remain inadequately defined. We previously reported that a toll-like receptor 9 (TLR9) antagonist, oligodeoxynucleotide 2088 (ODN 2088), administered intrathecally, improves the functional and histopathological outcomes of SCI. However, the molecular and cellular changes that occur at the injury epicenter following ODN 2088 treatment are not completely understood. Following traumatic SCI, a glial scar, consisting primarily of proliferating reactive astrocytes, forms at the injury epicenter and assumes both beneficial and detrimental roles. Increased production of chondroitin sulfate proteoglycans (CSPGs) by reactive astrocytes inhibits the regeneration of injured axons. Astrocytes express TLR9, which can be activated by endogenous ligands released by damaged cells. It is not yet known how TLR9 antagonism modifies astrocyte function at the glial scar and how this affects axonal preservation or re-growth following SCI. The present studies were undertaken to address these issues. We report that in female mice sustaining a severe mid-thoracic (T8) contusion injury, the number of proliferating astrocytes in regions rostral and caudal to the lesion border increased significantly by 30- and 24-fold, respectively, compared to uninjured controls. Intrathecal ODN 2088 treatment significantly reduced the number of proliferating astrocytes by 60% in both regions. This effect appeared to be, at least partly, mediated through the direct actions of ODN 2088 on astrocytes, since the antagonist decreased proliferation in pure SC astrocyte cultures by preventing the activation of the Erk/MAPK signaling pathway. In addition, CSPG immunoreactivity at the lesion border was more pronounced in vehicle-treated injured mice compared to uninjured controls and was significantly reduced following administration of ODN 2088 to injured mice. Moreover, ODN 2088 significantly decreased astrocyte migration in an in vitro scratch-wound assay. Anterograde tracing and quantification of corticospinal tract (CST) axons in injured mice, indicated that ODN 2088 preserves proximal axons. Taken together, these findings suggest that ODN 2088 modifies the glial scar and creates a milieu that fosters axonal protection at the injury site.
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Astrocitos/metabolismo , Axones/metabolismo , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Astrocitos/patología , Axones/inmunología , Axones/patología , Proliferación Celular , Femenino , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/administración & dosificación , Traumatismos de la Médula Espinal/patología , Receptor Toll-Like 9/antagonistas & inhibidoresRESUMEN
Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.
Asunto(s)
Antineoplásicos/farmacología , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Oligonucleótidos/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Inmunoterapia , Linfoma de Células B/patología , Linfoma de Células B/terapia , Ratones , Terapia Molecular Dirigida , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Transcripción Genética , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIMS/HYPOTHESIS: Innate immune effectors interact with the environment to contribute to the pathogenesis of the autoimmune disease, type 1 diabetes. Although recent studies have suggested that innate immune Toll-like receptors (TLRs) are involved in tissue development, little is known about the role of TLRs in tissue development, compared with autoimmunity. We aimed to fill the knowledge gap by investigating the role of TLR9 in the development and function of islet beta cells in type 1 diabetes, using NOD mice. METHODS: We generated Tlr9-/- NOD mice and examined them for type 1 diabetes development and beta cell function, including insulin secretion and glucose tolerance. We assessed islet and beta cell number and characterised CD140a expression on beta cells by flow cytometry. We also tested beta cell function in Tlr9-/- C57BL/6 mice. Finally, we used TLR9 antagonists to block TLR9 signalling in wild-type NOD mice to verify the role of TLR9 in beta cell development and function. RESULTS: TLR9 deficiency promoted pancreatic islet development and beta cell differentiation, leading to enhanced glucose tolerance, improved insulin sensitivity and enhanced first-phase insulin secretory response. This was, in part, mediated by upregulation of CD140a (also known as platelet-derived growth factor receptor-α [PDGFRα]). In the absence of TLR9, induced by either genetic targeting or treatment with TLR9 antagonists, which had similar effects on ontogenesis and function of beta cells, NOD mice were protected from diabetes. CONCLUSIONS/INTERPRETATION: Our study links TLR9 and the CD140a pathway in regulating islet beta cell development and function and indicates a potential therapeutic target for diabetes prevention and/or treatment.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Cloroquina/farmacología , Diabetes Mellitus Tipo 1/patología , Femenino , Citometría de Flujo , Prueba de Tolerancia a la Glucosa , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Mutantes , Oligodesoxirribonucleótidos/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/genéticaRESUMEN
The MYD88(L265P) mutation is found in 2% to 10% of chronic lymphocytic leukemia, 29% of activated B-cell type diffuse large B-cell lymphoma and 90% of Waldenström macroglobulinemia, making it conceptually attractive to treat these malignancies with inhibitors of endosomal Toll-like receptors (TLR9, TLR7) that activate MYD88. Here we show that genetic inhibition of endosomal TLRs has the opposite effect on accumulation of MYD88(L265P) B cells in vitro and in vivo. Activated mature B cells from wild-type, Unc93b1(3d/3d)-mutant, or Tlr9-deficient mice were transduced with retrovirus encoding MYD88(L265P) and analyzed either in vitro or after transplantation into Rag1(-/-) recipient mice. Unc93b1(3d/3d) mutation, which blocks TLR9 and TLR7 signaling, or Tlr9 deficiency suppressed MYD88(L265P) B-cell growth in vitro but paradoxically increased in vivo accumulation of MYD88(L265P) B cells as CD19(low) plasmablasts by 10- to 100-fold. These results reveal an unexpected, powerful inhibitory effect of TLR9 on MYD88(L265P) B-cell proliferation and differentiation that appears independent of TLR7, and they provide a preclinical indicator for caution in clinical trials of TLR7/9 inhibitors for MYD88(L265P) B-cell malignancies.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Proteínas de Transporte de Membrana/química , Factor 88 de Diferenciación Mieloide/metabolismo , Células Plasmáticas/inmunología , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Diferenciación Celular , Proliferación Celular , Proteínas de Transporte de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Transducción de Señal , Receptor Toll-Like 9/fisiologíaRESUMEN
Diabetic encephalopathy (DE) is one of the most common complications of diabetes. The major pathological variations include neurofibrillary tangles (NFTs), which are caused by tau hyperphosphorylation, and senile plaques (SPs) consisting of amyloid ß- protein(Aß) deposits. In recent years, DE research studies have focused on exploring the activation of the inflammatory signaling pathway in immune cells. Toll-like receptor 9 (TLR9) is well known to regulate the inflammatory reactions in immune processes. During the tau hyperphosphorylation process, TLR9 in microglia plays bidirectional roles. However, no studies have clearly demonstrated the relationship between TLR9 and tau hyperphosphorylation in neurons. Based on our experiments, we found significant increase in TLR9 expression in neurons and an increase in tau hyperphosphorylation in high-glucose media. However, these alterations can be reversed by TLR9 inhibitor. Furthermore, we specifically inhibited the activation of P38mitogenactivated protein kinase(P38MAPK) and found an effective decrease in tau hyperphosphorylation. This effect is likely related to Unc93b1. Meanwhile, High glucose levels can induce neuronal apoptosis via the TLR9 signaling pathway. Our studies are the first to reveal that high glucose can regulate tau hyperphosphorylation and neuronal apoptosis via TLR9-P38MAPK signaling pathway. These findings provide a new method for studying the mechanism underlying DE.
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Glucosa/toxicidad , Neuronas/efectos de los fármacos , Receptor Toll-Like 9/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas tau/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Regulación de la Expresión Génica , Hipocampo , Imidazoles/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Neuronas/citología , Neuronas/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Piridinas/farmacología , Transducción de Señal , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas tau/metabolismoRESUMEN
Heart failure is a leading cause of morbidity and mortality in industrialized countries. Although infection with microorganisms is not involved in the development of heart failure in most cases, inflammation has been implicated in the pathogenesis of heart failure. However, the mechanisms responsible for initiating and integrating inflammatory responses within the heart remain poorly defined. Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA. Mitochondria damaged by external haemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes. Here we show that mitochondrial DNA that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated inflammatory responses in cardiomyocytes and is capable of inducing myocarditis and dilated cardiomyopathy. Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions, but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload. Early in the pathogenesis, DNase II-deficient hearts showed infiltration of inflammatory cells and increased messenger RNA expression of inflammatory cytokines, with accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium. Administration of inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA, or ablation of Tlr9 attenuated the development of cardiomyopathy in DNase II-deficient mice. Furthermore, Tlr9 ablation improved pressure overload-induced cardiac dysfunction and inflammation even in mice with wild-type Dnase2a alleles. These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.
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Autofagia , ADN Mitocondrial/inmunología , ADN Mitocondrial/metabolismo , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Miocarditis/etiología , Miocarditis/inmunología , Alelos , Animales , Aorta/patología , Cardiomegalia/etiología , Constricción Patológica/complicaciones , Citocinas/genética , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Corazón/fisiopatología , Insuficiencia Cardíaca/inmunología , Insuficiencia Cardíaca/metabolismo , Lisosomas/enzimología , Lisosomas/metabolismo , Masculino , Ratones , Mitocondrias , Miocarditis/metabolismo , Miocarditis/patología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Presión , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/deficiencia , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismoRESUMEN
BACKGROUND: Aberrant toll-like receptors (TLRs) 7, 8, and 9 activation by self-nucleic acids is implicated in immune-mediated inflammatory diseases (IMIDs) such as psoriasis. In preclinical IMID models, blocking TLR-activation reduced disease severity. IMO-8400 is a first-in-class, oligonucleotide-based antagonist of TLRs 7, 8, and 9. We evaluated the short-term safety and proof-of-concept for efficacy of IMO-8400 in a first-in-patient phase 2 trial. METHODS: Forty-six psoriasis patients were randomly assigned to IMO-8400 in four dose levels or placebo for 12weeks. Post-treatment follow-up was seven weeks. Primary outcome was incidence of adverse events. Secondary, exploratory outcomes included changes in psoriasis area and severity index (PASI). RESULTS: IMO-8400 across all dose levels did not cause any serious or severe adverse events. The most common treatment-related adverse events were dose-dependent injection-site reactions. All IMO-8400 groups showed clinical improvement, but a clear dose-response relationship and statistically significant differences with placebo were not observed (P=0.26). Eleven (38%) of 29 subjects on IMO-8400 achieved ≥50% PASI-reduction, compared to 1 (11%) of 9 subjects on placebo. Five (17%) and 2 (7%) IMO-8400-treated subjects achieved PASI-75 and PASI-90, respectively, compared to none on placebo. CONCLUSIONS: Short-term IMO-8400-treatment was well tolerated and reduced psoriasis severity. These findings warrant further investigation of endosomal TLR-antagonism as a therapeutic approach in psoriasis and other TLR-mediated IMIDs. TRIAL REGISTRATION: EudraCT 2013-000164-28 and Clinicaltrials.govNCT01899729.
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Psoriasis/tratamiento farmacológico , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Adulto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Psoriasis/sangre , Psoriasis/patología , Índice de Severidad de la Enfermedad , Piel/efectos de los fármacos , Piel/patología , Resultado del Tratamiento , Adulto Joven , beta-DefensinasRESUMEN
Toll-like receptors (TLRs) are innate immune receptors that respond to both exogenous and endogenous stimuli and are suggested to contribute to the perpetuation of chronic inflammation associated with rheumatoid arthritis (RA). In particular, the endosomal TLRs 3, 7, 8, and 9 have more recently been postulated to be of importance in RA pathogenesis. In this study, pan inhibition of the endosomal TLRs by a phosphorothioate-modified inhibitory oligodeoxynucleotide (ODN) is demonstrated in primary human B cells, macrophages, and RA fibroblasts. Inhibition of TLR8 was of particular interest as TLR8 has been associated with RA pathogenesis in both human and murine arthritis models. ODN1411 competitively inhibited TLR8 signaling and was observed to directly bind to a purified TLR8 ectodomain, suggesting inhibition was through a direct interaction with the receptor. Addition of ODN1411 to human RA synovial membrane cultures significantly inhibited spontaneous cytokine production from these cultures, suggesting a potential role for one or more of the endosomal TLRs in inflammatory cytokine production in RA and the potential for inhibitory ODNs as novel therapies.
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Artritis Reumatoide/inmunología , Citocinas/biosíntesis , Oligodesoxirribonucleótidos/farmacología , Receptores Toll-Like/antagonistas & inhibidores , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Células Cultivadas , Endosomas/inmunología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Humanos , Inflamación , Ratones , Oligodesoxirribonucleótidos/inmunología , Membrana Sinovial/citología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/inmunología , Receptor Toll-Like 3/antagonistas & inhibidores , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/antagonistas & inhibidores , Receptor Toll-Like 8/inmunología , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
UNLABELLED: Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a "shock-and-kill" approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) (P= 0.005) and CXCL10 (P= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56(dim)CD16(+)NK cells (P= 0.001) as well as higher proportions of CD107a-positive (P= 0.002) and IFN-γ-producing (P= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4(+)and CD8(+)T cells. Furthermore, CD4(+)T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4(+)T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4(+)T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. IMPORTANCE: We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel "dumbbell-shape" structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4(+)T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4(+)T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.
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Linfocitos T CD4-Positivos/inmunología , ADN/farmacología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Células Asesinas Naturales/inmunología , Receptor Toll-Like 9/antagonistas & inhibidores , Transcripción Genética , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/virología , Estudios de Casos y Controles , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Citocinas/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Inmunomodulación/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/genética , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , ARN Viral , Carga Viral , Latencia del VirusRESUMEN
Activation of TLR7-9 has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. Thus, therapeutic applications of antagonists of these TLRs for such disorders are being investigated. Bortezomib (Velcade) is a proteasome inhibitor known to suppress activation of these TLRs. To identify novel TLR7-9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles of bortezomib-treated cells. These profiles were then used to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. A natural antibiotic, thiostrepton, was identified for study. Similar to bortezomib, thiostrepton effectively inhibits TLR7-9 activation in cell-based assays and in dendritic cells. In contrast to bortezomib, thiostrepton does not inhibit NF-κB activation induced by TNF-α, IL-1, and other TLRs, and it is less cytotoxic to dendritic cells. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms, which distinguish it from currently used TLR7-9 inhibitors. One mechanism is similar to the proteasome inhibitory function of bortezomib, whereas the other is through inhibition of endosomal acidification. Accordingly, in different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel TLR7-9 inhibitor, and compared with bortezomib, its inhibitory effect is more specific to these TLRs, suggesting the potential therapeutic applications of thiostrepton on immunologic disorders elicited by inappropriate activation of TLR7-9.
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Glicoproteínas de Membrana/antagonistas & inhibidores , Psoriasis/tratamiento farmacológico , Tioestreptona/farmacología , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Línea Celular , Humanos , Inflamación/inmunología , Inflamación/patología , Interleucina-1/inmunología , Glicoproteínas de Membrana/inmunología , Ratones , Psoriasis/inmunología , Psoriasis/patología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 9/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The development of therapeutic vaccines for treatment of established cancer has proven challenging. Cancer vaccines not only need to induce a robust tumor Ag-specific immune response but also need to overcome the tolerogenic and immunosuppressive microenvironments that exist within many solid cancers. ISCOMATRIX adjuvant (ISCOMATRIX) is able to induce both tumor Ag-specific cellular and Ab responses to protect mice against tumor challenge, but this is insufficient to result in regression of established solid tumors. In the current study, we have used B16-OVA melanoma, Panc-OVA pancreatic, and TRAMP-C1 prostate cancer mouse tumor models to test therapeutic efficacy of ISCOMATRIX vaccines combined with other immune modulators. The coadministration of an ISCOMATRIX vaccine with the TLR3 agonist, polyinosinic-polycytidylic acid, and TLR9 agonist, CpG, reduced tumor growth in all tumor models and the presence of ISCOMATRIX in the formulation was critical for the therapeutic efficacy of the vaccine. This vaccine combination induced a robust and multifunctional CD8(+) T cell response. Therapeutic protection required IFN-γ and CD8(+) T cells, whereas NK and CD4(+) T cells were found to be redundant. ISCOMATRIX vaccines combined with TLR3 and TLR9 agonists represent a promising cancer immunotherapy strategy.
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Linfocitos T CD8-positivos/efectos de los fármacos , Vacunas contra el Cáncer/administración & dosificación , Colesterol/administración & dosificación , Melanoma Experimental/terapia , Neoplasias Pancreáticas/terapia , Fosfolípidos/administración & dosificación , Neoplasias de la Próstata/terapia , Saponinas/administración & dosificación , Neoplasias Cutáneas/terapia , Adyuvantes Inmunológicos/administración & dosificación , Animales , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Combinación de Medicamentos , Humanos , Inmunoterapia/métodos , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/mortalidad , Ratones , Ratones Noqueados , Trasplante de Neoplasias , Oligodesoxirribonucleótidos/farmacología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/mortalidad , Poli I-C/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/mortalidad , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Análisis de Supervivencia , Receptor Toll-Like 3/antagonistas & inhibidores , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunología , Carga Tumoral/efectos de los fármacosRESUMEN
A subset of chronic lymphocytic leukemia (CLL) BCRs interacts with Ags expressed on apoptotic cells, suggesting that CLL BCRs have the potential to internalize apoptotic cell RNA- or DNA-containing fragments with resultant activation of TLR7 or TLR9, respectively. By blocking cAMP degradation, type 4 cAMP phosphodiesterase (PDE4) inhibitors activate cAMP-mediated signaling and induce apoptosis in CLL cells. In this study, we show that autologous irradiated leukemic cells induce proliferation in CLL cells and that such proliferation is blocked by a TLR7/8/9 inhibitor, by DNase, and by the PDE4 inhibitor rolipram. Rolipram also inhibited CLL cell proliferation induced by synthetic TLR7 and TLR9 agonists, as well as TLR agonist-induced costimulatory molecule expression and TNF-a (but not IL-6 or IL-10) production. Whereas treatment with a TLR9 agonist protected IgH V region unmutated, but not mutated, CLL cells from apoptosis, PDE4 inhibitors augmented apoptosis in both subtypes, suggesting that cAMP-mediated signaling may abrogate a TLR9-mediated survival signal in prognostically unfavorable IGHV unmutated CLL cells. Rolipram inhibited both TLR7/8- and TLR9-induced IFN regulatory factor 5 and NF-kB p65 nuclear translocation. PDE4 inhibitors also blocked TLR signaling in normal human immune cells. In PBMC and CD14-positive monocytes, PDE4 inhibitors blocked IFN-a or TNF-a (but not IL-6) production, respectively, following stimulation with synthetic TLR agonists or RNA-containing immune complexes. These results suggest that PDE4 inhibitors may be of clinical utility in CLL or autoimmune diseases that are driven by TLR-mediated signaling.