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1.
Nat Biotechnol ; 38(3): 320-332, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932728

RESUMO

Personalized cancer vaccines targeting patient-specific neoantigens are a promising cancer treatment modality; however, neoantigen physicochemical variability can present challenges to manufacturing personalized cancer vaccines in an optimal format for inducing anticancer T cells. Here, we developed a vaccine platform (SNP-7/8a) based on charge-modified peptide-TLR-7/8a conjugates that are chemically programmed to self-assemble into nanoparticles of uniform size (~20 nm) irrespective of the peptide antigen composition. This approach provided precise loading of diverse peptide neoantigens linked to TLR-7/8a (adjuvant) in nanoparticles, which increased uptake by and activation of antigen-presenting cells that promote T-cell immunity. Vaccination of mice with SNP-7/8a using predicted neoantigens (n = 179) from three tumor models induced CD8 T cells against ~50% of neoantigens with high predicted MHC-I binding affinity and led to enhanced tumor clearance. SNP-7/8a delivering in silico-designed mock neoantigens also induced CD8 T cells in nonhuman primates. Altogether, SNP-7/8a is a generalizable approach for codelivering peptide antigens and adjuvants in nanoparticles for inducing anticancer T-cell immunity.


Assuntos
Adjuvantes Imunológicos/química , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Melanoma Experimental/tratamento farmacológico , Animais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Melanoma Experimental/imunologia , Camundongos , Nanopartículas , Medicina de Precisão , Primatas , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Vacinação , Vacinas Conjugadas
2.
Immunology ; 159(2): 183-192, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31630392

RESUMO

The plant virus, cowpea mosaic virus (CPMV), has demonstrated a remarkable capacity to induce anti-tumour immune responses following direct administration into solid tumours. The molecular pathways that account for these effects and the capacity of CPMV to activate human cells are not well defined. Here, we examine the ability of CPMV particles to activate human monocytes, dendritic cells (DCs) and macrophages. Monocytes in peripheral blood mononuclear cell cultures and purified CD14+ monocytes were readily activated by CPMV in vitro, leading to induction of HLA-DR, CD86, PD-L1, IL-15R and CXCL10 expression. Monocytes released chemokines, CXCL10, MIP-1α and MIP-1ß into cell culture supernatants after incubation with CPMV. DC subsets (pDC and mDC) and monocyte-derived macrophages also demonstrated evidence of activation after incubation with CPMV. Inhibitors of spleen tyrosine kinase (SYK), endocytosis or endocytic acidification impaired the capacity of CPMV to activate monocytes. Furthermore, CPMV activation of monocytes was partially blocked by a TLR7/8 antagonist. These data demonstrate that CPMV activates human monocytes in a manner dependent on SYK signalling, endosomal acidification and with an important contribution from TLR7/8 recognition.


Assuntos
Comovirus/patogenicidade , Endossomos/virologia , Monócitos/virologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Endossomos/imunologia , Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia
3.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31591164

RESUMO

Toll-like receptors (TLR) trigger the immune system to mount a rapid innate response capable of protecting the host from a wide variety of bacterial and viral pathogens. There is interest in harnessing TLR agonists to reduce the susceptibility of at-risk populations to infection. However, the widespread prophylactic use of TLR agonists has been compromised by the need to administer them by parenteral injection. An exception is the TLR7/8 agonist R848, which can boost gastrointestinal and systemic immunity when administered orally. This work examines the effect of R848 on host susceptibility to Listeria monocytogenes in a murine challenge model and describes the underlying mechanisms. Results show that prophylactic administration of R848 significantly reduces susceptibility to infection of BALB/c mice, an effect that lasts 1 week. Oral R848 directly stimulated B cells to produce cytokines and Ig. In the absence of B cells, R848-mediated protection was lost. These findings support the use of oral R848 to reduce the susceptibility of at-risk individuals to infection and identify the critical role of B cells in TLR7-mediated resistance to bacterial infection.


Assuntos
Linfócitos B/imunologia , Imidazóis/farmacologia , Listeria monocytogenes/imunologia , Listeriose/prevenção & controle , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Listeriose/imunologia , Camundongos , Camundongos Endogâmicos BALB C
4.
Int J Mol Sci ; 20(18)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546763

RESUMO

Systemic lupus erythematosus (SLE) is a chronic, multifactorial autoimmune disease that predominantly affects young females. Dysregulation of different immune cell populations leads to self-tolerance breakdown and subsequent multiple organ damage as the disease develops. Plasmacytoid dendritic cells (pDCs) are potent producers of type I interferon (IFN), while myeloid dendritic cells (mDCs) are more specialized in antigen presentations. We have previously reported that bone-marrow (BM)-derived pDCs from the murine lupus model New Zealand black/white F1 (BWF1) possess abnormalities. Therefore, this study continues to investigate what aberrant properties peripheral pDCs and mDCs possess in BWF1 and how they mediate SLE progression, by comparing their properties in pre-symptomatic and symptomatic mice. Results showed that CD11chiCD11b+ myeloid DCs expanded during the disease state with down-regulation of co-stimulatory molecules and major histocompatibility complex class II molecules (MHC II), but their capacity to stimulate T cells was not hampered. During the disease state, this subset of mDCs displayed heightened toll-like receptors 7 and 9 (TLR 7/9) responses with increased interleukin 10 (IL-10) and C-X-C motif chemokine ligand 13 (CXCL13) expressions. Moreover, the expressions of myeloid differentiation primary response 88 (Myd88) and nuclear factor kappa B subunit 1 (Nfkb1) were higher in CD11chiCD11b+ DCs at the disease stage, leading to higher nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation activity. In summary, we reported aberrant phenotypic properties with enhanced TLR7/9 responses of CD11chiCD11b+ DCs in SLE mediated by aberrant NF-κB signaling pathway. Our findings add additional and novel information to our current understanding of the role of DCs in lupus immunopathogenesis. Lastly, molecular candidates in the NF-κB pathway should be exploited for developing therapeutic targets for SLE.


Assuntos
Antígenos CD11/imunologia , Antígeno CD11b/imunologia , Quimiocina CXCL13/imunologia , Células Dendríticas/imunologia , Interleucina-10/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/imunologia , Subunidade p50 de NF-kappa B/imunologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Animais , Antígenos CD11/genética , Antígeno CD11b/genética , Quimiocina CXCL13/genética , Células Dendríticas/patologia , Modelos Animais de Doenças , Feminino , Interleucina-10/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/patologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Subunidade p50 de NF-kappa B/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética
5.
Biomed Res Int ; 2019: 1401894, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31309100

RESUMO

Toll-like receptors (TLRs) play a key role in the innate immune response to numerous pathogens, including Acanthamoeba spp. The aim of this study was to determine the expression of TLR2 and TLR4 in the eyes of mice following intranasal infection with Acanthamoeba spp. in relation to the host's immunological status. Amoebae used in this study were isolated from the bronchial aspirate of a patient with acute myeloid leukemia (AML) and atypical symptoms of pneumonia. We found statistically significant differences in the expression of TLR2 and TLR4 in the eye of immunocompetent mice at 8, 16, and 24 days after Acanthamoeba spp. infection (dpi) compared to control group. Immunosuppressed mice showed significant differences in the expression of TLR2 at 16 and 24 dpi compared to uninfected animals. Our results indicate that TLR2 and TLR4 are upregulated in the eyes of mice in response to Acanthamoeba spp. We suggest that it is possible for trophozoites to migrate through the optic nerve from the brain to the eyes. The course of disseminated acanthamoebiasis may be influenced by the host's immunological status, and the observed changes in expression of TLR2 and TLR4 in the host's organs may indicate the role of these receptors in the pathomechanism of acanthamoebiasis.


Assuntos
Amebíase/imunologia , Proteínas do Olho/imunologia , Olho/imunologia , Regulação da Expressão Gênica/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologia , Acanthamoeba/imunologia , Amebíase/patologia , Animais , Olho/parasitologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C
6.
ACS Appl Mater Interfaces ; 11(30): 26637-26647, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31276378

RESUMO

Stimulation of Toll-like receptors (TLRs) and/or NOD-like receptors on immune cells initiates and directs immune responses that are essential for vaccine adjuvants. The small-molecule TLR7 agonist, imiquimod, has been approved by the FDA as an immune response modifier but is limited to topical application due to its poor pharmacokinetics that causes undesired adverse effects. Nanoparticles are increasingly used with innate immune stimulators to mitigate side effects and enhance adjuvant efficacy. In this study, a potent small-molecule TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), was conjugated to hollow silica nanoshells (NS). Proinflammatory cytokine (IL-6, IL-12) release by mouse bone-marrow-derived dendritic cells and human peripheral blood mononuclear cells revealed that the potency of silica nanoshells-TLR7 conjugates (NS-TLR) depends on nanoshell size and ligand coating density. Silica nanoshells of 100 nm diameter coated with a minimum of ∼6000 1V209 ligands/particle displayed 3-fold higher potency with no observed cytotoxicity when compared to an unconjugated TLR7 agonist. NS-TLR activated the TLR7-signaling pathway, triggered caspase activity, and stimulated IL-1ß release, while neither unconjugated TLR7 ligands nor silica shells alone produced IL-1ß. An in vivo murine immunization study, using the model antigen ovalbumin, demonstrated that NS-TLR increased antigen-specific IgG antibody induction by 1000× with a Th1-biased immune response, compared to unconjugated TLR7 agonists. The results show that the TLR7 ligand conjugated to silica nanoshells is capable of activating an inflammasome pathway to enhance both innate immune-stimulatory and adjuvant potencies of the TLR7 agonist, thereby broadening applications of innate immune stimulators.


Assuntos
Imiquimode/imunologia , Imunidade Inata/efeitos dos fármacos , Imunoconjugados/imunologia , Receptor 7 Toll-Like/imunologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Humanos , Imiquimode/química , Imiquimode/uso terapêutico , Imunidade Inata/genética , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Nanoconchas/química , Transdução de Sinais/efeitos dos fármacos , Dióxido de Silício/química , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/genética
7.
Cancer Immunol Immunother ; 68(7): 1073-1085, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31161238

RESUMO

Cryptotanshinone (CT), a purified compound initially isolated from the dried roots of Salvia militorrhiza. Bunge, exhibits cytotoxic antitumor effects on many tumors. We have shown that CT possesses the dual capacities to concomitantly inhibit the proliferation of lung cancer cells and promote the generation of antitumor immunity. In this study, we investigated whether CT could be used to treat hepatocellular carcinoma (HCC) using a mouse Hepa1-6 model. CT inhibited the proliferation of mouse hepatoma (Hepa1-6) cells in vitro by inducing Hepa1-6 cells apoptosis through the JAK2/STAT3 signaling pathway. In addition, CT activated macrophages and polarized mouse bone marrow-derived macrophages (BMM) toward an M1 phenotype in vitro, which depended on the TLR7/MyD88/NF-κB signaling pathway. Furthermore, CT significantly inhibited the growth of syngeneic Hepa1-6 hepatoma tumors, and, in combination with anti-PD-L1 cured Hepa1-6-bearing mice with the induction of long-term anti-Hepa1-6 specific immunity. Immunoprofiling of treated Hepa1-6-bearing mice revealed that CT-promoted activation of tumor-infiltrating macrophages and dendritic cells, induction of antitumor T cell response, and infiltration of effector/memory CD8 T cells in the tumor tissue. Importantly, the immunotherapeutic effects of CT and anti-PD-L1 depended on the presence of CD8 T cells. Thus, CT and anti-PD-L1 may provide an effective immunotherapeutic regimen for human HCC based on a combination of cytotoxic effects and induction of tumor-specific immunity.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Fenantrenos/farmacologia , Receptor 7 Toll-Like/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Antígeno B7-H1/antagonistas & inibidores , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Ativação Linfocitária , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fenantrenos/uso terapêutico , Salvia miltiorrhiza/química , Transdução de Sinais/efeitos dos fármacos , Receptor 7 Toll-Like/imunologia , Resultado do Tratamento
8.
J Immunol Res ; 2019: 1749803, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31093508

RESUMO

Plasmacytoid dendritic cells (pDCs) express high levels of the toll-like receptors (TLRs) TLR7 and TLR9. In response to TLR7 and TLR9 ligands, pDCs are primary producers of type I interferons. Our previous study demonstrated that pDCs activated by the TLR7 ligand imiquimod (IMQ) and the TLR9 ligand CpG A can kill breast cancer cells in vitro and inhibit tumor growth in vivo. Moreover, we observed a distinctive morphological, phenotypic change in pDCs after activation by IMQ and CpG A. However, the effect of other TLR7 and TLR9 ligands on pDCs remains less understood. In this study, we treat pDCs with the TLR7 ligand IMQ, TLR7/8 ligands (CL097 and CL075), and three TLR9 ligands (different types of CpGs). The size of pDCs increased significantly after activation by TLR7, or TLR7/8 ligands. TLR7, TLR7/8, and TLR9 ligands similarly modulated cytokine release, as well as protein expression of pDC markers, costimulatory molecules, and cytotoxic molecules. Interestingly, TLR7/8 ligands, especially CL097, induced stronger responses. These results are relevant to the further study of the role and mechanism of pDC-induced antitumor effects and may aid in the development of a new strategy for future tumor immunotherapy.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Imiquimode/farmacologia , Glicoproteínas de Membrana/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Animais , Feminino , Imidazóis/farmacologia , Indutores de Interferon/farmacologia , Interferon Tipo I/imunologia , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Quinolinas/farmacologia , Tiazóis/farmacologia
9.
J Immunol ; 202(9): 2529-2534, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30936294

RESUMO

Systemic lupus erythematosus severity correlates with elevated serum levels of type I IFNs, cytokines produced in large quantities by plasmacytoid dendritic cells (pDC) in response to engagement of TLR7 and TLR9 with endocytosed nucleic acids. B cell adaptor for PI3K (BCAP) promoted many aspects of TLR7-driven lupus-like disease, including Isg15 and Ifit1 expression in blood and an immature pDC phenotype associated with higher IFN production. BCAP-/- mice produced significantly less serum IFN-α than wild-type mice after injection of TLR9 agonist, and BCAP promoted TLR7 and TLR9-induced IFN-α production specifically in pDC. TLR-induced IFN-α production in pDC requires DOCK2-mediated activation of Rac1 leading to activation of IKKα, a mechanism we show was dependent on BCAP. BCAP-/- pDC had decreased actin polymerization and Rac1 activation and reduced IKKα phosphorylation upon TLR9 stimulation. We show a novel role for BCAP in promoting TLR-induced IFN-α production in pDC and in systemic lupus erythematosus pathogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Células Dendríticas/imunologia , Interferon-alfa/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/imunologia , Plasmócitos/imunologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/patologia , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/imunologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Interferon-alfa/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Plasmócitos/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética , Ubiquitinas/genética , Ubiquitinas/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia
10.
Nat Commun ; 10(1): 1780, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992428

RESUMO

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.


Assuntos
Plaquetas/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Ativação Plaquetária/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Complemento C3/imunologia , Complemento C3/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Humana/sangue , Influenza Humana/virologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neutrófilos/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo
11.
Microb Pathog ; 131: 65-74, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30926417

RESUMO

BACKGROUND AND AIM: Toll-like receptor 7 (TLR7) can recognize single-stranded RNA viruses like hepatitis C virus (HCV) with subsequent induction of different interferon (IFN) types including IFN lambda (IFNL), which activate an immediate anti-viral response. However, the role of TLR7 in inflammation and fibrosis, characteristics of HCV-induced liver injury, is still controversial. The present work was designed to investigate the potential role of TLR7 and IFNL1 in chronic hepatitis C (CHC) in relation to viral replication and liver injury. METHODS: Forty two treatment-naïve patients with CHC and 20 healthy subjects were enrolled in the study. TLR7 expression on peripheral blood CD14+ monocytes was studied by color flow cytometry and the frequency of TLR7+CD14+ cells was expressed as percentage of total monocyte count. Quantification of IFNL1 levels in serum was determined using enzyme-linked immunosorbant assay. Liver biopsies were examined for assessment of histological activity grade (A0-A3) and fibrosis stage (F0-F4) according to METAVIR scoring system as well as steatosis grade. Immunohistochemical staining was performed using human antibodies against TLR7 and IFNL1 and was scored semi-quantitatively (score 0-3). Hepatic expression of TLR7 and IFNL1 was further classified using a two-grade scale as low expression (score 0 or 1) and high expression (score 2 or 3). RESULTS: Percentages of circulating TLR7+CD14+ monocytes and serum IFNL1 levels were significantly higher in patients with CHC than in healthy controls (P = 0.025 and P < 0.001 respectively) and were positively correlated with corresponding hepatic TLR7 and IFNL1 expression (P < 0.001 and P = 0.010 respectively). Significantly lower peripheral blood and hepatic TLR7 expression and IFNL1 levels were found in patients with viral loads between 200,000-600,000 IU/ml and >600,000 IU/ml than in those with viral load <200,000 IU/ml (P < 0.05), in patients with severe necroinflammation than in those with mild-to-moderate necroinflammation (P < 0.05) and in patients with advanced fibrosis than in those with early fibrosis (P < 0.01). Also, changes in TLR7 expression and IFNL1 production in peripheral blood and the liver were inversely correlated with serum levels of aspartate and alanine aminotransferases (P < 0.05) and HCV RNA (P < 0.01), histological activity grade (P < 0.01) and fibrosis stage (P < 0.01). By plotting receiver operating characteristics (ROC) curve, serum IFNL1 showed higher sensitivity and specificity than percentages of circulating TLR7+CD14+ monocytes in discriminating patients with CHC according to the severity of hepatic necroinflammation (area under the curve (AUC) = 0.901 vs. 0.816 respectively) and fibrosis (AUC = 0.971 vs. 0.825 respectively) at a cut-off value of 44.75 pg/ml and 10.25% respectively. CONCLUSIONS: TLR7 activation and IFNL1 production in CHC may play an important role in controlling viral replication and limiting hepatic inflammation and fibrosis and their downregulation may result in viral persistence and disease progression. The immunoregulatory role of TLR7-IFNL1 pathway in the pathogenesis of chronic HCV infection should be further studied. Clinical trials with a large number of patients are needed to assess the usefulness of serum IFNL1 as a potential biomarker for severity of liver injury in chronic HCV infection and other liver diseases.


Assuntos
Hepatite C Crônica/metabolismo , Interleucinas/sangue , Interleucinas/metabolismo , Fígado/lesões , Fígado/metabolismo , Receptor 7 Toll-Like/sangue , Receptor 7 Toll-Like/metabolismo , Replicação Viral , Adulto , Alanina Transaminase/sangue , Antivirais/farmacologia , Ácido Aspártico/sangue , Biomarcadores/sangue , Citocinas/sangue , Egito , Feminino , Fibrose/patologia , Hepacivirus/patogenicidade , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/patologia , Humanos , Imuno-Histoquímica , Interferons , Interleucinas/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Receptor 7 Toll-Like/imunologia , Carga Viral , Adulto Jovem
12.
Dev Comp Immunol ; 95: 77-88, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30742850

RESUMO

TLR7 subfamily members are important pattern recognition receptors participating in the recognition of pathogen-associated molecular patterns. In this study, we successfully identified 3 members of TLR7 subfamily from the spiny eel Mastacembelus armatus (MaTLR7, MaTLR8 and MaTLR9). The amino acid sequence identities of MaTLR7 and MaTLR8 with Monopterus albus TLR7 were 87.2% and 76.5%, respectively and the identity of MaTLR9 with Seriola lalandi TLR9 was 74.7%. The phylogenetic analysis revealed MaTLRs showed close relationship to other species in Synbranchiformes or Perciformes. Quantitative real-time PCR analysis revealed that they were expressed in all tested tissues and higher expression was found in spleen or gill. After infection with Aeromonas veronii, expression of MaTLR7, MaTLR8 and MaTLR9 were all significantly downregulated in spleen and kidney. Evolutionary analysis suggested that the ancestral lineages of teleost TLR8 and TLR9 had been subject to positive selection pressures and multiple Maximum likelihood methods recovered 3 positively selected sites in teleost TLR7, 4 in TLR8 and 8 in TLR9. Domain distribution revealed most positively selected sites were located in leucine-rich repeat domain. Our results will contribute to better understanding the antibacterial mechanism of TLRs and their co-evolution with pathogens.


Assuntos
Evolução Molecular , Proteínas de Peixes/genética , Smegmamorpha/genética , Receptor 7 Toll-Like/genética , Aeromonas veronii/imunologia , Aeromonas veronii/patogenicidade , Sequência de Aminoácidos/genética , Animais , Coevolução Biológica/genética , Coevolução Biológica/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Filogenia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Seleção Genética , Smegmamorpha/imunologia , Smegmamorpha/microbiologia , Receptor 7 Toll-Like/imunologia
13.
J Exp Med ; 216(2): 384-406, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30674564

RESUMO

Glucocorticoids remain the most widely used immunosuppressive and anti-inflammatory drugs, yet substantial gaps exist in our understanding of glucocorticoid-mediated immunoregulation. To address this, we generated a pathway-level map of the transcriptional effects of glucocorticoids on nine primary human cell types. This analysis revealed that the response to glucocorticoids is highly cell type dependent, in terms of the individual genes and pathways affected, as well as the magnitude and direction of transcriptional regulation. Based on these data and given their importance in autoimmunity, we conducted functional studies with B cells. We found that glucocorticoids impair upstream B cell receptor and Toll-like receptor 7 signaling, reduce transcriptional output from the three immunoglobulin loci, and promote significant up-regulation of the genes encoding the immunomodulatory cytokine IL-10 and the terminal-differentiation factor BLIMP-1. These findings provide new mechanistic understanding of glucocorticoid action and emphasize the multifactorial, cell-specific effects of these drugs, with potential implications for designing more selective immunoregulatory therapies.


Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-10/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Glucocorticoides/imunologia , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/imunologia , Transcrição Genética/imunologia
14.
Viruses ; 11(1)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650519

RESUMO

Respiratory syncytial virus (RSV) is a major cause of respiratory infectious disease in infants and young children. Dendritic cells (DCs) and macrophages (MACs) are known to play important roles in RSV recognition, and in the production of type I interferons (IFNs) and pro-inflammatory cytokine in RSV infection. Toll-like receptor 7 (TLR7), myeloid differentiation primary response 88 (MyD88), and mitochondrial antiviral-signaling protein (MAVS) are known to be important for the RSV sensing pathway in DCs and MACs. However, despite the critical roles of type I IFNs in the anti-RSV immune response, the pattern recognition receptors (PRRs) that are required for RSV sensing in DCs and MACs remain unclear. Here, we investigate the pathway activated by RSV A2 strain infection using an IFN-ß/YFP reporter mouse model to visualize IFN-ß-producing cells and in vitro RSV infection in bone marrow-derived DCs (BM-DCs) and macrophages (BM-DMs). We present our finding that MyD88, but not TLR7, are important for RSV recognition and type I IFN and pro-inflammatory production in DCs and MACs. MAVS-deficient BM-DCs and BM-DMs show impaired induction of IFN-ß production upon RSV stimulation, and this effect is RSV replication-dependent. Our study provides information on cell type-specific PRR requirements in innate immune responses against RSV infection.


Assuntos
Células Dendríticas/imunologia , Imunidade Inata , Interferon beta/imunologia , Macrófagos/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Transdução de Sinais , Animais , Células Cultivadas , Células Dendríticas/virologia , Macrófagos/virologia , Glicoproteínas de Membrana/imunologia , Camundongos , Fator 88 de Diferenciação Mieloide/imunologia , Receptores de Reconhecimento de Padrão , Receptor 7 Toll-Like/imunologia , Replicação Viral
15.
Br J Dermatol ; 180(2): 297-305, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30171698

RESUMO

BACKGROUND: Topical immune response modifiers are established for actinic keratosis (AK) treatment and efforts are underway to make further improvements to their efficacy and safety. OBJECTIVES: To investigate the optimal dosing regimens of the Toll-like receptor 7/8 agonist resiquimod in terms of efficacy, safety and tolerability. METHODS: In a multicentre, partly placebo-controlled, double-blind clinical trial, we randomized 217 patients with AK lesions to 0·03% resiquimod gel once-daily application three times per week for 4 weeks or seven times within 2 weeks or five times for 1 week (arms 1/2/3) followed by a treatment-free interval of 8 weeks and one repetition of the cycle. In two additional arms (arms 4/5), patients applied either resiquimod gel 0·01% or 0·03% three times per week up to a biological end point defined by skin erosion or for a maximum duration of 8 weeks. Clearance was assessed clinically and histologically. RESULTS: Complete clinical clearance ranged from 56% to 85% with the highest rate observed in arm 2. Resiquimod 0·03% gel was more effective than 0·01% gel. Clearance rates in arms 1/2/3 were comparable and higher than with placebo and were reached with 24, 14 and 10 gel applications, respectively. Overall, 128 patients (59%) experienced treatment-related adverse reactions. CONCLUSIONS: Resiquimod 0·03% gel is more effective than 0·01% gel. From the perspectives of safety and tolerability, the lower concentration and shorter duration are preferable. The clinical response in arms 2/3 was reached with fewer gel applications. The dosing regimens that used the biological end point (arms 4/5) proved equally efficacious as predefined treatment durations and may therefore be suitable for personalized AK treatment.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Imidazóis/administração & dosagem , Ceratose Actínica/tratamento farmacológico , Adjuvantes Imunológicos/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Imidazóis/efeitos adversos , Ceratose Actínica/imunologia , Masculino , Pessoa de Meia-Idade , Placebos/administração & dosagem , Placebos/efeitos adversos , Fatores de Tempo , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/imunologia , Resultado do Tratamento
16.
J Immunol ; 202(3): 714-723, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578304

RESUMO

Optimal T cell activation requires Ag recognition through the TCR, engagement of costimulatory molecules, and cytokines. T cells can also directly recognize danger signals through the expression of TLRs. Whether TLR ligands have the capacity to provide costimulatory signals and enhance Ag-driven T cell activation is not well understood. In this study, we show that TLR2 and TLR7 ligands potently lower the Ag threshold for cytokine production in T cells. To investigate how TLR triggering supports cytokine production, we adapted the protocol for flow cytometry-based fluorescence in situ hybridization to mouse T cells. The simultaneous detection of cytokine mRNA and protein with single-cell resolution revealed that TLR triggering primarily drives de novo mRNA transcription. Ifng mRNA stabilization only occurs when the TCR is engaged. TLR2-, but not TLR7-mediated costimulation, can enhance mRNA stability at low Ag levels. Importantly, TLR2 costimulation increases the percentage of polyfunctional T cells, a hallmark of potent T cell responses. In conclusion, TLR-mediated costimulation effectively potentiates T cell effector function to suboptimal Ag levels.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária , Receptor 2 Toll-Like/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Interferon gama/metabolismo , Ligantes , Melanoma Experimental , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/imunologia , Organismos Livres de Patógenos Específicos , Receptor 2 Toll-Like/genética , Receptor 7 Toll-Like/imunologia
17.
Viral Immunol ; 32(1): 68-74, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30585774

RESUMO

Japanese encephalitis (JE) is a vector-borne viral disease with clinical manifestations ranging from asymptomatic to severe neurological symptoms and even leading to death. The exact pathophysiology for diverse clinical spectrum of the disease is complex and has not yet been defined. Studies have postulated that during JE infection, inflammatory cytokines and chemokines are produced after the initial recognition of viral antigens through the engagement of toll-like receptors (TLR) pathways. However, there is paucity of knowledge on the expression levels of chemokines and TLRs among mild and severely affected JE patients. Hence, to better understand disease pathogenesis, we examined the mRNA expression of chemokines, CCL2 and CCL5, and their respective receptors CCR2 and CCR5 along with TLRs viz. TLR3, TLR7, TLR8, and TLR9 in context of mild and severely Japanese encephalitis virus (JEV)-infected (n = 19) and healthy (n = 19) individuals. Our study showed significant downregulation of CCL2, CCL5, CCR2, CCR5, and TLR3 by log 0.87, 1.02, 0.82, 0.68, and 0.37-fold respectively, among mild cases compared with controls. Significant difference of gene expression among mild and severe JE cases for CCL2 (p < 0.001), CCL5 (p < 0.01), and TLR7 (p < 0.05) was observed. In conclusion, our results proposes that chemokines viz. CCL2 and CCL5 along with TLR7 may be associated with degree of pathogenesis of JE and could be putative therapeutic targets for preventing severe inflammation during viral encephalitis.


Assuntos
Quimiocinas/genética , Encefalite Japonesa/imunologia , Expressão Gênica , Receptores Toll-Like/genética , Adolescente , Adulto , Idoso , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocinas/imunologia , Criança , Regulação para Baixo , Encefalite Japonesa/patologia , Feminino , Humanos , Índia , Inflamação , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptores Toll-Like/imunologia , Adulto Jovem
18.
Front Immunol ; 9: 2677, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30515164

RESUMO

During aging the immune system is dysregulated. Especially plasmacytoid dendritic cells (pDCs) and myeloid DCs (mDCs) have reduced Toll like receptor (TLR)-mediated responses resulting in increased susceptibility to infections. Consumption of bovine lactoferrin (bLF) has been shown to reduce infections with viruses. Galacto-oligosacharides (GOS) and vitamin D are associated with reduced pro-inflammatory cytokine levels in serum, and increased TLR7/8 responses, respectively. A double-blind placebo-controlled nutritional intervention study in elderly women was performed, to investigate the potential of bLF, GOS, and vitamin D to restore TLR responsiveness of pDCs and mDCs and to reduce inflammatory markers in serum. The nutritional intervention group (n = 15) received bLF for 3 weeks, followed by 3 weeks of bLF + GOS, and subsequently 3 weeks of bLF + GOS + vitamin D. The placebo group (n = 15) received maltodextrin for 9 weeks. Every 3 weeks, blood was collected and TLR responses of pDCs and mDCs, and inflammation-related markers in serum were measured. After 3 weeks of bLF supplementation, increased TLR7/8 and TLR1/2 responses were observed in pDCs of the nutritional intervention group compared to the placebo group. When the effects of the entire nutritional intervention were investigated, increased TLR1/2 mediated responses in mDCs were observed, and in serum sVCAM tended to decrease. Finally, based on the RAND-36 questionnaire physical function tended to improve in the intervention group. Since especially TLR7-mediated responses in pDCs were enhanced after bLF supplementation compared to placebo, this suggests that bLF may contribute to antiviral responses mediated by pDC in elderly women.Clinical trial registry number: NCT03026244, clinicaltrials.gov.


Assuntos
Células Dendríticas/imunologia , Lactoferrina/administração & dosagem , Oligossacarídeos/administração & dosagem , Receptor 7 Toll-Like/imunologia , Vitamina D/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Animais , Bovinos , Feminino , Humanos
19.
Front Immunol ; 9: 2817, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30564235

RESUMO

Using an in vivo model of tolerance to TLR7-induced skin inflammation, we found a critical role for macrophage-derived MMP10 in mediating immune hypo-responsiveness. Cutaneous exposure to Imiquimod (IMQ), a TLR7 agonist, induced acute expression of pro-inflammatory factors (IL1ß, IL6, CXCL1) and neutrophil influx equally in both wildtype and Mmp10 -/- mice. However, whereas subsequent exposure (11 and 12 days later) to IMQ led to marked abrogation of pro-inflammatory factor expression in wildtype mice, Mmp10 -/- mice responded similarly as they did to the first application. In addition, the second exposure led to increased expression of negative regulators of TLR signaling (TNFAIP3, IRAK3) and immunosuppressive cytokines (IL10, TGFß1) in wildtype mice but not in Mmp10 -/- mice. In vitro studies demonstrated that prior exposure of IMQ to bone marrow-derived macrophages (BMDM) made wildtype cells refractory to subsequent stimulation but did not for Mmp10 -/- macrophages. These findings expand the critical roles MMP10 plays in controlling macrophage activation to indicate that the development of immune tolerance to TLR7 ligand is dependent on this macrophage-derived proteinase.


Assuntos
Tolerância Imunológica/imunologia , Macrófagos/imunologia , Metaloproteinase 10 da Matriz/imunologia , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Citocinas/imunologia , Feminino , Imiquimode/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Masculino , Glicoproteínas de Membrana/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/agonistas
20.
JCI Insight ; 3(23)2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30518690

RESUMO

The peptidylarginine deiminases PAD2 and PAD4 are implicated in the pathogenesis of several autoimmune diseases. PAD4 may be pathogenic in systemic lupus erythematosus (SLE) through its role in neutrophil extracellular trap (NET) formation that promotes autoantigen externalization, immune dysregulation, and organ damage. The role of this enzyme in mouse models of autoimmunity remains unclear, as pan-PAD chemical inhibitors improve clinical phenotype, whereas PAD4-KO models have given conflicting results. The role of PAD2 in SLE has not been investigated. The differential roles of PAD2 and PAD4 in TLR-7-dependent lupus autoimmunity were examined. Padi4-/- displayed decreased autoantibodies, type I IFN responses, immune cell activation, vascular dysfunction, and NET immunogenicity. Padi2-/- mice showed abrogation of Th subset polarization, with some disease manifestations reduced compared with WT but to a lesser extent than Padi4-/- mice. RNA sequencing analysis revealed distinct modulation of immune-related pathways in PAD-KO lymphoid organs. Human T cells express both PADs and, when exposed to either PAD2 or PAD4 inhibitors, displayed abrogation of Th1 polarization. These results suggest that targeting PAD2 and/or PAD4 activity modulates dysregulated TLR-7-dependent immune responses in lupus through differential effects of innate and adaptive immunity. Compounds that target PADs may have potential therapeutic roles in T cell-mediated diseases.


Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Desiminases de Arginina em Proteínas/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Receptor 7 Toll-Like/imunologia , Animais , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Armadilhas Extracelulares , Feminino , Regulação da Expressão Gênica , Histonas , Humanos , Hidrolases/genética , Hidrolases/imunologia , Hidrolases/metabolismo , Inflamação , Interferon Tipo I , Camundongos , Camundongos Knockout , Desiminases de Arginina em Proteínas/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Th1 , Células Th17 , Transcriptoma
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