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1.
Aging (Albany NY) ; 13(18): 21838-21854, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531331

RESUMO

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse ß-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.


Assuntos
COVID-19/virologia , Senescência Celular , SARS-CoV-2/patogenicidade , Receptor 3 Toll-Like/metabolismo , Envelhecimento , Animais , Apoptose , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Inflamação , Pulmão/metabolismo , Camundongos , Fenótipo , Proteínas Virais
2.
Biomed Khim ; 67(4): 331-337, 2021 Jul.
Artigo em Russo | MEDLINE | ID: mdl-34414891

RESUMO

The pathogenetic mechanisms associated with alcohol use include dysregulation of the innate immune system mechanisms in the brain. TLR3 expression is increased in the postmortem material of the prefrontal cortex of humans. An increase in the TLR3 signaling activity leads to the induction of interferons (IFN). IFN are associated with depressive symptoms and, therefore, may play a role in the pathogenesis of alcoholism; however, the exact mechanisms of intracellular signaling mediated by the influence of ethanol are not fully elucidated and their study was the purpose of this work. The experimental results showed that ethanol and the TLR3 agonist Poly (I:C) increased the content of TLR3, IFNß, and IFNγ mRNA in the prefrontal cortex. In addition, expression of the TRAIL encoding gene also increased, and this increase positively correlaed with the mRNA content of TLR3, IFNß and IFNγ both under alcoholization conditions and after injections of the TLR3 agonist. The data obtained may indicate that alcoholization is able to activate TLR3-TRAIL-IFN-signaling in the prefrontal cortex of the brain.


Assuntos
Interferons , Receptor 3 Toll-Like , Animais , Encéfalo/metabolismo , Poli I-C/farmacologia , Ratos , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo
3.
J Immunol ; 207(5): 1298-1309, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34362833

RESUMO

Intralesional therapy is a promising approach for remodeling the immunosuppressive tumor microenvironment while minimizing systemic toxicities. A combinatorial in situ immunomodulation (ISIM) regimen with intratumoral administration of Fms-like tyrosine kinase 3 ligand (Flt3L), local irradiation, and TLR3/CD40 stimulation induces and activates conventional type 1 dendritic cells in the tumor microenvironment and elicits de novo adaptive T cell immunity in poorly T cell-inflamed tumors. However, the impact of ISIM on myeloid-derived suppressor cells (MDSCs), which may promote treatment resistance, remains unknown. In this study, we examined changes in the frequencies and heterogeneity of CD11b+Ly-6CloLy-6G+ polymorphonuclear (PMN)-MDSCs and CD11b+Ly-6ChiLy-6G- monocytic (M)-MDSCs in ISIM-treated tumors using mouse models of triple-negative breast cancer. We found that ISIM treatment decreased intratumoral PMN-MDSCs, but not M-MDSCs. Although the frequency of M-MDSCs remained unchanged, ISIM caused a substantial reduction of CX3CR1+ M-MDSCs that express F4/80. Importantly, these ISIM-induced changes in tumor-residing MDSCs were not observed in Batf3-/- mice. ISIM upregulated PD-L1 expression in both M-MDSCs and PMN-MDSCs and synergized with anti-PD-L1 therapy. Furthermore, ISIM increased the expression of IFN regulatory factor 8 (IRF8) in myeloid cells, a known negative regulator of MDSCs, indicating a potential mechanism by which ISIM decreases PMN-MDSC levels. Accordingly, ISIM-mediated reduction of PMN-MDSCs was not observed in mice with conditional deletion of IRF8 in myeloid cells. Altogether, these findings suggest that ISIM holds promise as a multimodal intralesional therapy to alter both lymphoid and myeloid compartments of highly aggressive poorly T cell-inflamed, myeloid-enriched tumors resistant to anti-PD-L1 therapy.


Assuntos
Células Dendríticas/imunologia , Imunoterapia/métodos , Fatores Reguladores de Interferon/metabolismo , Neoplasias Mamárias Animais/terapia , Proteínas de Membrana/uso terapêutico , Células Supressoras Mieloides/imunologia , Linfócitos T/imunologia , Animais , Antígeno B7-H1 , Fatores de Transcrição de Zíper de Leucina Básica/genética , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Resistência a Medicamentos , Regulação da Expressão Gênica , Humanos , Injeções Intralesionais , Fatores Reguladores de Interferon/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Radioterapia , Proteínas Repressoras/genética , Receptor 3 Toll-Like/metabolismo , Microambiente Tumoral
4.
Viruses ; 13(6)2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207750

RESUMO

An inefficient immune response against the hepatitis C virus (HCV), combined with viral evasion mechanisms, is responsible for the chronicity of infection. The need to evaluate the innate mechanisms of the immune response, such as TLR3 and IFN-λ3, and their relationship with the virus-host interaction is important for understanding the pathogenesis of chronic hepatitis C. The present study aimed to investigate the gene expressions of TRL3 and IFNL3 in liver tissue, seeking to evaluate whether these could be potential biomarkers of HCV infection. A total of 23 liver biopsy samples were collected from patients with chronic HCV, and 8 biopsies were collected from healthy control patients. RNA extraction, reverse transcription and qPCR were performed to quantify the relative gene expressions of TLR3 and IFNL3. Data on the viral load; AST, ALT, GGT and AFP levels; and the viral genotype were collected from the patients' medical records. The intrahepatic expression of TLR3 (p = 0.0326) was higher in chronic HCV carriers than in the control group, and the expression of IFNL3 (p = 0.0037) was lower in chronic HCV carriers than in the healthy control group. The expression levels of TLR3 (p = 0.0030) and IFNL3 (p = 0.0036) were higher in the early stages of fibrosis and of necroinflammatory activity in the liver; in contrast, TLR3 and IFNL3 expressions were lower in the more advanced stages of fibrosis and inflammation. There was no correlation between the gene expression and the serum viral load. Regarding the initial METAVIR scale scores, liver transaminase levels were lower in patients with advanced fibrosis when correlated with TLR3 and IFNL3 gene expressions. The results suggest that in the early stages of the development of hepatic fibrosis, TLR3 and IFN-λ3 play important roles in the antiviral response and in the modulation of the tolerogenic liver environment because there is a decrease in the intrahepatic expressions of TLR3 and IFNL3 in the advanced stages of fibrosis, probably due to viral evasion mechanisms.


Assuntos
Hepacivirus , Hepatite C Crônica/complicações , Hepatite C Crônica/genética , Interferons/genética , Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Receptor 3 Toll-Like/genética , Biomarcadores , Biópsia , Estudos Transversais , Suscetibilidade a Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Genoma Viral , Genótipo , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Interferons/metabolismo , Masculino , Índice de Gravidade de Doença , Receptor 3 Toll-Like/metabolismo , Carga Viral
5.
Eur J Med Chem ; 224: 113684, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34256126

RESUMO

Respiratory syncytial virus (RSV) causes serious lower respiratory tract infections. Currently, the only clinical anti-RSV drug is ribavirin, but ribavirin has serious toxic side effect and can only be used by critically ill patients. A series of benzimidazole derivatives were synthesized starting from 1,4:3,6-dianhydro-d-fructose and a variety of o-phenylenediamines. Evaluation of their antiviral activity showed that compound a27 had the highest antiviral activity with a half maximal effective concentration (EC50) of 9.49 µM. Investigation of the antiviral mechanism of compound a27 indicated that it can inhibit the replication of RSV by inhibiting apoptosis and autophagy pathways. Retinoic acid-inducible gene (RIG)-I, TNF receptor associated factor (TRAF)-3, TANK binding kinase (TBK)-1, interferon regulatory factor (IRF)-3, nuclear factor Kappa-B (NF-κB), interferon (IFN)-ß, Toll-like receptor (TLR)-3, interleukin (IL)-6 were suppressed at the cellular level. Mouse lung tissue was subjected to hematoxylin and eosin (HE) staining and immunohistochemistry, which showed that RSV antigen and M gene expression could be reduced by compound a27. Decreased expression of RIG-I, IRF-3, IFN-ß, TLR-3, IL-6, interleukin (IL)-8, interleukin (IL)-10, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α was also found in vivo.


Assuntos
Antivirais/síntese química , Benzimidazóis/química , Desenho de Fármacos , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Apoptose/efeitos dos fármacos , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular , Citocinas/metabolismo , Humanos , Isomerismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Conformação Molecular , Espécies Reativas de Oxigênio/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/fisiologia , Relação Estrutura-Atividade , Receptor 3 Toll-Like/metabolismo , Replicação Viral/efeitos dos fármacos
6.
PLoS Pathog ; 17(7): e1009781, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280250

RESUMO

Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO's in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.


Assuntos
Doença dos Legionários/imunologia , Macrófagos/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteínas de Bactérias/imunologia , Humanos , Legionella pneumophila/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Padrões Moleculares Associados a Patógenos/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
7.
PLoS Negl Trop Dis ; 15(7): e0009638, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34310619

RESUMO

BACKGROUND: The leishmaniases are a group of sandfly-transmitted diseases caused by species of the protozoan parasite, Leishmania. With an annual incidence of 1 million cases, 1 billion people living in Leishmania-endemic regions, and nearly 30,000 deaths each year, leishmaniasis is a major global public health concern. While phlebotomine sandflies are well-known as vectors of Leishmania, they are also the vectors of various phleboviruses, including Sandfly Fever Sicilian Virus (SFSV). Cutaneous leishmaniasis (CL), caused by Leishmania major (L. major), among other species, results in development of skin lesions on the infected host. Importantly, there exists much variation in the clinical manifestation between individuals. We propose that phleboviruses, vectored by and found in the same sandfly guts as Leishmania, may be a factor in determining CL severity. It was reported by our group that Leishmania exosomes are released into the gut of the sandfly vector and co-inoculated during blood meals, where they exacerbate CL skin lesions. We hypothesized that, when taking a blood meal, the sandfly vector infects the host with Leishmania parasites and exosomes as well as phleboviruses, and that this viral co-infection results in a modulation of leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, we observed modulation by SFSV in MAP kinase signaling as well as in the IRF3 pathway that resulted in a pro-inflammatory phenotype. Additionally, we found that SFSV and L. major co-infection resulted in an exacerbation of leishmaniasis in vivo, and by using endosomal (Toll-like receptor) TLR3, and MAVS knock-out mice, deduced that SFSV's hyperinflammatory effect was TLR3- and MAVS-dependent. Critically, we observed that L. major and SFSV co-infected C57BL/6 mice demonstrated significantly higher parasite burden than mice solely infected with L. major. Furthermore, viral presence increased leukocyte influx in vivo. This influx was accompanied by elevated total extracellular vesicle numbers. Interestingly, L. major displayed higher infectiveness with coincident phleboviral infection compared to L. major infection alone. CONCLUSION/SIGNIFICANCE: Overall our work represents novel findings that contribute towards understanding the causal mechanisms governing cutaneous leishmaniasis pathology. Better comprehension of the potential role of viral co-infection could lead to treatment regimens with enhanced effectiveness.


Assuntos
Infecções por Bunyaviridae/complicações , Leishmaniose Cutânea/complicações , Macrófagos/metabolismo , Células Mieloides/metabolismo , Phlebovirus , Animais , Linhagem Celular , Coinfecção , Feminino , Imunidade Inata , Inflamação , Fator Regulador 3 de Interferon , Leishmania major , Sistema de Sinalização das MAP Quinases , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/parasitologia , Células Mieloides/virologia , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo
8.
Front Immunol ; 12: 669812, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220816

RESUMO

Crimean-Congo hemorrhagic fever (CCHF) is a highly severe and virulent viral disease of zoonotic origin, caused by a tick-born CCHF virus (CCHFV). The virus is endemic in many countries and has a mortality rate between 10% and 40%. As there is no licensed vaccine or therapeutic options available to treat CCHF, the present study was designed to focus on application of modern computational approaches to propose a multi-epitope vaccine (MEV) expressing antigenic determinants prioritized from the CCHFV genome. Integrated computational analyses revealed the presence of 9 immunodominant epitopes from Nucleoprotein (N), RNA dependent RNA polymerase (RdRp), Glycoprotein N (Gn/G2), and Glycoprotein C (Gc/G1). Together these epitopes were observed to cover 99.74% of the world populations. The epitopes demonstrated excellent binding affinity for the B- and T-cell reference set of alleles, the high antigenic potential, non-allergenic nature, excellent solubility, zero percent toxicity and interferon-gamma induction potential. The epitopes were engineered into an MEV through suitable linkers and adjuvating with an appropriate adjuvant molecule. The recombinant vaccine sequence revealed all favorable physicochemical properties allowing the ease of experimental analysis in vivo and in vitro. The vaccine 3D structure was established ab initio. Furthermore, the vaccine displayed excellent binding affinity for critical innate immune receptors: TLR2 (-14.33 kcal/mol) and TLR3 (-6.95 kcal/mol). Vaccine binding with these receptors was dynamically analyzed in terms of complex stability and interaction energetics. Finally, we speculate the vaccine sequence reported here has excellent potential to evoke protective and specific immune responses subject to evaluation of downstream experimental analysis.


Assuntos
Antígenos Virais/farmacologia , Biologia Computacional , Desenho Assistido por Computador , Desenvolvimento de Medicamentos , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Febre Hemorrágica da Crimeia/prevenção & controle , Epitopos Imunodominantes , Carrapatos/virologia , Vacinologia , Vacinas Virais/farmacologia , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/imunologia , Febre Hemorrágica da Crimeia/virologia , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/metabolismo , Vacinas de DNA/farmacologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinas Virais/metabolismo
9.
Front Immunol ; 12: 686060, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34211474

RESUMO

Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3' ends of mouse Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.


Assuntos
Epitopos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/fisiologia , Neurônios/metabolismo , Receptor 3 Toll-Like/fisiologia , Receptor 7 Toll-Like/fisiologia , Animais , Quimiocinas/metabolismo , Citocinas/metabolismo , Epitopos/imunologia , Feminino , Perfilação da Expressão Gênica , Imunidade Inata , Macrófagos/imunologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/fisiologia , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo
10.
J Biol Chem ; 297(2): 100925, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34214498

RESUMO

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.


Assuntos
Famotidina/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Receptor 3 Toll-Like/metabolismo , Células A549 , Sítios de Ligação , Células CACO-2 , Quimiocina CCL2/metabolismo , Proteases 3C de Coronavírus/metabolismo , Células HeLa , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interleucina-6/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Ligação Proteica , SARS-CoV-2/fisiologia , Transdução de Sinais , Receptor 3 Toll-Like/química , Replicação Viral
11.
Mol Immunol ; 136: 110-117, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34098343

RESUMO

Reticuloendothelial virus (REV) is widely found in many domestic poultry areas and results in severe immunosuppression of infected chickens. This increases the susceptibility to other pathogens, which causes economic losses to the poultry industry. The aim of our study was to determine whether polyinosinic-polycytidylic acid [Poly (I: C)] treatment could inhibit REV replication in chicken macrophage-like cell line, HD11. We found that Poly (I: C) treatment could markedly inhibit REV replication in HD11 from 24 to 48 h post infection (hpi). Additionally, Poly (I: C) treatment could switch HD11 from an inactive type into M1-like polarization from 24 to 48 hpi. Furthermore, Poly (I: C) treatment promoted interferon-ß secretion from HD11 post REV infection. Moreover, Toll-like receptor-3 (TLR-3) mRNA and protein levels in HD11 treated with Poly (I: C) were markedly increased compared to those of HD11 not treated with Poly (I: C). The above results suggested that Poly (I: C) treatment switches HD11 into M1-like polarization to secret more interferon-ß and activate TLR-3 signaling, which contributes to block REV replication. Our findings provide a theoretical reference for further studying the underlying pathogenic mechanism of REV and Poly (I: C) as a potential therapeutic intervention against REV infection.


Assuntos
Antivirais/farmacologia , Indutores de Interferon/farmacologia , Poli I-C/farmacologia , Vírus da Reticuloendoteliose Aviária/crescimento & desenvolvimento , Receptor 3 Toll-Like/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Galinhas , Interferon beta/biossíntese , Interferon beta/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Vírus da Reticuloendoteliose Aviária/efeitos dos fármacos , Infecções por Retroviridae/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Infecções Tumorais por Vírus/tratamento farmacológico
12.
J Microbiol Biotechnol ; 31(7): 942-948, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34099596

RESUMO

Canine influenza virus (CIV) induces acute respiratory disease in dogs. In this study, we aimed to determine the signaling pathways leading to the induction of IFN-ß in a canine respiratory epithelial cell line (KU-CBE) infected with the H3N2 subtype of CIV. Small interfering RNAs (siRNAs) specific to pattern recognition receptors (PRRs) and transcription factors were used to block the IFN-ß induction signals in H3N2 CIV-infected KU-CBE cells. Among the PRRs, only the TLR3 and RIG-I expression levels significantly (p < 0.001) increased in CIV-infected cells. Following transfection with siRNA specific to TLR3 (siTLR3) or RIG-I (siRIG-I), the mRNA expression levels of IFN-ß significantly (p < 0.001) decreased, and the protein expression of IFN-ß also decreased in infected cells. In addition, co-transfection with both siTLR3 and siRIG-I significantly reduced IRF3 (p < 0.001) and IFN-ß (p < 0.001) mRNA levels. Moreover, the protein concentration of IFN-ß was significantly (p < 0.01) lower in cells co-transfected with both siTLR3 and siRIG-I than in cells transfected with either siTLR3 or siRIGI alone. Also, the antiviral protein MX1 was only expressed in KU-CBE cells infected with CIV or treated with IFN-ß or IFN-α. Thus, we speculate that IFN-ß further induces MX1 expression, which might suppress CIV replication. Taken together, these data indicate that TLR3 and RIG-I synergistically induce IFN-ß expression via the activation of IRF3, and the produced IFN-ß further induces the production of MX1, which would suppress CIV replication in CIV-infected cells.


Assuntos
Proteína DEAD-box 58/metabolismo , Células Epiteliais/virologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Interferon beta/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58/genética , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Interferon beta/farmacologia , Proteínas de Resistência a Myxovirus/metabolismo , RNA Interferente Pequeno/farmacologia , Mucosa Respiratória/citologia , Transdução de Sinais , Receptor 3 Toll-Like/genética , Regulação para Cima/genética , Replicação Viral/efeitos dos fármacos
13.
Am J Hum Genet ; 108(6): 1012-1025, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34015270

RESUMO

The human genetic dissection of clinical phenotypes is complicated by genetic heterogeneity. Gene burden approaches that detect genetic signals in case-control studies are underpowered in genetically heterogeneous cohorts. We therefore developed a genome-wide computational method, network-based heterogeneity clustering (NHC), to detect physiological homogeneity in the midst of genetic heterogeneity. Simulation studies showed our method to be capable of systematically converging genes in biological proximity on the background biological interaction network, and capturing gene clusters harboring presumably deleterious variants, in an efficient and unbiased manner. We applied NHC to whole-exome sequencing data from a cohort of 122 individuals with herpes simplex encephalitis (HSE), including 13 individuals with previously published monogenic inborn errors of TLR3-dependent IFN-α/ß immunity. The top gene cluster identified by our approach successfully detected and prioritized all causal variants of five TLR3 pathway genes in the 13 previously reported individuals. This approach also suggested candidate variants of three reported genes and four candidate genes from the same pathway in another ten previously unstudied individuals. TLR3 responsiveness was impaired in dermal fibroblasts from four of the five individuals tested, suggesting that the variants detected were causal for HSE. NHC is, therefore, an effective and unbiased approach for unraveling genetic heterogeneity by detecting physiological homogeneity.


Assuntos
Biologia Computacional/métodos , Encefalite por Herpes Simples/genética , Encefalite por Herpes Simples/patologia , Fibroblastos/imunologia , Redes Reguladoras de Genes , Heterogeneidade Genética , Predisposição Genética para Doença , Estudos de Casos e Controles , Encefalite por Herpes Simples/imunologia , Fibroblastos/metabolismo , Humanos , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 3 Toll-Like/metabolismo , Sequenciamento Completo do Exoma
14.
J Nutr Biochem ; 95: 108761, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33965533

RESUMO

Inflammation in insulin-sensitive tissues (e.g., liver, visceral adipose tissue [VAT]) plays a major role in obesity and insulin resistance. Recruitment of innate immune cells drives the dysregulation of glucose and lipid metabolism. We aimed to seek the role of Toll like receptor 3 (TLR3), a pattern recognition receptor involved in innate immunity, obesity and the metabolic disorder. TLR3 expression in liver and VAT from diet induced obese mice and in VAT from overweight women was examined. Body weight, glucose homeostasis and insulin sensitivity were evaluated in TLR3 wild-type and knockout (KO) mice on a chow diet (CD) or high-fat diet for 15 weeks. At euthanasia, blood was collected, and plasma biochemical parameters and adipokines were determined with commercial kits. Flow cytometry was used to measure macrophage infiltration and activation in VAT. Standard western blot, immunohistochemistry and quantative PCR were used to assess molecules in pathways about lipid and glucose metabolism, insulin and inflammation in tissues of liver and VAT. Utilizing human and animal samples, we found that expression of TLR3 was upregulated in the liver and VAT in obese mice as well as VAT in overweight women. TLR3-deficiency protected against high-fat diet induced obesity, glucose intolerance, insulin resistance and lipid accumulation. Lipolysis was enhanced in VAT and hepatic lipogenesis was inhibited in TLR3 KO animals. Macrophages infiltration into adipose tissue was attenuated in TLR3 KO mice, accompanied with inhibition of NF-κB-dependent AMPK/Akt signaling pathway. These findings demonstrated that TLR3 ablation prevented obesity and metabolic disorders, thereby providing new mechanistic links between inflammation and obesity and associated metabolic abnormalities in lipid/glucose metabolism.


Assuntos
Gordura Intra-Abdominal/metabolismo , Receptor 3 Toll-Like/metabolismo , Adulto , Animais , Dieta Hiperlipídica , Feminino , Regulação da Expressão Gênica/fisiologia , Intolerância à Glucose , Transtornos do Metabolismo de Glucose , Humanos , Metabolismo dos Lipídeos , Fígado/metabolismo , Macrófagos/fisiologia , Masculino , Síndrome Metabólica , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Obesidade , Receptor 3 Toll-Like/genética
15.
Sci Rep ; 11(1): 10609, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011952

RESUMO

In cancer cells only, TLR3 acquires death receptor properties by efficiently triggering the extrinsic pathway of apoptosis with Caspase-8 as apical protease. Here, we demonstrate that in the absence of Caspase-8, activation of TLR3 can trigger a form of programmed cell death, which is distinct from classical apoptosis. When TLR3 was activated in the Caspase-8 negative neuroblastoma cell line SH-SY5Y, cell death was accompanied by lysosomal permeabilization. Despite caspases being activated, lysosomal permeabilization as well as cell death were not affected by blocking caspase-activity, positioning lysosomal membrane permeabilization (LMP) upstream of caspase activation. Taken together, our data suggest that LMP with its deadly consequences represents a "default" death mechanism in cancer cells, when Caspase-8 is absent and apoptosis cannot be induced.


Assuntos
Apoptose , Caspase 8/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Receptor 3 Toll-Like/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Interferon Tipo I/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Necroptose/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Poli I-C/farmacologia
16.
Nat Commun ; 12(1): 2935, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006895

RESUMO

With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic-co-glycolic acid) (PLGA) particles containing antigens and toll-like receptor ligands induces vigorous antitumor immune responses in vivo. Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8+ T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy.


Assuntos
Vacinas Anticâncer/imunologia , Proteína DEAD-box 58/imunologia , Inibidores de Checkpoint Imunológico/imunologia , Imunoterapia/métodos , Neoplasias Experimentais/terapia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/imunologia , Receptores Imunológicos/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Sinergismo Farmacológico , Feminino , Humanos , Inibidores de Checkpoint Imunológico/administração & dosagem , Ligantes , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Nanopartículas/química , Nanopartículas/ultraestrutura , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Receptores Imunológicos/metabolismo , Células THP-1 , Receptor 3 Toll-Like/metabolismo , Resultado do Tratamento
17.
Front Immunol ; 12: 677905, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025676

RESUMO

Most blood transfusion-related adverse reactions involve the immunologic responses of recipients to exogenous blood components. Extracellular vesicles isolated from packed red cells can affect the recipient's immune system. Mast cells are traditionally known as effector cells for allergic transfusion reactions. However, growing evidence supports the notion that activated mast cells might disturb host innate immunologic responses. Exosomes are a type of extracellular vesicle. To determine the effect of exosomes on mast cells, we enriched exosomes derived from volunteer plasma (EXs-nor) and packed red cells (EXs-RBCs) using ultracentrifugation and incubated them with a human mast cell line (HMC-1). We found that EXs-RBC exposure increased the expression of tryptase-1 and prostaglandin D2, the production of multiple inflammatory mediators, and the levels of Toll-like receptor-3 (TLR-3) and phospho-mitogen-activated protein kinase (MAPK) in HMC-1 cells. MAPK inhibitors (SB203580, PD98059, and SP600125) and a TLR-3/dsRNA complex inhibitor reduced the EXs-RBC-stimulated production of inflammatory mediators in HMC-1 cells, whereas the TLR-3 agonist [poly (A:U)] elevated the production of these mediators. These results indicate that EXs-RBCs activate HMC-1 cells and elicit the production of multiple inflammatory mediators, partly via the TLR-3 and MAPK pathways. Mast cells activated by EXs-RBCs exhibit complex inflammatory properties and might play a potential role in transfusion-related adverse reactions.


Assuntos
Eritrócitos/imunologia , Exossomos/imunologia , Imunidade Inata , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Mastócitos/imunologia , Adolescente , Adulto , Antracenos/farmacologia , Doadores de Sangue , Transfusão de Sangue/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Piridinas/farmacologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 3 Toll-Like/metabolismo , Adulto Jovem
18.
Front Immunol ; 12: 667454, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33986756

RESUMO

Toll-like receptor 3 (TLR3) is an important member of the TLR family, which is an important group of pathogen-associated molecular patterns. TLR3 can recognize double-stranded RNA and induce activation of NF-κB and the production of type I interferons. In addition to its immune-associated role, TLR3 has also been detected in some tumors. However TLR3 can play protumor or antitumor roles in different tumors or cell lines. Here, we review the basic signaling associated with TLR3 and the pro- or antitumor roles of TLR3 in different types of tumors and discuss the possible reasons for the opposing roles of TLR3 in tumors.


Assuntos
Neoplasias/metabolismo , RNA de Cadeia Dupla/farmacologia , Receptor 3 Toll-Like/metabolismo , Linhagem Celular Tumoral , Humanos , Interferon Tipo I/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 3 Toll-Like/agonistas
19.
Sci Rep ; 11(1): 10447, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001933

RESUMO

Microglia, CNS resident innate immune cells, respond strongly to activation of TLR3 and TLR4, which recognize viral dsRNA poly(I:C) and bacterial endotoxin LPS, respectively. However, few studies have thoroughly and parallelly compared functional phenotypes and downstream mechanisms between LPS- and poly(I:C)-exposed primary microglia. Here, we investigated the responses of mouse primary microglia upon LPS and poly(I:C) stimulation by detecting various phenotypes ranging from morphology, proliferation, secretion, chemotaxis, to phagocytosis. Furthermore, we explored their sequential gene expression and the downstream signal cascades. Interestingly, we found that the microglial activation pattern induced by LPS was distinguished from that induced by poly(I:C). Regarding microglial morphology, LPS caused an ameboid-like shape while poly(I:C) induced a bushy shape. Microglial proliferation was also facilitated by LPS but not by poly(I:C). In addition, LPS and poly(I:C) modulated microglial chemotaxis and phagocytosis differently. Furthermore, genome-wide analysis provided gene-level support to these functional differences, which may be associated with NF-κb and type I interferon pathways. Last, LPS- and poly(I:C)-activated microglia mediated neurotoxicity in a co-culture system. This study extends our understanding of TLR roles in microglia and provides insights into selecting proper inflammatory microglial models, which may facilitate identification of new targets for therapeutic application.


Assuntos
Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Poli I-C/farmacologia , Animais , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Técnicas de Cocultura , Feminino , Interferon Tipo I/metabolismo , Camundongos , Microglia/imunologia , NF-kappa B/metabolismo , Neurônios , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo
20.
J Biol Chem ; 297(1): 100828, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34048712

RESUMO

Cathelicidins such as the human 37-amino acid peptide (LL-37) are peptides that not only potently kill microbes but also trigger inflammation by enabling immune recognition of endogenous nucleic acids. Here, a detailed structure-function analysis of LL-37 was performed to understand the details of this process. Alanine scanning of 34-amino acid peptide (LL-34) showed that some variants displayed increased antimicrobial activity against Staphylococcus aureus and group A Streptococcus. In contrast, different substitutions clustered on the hydrophobic face of the LL-34 alpha helix inhibited the ability of those variants to promote type 1 interferon expression in response to U1 RNA or to present U1 to the scavenger receptor (SR) B1 on the keratinocyte cell surface. Small-angle X-ray scattering experiments of the LL-34 variants LL-34, F5A, I24A, and L31A demonstrated that these peptides form cognate supramolecular structures with U1 characterized by inter-dsRNA spacings of approximately 3.5 nm, a range that has been previously shown to activate toll-like receptor 3 by the parent peptide LL-37. Therefore, while alanine substitutions on the hydrophobic face of LL-34 led to loss of binding to SRs and the complete loss of autoinflammatory responses in epithelial and endothelial cells, they did not inhibit the ability to organize with U1 RNA in solution to associate with toll-like receptor 3. These observations advance our understanding of how cathelicidin mediates the process of innate immune self-recognition to enable inert nucleic acids to trigger inflammation. We introduce the term "innate immune vetting" to describe the capacity of peptides such as LL-37 to enable certain nucleic acids to become an inflammatory stimulus through SR binding prior to cell internalization.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Inflamação/patologia , RNA de Cadeia Dupla/metabolismo , Receptores Depuradores/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Fenômenos Biofísicos , Linhagem Celular , Membrana Celular/metabolismo , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/genética , Interferon Tipo I/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Ligação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Receptor 3 Toll-Like/metabolismo , Transcrição Genética
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