RESUMEN
Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)stimulated gene (ISG)60 in noncancerous bronchial epithelial BEAS2B cells exposed to a Tolllike receptor 3 agonist. BEAS2B cells were treated with a synthetic TLR3 ligand, polyinosinicpolycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcriptionquantitative PCR and western blotting, respectively. The levels of CXC motif chemokine ligand 10 (CXCL10) were examined using an enzymelinked immunosorbent assay, and the effects of knockdown of IFNß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFNß also induced ISG60 expression. By contrast, knockdown of IFNß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.
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Bronquios , Quimiocina CXCL10 , Células Epiteliales , Poli I-C , Transducción de Señal , Receptor Toll-Like 3 , Humanos , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Bronquios/citología , Bronquios/metabolismo , Línea Celular , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Innata , Interferón beta/metabolismo , Interferón beta/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Poli I-C/farmacología , Proteínas de Unión al ARN , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genéticaRESUMEN
BACKGROUND: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. METHODS AND RESULTS: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation in vitro (P<0.01). CONCLUSIONS: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.
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Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila , Receptor Toll-Like 3 , Trombosis de la Vena , Animales , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , Trombosis de la Vena/metabolismo , Trombosis de la Vena/genética , Trombosis de la Vena/patología , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Transducción de Señal , Células HEK293 , Lesiones del Sistema Vascular/metabolismo , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patología , Neutrófilos/metabolismo , ARN/genética , Masculino , Ratones , Poli I-C/farmacología , Coagulación SanguíneaAsunto(s)
Inflamación , Receptor Toll-Like 3 , Tromboembolia Venosa , Humanos , Tromboembolia Venosa/genética , Tromboembolia Venosa/inmunología , Tromboembolia Venosa/sangre , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Inflamación/metabolismo , Inflamación/genética , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , AnimalesRESUMEN
AIM: To determine variations in allele and genotype frequencies between keratoacanthoma (KA) and common warts (CW), compared with the control group, in three single nucleotide polymorphisms (SNPs) within the TLR2, TLR3, and TLR9 genes. METHODS: This case-control study involved samples from 161 patients with KA, 152 patients with CW, and 469 controls. DNA was isolated from formalin-fixed paraffin-embedded tissue sections. Three SNPs - rs4696480 in TLR2, rs7657186 in TLR9, and rs35213 in TLR3 - were genotyped with TaqMan Genotyping Assays on the 7500 Real-Time PCR System. RESULTS: TLR2 rs4696480 and TLR3 rs7657186 were significantly overrepresented in KA and CW compared with controls (P<0.001). The association was stronger for CW than for KA, as evidenced by higher frequencies of the A allele and AA genotype for rs4696480. Both KA and CW patients had higher frequencies of the G allele and GG genotype for rs7657186 than controls. rs7657186 was moderately associated with KA and CW, with the G allele and GG genotype being more prevalent in CW cases, where no AA homozygotes were found. CONCLUSION: Genetic variants in TLR2 (rs4696480) and TLR3 (rs7657186) genes may affect KA and CW development, influencing immune responses and susceptibility to these skin lesions. Further research is required to elucidate TLR expression patterns and their role in KA development.
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Queratoacantoma , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 2 , Receptor Toll-Like 3 , Verrugas , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Alelos , Estudios de Casos y Controles , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Queratoacantoma/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 3/genética , Verrugas/genéticaRESUMEN
BACKGROUND: A distinct phenotype in Coronavirus disease 2019 (Covid-19) was observed in severe patients, consisting of a highly impaired interferon (IFN) type I response, an exacerbated inflammatory response. OBJECTIVE: The aim of the present study was to investigate a possible association of single nucleotide polymorphisms (SNPs), in five genes related to the immune response, rs3775291 in TLR3; rs2292151 in TICAM1; rs1758566 in IFNA1; rs1800629 in TNF, and rs1800795 in IL6 with the severity of Covid-19. METHODS: A cross-sectional study was performed, with non-severe and severe/critical patients diagnosed with Covid-19, by two public hospitals in Brazil. In total, 300 patients were genotyped for the SNPs, 150 with the non-severe form of the disease and 150 with severe/critical form. RESULTS: The T/T genotype of TLR3 in recessive model shows 58% of protection against severe/critical Covid-19; as well as the genotypes G/A+A/A of TICAM1 in dominant model with 60% of protection, and in a codominant model G/A with 57% and A/A with 71% of protection against severe/critical Covid-19. Comparing severe and critical cases, the T/C genotype of IFNA1 in the codominant model and TC+C/C in the dominant model showed twice the risk of critical Covid-19. CONCLUSION: We can conclude that rs3775291, rs2292151 and rs1758566 can influence the COVID-19 severity.
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COVID-19 , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Receptor Toll-Like 3 , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Brasil/epidemiología , COVID-19/genética , COVID-19/virología , Estudios Transversales , Genotipo , Interferón Tipo I/genética , Interferón-alfa , SARS-CoV-2/genética , Receptor Toll-Like 3/genéticaRESUMEN
Viral infections in tubular epithelial cells lead to the production of inflammatory cytokines by innate immunity, causing tubulointerstitial nephritis. TLR3 recognizes viral infections and acts via the activation of interferon (IFN)/IFN-stimulated genes (ISGs). This study investigates the role of ISG56, a representative ISG, in TLR3 signaling in cultured human renal proximal tubular epithelial cells (hRPTECs). To this end, hRPTECs were stimulated by a synthetic TLR3 ligand, polyinosinic-polycytidylic acid (poly IC), recombinant human interferon-ß [r(h)IFN-ß] or Japanese encephalitis virus (JEV) infection and assayed for inflammatory cytokine mRNA expression by RT-qPCR, and protein expression via western blotting or ELISA. ISG56 was expressed by poly IC or r(h)IFN-ß and IFN-ß knockdown reduced poly IC-induced expression of ISG56 and CXCL10. Moreover, ISG56 knockdown reduced poly IC- or r(h)IFN-ß-induced expression of CXCL10 at the same time as increasing JEV growth and reducing CXCL10 expression induced by JEV infection. Overall, TLR3 signaling induced IFN-ß-dependent expression of ISG56 and CXCL10. We show that ISG56 possibly plays a critical role in antiviral immunity of hRPTECs by positive regulation of IFN-ß-mediated CXCL10 expression downstream of TLR3.
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Quimiocina CXCL10 , Células Epiteliales , Interferón beta , Túbulos Renales Proximales , Receptor Toll-Like 3 , Humanos , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/citología , Células Epiteliales/metabolismo , Interferón beta/metabolismo , Interferón beta/genética , Poli I-C/farmacología , Transducción de Señal , Células Cultivadas , Inmunidad Innata , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
The presence of moieties denoting viral infection is crucial to mount powerful cytotoxic T-cell immune responses acting through innate receptors such as Toll-like receptor 3. For cancer immunotherapy, several safe analogues of viral double-stranded RNA are under clinical development following compelling evidence for efficacy in mouse models. See related article by van Eijck et al., p. 3447.
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Inmunoterapia , Neoplasias , ARN Bicatenario , Virosis , Humanos , Neoplasias/terapia , Neoplasias/inmunología , ARN Bicatenario/inmunología , Inmunoterapia/métodos , Animales , Virosis/inmunología , Receptor Toll-Like 3/genética , Ratones , Linfocitos T Citotóxicos/inmunología , ARN ViralRESUMEN
BACKGROUND: Toll-Like receptors (TLRs) play an important role in the immune response during hepatitis B virus (HBV) infection. In this study, we evaluated the association between two SNP variants (TLR3 rs3775290 and TLR4 rs4986790) and susceptibility to chronic HBV infection in Mauritania. SUBJECTS AND METHODS: A total of 188 subjects were recruited for this study: 102 chronically infected patients and 86 individuals with spontaneously resolved HBV infection who were considered controls. Targeted PCR products were sequenced using Sanger sequencing. RESULTS: We found that TLR3 rs3775290 was significantly more frequent in patients with chronic HBV than in the control population (p = 0.03). However, no association was found between the TLR4 rs3775290 polymorphism and chronic infection. CONCLUSION: Our results suggest that the TLR3 rs3775290 polymorphism may be a risk factor for susceptibility to chronic HBV infection in the Mauritanian population.
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Predisposición Genética a la Enfermedad , Hepatitis B Crónica , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 3 , Humanos , Receptor Toll-Like 3/genética , Masculino , Femenino , Estudios de Casos y Controles , Adulto , Hepatitis B Crónica/genética , Hepatitis B Crónica/virología , Persona de Mediana Edad , Mauritania , Adulto Joven , Virus de la Hepatitis B/genéticaRESUMEN
Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2, in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes.
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Encefalitis por Herpes Simple , Herpesvirus Humano 1 , Mutación , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Femenino , Encefalitis por Herpes Simple/genética , Lactante , Herpesvirus Humano 1/genética , Células Madre Pluripotentes Inducidas/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Ubiquitinación , Neuronas/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/virología , Sistemas CRISPR-CasRESUMEN
Seipin plays a crucial role in lipid metabolism and is involved in neurological disorders. However, the function and mechanism of action of seipin in acute ischemic stroke have not yet been elucidated. Here, we aimed to investigate the effect of seipin on neuroinflammation induced by oxygen-glucose deprivation/reoxygenation (OGD/R) and further explore the molecular mechanism by functional experiments. Our results revealed a significant decrease in seipin mRNA levels, accompanied by enhanced expression of TNF-α in patients with AIS, and a significant negative correlation between seipin and TNF-α was observed. Additionally, there was a negative correlation between seipin levels and the National Institutes of Health Stroke Scale (NIHSS) score. Furthermore, seipin levels were also decreased in middle cerebral artery occlusion/reperfusion (MCAO/R) mice and OGD/R-treated BV2 cells. RNA sequencing analysis showed that seipin knockdown altered the Toll-like receptor 3 (TLR3) signaling pathway. It was further confirmed in vitro that seipin knockdown caused significantly increased secretion of inflammatory factors including TNF-α, interleukin (IL)-1ß, and interferon (IFN)-ß. Meanwhile, seipin knockdown activated the Tlr3 signal pathway while this effect could be reversed by Tlr3 inhibitor in OGD/R treated BV2 cells. Furthermore, neuroinflammation induced by OGD/R was significantly reduced by seipin overexpression. Overall, our study demonstrate that seipin deficiency aggravates neuroinflammation by activating the TLR3/TRAF3/NF-κB signaling pathway after OGD/R stimuli, and suggest that seipin may be a potential therapeutic target for AIS.
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Glucosa , FN-kappa B , Enfermedades Neuroinflamatorias , Oxígeno , Transducción de Señal , Factor 3 Asociado a Receptor de TNF , Receptor Toll-Like 3 , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Línea Celular , Modelos Animales de Enfermedad , Glucosa/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/inmunología , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/inmunología , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/metabolismo , FN-kappa B/metabolismo , Oxígeno/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genéticaRESUMEN
The study aimed to explore the pontential impact of 10 polymorphisms within IFN-α, IFN-ß1, IFN-γ and TLR3 genes on SLE phenotype and susceptibility and to study the relationship between specific genotypes and clinics. Whole blood samples from SLE patients and healthy controls was obtained. DNA was extracted from the peripheral blood by the QIAamp DNA Blood Mini Kit (Qiagen). The quality and quantity of isolated DNA was estimated by the Quawell Q5000 spectrophotometer. We genotyped SLE patients and healthy subjects using real-time PCR (QuantStudio 5 thermocycler). The study suggests that IFN-γ rs2069705, IFN-γ rs2069718 and IFN-α rs3758236 polymorphisms have a protective role in SLE. We observed relations between TLR3 rs3775292, IFN-ß1 rs7873167, IFN-γ rs2069705, TLR3 rs3775291 and TLR3 rs5743305 polymorphisms and clinical picture of SLE patients. We found associations between the IFN-α rs3758236, IFN-γ rs2069705, IFN-γ rs2069718, IFN-γ rs1861493 and IFN-ß1 rs10964831 polymorphisms and the clinical manifestation of the SLE and/or its comorbidities. We perceived links between IFN-γ rs2069705, IFN-γ rs2069718, IFN-γ rs1861493, TLR3 rs3775291, TLR3 rs3775292 and TLR3 rs5743305 polymorphisms and the occurrence of autoantibodies. Our study presented the relationship between IFN and TLR gene polymorphisms with SLE susceptibility, phenotype and autoantibodies profile. This study propose that polymorphisms within interferons and TLR3 genes can be engaged in the SLE pathogenesis and course.
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Predisposición Genética a la Enfermedad , Genotipo , Lupus Eritematoso Sistémico , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 3 , Humanos , Lupus Eritematoso Sistémico/genética , Receptor Toll-Like 3/genética , Femenino , Masculino , Adulto , Persona de Mediana Edad , Frecuencia de los Genes , Alelos , Estudios de Casos y Controles , Interferones/genética , Estudios de Asociación GenéticaRESUMEN
Toll-like receptors (TLRs) play an important role in innate immunity. Previous studies have shown that single nucleotide polymorphisms (SNPs) in the genes coding for these innate immune molecules can affect susceptibility to and the outcome of certain diseases. The aim of the present study was to examine the clinical relevance of well-studied TLR1-4 SNPs in individuals who are prone to infections. Four functional SNPs, TLR1 rs5743618 (1805C > A, Ser602Ile), TLR2 rs5743708 (2258G > A, Arg753Gln), TLR3 rs3775291 (1234C > T, Leu412Phe) and TLR4 rs4986790 (896A > G, Asp299Gly), were analysed in 155 patients with recurrent respiratory infections (n = 84), severe infections (n = 15) or common variable immunodeficiency (n = 56), and in 262 healthy controls, using the High Resolution Melting Analysis method. Polymorphisms of TLR2 rs5743708 (odds ratio [OR] 3.16; 95% confidence interval [CI] 1.45-6.83, p = .004, ap = .016) and TLR4 rs4986790 (OR 1.8; 95% CI 1.05-3.12, p = .028, ap = .112) were more frequent in patients with recurrent or severe infections than in controls. Interestingly, seven patients were found to carry both variant genotypes of TLR2 and TLR4, whereas none of the control group carried such genotypes (p ≤ .0001). Moreover, TLR2 polymorphism was associated with increased risk for acute otitis media episodes (OR, 3.02; 95% CI 1.41-6.47; p = .012). This study indicates that children and adults who are more prone to recurrent or severe respiratory infections carry one or both variant types of TLR2 and TLR4 more often than control subjects. Genetic variations of TLRs help explain why some children are more susceptible to respiratory infections.
Asunto(s)
Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 1 , Receptor Toll-Like 2 , Receptor Toll-Like 3 , Receptor Toll-Like 4 , Humanos , Masculino , Femenino , Receptor Toll-Like 4/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 1/genética , Niño , Adulto , Infecciones del Sistema Respiratorio/genética , Preescolar , Adolescente , Recurrencia , Persona de Mediana Edad , Genotipo , Frecuencia de los Genes , Estudios de Casos y ControlesRESUMEN
BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.
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Condrocitos , Células Madre Mesenquimatosas , Humanos , Ratas , Animales , Condrocitos/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Células Cultivadas , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Ratas Sprague-Dawley , Células Madre Mesenquimatosas/metabolismo , Receptores Toll-Like/metabolismo , Fenotipo , Poli I/metabolismo , Poli I/farmacologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) infection can result in HIV-associated neurocognitive disorder (HAND), a spectrum of disorders characterized by neurological impairment and chronic inflammation. Combined antiretroviral therapy (cART) has elicited a marked reduction in the number of individuals diagnosed with HAND. However, there is continual, low-level viral transcription due to the lack of a transcription inhibitor in cART regimens, which results in the accumulation of viral products within infected cells. To alleviate stress, infected cells can release accumulated products, such as TAR RNA, in extracellular vesicles (EVs), which can contribute to pathogenesis in neighboring cells. Here, we demonstrate that cART can contribute to autophagy deregulation in infected cells and increased EV release. The impact of EVs released from HIV-1 infected myeloid cells was found to contribute to CNS pathogenesis, potentially through EV-mediated TLR3 (Toll-like receptor 3) activation, suggesting the need for therapeutics to target this mechanism. Three HIV-1 TAR-binding compounds, 103FA, 111FA, and Ral HCl, were identified that recognize TAR RNA and reduce TLR activation. These data indicate that packaging of viral products into EVs, potentially exacerbated by antiretroviral therapeutics, may induce chronic inflammation of the CNS observed in cART-treated patients, and novel therapeutic strategies may be exploited to mitigate morbidity.
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Autofagia , Vesículas Extracelulares , Infecciones por VIH , VIH-1 , Receptor Toll-Like 3 , Vesículas Extracelulares/metabolismo , Humanos , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , VIH-1/fisiología , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , Autofagia/efectos de los fármacos , ARN Viral/metabolismo , ARN Viral/genéticaRESUMEN
Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-ß. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-ß to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.
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Escherichia coli , Levivirus , ARN Bicatenario , Escherichia coli/virología , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Bicatenario/metabolismo , Humanos , Levivirus/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , Inmunidad Innata , Interferón beta/metabolismo , Interferón beta/genética , FN-kappa B/metabolismo , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Transducción de Señal , Factor 7 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Receptores Inmunológicos , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genéticaRESUMEN
Deubiquitinating enzyme A (DUBA), a member of the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs), is recognized for its negative regulatory role in type I interferon (IFN) expression downstream of Toll-like receptor 3 (TLR3). However, its involvement in the TLR3 signaling pathway in fish remains largely unexplored. In this study, we investigated the regulatory role of DUBA (OmDUBA) in the TLR3 response in rainbow trout (Oncorhynchus mykiss). OmDUBA features a conserved OTU domain, and its expression increased in RTH-149 cells following stimulation with the TLR3 agonist poly(I:C). Gain- and loss-of-function experiments demonstrated that OmDUBA attenuated the activation of TANK-binding kinase 1 (TBK1), resulting in a subsequent reduction in type I IFN expression and IFN-stimulated response element (ISRE) activation in poly(I:C)-stimulated cells. OmDUBA interacted with TRAF3, a crucial mediator in TLR3-mediated type I IFN production. Under poly(I:C) stimulation, there was an augmentation in the K63-linked polyubiquitination of TRAF3, a process significantly inhibited upon OmDUBA overexpression. These findings suggest that OmDUBA may function similarly to its mammalian counterparts in downregulating the poly(I:C)-induced type I IFN response in rainbow trout by removing the K63-linked ubiquitin chain on TRAF3. Our study provides novel insights into the role of fish DUBA in antiviral immunity.
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Proteínas de Peces , Interferón Tipo I , Oncorhynchus mykiss , Poli I-C , Transducción de Señal , Factor 3 Asociado a Receptor de TNF , Animales , Oncorhynchus mykiss/inmunología , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/inmunología , Interferón Tipo I/inmunología , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Transducción de Señal/inmunología , Poli I-C/farmacología , Inmunidad Innata , Regulación de la Expresión Génica/inmunología , Ubiquitinación , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/inmunologíaRESUMEN
BACKGROUND: Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to infection or injury. Recent studies have uncovered their intriguing functions as moonlighting proteins involved in various biological processes, including development, learning, and memory. However, the specific functions of individual TLRs are still largely unknown. AIMS: We investigated the effects of TLR3 and TLR9 receptor deficiency on motor, cognitive, and behavioral functions during development using genetically modified male mice of different ages. METHODS: We evaluated the motor coordination, anxiety-like behavior, spatial learning, and working memory of male mice lacking the TLR3 and TLR9 genes at different ages (two, four, six, and eight months) using the rotarod, open field, water maze, and T-maze tests. RESULTS: We observed that the deletion of either TLR3 or TLR9 resulted in impaired motor performance. Furthermore, young TLR3-deficient mice exhibited reduced anxiety-like behavior and spatial learning deficits; however, their working memory was unaffected. In contrast, young TLR9-knockout mice showed hyperactivity and a tendency toward decreased working memory. CONCLUSIONS: These findings provide valuable insights into the broader roles of the TLR system beyond the innate immune response, revealing its involvement in pathways associated with the central nervous system. Importantly, our results establish a strong association between the endosomal receptors TLR3 and TLR9 and the performance of motor, cognitive, and behavioral tasks that change over time. This study contributes to the growing body of research on the multifaceted functions of TLRs and enhances our understanding of their participation in non-immune-related processes.
Asunto(s)
Receptor Toll-Like 3 , Receptor Toll-Like 9 , Animales , Masculino , Ratones , Cognición , Ratones Noqueados , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 9/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.
Asunto(s)
Asma , Citocinas , Estrés del Retículo Endoplásmico , Células Epiteliales , Linfopoyetina del Estroma Tímico , Receptor Toll-Like 3 , Respuesta de Proteína Desplegada , Humanos , Citocinas/metabolismo , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , Asma/metabolismo , Asma/patología , Asma/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Factor de Transcripción CHOP/metabolismo , Factor de Transcripción CHOP/genética , Transducción de Señal , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Bronquios/metabolismo , Bronquios/patología , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , Células Cultivadas , Femenino , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Tripartite-motif protein-56 (TRIM56) positively regulates the induction of type I interferon response via the TLR3 pathway by enhancing IRF3 activation and depends on its C-terminal residues 621-750 for interacting with the adaptor TRIF. However, the precise underlying mechanism and detailed TRIM56 determinants remain unclear. Herein, we show ectopic expression of murine TRIM56 also enhances TLR3-dependent interferon-ß promoter activation, suggesting functional conservation. We found that endogenous TRIM56 and TRIF formed a complex early (0.5-2 h) after poly-I:C stimulation and that TRIM56 overexpression also promoted activation of NF-κB by poly-I:C but not that by TNF-α or IL-1ß, consistent with a specific effect on TRIF prior to the bifurcation of NF-κB and IRF3. Using transient transfection and Tet-regulated cell lines expressing various TRIM56 mutants, we demonstrated the Coiled-coil domain and a segment spanning residues â¼434-610, but not the B-box or residues 355-433, were required for TRIM56 augmentation of TLR3 signaling. Moreover, alanine substitution at each putative phosphorylation site, Ser471, Ser475, and Ser710, abrogated TRIM56 function. Concordantly, mutants bearing Ser471Ala, Ser475Ala, or Ser710Ala, or lacking the Coiled-coil domain, all lost the capacity to enhance poly-I:C-induced establishment of an antiviral state. Furthermore, the Ser710Ala mutation disrupted the TRIM56-TRIF association. Using phospho-specific antibodies, we detected biphasic phosphorylation of TRIM56 at Ser471 and Ser475 following TLR3 stimulation, with the early phase occurring at â¼0.5 to 1 h, prior to IRF3 phosphorylation. Together, these data reveal novel molecular details critical for the TRIM56 augmentation of TLR3-dependent antiviral response and highlight important roles for TRIM56 scaffolding and phosphorylation.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Inmunidad Innata , Receptor Toll-Like 3 , Proteínas de Motivos Tripartitos , Animales , Humanos , Ratones , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Células HEK293 , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , FN-kappa B/metabolismo , Fosforilación , Poli I-C/farmacología , Dominios Proteicos , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Urothelial damage and barrier dysfunction emerge as the foremost mechanisms in Hunner-type interstitial cystitis/bladder pain syndrome (HIC). Although treatments aimed at urothelial regeneration and repair have been employed, their therapeutic effectiveness remains limited due to the inadequate understanding of specific cell types involved in damage and the lack of specific molecular targets within these mechanisms. Therefore, we harnessed single-cell RNA sequencing to elucidate the heterogeneity and developmental trajectory of urothelial cells within HIC bladders. Through reclustering, we identified eight distinct clusters of urothelial cells. There was a significant reduction in UPK3A+ umbrella cells and a simultaneous increase in progenitor-like pluripotent cells (PPCs) within the HIC bladder. Pseudotime analysis of the urothelial cells in the HIC bladder revealed that cells faced challenges in differentiating into UPK3A+ umbrella cells, while PPCs exhibited substantial proliferation to compensate for the loss of UPK3A+ umbrella cells. The urothelium in HIC remains unrepaired, despite the substantial proliferation of PPCs. Thus, we propose that inhibiting the pivotal signaling pathways responsible for the injury to UPK3A+ umbrella cells is paramount for restoring the urothelial barrier and alleviating lower urinary tract symptoms in HIC patients. Subsequently, we identified key molecular pathways (TLR3 and NR2F6) associated with the injury of UPK3A+ umbrella cells in HIC urothelium. Finally, we conducted in vitro and in vivo experiments to confirm the potential of the TLR3-NR2F6 axis as a promising therapeutic target for HIC. These findings hold the potential to inhibit urothelial injury, providing promising clues for early diagnosis and functional bladder self-repair strategies for HIC patients. © 2024 The Pathological Society of Great Britain and Ireland.