RESUMEN
Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-ß production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-ß induction, prompting a negative feedback regulatory mechanism that represses IFN-ß expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.
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Factor 4E Eucariótico de Iniciación/inmunología , Inmunidad Innata , MicroARNs/inmunología , Biosíntesis de Proteínas/inmunología , Infecciones por Virus ARN/inmunología , Virus ARN/inmunología , Animales , Factor 4E Eucariótico de Iniciación/genética , Células HEK293 , Humanos , Interferón beta/genética , Interferón beta/inmunología , Ratones , Ratones Transgénicos , MicroARNs/genética , Infecciones por Virus ARN/genética , Virus ARN/genéticaRESUMEN
Inflammasome-activated caspase-1 cleaves gasdermin D to unmask its pore-forming activity, the predominant consequence of which is pyroptosis. Here, we report an additional biological role for gasdermin D in limiting cytosolic DNA surveillance. Cytosolic DNA is sensed by Aim2 and cyclic GMP-AMP synthase (cGAS) leading to inflammasome and type I interferon responses, respectively. We found that gasdermin D activated by the Aim2 inflammasome suppressed cGAS-driven type I interferon response to cytosolic DNA and Francisella novicida in macrophages. Similarly, interferon-ß (IFN-ß) response to F. novicida infection was elevated in gasdermin D-deficient mice. Gasdermin D-mediated negative regulation of IFN-ß occurred in a pyroptosis-, interleukin-1 (IL-1)-, and IL-18-independent manner. Mechanistically, gasdermin D depleted intracellular potassium (K+) via membrane pores, and this K+ efflux was necessary and sufficient to inhibit cGAS-dependent IFN-ß response. Thus, our findings have uncovered an additional interferon regulatory module involving gasdermin D and K+ efflux.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Francisella/fisiología , Infecciones por Bacterias Gramnegativas/inmunología , Inflamasomas/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Interferón Tipo I/metabolismo , Interleucina-1/metabolismo , Interleucina-18/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Proteínas de Unión a Fosfato , Potasio/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Lumpy skin disease (LSD) is a severe animal infectious disease caused by lumpy skin disease virus (LSDV), inducing extensive nodules on the cattle mucosa or the scarfskin. LSDV genome encodes multiple proteins to evade host innate immune response. However, the underlying molecular mechanisms are poorly understood. In this study, we found that LSDV could suppress the expression of IFN-ß and interferon-stimulated genes (ISGs) in MDBK cells during the early stage of infection. Subsequently, an unbiased screen was performed to screen the LSDV genes with inhibitory effects on the type I interferon (IFN-I) production. ORF127 protein was identified as one of the strongest inhibitory effectors on the expression of IFN-ß and ISGs, meanwhile, the 1-43 aa of N-terminal of ORF127 played a vital role in suppressing the expression of IFN-ß. Overexpression of ORF127 could significantly promote LSDV replication through inhibiting the production of IFN-ß and ISGs in MDBK cells. Mechanism study showed that ORF127 specifically interacted with TBK1 and decreased the K63-linked polyubiquitination of TBK1 which suppressed the phosphorylation of TBK1 and ultimately decreased the production of IFN-ß. In addition, truncation mutation analysis indicated that the 1-43 aa of N-terminal of ORF127 protein was the key structural domain for its interaction with TBK1. In short, these results validated that ORF127 played a negative role in regulating IFN-ß expression through cGAS-STING signaling pathway. Taken together, this study clarified the molecular mechanism of ORF127 gene antagonizing IFN-I-mediated antiviral, which will helpfully provide new strategies for the treatment and prevention of LSD.
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Interacciones Huésped-Patógeno , Interferón Tipo I , Virus de la Dermatosis Nodular Contagiosa , Proteínas Serina-Treonina Quinasas , Animales , Bovinos , Inmunidad Innata , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Interferón beta/metabolismo , Virus de la Dermatosis Nodular Contagiosa/metabolismo , Transducción de Señal , Ubiquitinación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.
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Autofagia , Proteína 58 DEAD Box , Glaucoma , Presión Intraocular , Malla Trabecular , Animales , Femenino , Humanos , Masculino , Ratones , Enfermedades de la Aorta , Autofagia/efectos de los fármacos , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Hipoplasia del Esmalte Dental , Glaucoma/patología , Glaucoma/metabolismo , Glaucoma/genética , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/patología , Pérdida Auditiva Sensorineural/metabolismo , Interferón beta/metabolismo , Presión Intraocular/genética , Metacarpo/anomalías , Ratones Endogámicos C57BL , Enfermedades Musculares , Mutación , Odontodisplasia , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Atrofia Óptica/patología , Osteoporosis , Linaje , Receptores Inmunológicos , Malla Trabecular/metabolismo , Malla Trabecular/efectos de los fármacos , Calcificación VascularRESUMEN
Zika virus (ZIKV) is a re-emerging RNA virus and causes major public health events due to its link to severe neurological complications in foetuses and neonates. The cGAS-STING signalling pathway regulates innate immunity and plays an important role in the invasion of DNA and RNA viruses. This study reveals a distinct mechanism by which ZIKV restricts the cGAS-STING signalling to repress IFN-ß expression. ZIKV attenuates IFN-ß expression induced by DNA viruses (herpes simplex virus type 1, HSV-1) or two double-stranded DNAs (dsDNA90 and HSV120) in mouse embryonic fibroblasts (MEFs). Notably, ZIKV NS5, the viral RNA-dependent RNA polymerase, was responsible for the repression of IFN-ß. NS5 interacts with STING in the cytoplasm, suppresses IRF3 phosphorylation and nucleus localization and promotes the cleavage of STING K48-linked polyubiquitination. Furthermore, the NS5 methyltransferase (MTase) domain interacts with STING to restrict STING-induced IFN-ß expression. Interestingly, point mutation analyses of conserved methyltransferase active site residue D146 indicate that it is critical for repressing IFN-ß expression induced by STING stimulation in cGAS-STING signalling.
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Infección por el Virus Zika , Virus Zika , Animales , Ratones , Dominio Catalítico , ADN , Fibroblastos/metabolismo , Inmunidad Innata , Interferones , Metiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Virus Zika/fisiologíaRESUMEN
IMPORTANCE: Type I interferon (IFN-I), produced by the innate immune system, plays an essential role in host antiviral responses. Proper regulation of IFN-I production is required for the host to balance immune responses and prevent superfluous inflammation. IFN regulatory factor 3 (IRF3) and subsequent sensors are activated by RNA virus infection to induce IFN-I production. Therefore, proper regulation of IRF3 serves as an important way to control innate immunity and viral replication. Here, we first identified Prohibitin1 (PHB1) as a negative regulator of host IFN-I innate immune responses. Mechanistically, PHB1 inhibited the nucleus import of IRF3 by impairing its binding with importin subunit alpha-1 and importin subunit alpha-5. Our study demonstrates the mechanism by which PHB1 facilitates the replication of multiple RNA viruses and provides insights into the negative regulation of host immune responses.
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Proteína 58 DEAD Box , Prohibitinas , Virus ARN , Receptores Inmunológicos , Transducción de Señal , Replicación Viral , Proteína 58 DEAD Box/antagonistas & inhibidores , Proteína 58 DEAD Box/metabolismo , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Carioferinas/metabolismo , Prohibitinas/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Virus ARN/crecimiento & desarrollo , Virus ARN/inmunología , Virus ARN/metabolismoRESUMEN
IMPORTANCE: Coxsackievirus A6 (CV-A6) is a major emerging pathogen associated with atypical hand, foot, and mouth disease and can cause serious complications such as encephalitis, acute flaccid paralysis, and neurorespiratory syndrome. Therefore, revealing the associated pathogenic mechanisms could benefit the control of CV-A6 infections. In this study, we demonstrate that the nonstructural 2CCV-A6 suppresses IFN-ß production, which supports CV-A6 infection. This is achieved by depleting RNA sensors such as melanoma differentiation-associated gene 5 and retinoic acid-inducible gene I (RIG-I) through the lysosomal pathway. Such a function is shared by 2CEV-A71 and 2CCV-B3 but not 2CCV-A16, suggesting the latter might have an alternative way to promote viral replication. This study broadens our understanding of enterovirus 2C protein regulation of the RIG-I-like receptor signaling pathway and reveals a novel mechanism by which CV-A6 and other enteroviruses evade the host innate immune response. These findings on 2C may provide new therapeutic targets for the development of effective inhibitors against CV-A6 and other enterovirus infections.
Asunto(s)
Infecciones por Coxsackievirus , Humanos , Enterovirus Humano A/genética , Infecciones por Enterovirus/metabolismo , Infecciones por Enterovirus/virología , Enfermedad de Boca, Mano y Pie/virología , Inmunidad Innata , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Interferón beta/metabolismoRESUMEN
Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the Coronaviridae family and can cause fatal watery diarrhea in piglets, causing significant economic losses. Heterogeneous nuclear protein U (HNRNPU) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. However, it remains elusive whether and how cytoplasmic PEDV can be sensed by the RNA sensor HNRNPU. In this study we determined that HNRNPU was the binding partner of Nsp13 by immunoprecipitation-liquid chromatography-tandem mass spectrometry (IP/LC-MS/MS) analysis. The interaction between Nsp13 and HNRNPU was demonstrated by using coimmunoprecipitation and confocal immunofluorescence. Next, we identified that HNRNPU expression is significantly increased during PEDV infection, whereas the transcription factor hepatocyte nuclear factor 1α (HNF1A) could negatively regulate HNRNPU expression. HNRNPU was retained in the cytoplasm by interaction with PEDV Nsp13. We found that HNRNPU overexpression effectively facilitated PEDV replication, while knockdown of HNRNPU impaired viral replication, suggesting a promoting function of HNRNPU to PEDV infection. Additionally, HNRNPU was found to promote PEDV replication by affecting TRAF3 degradation at the transcriptional level to inhibit PEDV-induced beta interferon (IFN-ß) production. Mechanistically, HNRNPU downregulates TRAF3 mRNA levels via the METTL3-METTL14/YTHDF2 axis and regulates immune responses through YTHDF2-dependent mRNA decay. Together, our findings reveal that HNRNPU serves as a negative regulator of innate immunity by degrading TRAF3 mRNA in a YTHDF2-dependent manner and consequently facilitating PEDV propagation. Our findings provide new insights into the immune escape of PEDV. IMPORTANCE PEDV, a highly infectious enteric coronavirus, has spread rapidly worldwide and caused severe economic losses. During virus infection, the host regulates innate immunity to inhibit virus infection. However, PEDV has evolved a variety of different strategies to suppress host IFN-mediated antiviral responses. Here, we identified that HNRNPU interacted with viral protein Nsp13. HNRNPU protein expression was upregulated, and the transcription factor HNF1A could negatively regulate HNRNPU expression during PEDV infection. HNRNPU also downregulated TRAF3 mRNA through the METTL3-METTL14/YTHDF2 axis to inhibit the production of IFN-ß and downstream antiviral genes in PEDV-infected cells, thereby promoting viral replication. Our findings reveal a new mechanism with which PEDV suppresses the host antiviral response.
Asunto(s)
Infecciones por Coronavirus , Proteínas Nucleares , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Replicación Viral , Animales , Línea Celular , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Proteínas Nucleares/metabolismo , Virus de la Diarrea Epidémica Porcina/fisiología , ARN Mensajero/metabolismo , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Factor 3 Asociado a Receptor de TNF/metabolismo , Factores de Transcripción/metabolismo , Replicación Viral/fisiologíaRESUMEN
Pseudorabies virus (PRV), the causative pathogen of Aujeszky's disease, is one of the most important pathogens threatening the global pig industry. Although vaccination has been used to prevent PRV infection, the virus cannot be eliminated in pigs. Thus, novel antiviral agents as complementary to vaccination are urgently needed. Cathelicidins (CATHs) are host defense peptides that play an important role in the host immune response against microbial infections. In the study, we found that the chemical synthesized chicken cathelicidin B1 (CATH-B1) could inhibit PRV regardless of whether CATH-B1 was added pre-, co-, or post-PRV infection in vitro and in vivo. Furthermore, coincubation of CATH-B1 with PRV directly inactivated virus infection by disrupting the virion structure of PRV and mainly inhibited virus binding and entry. Importantly, pretreatment of CATH-B1 markedly strengthened the host antiviral immunity, as indicated by the increased expression of basal interferon-ß (IFN-ß) and several IFN-stimulated genes (ISGs). Subsequently, we investigated the signaling pathway responsible for CATH-B1-induced IFN-ß production. Our results showed that CATH-B1 induced phosphorylation of interferon regulatory transcription factor 3 (IRF3) and further led to production of IFN-ß and reduction of PRV infection. Mechanistic studies revealed that the activation of Toll-like receptor 4 (TLR4), endosome acidification, and the following c-Jun N-terminal kinase (JNK) was responsible for CATH-B1-induced IRF3/IFN-ß pathway activation. Collectively, CATH-B1 could markedly inhibit PRV infection via inhibiting virus binding and entry, direct inactivation, and regulating host antiviral response, which provided an important theoretical basis for the development of antimicrobial peptide drugs against PRV infection. IMPORTANCE Although the antiviral activity of cathelicidins could be explained by direct interfering with the viral infection and regulating host antiviral response, the specific mechanism of cathelicidins regulating host antiviral response and interfering with pseudorabies virus (PRV) infection remains elusive. In this study, we investigated the multiple roles of cathelicidin CATH-B1 against PRV infection. Our study showed that CATH-B1 could suppress the binding and entry stages of PRV infection and direct disrupt PRV virions. Remarkably, CATH-B1 significantly increased basal interferon-ß (IFN-ß) and IFN-stimulated gene (ISG) expression levels. Furthermore, TLR4/c-Jun N-terminal kinase (JNK) signaling was activated and involved in IRF3/IFN-ß activation in response to CATH-B1. In conclusion, we elucidate the mechanisms by which the cathelicidin peptide direct inactivates PRV infection and regulates host antiviral IFN-ß signaling.
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Herpesvirus Suido 1 , Seudorrabia , Porcinos , Animales , Herpesvirus Suido 1/metabolismo , Catelicidinas/uso terapéutico , Receptor Toll-Like 4 , Interferón beta/metabolismo , Antivirales/farmacologíaRESUMEN
Preclinical data suggest that type I interferon (IFN) responsiveness is essential for the antitumor effects of radiotherapy (RT). However, its clinical value remains unclear. This study aimed to explore this from a clinical perspective. In cohort 1, data from 152 hepatocellular carcinoma (HCC) patients who received RT were analyzed. Blood samples were taken 1 day before and 2 weeks after RT. RT was found to increase serum levels of IFN-ß (a subtype of IFN-I) in HCC patients (3.42â ±â 1.57 to 5.51â ±â 2.11 pg/ml, Pâ <â 0.01), particularly in those with favorable responses. Higher post-RT serum IFN-ß levels (≥4.77 pg/ml) were associated with better progression-free survival (HRâ =â 0.58, Pâ <â 0.01). Cohort 2 included 46 HCC patients, including 23 who underwent preoperative RT and 23 matched control HCC who received surgical resection without RT. Formalin-fixed paraffin-embedded samples were obtained. Neoadjuvant RT significantly increased IFN-ß expression in tumor tissues compared to direct surgery (8.13%â ±â 5.19% to 15.10%â ±â 5.89%, Pâ <â 0.01). Higher post-RT IFN-ß (>median) indicated better disease-free survival (Pâ =â 0.049). Additionally, increased CD11c+MHCII+CD141+ antigen-presenting cell subsets and CD103+CD39+CD8+ tumor-infiltrating lymphocytes were found in the higher IFN-ß group (Pâ =â 0.02, Pâ =â 0.03), which may contribute to the favorable prognosis in higher IFN-ß group. Collectively, these findings suggest that IFN-ß response activated by radiation may serve as a prognostic biomarker for HCC patients undergoing RT.
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Carcinoma Hepatocelular , Interferón beta , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Masculino , Femenino , Persona de Mediana Edad , Interferón beta/uso terapéutico , Anciano , Pronóstico , AdultoRESUMEN
Type I interferons (IFN) induce powerful antiviral and innate immune responses via the transcription factor, IFN-stimulated gene factor (ISGF3). However, in some pathological contexts, type I IFNs are responsible for exacerbating inflammation. Here, we show that a high dose of IFN-ß also activates an inflammatory gene expression program in contrast to IFN-λ3, a type III IFN, which elicits only the common antiviral gene program. We show that the inflammatory gene program depends on a second, potentiated phase in ISGF3 activation. Iterating between mathematical modeling and experimental analysis, we show that the ISGF3 activation network may engage a positive feedback loop with its subunits IRF9 and STAT2. This network motif mediates stimulus-specific ISGF3 dynamics that are dependent on ligand, dose, and duration of exposure, and when engaged activates the inflammatory gene expression program. Our results reveal a previously underappreciated dynamical control of the JAK-STAT/IRF signaling network that may produce distinct biological responses and suggest that studies of type I IFN dysregulation, and in turn therapeutic remedies, may focus on feedback regulators within it.
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Regulación de la Expresión Génica , Factores de Transcripción , Retroalimentación , Antivirales , Transducción de SeñalRESUMEN
Highly malignant brain tumors, glioblastomas (GBM), are immunosuppressive, thereby limiting current promising immunotherapeutic approaches. In this study, we created interferon receptor 1 knockout allogeneic mesenchymal stem cells (MSC) to secrete dual-function pro-apoptotic and immunomodulatory interferon (IFN) ß (MSCKO-IFNß) using a single lentiviral vector CRISPR/Cas9 system. We show that MSCKO-IFNß induces apoptosis in GBM cells and upregulates the cell surface expression of programmed death ligand-1 in tumor cells. Next, we engineered MSCKO to release a secretable single-chain variable fragment (scFv) to block programmed death (PD)-1 and show the ability of MSCKO-scFv-PD1 to enhance T-cell activation and T-cell-mediated tumor cell killing. To simultaneously express both immune modulators, we engineered MSCKO-IFNß to co-express scFv-PD1 (MSCKO-IFNß-scFv-PD1) and show the expression of both IFNß and scFv-PD1 in vitro leads to T-cell activation and lowers the viability of tumor cells. Furthermore, to mimic the clinical scenario of GBM tumor resection and subsequent treatment, we show that synthetic extracellular matrix (sECM) encapsulated MSCKO-IFNß-scFv-PD1 treatment of resected tumors results in the increase of CD4+ and CD8+ T cells, mature conventional dendritic cells type II and activation of microglia as compared to the control treatment group. Overall, these results reveal the ability of MSCKO-IFNß-scFv-PD1 to shape the tumor microenvironment and enhance therapeutic outcomes in GBM.
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Glioblastoma , Interferón beta , Células Madre Mesenquimatosas , Receptor de Muerte Celular Programada 1 , Anticuerpos de Cadena Única , Microambiente Tumoral , Glioblastoma/terapia , Glioblastoma/patología , Glioblastoma/metabolismo , Humanos , Receptor de Muerte Celular Programada 1/metabolismo , Anticuerpos de Cadena Única/genética , Interferón beta/metabolismo , Células Madre Mesenquimatosas/metabolismo , Línea Celular Tumoral , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patología , Animales , Apoptosis , RatonesRESUMEN
Human immunodeficiency virus-1 (HIV-1) infects the central nervous system (CNS) and causes HIV-associated neurocognitive disorders (HAND) in about half of the population living with the virus despite combination anti-retroviral therapy (cART). HIV-1 activates the innate immune system, including the production of type 1 interferons (IFNs) α and ß. Transgenic mice expressing HIV-1 envelope glycoprotein gp120 (HIVgp120tg) in the CNS develop memory impairment and share key neuropathological features and differential CNS gene expression with HIV patients, including the induction of IFN-stimulated genes (ISG). Here we show that knocking out IFNß (IFNßKO) in HIVgp120tg and non-tg control mice impairs recognition and spatial memory, but does not affect anxiety-like behavior, locomotion, or vision. The neuropathology of HIVgp120tg mice is only moderately affected by the KO of IFNß but in a sex-dependent fashion. Notably, in cerebral cortex of IFNßKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNßKO results in the hippocampal CA1 region of both male and female HIVgp120tg mice in an ameliorated loss of neuronal presynaptic terminals but no protection of neuronal dendrites. Only female IFNß-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNß-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNßKO abrogates or diminishes the induction of MX1, DDX58, IRF7 and IRF9 in HIVgp120tg brains of both sexes. Expression analysis of neurotransmission related genes reveals an influence of IFNß on multiple components with more pronounced changes in IFNßKO females. In contrast, the effects of IFNßKO on MAPK activities are independent of sex with pronounced reduction of active ERK1/2 but also of active p38 in the HIVgp120tg brain. In summary, our findings show that the absence of IFNß impairs memory dependent behavior and modulates neuropathology in HIVgp120tg brains, indicating that its absence may facilitate development of HAND. Moreover, our data suggests that endogenous IFNß plays a vital role in maintaining neuronal homeostasis and memory function.
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Infecciones por VIH , VIH-1 , Interferón beta , Animales , Femenino , Masculino , Ratones , Encéfalo/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Interferón beta/metabolismo , Ratones TransgénicosRESUMEN
Stimulator of interferon genes (STING) is an essential signaling protein that is located on the endoplasmic reticulum (ER) and triggers the production of type I interferons (IFN) and proinflammatory cytokines in response to pathogenic DNA. Aberrant activation of STING is linked to autoimmune diseases. The mechanisms underlying homeostatic regulation of STING are unclear. Here, we report that UNC13D, which is associated with familial hemophagocytic lymphohistiocytosis (FHL3), is a negative regulator of the STING-mediated innate immune response. UNC13D colocalizes with STING on the ER and inhibits STING oligomerization. Cellular knockdown and knockout of UNC13D promote the production of interferon-ß (IFN-ß) induced by DNA viruses, but not RNA viruses. Moreover, UNC13D deficiency also increases the basal level of proinflammatory cytokines. These effects are diminished by an inhibitor of STING signaling. Furthermore, the domains involved in the UNC13D/STING interaction on both proteins are mapped. Our findings provide insight into the regulatory mechanism of STING, the previously unknown cellular function of UNC13D and the potential pathogenesis of FHL3.
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Retículo Endoplásmico , Interferón Tipo I , Retículo Endoplásmico/metabolismo , Transducción de Señal , Inmunidad Innata , Interferón beta/genéticaRESUMEN
Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.
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Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Lubina , Enfermedades de los Peces , Proteínas de Peces , FN-kappa B , Filogenia , Animales , Lubina/inmunología , Lubina/genética , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/inmunología , Enfermedades de los Peces/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Transducción de Señal/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Alineación de Secuencia/veterinaria , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/química , Perfilación de la Expresión Génica/veterinaria , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Secuencia de BasesRESUMEN
BACKGROUND: Bronchial epithelial cells are at the front line of viral infections. Toll-like receptor 3 (TLR3) cascade causes the expression of interferon (IFN)-ß and IFN-stimulated genes (ISGs), which in turn induce an antiviral response. Members of the transmembrane protein (TMEM) family are expressed in various cell types. Although the prognostic value of TMEM2 in various cancers has been reported, its association with infectious diseases remains unknown. In this study, we investigated the effects of TMEM2 on antiviral immunity in BEAS-2B bronchial epithelial cells. METHODS AND RESULTS: TMEM2 protein was found in the cytoplasm of normal human bronchial epithelial cells and differed between organs using immunohistochemistry. Cultured BEAS-2B cells were transfected with TMEM2 siRNA, followed by administration of TLR3 ligand polyinosinic-polycytidylic acid (poly IC) or recombinant human (r(h)) IFN-ß. The expression of TMEM2, IFN-ß, ISG56, C-X-C motif chemokine ligand 10 (CXCL10) and hyaluronan were evaluated appropriately by western blotting, quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. TMEM2 expression was not altered by poly IC stimulation. Knockdown of TMEM2 increased poly IC-induced expression of IFN-ß, CXCL10, and ISG56, while IFN-ß-induced expression of ISG56 and CXCL10 were not changed by TMEM2 knockdown. The hyaluronan concentration in the medium was decreased by either TMEM2 knockdown or poly IC, but additive or synergistic effects were not observed. CONCLUSIONS: TMEM2 knockdown enhanced TLR3-mediated IFN-ß, CXCL10, and ISG56 expression in BEAS-2B cells. This implies that TMEM2 suppresses antiviral immune responses and prevents tissue injury in bronchial epithelial cells.
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Ácido Hialurónico , Receptor Toll-Like 3 , Humanos , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Ligandos , Poli I-C/farmacología , Células Epiteliales/metabolismo , Células Cultivadas , Quimiocina CXCL10/genéticaRESUMEN
PARP7 has been proven to play an important role in immunity. Substantial upregulation of PARP7 is observed in numerous cancerous cell types, consequently resulting in the inhibition of type â interferon signaling pathways. Therefore, inhibiting the activity of PARP7 can enhance type â interferon signaling to exert an anti-tumor immune response. In this study, we reported the identification of a newly found PARP7 inhibitor (XLY-1) with higher inhibitory activity (IC50 = 0.6 nM) than that of RBN-2397 (IC50 = 6.0 nM). Additionally, XYL-1 displayed weak inhibitory activity on PARP1 (IC50 > 1.0 µM). Mechanism studies showed that XYL-1 could enhance the type â interferon signaling in vitro. Pharmacodynamic experiments showed that 50 mg/kg XYL-1 could significantly inhibit tumor growth (TGI: 76.5 %) and related experiments showed that XYL-1 could restore type â interferon signaling and promote T cell infiltration in tumor tissues. Taken together, XYL-1 shows promise as a potential candidate for developing cancer immunotherapy agents.
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Antineoplásicos , Proliferación Celular , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Inmunoterapia , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis química , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Estructura Molecular , Animales , Ratones , Proliferación Celular/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Línea Celular Tumoral , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Ratones Endogámicos BALB CRESUMEN
Apobec-1 complementation factor (A1CF) functions as an RNA-binding cofactor for APO-BEC1-mediated C-to-U conversion during RNA editing and as a hepatocyte-specific regulator in the alternative pre-mRNA splicing of metabolic enzymes. Its role in RNA editing has not been clearly established. Western blot, co-immunoprecipitation (Co-IP), immunofluorescence (IF), methyl thiazolyl tetrazolium (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to examine the role of A1CF beyond RNA editing in renal carcinoma cells. We demonstrated that A1CF interacts with NKRF, independent of RNA and DNA, without affecting its expression or nuclear translocation; however, it modulates p65(Ser536) phosphorylation and IFN-ß levels. Truncation of A1CF or deletion on NKRF revealed that the RRM1 domain of A1CF and the p65 binding motif of NKRF are required for their interaction. Deletion of RRM1 on A1CF abrogates NKRF binding, and the decrease in IFN-ß expression and p65(Ser536) phosphorylation was induced by A1CF. Moreover, full-length A1CF, but not an RRM1 deletion mutant, promoted cell proliferation in renal carcinoma cells. Perturbation of A1CF levels in renal carcinoma cells altered anchorage-independent growth and tumor progression in nude mice. Moreover, p65(Ser536) phosphorylation and IFN-ß expression were lower, but ki67 was higher in A1CF-overexpressing tumor tissues of a xenograft mouse model. Notably, primary and metastatic samples from renal cancer patients exhibited high A1CF expression, low p65(Ser536) phosphorylation, and decreased IFN-ß levels in renal carcinoma tissues compared with the corresponding paracancerous tissues. Our results indicate that A1CF-decreased p65(Ser536) phosphorylation and IFN-ß levels may be caused by A1CF competitive binding to the p65-combined site on NKRF and demonstrate the direct binding of A1CF independent of RNA or DNA in signal pathway regulation and tumor promotion in renal carcinoma cells.
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Carcinoma de Células Renales , Neoplasias Renales , Animales , Humanos , Ratones , Desaminasas APOBEC-1 , Carcinoma de Células Renales/genética , Modelos Animales de Enfermedad , ADN , Neoplasias Renales/genética , Ratones Desnudos , Fosforilación , ARN , Proteínas de Unión al ARN , Interferón betaRESUMEN
Alcoholic liver disease (ALD) is a form of hepatic inflammation. ALD is mediated by gut leakiness. This study evaluates the anti-inflammatory effects of ASCs overexpressing interferon-beta (ASC-IFN-ß) on binge alcohol-induced liver injury and intestinal permeability. In vitro, ASCs were transfected with a non-viral vector carrying the human IFN-ß gene, which promoted hepatocyte growth factor (HGF) secretion in the cells. To assess the potential effects of ASC-IFN-ß, C57BL/6 mice were treated with three oral doses of binge alcohol and were administered intraperitoneal injections of ASC-IFN-ß. Mice treated with binge alcohol and administered ASC-IFN-ß showed reduced liver injury and inflammation compared to those administered a control ASC. Analysis of intestinal tissue from ethanol-treated mice administered ASC-IFN-ß also indicated decreased inflammation. Additionally, fecal albumin, blood endotoxin, and bacterial colony levels were reduced, indicating less gut leakiness in the binge alcohol-exposed mice. Treatment with HGF, but not IFN-ß or TRAIL, mitigated the ethanol-induced down-regulation of cell death and permeability in Caco-2 cells. These results demonstrate that ASCs transfected with a non-viral vector to induce IFN-ß overexpression have protective effects against binge alcohol-mediated liver injury and gut leakiness via HGF.
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Etanol , Interferón beta , Hepatopatías Alcohólicas , Células Madre Mesenquimatosas , Ratones Endogámicos C57BL , Permeabilidad , Animales , Humanos , Interferón beta/metabolismo , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Hepatopatías Alcohólicas/genética , Ratones , Células Madre Mesenquimatosas/metabolismo , Etanol/efectos adversos , Células CACO-2 , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genética , Masculino , Tejido Adiposo/metabolismo , Hígado/metabolismo , Hígado/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patologíaRESUMEN
The aim of the present study was to investigate the impact of CCR5 Δ32 and CTLA-4 polymorphisms on the response to IFN-ß treatment in our cohort of MS patients from Croatia and Slovenia. Genomic DNA was obtained from 295 MS patients (230 female; 65 male) classified as responders (n = 173) and non-responders (n = 122) based on clinical criteria for treatment efficacy. Genotyping was performed via PCR/PCR-RFLP. No significant differences in the genotype/allele frequencies of CCR5Δ32 and CTLA-4 +49 A/G were detected between male responders and non-responders. A significantly higher prevalence (p = 0.039) of the CTLA-4 +49 AA genotype was found in female responders (42.1%) compared to non-responders (28.9%). Using multiple forward regression analysis, the CTLA-4 +49 AA genotype significantly predicted a positive response to IFN-ß therapy in females (p = 0.011) and contributed to 4.5% of response variability. Furthermore, the combined presence of the CCR5Δ32 wtwt/CTLA-4 +49 AA genotype significantly predicted a positive response to treatment in females (p = 0.025). The age at disease onset, pretreatment relapse rate, and baseline EDSS score were not reliable predictors of treatment response in MS patients. Our results indicate that the presence of the CCR5Δ32 polymorphism was not associated with the response to IFN-ß treatment, whereas the CTLA-4 +49 polymorphism showed a positive correlation with an optimal response in female patients.