Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.903
Filtrar
1.
Sci Rep ; 12(1): 6972, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35484173

RESUMO

Common alphacoronaviruses and human rhinoviruses (HRV) induce type I and III interferon (IFN) responses important to limiting viral replication in the airway epithelium. In contrast, highly pathogenic betacoronaviruses including SARS-CoV-2 may evade or antagonize RNA-induced IFN I/III responses. In airway epithelial cells (AECs) from children and older adults we compared IFN I/III responses to SARS-CoV-2 and HRV-16, and assessed whether pre-infection with HRV-16, or pretreatment with recombinant IFN-ß or IFN-λ, modified SARS-CoV-2 replication. Bronchial AECs from children (ages 6-18 years) and older adults (ages 60-75 years) were differentiated ex vivo to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 or HRV-16, and RNA and protein was harvested from cell lysates 96 h. following infection and supernatant was collected 48 and 96 h. following infection. In additional experiments cultures were pre-infected with HRV-16, or pre-treated with recombinant IFN-ß1 or IFN-λ2 before SARS-CoV-2 infection. In a subset of experiments a range of infectious concentrations of HRV-16, SARS-CoV-2 WA-01, SARS-CoV-2 Delta variant, and SARS-CoV-2 Omicron variant were studied. Despite significant between-donor heterogeneity SARS-CoV-2 replicated 100 times more efficiently than HRV-16. IFNB1, INFL2, and CXCL10 gene expression and protein production following HRV-16 infection was significantly greater than following SARS-CoV-2. IFN gene expression and protein production were inversely correlated with SARS-CoV-2 replication. Treatment of cultures with recombinant IFNß1 or IFNλ2, or pre-infection of cultures with HRV-16, markedly reduced SARS-CoV-2 replication. In addition to marked between-donor heterogeneity in IFN responses and viral replication, SARS-CoV-2 (WA-01, Delta, and Omicron variants) elicits a less robust IFN response in primary AEC cultures than does rhinovirus, and heterologous rhinovirus infection, or treatment with recombinant IFN-ß1 or IFN-λ2, reduces SARS-CoV-2 replication, although to a lesser degree for the Delta and Omicron variants.


Assuntos
COVID-19 , Interferons , Adolescente , Idoso , Antivirais , COVID-19/tratamento farmacológico , Criança , Humanos , Interferons/farmacologia , Pessoa de Meia-Idade , RNA , Rhinovirus , SARS-CoV-2
2.
J Tradit Chin Med ; 42(2): 221-226, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35473342

RESUMO

OBJECTIVE: To examine the efficacy of Qinghuayin (, QHY) in rat chronic atrophic gastritis (CAG) models and explored the molecular mechanism of QHY in treating CAG. METHODS: In total, 65 Wistar rats were randomly divided into the control (= 10) and CAG groups ( = 55). CAG model rats were further divided into five groups: model ( = 10), vitacoenzyme ( = 10), low-dose QHY ( = 10), medium-dose QHY ( = 10), and high-dose QHY groups ( = 10). We analyzed histopathological changes using hematoxylin and eosin staining and measured interleukin (IL)-6 and IL-8 levels in serum using enzyme-linked immunosorbent assay (ELISA) (Boster Bio, Pleasanton, USA). In addition, gastrin (GAS), pepsinogen I (PGI), and PGII expressions were evaluated using ELISA. The protein and mRNA expression of toll-like receptor 4 (TLR4) and toll or interleukin-1 receptor domain-containing adaptor inducing interferon-ß (TRIF) was detected by Western blotting and quantitative reverse transcription-polymerase chain reaction, respectively. RESULTS: Our results revealed that histopathological changes in CAG model rates could be restored by low-, medium-, and high-dose QHY. The changes in GAS and PGI/II expression demonstrated that QHY improved CAG. Serum IL-6 and IL-levels were decreased by QHY administration. TLR4 and TRIF were upregulated at the mRNA and protein levels in the model group but downregulated by QHY administration. CONCLUSION: We concluded that QHY could effectively improve the histopathological changes of the gastric mucosa induced by CAG in rats. The therapeutic mechanism of QHY may be related to inhibition of the inflammatory factors IL-6 and IL-8 and suppression of TLR4/TRIF mRNA and protein expression.


Assuntos
Gastrite Atrófica , Interferons , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/farmacologia , Animais , Gastrite Atrófica/tratamento farmacológico , Gastrite Atrófica/genética , Humanos , Interferon beta/metabolismo , Interferon beta/farmacologia , Interferons/farmacologia , Interleucina-6/genética , Interleucina-8/genética , RNA Mensageiro , Ratos , Ratos Wistar , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
3.
PLoS One ; 17(4): e0266412, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35436306

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.


Assuntos
COVID-19 , Infecções por Vírus Respiratório Sincicial , Antivirais/farmacologia , Células Epiteliais/metabolismo , Humanos , Interferons/metabolismo , Interferons/farmacologia , Janus Quinases/metabolismo , SARS-CoV-2 , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais
4.
J Virol ; 96(7): e0170521, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35262371

RESUMO

The coronavirus SARS-CoV-2 caused the COVID-19 global pandemic leading to 5.3 million deaths worldwide as of December 2021. The human intestine was found to be a major viral target which could have a strong impact on virus spread and pathogenesis since it is one of the largest organs. While type I interferons (IFNs) are key cytokines acting against systemic virus spread, in the human intestine type III IFNs play a major role by restricting virus infection and dissemination without disturbing homeostasis. Recent studies showed that both type I and III IFNs can inhibit SARS-CoV-2 infection, but it is not clear whether one IFN controls SARS-CoV-2 infection of the human intestine better or with a faster kinetics. In this study, we could show that type I and III IFNs both possess antiviral activity against SARS-CoV-2 in human intestinal epithelial cells (hIECs); however, type III IFN is more potent. Shorter type III IFN pretreatment times and lower concentrations were required to efficiently reduce virus load compared to type I IFNs. Moreover, type III IFNs significantly inhibited SARS-CoV-2 even 4 h postinfection and induced a long-lasting antiviral effect in hIECs. Importantly, the sensitivity of SARS-CoV-2 to type III IFNs was virus specific since type III IFN did not control VSV infection as efficiently. Together, these results suggest that type III IFNs have a higher potential for IFN-based treatment of SARS-CoV-2 intestinal infection compared to type I IFNs. IMPORTANCE SARS-CoV-2 infection is not restricted to the respiratory tract and a large number of COVID-19 patients experience gastrointestinal distress. Interferons are key molecules produced by the cell to combat virus infection. Here, we evaluated how two types of interferons (type I and III) can combat SARS-CoV-2 infection of human gut cells. We found that type III interferons were crucial to control SARS-CoV-2 infection when added both before and after infection. Importantly, type III interferons were also able to produce a long-lasting effect, as cells were protected from SARS-CoV-2 infection up to 72 h posttreatment. This study suggested an alternative treatment possibility for SARS-CoV-2 infection.


Assuntos
COVID-19 , Interferon Tipo I , Interferons , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Células Cultivadas , Células Epiteliais , Humanos , Interferon Tipo I/farmacologia , Interferons/farmacologia , SARS-CoV-2/efeitos dos fármacos
5.
Arch Virol ; 167(5): 1293-1300, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35322318

RESUMO

Hepatitis E virus (HEV) infection is the leading cause of acute hepatitis worldwide. The mitochondrial antiviral signaling protein (MAVS)-mediated interferon (IFN) response plays a pivotal role in hepatic antiviral immunity. However, little is known about the effect of overexpression of MAVS on HEV infection. Full-length MAVS (FL-MAVS) is the main form of MAVS that increases the production of IFNs. Here, we studied the effect of FL-MAVS on HEV infection. We found that overexpression of FL-MAVS profoundly inhibited HEV replication. Furthermore, we showed that the anti-HEV effect of FL-MAVS is largely dependent on JAK-STAT signaling activation.


Assuntos
Vírus da Hepatite E , Hepatite E , Antivirais/farmacologia , Humanos , Imunidade Inata , Interferons/farmacologia , Replicação Viral
6.
Nat Cancer ; 3(3): 355-370, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35301507

RESUMO

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia , Interferons/farmacologia , Melanoma , Proteínas Repressoras/uso terapêutico , Neoplasias Cutâneas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
7.
Front Immunol ; 13: 821816, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35251003

RESUMO

In solid tumors, as the tumor grows and the disease progresses, hypoxic regions are often generated, but in contrast to most normal cells which cannot survive under these conditions, tumour cells adapt to hypoxia by HIF-driven mechanisms. Hypoxia can further promote cancer development by generating an immunosuppressive environment within the tumour mass, which allows tumour cells to escape the immune system recognition. This is achieved by recruiting immunosuppressive cells and by upregulating molecules which block immune cell activation. Hypoxia can also confer resistance to antitumor therapies by inducing the expression of membrane proteins that increase drug efflux or by inhibiting the apoptosis of treated cells. In addition, tumor cells require an active interferon (IFN) signalling pathway for the success of many anticancer therapies, such as radiotherapy or chemotherapy. Therefore, hypoxic effects on this pathway needs to be addressed for a successful treatment.


Assuntos
Interferons , Neoplasias , Apoptose , Humanos , Hipóxia/metabolismo , Interferons/farmacologia , Transdução de Sinais
8.
Sheng Wu Gong Cheng Xue Bao ; 38(3): 893-902, 2022 Mar 25.
Artigo em Chinês | MEDLINE | ID: mdl-35355462

RESUMO

Hepatitis B virus (HBV) infection is one of the most serious public health problems. HBV infection could lead to hepatitis B, and even further develop into hepatic cirrhosis and hepatocellular carcinoma. Interferon lambda (IFN-λ) is a member of the interferon (IFN) family and an important cytokine for antiviral defense. There are four members in IFN-λ family, including IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4. The genetic polymorphisms in the IFN-λ genes are associated with HBV replication and treatment response of HBV patients. In this review, we summarized the roles of genetic polymorphisms of the IFN-λ genes played in HBV infection, disease progression and treatment, with the aim to better understand their function. This review could serve as a reference for the HBV prevention and treatment of HBV patients, as well as for future clinical usage.


Assuntos
Hepatite B , Neoplasias Hepáticas , Antivirais/farmacologia , Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Interferons/genética , Interferons/farmacologia , Polimorfismo Genético , Replicação Viral/genética
9.
Cell Rep ; 38(7): 110396, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35172151

RESUMO

Fasciculation and elongation protein zeta-1 (FEZ1) is a multifunctional kinesin adaptor involved in processes ranging from neurodegeneration to retrovirus and polyomavirus infection. Here, we show that, although modulating FEZ1 expression also impacts infection by large DNA viruses in human microglia, macrophages, and fibroblasts, this broad antiviral phenotype is associated with the pre-induction of interferon-stimulated genes (ISGs) in a STING-independent manner. We further reveal that S58, a key phosphorylation site in FEZ1's kinesin regulatory domain, controls both binding to, and the nuclear-cytoplasmic localization of, heat shock protein 8 (HSPA8), as well as ISG expression. FEZ1- and HSPA8-induced changes in ISG expression further involved changes in DNA-dependent protein kinase (DNA-PK) accumulation in the nucleus. Moreover, phosphorylation of endogenous FEZ1 at S58 was reduced and HSPA8 and DNA-PK translocated to the nucleus in cells stimulated with DNA, suggesting that FEZ1 is a regulatory component of the recently identified HSPA8/DNA-PK innate immune pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSC70/metabolismo , Interferons/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Vírus de DNA/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Reguladores de Interferon/metabolismo , Proteínas de Membrana/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Células Vero
10.
Nat Commun ; 13(1): 679, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35115549

RESUMO

Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin-converting enzyme 2 (ACE2) expression. These macrophages are significantly enriched in the lungs of macaques at 3dpi and harbor SARS-CoV-2 while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery.


Assuntos
COVID-19/imunologia , Interferons/farmacologia , Células Mieloides/imunologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais , Lavagem Broncoalveolar , Modelos Animais de Doenças , Humanos , Imunidade Inata , Inflamação , Interferon Tipo I/genética , Interferon Tipo I/farmacologia , Interferons/genética , Pulmão/imunologia , Pulmão/patologia , Macaca mulatta , Macrófagos/imunologia , Linfócitos T/imunologia
11.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34983841

RESUMO

Macrophages induce a number of inflammatory response genes in response to stimulation with microbial ligands. In response to endotoxin Lipid A, a gene-activation cascade of primary followed by secondary-response genes is induced. Epigenetic state is an important regulator of the kinetics, specificity, and mechanism of gene activation of these two classes. In particular, SWI/SNF chromatin-remodeling complexes are required for the induction of secondary-response genes, but not primary-response genes, which generally exhibit open chromatin. Here, we show that a recently discovered variant of the SWI/SNF complex, the noncanonical BAF complex (ncBAF), regulates secondary-response genes in the interferon (IFN) response pathway. Inhibition of bromodomain-containing protein 9 (BRD9), a subunit of the ncBAF complex, with BRD9 bromodomain inhibitors (BRD9i) or a degrader (dBRD9) led to reduction in a number of interferon-stimulated genes (ISGs) following stimulation with endotoxin lipid A. BRD9-dependent genes overlapped highly with a subset of genes differentially regulated by BET protein inhibition with JQ1 following endotoxin stimulation. We find that the BET protein BRD4 is cobound with BRD9 in unstimulated macrophages and corecruited upon stimulation to ISG promoters along with STAT1, STAT2, and IRF9, components of the ISGF3 complex activated downstream of IFN-alpha receptor stimulation. In the presence of BRD9i or dBRD9, STAT1-, STAT2-, and IRF9-binding is reduced, in some cases with reduced binding of BRD4. These results demonstrate a specific role for BRD9 and the ncBAF complex in ISG activation and identify an activity for BRD9 inhibitors and degraders in dampening endotoxin- and IFN-dependent gene expression.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Interferons/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Antivirais/farmacologia , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Humanos , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Interferon-alfa/farmacologia , Interferons/genética , Interferons/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Domínios Proteicos , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos
12.
Inflammation ; 45(3): 1348-1361, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35044570

RESUMO

The oral mucosa is one of the first lines of the innate host defense system against microbial invasion. Interferon (IFN) lambda-1 (IFN-λ1), a type III IFN, exhibits type I IFN-like antiviral activity. In contrast to ubiquitously expressed type I IFN receptors, IFN-λ receptor 1 (IFN-λR1), which has higher affinity for type III IFNs than low-affinity interleukin (IL)-10 receptor 2, is mainly expressed on epithelial cells. Although IFN-λ1 has been shown to exert antiviral effects in the respiratory tract, gastrointestinal tract, and skin, the regulation of type III IFN receptor expression and its functions in the oral mucosa remain unclear. We herein showed the expression of IFN-λR1 in human gingival keratinocytes. The expression of IL-6, angiotensin-converting enzyme 2 (a critical molecule for severe acute respiratory syndrome coronavirus 2 infection), and IL-8 in human primary gingival keratinocytes (HGK) were significantly higher following treatments with either type I IFN (IFN-ß) or type II IFN (IFN-γ) than with IFN-λ1. However, the IFN-λ1 treatment strongly induced toll-like receptor (TLR) 3 and retinoic acid-inducible gene I (RIG-I), which mainly recognize viral nucleic acids, via the STAT1-mediated pathway. Furthermore, a stimulation with a RIG-I or TLR3 agonist promoted the production of IL-6, IL-8, and IFN-λ in HGK, which was significantly enhanced by a pretreatment with IFN-λ1. These results suggest that IFN-λ1 may contribute to the activation of innate immune responses to oral viral infections by up-regulating the expression of RIG-I and TLR3 and priming their functions in keratinocytes.


Assuntos
COVID-19 , Receptor 3 Toll-Like , Antivirais/farmacologia , Humanos , Interferons/farmacologia , Interleucina-6 , Interleucina-8 , Mucosa Bucal/metabolismo , Receptor 3 Toll-Like/genética
13.
Adv Drug Deliv Rev ; 182: 114112, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35085624

RESUMO

Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and elimination, and thus, there is a long clinical history associated with the use of recombinant cytokines to treat cancer. However, the use of cytokines as therapeutics has been limited by cytokine pleiotropy, complex biology, poor drug-like properties, and severe dose-limiting toxicities. Nevertheless, cytokines are crucial mediators of innate and adaptive antitumor immunity and have the potential to enhance immunotherapeutic approaches to treat cancer. Development of immune checkpoint inhibitors and combination immunotherapies has reinvigorated interest in cytokines as therapeutics, and a variety of engineering approaches are emerging to improve the safety and effectiveness of cytokine immunotherapy. In this review we highlight recent advances in cytokine biology and engineering for cancer immunotherapy.


Assuntos
Bioengenharia/métodos , Interferons/farmacologia , Interleucinas/farmacologia , Neoplasias/patologia , Biomimética , Sistemas de Liberação de Medicamentos/métodos , Engenharia Genética/métodos , Humanos , Concentração de Íons de Hidrogênio , Interferons/efeitos adversos , Interferons/metabolismo , Interferons/farmacocinética , Interleucinas/efeitos adversos , Interleucinas/metabolismo , Interleucinas/farmacocinética , Neoplasias/tratamento farmacológico
14.
J Virol ; 96(1): e0168221, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-34643436

RESUMO

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.


Assuntos
Vetores de Doenças , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/virologia , Ixodes/virologia , Animais , Células Cultivadas , Vírus da Encefalite Transmitidos por Carrapatos/classificação , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/transmissão , Células Endoteliais , Ordem dos Genes , Genoma Viral , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferons/farmacologia , Pericitos/virologia , Filogenia , Replicação Viral/efeitos dos fármacos
15.
Front Immunol ; 12: 767666, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34899719

RESUMO

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNß or IFNλ increased PD-L1 and PD-L2, and IFNß or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C-induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C-induced PD-L1. IC87114 enhanced poly I:C-induced gene expression of IFNß, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C-induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C-induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus-infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.


Assuntos
Adenina/análogos & derivados , Antígeno B7-H1/imunologia , Células Epiteliais/imunologia , Metapneumovirus/imunologia , Poli I-C/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Quinazolinas/farmacologia , Adenina/farmacologia , Asma/genética , Asma/imunologia , Asma/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Brônquios/citologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferons/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação/efeitos dos fármacos , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , /imunologia , /metabolismo
16.
mBio ; 12(6): e0275621, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34724828

RESUMO

Outbreaks of emerging viral pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a major medical challenge. There is a pressing need for antivirals that can be rapidly deployed to curb infection and dissemination. We determined the efficacy of interferon lambda-1 (IFN-λ) as a broad-spectrum antiviral agent to inhibit SARS-CoV-2 infection and reduce pathology in a mouse model of disease. IFN-λ significantly limited SARS-CoV-2 production in primary human bronchial epithelial cells in culture. Pretreatment of human lung cells with IFN-λ completely blocked infectious virus production, and treatment with IFN-λ at the time of infection inhibited virus production more than 10-fold. To interrogate the protective effects of IFN-λ in response to SARS-CoV-2 infection, transgenic mice expressing the human angiotensin-converting enzyme 2 (ACE-2) were tested. One dose of IFN-λ administered intranasally was found to reduce animal morbidity and mortality. Our study with SARS-CoV-2 also revealed a sex differential in disease outcome. Male mice had higher mortality, reflecting the more severe symptoms and mortality found in male patients infected with SARS-CoV-2. The results indicate that IFN-λ potentially can treat early stages of SARS-CoV-2 infection and decrease pathology, and this murine model can be used to investigate the sex differential documented in COVID-19. IMPORTANCE The COVID-19 pandemic has claimed millions of lives worldwide. In this report, we used a preclinical mouse model to investigate the prophylactic and therapeutic value of intranasal IFN-λ for this acute respiratory disease. Specific vaccines have been responsible for curbing the transmission of SARS-CoV-2 in developed nations. However, vaccines require time to generate and keep pace with antigenic variants. There is a need for broad-spectrum prophylactic and therapeutic agents to combat new emerging viral pathogens. Our mouse model suggests IFN-λ has clinical utility, and it reflects the well-documented finding that male COVID-19 patients manifest more severe symptoms and mortality. Understanding this sex bias is critical for considering therapeutic approaches to COVID-19.


Assuntos
Antivirais/uso terapêutico , COVID-19/imunologia , COVID-19/terapia , Células Epiteliais/efeitos dos fármacos , Interferons/imunologia , Interferons/farmacologia , SARS-CoV-2/imunologia , Administração Intranasal , Enzima de Conversão de Angiotensina 2/genética , Animais , Antivirais/farmacologia , Brônquios/citologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Células HEK293 , Humanos , Interferons/classificação , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Risco , SARS-CoV-2/efeitos dos fármacos , Fatores Sexuais
17.
Biochem Biophys Res Commun ; 585: 162-168, 2021 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-34808499

RESUMO

tRNase ZS (ELAC1) and TRNT1 function in tRNA recycling. Recently, we have shown that these genes are upregulated in the cells infected with Theiler's mouse encephalitis virus (TMEV), implying that tRNA recycling functions in response to viral infection. To address the molecular mechanism underlying the ELAC1 upregulation in the cells infected with TMEV, we performed luciferase assays using various plasmid constructs harboring the ELAC1 promoter region. The luciferase expression from a construct containing the full-length ELAC1 promoter was augmented by TMEV, poly IC, IFN-ß, or IFN-γ. We identified four IFN-stimulated responsible elements (ISREs) in the proximal promoter region. The luciferase expression from the constructs that lack all the ISREs was strongly reduced compared with that from the constructs with the four ISREs in the presence of IFN-ß or IFN-γ. The observation that the ISREs from the ELAC1 promoter are essential for the gene upregulation by IFN-ß or IFN-γ suggests that the ELAC1 gene is upregulated by IFNs.


Assuntos
Interferons/farmacologia , Regiões Promotoras Genéticas/genética , RNA de Transferência/genética , Transcrição Genética , Proteínas Supressoras de Tumor/genética , Regulação para Cima/efeitos dos fármacos , Antivirais/farmacologia , Sequência de Bases , Células HeLa , Humanos , Interferon beta/farmacologia , Interferon gama/farmacologia , RNA de Transferência/metabolismo , Elementos de Resposta/genética , Theilovirus/efeitos dos fármacos , Theilovirus/fisiologia , Regulação para Cima/genética
18.
Bull Exp Biol Med ; 172(1): 53-56, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34791556

RESUMO

The antiviral activity of recombinant human IFN-lambda type 1 (IFNλ-1) against culture strain of SARS-CoV-2 virus was determined by infecting a highly sensitive VeroE6 coronavirus cell culture after preincubation test (the cell monolayer was incubated with 4-fold dilutions of IFNλ-1 in a concentration range of 0.16-42,500 ng/ml in a culture medium for 12 h at 37°C) and without preincubation (simultaneous addition of different concentrations of IFNλ-1 and SARS-CoV-2 infection in a dose of 102 TCID50). The created recombinant human IFNλ-1 demonstrated obvious antiviral activity against SARS-CoV-2 virus in vitro. In the tests with and without preincubation, IFNλ-1 exhibited significant activity, although somewhat lower in variant with simultaneous addition of IFNλ-1 and virus to the cell culture. It should be noted that the antiviral effect of IFNλ-1 was observed in a wide range of concentrations.


Assuntos
Antivirais/farmacologia , Interferons/farmacologia , Proteínas Recombinantes/farmacologia , SARS-CoV-2/efeitos dos fármacos , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , COVID-19/tratamento farmacológico , COVID-19/virologia , Chlorocebus aethiops , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Interferons/biossíntese , Interferons/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/crescimento & desenvolvimento , Células Vero , Carga Viral/genética
19.
Front Immunol ; 12: 745802, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34671360

RESUMO

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.


Assuntos
Antivirais/farmacologia , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Poli dA-dT/farmacologia , Receptores de Superfície Celular/agonistas , Receptores de Reconhecimento de Padrão/agonistas , Receptores Virais/agonistas , Animais , Antivirais/uso terapêutico , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Citosol/virologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Hepatite B/imunologia , Hepatite B/virologia , Vírus da Hepatite B da Marmota/fisiologia , Hepatócitos/virologia , Imunidade Inata , Interferons/farmacologia , Fígado/efeitos dos fármacos , Fígado/virologia , Marmota , Poli dA-dT/uso terapêutico , Pteridinas/farmacologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Reconhecimento de Padrão/biossíntese , Receptores de Reconhecimento de Padrão/genética , Receptores Virais/biossíntese , Receptores Virais/genética , Replicação Viral/efeitos dos fármacos
20.
Science ; 374(6571): 1099-1106, 2021 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-34648371

RESUMO

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.


Assuntos
RNA Viral/genética , Replicon/fisiologia , SARS-CoV-2/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antivirais/farmacologia , Linhagem Celular , Humanos , Interferons/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Plasmídeos , RNA Viral/metabolismo , Replicon/genética , Genética Reversa , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Saccharomyces cerevisiae/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Vírion/genética , Vírion/fisiologia , Replicação Viral
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...