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1.
Nature ; 606(7914): 576-584, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35385861

RESUMEN

SARS-CoV-2 can cause acute respiratory distress and death in some patients1. Although severe COVID-19 is linked to substantial inflammation, how SARS-CoV-2 triggers inflammation is not clear2. Monocytes and macrophages are sentinel cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D, leading to inflammatory death (pyroptosis) and the release of potent inflammatory mediators3. Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2. Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors. The plasma of vaccine recipients does not promote antibody-dependent monocyte infection. SARS-CoV-2 begins to replicate in monocytes, but infection is aborted, and infectious virus is not detected in the supernatants of cultures of infected monocytes. Instead, infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes, caspase-1 and gasdermin D. Moreover, tissue-resident macrophages, but not infected epithelial and endothelial cells, from lung autopsies from patients with COVID-19 have activated inflammasomes. Taken together, these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis.


Asunto(s)
COVID-19 , Inflamación , Monocitos , Receptores de IgG , SARS-CoV-2 , COVID-19/virología , Caspasa 1/metabolismo , Proteínas de Unión al ADN , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Inflamación/virología , Monocitos/metabolismo , Monocitos/virología , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas de Unión a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Receptores de IgG/metabolismo
2.
J Immunol ; 205(9): 2479-2488, 2020 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-32978279

RESUMEN

The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Expresión Génica/genética , Linfotoxina-alfa/genética , Factor de Necrosis Tumoral alfa/genética , Acetilación , Sitios de Unión/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Secuencia Conservada/genética , Elementos de Facilitación Genéticos/genética , Histonas/genética , Humanos , Leucocitos Mononucleares/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , ARN Polimerasa II/genética , ARN Mensajero/genética , Células THP-1/metabolismo , Transcripción Genética/genética
3.
Immunol Rev ; 264(1): 74-87, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25703553

RESUMEN

The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T-cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4(+) and CD8(+) T-cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis-specific T-cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T-cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis-specific adaptive immunity during human immunodeficiency virus co-infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection.


Asunto(s)
Inmunidad Adaptativa , Interacciones Huésped-Patógeno/inmunología , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Tuberculosis/inmunología , Vacuna BCG/inmunología , Coinfección , Citocinas/metabolismo , Infecciones por VIH/inmunología , Humanos , Inmunomodulación , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/metabolismo , Tuberculosis/metabolismo , Tuberculosis/microbiología , Tuberculosis/prevención & control
4.
Methods ; 112: 91-104, 2017 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-27642004

RESUMEN

Imaging flow cytometry has been applied to address questions in infection biology, in particular, infections induced by intracellular pathogens. This methodology, which utilizes specialized analytic software makes it possible to analyze hundreds of quantified features for hundreds of thousands of individual cellular or subcellular events in a single experiment. Imaging flow cytometry analysis of host cell-pathogen interaction can thus quantitatively addresses a variety of biological questions related to intracellular infection, including cell counting, internalization score, and subcellular patterns of co-localization. Here, we provide an overview of recent achievements in the use of fluorescently labeled prokaryotic or eukaryotic pathogens in human cellular infections in analysis of host-pathogen interactions. Specifically, we give examples of Imagestream-based analysis of cell lines infected with Toxoplasma gondii or Mycobacterium tuberculosis. Furthermore, we illustrate the capabilities of imaging flow cytometry using a combination of standard IDEAS™ software and the more recently developed Feature Finder algorithm, which is capable of identifying statistically significant differences between researcher-defined image galleries. We argue that the combination of imaging flow cytometry with these software platforms provides a powerful new approach to understanding host control of intracellular pathogens.


Asunto(s)
Citometría de Flujo/métodos , Interacciones Huésped-Patógeno , Citometría de Imagen/métodos , Mycobacterium tuberculosis/metabolismo , Programas Informáticos , Toxoplasma/metabolismo , Algoritmos , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Citometría de Flujo/instrumentación , Colorantes Fluorescentes/química , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Citometría de Imagen/instrumentación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mycobacterium tuberculosis/ultraestructura , Fagocitosis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Coloración y Etiquetado/métodos , Células THP-1 , Toxoplasma/ultraestructura , Proteína Fluorescente Roja
5.
Thorax ; 70(12): 1181-8, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26506854

RESUMEN

BACKGROUND: In Africa, fewer than half of patients receiving therapy for multidrug-resistant TB (MDR TB) are successfully treated, with poor outcomes reported for HIV-coinfected patients. METHODS: A standardised second-line drug (SLD) regimen was used in a non-governmental organisation-Ministry of Health (NGO-MOH) collaborative community and hospital-based programme in Ethiopia that included intensive side effect monitoring, adherence strategies and nutritional supplementation. Clinical outcomes for patients with at least 24 months of follow-up were reviewed and predictors of treatment failure or death were evaluated by Cox proportional hazards models. RESULTS: From February 2009 to December 2014, 1044 patients were initiated on SLD. 612 patients with confirmed or presumed MDR TB had ≥ 24 months of follow-up, 551 (90.0%) were confirmed and 61 (10.0%) were suspected MDR TB cases. 603 (98.5%) had prior TB treatment, 133 (21.7%) were HIV coinfected and median body mass index (BMI) was 16.6. Composite treatment success was 78.6% with 396 (64.7%) cured, 85 (13.9%) who completed treatment, 10 (1.6%) who failed, 85 (13.9%) who died and 36 (5.9%) who were lost to follow-up. HIV coinfection (adjusted HR (AHR): 2.60, p<0.001), BMI (AHR 0.88/kg/m(2), p=0.006) and cor pulmonale (AHR 3.61, p=0.003) and confirmed MDR TB (AHR 0.50, p=0.026) were predictive of treatment failure or death. CONCLUSIONS: We report from Ethiopia the highest MDR TB treatment success outcomes so far achieved in Africa, in a setting with severe resource constraints and patients with advanced disease. Intensive treatment of adverse effects, nutritional supplementation, adherence interventions and NGO-MOH collaboration were key strategies contributing to success. We argue these approaches should be routinely incorporated into programmes.


Asunto(s)
Tuberculosis Resistente a Múltiples Medicamentos/terapia , Adulto , Coinfección , Etiopía , Femenino , Infecciones por VIH/epidemiología , Humanos , Masculino , Cumplimiento de la Medicación , Modelos de Riesgos Proporcionales , Resultado del Tratamiento , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/mortalidad , Adulto Joven
6.
Clin Infect Dis ; 59(3): 435-45, 2014 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-24759827

RESUMEN

BACKGROUND: Shortening the interval between antituberculosis treatment onset and initiation of antiretroviral therapy (ART) reduces mortality in severely immunocompromised human immunodeficiency virus (HIV)-infected patients with tuberculosis. A better understanding of causes and determinants of death may lead to new strategies to further enhance survival. METHODS: We assessed mortality rates, causes of death, and factors of mortality in Cambodian HIV-infected adults with CD4 count ≤200 cells/µL and tuberculosis, randomized to initiate ART either 2 weeks (early ART) or 8 weeks (late ART) after tuberculosis treatment onset in the CAMELIA clinical trial. RESULTS: Six hundred sixty-one patients enrolled contributed to 1366.1 person-years of follow-up; 149 (22.5%) died. There were 8.3 deaths per 100 person-years (95% confidence interval [CI], 6.4-10.7) in the early-ART group and 13.8 deaths per 100 person-years (95% CI, 11.2-16.9) in the late-ART group (P = .002). Tuberculosis was the primary cause of death (28%), followed by other HIV-associated conditions (19%). Factors independently associated with mortality in the first 26 weeks were the age, body mass index, hemoglobin, interrupted or ineffective tuberculosis treatment before identification of drug resistance, disseminated tuberculosis, and nontuberculous mycobacterial disease. After 50 weeks in the trial, the most frequent causes of death were non-HIV related or tuberculosis related, including drug toxicity; factors associated with mortality were late ART, loss to follow-up, and absence of cotrimoxazole prophylaxis. CONCLUSIONS: Despite ART introduction, mortality remained high, with tuberculosis as the leading cause of death. Reducing tuberculosis-related mortality remains a challenge in resource-limited settings and requires innovative strategies. Clinical Trials Registration. NCT00226434.


Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Antituberculosos/uso terapéutico , Infecciones por VIH/mortalidad , Tuberculosis/mortalidad , Adulto , Antiinfecciosos/uso terapéutico , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Humanos , Huésped Inmunocomprometido , Masculino , Combinación Trimetoprim y Sulfametoxazol/uso terapéutico , Tuberculosis/complicaciones , Tuberculosis/tratamiento farmacológico
7.
N Engl J Med ; 365(16): 1471-81, 2011 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-22010913

RESUMEN

BACKGROUND: Tuberculosis remains an important cause of death among patients infected with the human immunodeficiency virus (HIV). Robust data are lacking with regard to the timing for the initiation of antiretroviral therapy (ART) in relation to the start of antituberculosis therapy. METHODS: We tested the hypothesis that the timing of ART initiation would significantly affect mortality among adults not previously exposed to antiretroviral drugs who had newly diagnosed tuberculosis and CD4+ T-cell counts of 200 per cubic millimeter or lower. After beginning the standard, 6-month treatment for tuberculosis, patients were randomly assigned to either earlier treatment (2 weeks after beginning tuberculosis treatment) or later treatment (8 weeks after) with stavudine, lamivudine, and efavirenz. The primary end point was survival. RESULTS: A total of 661 patients were enrolled and were followed for a median of 25 months. The median CD4+ T-cell count was 25 per cubic millimeter, and the median viral load was 5.64 log(10) copies per milliliter. The risk of death was significantly reduced in the group that received ART earlier, with 59 deaths among 332 patients (18%), as compared with 90 deaths among 329 patients (27%) in the later-ART group (hazard ratio, 0.62; 95% confidence interval [CI]; 0.44 to 0.86; P=0.006). The risk of tuberculosis-associated immune reconstitution inflammatory syndrome was significantly increased in the earlier-ART group (hazard ratio, 2.51; 95% CI, 1.78 to 3.59; P<0.001). Irrespective of the study group, the median gain in the CD4+ T-cell count was 114 per cubic millimeter, and the viral load was undetectable at week 50 in 96.5% of the patients. CONCLUSIONS: Initiating ART 2 weeks after the start of tuberculosis treatment significantly improved survival among HIV-infected adults with CD4+ T-cell counts of 200 per cubic millimeter or lower. (Funded by the French National Agency for Research on AIDS and Viral Hepatitis and the National Institutes of Health; CAMELIA ClinicalTrials.gov number, NCT01300481.).


Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Antirretrovirales/administración & dosificación , Antituberculosos/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Tuberculosis/tratamiento farmacológico , Adulto , Antirretrovirales/efectos adversos , Recuento de Linfocito CD4 , Esquema de Medicación , Femenino , Estudios de Seguimiento , Infecciones por VIH/complicaciones , Infecciones por VIH/mortalidad , Humanos , Estimación de Kaplan-Meier , Masculino , Tuberculosis/complicaciones , Carga Viral
8.
PLoS Pathog ; 8(4): e1002620, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22496647

RESUMEN

Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.


Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/fisiología , Mycobacterium tuberculosis/metabolismo , Transducción de Señal/inmunología , Receptores Toll-Like/metabolismo , Factores de Transcripción/metabolismo , Tuberculosis/metabolismo , Replicación Viral/inmunología , Células Cultivadas , Femenino , Regulación de la Expresión Génica/inmunología , Infecciones por VIH/inmunología , Humanos , Inmunidad Innata , Quinasas Asociadas a Receptores de Interleucina-1/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Masculino , Mycobacterium tuberculosis/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Regiones Promotoras Genéticas/inmunología , Receptores Toll-Like/inmunología , Factores de Transcripción/inmunología , Tuberculosis/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo
9.
J Biol Chem ; 286(51): 44126-44133, 2011 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-22027829

RESUMEN

Regulation of TNF gene expression is cell type- and stimulus-specific. We have previously identified highly conserved noncoding regulatory elements within DNase I-hypersensitive sites (HSS) located 9 kb upstream (HSS-9) and 3 kb downstream (HSS+3) of the TNF gene, which play an important role in the transcriptional regulation of TNF in T cells. They act as enhancers and interact with the TNF promoter and with each other, generating a higher order chromatin structure. Here, we report a novel monocyte-specific AT-rich DNase I-hypersensitive element located 7 kb upstream of the TNF gene (HSS-7), which serves as a matrix attachment region in monocytes. We show that HSS-7 associates with topoisomerase IIα (Top2) in vivo and that induction of endogenous TNF mRNA expression is suppressed by etoposide, a Top2 inhibitor. Moreover, Top2 binds to and cleaves HSS-7 in in vitro analysis. Thus, HSS-7, which is selectively accessible in monocytes, can tether the TNF locus to the nuclear matrix via matrix attachment region formation, potentially promoting TNF gene expression by acting as a Top2 substrate.


Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Monocitos/citología , Secuencia de Bases , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Células HeLa , Histonas , Humanos , Datos de Secuencia Molecular , Proteínas de Unión a Poli-ADP-Ribosa , Linfocitos T/citología , Transcripción Genética
10.
bioRxiv ; 2022 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-35169796

RESUMEN

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

11.
Am J Respir Cell Mol Biol ; 45(6): 1116-24, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21852682

RESUMEN

In this review, we examine how a subset of signal transduction cascades initiated by Mycobacterium tuberculosis (Mtb) infection modulates transcription mediated by the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). We describe two distinct phases of signaling that target transcription factors known to bind the HIV-1 LTR, and thus drive viral transcription and replication, in cells of the Mtb-infected host. First, Mtb-derived molecules, including cell wall components and DNA, interact with a number of host pattern recognition receptors. Second, cytokines and chemokines secreted in response to Mtb infection initiate signal transduction cascades through their cognate receptors. Given the variation in cell wall components among distinct clinical Mtb strains, the initial pattern recognition receptor interaction leading to direct LTR activation and differential cytokine and chemokine production is likely to be an important aspect of Mtb strain-specific regulation of HIV-1 transcription and replication. Improved understanding of these molecular mechanisms in the context of bacterial and host genetics should provide key insights into the accelerated viral replication and disease progression characteristic of HIV/TB coinfection.


Asunto(s)
Infecciones por VIH , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , Mycobacterium tuberculosis/metabolismo , Tuberculosis , Replicación Viral/fisiología , Animales , Citocinas/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Infecciones por VIH/microbiología , Humanos , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética , Tuberculosis/complicaciones , Tuberculosis/metabolismo , Tuberculosis/virología
12.
Curr Dir Autoimmun ; 11: 27-60, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20173386

RESUMEN

The cytokine TNF is a critical mediator of immune and inflammatory responses. The TNF gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving the recruitment of distinct sets of transcription factors to a compact and modular promoter region. In this review, we describe our current understanding of the mechanisms through which TNF transcription is specifically activated by a variety of extracellular stimuli in multiple cell types, including T cells, B cells, macrophages, mast cells, dendritic cells, and fibroblasts. We discuss the role of nuclear factor of activated T cells and other transcription factors and coactivators in enhanceosome formation, as well as the contradictory evidence for a role for nuclear factor kappaB as a classical activator of the TNF gene. We describe the impact of evolutionarily conserved cis-regulatory DNA motifs in the TNF locus upon TNF gene transcription, in contrast to the neutral effect of single nucleotide polymorphisms. We also assess the regulatory role of chromatin organization, epigenetic modifications, and long-range chromosomal interactions at the TNF locus.


Asunto(s)
Factor de Necrosis Tumoral alfa/genética , Animales , Secuencia de Bases , Ensamble y Desensamble de Cromatina , ADN/genética , Epigénesis Genética , Genes Inmediatos-Precoces , Humanos , Ratones , Datos de Secuencia Molecular , FN-kappa B/inmunología , Factores de Transcripción NFATC/inmunología , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Factores de Transcripción/inmunología , Transcripción Genética , Activación Transcripcional , Factor de Necrosis Tumoral alfa/fisiología
13.
Proc Natl Acad Sci U S A ; 105(50): 19637-42, 2008 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-19060202

RESUMEN

The transcription factor NFATp integrates multiple signal transduction pathways through coordinate binding with basic-region leucine zipper (bZIP) proteins and other transcription factors. The NFATp monomer, even in the absence of its activation domains, recruits bZIP proteins to canonical NFAT-bZIP composite DNA elements. By contrast, the NFATp dimer and its bZIP partner bind noncooperatively to the NFAT-bZIP element of the tumor necrosis factor (TNF) gene promoter. This observation raises the possibility that the function of the activation domains of NFATp is dimer-specific. Here, we determine the consensus DNA binding site of the NFATp dimer, describe monomer- and dimer-specific NFATp-DNA contact patterns, and demonstrate that NFATp dimerization and dimer-specific activation subdomains are required for transcriptional activation from the TNF NFAT-bZIP element. We also show that these NFATp subdomains interact with the coactivator CBP (CREB-binding protein), which is required for NFATp-dependent TNF gene transcription. Thus, the context-specific function of the activation domains of NFAT can be potentiated by DNA-directed dimerization.


Asunto(s)
Factores de Transcripción NFATC/metabolismo , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteína de Unión a CREB/metabolismo , Secuencia de Consenso , ADN/metabolismo , Dimerización , Ensayo de Cambio de Movilidad Electroforética , Factores de Transcripción NFATC/química , Factores de Transcripción NFATC/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Transcripción Genética , Factor de Necrosis Tumoral alfa/genética
14.
medRxiv ; 2021 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-33758872

RESUMEN

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in some patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes are sentinel blood cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that ~10% of blood monocytes in COVID-19 patients are dying and infected with SARS-CoV-2. Monocyte infection, which depends on antiviral antibodies, activates NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD cleavage and relocalization. Signs of pyroptosis (IL-1 family cytokines, LDH) in the plasma correlate with development of severe disease. Moreover, expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes triggers inflammation that contributes to severe COVID-19 disease pathogenesis. ONE SENTENCE SUMMARY: Antibody-mediated SARS-CoV-2 infection of monocytes activates inflammation and cytokine release.

15.
Res Sq ; 2021 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-34401873

RESUMEN

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in a minority of patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes and macrophages are sentinel immune cells in the blood and tissue, respectively, that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). We find that about 10% of blood monocytes in COVID-19 patients are infected with SARS-CoV-2. Monocyte infection depends on viral antibody opsonization and uptake of opsonized virus by the Fc receptor CD16. After uptake, SARS-CoV-2 begins to replicate in monocytes, as evidenced by detection of double-stranded RNA and subgenomic RNA and expression of a fluorescent reporter gene. However, infection is aborted, and infectious virus is not detected in infected monocyte supernatants or patient plasma. Instead, infected cells undergo inflammatory cell death (pyroptosis) mediated by activation of the NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD. Moreover, tissue-resident macrophages, but not infected epithelial cells, from COVID-19 lung autopsy specimens showed evidence of inflammasome activation. These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes/macrophages triggers inflammatory cell death that aborts production of infectious virus but causes systemic inflammation that contributes to severe COVID-19 disease pathogenesis.

16.
iScience ; 22: 299-313, 2019 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-31805434

RESUMEN

To establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

17.
iScience ; 19: 1279-1290, 2019 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-31402258

RESUMEN

Here, we show that the US Food and Drug Administration-approved oral drug nitazoxanide (NTZ) broadly amplifies the host innate immune response to viruses and inhibits Ebola virus (EBOV) replication. We find that NTZ enhances retinoic-acid-inducible protein I (RIG-I)-like-receptor, mitochondrial antiviral signaling protein, interferon regulatory factor 3, and interferon activities and induces transcription of the antiviral phosphatase GADD34. NTZ significantly inhibits EBOV replication in human cells through its effects on RIG-I and protein kinase R (PKR), suggesting that it counteracts EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV. NTZ also inhibits a second negative-strand RNA virus, vesicular stomatitis virus (VSV), through RIG-I and GADD34, but not PKR, consistent with VSV's distinct host innate immune evasion mechanisms. Thus, NTZ counteracts varied virus-specific immune evasion strategies by generally enhancing the RNA sensing and interferon axis that is triggered by foreign cytoplasmic RNA exposure, and holds promise as an oral therapy against EBOV.

18.
PLoS Pathog ; 2(12): e130, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17173480

RESUMEN

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.


Asunto(s)
VIH-1/fisiología , Macrófagos/virología , Interferencia de ARN , Secuencias Repetidas Terminales/fisiología , Factores de Transcripción/fisiología , Replicación Viral/fisiología , Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Línea Celular , Células Cultivadas , ADN Viral , Progresión de la Enfermedad , Regulación de la Expresión Génica , VIH-1/genética , VIH-1/patogenicidad , Células HeLa , Humanos , Macrófagos/fisiología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetidas Terminales/genética , Factores de Transcripción/genética , Replicación Viral/genética
19.
Curr Opin HIV AIDS ; 13(6): 478-485, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30286039

RESUMEN

PURPOSE OF REVIEW: This review aims to describe the key principles in treatment of drug-resistant tuberculosis (TB) in people living with HIV, including early access to timely diagnostics, linkage into care, TB treatment strategies including the use of new and repurposed drugs, co-management of HIV disease, and treatment complications and programmatic support to optimize treatment outcomes. These are necessary strategies to decrease the likelihood of poor treatment outcomes including lower treatment completion rates and higher mortality. RECENT FINDINGS: Diagnosis of drug-resistant TB is the gateway into care; yet understanding the utility and the limitations of genotypic methods in this population is necessary. The principles of TB treatment in HIV-infected individuals are similar to those without HIV co-infection, with few exceptions. However, adverse effects with potential significant morbidity may emerge during treatment, and timely antiretroviral therapy is essential to improve mortality in this patient population. Emerging data on the use of new and repurposed drugs and short course multidrug-resistant TB regimens and adherence strategies benefiting this population are reviewed. SUMMARY: The clinical complexity of co-managing drug-resistant TB and HIV, and the higher rate of poor treatment outcomes in this population demand careful clinical management strategies, and multidisciplinary and comprehensive programmatic interventions to optimize treatment success in this vulnerable group.


Asunto(s)
Antituberculosos/administración & dosificación , Infecciones por VIH/complicaciones , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Animales , Coinfección/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Mycobacterium tuberculosis/fisiología , Resultado del Tratamiento , Tuberculosis Resistente a Múltiples Medicamentos/complicaciones , Tuberculosis Resistente a Múltiples Medicamentos/microbiología
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