RESUMO
Many functionally important interactions between genes and proteins involved in immunological diseases and processes are unknown. The exponential growth in public high-throughput data offers an opportunity to expand this knowledge. To unlock human-immunology-relevant insight contained in the global biomedical research effort, including all public high-throughput datasets, we performed immunological-pathway-focused Bayesian integration of a comprehensive, heterogeneous compendium comprising 38,088 genome-scale experiments. The distillation of this knowledge into immunological networks of functional relationships between molecular entities (ImmuNet), and tools to mine this resource, are accessible to the public at http://immunet.princeton.edu. The predictive capacity of ImmuNet, established by rigorous statistical validation, is easily accessed by experimentalists to generate data-driven hypotheses. We demonstrate the power of this approach through the identification of unique host-virus interaction responses, and we show how ImmuNet complements genetic studies by predicting disease-associated genes. ImmuNet should be widely beneficial for investigating the mechanisms of the human immune system and immunological diseases.
Assuntos
Biologia Computacional/métodos , Doenças do Sistema Imunitário/imunologia , Sistema Imunitário/imunologia , Mapeamento de Interação de Proteínas/métodos , Transdução de Sinais/imunologia , Algoritmos , Teorema de Bayes , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Sistema Imunitário/metabolismo , Doenças do Sistema Imunitário/genética , Internet , Mapas de Interação de Proteínas/genética , Mapas de Interação de Proteínas/imunologia , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Máquina de Vetores de Suporte , Transcriptoma/genética , Transcriptoma/imunologia , Viroses/genética , Viroses/imunologia , Viroses/virologiaRESUMO
A major challenge in gene expression analysis is to accurately infer relevant biological insights, such as variation in cell-type proportion or pathway activity, from global gene expression studies. We present pathway-level information extractor (PLIER) ( https://github.com/wgmao/PLIER and http://gobie.csb.pitt.edu/PLIER ), a broadly applicable solution for this problem that outperforms available cell proportion inference algorithms and can automatically identify specific pathways that regulate gene expression. Our method improves interstudy replicability and reveals biological insights when applied to trans-eQTL (expression quantitative trait loci) identification.
Assuntos
Regulação da Expressão Gênica , Armazenamento e Recuperação da Informação , Algoritmos , Humanos , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions, are not merely extruded to maintain homeostatic cell numbers, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4+ T-cell activation. A common 'suppression of inflammation' signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4+ T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.
Assuntos
Apoptose , Células Epiteliais/citologia , Células Epiteliais/imunologia , Homeostase , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Fagócitos/citologia , Fagócitos/imunologia , Aminoácidos/metabolismo , Animais , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Cadeias alfa de Integrinas/metabolismo , Metabolismo dos Lipídeos , Linfonodos/imunologia , Ativação Linfocitária , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Fagócitos/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Transcrição GênicaRESUMO
A key unmet challenge in interpreting omics experiments is inferring biological meaning in the context of public functional genomics data. We developed a computational framework, Your Evidence Tailored Integration (YETI; http://yeti.princeton.edu/ ), which creates specialized functional interaction maps from large public datasets relevant to an individual omics experiment. Using this tailored integration, we predicted and experimentally confirmed an unexpected divergence in viral replication after seasonal or pandemic human influenza virus infection.
Assuntos
Interpretação Estatística de Dados , Redes Reguladoras de Genes , Genômica/métodos , Influenza Humana/genética , Orthomyxoviridae/fisiologia , Proteínas Virais/genética , Replicação Viral , Algoritmos , Células Cultivadas , Conjuntos de Dados como Assunto , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Influenza Humana/metabolismo , Influenza Humana/virologiaRESUMO
OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections cause coronavirus disease 2019 (COVID-19) and induce a specific antibody response. Serological assays detecting IgG against the receptor binding domain (RBD) of the spike (S) protein are useful to monitor the immune response after infection or vaccination. The objective of our study was to evaluate the clinical performance of the Siemens SARS-CoV-2 IgG (sCOVG) assay. METHODS: Sensitivity and specificity of the Siemens sCOVG test were evaluated on 178 patients with SARS-CoV-2-infection and 160 pre-pandemic samples in comparison with its predecessor test COV2G. Furthermore, correlation with virus neutralization titers was investigated on 134 samples of convalescent COVID-19 patients. RESULTS: Specificity of the sCOVG test was 99.4% and sensitivity was 90.5% (COV2G assay 78.7%; p<0.0001). S1-RBD antibody levels showed a good correlation with virus neutralization titers (r=0.843; p<0.0001) and an overall qualitative agreement of 98.5%. Finally, median S1-RBD IgG levels increase with age and were significantly higher in hospitalized COVID-19 patients (median levels general ward: 25.7 U/mL; intensive care: 59.5 U/mL) than in outpatients (3.8 U/mL; p<0.0001). CONCLUSIONS: Performance characteristics of the sCOVG assay have been improved compared to the predecessor test COV2G. Quantitative SARS-CoV-2 S1-RBD IgG levels could be used as a surrogate for virus neutralization capacity. Further harmonization of antibody quantification might assist to monitor the humoral immune response after COVID-19 disease or vaccination.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/diagnóstico , Imunoglobulina G/imunologia , Testes de Neutralização , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/patologia , COVID-19/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas/imunologia , Kit de Reagentes para Diagnóstico , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Adulto JovemRESUMO
The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-ß) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans.IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.
Assuntos
Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Proteínas não Estruturais Virais/genética , Animais , Aves , Células Dendríticas/imunologia , Células Dendríticas/virologia , Cães , Células Epiteliais/virologia , Regulação da Expressão Gênica , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H7N2/classificação , Vírus da Influenza A Subtipo H7N2/genética , Vírus da Influenza A Subtipo H7N2/imunologia , Subtipo H7N9 do Vírus da Influenza A/classificação , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Células Madin Darby de Rim Canino , Filogenia , Cultura Primária de Células , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas não Estruturais Virais/classificação , Proteínas não Estruturais Virais/imunologiaRESUMO
Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread.IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/metabolismo , Interferons/biossíntese , Interleucinas/biossíntese , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Interferons/genética , Interleucinas/genética , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Análise de Célula Única , Proteínas não Estruturais Virais/genéticaRESUMO
Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Gonadotrofos/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , Animais , Soluções Tampão , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Genes Precoces , Heterogeneidade Genética , Gonadotrofos/citologia , Gonadotrofos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Análise de Célula Única/normas , Ativação Transcricional/efeitos dos fármacos , TranscriptomaRESUMO
UNLABELLED: Influenza viruses continue to present global threats to human health. Antigenic drift and shift, genetic reassortment, and cross-species transmission generate new strains with differences in epidemiology and clinical severity. We compared the temporal transcriptional responses of human dendritic cells (DC) to infection with two pandemic (A/Brevig Mission/1/1918, A/California/4/2009) and two seasonal (A/New Caledonia/20/1999, A/Texas/36/1991) H1N1 influenza viruses. Strain-specific response differences included stronger activation of NF-κB following infection with A/New Caledonia/20/1999 and a unique cluster of genes expressed following infection with A/Brevig Mission/1/1918. A common antiviral program showing strain-specific timing was identified in the early DC response and found to correspond with reported transcript changes in blood during symptomatic human influenza virus infection. Comparison of the global responses to the seasonal and pandemic strains showed that a dramatic divergence occurred after 4 h, with only the seasonal strains inducing widespread mRNA loss. IMPORTANCE: Continuously evolving influenza viruses present a global threat to human health; however, these host responses display strain-dependent differences that are incompletely understood. Thus, we conducted a detailed comparative study assessing the immune responses of human DC to infection with two pandemic and two seasonal H1N1 influenza strains. We identified in the immune response to viral infection both common and strain-specific features. Among the stain-specific elements were a time shift of the interferon-stimulated gene response, selective induction of NF-κB signaling by one of the seasonal strains, and massive RNA degradation as early as 4 h postinfection by the seasonal, but not the pandemic, viruses. These findings illuminate new aspects of the distinct differences in the immune responses to pandemic and seasonal influenza viruses.
Assuntos
Células Dendríticas/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Pandêmica, 1918-1919/história , Influenza Humana/epidemiologia , Pandemias , Vírus Reordenados/imunologia , Variação Antigênica , Células Dendríticas/virologia , Europa (Continente)/epidemiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , História do Século XX , História do Século XXI , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Influenza Humana/história , Influenza Humana/imunologia , Interferons/genética , Interferons/imunologia , Epidemiologia Molecular , NF-kappa B/genética , NF-kappa B/imunologia , Vírus Reordenados/genética , Recombinação Genética , Estações do Ano , Transdução de Sinais , Fatores de Tempo , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Comparative analysis of genome-wide expression profiles are increasingly being used to study virus-specific host interactions. In order to gain mechanistic insights, gene expression profiles can be combined with information on DNA-binding sites of transcription factors to detect transcription factor activity (by analysis of target gene sets) during viral infections. Here, we apply this approach to study mechanisms of immune antagonism elicited by Influenza A virus (New Caledonia/20/1999) by comparing the transcriptional response with the non-pathogenic Newcastle disease virus (NDV), which lacks human immune antagonism. RESULTS: Existing gene set approaches do not quantify activity in a way that can be statistically compared between responses. We thus developed a new method for Bayesian Estimation of Transcription factor Activity (BETA) that allows for such quantification and comparative analysis across multiple responses. BETA predicted decreased ISGF3 activity during influenza A infection of human dendritic cells (reflected in lower expression of Interferon Stimulated Genes, ISGs). This prediction was confirmed through a combination of mathematical modeling and experiments at different multiplicities of infection to show that ISGs were specifically blocked in infected cells. Suppression of the transcription factor SATB1 was also predicted as a novel effect of influenza-mediated immune antagonism, and validated experimentally. CONCLUSIONS: Comparative analysis of genome-wide transcriptional profiles can reveal new effects of viral immune antagonism. We have developed a computational framework (BETA) that enables quantitative comparative analysis of transcription factor activities. This method will aid future studies to identify mechanistic differences in the host-pathogen interactions. Application of BETA to genome-wide transcriptional profiling data from human DCs identified SATB1 as a novel effect of influenza antagonism.
Assuntos
Perfilação da Expressão Gênica , Vírus da Influenza A/imunologia , Influenza Humana/genética , Influenza Humana/imunologia , Transcriptoma/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Interferons/farmacologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Modelos Imunológicos , Vírus da Doença de Newcastle/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) infection is chronic and presently still incurable. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 infection. Purinergic receptors are distributed throughout a wide variety of tissue types and detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in the transmission of HIV-1 from cell to cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection did not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, we observed CXCR4 colocalization with the newly internalized virus particles within target lymphocytes and found that the purinergic antagonists did not impair the recruitment of the coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y-selective receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target and that purinergic antagonists may have potent activity against viral infection of CD4+ T lymphocytes by both cell-free and cell-to-cell transmission. IMPORTANCE: This study identifies purinergic antagonists to be potent inhibitors of HIV-1 cell-free and cell-to-cell-mediated infection and provides a stepwise determination of when these compounds inhibit HIV-1 infection. These data provide a rationale for the development of novel antiretroviral therapies that have a dual role in both direct antiviral activity and the reduction of HIV-associated inflammation. Purinergic antagonists are shown here to have equivalent efficacy in inhibiting HIV infection via cell-free and cell-to-cell infection, and it is shown that purinergic receptors could provide an attractive therapeutic anti-HIV target that might avoid resistance by targeting a host signaling pathway that potently regulates HIV infection. The high-throughput screen of HIV-1 fusion inhibitors further defines P2X-selective compounds among the purinergic compounds as being the most potent HIV entry inhibitors. Clinical studies on these drugs for other inflammatory indications suggest that they are safe, and thus, if developed for use as anti-HIV agents, they could reduce both HIV replication and HIV-related inflammation.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Antagonistas Purinérgicos/farmacologia , Receptores Purinérgicos P2X/genética , Vírion/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Fusão Celular , Linhagem Celular , Células HEK293 , HIV-1/fisiologia , Humanos , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Purinérgicos P2X/metabolismo , Relação Estrutura-Atividade , Vírion/fisiologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
We show that influenza A H1N1 virus infection leads to very low infectivity in mouse dendritic cells (DCs) in vitro compared with that in human DCs. This holds when H3 or H5 replaces H1 in recombinant viruses. Viruslike particles confirm the difference between mouse and human, suggesting that reduced virus entry contributes to lower mouse DC infectivity. Low infectivity of mouse DCs should be considered when they are used to study responses of DCs that are actually infected.
Assuntos
Células Dendríticas/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Internalização do Vírus , Animais , Células Cultivadas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , CamundongosRESUMO
An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible "parental" strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a "stealth" strategy to evade detection by the host immune system.
Assuntos
Proteínas de Bactérias/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/tratamento farmacológico , Resistência a Vancomicina/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Mutação da Fase de Leitura , Perfilação da Expressão Gênica , Genótipo , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Vancomicina/farmacologiaRESUMO
BACKGROUND: During infection, neutrophil extracellular traps (NETs) are associated with severity of pulmonary diseases such as acute respiratory disease syndrome. NETs induce subsequent immune responses, are directly cytotoxic to pulmonary cells, and are highly procoagulant. Anticoagulation treatment was shown to reduce in-hospital mortality, indicating thromboinflammatory complications. However, data are sparsely available on the involvement of NETs in secondary events after virus clearance, which can lead to persistent lung damage and postacute sequelae with chronic fatigue and dyspnea. OBJECTIVES: This study focuses on late-phase events using a murine model of viral lung infection with postacute sequelae after virus resolution. METHODS: C57BL/6JRj mice were infected intranasally with the betacoronavirus murine coronavirus (MCoV, strain MHV-A95), and tissue samples were collected after 2, 4, and 10 days. For NET modulation, mice were pretreated with OM-85 or GSK484 and DNase I were administered intraperitoneally between days 2 to 5 and days 4 to 7, respectively. RESULTS: Rapid, platelet-attributed thrombus formation was followed by a second, late phase of thromboinflammation. This phase was characterized by negligible virus titers but pronounced tissue damage, apoptosis, oxidative DNA damage, and presence of NETs. Inhibition of NETs during the acute phase did not impact virus burden but decreased lung cell apoptosis by 67% and oxidative stress by 94%. Prevention of neutrophil activation by immune training before virus infection reduced damage by 75%, NETs by 31%, and pulmonary thrombi by 93%. CONCLUSION: NETs are detrimental inducers of tissue damage during respiratory virus infection but do not contribute to virus clearance.
Assuntos
Infecções por Coronavirus , Coronavirus , Armadilhas Extracelulares , Trombose , Animais , Camundongos , Neutrófilos , Tromboinflamação , Modelos Animais de Doenças , Inflamação/complicações , Trombose/complicações , Camundongos Endogâmicos C57BL , Pulmão , Infecções por Coronavirus/complicaçõesRESUMO
The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.
Assuntos
COVID-19 , Modelos Animais de Doenças , Proteína Ligante Fas , SARS-CoV-2 , Animais , Camundongos , Líquido da Lavagem Broncoalveolar , COVID-19/patologia , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/virologia , COVID-19/mortalidade , Proteína Ligante Fas/metabolismo , Inflamação/patologia , Inflamação/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Pulmão/patologia , Pulmão/virologia , Pulmão/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: H1N1 influenza viruses were responsible for the 1918 pandemic that caused millions of deaths worldwide and the 2009 pandemic that caused approximately twenty thousand deaths. The cellular response to such virus infections involves extensive genetic reprogramming resulting in an antiviral state that is critical to infection control. Identifying the underlying transcriptional network driving these changes, and how this program is altered by virally-encoded immune antagonists, is a fundamental challenge in systems immunology. RESULTS: Genome-wide gene expression patterns were measured in human monocyte-derived dendritic cells (DCs) infected in vitro with seasonal H1N1 influenza A/New Caledonia/20/1999. To provide a mechanistic explanation for the timing of gene expression changes over the first 12 hours post-infection, we developed a statistically rigorous enrichment approach integrating genome-wide expression kinetics and time-dependent promoter analysis. Our approach, TIme-Dependent Activity Linker (TIDAL), generates a regulatory network that connects transcription factors associated with each temporal phase of the response into a coherent linked cascade. TIDAL infers 12 transcription factors and 32 regulatory connections that drive the antiviral response to influenza. To demonstrate the generality of this approach, TIDAL was also used to generate a network for the DC response to measles infection. The software implementation of TIDAL is freely available at http://tsb.mssm.edu/primeportal/?q=tidal_prog. CONCLUSIONS: We apply TIDAL to reconstruct the transcriptional programs activated in monocyte-derived human dendritic cells in response to influenza and measles infections. The application of this time-centric network reconstruction method in each case produces a single transcriptional cascade that recapitulates the known biology of the response with high precision and recall, in addition to identifying potentially novel antiviral factors. The ability to reconstruct antiviral networks with TIDAL enables comparative analysis of antiviral responses, such as the differences between pandemic and seasonal influenza infections.
Assuntos
Biologia Computacional/métodos , Células Dendríticas/metabolismo , Regulação Viral da Expressão Gênica , Redes Reguladoras de Genes , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/genética , Influenza Humana/imunologia , Software , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Humanos , Influenza Humana/fisiopatologia , Influenza Humana/virologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the 'sensitivity improved two-test' or 'SIT²' algorithm. METHODS: SIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives). RESULTS: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. CONCLUSION: For SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Estudos Soroepidemiológicos , Técnicas de Laboratório Clínico/métodos , Teste para COVID-19 , Sensibilidade e EspecificidadeAssuntos
Arachis/imunologia , Basófilos/imunologia , Hipersensibilidade a Amendoim/sangue , Hipersensibilidade a Amendoim/imunologia , Alérgenos/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Citometria de Fluxo/métodos , Humanos , Imunoglobulina E/imunologia , Contagem de Leucócitos/métodos , Espectrometria de Massas/métodosRESUMO
Training of the innate immune system with orally ingested bacterial extracts was demonstrated to have beneficial effects on infection clearance and disease outcome. The aim of our study was to identify cellular and molecular processes responsible for these immunological benefits. We used a murine coronavirus (MCoV) A59 mouse model treated with the immune activating bacterial extract Broncho-Vaxom (BV) OM-85. Tissue samples were analysed with qPCR, RNA sequencing, histology, and flow cytometry. After BV OM-85 treatment, interstitial macrophages accumulated in lung tissue leading to a faster response of type I interferon (IFN) signalling after MCoV infection resulting in overall lung tissue protection. Moreover, RNA sequencing showed that lung tissue from mice receiving BV OM-85 resembled an intermediate stage between healthy and viral infected lung tissue at day 4, indicating a faster return to normal tissue homoeostasis. The pharmacologic effect was mimicked by adoptively transferring naive lung macrophages into lungs from recipient mice before virus infection. The beneficial effect of BV OM-85 was abolished when inhibiting initial type I IFN signalling. Overall, our data suggest that BV OM-85 enhances lung macrophages allowing for a faster IFN response towards a viral challenge as part of the oral-induced innate immune system training.
Assuntos
Adjuvantes Imunológicos , Betacoronavirus , Animais , Bactérias , Imunidade Inata , Pulmão , Macrófagos , CamundongosRESUMO
The presence of neutralizing antibodies against SARS-CoV-2 in a large number of people is - besides cellular immunity - important to overcome the SARS-CoV-2 pandemic. While determination of neutralizing antibodies via virus neutralization tests are laborious, assays to determine the antibody levels serologically are fully automated and widely available. Correlations between these methodologies were recently given by the manufacturers, however performance in samples close to the cut off value have not yet been fully validated. Thus, we analysed 22 borderline and low positive (<100 BAU/ml) samples and 9 high positive (≥ 100 BAU/ml) from infected and/or vaccinated individuals and compared the SARS-CoV-2 IgG II Quant assay (Abbott), LIAISON SARS-CoV-2 TrimericS IgG (Diasorin), Elecsys Anti-SARS-CoV-2 S (Roche), and SARS-CoV-2 IgG (Siemens) with results obtained from a virus neutralization test. Based on the cut off values given by Abbott, Diasorin, Roche, and Siemens, the positive serologic results were concordant with the virus neutralization test in 100%, 76%, 88%, and 71%, respectively, while in turn, negative ones were in agreement in 29%, 79%, 93%, and 86%, respectively. In conclusion, weakly positive, serologic results are challenging to correctly predict the presence of neutralizing antibodies. Our study suggests, that different cut off values (for positivity vs. presence of neutralizing antibodies) could improve the test's performance, but determination thereof requires more samples to be analysed.