RESUMEN
Whole blood transcriptional signatures distinguishing active tuberculosis patients from asymptomatic latently infected individuals exist. Consensus has not been achieved regarding the optimal reduced gene sets as diagnostic biomarkers that also achieve discrimination from other diseases. Here we show a blood transcriptional signature of active tuberculosis using RNA-Seq, confirming microarray results, that discriminates active tuberculosis from latently infected and healthy individuals, validating this signature in an independent cohort. Using an advanced modular approach, we utilise the information from the entire transcriptome, which includes overabundance of type I interferon-inducible genes and underabundance of IFNG and TBX21, to develop a signature that discriminates active tuberculosis patients from latently infected individuals or those with acute viral and bacterial infections. We suggest that methods targeting gene selection across multiple discriminant modules can improve the development of diagnostic biomarkers with improved performance. Finally, utilising the modular approach, we demonstrate dynamic heterogeneity in a longitudinal study of recent tuberculosis contacts.
Asunto(s)
Interferón gamma/metabolismo , Proteínas de Dominio T Box/metabolismo , Transcriptoma , Tuberculosis Pulmonar/metabolismo , Adulto , Anciano , Área Bajo la Curva , Biomarcadores/metabolismo , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , Inmunosupresores/química , Interferón gamma/genética , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Curva ROC , Riesgo , Análisis de Secuencia de ARN , Proteínas de Dominio T Box/genética , Transcripción Genética , Tuberculosis Pulmonar/genéticaRESUMEN
BACKGROUND: Mycobacterium tuberculosis infection is a leading cause of infectious death worldwide. Gene-expression microarray studies profiling the blood transcriptional response of tuberculosis (TB) patients have been undertaken in order to better understand the host immune response as well as to identify potential biomarkers of disease. To date most of these studies have focused on pulmonary TB patients with gene-expression profiles of extra-pulmonary TB patients yet to be compared to those of patients with pulmonary TB or sarcoidosis. METHODS: A novel cohort of patients with extra-pulmonary TB and sarcoidosis was recruited and the transcriptional response of these patients compared to those with pulmonary TB using a variety of transcriptomic approaches including testing a previously defined 380 gene meta-signature of active TB. RESULTS: The 380 meta-signature broadly differentiated active TB from healthy controls in this new dataset consisting of pulmonary and extra-pulmonary TB. The top 15 genes from this meta-signature had a lower sensitivity for differentiating extra-pulmonary TB from healthy controls as compared to pulmonary TB. We found the blood transcriptional responses in pulmonary and extra-pulmonary TB to be heterogeneous and to reflect the extent of symptoms of disease. CONCLUSIONS: The transcriptional signature in extra-pulmonary TB demonstrated heterogeneity of gene expression reflective of symptom status, while the signature of pulmonary TB was distinct, based on a higher proportion of symptomatic individuals. These findings are of importance for the rational design and implementation of mRNA based TB diagnostics.
Asunto(s)
Transcriptoma , Tuberculosis Pulmonar/diagnóstico , Tuberculosis/diagnóstico , Recuento de Células Sanguíneas , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mycobacterium tuberculosis/aislamiento & purificación , Sarcoidosis/diagnóstico , Sarcoidosis/genética , Sarcoidosis/metabolismo , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/metabolismo , Regulación hacia ArribaRESUMEN
The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.
Asunto(s)
Sangre/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Humanos , Técnicas In Vitro , Lipopéptidos/metabolismo , Lipopolisacáridos/metabolismo , Análisis por Micromatrices , FN-kappa B/metabolismo , Factores de TiempoRESUMEN
RATIONALE: New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. OBJECTIVES: To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. METHODS: We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. MEASUREMENTS AND MAIN RESULTS: An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. CONCLUSIONS: Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.
Asunto(s)
Neoplasias Pulmonares/sangre , Neumonía/sangre , Sarcoidosis Pulmonar/sangre , Transcriptoma , Tuberculosis Pulmonar/sangre , Antituberculosos/uso terapéutico , Biomarcadores/sangre , Estudios de Casos y Controles , Glucocorticoides/uso terapéutico , Humanos , Mediadores de Inflamación/sangre , Interferones/fisiología , Neoplasias Pulmonares/diagnóstico , Neutrófilos/metabolismo , Neumonía/diagnóstico , Neumonía/tratamiento farmacológico , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , Sarcoidosis Pulmonar/diagnóstico , Sarcoidosis Pulmonar/tratamiento farmacológico , Transcripción Genética , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/tratamiento farmacológicoRESUMEN
There are 9 million cases of active tuberculosis reported annually; however, an estimated one-third of the world's population is infected with Mycobacterium tuberculosis and remains asymptomatic. Of these latent individuals, only 5-10% will develop active tuberculosis disease in their lifetime. CD4(+) T cells, as well as the cytokines IL-12, IFN-γ, and TNF, are critical in the control of Mycobacterium tuberculosis infection, but the host factors that determine why some individuals are protected from infection while others go on to develop disease are unclear. Genetic factors of the host and of the pathogen itself may be associated with an increased risk of patients developing active tuberculosis. This review aims to summarize what we know about the immune response in tuberculosis, in human disease, and in a range of experimental models, all of which are essential to advancing our mechanistic knowledge base of the host-pathogen interactions that influence disease outcome.
Asunto(s)
Tuberculosis Pulmonar/inmunología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Predisposición Genética a la Enfermedad , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Mycobacterium tuberculosis/inmunología , Resultado del Tratamiento , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/epidemiologíaRESUMEN
RATIONALE: Globally there are approximately 9 million new active tuberculosis cases and 1.4 million deaths annually. Effective antituberculosis treatment monitoring is difficult as there are no existing biomarkers of poor adherence or inadequate treatment earlier than 2 months after treatment initiation. Inadequate treatment leads to worsening disease, disease transmission and drug resistance. OBJECTIVES: To determine if blood transcriptional signatures change in response to antituberculosis treatment and could act as early biomarkers of a successful response. METHODS: Blood transcriptional profiles of untreated active tuberculosis patients in South Africa were analysed before, during (2 weeks and 2 months), at the end of (6 months) and after (12 months) antituberculosis treatment, and compared to individuals with latent tuberculosis. An active-tuberculosis transcriptional signature and a specific treatment-response transcriptional signature were derived. The specific treatment response transcriptional signature was tested in two independent cohorts. Two quantitative scoring algorithms were applied to measure the changes in the transcriptional response. The most significantly represented pathways were determined using Ingenuity Pathway Analysis. RESULTS: An active tuberculosis 664-transcript signature and a treatment specific 320-transcript signature significantly diminished after 2 weeks of treatment in all cohorts, and continued to diminish until 6 months. The transcriptional response to treatment could be individually measured in each patient. CONCLUSIONS: Significant changes in the transcriptional signatures measured by blood tests were readily detectable just 2 weeks after treatment initiation. These findings suggest that blood transcriptional signatures could be used as early surrogate biomarkers of successful treatment response.
Asunto(s)
Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Análisis por Conglomerados , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Evaluación de Resultado en la Atención de Salud/métodos , Factores de Tiempo , Transcriptoma/genética , Tuberculosis/sangre , Tuberculosis/genética , Adulto JovenRESUMEN
Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that persists lifelong in the host. In â¼4% of infected people, HTLV-1 causes a chronic disabling neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is unknown and treatment remains ineffective. We used gene expression microarrays followed by flow cytometric and functional assays to investigate global changes in blood transcriptional profiles of HTLV-1-infected and seronegative individuals. We found that perturbations of the p53 signaling pathway were a hallmark of HTLV-1 infection. In contrast, a subset of interferon (IFN)-stimulated genes was over-expressed in patients with HAM/TSP but not in asymptomatic HTLV-1 carriers or patients with the clinically similar disease multiple sclerosis. The IFN-inducible signature was present in all circulating leukocytes and its intensity correlated with the clinical severity of HAM/TSP. Leukocytes from patients with HAM/TSP were primed to respond strongly to stimulation with exogenous IFN. However, while type I IFN suppressed expression of the HTLV-1 structural protein Gag it failed to suppress the highly immunogenic viral transcriptional transactivator Tax. We conclude that over-expression of a subset of IFN-stimulated genes in chronic HTLV-1 infection does not constitute an efficient host response but instead contributes to the development of HAM/TSP.
Asunto(s)
Regulación de la Expresión Génica , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Interferón Tipo I/metabolismo , Leucocitos/metabolismo , Paraparesia Espástica Tropical/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Productos del Gen gag/metabolismo , Productos del Gen tax/metabolismo , Humanos , Leucocitos/virología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Biología de Sistemas/métodosRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the world's largest infectious disease problems. Despite decades of intensive study, the immune response to Mtb is incompletely characterised, reflecting the extremely complex interaction between pathogen and host. Pathways that may alter the balance between host protection and pathogenesis are therefore of great interest. One pathway shown to play a role in the pathogenesis of chronic infections, including TB, is the programmed death-1 (PD-1) pathway. We show here that the expression of the programmed death ligand 1 (PD-L1), which interacts with PD-1, is increased in whole blood from active TB patients compared with whole blood from healthy controls or Mtb-exposed individuals, and that expression by neutrophils is largely responsible for this increase.
Asunto(s)
Antígenos CD/sangre , Mycobacterium tuberculosis/inmunología , Neutrófilos/inmunología , Tuberculosis/inmunología , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Antígeno B7-H1 , Citometría de Flujo , Humanos , Análisis por Micromatrices , Receptor de Muerte Celular Programada 1 , Tuberculosis/sangreRESUMEN
Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is a major cause of morbidity and mortality worldwide. Efforts to control it are hampered by difficulties with diagnosis, prevention and treatment. Most people infected with M. tuberculosis remain asymptomatic, termed latent TB, with a 10% lifetime risk of developing active TB disease. Current tests, however, cannot identify which individuals will develop disease. The immune response to M. tuberculosis is complex and incompletely characterized, hindering development of new diagnostics, therapies and vaccines. Here we identify a whole-blood 393 transcript signature for active TB in intermediate and high-burden settings, correlating with radiological extent of disease and reverting to that of healthy controls after treatment. A subset of patients with latent TB had signatures similar to those in patients with active TB. We also identify a specific 86-transcript signature that discriminates active TB from other inflammatory and infectious diseases. Modular and pathway analysis revealed that the TB signature was dominated by a neutrophil-driven interferon (IFN)-inducible gene profile, consisting of both IFN-gamma and type I IFN-alphabeta signalling. Comparison with transcriptional signatures in purified cells and flow cytometric analysis suggest that this TB signature reflects changes in cellular composition and altered gene expression. Although an IFN-inducible signature was also observed in whole blood of patients with systemic lupus erythematosus (SLE), their complete modular signature differed from TB, with increased abundance of plasma cell transcripts. Our studies demonstrate a hitherto underappreciated role of type I IFN-alphabeta signalling in the pathogenesis of TB, which has implications for vaccine and therapeutic development. Our study also provides a broad range of transcriptional biomarkers with potential as diagnostic and prognostic tools to combat the TB epidemic.