RESUMEN
This study aims to identify RNA biomarkers distinguishing neuromyelitis optica (NMO) from relapsing-remitting multiple sclerosis (RRMS) and explore potential therapeutic applications leveraging machine learning (ML). An ensemble approach was developed using differential gene expression analysis and competitive ML methods, interrogating total RNA-sequencing data sets from peripheral whole blood of treatment-naïve patients with RRMS and NMO and healthy individuals. Pathway analysis of candidate biomarkers informed the biological context of disease, transcription factor activity, and small-molecule therapeutic potential. ML models differentiated between patients with NMO and RRMS, with the performance of certain models exceeding 90% accuracy. RNA biomarkers driving model performance were associated with ribosomal dysfunction and viral infection. Regulatory networks of kinases and transcription factors identified biological associations and identified potential therapeutic targets. Small-molecule candidates capable of reversing perturbed gene expression were uncovered. Mitoxantrone and vorinostat-two identified small molecules with previously reported use in patients with NMO and experimental autoimmune encephalomyelitis-reinforced discovered expression signatures and highlighted the potential to identify new therapeutic candidates. Putative RNA biomarkers were identified that accurately distinguish NMO from RRMS and healthy individuals. The application of multivariate approaches in analysis of RNA-sequencing data further enhances the discovery of unique RNA biomarkers, accelerating the development of new methods for disease detection, monitoring, and therapeutics. Integrating biological understanding further enhances detection of disease-specific signatures and possible therapeutic targets.
Asunto(s)
Biomarcadores , Aprendizaje Automático , Neuromielitis Óptica , Análisis de Secuencia de ARN , Neuromielitis Óptica/genética , Neuromielitis Óptica/diagnóstico , Neuromielitis Óptica/tratamiento farmacológico , Humanos , Femenino , Biomarcadores/sangre , Análisis de Secuencia de ARN/métodos , Masculino , Mitoxantrona/uso terapéutico , Adulto , Diagnóstico Diferencial , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/genética , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Perfilación de la Expresión Génica/métodos , Esclerosis Múltiple/genética , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/sangreRESUMEN
OBJECTIVE: Long non-coding RNAs (lncRNAs) are emerging as novel biomarkers for a variety of chronic conditions including autoimmune disease. PAXgene Blood RNA tubes are routinely used in clinical research and molecular diagnostic development to capture RNA profiles in peripheral whole blood. While the stability of mRNA expression profiles captured using PAXgene tubes has been documented previously, no previous work has determined the stability of lncRNA expression profiles observed in PAXgene tubes stored at - 80 °C. Here we sought to determine the effects on lncRNA expression profiles following - 80 °C storage of total RNA templates, cDNA synthesized using fresh or frozen total RNA template, and the impact of freeze-thaw cycles on both total RNA and cDNA obtained from PAXgene tubes. RESULTS: We find that storage of whole blood in PAXgene tubes, total RNA and cDNA for up to 1 year at - 80 °C or up to ten total RNA or cDNA freeze-thaw cycles do not significantly alter lncRNA expression profiles compared to baseline. As monthly expression profiles were determined, some month to month lncRNA expression variability was observed. However, all monthly observations fell within the 95% confidence interval calculated at baseline.
Asunto(s)
ARN Largo no Codificante , Conservación de la Sangre , Recolección de Muestras de Sangre , Perfilación de la Expresión Génica , Humanos , ARN , Estabilidad del ARN , ARN Largo no Codificante/genética , TranscriptomaRESUMEN
OBJECTIVE: We tested the hypothesis that the expression of IL-33 in MS is dynamic and is likely to reflect the clinical and radiological changes during the course of RRMS. METHODS: MS with either clinical or radiological relapses were recruited for the study and followed for one year. IL-33 and a panel of genes was measured by q PCR and flow cytometry at different time points. RESULTS: Among 22 RRMS patients, 4 patients showed highest levels of IL-33 at the time they were recruited to the study (Month 0); in 14 patients highest levels of IL-33 were seen between 6-11 months after relapse and in 4 patients maximal levels of IL-33 were seen 12 months after relapse. A similar pattern of IL-33 kinetics was seen when IL-33 was measured by flow cytometry in an additional cohort of 12 patients. The timing of the improvement clinically did not correlate with IL-33 expression with highest expression levels either preceding or following clinical recovery. From our whole genome RNA-sequencing data we found a strong correlation between expression levels of IL-33 and a ~2000 mRNA genes. However, none of these genes encoded proteins involved in either innate or adaptive immunity. Rather, many of the genes that correlated highly with IL-33 encoded to proteins involved in DNA repair or mitochondrial function and mRNA splicing pathways. INTERPRETATION: Given the neuro-reparative and remodeling functions attributed to IL-33, it is likely that some of the novel genes we have uncovered may be involved in repair and recovery of the CNS in MS.
Asunto(s)
Interleucina-33/sangre , Esclerosis Múltiple Recurrente-Remitente/sangre , Adulto , Biomarcadores/sangre , Estudios Transversales , Progresión de la Enfermedad , Femenino , Expresión Génica , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/diagnóstico por imagen , Esclerosis Múltiple Recurrente-Remitente/terapia , ARN Mensajero/sangre , Adulto JovenRESUMEN
Long non-coding RNAs (lncRNAs) possess a diverse array of regulatory functions including activation and silencing of gene transcription, regulation of splicing, and coordinating epigenetic modifications. GATA3-AS1 is a divergent lncRNA gene neighboring GATA3. GATA3 is considered the master regulator of TH2 lineage commitment enabling TH2 effector cells to efficiently transcribe genes encoding cytokines IL-4, IL-5, and IL-13. Here, we show that the GATA3-AS1 lncRNA is selectively expressed under TH2 polarizing conditions and is necessary for efficient transcription of GATA3, IL5, and IL13 genes, while being sufficient for GATA3 transcription. GATA3-AS1 is required for formation of permissive chromatin marks, H3K27 acetylation and H3K4 di/tri-methylation, at the GATA3-AS1-GATA3 locus. Further, GATA3-AS1 binds components of the MLL methyltransferase and forms a DNA-RNA hybrid (R-loop) thus tethering the MLL methyltransferase to the gene locus. Our results indicate a novel regulatory function for a divergent lncRNA and provide new insight into the function of lncRNAs in T helper cell differentiation.
Asunto(s)
Factor de Transcripción GATA3/genética , ARN Largo no Codificante/genética , Diferenciación Celular/genética , Línea Celular , Cromatina/genética , Citocinas/genética , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células Th2 , Transcripción Genética/genéticaRESUMEN
Pulmonary fibrosis is a progressive inflammatory disease with high mortality and limited therapeutic options. Previous genetic and immunologic investigations suggest common intersections between idiopathic pulmonary fibrosis (IPF), sarcoidosis, and murine models of pulmonary fibrosis. To identify immune responses that precede collagen deposition, we conducted molecular, immunohistochemical, and flow cytometric analysis of human and murine specimens. Immunohistochemistry revealed programmed cell death-1 (PD-1) up-regulation on IPF lymphocytes. PD-1+CD4+ T cells with reduced proliferative capacity and increased transforming growth factor-ß (TGF-ß)/interleukin-17A (IL-17A) expression were detected in IPF, sarcoidosis, and bleomycin CD4+ T cells. PD-1+ T helper 17 cells are the predominant CD4+ T cell subset expressing TGF-ß. Coculture of PD-1+CD4+ T cells with human lung fibroblasts induced collagen-1 production. Strikingly, ex vivo PD-1 pathway blockade resulted in reductions in TGF-ß and IL-17A expression from CD4+ T cells, with concomitant declines in collagen-1 production from fibroblasts. Molecular analysis demonstrated PD-1 regulation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Chemical blockade of STAT3, using the inhibitor STATTIC, inhibited collagen-1 production. Both bleomycin administration to PD-1 null mice or use of antibody against programmed cell death ligand 1 (PD-L1) demonstrated significantly reduced fibrosis compared to controls. This work identifies a critical, previously unrecognized role for PD-1+CD4+ T cells in pulmonary fibrosis, supporting the use of readily available therapeutics that directly address interstitial lung disease pathophysiology.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Fibrosis Pulmonar Idiopática/inmunología , Fibrosis Pulmonar Idiopática/patología , Interleucina-17/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Regulación hacia Arriba , Adulto , Anciano , Animales , Bleomicina , Proliferación Celular , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/genética , Masculino , Ratones , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/genética , Sarcoidosis/inmunología , Sarcoidosis/patología , Células Th17/metabolismoRESUMEN
Investigation of the Th1 immune response in sarcoidosis CD4+ T cells has revealed reduced proliferative capacity and cytokine expression upon TCR stimulation. In other disease models, such cellular dysfunction has been associated with a step-wise, progressive loss of T cell function that results from chronic antigenic stimulation. T cell exhaustion is defined by decreased cytokine production upon TCR activation, decreased proliferation, increased expression of inhibitory cell surface receptors, and increased susceptibility to apoptosis. We characterized sarcoidosis CD4+ T cell immune function in systemic and local environments among subjects undergoing disease progression compared to those experiencing disease resolution. Spontaneous and TCR-stimulated Th1 cytokine expression and proliferation assays were performed in 53 sarcoidosis subjects and 30 healthy controls. PD-1 expression and apoptosis were assessed by flow cytometry. Compared to healthy controls, sarcoidosis CD4+ T cells demonstrated reductions in Th1 cytokine expression, proliferative capacity (p < 0.05), enhanced apoptosis (p < 0.01), and increased PD-1 expression (p < 0.001). BAL-derived CD4+ T cells also demonstrated multiple facets of T cell exhaustion (p < 0.05). Reversal of CD4+ T cell exhaustion was observed in subjects undergoing spontaneous resolution (p < 0.05). Sarcoidosis CD4+ T cells exhibit loss of cellular function during progressive disease that follows the archetype of T cell exhaustion.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Sarcoidosis Pulmonar/inmunología , Células TH1/inmunología , Adulto , Anciano , Apoptosis , Proliferación Celular , Células Cultivadas , Anergia Clonal , Citocinas/genética , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Adulto JovenRESUMEN
We employed whole-genome RNA-sequencing to profile mRNAs and both annotated and novel long noncoding RNAs (lncRNAs) in human naive, central memory, and effector memory CD4+ T cells. Loci transcribing both lineage-specific annotated and novel lncRNA are adjacent to lineage-specific protein-coding genes in the genome. Lineage-specific novel lncRNA loci are transcribed from lineage-specific typical- and supertranscriptional enhancers and are not multiexonic, thus are more similar to enhancer RNAs. Novel enhancer-associated lncRNAs transcribed from the IFNG locus bind the transcription factor NF-κB and enhance binding of NF-κB to the IFNG genomic locus. Depletion of the annotated lncRNA, IFNG-AS1, or one IFNG enhancer-associated lncRNA abrogates IFNG expression by memory T cells, indicating these lncRNAs have biologic function.
Asunto(s)
Memoria Inmunológica , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Linfocitos T/inmunología , Linaje de la Célula , Genoma Humano , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , FN-kappa B/metabolismo , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARNRESUMEN
Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Fosfatidilinositol 3-Quinasa/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sarcoidosis/inmunología , Sarcoidosis/patología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Proliferación Celular , Demografía , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Sarcoidosis/metabolismo , Adulto JovenRESUMEN
RATIONALE: Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. OBJECTIVES: To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. METHODS: Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. MEASUREMENTS AND MAIN RESULTS: Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. CONCLUSIONS: Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.