The Impact of Transcriptomics on the Fight against Tuberculosis: Focus on Biomarkers, BCG Vaccination, and Immunotherapy

In 1882 Robert Koch identified Mycobacterium tuberculosis as the causative agent of tuberculosis (TB), a disease as ancient as humanity. Although there has been more than 125 years of scientific effort aimed at understanding the disease, serious problems in TB persist that contribute to the estimated 1/3 of the world population infected with this pathogen. Nonetheless, during the first decade of the 21st century, there were new advances in the fight against TB. The development of high-throughput technologies is one of the major contributors to this advance, because it allows for a global vision of the biological phenomenon. This paper analyzes how transcriptomics are supporting the translation of basic research into therapies by resolving three key issues in the fight against TB: (a) the discovery of biomarkers, (b) the explanation of the variability of protection conferred by BCG vaccination, and (c) the development of new immunotherapeutic strategies to treat TB

Development of Type 1 Diabetes Mellitus in Nonobese Diabetic Mice Follows Changes in Thymocyte and Peripheral T Lymphocyte Transcriptional Activity

As early as one month of age, nonobese diabetic (NOD) mice feature pancreatic infiltration of autoreactive T lymphocytes, which destruct insulin-producing beta cells, producing autoimmune diabetes mellitus (T1D) within eight months. Thus, we hypothesized that during the development of T1D, the transcriptional modulation of immune reactivity genes may occur as thymocytes mature into peripheral T lymphocytes. The transcriptome of thymocytes and peripheral CD3+ T lymphocytes from prediabetic or diabetic mice analyzed through microarray hybridizations identified 2,771 differentially expressed genes. Hierarchical clustering grouped mice according to age/T1D onset and genes according to their transcription profiling. The transcriptional activity of thymocytes developing into peripheral T lymphocytes revealed sequential participation of genes involved with CD4+/CD8+ T-cell differentiation (Themis), tolerance induction by Tregs (Foxp3), and apoptosis (Fasl) soon after T-cell activation (IL4), while the emergence of T1D coincided with the expression of cytotoxicity (Crtam) and inflammatory response genes (Tlr) by peripheral T lymphocytes

Abnormal T-Cell Development in the Thymus of Non-obese Diabetic Mice: Possible Relationship With the Pathogenesis of Type 1 Autoimmune Diabetes

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing cells in the pancreas, by direct interactions with autoreactive pancreas infiltrating T lymphocytes (PILs). One of the most important animal models for this disease is the non-obese diabetic (NOD) mouse. Alterations in the NOD mouse thymus during the pathogenesis of the disease have been reported. From the initial migratory disturbances to the accumulation of mature thymocytes, including regulatory Foxp3+ T cells, important mechanisms seem to regulate the repertoire of T cells that leave the thymus to settle in peripheral lymphoid organs. A significant modulation of the expression of extracellular matrix and soluble chemoattractant molecules, in addition to integrins and chemokine receptors, may contribute to the progressive accumulation of mature thymocytes and consequent formation of giant perivascular spaces (PVS) that are observed in the NOD mouse thymus. Comparative large-scale transcriptional expression and network analyses involving mRNAs and miRNAs of thymocytes, peripheral T CD3+ cells and PILs provided evidence that in PILs chemokine receptors and mRNAs are post-transcriptionally regulated by miR-202-3p resulting in decreased activity of these molecules during the onset of T1D in NOD mice. In this review, we discuss the abnormal T-cell development in NOD mice in the context of intrathymic expression of different migration-related molecules, peptides belonging to the family of insulin and insulin-like growth factors as well as the participation of miRNAs as post-transcriptional regulators and their possible influence on the onset of aggressive autoimmunity during the pathogenesis of T1D

The Impact of Transcriptomics on the Fight against Tuberculosis: Focus on Biomarkers, BCG Vaccination, and Immunotherapy

In 1882 Robert Koch identified Mycobacterium tuberculosis as the causative agent of tuberculosis (TB), a disease as ancient as humanity. Although there has been more than 125 years of scientific effort aimed at understanding the disease, serious problems in TB persist that contribute to the estimated 1/3 of the world population infected with this pathogen. Nonetheless, during the first decade of the 21st century, there were new advances in the fight against TB. The development of high-throughput technologies is one of the major contributors to this advance, because it allows for a global vision of the biological phenomenon. This paper analyzes how transcriptomics are supporting the translation of basic research into therapies by resolving three key issues in the fight against TB: (a) the discovery of biomarkers, (b) the explanation of the variability of protection conferred by BCG vaccination, and (c) the development of new immunotherapeutic strategies to treat TB

Promiscuous Gene Expression in the Thymus: The Root of Central Tolerance

The thymus is a complex organ with an epithelium formed by two main cell types, the cortical thymic epithelial (cTECs) and medullary thymic epithelial cells (mTECs), referred to as stroma. Immature thymocytes arising from the bone marrow, macrophages and dendritic cells also populate the thymus. Thymocytes evolve to mature T cells featuring cell differentiation antigens (CDs), which characterize the phenotypically distinct stages, defined as double-negative (DN), double positive (DP) and single positive (SP), based on expression of the coreceptors CD4 and CD8. The thymus is therefore implicated in T cell differentiation and during development into T cells thymocytes are in close association with the stroma. Recent evidence showed that mTECs express a diverse set of genes coding for parenchymal organ specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has led to the reconsideration of the role of the thymus in central T cell tolerance to self-antigens, which prevents autoimmunity. The evidence of PGE is causing a reanalysis in the scope of central tolerance understanding. We summarize the evidence of PGE in the thymus, focusing particularly the use of cDNA microarray technology for the broad characterization of gene expression and demarcation of PGE emergence during thymus ontogeny

Identification of Cell-Free Circulating MicroRNAs for the Detection of Early Breast Cancer and Molecular Subtyping

Early detection is crucial for achieving a reduction in breast cancer mortality. Analysis of circulating cell-free microRNAs present in the serum of cancer patients has emerged as a promising new noninvasive biomarker for early detection of tumors and for predicting their molecular classifications. The rationale for this study was to identify subtype-specific molecular profiles of cell-free microRNAs for early detection of breast cancer in serum. Fifty-four early-stage breast cancers with 27 age-matched controls were selected for circulating microRNAs evaluation in the serum. The 54 cases were molecularly classified (luminal A, luminal B, luminal B Her2 positive, Her-2, triple negative). NanoString platform was used for digital detection and quantitation of 800 tagged microRNA probes and comparing the overall differences in serum microRNA expression from breast cancer cases with controls. We identified the 42 most significant (P ≤ 0.05, 1.5-fold) differentially expressed circulating microRNAs in each molecular subtype for further study. Of these microRNAs, 19 were significantly differentially expressed in patients presenting with luminal A, eight in the luminal B, ten in luminal B HER 2 positive, and four in the HER2 enriched subtype. AUC is high with suitable sensitivity and specificity. For the triple negative subtype miR-25-3p had the best accuracy. Predictive analysis of the mRNA targets suggests they encode proteins involved in molecular pathways such as cell adhesion, migration, and proliferation. This study identified subtype-specific molecular profiles of cell-free microRNAs suitable for early detection of breast cancer selected by comparison to the microRNA profile in serum for female controls without apparent risk of breast cancer. This molecular profile should be validated using larger cohort studies to confirm the potential of these miRNA for future use as early detection biomarkers that could avoid unnecessary biopsy in patients with a suspicion of breast cancer.Foundation for Research Support of the State of São Paulo (FAPESP process 2015/21082-0) and Public Ministry of Labor Campinas (Research, Prevention, and Education of Occupational Cancer)

Cigarette smoke induces miR-132 in Th17 cells that enhance osteoclastogenesis in inflammatory arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint destruction and severe morbidity. Cigarette smoking (CS) can exacerbate the incidence and severity of RA. Although Th17 cells and the Aryl hydrocarbon receptor (AhR) have been implicated, the mechanism by which CS induces RA development remains unclear. Here, using transcriptomic analysis, we show that microRNA-132 is specifically induced in Th17 cells in the presence of either AhR agonist or CS-enriched medium. miRNA-132 thus induced is packaged into extracellular vesicles produced by Th17 and acts as a proinflammatory mediator increasing osteoclastogenesis through the down-regulation of COX2. In vivo, articular knockdown of miR-132 in murine arthritis models reduces the number of osteoclasts in the joints. Clinically, RA patients express higher levels of miR-132 than do healthy individuals. This increase is further elevated by cigarette smoking. Together, these results reveal a hitherto unrecognized mechanism by which CS could exacerbate RA and further advance understanding of the impact of environmental factors on the pathogenesis of chronic inflammatory diseases

The AIRE-230Y Polymorphism Affects AIRE Transcriptional Activity: Potential Influence on AIRE Function in the Thymus

Background The autoimmune regulator (AIRE) is expressed in the thymus, particularly in thymic medullary epithelial cells (mTECs), and is required for the ectopic expression of a diverse range of peripheral tissue antigens by mTECs, facilitating their ability to perform negative selection of auto-reactive immature T-cells. The expression profile of peripheral tissue antigens is affected not only by AIRE deficiency but also with variation of AIRE activity in the thymus. Method and Results Therefore we screened 591bp upstream of the AIRE transcription start site including AIRE minimal promoter for single nucleotide polymorphism (SNPs) and identified two SNPs -655R (rs117557896) and -230Y (rs751032) respectively. To study the effect of these variations on AIRE promoter activity we generated a Flp-In host cell line which was stably transfected with a single copy of the reporter vector. Relative promoter activity was estimated by comparing the luciferase specific activity for lysates of the different reporter AIRE promoterreporter gene constructs including AIRE-655G AIRE-230C, AIRE-655G AIRE-230T and AIRE-655A AIRE-230C. The analysis showed that the commonest haplotype AIRE-655G AIRE-230C has the highest luciferase specific activity (p<0.001). Whereas AIRE-655G AIRE-230T has a luciferase specific activity value that approaches null. Both AIRE promoter polymorphic sites have one allele that forms a CpG methylation site which we determined can be methylated in methylation assays using the M.SssI CpG methyltransferase. Conclusion AIRE-230Y is in a conserved region of the promoter and is adjacent to a predicted WT1 transcription factor binding site, suggesting that AIRE-230Y affects AIRE expression by influencing the binding of biochemical factors to this region. Our findings show that AIRE655GAIRE-230T haplotype could dramatically alter AIRE transcription and so have an effect on the process of negative selection and affect susceptibility to autoimmune conditions

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen