Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers

CITATION: Esterhuyse, M. M. et al. 2015. Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers. mBio, 6(5):e01187-15, doi:10.1128/mBio.01187-15.The original publication is available at http://mbio.asm.orgAn estimated one-third of the world’s population is currently latently infected with Mycobacterium tuberculosis. Latent M. tuberculosis infection (LTBI) progresses into active tuberculosis (TB) disease in ~5 to 10% of infected individuals. Diagnostic and prognostic biomarkers to monitor disease progression are urgently needed to ensure better care for TB patients and to decrease the spread of TB. Biomarker development is primarily based on transcriptomics. Our understanding of biology combined with evolving technical advances in high-throughput techniques led us to investigate the possibility of additional platforms (epigenetics and proteomics) in the quest to (i) understand the biology of the TB host response and (ii) search for multiplatform biosignatures in TB. We engaged in a pilot study to interrogate the DNA methylome, transcriptome, and proteome in selected monocytes and granulocytes from TB patients and healthy LTBI participants. Our study provides first insights into the levels and sources of diversity in the epigenome and proteome among TB patients and LTBI controls, despite limitations due to small sample size. Functionally the differences between the infection phenotypes (LTBI versus active TB) observed in the different platforms were congruent, thereby suggesting regulation of function not only at the transcriptional level but also by DNA methylation and microRNA. Thus, our data argue for the development of a large-scale study of the DNA methylome, with particular attention to study design in accounting for variation based on gender, age, and cell type.http://mbio.asm.org/content/6/5/e01187-15.abstract?sid=fe0ea1c7-6da2-4e53-b4a4-5cd8233777c7Publisher's versio

DNA methylation in glioblastoma: impact on gene expression and clinical outcome

International audienceBACKGROUND: Changes in promoter DNA methylation pattern of genes involved in key biological pathways have been reported in glioblastoma. Genome-wide assessments of DNA methylation levels are now required to decipher the epigenetic events involved in the aggressive phenotype of glioblastoma, and to guide new treatment strategies. RESULTS: We performed a whole-genome integrative analysis of methylation and gene expression profiles in 40 newly diagnosed glioblastoma patients. We also screened for associations between the level of methylation of CpG sites and overall survival in a cohort of 50 patients uniformly treated by surgery, radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP protocol). The methylation analysis identified 616 CpG sites differentially methylated between glioblastoma and control brain, a quarter of which was differentially expressed in a concordant way. Thirteen of the genes with concordant CpG sites displayed an inverse correlation between promoter methylation and expression level in glioblastomas: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. Survival analysis identified six CpG sites associated with overall survival. SOX10 promoter methylation status (two CpG sites) stratified patients similarly to MGMT status, but with a higher Area Under the Curve (0.78 vs. 0.71, p-value < 5e-04). The methylation status of the FNDC3B, TBX3, DGKI, and FSD1 promoters identified patients with MGMT-methylated tumors that did not respond to STUPP treatment (p-value < 1e-04). CONCLUSIONS: This study provides the first genome-wide integrative analysis of DNA methylation and gene expression profiles obtained from the same GBM cohort. We also present a methylome-based survival analysis for one of the largest uniformly treated GBM cohort ever studied, for more than 27,000 CpG sites. We have identified genes whose expression may be tightly regulated by epigenetic mechanisms and markers that may guide treatment decisions

Interleukin-15 and NKG2 receptors modulate CD8 T cell responses in the central nervous system during multiple sclerosis

Multiple sclerosis (MS) is a demyelinating inflammatory disease affecting the central nervous system (CNS). The pathology of MS is characterized by the infiltration of immune cells into the CNS and by the destruction of myelin-producing cells, the oligodendrocytes. CD8 T cells constitute a prominent fraction of CNS-infiltrating immune cells. They have an effector memory phenotype and can persist as clonal expansions for years in the CNS of MS patients sug gesting their active participation in the local immune response. The work presented in this thesis emanates from the hypothesis that the microenvironment of MS lesions favors and enhances the effector functions of CD8 T cells. First, we show that glial cells, especially astrocytes, expressed elevated levels of the pro-inflammatory cytokine interleukin (IL)-15 in MS lesions. Treatment of astrocytes in vitro with interferon-g, interleukin-1b or tumor necrosis factor resulted in elevated levels of IL-15. This IL-15 activated co-cultured antigen-specific CD8 T cell lines and augmented their lytic enzyme content and antigen-restricted cytotoxicity. These results prove that IL-15 provided by astrocytes in MS lesions may enhance the effector function of CD8 T cells present in MS lesions and consequently exacerbate the observed tissue damage. Second, we showed that oligodendrocytes in active MS lesions expressed elevated levels of the ligands of NKG2D, an activating receptor expressed by CD8 T cells, gd T cells and natural killer (NK) cells. NKG2D ligands were expressed by primary human adult oligodendrocytes in vitro and increased their susceptibility to cytotoxicity mediated by all NKG2D-expressing cells. These results prove that oligodendrocytes in MS lesions may have an increased susceptibility to immune-mediated cytotoxicity because they express NKG2D ligands. Third, we evaluated the contribution of human leukocyte antigen (HLA) -E to the dialogue between immune cells and oLa sclérose en plaques (SEP) est une maladie inflammatoire qui affecte le système nerveux central (SNC). La pathologie de la SEP est caractérisée par la destruction de la gaine de la myéline et des cellules qui la produisent, les oligodendrocytes ainsi que par une perte axonale. Les lésions observées au sein du SNC contiennent de nombreuses cellules immunitaires ainsi que des cellules gliales (microglies et astrocytes) démontrant un phénotype activé. De nombreuses études ont démontré que les cellules immunitaires détectées au sein du SNC pendant la SEP sont impliquées dans la pathogenèse de cette maladie. En particulier, les cellules T CD8 sont nombreuses dans les lésions SEP et leur phénotype suggère une participation active dans les dommages infligés aux oligodendrocytes. Les résultats présentés dans cette thèse émanent de l'hypothèse que l'environnement des lésions SEP favorise l'activation des cellules T CD8 ainsi que leur cytotoxicité envers les oligodendrocytes. Nous avons démontré que les astrocytes dans les lésions SEP expriment des niveaux élevés d'interleukine (IL) -15, une cytokine impliquée dans l'activation et la préservation des cellules mémoire T CD8 (entre autres). La co-culture in vitro d'astrocytes humains exprimant de hauts niveaux d'IL-15 avec des lignées T CD8 a prouvé que l'IL-15 fourni par les astrocytes augmente l'expression d'enzymes lytiques par les cellules CD8 T. Ces conditions de co-cultures amplifient aussi leur cytotoxicité envers des cellules cibles. Ces résultats suggèrent que l'IL-15 présente au sein des lésions de SEP peut augmenter la capacité des cellules CD8 T à tuer des cellules cibles tels que les oligodendrocytes.Le second chapitre de cette thèse démontre l'expression des ligands NKG2D par les oligodendrocytes dans les lésions SEP. Ces ligands, habituellement exprimés par des cellules endommagées, se lient au récepteur NKG2D, prés

Contribution of CD8 T lymphocytes to the immuno-pathogenesis of multiple sclerosis and its animal models

AbstractMultiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by multi-focal demyelination, axonal loss, and immune cell infiltration. Numerous immune mediators are detected within MS lesions, including CD4+ and CD8+ T lymphocytes suggesting that they participate in the related pathogenesis. Although CD4+ T lymphocytes are traditionally considered the main actors in MS immunopathology, multiple lines of evidence suggest that CD8+ T lymphocytes are also implicated in the pathogenesis. In this review, we outline the recent literature pertaining to the potential roles of CD8+ T lymphocytes both in MS and its animal models. The CD8+ T lymphocytes detected in MS lesions demonstrate characteristics of activated and clonally expanded cells supporting the notion that these cells actively contribute to the observed injury. Moreover, several experimental in vivo models mediated by CD8+ T lymphocytes recapitulate important features of the human disease. Whether the CD8+ T cells can induce or aggravate tissue destruction in the CNS needs to be fully explored. Strengthening our understanding of the pathogenic potential of CD8+ T cells in MS should provide promising new avenues for the treatment of this disabling inflammatory disease

From the core to beyond the margin: a genomic picture of glioblastoma intratumor heterogeneity

PMID 25940437International audienceGlioblastoma (GB) is a highly invasive primary brain tumor that almost systematically recurs despite aggressive therapies. One of the most challenging problems in therapy of GB is its extremely complex and heterogeneous molecular biology. To explore this heterogeneity, we performed a genome-wide integrative screening of three molecular levels: genome, transcriptome, and methylome. We analyzed tumor biopsies obtained by neuro-navigation in four distinct areas for 10 GB patients (necrotic zone, tumor zone, interface, and peripheral brain zone). We classified samples and deciphered a key genes signature of intratumor heterogeneity by Principal Component Analysis and Weighted Gene Co-expression Network Analysis. At the genome level, we identified common GB copy number alterations and but a strong interindividual molecular heterogeneity. Transcriptome analysis highlighted a pronounced intratumor architecture reflecting the surgical sampling plan of the study and identified gene modules associated with hallmarks of cancer. We provide a signature of key cancer-heterogeneity genes highly associated with the intratumor spatial gradient and show that it is enriched in genes with correlation between methylation and expression levels. Our study confirms that GBs are molecularly highly diverse and that a single tumor can harbor different transcriptional GB subtypes depending on its spatial architecture

Gene Expression Meta-Analysis Identifies Grading and Survival Markers in Anaplastic Glioma.

International audiencePurpose: Although molecular analyses have been shown to be useful for classifying tumors and formulating a prognosis for patients, current methods applied to glioma are mostly based on histopathology. To search for robust diagnostic and prognostic markers of high-grade gliomas (HGGs), we applied a meta-analysis that extracts the most reliable information from the available gene expression data. Methods: We obtained data sets from three studies in HGGs. We selected a consensus set of 267 patients. Differential analyses were performed separately for each study and we applied a meta-analysis approach based on nonparametric rank products to evaluate the combined data. The genes selected in both analyses were used to construct a gene classifier by logistic regression modeling. We performed survival analyses on 144 patients by fitting Cox proportional hazard model. Genes identified as both differentially expressed and correlated to survival were used to build an optimal survival model. Performances were evaluated on an independent data set comprising 54 patients. Several genes were validated using RT, Q-PCR, and immunohistochemistry on a local cohort of 130 patients. Results: This interstudy cross-validation approach generated a set of 65 genes consistently and specifically differentially expressed in GBM. Functional annotation revealed a clear association with the nervous system development and the cell communication. The genes significantly associated with grading were mostly related to the extracellular matrix. The optimal survival model was built on a four genes signature. Kaplan-Meier curves and the log rank test (training: p52 e-11, testing: p51 e-4) indicated the high survival prognostic potential for this classifier. Finally, applied only to GBM it clearly outperformed previous reports, by grouping GBM in two subtypes with significant different prognosis (p53 e-4). Conclusions: Meta-analysis allows the identification and validation of HGGs biomarkers that might represent good candidates for novel diagnostic and prognostic approaches in HGGs

Integration of Genomic Alterations and Expression Profiling in Glioblastoma Multiforme.

International audiencePurpose: Glioblastomas (GBM) are highly malignant and heterogeneous gliomas with very poor prognosis. The biological and molecular characterization of these tumors is still challenging and impacts their therapeutic management. Previous genomic surveys have revealed the highly rearranged nature of GBM genome and transcriptome. However, the impacts of tumor DNA aberrations on gene expression remain unclear. Methods: We investigated copy number alterations (CNA) and gene expression to identify causal genetic events in GBM. High-resolution maps of somatic chromosomal alterations were obtained for 20 GBM. Gene expression profiling was carried out on the same tumor samples, and compared to those obtained on nonneoplastic brain samples. Concordance between CNA and gene expression was identified by two complementary approaches (correlated or targeted probes). The resulting GBM signature was validated with an independent microarray data set of 81 GBM and 23 normal brains. Results: Loci targeted for high-priority minimal common regions (MCR) of recurrent CNA were defined and combined with gene expression profiles performed on the same tumor samples. Genes with concordant changes in CNA and expression levels were defined as over/underexpressed genes located in amplified/deleted regions, or as MCR genes with expression correlated to the corresponding genomic state. After validation, we found that the expression of 318 genes was significantly affected by CNA. Associated enriched GO process annotations were related to cell cycle disorder, cellular adhesion and angiogenesis. The gene signature included well-known GBM genes such as EGFR, PDGFA, and p16INK4 but also novel candidate genes. Two tumor suppressor genes PCDH9 and STARD13, involved in tumor invasiveness and resistance to etoposide, were validated by qPCR in an independent set of 57 glioblastoma. Conclusion: This study shows the power of combining genomic alterations and gene expression to identify robust transcriptome signature and putative tumor biomarkers in GBM

Mucosal BCG Vaccination Induces Protective Lung-Resident Memory T Cell Populations against Tuberculosis

Mycobacterium bovis Bacille Calmette-Guééérin (BCG) is the only licensed vaccine against tuberculosis (TB), yet its moderate efficacy against pulmonary TB calls for improved vaccination strategies. Mucosal BCG vaccination generates superior protection against TB in animal models; however, the mechanisms of protection remain elusive. Tissue-resident memory T (TRM) cells have been implicated in protective immune responses against viral infections, but the role of TRM cells following mycobacterial infection is unknown. Using a mouse model of TB, we compared protection and lung cellular infiltrates of parenteral and mucosal BCG vaccination. Adoptive transfer and gene expression analyses of lung airway cells were performed to determine the protective capacities and phenotypes of different memory T cell subsets. In comparison to subcutaneous vaccination, intratracheal and intranasal BCG vaccination generated T effector memory and TRM cells in the lung, as defined by surface marker phenotype. Adoptive mucosal transfer of these airway-resident memory T cells into naive mice mediated protection against TB. Whereas airway-resident memory CD4+ T cells displayed a mixture of effector and regulatory phenotype, airway-resident memory CD8+ T cells displayed prototypical TRM features. Our data demonstrate a key role for mucosal vaccination-induced airway-resident T cells in the host defense against pulmonary TB. These results have direct implications for the design of refined vaccination strategies