Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

Immunity to intestinal helminth infections requires the rapid activation of T helper 2 cells (Th2 cells). However, simultaneous expansion of CD4 + Foxp3 + regulatory T cells (T reg cells) impedes protective responses, resulting in chronic infections. The ratio between T reg and effector T cells can therefore determine the outcome of infection. The redifferentiation of T reg cells into Th cells has been identified in hyperinflammatory diseases. In this study, we asked whether ex–T reg Th2 cells develop and contribute to type-2 immunity. Using multigene reporter and fate-reporter systems, we demonstrate that a significant proportion of Th2 cells derive from Foxp3 + cells after Heligmosomoides polygyrus infection and airway allergy. Ex-Foxp3 Th2 cells exhibit characteristic Th2 effector functions and provide immunity to H. polygyrus . Through selective deletion of Il4ra on Foxp3 + cells, we further demonstrate IL-4 is required for the development of ex-Foxp3 Th2 cells. Collectively, our findings indicate that converting T reg cells into Th2 cells could concomitantly enhance Th2 cells and limit T reg cell–mediated suppression

Coordinated changes of histone modifications and HDAC mobilization regulate the induction of MHC class II genes by Trichostatin A

The deacetylase inhibitor Trichostatin A (TSA) induces the transcription of the Major Histocompatibility Class II (MHC II) DRA gene in a way independent of the master coactivator CIITA. To analyze the molecular mechanisms by which this epigenetic regulator stimulates MHC II expression, we used chromatin immunoprecipitation (ChIP) assays to monitor the alterations in histone modifications that correlate with DRA transcription after TSA treatment. We found that a dramatic increase in promoter linked histone acetylation is followed by an increase in Histone H3 lysine 4 methylation and a decrease of lysine 9 methylation. Fluorescence recovery after photobleaching (FRAP) experiments showed that TSA increases the mobility of HDAC while decreasing the mobility of the class II enhanceosome factor RFX5. These data, in combination with ChIP experiments, indicate that the TSA-mediated induction of DRA transcription involves HDAC relocation and enhanceosome stabilization. In order to gain a genome-wide view of the genes responding to inhibition of deacetylases, we compared the transcriptome of B cells before and after TSA treatment using Affymetrix microarrays. This analysis showed that in addition to the DRA gene, the entire MHC II family and the adjacent histone cluster that are located in chromosome 6p21-22 locus are strongly induced by TSA. A complex pattern of gene reprogramming by TSA involves immune recognition, antiviral, apoptotic and inflammatory pathways and extends the rationale for using Histone Deacetylase Inhibitors (HDACi) to modulate the immune response

Activation of the Aryl Hydrocarbon Receptor Interferes with Early Embryonic Development.

The transcriptional program of early embryonic development is tightly regulated by a set of well-defined transcription factors that suppress premature expression of differentiation genes and sustain the pluripotent identity. It is generally accepted that this program can be perturbed by environmental factors such as chemical pollutants; however, the precise molecular mechanisms remain unknown. The aryl hydrocarbon receptor (AHR) is a widely expressed nuclear receptor that senses environmental stimuli and modulates target gene expression. Here, we have investigated the AHR interactome in embryonic stem cells by mass spectrometry and show that ectopic activation of AHR during early differentiation disrupts the differentiation program via the chromatin remodeling complex NuRD (nucleosome remodeling and deacetylation). The activated AHR/NuRD complex altered the expression of differentiation-specific genes that control the first two developmental decisions without affecting the pluripotency program. These findings identify a mechanism that allows environmental stimuli to disrupt embryonic development through AHR signaling

Aryl hydrocarbon receptor is required for optimal B-cell proliferation

The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up‐regulated upon B‐cell receptor (BCR) engagement and IL‐4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR‐deficient (Ahr (−/−)) B cells proliferate less than AhR‐sufficient (Ahr (+/+)) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr (−/−) B cells are outcompeted by Ahr (+/+) cells. Transcriptome comparison of AhR‐deficient and AhR‐sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency

Multiple Histone Methyl and Acetyltransferase Complex Components Bind the HLA-DRA Gene

Major histocompatibility complex class II (MHC-II) genes are fundamental components that contribute to adaptive immune responses. While characterization of the chromatin features at the core promoter region of these genes has been studied, the scope of histone modifications and the modifying factors responsible for activation of these genes are less well defined. Using the MHC-II gene HLA-DRA as a model, the extent and distribution of major histone modifications associated with active expression were defined in interferon-γ induced epithelial cells, B cells, and B-cell mutants for MHC-II expression. With active transcription, nucleosome density around the proximal regulatory region was diminished and histone acetylation and methylation modifications were distributed throughout the gene in distinct patterns that were dependent on the modification examined. Irrespective of the location, the majority of these modifications were dependent on the binding of either the X-box binding factor RFX or the class II transactivator (CIITA) to the proximal regulatory region. Importantly, once established, the modifications were stable through multiple cell divisions after the activating stimulus was removed, suggesting that activation of this system resulted in an epigenetic state. A dual crosslinking chromatin immunoprecipitation method was used to detect histone modifying protein components that interacted across the gene. Components of the MLL methyltransferase and GCN5 acetyltransferase complexes were identified. Some MLL complex components were found to be CIITA independent, including MLL1, ASH2L and RbBP5. Likewise, GCN5 containing acetyltransferase complex components belonging to the ATAC and STAGA complexes were also identified. These results suggest that multiple complexes are either used or are assembled as the gene is activated for expression. Together the results define and illustrate a complex network of histone modifying proteins and multisubunit complexes participating in MHC-II transcription

Histone deacetylase inhibitors: potential targets responsible for their anti-cancer effect

The histone deacetylase inhibitors (HDACi) have demonstrated anticancer efficacy across a range of malignancies, most impressively in the hematological cancers. It is uncertain whether this clinical efficacy is attributable predominantly to their ability to induce apoptosis and differentiation in the cancer cell, or to their ability to prime the cell to other pro-death stimuli such as those from the immune system. HDACi-induced apoptosis occurs through altered expression of genes encoding proteins in both intrinsic and extrinsic apoptotic pathways; through effects on the proteasome/aggresome systems; through the production of reactive oxygen species, possibly by directly inducing DNA damage; and through alterations in the tumor microenvironment. In addition HDACi increase the immunogenicity of tumor cells and modulate cytokine signaling and potentially T-cell polarization in ways that may contribute the anti-cancer effect in vivo. Here, we provide an overview of current thinking on the mechanisms of HDACi activity, with attention given to the hematological malignancies as well as scientific observations arising from the clinical trials. We also focus on the immune effects of these agents

Prenatal exposures and exposomics of asthma

This review examines the causal investigation of preclinical development of childhood asthma using exposomic tools. We examine the current state of knowledge regarding early-life exposure to non-biogenic indoor air pollution and the developmental modulation of the immune system. We examine how metabolomics technologies could aid not only in the biomarker identification of a particular asthma phenotype, but also the mechanisms underlying the immunopathologic process. Within such a framework, we propose alternate components of exposomic investigation of asthma in which, the exposome represents a reiterative investigative process of targeted biomarker identification, validation through computational systems biology and physical sampling of environmental medi

Epigenetic control of the immune response: Nuclear organization and chromatin

Transcription of the Class II genes of the Major Histocompatibility Complex (MHCII) requires a complex of proteins that synergistically bind to the promoter and constitute the MHCII enhanceosome. Although necessary, these proteins are not sufficient to drive transcription. The master coactivator CIITA is required for efficient transcriptional initiation and elongation. The deacetylase inhibitor Trichostatin A (TSA) induces the transcription of the MHCII DRA gene in a CIITA-independent manner. To analyze the molecular mechanisms by which this epigenetic regulator stimulates MHCII expression, chromatin immunoprecipitation (ChIP) assays were employed to monitor the alterations in histone modifications that correlate with DRA transcription after TSA treatment. A dramatic increase in promoter linked histone acetylation and Histone H3 lysine 4 methylation followed by concurrent decrease of lysine 9 methylation were found. Fluorescence recovery after photobleaching (FRAP) experiments showed that TSA treatment increased the mobility of HDAC while decreasing the mobility of the class II enhanceosome factor RFX5. These data, in combination with ChIP experiments, indicate that the TSA-mediated induction of DRA transcription involves HDAC relocation and enhanceosome stabilization. In order to gain a genome-wide view of the genes responding to inhibition of deacetylases, we compared the transcriptome of B cells before and after TSA treatment using Affymetrix microarrays. This analysis showed that in addition to the DRA gene, the entire MHCII family and the adjacent histone cluster, located in chromosome 6p21-22, are strongly induced by TSA. A complex pattern of gene reprogramming by TSA involves immune recognition, antiviral, apoptotic and inflammatory pathways and extends the rationale for using Histone Deacetylase Inhibitors (HDACi) to modulate the immune response. Since many of these genes are direct targets of STAT1 transcription factor we monitored its expression after TSA treatment and found it to be induced. This induction was mediated by the simultaneous down-regulation of c-Myc oncogene expression thus revealing a negative feedback loop between the two genes. Concurrent activation by tyrosine phosphorylation of the produced STAT1 protein prompted for the examination of interferon (IFN) production. Its presence in the culture supernatant was confirmed and was identified as type I IFN. The whole antiviral response of the cell was found to be activated by TSA. The presence of Epstein Barr virus (EBV) was crucial for the initiation of this response. Adaptation is one essential property of the immune response conferred by immunological memory. The question whether IFN-gamma (IFNβ) inducible transcription generates memory that sensitizes cells to a secondary stimulus was addressed. The MHCII gene DRA was found to relocate to Promyelocytic leukemia nuclear bodies (PML-NB) upon induction with IFNβ and this topology was maintained long after transcription shut off. Concurrent interaction of PML protein with Mixed Lineage Leukemia (MLL) generates a prolonged permissive chromatin state on the DRA gene characterized by high promoter histone H3 K4 dimethylation that facilitates rapid expression upon restimulation. Primary signal-induced transcription generates spatial and epigenetic memory that is maintained through several cell generations and endows the immune system with increased responsiveness to future activation signals

Gamma Interferon-Dependent Transcriptional Memory via Relocalization of a Gene Locus to PML Nuclear Bodies▿ †

Memory of past cellular responses is an essential adaptation to repeating environmental stimuli. We addressed the question of whether gamma interferon (IFN-γ)-inducible transcription generates memory that sensitizes cells to a second stimulus. We have found that the major histocompatibility complex class II gene DRA is relocated to promyelocytic leukemia (PML) nuclear bodies upon induction with IFN-γ, and this topology is maintained long after transcription shut off. Concurrent interaction of PML protein with mixed-lineage leukemia generates a prolonged permissive chromatin state on the DRA gene characterized by high promoter histone H3 K4 dimethylation that facilitates rapid expression upon restimulation. We propose that the primary signal-induced transcription generates spatial and epigenetic memory that is maintained through several cell generations and endows the cell with increased responsiveness to future activation signals

Τεχνολογίες φασματικής απεικόνισης και μηχανικής μάθησης για την αυτοματοποίηση της διαγνωστικής μικροσκοπίας

Summarization: Microscopy for years is an instrumental technology for analyzing tissue samples and locating cancerous cells. The biopsy is a process that can last for several days and is crucial, since the doctor will decide on the most suitable treatment depending on the results. The goal of this thesis is to speed up the process of analyzing a biopsy by using image stitching algorithms. By creating high-resolution mosaics of the samples, it will be easier for different doctors to examine the same sample, while being located in a different area or re-examine the same sample, if needed. The chosen algorithm for stitching is SIFT, which is distinguishable among other algorithms due to its high accuracy. At the same time, the main disadvantage that needs to be mended is the time needed to complete a stitching due to its high complexity. Using various techniques aiming to reduce the elapsed time, like reducing the image size to be analyzed each time and defining regions of interest in the images, can reduce the time needed. By applying those techniques, it is possible to speed up the average time of a single horizontal stitch by approximately four times. These results suggest that it will be viable for the algorithm to be used in a microscope, reducing the time of analysis of the samples.Περίληψη: Η μικροσκοπία εδώ και αρκετά χρόνια αποτελεί σημαντικό εργαλείο στην ανάλυση δειγμάτων ιστού και στον εντοπισμό καρκινικών κυττάρων. Η βιοψία είναι μια διαδικασία που μπορεί να χρειαστούν αρκετές μέρες για να ολοκληρωθεί και είναι κρίσιμης σημασίας καθώς ο γιατρός θα χρειαστεί να επιλέξει την κατάλληλη θεραπεία σύμφωνα με τα αποτελέσματα. Στόχος της συγκεκριμένης διπλωματικής είναι να επιταχύνει τη διαδικασία ανάλυσης μιας βιοψίας αξιοποιώντας αλγορίθμους image stitching. Κατασκευάζοντας ένα μωσαϊκό εικόνων του δείγματος υψηλής ανάλυσης, θα είναι ευκολότερο για διαφορετικούς γιατρούς να εξετάσουν το ίδιο δείγμα ενώ βρίσκονται σε διαφορετικές περιοχές είτε να γίνει επανεξέταση του ίδιο δείγματος σε περίπτωση που κριθεί απαραίτητο. Ο αλγόριθμος που επιλέχθηκε για αυτή την διαδικασία ονομάζεται SIFT, ο οποίος ξεχωρίζει ανάμεσα σε άλλους αλγόριθμους του ίδιου είδους λόγω της υψηλής του ακρίβειας. Την ίδια στιγμή, το βασικό μειονέκτημα που χρειάζεται να βελτιωθεί είναι ο χρόνος που χρειάζεται να ολοκληρωθεί ενα stitching, λόγω της υψηλής του πολυπλοκότητας. Χρησιμοποιώντας διάφορες τεχνικές που στοχεύουν στην μείωση του χρόνου ολοκλήρωσης μίας διαδικασίας stitching, όπως η μείωση του μεγέθους της εικόνας που θα αναλυθεί ή ο ορισμός περιοχής ενδιαφέροντος, είναι δυνατόν να μειωθεί ο χρόνος που απαιτείται. Εφαρμόζοντας αυτές τις τεχνικές είναι δυνατό να επιταχύνουμε το χρόνο που απαιτείται για να ολοκληρωθεί ένα οριζόντιο stitch, κατά σχεδόν τέσσερις φορές. Αυτά τα αποτελέσματα υποδεικνύουν ότι είναι εφικτή η χρήση του αλγόριθμου στο μικροσκόπιο, μειώνοντας το χρόνο που απαιτείται για την ανάλυση των δειγμάτων