Heat shock proteins in leukaemia cell differentiation and cell death

When HL60 cells were induced to differentiate to granulocyte-like cells with the agents N-methylformamide and tunicamycin an concentrations marginally below those which were cytotoxic, there was a decrease in the synthesis of the glucose- regulated proteins which preceded the expression of markers of a differentiated phenotype. There was a transient increase in the amount of hsp70 after 36 hours in NMF treated cells but in differentiated cells negligible amounts were detected. Inducers which were known to modulate hsp70 such as azetadine carboxylic acid did not induce differentiation suggesting early changes in the endoplasmic reticulum may be involved in the commitment to terminal differentiation of HL60 cells. These changes in group synthesis were not observed when K562 human chronic myelogenous leukemia cells were induced to differentiate to erythroid-like cells but there was a comparable increase in amounts of hsp70. When cells were treated with concentrations of drugs which brought about a loss in cell viability there was an early increase in the amount of hsp70 protein in the absence of any increase in synthesis. HL60 cells were treated with NMF (225mM), Adriamycin (1μM), or CB3717 (5μM) and there was an increase in the amounts of hsp70, in the absence of any new synthesis, which preceded any loss of membrane integrity and any significant changes in cell cycle but was concomitant with a later loss in viability of > 50% and a loss in proliferative potential. The amounts of hsp70 in the cell after treatment with any of the drugs was comparable to that obtained after a heat shock. Following a heat shock hsp70 was translocated from the cytoplasm to the nucleus, but treatment with toxic concentrations of drug caused hsp70 to remain localised in the cytoplasm. Changes in hsp70 turn-over was observed after a heat shock compared to NMF-treated cells. Morphological studies suggested that cells that had been treated with NMF and CB3717 were undergoing necrosis whereas the Adriamycin cells showed characteristics that were indicative of apoptosis. The data supports the hypothesis that an increase in amounts of hsp70 is an early marker of cell death.

Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, glucose metabolism, and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff-cell glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Analysis of Histone Deacetylase 7 (HDAC7) Alternative Splicing and Its Role in Embryonic Stem Cell Differentiation Toward Smooth Muscle Lineage

Histone deacetylases (HDACs) have a central role in the regulation of gene expression, which undergoes alternative splicing during embryonic stem cell (ES) cell differentiation. Alternative splicing gives rise to vast diversity over gene information, arousing public concerns in the last decade. In this chapter, we describe a strategy to detect HDAC7 alternative splicing and analyze its function on ES cell differentiation.

Erythropoietin reduces pathogenic humoral immunity by inhibiting T Follicular Helper cell differentiation and function

A large fraction of organ transplant recipients develop anti-donor antibodies (DSA), with accelerated graft loss and increased mortality. We tested the hypothesis that erythropoietin (EPO) reduces DSA formation by inhibiting T follicular helper (TFH) cells. We measured DSA levels, splenic TFH, TFR cells, germinal center (GC), and class switched B cells, in murine models of allogeneic sensitization, allogeneic transplantation and in parent-to-F1 models of graft versus host disease (GVHD). We quantified the same cell subsets and specific antibodies, upon EPO or vehicle treatment, in wild type mice and animals lacking EPO receptor selectively on T or B cells, immunized with T-independent or T-dependent stimuli. In vitro, we tested the EPO effect on TFH induction. We isolated TFH and TFR cells to perform in vitro assay and clarify their role. EPO reduced DSA levels, GC, class switched B cells, and increased the TFR/TFH ratio in the heart transplanted mice and in two GVHD models. EPO did also reduce TFH and GC B cells in SRBC-immunized mice, while had no effect in TNP-AECM-FICOLL-immunized animals, indicating that EPO inhibits GC B cells by targeting TFH cells. EPO effects were absent in T cells EPOR conditional KO mice, confirming that EPO affects TFH in vivo through EPOR. In vitro, EPO affected TFH induction through an EPO-EPOR-STAT5-dependent pathway. Suppression assay demonstrated that the reduction of IgG antibodies was dependent on TFH cells, sustaining the central role of the subset in this EPO-mediated mechanism. In conclusion, EPO prevents DSA formation in mice through a direct suppression of TFH. Development of DSA is associated with high risk of graft rejection, giving our data a strong rationale for studies testing the hypothesis that EPO administration prevents their formation in organ transplant recipients. Our findings provide a foundation for testing EPO as a treatment of antibody mediated disease processes.

TAGLN expression is deregulated in endometriosis and may be involved in cell invasion, migration, and differentiation

We found an increased expression of the TAGLN gene in endometriotic lesions compared with the eutopic endometrium of the same patients by real-time polymerase chain reaction. It is possible that this deregulation contributes to the development and maintenance of endometriosis by being involved in the pathways of organization of cytoskeletal architecture. (Fertil Steril (R) 2011;96:700-3. (C)2011 by American Society for Reproductive Medicine.)

Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory gd T cell subsets

Two distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27(-) γδ T cells) or interferon-γ (IFN-γ) (CD27(+) γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27(+) γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27(-) γδ T cells displayed permissive chromatin configurations at loci encoding both cytokines and their regulatory transcription factors and differentiated into cells that produced both IL-17 and IFN-γ in a tumor microenvironment.

Protein kinase C-activating tumor promoters enhance the differentiation of astrocytes in aggregating fetal brain cell cultures.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a marked, rapid, and sustained increase in the activity of the astrocyte-specific enzyme glutamine synthetase (GS). This effect was accompanied by a small increase in RNA synthesis and a progressive reduction in DNA synthesis. Only mitotically active cultures were responsive to PMA treatments. Since in aggregate cultures astrocytes are the preponderant cell type, both in number and mitotic activity, it can be concluded that PMA induces and/or enhances the terminal differentiation of astrocytes. The developmental expression of GS was also greatly stimulated by mezerein, a potent nonphorbol tumor promoter, but not by 4 alpha-phorbol 12,13-didecanoate, a nonpromoting phorbol ester. Since both tumor promoters, PMA and mezerein, are potent and specific activators of C-kinase, it is suggested that C-kinase plays a regulatory role in the growth and differentiation of normal astrocytes.

Notch signaling is required for normal prostatic epithelial cell proliferation and differentiation.

Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis.

An HMG-box-containing T-cell factor required for thymocyte differentiation.

Two candidate genes for controlling thymocyte differentiation, T-cell factor-1 (Tcf-1) and lymphoid enhancer-binding factor (Lef-1), encode closely related DNA-binding HMG-box proteins. Their expression pattern is complex and largely overlapping during embryogenesis, yet restricted to lymphocytes postnatally. Here we generate two independent germline mutations in Tcf-1 and find that thymocyte development in (otherwise normal) mutant mice is blocked at the transition from the CD8+, immature single-positive to the CD4+/CD8+ double-positive stage. In contrast to wild-type mice, most of the immature single-positive cells in the mutants are not in the cell cycle and the number of immunocompetent T cells in peripheral lymphoid organs is reduced. We conclude that Tcf-1 controls an essential step in thymocyte differentiation.

Epidermal growth factor and bovine growth hormone stimulate differentiation and myelination of brain cell aggregates in culture.

Bovine growth hormone (bGH) and epidermal growth factor (EGF) increased the activity of ornithine decarboxylase (ODC) in brain cell aggregates cultured in a serum-free chemically defined medium. ODC is considered as a marker of cell growth and differentiation. The effect of bGH and EGF on myelination was investigated by measuring two myelin markers, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin basic protein (MBP). EGF treatment at days 2 and 5 caused a dose-dependent increase of both myelin markers at culture day 12. This increase could still be observed at culture day 19, indicating a prolonged action of EGF. The continual presence of bGH in the culture medium produced a large accumulation of MBP at day 19. This effect was dose-dependent and required the presence of triiodothyronine (T3). In contrast, the effect of bGH on CNP activity did not require the presence of T3. This is the first report showing a direct effect of bGH on CNS myelination in vitro and of EGF on both MBP accumulation and ODC activity.

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

Unicellular organisms, such as the protozoan parasite Leishmania, can be stimulated to show some morphological and biochemical features characteristic of mammalian apoptosis. This study demonstrates that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enzymes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa.

Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC self-renewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis.