Transcriptome analysis of HSPC, THP1 and K562 cells under IFNɑ treatment
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ABSTRACT: HSPC harbor constitutively active antiviral blocks that can be induced by type I IFN in certain cell types such as THP-1 cells but not in the K562 cell line. In the effort to identify such factors we compared trascriptomes from HSPC, THP1 and K562 stimulated or not with type I IFN
Project description:Macrophage activation is required for the control of innate and adaptive immune responses. The classification in M1 and M2 macrophages based on a combination of small numbers of membrane and soluble markers is operational in murine and human macrophages. If this classification may be extended to circulating monocytes is not elucidated. To answer such question, human monocytes were stimulated for 6 and 18 hours with IFN-gamma and IL-4, two canonical agonists of M1/M2 polarization in macrophages, and gene expression programs were investigated with whole genome microarrays. The temporal analysis of these programs showed marked differences to both IFN-gamma and IL-4. In 6-h stimulated monocytes, gene categories related to inflammatory and immune responses were enriched, and these monocytes exhibited a M1 and M2 polarization in response to IFN-gamma and IL-4, respectively, as found in macrophages. In 18-h stimulated monocytes, the categories related to innate immunity and metabolic pathways were enriched in response to IFN-gamma and IL-4, whereas PPAR signaling pathway was specifically enriched in response to IL-4. In addition, the M1 and M2 polarization induced by IFN-gamma and IL-4, respectively, was replaced by an original transcriptional program that did not depend on IFN-gamma and IL-4. This program appeared as networks around chemokines, NF-kappaB/MAP kinase pathways and MHC class II molecules. These results clearly demonstrated that monocyte activation consisted of an early polarized stage likely involved in effector responses and a delayed stage that may regulate host responses. The establishment of databases on human circulating monocytes using high throughput methods may be critical for pathophysiological and clinical non-invasive studies. Peripheral blood mononuclear cells (PBMCs) were isolated from leukopacks from normal blood donor buffy coats (Etablissement Français du Sang, Marseille, France) by Ficoll density gradient
Project description:In the early stages of wound healing, keratinocytes become “activated” and release inflammatory molecules such as interleukin-1 and interleukin-8 that are linked to innate immune responses and neutrophil recruitment. It is unclear, however, whether keratinocytes release molecules linked to adaptive immune responses, e.g. CCL20, in their early state of activation without signals from infiltrating T cells. This study aims to isolate the immediate alterations in protective and inflammatory gene expression that occur in epidermal keratinocytes, with a particular focus on molecules associated with cell-mediated immunity. We used dispase-separated epidermis, followed by intercellular disassociation by trypsinization, as a model for epidermal injury. We obtained a pure population of keratinocytes using flow cytometry. As a control for uninjured epidermis, we performed laser capture microdissection on normal human skin. Sorted keratinocytes had an early burst of upregulated gene expression, which included CCL20, IL-15, IL-23A, IFN-κ, and several antimicrobial peptides. Our results provide insight into the potential role of keratinocytes as contributors to cell-mediated inflammation, and expand knowledge about gene modulation that occurs during early wound healing. Our findings may be relevant to cutaneous diseases such as psoriasis, where micro-injury can trigger the formation of psoriatic plaques at the site of trauma. Compare keratinocyte response to cell disassociation with (1) epidermal samples isolated by laser caputre microscopy and (2) cultured KCs
Project description:Macrophages are known to be polarized into inflammatory (M1) and immunoregulatory (M2) cells when they are stimulated by agonists such as IFN-gamma and IL-4, respectively. If circulating monocytes may be polarized in response to T cell signals is often misguidedly deduced from macrophage results. Here the transcriptional responses of human CD14+ monocytes to IFN-gamma and IL-4 were analyzed using whole genome microarrays. A principal component analysis and hierarchical clustering showed that monocyte and macrophage responses were distinct. Monocytes stimulated with IFN-gamma and IL-4 for 6 hours exhibited some features of macrophage polarization. Indeed, when 80 genes considered as M1 and M2 genes were analyzed, we found that M1 genes were modulated in response to IFN-gamma and that M2 genes were modulated in response to IL-4. The M1 polarization of monocytes was transient because only M2 genes were modulated when monocytes were stimulated with IFN-gamma and IL-4 for 18 hours. However, the activation of monocytes by IFN-gamma and IL-4 could not be reduced to M1/M2 polarization status. Indeed, monocytes exhibited early specific signatures composed of 46 and 39 up-regulated genes in response to IFN-gamma and IL-4, respectively, and a late signature common to both molecules that consisted of 57 up-regulated genes. Taken together, these results demonstrated the extreme plasticity of human monocytes and suggested the existence of a core transcriptional termination program. Using early and late signatures might be pertinent to investigate monocyte activation in inflammatory or infectious diseases. Monocytes were stimulated with IFN-gamma (20ng/mL) or IL-4 (20ng/mL) for 6 and 18 hours or culture for 6 and 18 hours without agonist (Unstimulated samples). Monocytes-derived-macrophages (MDM) stimulated with IFN-gamma and IL-4 for 18 hours were used as controls. Each microarray is derived from a single biological sample.
Project description:Acute myeloid leukemia (AML) is a hematological malignancy, associated with unfavorable patient outcome primarily due to disease relapse. Since specific early leukemic hematopoietic stem and progenitor cells (HSPCs) are suggested to be responsible for AML propagation, the present study used single cell analysis (SCA) to detect and explore rare relapse-initiating HSPC clones, appearing already at diagnosis. To address inherent SCA limitations, we developed a unique high-resolution technique capable to follow single cell-derived subclones of heterogeneous HSPC subpopulations during AML evolution. Each of these subclones was evaluated for chemo-resistance, in-vivo leukemogenic potential, mutational profile, and the subclone cell of origin identified using reconstruction of phylogenetic trees. This study, employing combined functional and genomic analyses, unraveled the patient-specific HSPC subpopulations involved in chemo-resistance and determined, at time of diagnosis, the phenotype of the relapse-initiating clone, allowing early prediction of AML recurrence and suggesting novel precise therapeutic targets for relapse prevention.
Project description:The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-?) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-? bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-?/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-?/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system. polyA RNA profiling of Neural Stem/Progenitor cells (NPCs) cultured in basal/Th1/Th2 conditions, of Exosomes derived from NPCs cultured in basal/Th1/Th2 conditions and of EVs derived from NPCs cultured in Basal/Th1/Th2 conditions. Total RNA was purified using Trizol. Purity and integrity were confirmed by BioAnalyser (Agilent). Paired End library construction and poly-A selection were performed by EASIH (The Eastern Sequence and Informatics Hub, University of Cambridge, Cambridge) according to the Illumina standard protocol. Sequencing was performed by EASIH using Illumina GAII.
Project description:A growing body of evidence suggests that inflammatory cytokines have a dualistic role in immunity. In this study, we sought to determine the direct effects IFN-gamma on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (moDC). Here, we report that following differentiation of human peripheral-blood monocytes into moDCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, interferon-gamma (IFN-gamma) induces moDC maturation and up-regulates the co-stimulatory markers CD80, CD86, CD95, and MHC Class I, enabling moDCs to effectively generate antigen-specific CD4+ and CD8+ T cell responses for multiple viral and tumor antigens. Interestingly, early exposure of monocytes to high concentrations of IFN-gamma promotes monocyte differentiation into macrophages, despite the presence of GM-CSF and IL-4. However, under low concentrations of IFN-gamma, monocytes continue to differentiate into dendritic cells possessing a unique gene-expression profile, resulting in impairments in subsequent maturation by IFN-gamma and an inability to generate effective antigen-specific CD4+ and CD8+ T cell responses compared to standard moDCs. Monocytes differentiated in the presence of low levels of IFN-gamma downregulate IFN-gamma receptor expression, impairing their response to an inflammatory rechallenge. These findings demonstrate the ability of IFN-gamma to impart differential programs on human moDCs which shape the antigen-specific T cell responses they induce. Timing and intensity of exposure to IFN-gamma can thus determine whether moDCs are tolerogenic or immunostimulating. Human monocyte-derived dendritic cells from 4 healthy donors were differentiated with either GM-CSF and IL-4 (n=4) or GM-CSF, IL-4, and IFN-gamma (n=4). These samples were subsequently hybridized to arrays as 4 biological repeats for each of the two treatment conditions.
Project description:The source of IFN-γ in ovarian cancer microenvironment and its biological effect to the tumor cells is unclear. The immortalized human ovarian surface epithelial cell line, HOSE-E7/hTERT (HOSE) was treated with IFN-γ and expression microarray analysis was performed, and probes showing significantly higher values in IFN-γ-added group were termed â??IFN-γ signature genes (295 probes)â??. We then applied this signature to our ovarian cancer microarray data, which included 75 ovarian cancer clinical samples, by means of ss-GSEA. IFN-γ signature score was strongly correlated to the number of infiltrating CD4-positive or CD8-positive lymphocytes in the tumors. These data suggest that the IFN-γ in the ovarian cancer microenvironment is derived from lymphocytes, and an IFN-γ-rich microenvironment is strongly correlated to a lymphocyte-rich microenvironment. HOSE cells were incubated in 8 separate culture dishes, 4 dishes with and 4 dishes without 500 IU/ml recombinant human IFN-γ (R&D Systems) in the culture medium for 6 hours prior to the analysis.
Project description:To identify gene expression changes and pathways induced by interferon-? (IFN-?) in B cells, we studied the in vitro response of EBV-transformed B cells (lymphoblast cell lines-LCLs). LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-? exposure, with over 50 novel IFN-? response genes. Pathways analysis demonstrated that IFN-? affected LCLs in a similar manner to other cell types by activating known IFN-? canonical pathways. Additionally, IFN-? increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Several of the novel response genes were tested and validated as IFN-? response genes in human primary B cells. Total RNA from LCL samples treated for four hours with interferon beta (100u/ml) was compared to RNA from untreated LCLs (paired analysis). LCL samples were from patients with MS.