Unknown,Transcriptomics,Genomics,Proteomics

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Dendritic Cells

ABSTRACT: Dendritic cells (DC) play a crucial role in initiating, shaping and controlling the immune response. They are the most important antigen presenting cells in the immune system. DC can be divided in immature and mature DC. DCs reside in an immature state in most tissues or they circulate in the blood, where they recognize and phagocytose pathogens and other antigens. Direct contact with many pathogens leads to the maturation of DCs. The property to stimulate T cells is reserved for mature DC (O`Doherty, U. 1994). This functional maturation results in an up-regulation of the surface molecule MHC class II, adhesion molecules (CD54 and CD58) and co-stimulatory receptors (CD80 and CD86) as well as decreased antigen uptake capacity (CD206). (Banchereau, J. and Steinman, R.M. 1998). Immunosuppressive drugs have revolutionized organ transplantation and improved the therapeutic management of autoimmune diseases. These drugs interfere on lymphocytes but they also act at the earliest stage of immune response. They are targeting key functions of dendritic cells (Hackstein H. and Thomson A.W., 2004 review). Sanglifehrin A (SFA) is a new immunosuppressive drug. It is a representative of a class of macrolides produced by the actinomycetes strain Streptomyces A92-308110 that bind to cyclophilin A (CypA), the binding protein of cyclosporine A (CsA). Most studies about immunosuppressive effects of SFA took place on T and B lymphocytes. Unlike CsA, the cyclophilin-SFA-complex shows no effect on the calcium-dependent protein phosphatase calcineurin (Zenke, G. 2001). Unlike CsA and FK506, SFA shows no inhibition of IL-2 expression (Zenke, G. 2001; Zhang, L.H. 2001), nor does it suppress IL-2 receptor transcription (Zhang, L.H. 2001) in T lymphocytes. It could be shown that SFA reduces the Interleukin 12 production in dendritic cells (Steinschulte, C. 2003). IL-12p70 plays an important role in the pathogenesis of inflammation and autoimmune diseases. DCs generated in the presence of SFA show reduced macropinocytosis and lectin-mediated endocytosis. In contrast, CD89 und CD32 were increased by SFA. The effect of SFA on the expression of Ag uptake receptors and Ag capture by DCs makes SFA unique among other immunophilin-binding immunosuppressive drugs (Woltman, A. 2004). Our goal is to investigate the gene expression profile of SFA treated mature human monocyte-derived dendritic cells. Comparison of SFA-stimulated versus unstimulated cells. 7 biological replicates

ORGANISM(S): Homo sapiens

SUBMITTER: Jochen Wilhelm

PROVIDER: E-GEOD-15956 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Dendritic cells (DC) play a crucial role in initiating, shaping and controlling the immune response. They are the most important antigen presenting cells in the immune system. DC can be divided in immature and mature DC. DCs reside in an immature state in most tissues or they circulate in the blood, where they recognize and phagocytose pathogens and other antigens. Direct contact with many pathogens leads to the maturation of DCs. The property to stimulate T cells is reserved for mature DC (O`Doherty, U. 1994). This functional maturation results in an up-regulation of the surface molecule MHC class II, adhesion molecules (CD54 and CD58) and co-stimulatory receptors (CD80 and CD86) as well as decreased antigen uptake capacity (CD206). (Banchereau, J. and Steinman, R.M. 1998). Immunosuppressive drugs have revolutionized organ transplantation and improved the therapeutic management of autoimmune diseases. These drugs interfere on lymphocytes but they also act at the earliest stage of immune response. They are targeting key functions of dendritic cells (Hackstein H. and Thomson A.W., 2004 review). Sanglifehrin A (SFA) is a new immunosuppressive drug. It is a representative of a class of macrolides produced by the actinomycetes strain Streptomyces A92-308110 that bind to cyclophilin A (CypA), the binding protein of cyclosporine A (CsA). Most studies about immunosuppressive effects of SFA took place on T and B lymphocytes. Unlike CsA, the cyclophilin-SFA-complex shows no effect on the calcium-dependent protein phosphatase calcineurin (Zenke, G. 2001). Unlike CsA and FK506, SFA shows no inhibition of IL-2 expression (Zenke, G. 2001; Zhang, L.H. 2001), nor does it suppress IL-2 receptor transcription (Zhang, L.H. 2001) in T lymphocytes. It could be shown that SFA reduces the Interleukin 12 production in dendritic cells (Steinschulte, C. 2003). IL-12p70 plays an important role in the pathogenesis of inflammation and autoimmune diseases. DCs generated in the presence of SFA show reduced macropinocytosis and lectin-mediated endocytosis. In contrast, CD89 und CD32 were increased by SFA. The effect of SFA on the expression of Ag uptake receptors and Ag capture by DCs makes SFA unique among other immunophilin-binding immunosuppressive drugs (Woltman, A. 2004). Our goal is to investigate the gene expression profile of SFA treated mature human monocyte-derived dendritic cells.

Project description:Pro-inflammation triggered by microbial lipopolysaccharide (LPS) through Toll-like receptor (TLR) 4 in the presence of interferon (IFN)-g induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized by a dynamic immune activating but also tolerance inducing phenotype associated with irreversible down-modulation of cytokines. CD40L on activated T cells further modifies DC differentiation. Using DNA micro arrays we showed down-regulated mRNA levels of TLR signaling molecules while CD40/CD40L signaling molecules were up-regulated at a time when LPS/IFN-g activated DCs have ceased cytokine expression. Accordingly we demonstrated that CD40/CD40L but not TLR4 or TLR3 signaling mediated by LPS or poly (cytidylic-inosinic) acid (poly I:C) and dsRNA re-established the capacity to secret interleukin (IL)-12 in LPS/IFN-g activated DCs, which have exhausted their potential for cytokine secretion. This resulting TH1 polarizing DC phenotype â which lacked accompanying secretion of the crucial immune suppressive IL-10 - enhanced activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T-cell mediated stimulus at an exhausted DC state which prevents an immune tolerant DC phenotype. These findings impacts on the rational design of TLR activated DC-based cancer vaccines for the induction of anti-tumoral CTL responses. Experiment Overall Design: Monocyte derived DCs were matured for 6 hours with 30 ng/ml lipopolysaccharide (LPS, E. coli strain O111:B4, Calbiochem, San Diego, CA) and 1.000 U/ml IFN-g (Imukin, Boehringer Ingelheim, Austria). RNA of DCs was isolated after 6, 12, 24, or 48 hours of maturation using the RNeasy kit (Qiagen, Hilden, Germany) and analyzed by the RZPD (German Resource Centre for Genome Research, Berlin) using the Affymetrix DNA micro array platform (U133 plus 2.0). DNA array data from different maturation-time experiments were compared to controls that were kept in culture for the same time without stimulation.

Project description:Dendritic cells (DCs) in lymphoid tissue comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that develop from common DC progenitors (CDPs). CDPs are Flt3+c-kitintM-CSFR+ and reside in bone marrow. Here we describe a two-step culture system that recapitulates DC development from c-kithiFlt3-/lo multipotent progenitors (MPPs) into CDPs and further into cDC and pDC subsets. MPPs and CDPs are amplified in vitro with Flt3 ligand, stem cell factor, hyper-IL-6 and insulin- like growth factor-1. The four-factor cocktail readily induces self-renewal of MPPs and their progression into CDPs and has no self-renewal activity on CDPs. The amplified CDPs respond to all known DC poietins and generate all lymphoid tissue DCs in vivo and in vitro. Additionally, in vitro CDPs recapitulate the cell surface marker and gene expression profile of in vivo CDPs and possess a DC-primed transcription profile. Transforming growth factor-Î²1 (TGF-Î²1) impacts on CDPs and directs their differentiation towards cDCs. Genome-wide gene expression profiling of TGF-Î²1-induced genes identified transcription factors, such as interferon regulatory factor-4 (IRF-4) and RelB, that are implicated as instructive factors for cDC subset specification. TGF-Î²1 also induced the transcription factor inhibitor of differentiation/DNA binding 2 (Id2) that suppresses pDC development. Thus, TGF-Î²1 directs CDP differentiation into cDC by inducing both cDC instructive factors and pDC inhibitory factors. 20 samples in total. Multipotent progenitor - MPP_1 - MPP_2 Common dendritic cell progenitor - CDP_1 - CDP_2 Plasmacytoid dendritic cell - pDC_1 - pDC_2 Conventional dendritic cell - cDC_1 - cDC_2 In vivo common dendritic cell progenitor - In vivo CDP_1 - In vivo CDP_2 Untreated common dendritic cell progenitor (CDP) - CDP_0h_1 - CDP_0h_2 TGF-beta1 treated (4 hours) CDP - CDP_4h_1 - CDP_4h_2 TGF-beta1 treated (8 hours) CDP - CDP_8h_1 - CDP_8h_2 TGF-beta1 treated (12 hours) CDP - CDP_12h_1 - CDP_12h_2 TGF-beta1 treated (24 hours) CDP - CDP_24h_1 - CDP_24h_2

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Dendritic Cells

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