Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human monocyte derived dendritic cells treated with DC-SIGN initiates an immature dendritic cell phenotype triggering Rho activation that is utilised by HIV-1

ABSTRACT: DC-SIGN is a C-type lectin expressed by dendritic cells (DCs) that binds HIV-1, sequestering it within multivesicular bodies to facilitate transmission to CD4+ T cells. Here we characterize the molecular basis of signalling through DC-SIGN by large-scale gene expression profiling and phosphoproteome analysis. Solitary DC-SIGN activation leads to a phenotypically disparate transcriptional program from Toll-like receptor (TLR) triggering with downregulation of MHC II, CD86, and interferon response genes and with induction of the TLR negative regulator ATF3. Phosphoproteome analysis reveals DC-SIGN signals through the leukemia-associated Rho guanine nucleotide exchange factor (LARG) to induce Rho activity. This LARG activation also occurs on DC HIV exposure and is required for effective HIV viral synapse formation. Taken together HIV mediated DC-SIGN signalling provides a mechanism by which HIV evades the immune response yet induces viral spread. Experiment Overall Design: Circulating monocyte derived DCs were isolated from buffy coats by adherence and culture in IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). DC preparations analyzed were more than 98% pure. At day four 10 million immature DCs were either left unstimulated or stimulated using plate bound anti-DC-SIGN antibody for 2 hr. Three replicates of non-stimulated or stimulated cells were taken and used to extract total RNA.

ORGANISM(S): Homo sapiens

SUBMITTER: Dilair Baban

PROVIDER: E-GEOD-6090 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Activation of the lectin DC-SIGN induces an immature dendritic cell phenotype triggering Rho-GTPase activity required for HIV-1 replication.

Hodges Ashleigh A Sharrocks Katherine K Edelmann Mariola M Baban Dilair D Moris Arnaud A Schwartz Olivier O Drakesmith Hal H Davies Kay K Kessler Benedikt B McMichael Andrew A Simmons Alison A

Nature immunology 20070513 6

DC-SIGN, a C-type lectin expressed on dendritic cells (DCs), can sequester human immunodeficiency virus (HIV) virions in multivesicular bodies. Here, using large-scale gene expression profiling and tyrosine-phosphorylated proteome analyses, we characterized signaling mediated by DC-SIGN after activation by either HIV or a DC-SIGN-specific antibody. Activation of DC-SIGN resulted in downregulation of genes encoding major histocompatibility complex class II, Jagged 1 and interferon-response molecu ...[more]

PMID: 17496896

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Project description:Toll-like receptor (TLR)-induced maturation of dendritic cells (DCs) leads to the production of proinflammatory cytokines as well as the upregulation of various molecules involved in T cell activation. These are believed to be the critical events that account for the induction of the adaptive immune response. Here, we have examined the role of micro-RNA-155 (miR-155) in DC function and the induction of immunity. Using a model where the transfer of self-antigen-pulsed, TLR-matured DCs can induce a functional CD8 T cell response and autoimmunity, we find that DCs lacking miR-155 have an impaired ability to break immune tolerance. Importantly, transfer of self- antigen-pulsed DCs overexpressing miR-155 was sufficient to break tolerance in the absence of TLR stimuli. Although these unstimulated DCs induced T cell function in vivo, there was no evidence for the upregulation of costimulatory ligands or cytokine secretion. Further analysis showed that miR-155 influenced the level of the phosphatase SHIP1 in DCs, and that the lack of SHIP1 in DCs was sufficient to break T cell tolerance in vivo, again in the absence of TLR induced DC maturation. Our study demonstrates that the overexpression of miR-155 in DCs is a critical event that is alone sufficient to break self tolerance and promote a CD8-mediated autoimmune response in vivo. This process is independent of the induction of conventional DC maturation markers, indicating that miR-155 regulation of SHIP represents a unique axis that regulates DC function in vivo. Dendritic cell culture: Bone marrow was flushed from tibias and femurs of mice with HBSS. Bone marrow cells were cultured at 2x106 cells/ml in 10 ml non-tissue culture treated dishes in RPMI 1640 containing 10% LPS-free FBS, Penicillin:streptomycin glutamine 2-mercaptoethanol (cRPMI), with 40ng/ml murine GM-CSF (Peprotech). On day 3 of cultures 10ml of fresh media was added also containing 40ng/ml GM-CSF. On days 6 and 8, 10ml of media was removed and centrifuged to collect cells, which were resuspended in 10ml fresh media containing GM-CSF and were added back to dishes. Non-adherent cells were isolated on day 9. DCs were plated in 24-well plates at 2x106 cells/ml in 1ml of cRPMI with or without the class B CpG ODN1826 (ACGT DNA Technologies corporation, Toronto, ON, Canada) at 10?M final concentration overnight. Array analysis of mi-RNA expression: BMDCs from WT mice were either stimulated with CpG or left in media alone overnight. Whole RNA was isolated using mirVana miRNA isolation kit following the manufacturers instructions. RNA samples were labeled and hybridized to Agilent mouse miRNA 8x15K arrays (Agilent). Data was generated from 8 individual samples (4 unstimulated and 4 CpG-treated).

Dataset Information

Transcription profiling of human monocyte derived dendritic cells treated with DC-SIGN initiates an immature dendritic cell phenotype triggering Rho activation that is utilised by HIV-1

Publications

Activation of the lectin DC-SIGN induces an immature dendritic cell phenotype triggering Rho-GTPase activity required for HIV-1 replication.

Similar Datasets

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