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Monocyte-Derived Dendritic Cells Differ in Cytoskeleton and Cell Adhesion-Related Expression Depending on VEGF Exposure

ABSTRACT: The inadequate function of the antigen presenting cells, such as dendritic cells (DCs) in cancer is one of the major factors leading to compromised anti-tumor immune response. Cell-to-cell interactions and cell migration are essential features of the immunological response in cancer. Vascular endothelial growth factor (VEGF) has been shown to affect DCs differentiation and maturation processes, thus leading to impaired DCs function. This study aimed to compare the expression of adherence and cytoskeleton-related genes between mature monocyte-derived dendritic cells (mDCs) and mDCs previously exposed to VEGF (mDCVs). VEGF is known to stimulate cell migration and actin rearrangement, but its association with adhesion molecules has not been largely explored in the context of DCs. Our results show that immature human DCs exposed to VEGF up-regulated CD80 and CD86, typical cell surface markers of DC maturation. However, unlike mDCs, mDCVs showed alterations in cell morphology, with broad changes in the characteristic cytoplasmic processes. DNA microarray analysis revealed that mDCVs up-regulated a significant subset of genes related to cell migration, including cell adhesion molecules and related signaling pathways as well as cytoskeleton-related genes. Our results suggest that VEGF, known to be involved in the inhibition of DC maturation, interferes in cell mechanisms leading to cytoskeleton reorganization and the expression of cell adhesion molecules. Cell morphology is associated with DC maturation and migration, both impaired in pathological conditions such as cancer. Keywords: Gene expression profiling

ORGANISM(S): Homo sapiens

PROVIDER: GSE16072 | GEO | 2011/12/30

SECONDARY ACCESSION(S): PRJNA115497

REPOSITORIES: GEO

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Project description:The inadequate function of the antigen presenting cells, such as dendritic cells (DCs) in cancer is one of the major factors leading to compromised anti-tumor immune response. Cell-to-cell interactions and cell migration are essential features of the immunological response in cancer. Vascular endothelial growth factor (VEGF) has been shown to affect DCs differentiation and maturation processes, thus leading to impaired DCs function. This study aimed to compare the expression of adherence and cytoskeleton-related genes between mature monocyte-derived dendritic cells (mDCs) and mDCs previously exposed to VEGF (mDCVs). VEGF is known to stimulate cell migration and actin rearrangement, but its association with adhesion molecules has not been largely explored in the context of DCs. Our results show that immature human DCs exposed to VEGF up-regulated CD80 and CD86, typical cell surface markers of DC maturation. However, unlike mDCs, mDCVs showed alterations in cell morphology, with broad changes in the characteristic cytoplasmic processes. DNA microarray analysis revealed that mDCVs up-regulated a significant subset of genes related to cell migration, including cell adhesion molecules and related signaling pathways as well as cytoskeleton-related genes. Our results suggest that VEGF, known to be involved in the inhibition of DC maturation, interferes in cell mechanisms leading to cytoskeleton reorganization and the expression of cell adhesion molecules. Cell morphology is associated with DC maturation and migration, both impaired in pathological conditions such as cancer. Keywords: Gene expression profiling Gene expression profiling was carried out in freshly isolated CD14+ cells prior to cultivation (“D0” samples) and in DCs after 6 days (“D6” samples) and 7 days in culture, with or without VEGF treatment (“D7V” and “D7” samples, respectively). Blood samples were collected on the same day and processed concomitantly. Cell culture, RNA extraction, and microarray hybridization were also carried out simultaneously in an attempt to minimize technical inconsistencies. A total of 14 independent microarray hybridizations were carried out with oligonucleotide microarrays, covering approximately 55,000 human transcripts (Whole Genome CodeLink™ Bioarrays, GE Healthcare). Target preparation and hybridization procedures strictly followed protocols provided by the manufacturer.Arrays were scanned following the recommended scanning procedure and settings for use with CodeLink bioarrays (GE Healthcare) on GenePix 4000B Array Scanner/GenePix Pro 4.1 software (Axon Instruments).

Project description:The maturation of dendritic cells (DCs) after exposure to microbial products or inflammatory mediators plays a critical role in initiating the immune response. We found that maturation can also occur under steady state conditions, triggered by alterations in E-cadherin-mediated DC-DC adhesion. Selective disruption of these interactions induced the typical features of DC maturation including the upregulation of costimulatory molecules, MHC class II, and chemokine receptors. These events were triggered at least in part by activation of the b-catenin pathway. However, unlike maturation induced by microbial products, E-cadherin-stimulated DCs failed to release immunostimulatory cytokines, exhibiting an entirely different transcriptional profile. As a result, E-cadherin-stimulated DCs elicited an entirely different T cell response in vivo, generating T cells with a regulatory as opposed to an effector phenotype. These DCs induced tolerance in vivo and may thus contribute to the elusive steady state âtolerogenic DCsâ. Experiment Overall Design: We performed a genome-wide microarray analysis to study the expression profiles of DCs matured by CD as opposed to a conventional TLR agonist (E. coli, which stimulates multiple TLRs). RNA was isolated from human CD34+ DCs at various times after stimulation and used to probe Affymetrix U95Av2 chips. A time course was followed after various stimuli, with single chips used per time point. >700 genes were found differentially regulated upon maturation by either CD or bacterial stimulation. Cluster analysis revealed that after an early phase (1-3 hr) of similarity, expression profiles exhibited by the two sets of DCs diverged dramatically at later time points (>6 hr). A number of transcripts were markedly upregulated in the bacteria-stimulated set that remained relatively unchanged or actually decreased in the cluster-disrupted set. There were some transcripts upregulated in cluster-disrupted cells, however, with at least some of these increases prevented by adding anti-E-cadherin mAb under conditions that blocked maturation. Clearly, the transcriptional events induced by alteration of E-cadherin-mediated adhesion were quite distinct from those induced by TLR activation.

Project description:Background: It has been shown that intracellular pathogens hijack DC functions to evade immune defense mechanisms. In this study, we investigated the responses of human monocyte derived DCs to four intracellular bacteria, Tropheryma whipplei, Coxiella burnetii, Brucella abortus and Orientia tsutsugamushi, responsible for human infectious diseases and known to infect myeloid cells. Methods: Whole genome microarrays were assessed to define common and specific transcriptionnal responses to bacterial pathogen. Bacterial pathogen ability to affect DC maturation was assessed by measuring lymphoproliferation and endocytosis as well as phenotypic maturation markers (CD80, CD83, CD86 and HLA-DR) expression. Results: We found that Coxiella burnetii, Orientia tsutsugamushi and Brucella abortus induced DC maturation assessed through decreased endocytosis ability, triggering lymphoproliferation, surface expression of HLA class II molecules and phenotypic changes, whereas Tropheryma whipplei did not induce DC maturation. As revealed by microarray analysis, the response of DCs to these bacteria consisted of a core associated with the maturation of DCs and signatures specific for each pathogen. The core response represented 10% of genes modulated in response to pathogens and consisted of general cellular processes including nucleotide binding, protein transport, cell fraction, protein kinase, cell cycle, mitochondrial membrane and cytoskeleton. The specific transcriptional signature induced by C. burnetii is associated with the communication between innate and adaptive immune cells and DC maturation. B. abortus signature specifically involved arachidonic acid and lipooxygenase pathways and O. tsutsugamushi signature involved type I and type III IFN responses. Conclusion: This study demonstrates that intracellular bacteria use multifaceted pathways to induce DC maturation which may lead to unadapted immune response. The understanding of these pathways may be useful to improve our knowledge of bacterial recognition by the immune system but also intracellular bacterial diseases. IL4/GM-CSF monocyte-derived dendritic cells were stimulated by T. whipplei, B. abortus, C. burnetii, O. tsutsugamushi and LPS during 6 hours. Common and specific signatures were determined by comparison with uninfected DCs. moDCs (5.10^6) were plated in 6 well plates and stimulated with bacteria or LPS for 6 hours, and total RNA was extracted using the RNeasy minikit (Qiagen, adresse CA) and DNase treatment. The Agilent-014850 4X44k Human Whole Genome microarrays (Agilent Technologies, CA) representing 44,000 probes were used as recently described. Reverse transcription, sample labeling and hybridization were performed according to protocols specified by the manufacturer (One-Color Microarray-Based Gene Expression Analysis). Three samples per experimental condition were included in the analysis.

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Monocyte-Derived Dendritic Cells Differ in Cytoskeleton and Cell Adhesion-Related Expression Depending on VEGF Exposure

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