Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome of inflammatory myeloid DCs in psoriasis

ABSTRACT: Background: Previous work has identified CD11c+CD1c- dendritic cells (DCs) as the major 'inflammatory' dermal DC population in psoriasis vulgaris and CD1c+ DCs as the 'resident' cutaneous DC population. Objective: To further define molecular differences between these two myeloid dermal DC populations. Methods: Inflammatory and resident DCs were single-cell sorted from psoriasis lesional skin biopsies, and gene array expression profiling was performed. Results were confirmed with RT-PCR, flow cytometry, immunohistochemistry, and double label immunofluorescence. Pooled human keratinocytes were cultured for functional studies. Results: TNF-related apoptosis-inducing ligand (TRAIL), Toll-like receptors (TLRs) 1 and 2, S100A12/EN-RAGE, CD32, and many other inflammatory products were selectively expressed in inflammatory DCs than in resident DCs. Flow cytometry and immunofluorescence confirmed higher protein expression on CD1c- versus CD1c+ DCs. TRAIL receptor, death receptor 4 (DR4), was expressed on basal keratinocytes and blood vessels, and in vitro culture of keratinocytes with rh-TRAIL induced CCL20 leukocyte chemokine. Conclusion: CD11c+CD1c- inflammatory DCs in psoriatic lesional skin express a wide range of inflammatory molecules compared to skin resident CD1c+ DCs. Some molecules made by inflammatory DCs, including TRAIL, could have direct effects on keratinocytes or other skin cell types to promote disease pathogenesis. Shave biopsies were cultured overnight in dispase to remove the epidermis. Dermis was cultured for 36-48 hours to obtain dermal single cell suspensions. These cells were stained with HLA-DR, CD11c, BDCA-1 (DCs) and FACS-sorted into HLA-DR+11c+BDCA-1+, HLA-DR+11c+BDCA-1- populations into Trizol. RNA was extracted and processed for microarray. 2 dependent groups.

ORGANISM(S): Homo sapiens

SUBMITTER: Lisa Zaba

PROVIDER: E-GEOD-20264 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification of TNF-related apoptosis-inducing ligand and other molecules that distinguish inflammatory from resident dendritic cells in patients with psoriasis.

Zaba Lisa C LC Fuentes-Duculan Judilyn J Eungdamrong Narat John NJ Johnson-Huang Leanne M LM Nograles Kristine E KE White Traci R TR Pierson Katherine C KC Lentini Tim T Suárez-Fariñas Mayte M Lowes Michelle A MA Krueger James G JG

The Journal of allergy and clinical immunology 20100514 6

<h4>Background</h4>Previous work has identified CD11c(+)CD1c(-) dendritic cells (DCs) as the major "inflammatory" dermal DC population in patients with psoriasis vulgaris and CD1c(+) DCs as the "resident" cutaneous DC population.<h4>Objective</h4>We sought to further define molecular differences between these 2 myeloid dermal DC populations.<h4>Methods</h4>Inflammatory and resident DCs were single-cell sorted from lesional skin biopsy specimens of patients with psoriasis, and the transcriptome o ...[more]

PMID: 20471070

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Project description:In this study transcriptome profiling of dendritic cell subtypes was performed using various human dendritic cells. Monocyte-derived DC: CD14+ monocytes were obtained from buffy coats by Ficoll - gradient centrifugation and immunomagnetic cell separation using anti-CD14-conjugated microbeads. Monocytes were resuspended into 6-well culture dishes at a density of 1.5 x 10 million cells/ml and cultured in RPMI 1640 supplemented with 10% FBS containing 800 U/ml GM-CSF and 500 U/ml IL-4. Cells were cultured for 5 days and the IL-4 and GM-CSF addition was repeated at day 2. Dermal DC (DDC) and Langerhans Cells (LC) from skin: Human skin specimens were obtained from healthy donors undergoing corrective breast or abdominal plastic surgery after informed consent. Thick slices (3 mm) of skin containing the epidermis and the dermis were cut by use of a dermatome. Slices of skin were cut in pieces of 1 cm2 and incubated with Dispase II for 30â60 min at 37Â°C. The epidermis and dermis were separated with tweezers and washed with PBS. To isolate LC, the epidermal sheets were incubated with PBS with 0.05% trypsin for 10 min at 37Â°C, and a single-cell suspension was prepared by pushing the tissue through 100 Âµm pore nylon cell strainers with a plunger of a 2-ml syringe. Epidermal cell suspension was enriched for LC by density centrifugation over Lymphoprep and CD1a-guided MACS (Miltenyi Biotec). To isolate DDC, the dermis was incubated with PBS containing Dispase and Collagenase A at 37Â°C for 2 h, after which, a single-cell suspension was prepared by pushing through 100 Âµm pore nylon cell strainers with a plunger of a 2-ml syringe. Cell suspension was enriched for DDC by CD1a-guided MACS. Blood Cd1c+ DC: Blood CD1c+ DC were isolated from periferal blood. BDCA1+ (CD1c) DCs were incubated with a lineage-specific PE-labeled Ab mixture, HLA-DR-allophycocyanin, and BDCA1-FITC. Lineage-negative and HLA-DRhigh DCs, positive for BDCA1 were sorted on a FACS and collected in tubes containing 1 ml of 100% FBS. A total of 40,000â100,000 cells was isolated (>98% pure). Dendritic cell subtypes were differentiated in vitro from monocytes and primary dendritic cells were isolated from human skin and blood.

Dataset Information

Transcriptome of inflammatory myeloid DCs in psoriasis

Publications

Identification of TNF-related apoptosis-inducing ligand and other molecules that distinguish inflammatory from resident dendritic cells in patients with psoriasis.

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