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Gene expression in Mouse thrombomoudlin+ and thrombomodulin- dendritic cell

ABSTRACT: Previously we had shown in a mouse model of bronchial asthma that thrombomodulin (TM; CD141; BDCA3) can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on the cell surface. Thrombomodulin+ dendritic cells are tolerogenic while thrombomodulin- dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin+ and thrombomodulin- dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while cell cycle genes were increased in thrombomodulin-treated and thrombomodulin+ dendritic cells compared to control dendritic cells and thrombomodulin- dendritic cells. We compared the effects on global gene expression of treating dendritic cells with soulble thrombomodulin to no treatment, and dendritic cells separated into thrombomodulin+ and thrombomodulin- dendritic cell subsets.

ORGANISM(S): Mus musculus

PROVIDER: GSE45652 | GEO | 2014/12/31

SECONDARY ACCESSION(S): PRJNA195538

REPOSITORIES: GEO

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Project description:Abstract Two major dendritic cell (DC) subsets have been described in the islets of mice: The immunogenic CD8Î±-CD11b+ DCs and the tolerogenic CD8Î±+CD103+ DCs. We have recently reported on reduced numbers of the minor population of tolerogenic CD8Î±+CD103+ DCs in the pancreas of 5 week old pre-diabetic non-obese diabetic (NOD) mice. Aim: To analyze also the larger subset of CD11c+CD8Î±- DCs isolated from the pancreas of pre-diabetic NOD mice 1) for maturation and tolerance inducing molecules found abnormally expressed on CD8Î±+CD103+ DCs, and 2) for genome-wide gene expression to further elucidate abnormalities in underlying gene expression networks. Methods: CD11c+CD8Î±- DCs were isolated from 5 week old C57BL/6 and NOD pancreas. Expression of cell surface markers including CD86, CCR5, CD11b, CD103, Clec9a, CD24 and CD200R3 were measured by FACS. Genome-wide gene expression by microarray was assessed during the steady state and after in vitro LPS stimulation. Results: The steady state pancreatic CD11c+ CD8Î±- DCs during the pre-diabetic stage showed: 1) A reduced expression of several gene networks important for the prime functions of the cell, such as for cell renewal, immune stimulation and immune tolerance induction, for migration and for the provision of growth factors for beta cell regeneration. This general deficiency state was corroborated by a reduced in vivo proliferation (BrdU incorporation) of the cells and the reduced expression in FACS analysis of CD86, CCR5, CD103, Clec9a, CD24 and CD200R3 on the cells. 2) A hyper reactivity of these cells to LPS correlated with an enhanced pro-inflammatory state characterized by altered expression of a number of classical pro-inflammatory factors and cytokines. Conclusion: The NOD CD11c+CD8Î±- DCs seem to be Janus-faced depending on the conditions: Deficient in steady state with reduced immune stimulation capabilities also for tolerance induction; over-inflammatory with a molecular profile suggesting a preferential stimulatory capacity for Th1 cells when encountering a Pathogen-Associated Molecular Pattern (PAMP) in the form of LPS. We used microarray gene expression analysis to explain the abnormal expression of several cell surface markers involved in tolerace, migration and maturation in the steady-state and to measure the effect of a PAMP such as LPS We isolated RNA from FACS sorted CD11c+CD8Î±- DCs in 10 pooled pancreases from pre-diabetic NOD and non-diabetic C57BL/6 mice at 5 weeks. In addition, we treated in another experiment the isolated pancreas DCs with LPS (and PBS), incubated for 18h and measured gene expression. We compared gene expression between strains NOD vs C57BL/6 under steady-state and after in-vitro LPS/PBS stimulation.

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Gene expression in Mouse thrombomoudlin+ and thrombomodulin- dendritic cell

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