Unknown,Transcriptomics,Genomics,Proteomics

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Transcript abundance comparison between uninfected DCs and DCs housing L. amazonensis

ABSTRACT: To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes Dendritic cells were generated in vitro from bone marrow progenitors. Leishmania amazonensis amastigotes were purified from mouse cutaneous lesions and were added to DC cultures. After 24h, and following a sorting procedure, only BMDCs housing living amastigotes were selected for total RNA extraction.

ORGANISM(S): Mus musculus

SUBMITTER: Emilie De La Llave

PROVIDER: E-GEOD-16644 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Dendritic cells (DCs) are a special class of leukocytes able to activate both innate and adaptive immune responses. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. We have observed that following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. Indeed, LPS induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. According with this observation CD14-deficient DCs do not die following LPS stimulation. Nevertheless, CD14-deficient DC death following LPS activation can be restored by co-stimulating DCs with LPS and thapsigargin. Thapsigargin empties the intracellular calcium stores by blocking calcium pumping into the sarcoplasmic and endoplasmic reticulum and thereby activates plasma membrane calcium channels. This, in turn, allows an influx of calcium into the cytosol and NFAT activation. To identify the NFAT controlled apoptosis genes in LPS activated DCs we have performed a kinetic microarray analysis (0, 48 and 60 h) in conditions allowing or inhibiting NFAT activation. Four genes have been selected: Nur77, Gadd45g, Ddit3/Gadd153/Chop-10 and Tia1. To identify apoptosis genes selectively modulated by NFAT, a comparative kinetic (time points 0, 48 and 60 h) microarray analysis was performed in the following conditions: 1) wild type bone marrow derived DCs (wtBMDCs) stimulated with LPS; 2) CD14-deficient BMDCs stimulated with LPS; 3) wtBMDCs stimulated with LPS in presence of thapsigargin; 4) CD14-deficient BMDCs stimulated with LPS in presence of thapsigargin.

Project description:In mammals, resident dermal macrophages (MÎ¦s) are subverted by Leishmania (L.) amazonensis amastigotes as host cells permissive for parasite multiplication. These Leishmania are living within a communal parasitophorous vacuole (PV) and are expected to trigger unique MÎ¦ transcriptional signatures. We performed a transcription profiling of mouse MÎ¦s harboring amastigotes to get insights into their reprogramming as host cells for parasite multiplication. BALB/c mouse bone marrow-derived MÎ¦s were either loaded or not with four amastigotes on average. Twenty four hours later, when amastigotes multiply, total RNA from MÎ¦ cultures was prepared, amplified and hybridized onto Affymetrix Mouse430_2 GeneChipsÂ®. The outcome recorded a total of 1,248 probe-sets showing significant differential expression. Comparable fold-change values for a handful of genes were obtained between Affymetrix technology and the more sensitive RTqPCR method. Ingenuity Pathway Analysis softwareÂ® pinpointed the up-regulation of the sterol biosynthesis pathway (P-value = 1.31e-02) involving several genes (1.95 to 4.30 fold-change values), and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signaling. Our findings suggest that amastigotes exploit the MÎ¦ lipid and polyamine pathways to multiply efficiently, and induce a counter-inflammatory environment to expand their dermis niche. Experiment Overall Design: Mice, MÎ¦s and amastigotes: Experiment Overall Design: Swiss nu/nu and BALB/c mice were used (following National Scientific Ethics Committee guidelines) for L. amazonensis (LV79 strain, MPRO/BR/1972/M1841) amastigote propagation and to prepare bone marrow-derived MÎ¦s, respectively. Amastigotes were added at a multiplicity of 4 amastigotes per MÎ¦. Parasite-harboring MÎ¦s (>98%; samples I1, I2 and I3) and parasite-free ones (samples UI1, UI2 and UI3) were cultured at 34Â°C (LV79 permissive temperature) for 24h. Experiment Overall Design: Real-time quantitative PCR: Experiment Overall Design: Total RNA from various biological samples including those used for hybridization experiments were reverse transcribed into cDNA using random hexamers (Roche Diagnostics) and Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen Life Technologies). RTqPCR was performed using LightCycler-480 system (Roche) with primers designed with LightCycler Probe Design 1.0 software.Target genes primer sequences, amplicon melting temperatures and amplification efficiencies are available upon request. Gene expression analysis using qBase program allowed determining the normalized relative quantities between parasite-free and parasite-harboring MÎ¦s.

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Transcript abundance comparison between uninfected DCs and DCs housing L. amazonensis

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