ABSTRACT: Distinct Transcriptional Signatures of Bone Marrow-Derived C57BL/6 and DBA/2 Dendritic Leucocytes Hosting Live Leishmania amazonensis Amastigotes
Project description:To determine the modulation of gene expression of C57BL/6 and DBA/2 BMDLs in the presence of living intracellular Leishmania amazonensis amastigotes A genome-wide transcriptional analysis was performed by comparing the gene expression profiles of control DLs and live amastigote-hosting DLs from both mouse strains. Dendritic Leucocytes were generated in vitro from bone marrow progenitors (C57Bl/6 and DBA/2 mice). Leishmania amazonensis amastigotes were purified from mouse cutaneous lesions and were added to DL cultures. After 24h, and following a sorting procedure, only BMDls housing living amastigotes were selected for total RNA extraction. Three Biological replicates per condition were run.
Project description:To determine the modulation of gene expression of C57BL/6 and DBA/2 BMDLs in the presence of living intracellular Leishmania amazonensis amastigotes
Project description: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.
Project description:To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes
Project description:To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes at 24 hr post infection.
Project description:In mammals, resident dermal macrophages (MΦs) 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 MΦ transcriptional signatures. We performed a transcription profiling of mouse MΦs harboring amastigotes to get insights into their reprogramming as host cells for parasite multiplication. BALB/c mouse bone marrow-derived MΦs were either loaded or not with four amastigotes on average. Twenty four hours later, when amastigotes multiply, total RNA from MΦ cultures was prepared, amplified and hybridized onto Affymetrix Mouse430_2 GeneChips®. 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 software® 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 MΦ lipid and polyamine pathways to multiply efficiently, and induce a counter-inflammatory environment to expand their dermis niche.
Project description:In mammals, resident dermal macrophages (MΦs) 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 MΦ transcriptional signatures. We performed a transcription profiling of mouse MΦs harboring amastigotes to get insights into their reprogramming as host cells for parasite multiplication. BALB/c mouse bone marrow-derived MΦs were either loaded or not with four amastigotes on average. Twenty four hours later, when amastigotes multiply, total RNA from MΦ cultures was prepared, amplified and hybridized onto Affymetrix Mouse430_2 GeneChips®. 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 software® 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 MΦ lipid and polyamine pathways to multiply efficiently, and induce a counter-inflammatory environment to expand their dermis niche. Experiment Overall Design: Mice, MΦs 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 MΦs, respectively. Amastigotes were added at a multiplicity of 4 amastigotes per MΦ. Parasite-harboring MΦs (>98%; samples I1, I2 and I3) and parasite-free ones (samples UI1, UI2 and UI3) were cultured at 34°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 MΦs.
Project description:During its life cycle Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. In mammals the parasites replicate within parasitophorous vacuoles of macrophages, compartments that contain low concentrations of iron. Here we show that stimulation of iron transport, which is induced in Leishmania amazonensis by an iron-poor environment, triggers the differentiation of avirulent promastigotes into virulent amastigotes. Iron depletion from the culture medium triggered expression of the LIT1 ferrous iron transporter, growth arrest, and differentiation of wild type promastigotes into infective amastigotes. In contrast, LIT1 null promastigotes showed continued exponential growth in iron-poor media, followed by massive cell death. Iron depletion from the medium and LIT1 upregulation increased iron superoxide dismutase activity (FeSOD) in wild type, but not in LIT1 null parasites. Notably, the superoxide-generating drug menadione or H2O2 were sufficient to trigger differentiation of wild type promastigotes into fully infective amastigotes. On the other hand, LIT1 null promastigotes accumulated superoxide radical and initiated amastigote differentiation after exposure to H2O2, but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species (ROS) in orchestrating the differentiation of Leishmania infective stages, in a process regulated by iron availability. Four samples were obtained in total from wild-type L. amazonensis promastigotes grown for 24 hours in culture. Two of these samples derived from medium with iron and two from mediam without iron.These two samples derived from each culture served as biological replicates.
Project description:During its life cycle Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. In mammals the parasites replicate within parasitophorous vacuoles of macrophages, compartments that contain low concentrations of iron. Here we show that stimulation of iron transport, which is induced in Leishmania amazonensis by an iron-poor environment, triggers the differentiation of avirulent promastigotes into virulent amastigotes. Iron depletion from the culture medium triggered expression of the LIT1 ferrous iron transporter, growth arrest, and differentiation of wild type promastigotes into infective amastigotes. In contrast, LIT1 null promastigotes showed continued exponential growth in iron-poor media, followed by massive cell death. Iron depletion from the medium and LIT1 upregulation increased iron superoxide dismutase activity (FeSOD) in wild type, but not in LIT1 null parasites. Notably, the superoxide-generating drug menadione or H2O2 were sufficient to trigger differentiation of wild type promastigotes into fully infective amastigotes. On the other hand, LIT1 null promastigotes accumulated superoxide radical and initiated amastigote differentiation after exposure to H2O2, but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species (ROS) in orchestrating the differentiation of Leishmania infective stages, in a process regulated by iron availability.