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Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes.

ABSTRACT: BACKGROUND: Mammal macrophages (MPhi) display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L). Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MPhi. RESULTS: Using BALB/c mouse bone marrow-derived MPhi loaded or not with amastigotes, we analyzed the transcriptional signatures of MPhi 24 h later, when the amastigote population was growing. Total RNA from MPhi cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR). A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the 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 signalling. CONCLUSION: Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MPhi lipid and polyamine pathways. Moreover, these MPhi hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

SUBMITTER: Osorio y Fortea J

PROVIDER: S-EPMC2666765 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes.

Osorio y Fortéa José J de La Llave Emilie E Regnault Béatrice B Coppée Jean-Yves JY Milon Geneviève G Lang Thierry T Prina Eric E

BMC genomics 20090320

<h4>Background</h4>Mammal macrophages (MPhi) display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L). Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MPhi.<h4>Results</h4>Using BALB/c mouse bone marrow-derived ...[more]

PMID: 19302708

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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.

Project description:Background/objectivesThe inoculation of a low number (10(4)) of L. amazonensis metacyclic promastigotes into the dermis of C57BL/6 and DBA/2 mouse ear pinna results in distinct outcome as assessed by the parasite load values and ear pinna macroscopic features monitored from days 4 to 22-phase 1 and from days 22 to 80/100-phase 2. While in C57BL/6 mice, the amastigote population size was increasing progressively, in DBA/2 mice, it was rapidly controlled. This latter rapid control did not prevent intracellular amastigotes to persist in the ear pinna and in the ear-draining lymph node/ear-DLN. The objectives of the present analysis was to compare the dendritic leukocytes-dependant immune processes that could account for the distinct outcome during the phase 1, namely, when phagocytic dendritic leucocytes of C57BL/6 and DBA/2 mice have been subverted as live amastigotes-hosting cells.Methodology/principal findingsBeing aware of the very low frequency of the tissues' dendritic leucocytes/DLs, bone marrow-derived C57BL/6 and DBA/2 DLs were first generated and exposed or not to live DsRed2 expressing L. amazonensis amastigotes. Once sorted from the four bone marrow cultures, the DLs were compared by Affymetrix-based transcriptomic analyses and flow cytometry. C57BL/6 and DBA/2 DLs cells hosting live L. amazonensis amastigotes do display distinct transcriptional signatures and markers that could contribute to the distinct features observed in C57BL/6 versus DBA/2 ear pinna and in the ear pinna-DLNs during the first phase post L. amazonensis inoculation.Conclusions/significanceThe distinct features captured in vitro from homogenous populations of C57BL/6 and DBA/2 DLs hosting live amastigotes do offer solid resources for further comparing, in vivo, in biologically sound conditions, functions that range from leukocyte mobilization within the ear pinna, the distinct emigration from the ear pinna to the DLN of live amastigotes-hosting DLs, and their unique signalling functions to either naive or primed T lymphocytes.

Dataset Information

Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes.

Publications

Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes.

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