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.

Figure 1

Time course of intracellular amastigote population size increase and MΦ culture imaging. A: time course experiment showing the evolution of the amastigote population within MΦ. Mean number of amastigotes per MΦ were plotted against the time points selected. Ten microscope fields split up into biological duplicates were visualized and more than 200 MΦ nuclei were counted. B: L. amazonensis-housing bone marrow-derived MΦ imaged 24 h post amastigote (4 parasites per MΦ) addition. Nuclei were stained with Hoechst (blue) and amastigote with 2A3.26 mAb and Texas Red-labelled conjugate (red). Image acquisition was performed using an immunofluorescence and differential interference contrast inverted microscope (Zeiss Axiovert 200 M). Asterisk: Parasitophorous vacuoles; arrow heads: Amastigotes.

Figure 2

Affymetrix outcome. A: Volcano plot. 1,248 probe-sets showed differential expression at the 0.05 threshold (green line): 605 positive and 643 negative FC values of which 454 in the right and 507 in the left upper corners (± 1.75 FC threshold, red lines, blue circles). B: Fold-change distribution of the 1,248 probe sets.

Figure 3

Modulation of the sterol biosynthesis pathway in L. amazonensis-hosting MΦ. L. amazonensis-hosting MΦ display an up-regulation of several genes involved in sterol biosynthesis (*, at least 2 probe-sets modulated).

Figure 4

Modulation of the polyamine biosynthesis pathways in L. amazonensis-hosting MΦ. L. amazonensis-hosting MΦ display a gene expression coordination of several genes involved in polyamine biosynthesis (*, at least 2 probe-sets modulated; blue values determined by RTQPCR).