Tunisian strain of L. major promastigotes (MHOM/TN/95/GLC94 zymodeme MON25) were grown at 26°C in RPMI 1640, supplemented with 2 mM L-glutamine, 10% heat inactivated foetal calf serum, penicillin (100 U/ml) and streptomycin (10 mg/ml). Metacyclic rich fraction obtained using Ficoll gradient were used in all experiments. Briefly, stationary phase cultures of Leishmania were centrifuged at 5,000 g for 10 min at room temperature and resuspended in 2 ml of PBS. The cell suspensions were then loaded onto a Ficoll gradient composed, from the bottom of 2 ml of 20%, 5 ml of 10% and 5 ml of 5% Ficoll diluted in PBS. The gradient was next centrifuged at 1,300 g for 10 min at room temperature. The metacyclic promastigotes were recovered on the top of 10% Ficoll layer.

BALB/c and C57BL/6 mice (Elevage Janvier) were killed and hind legs removed for BMdM isolation. Briefly, femurs and tibias were flushed with RPMI 1640 using a 25-gauge needle. Contaminating erythrocytes were lysed through the addition of Geys lysis solution (ammonium chloride 1.5 M, EDTA 0.1 mM, pH 7.3). All cells were incubated in T75 culture flasks at 1.5 106 cell per ml in RPMI 1640 media supplemented with 2 mM L-glutamine, 10% heat inactivated foetal calf serum (Perbio science, Brebières, France), penicillin (100 U/ml) and streptomycin (10 mg/ml) and 80 ng/ml M-CSF (Peprotech, Neuilly sur Seine, France) overnight for stromal cell elimination. Non-adherent, immature macrophages were transferred to fresh culture-treated Petri dishes (Nunc, USA) and grown for 7 days, with re-feeding on day 3, to induce macrophage differentiation. BMdM purity was analyzed through the evaluation of phenotypic expression of specific macrophage subset surface marker (F4/80) by Flow cytometry. Generated macrophages were assessed by flow cytometry for expression of F4/80 (80-90% were positive).

All mouse work was done according to the directive 86/609/EEC of the European parliament and of the council on the protection of animals used for scientific purposes. Approval for mice experiments was obtained from the ethic committee of Institute Pasteur of Tunis with ethics approval number 1204.

10⁶ BMdM Cells were seeded in 1 ml complete media on 24 well plates and subjected to adhere overnight at 37°C in 5% CO2. They are afterwards, incubated at a parasite to cell ratio of approximately 10:1 with Ficoll purified metacyclic promastigotes of L. major. After the desired time of incubation, the extracellular parasites were washed out and the cells were harvested to prepare samples. The macrophages were fixed, Giemsa-stained and counted to calculate the number of amastigotes per 100 macrophages to insure for homogenate cell infection under the different conditions.

The RNA isolation and quantification, the hybridization to the GeneChip Mouse Gene 1.0 ST array (Affymetrix, Santa Clara, CA) were performed as previously described 6 . Each infection and control time points were performed in triplicate, using different preparations of BMdMs, and processed independently to give three biological replicates. QC analysis was performed before and after normalization using BoxPlot of total intensities, MAPlots for all replicates and PCAplots. All microarrays of this study passed the quality control.

The intrachip and interchip normalisation were performed as previously described 6 . Expression analysis used the R Bioconductor package Limma 13 to identify genes that met statistical (P < 0.05 after adjustment according to the method of Benjamini and Hochberg and fold-change criteria (at least a 1.5-fold change) for differential expression using the following contrasts: macrophages infected with live parasites at a given time point versus non infected macrophages incubated with vehicule (media) for the same time. The same contrast was used for heat-killed L. major-infected macrophages. Macrophage genes modulated during the kinetics were detected.

In accordance with MIAME (Minimum Information About a Microarray Experiments) regulations 14 , all data were deposited into GEO (Gene Expression Omnibus) database at http://www.ncbi.nlm.nih.gov/geo under the accession number GSE31995 and GSE31996.

Original data represented as matrix was subject to a normalization pipeline, which consists in i) merging different triplicates for each condition into a single data point reducing the matrix columns from 45 columns to 15 columns and one additional column for non infected at t = 0 h. ii) To avoid gene duplicates, we merged probes with same gene ID which automatically shrinks the matrix so that all rows of probes from the same gene are merged into one average row using only gene ID as unique identifier. iii) In order to remove systematic variations that may occur because of reasons other than biological differences between RNA samples, a Quantile Normalization has been done on the expression data. iv) A fold change of 2 was used as a cutoff to further reduce the data and keep highly differential genes and a final standardization of the data was done on the mean (0) and standard deviation (1) of the final gene set on the expression matrix.

To compare conditions in order to extract highly differentially expressed genes, our strategy consists on the use of a supervised grouping using T-test as a statistical approach to extract significantly variable genes. Similar strategy was used for either BALB/c or C57BL/6. We first compared the group of uninfected conditions (NI) (at the different time points (1, 3, 6, 12 and 24 h)) to the infected group (P) (at the same time points). Similar comparison was performed between the parasite infected (P) and killed parasite (Kp) infected group. Selected probes obey the p-value cutoff of 0.05 using FDR as multiple tests correction. The outcome of these steps is groups of genes that are down-regulated or up-regulated depending on their expression values.

In order to locate in which pathway our up-regulated and down-regulated genes are enriched, we relied on the usage of KEGG pathway for this analysis. We used a stringent p-value cutoff of 1E-4 and the condition of having at least 4 genes within each enriched pathway. The results consist in a set of enriched pathways for up-regulated genes and down-regulated genes. Gene lists of each pathway found are extracted for further analysis. We run an additional pathway enrichment analysis based on gene occurrence in both BALB/c and C57BL/6 as well as genes that are unique to each of them. The goal behind this additional analysis is to detect pathways that are specific to each mice strain under the same infection conditions. The analysis of specific genes may inform on interconnected pathways that may be activated or deactivated depending on the gene sets involved.

In order to discover relationships between genes products in our up-regulated and down-regulated sets we used STRING DB as it contains an accurate and updated data on physical and functional interactions. STRING also allows to predict activation/repression relationships between different nodes of the same graph 15 .

In furtherance of highlighting relationship between differentially expressed genes and corresponding pathways, we used all up-regulated genes in BALB/c and C57BL/6 and studied genes interaction within the same pathway and genes interactions across different pathways. The analysis was done using GENMAPP-CS cytoscape plugin (http://www.genmapp.org/beta/genmappcs/). Orphan nodes were removed from the network to highlight the direct interactions between different genes.

We first established cell culture and infection conditions to ensure that the levels of infection of the bone marrow derived macrophages (BMdM) isolated from the two mice strains were equivalent. The same cell/parasite ratio (1:10) was found to give similar infection rate and parasite load. Light microscopy on Giemsa stained chamber slides of infected BMMs showed that the percentage of infected cells and the mean amastigote loads were comparable between L. major infected C57BL/6 and BALB/c BMdMs in each of the three experimental replicates used in this study (Table 1). Subsequent microarray analysis was carried out on each of the three biological replicates.

BMdMs were plated on chamber slides and infected for 24 h; washed thoroughly; The percentage of cells infected and the mean number of parasites per cell were determined following staining with Giemsa according the manufacturer’s instructions.

GeneChip Mouse Gene 1.0 ST arrays were used to analyse global changes in gene transcripts to generate a pool of genes that was statistically significant (p-value < 0.05) and a fold change cut-off of 2. Of the 18 899 genes represented on the array, our analysis of mRNA expression in mice BMdM infected by live parasites showed that a total of 1175 genes were expressed differentially over the time course (with 658 down- and 517 up-regulated) in BALB/c BMdM and approximately the same number of genes (1174) was differentially modulated (663 down- and 511 up-regulated) in C57BL/6 BMdM. The microarrays analysis also revealed that as a result of Leishmania infection, the expression of 768 genes were shared between BALB/c and C57BL/6 BMdMs, with 434 genes down-regulated and 334 up-regulated (Figure 1). The relative expression of selected differentially expressed genes from the microarray data was further examined by RT-qPCR on the same samples that those analyzed by microarray analysis. Data from RT-qPCR analysis (Additional file 1: Table S1) were consistent with the results obtained by microarrays, albeit with magnitudes different from, and often higher than, those recorded by the microarray analysis.

Further analysis showed that 768 genes among the commonly altered in BALB/c and C57BL/6 BMdMs in response to infection, fall into 45 KEGG pathways. The genes unique to BALB/c correspond to 22 and those unique to C57BL/6 correspond to 25 pathways with a p-value less than 0.05 (Additional file 2: Table S2).

Functional analysis determined that the shared genes were primarily associated with metabolic pathways (including glycolysis/Gluconeogenesis, Galactose, Fructose and mannose metabolism, and N-Glycan biosynthesis). Analysis and biological validation of a set of those metabolic pathways have been previously reported 6 . L. major also modulates different other genes implicated in Fc gamma R-mediated phagocytosis, chemokines, and Toll like receptor signaling pathway. The expression of different genes involved in host cell defense pathways and especially in iron metabolism is also altered by the infection. Indeed, almost all the intracellular pathogens require iron to develop a productive infection and restricting the availability of iron is considered as an important strategy for defense against infections. L. major inhibits the mRNA expression of the genes encoding transferring, holotransferrin receptor and Ferroportin 1 (Slc40a1) which suggest that the parasite limits the uptake and the export of iron. L. major also enhances the induction of the mRNA of Nramp2 a protein implicated in the iron efflux from the endosomes suggesting more iron accumulation inside the cytosol. Otherwise, in both susceptible and resistant Leishmania major infected macrophages, the parasite induces the transcription down-regulation of most lysosomal proteins. These include different Glycosidases (Gusb, Galc), Sulfatases (Sgsh), membrane proteins (Laptm5, Lamp1), most V-ATPases and others lysosomal proteases such cathepsin.

We further analyzed the expression and kinetics of a panel of cytokines and chemokines during L. major infection in a comparative study of genetically resistant C57BL/6 and susceptible BALB/c mice. Among the chemokines responsible of leukocytes recruitment, L. major induces the mRNA expression of Ccl2 (MCP1), macrophage inflammatory protein-1α (Mip-1α/Ccl3) and Mip-1β/Ccl4. The infection also activates the transcription of Cxcl1, Cxcl2 that regulate the influx of PMNs, Cxcl3 that controls migration of monocytes, Cxcl9 the Th1-attracting protein and the interferon-γ - inducible protein-10 (IP-10/Ccxl10). The expression of Cxcl1, Cxcl2 and Cxcl3 mRNA induced by both live and killed parasites is not modulated to the same extent in the two mice strains BMdM. Indeed, as assessed by qRT-PCR, the mRNA induction of these chemokines is respectively six, five and four times higher in the C57BL/6 BMdM (Additional file 1: Table S1). Moreover, in the macrophage derived from susceptible mice, the transcription induced by live parasites seems actively repressed when compared to the one induced by killed parasites. The mRNA expression of Ccl3 is similarly regulated. L. major promastigotes induce rapid and transient expression of murine Ccl2 that besides attracting monocytes and macrophages, can attract other cells such as NK and DCs expressing the chemokine receptor Ccr2. However, the infection represses the transcription of Ccr2 receptor. By contrast, parasites activate the mRNA expression of Ccrl2 in macrophages derived from the two mice strain. This expression is however 5 times more important in resistant mice. The mRNA expression of Cxcl9, slightly increases in response to L. major during early infection in BMdM derived from the two mice strains. However, while this expression is quiet back to the baseline 24 hpi in BALB/c BMdM, it starts to significantly be enhanced beginning from 12 hpi in the C57BL/6 BMdM.

We next identified differentially expressed genes unique to BALB/c and C57BL/6 derived macrophages and performed enrichment analysis of these genes based on KEGG database (Additional file 2: Table S2). Pathways with p-values less than 0.05 were considered statistically significant. This analysis clearly identifies signaling pathways among the most relevant pathways modulated by L. major promastigotes. Indeed, the results show that down-regulated genes unique to macrophages derived from resistant mice were related among others to MAPK signaling pathways whereas a set of up-regulated genes were involved in the mTOR signaling pathway, Erbb and Insulin signaling pathways (Additional file 2: Table S2). Among the down-regulated genes unique to susceptible mice we listed those implicated in the Notch and chemokine signaling pathways. The up-regulated genes unique to BALB/c derived macrophages are implicated in p53 signaling pathway (Additional file 2: Table S2). Among these p53-dependent genes modulated by L. major, we found the mouse double minute 2 (Mdm2) an inhibitor of p53, the insulin growth factor 1 (Igf1), some genes implicated in cell cycle genes such Gadd45a or b, and genes implicated in the apoptotic pathway (Bid).

Furthermore, we investigated whether, in a given mice strain derived macrophages, these coordinately regulated unique genes may be interconnected. We focused on up-regulated genes and up-loaded on GENMAPP-CS the lists of the genes unique to BALB/c and C57BL/6 derived macrophages. This analysis shows (Figure 2) that after the removing of orphan nodes, the genes (and pathways) identified to be significantly induced by L. major in macrophages derived from susceptible mice, mostly unique to BALB/c appeared to be interconnected except for the p53 signaling pathway (Figure 2). These fall into several pathways including glycolysis/Gluconeogenesis (AldoC, Eno2, Pdhb), antigen processing, cellular pH regulation (VATP-ases, Cathepsin), immune response (TNFα, IL-1α). Our results also show that several genes and pathways induced by L. major in C57BL/6 macrophages are linked to the mammalian Target of Rapamycine (mTOR) signaling pathway (Figure 3). These pathways include the chemokine, the toll like receptors and the insulin signaling pathways.

As p53 and mTOR signaling pathways are known to be interconnected, the list of L. major-activated genes belonging to these two pathways were uploaded on STRING database. As illustrated in Figure 4, protein-protein interaction analysis, show that these two pathways are independent with Igf1, a p53 target gene, showing up as a major node in this network.