A comparison of two distinct murine macrophage gene expression profiles in response to Leishmania amazonensis infection

Abstract

Background: The experimental murine model of leishmaniasis has been widely used to characterize the immune response against Leishmania. CBA mice develop severe lesions, while C57BL/6 present small chronic lesions under L. amazonensis infection. Employing a transcriptomic approach combined with biological network analysis, the gene expression profiles of C57BL/6 and CBA macrophages, before and after L. amazonensis infection in vitro, were compared. These strains were selected due to their different degrees of susceptibility to this parasite.

Results: The genes expressed by C57BL/6 and CBA macrophages, before and after infection, differ greatly, both with respect to absolute number as well as cell function. Uninfected C57BL/6 macrophages express genes involved in the deactivation pathway of macrophages at lower levels, while genes related to the activation of the host immune inflammatory response, including apoptosis and phagocytosis, have elevated expression levels. Several genes that participate in the apoptosis process were also observed to be up-regulated in C57BL/6 macrophages infected with L. amazonensis, which is very likely related to the capacity of these cells to control parasite infection. By contrast, genes involved in lipid metabolism were found to be up-regulated in CBA macrophages in response to infection, which supports the notion that L. amazonensis probably modulates parasitophorous vacuoles in order to survive and multiply in host cells.

Conclusion: The transcriptomic profiles of C57BL/6 macrophages, before and after infection, were shown to be involved in the macrophage pathway of activation, which may aid in the control of L. amazonensis infection, in contrast to the profiles of CBA cells.

Figure 1

Comparison of differentially expressed genes using microarray and RT-qPCR techniques. RT-qPCR was used to verify the differential expression of randomly selected genes (n = 27) by uninfected C57BL/6 and CBA macrophages (A), by L. amazonensis-infected C57BL/6 macrophages in comparison to uninfected cells (n = 7) (B), and by L. amazonensis-infected CBA macrophages in comparison to uninfected cells (n = 2) (C). Figure 1 (A-C) depicts only genes that were successfully verified using RT-qPCR. Resulting comparison values are expressed as mean values of log₂ ± SE from two independent experiments in comparison (A), and three independent experiments in comparisons (B) and (C), all performed in duplicate. The nonparametric Mann-Whitney test was used for comparison between uninfected cells, and Stouffer method [29] was used to integrate the results from independent microarray and RT-qPCR analyses to determine significant differences between infected and uninfected cells (level of significance, p ≤ 0.05)

Figure 2

Networks built using differentially expressed genes in uninfected macrophages from C57BL/6 and CBA mice. C57BL/6 and CBA macrophages were cultured separately and then processed for microarray analysis as described in Materials and Methods. The cell death and lipid metabolism network (A) and the cell-cell signaling and interaction network (B) were modeled using Ingenuity Pathway Analysis software v8.8 (IPA-Ingenuity Systems^®). The above networks are displayed as a series of nodes (genes or gene products) and edges (or lines, corresponding to biological relationships between nodes). Nodes are displayed using shapes that represent the functional class of the gene product as indicated in the key. Nodes marked in green were found to be highly expressed in C57BL/6 macrophages in comparison to CBA. Nodes marked in red were found to be highly expressed in CBA macrophages compared to C57BL/6. The unmarked nodes were not identified in our samples; however, IPA^® added them to the networks due to their high probability of involvement in a given network. The node color intensity is an indication of the degree of up-(green) or down-(red) regulation of genes observed in the biological network analysis from uninfected C57BL/6 macrophages compared to CBA cells. Solid lines denote direct interactions, whereas dotted lines represent indirect interactions between the genes represented in this network.

Figure 3

Networks built using differentially expressed genes in L. amazonensis-infected and uninfected macrophages. C57BL/6 or CBA macrophages were cultured, infected and processed for microarray analysis as described in Materials and Methods. Considering the modulated genes in C57BL/6 infected macrophages, the immunological disease and cell morphology network (A), as well as the protein synthesis, cellular development and cell death network (B) were modeled by IPA^®. Considering the modulated genes in CBA infected macrophages, the lipid metabolism, cellular movement, and small molecule biochemistry network was built by IPA^® (C). C57BL/6 and CBA macrophages were cultured separately, then infected and processed for microarray analysis as described in Materials and Methods. Similar to Figure 2, the above networks are displayed as a series of nodes (genes or gene products) and edges (or lines, corresponding to biological relationships between nodes). Nodes are displayed using shapes as indicated in the key. Nodes marked in red were found to be highly expressed in infected macrophages. Nodes marked in green were found to be highly expressed in uninfected macrophages. Unmarked nodes were added by IPA^® due to a high degree of probability of involvement in a given network. The node color intensity is an indication of the degree of up-(red) or down-(green) regulation of genes observed in the biological network analysis from both C57BL/6 and CBA macrophages in response to infection. Solid lines denote direct interactions, whereas dotted lines represent indirect interactions between the genes represented in this network.