Differential immune response modulation in early Leishmania amazonensis infection of BALB/c and C57BL/6 macrophages based on transcriptome profiles

Abstract

The fate of Leishmania infection can be strongly influenced by the host genetic background. In this work, we describe gene expression modulation of the immune system based on dual global transcriptome profiles of bone marrow-derived macrophages (BMDMs) from BALB/c and C57BL/6 mice infected with Leishmania amazonensis. A total of 12,641 host transcripts were identified according to the alignment to the Mus musculus genome. Differentially expressed genes (DEGs) profiling revealed a differential modulation of the basal genetic background between the two hosts independent of L. amazonensis infection. In addition, in response to early L. amazonensis infection, 10 genes were modulated in infected BALB/c vs. non-infected BALB/c macrophages; and 127 genes were modulated in infected C57BL/6 vs. non-infected C57BL/6 macrophages. These modulated genes appeared to be related to the main immune response processes, such as recognition, antigen presentation, costimulation and proliferation. The distinct gene expression was correlated with the susceptibility and resistance to infection of each host. Furthermore, upon comparing the DEGs in BMDMs vs. peritoneal macrophages, we observed no differences in the gene expression patterns of Jun, Fcgr1 and Il1b, suggesting a similar activation trends of transcription factor binding, recognition and phagocytosis, as well as the proinflammatory cytokine production in response to early L. amazonensis infection. Analysis of the DEG profile of the parasite revealed only one DEG among the 8,282 transcripts, indicating that parasite gene expression in early infection does not depend on the host genetic background.

Figure 1

Transcriptome profiles of BMDMs from BALB/c and C57BL/6 mice infected with L. amazonensis. Differential gene expression profiles of BMDMs from BALB/c and C57BL/6 mice infected with L. amazonensis, presented as the numbers of upregulated (light gray) and downregulated (dark gray) transcripts in the following comparisons: non-infected BALB/c vs. non-infected C57BL/6 macrophages; infected BALB/c vs. non-infected BALB/c macrophages; infected C57BL/6 vs. non-infected C57BL/6 macrophages; and infected BALB/c vs. infected C57BL/6 macrophages. The data are from five independent biological replicates, considering a fold change ≥ 2 and a p-value < 0.05. La, L. amazonensis.

Figure 2

GO enrichment analysis of DEGs in BMDMs from BALB/c and C57BL/6 mice in response to L. amazonensis infection. The GO enrichment analysis results are presented as numbers of transcripts distributed in three main categories: biological process, molecular function and cellular component. Immune system processes were the focus of this work.

Figure 3

Immune response analysis of DEGs in BMDMs from BALB/c and C57BL/6 mice in response to L. amazonensis infection. Pie chart of the modulated molecules involved in the immune response processes grouped into main immune signaling pathways.

Figure 4

Venn diagram analysis of DEGs in BMDMs from BALB/c and C57BL/6 mice in response to L. amazonensis infection. (A) Venn diagram of the 361 DEGs involved in the immune response processes, showing the numbers of exclusively and common genes for each comparison. (B) List of exclusively and common genes according for each comparison in the Venn diagram.

Figure 5

DEGs profile of the exclusively modulated genes involved in the immune response processes in infected C57BL/6 vs. non-infected C57BL/6 BMDMs. The profiles of DEGs are presented as the log₂-fold changes in the expression of the 39 exclusively modulated genes involved in the immune response processes in BMDMs from C57BL/6 infected with L. amazonensis vs. non-infected C57BL/6 BMDMs. The genes were classified by their involvement in main immune response signaling pathways or by their identities as regulatory molecules of the immune response pathways. La, L. amazonensis.

Figure 6

RT-qPCR validation of some modulated genes in BALB/c and C57BL/6 BMDMs in response to L. amazonensis infection. Comparative analysis of the relative expression levels of selected genes determined by RNA-seq and validated by RT-qPCR. The bars represent the mean ± SD values of the fold changes in Il1b, Fcgr1, Ccr5, Smad6, Jun and Mapk14 expression determined with five independent biological replicates analyzed in duplicate. The fold changes were calculated through relative quantification using the ΔΔCt method. The data were normalized to Gapdh expression and the relative gene expression was set to 1 for the control (non-infected) samples. Statistical analysis was performed using the t-tests, and no significant differences were observed (p-value < 0.05) between the RT-qPCR and RNA-seq results for the BALB/c_La and C57BL/6_La groups. The bars for Amastin-like (LmxM.33.0960) show the mean after normalization to Gapdh in L. amazonensis infecting BALB/c and L. amazonensis infecting C57BL/6 macrophages. La, L. amazonensis.

Figure 7

Schematic representation of the exclusive genes and DEGs in BALB/c and C57BL/6 BMDMs in response to L. amazonensis infection. Summary of the data of the exclusive genes and DEGs in BMDMs derived from BALB/c and C657BL/6 mice in response to early L. amazonensis infection.