Transcriptomic Profile of Canine DH82 Macrophages Infected by Leishmania infantum Promastigotes with Different Virulence Behavior

Abstract

Zoonotic visceral leishmaniosis caused by Leishmania infantum is an endemic disease in the Mediterranean Basin affecting mainly humans and dogs, the main reservoir. The leishmaniosis outbreak declared in the Community of Madrid (Spain) led to a significant increase in human disease incidence without enhancing canine leishmaniosis prevalence, suggesting a better adaptation of the outbreak's isolates by other host species. One of the isolates obtained in the focus, IPER/ES/2012/BOS1FL1 (BOS1FL1), has previously demonstrated a different phenotype than the reference strain MCAN/ES/1996/BCN150 (BCN150), characterized by a lower infectivity when interacting with canine macrophages. Nevertheless, not enough changes in the cell defensive response were found to support their different behavior. Thus, we decided to investigate the molecular mechanisms involved in the interaction of both parasites with DH82 canine macrophages by studying their transcriptomic profiles developed after infection using RNA sequencing. The results showed a common regulation induced by both parasites in the phosphoinositide-3-kinase-protein kinase B/Akt and NOD-like receptor signaling pathways. However, other pathways, such as phagocytosis and signal transduction, including tumor necrosis factor, mitogen-activated kinases and nuclear factor-κB, were only regulated after infection with BOS1FL1. These differences could contribute to the reduced infection ability of the outbreak isolates in canine cells. Our results open a new avenue to investigate the true role of adaptation of L. infantum isolates in their interaction with their different hosts.

Keywords: DH82 cells; Leishmania infantum; RNA-seq; canine macrophage; virulence.

Figure 1

Leishmania infantum BCN150 and BOS1FL1 infection capacity in canine DH82 macrophages. DH82 cells were infected with L. infantum BCN150 or BOS1FL1 at a ratio of 5:1 parasites:cells. The percentage of infected cells (A) and the mean number of amastigotes per infected cell (intensity of infection) (B) were determined at 4, 24 and 72 h post-infection (pi). Data (mean ± SD) from four independent experiments are shown. Statistical analysis was performed with a parametric Student’s t test using GraphPad Prism software version 8.30. Asterisks (*) indicate statistically significant differences (* p < 0.05, ** p < 0.01).

Figure 2

Correlation analysis in DH82 L. infantum-infected and uninfected samples. Principal component analysis (PCA) (A) was performed to observe variation between samples. The percentage variability captured by the two principal components is displayed across PC1 and PC2 represented on the X and Y axes. Reproducibility between RNA sequencing data replicates (B) was evaluated based on the Pearson correlation coefficient according to all gene expression levels (fragments per kilobase of transcript per million mapped reads, FPKM) of each sample.

Figure 3

Differentially expressed genes in DH82 cells infected with L. infantum BCN150 and BOS1FL1 versus noninfected cells for 4 h. The total number of differentially expressed genes (DEGs) (A) in L. infantum-infected macrophages relative to uninfected controls is represented as horizontal bar plots. Genes were considered DEGs when they presented a false discovery rate value (FDR) ≤ 0.05. Box lengths depict the numbers of genes down-regulated and up-regulated. (B) Venn diagram showing the number of unique and shared DEGs in BCN-NI and BOS-NI comparisons. The results were obtained from three biological replicates for each condition.

Figure 4

Gene ontology (GO) terms enriched in DEGs between DH82 cells infected with L. infantum BCN150 and BOS1FL1 and noninfected cells. The graph shows the most relevant GO terms enriched from DEGs in the BCN-NI and BOS-NI comparisons. The x-axis represents the number of DEGs associated with each GO term. Dark bars indicate up-regulated genes and light bars indicate down-regulated genes. Asterisks indicate enriched GO terms considered statistically significant based on an FDR value ≤ 0.05. R, regulation; PR, positive regulation; NR, negative regulation; D, differentiation; Rsp, response.

Figure 5

Heatmap of differentially expressed genes in canine DH82 macrophages. The graph represents a selection of DEGs showing row Z-scores based on expression data of three replicates of DH82 cells infected with L. infantum BCN150 (BCN1–BCN3) and BOS1FL1 (BOS1–BOS3) and noninfected cells (NI1–NI3). Colored segments show the pathways in which DEGs are involved. The heatmap was generated using Heatmapper (http://www2.heatmapper.ca, accessed on 16 June 2021).

Figure 6

Phosphoinositide-3-kinase–protein kinase B/Akt (PI3K-Akt) KEGG pathway map. Graphic representation of PI3K-Akt signaling pathway (modified based on KEGG database) in which DEGs that were found in both BCN150-infected cells and BOS1FL1-infected cells with respect to the control are highlighted in green (up-regulated genes) and red (down-regulated genes). Black stars indicate those genes found exclusively in BOS1FL1-infected cells.