Transcriptomic Analysis in Human Macrophages Infected with Therapeutic Failure Clinical Isolates of Leishmania infantum

Abstract

Leishmaniasis is one of the neglected tropical diseases with a worldwide distribution, affecting humans and animals. In the absence of an effective vaccine, current treatment is through the use of chemotherapy; however, existing treatments have frequent appearance of drug resistance and therapeutic failure (TF). The identification of factors that contribute to TF in leishmaniasis will provide the basis for a future therapeutic strategy more efficient for the control of this disease. In this article, we have evaluated the transcriptomic changes in the host cells THP-1 after infection with clinical Leishmania infantum isolates from leishmaniasis patients with TF. Our results show that distinct L. infantum isolates differentially modulate host cell response, inducing phenotypic changes that probably may account for parasite survival and TF of patients. Analysis of differential expression genes (DEGs), with a statistical significance threshold of a fold change ≥ 2 and a false discovery rate value ≤ 0.05, revealed a different number of DEGs according to the Leishmanialine. Globally, there was a similar number of genes up- and downregulated in all the infected host THP-1 cells, with exception of Hi-L2221, which showed a higher number of downregulated DEGs. We observed a total of 58 DEGs commonly modulated in all infected host cells, including upregulated (log₂FC ≥ 1) and downregulated (log₂FC ≤ -1) genes. Based on the results obtained from the analysis of RNA-seq, volcano plot, and GO enrichment analysis, we identified the most significant transcripts of relevance for their possible contribution to the TF observed in patients with leishmaniasis.

Keywords: Leishmania infantum; human macrophages; infection; modulation of host cells; therapeutic failure clinical parasites; transcriptomic analysis.

Figure 1

Transcriptome profiles of THP-1 cells infected with different L. infantum lines. Differential gene expression profiles are presented as the numbers of upregulated (black) and downregulated (dark gray) transcripts after comparisons of the different infected cell lines vs non-infected cells (the unspecific phagocytosis background has been subtracted previously). The data are from three independent biological replicates, considering a fold change ≥ 2 and an FDR value ≤ 0.05.

Figure 2

Volcano plots from RNAseq data of THP-1 cells infected with different L. infantum lines. The log₂FC is plotted on the x-axis, and the negative log₁₀FDR (adjusted p-value) is plotted on the y-axis. The represented genes have an FDR lower than 0.05. Some relevant DEGs are indicated with an arrow.

Figure 3

GO term enrichment analysis of DEGs including in the category Biological Processes. Enriched GO terms for most relevant DEGs of lines Hi-L2221, Hi-L2165, Hi-L2070 and Hi-L2255. The number of DEGs is expressed in the x-axis and colors represent the significance level (FDR) of the enrichment.

Figure 4

Venn diagram analysis of DEGs in THP-1 cells in response to infection with L2221, L2070, L2165, and L2255 L. infantum lines. Venn diagram of the total upregulated and downregulated DEGs with Log₂FC ≥ 1 and Log₂FC ≤ −1, respectively. The number of exclusive and common genes for each comparison are shown.

Figure 5

Comparative analysis of the relative expression levels of selected genes determined by RNA-seq (black) and validated by RT-qPCR (dark gray). The bars represent the mean ± SD values of fold-change expression of ABCG1, ABCG2, ANO5, AQP9, BDH1, COL4A1, CYP19A1, DHDH, FBP1, HMOX1, PDK4 and SUCNR1 determined from three independent biological replicates analyzed in triplicate. RT-qPCR expression values of the genes in each line were normalized with the expression of ACTB. The relative expression of each gene was calculated as the fold change between Hi-L2126, Hi-L2221, Hi-L2070, or Hi-L2255 and macrophages infected with heat-inactivated parasites (which was set to 1.0).