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 Leishmania line. 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 (log 2 FC ≥ 1) and downregulated (log 2 FC ≤ −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.

Transcriptome Analysis of Intracellular Amastigotes of Clinical Leishmania infantum Lines from Therapeutic Failure Patients after Infection of Human Macrophages

“…The last four L. infantum lines were isolated from TF HIV patients with VL unsuccessfully treated with liposomal AmB (from the WHO Collaborating Center for Leishmaniasis, Instituto de Salud Carlos III; Dr. F. Javier Moreno). The sensitivity of all the above Leishmania lines to the different anti-leishmanial drugs has been recently published [ 28 , 29 ].…”

“…THP-1 cells (3 × 10 6 cells in 25 cm 2 flasks or 5 × 10 5 cells/well in 24-well plates) were differentiated to macrophages and infected with different L. infantum stationary-phase promastigotes incubated for 72 h in an acid medium plus 10% hiFBS at a macrophage/parasite ratio of 1:10 [ 28 , 29 ]. Infected macrophages were incubated in an RPMI-1640 medium plus 10% hiFBS at 37 °C and 5% CO 2 for 96 h. Finally, the samples were collected in Qiazol (Qiagen, Hilden, Germany), and total RNA was isolated at the Genomic Unit of GENyO facilities (Granada, Spain), as described later.…”

“…The protocols used for the RNA isolation of the samples and the generation of cDNA libraries were described previously [ 28 , 29 ]. Briefly, eight different group of samples, defined as LEM2126, LEM3323, LEM5159, LLM2221, LLM2255, LLM2165, LLM2070, and LJPC, with three independent biological replicate experiments each, were mRNA-extracted using a QIAamp RNA miRNeasy Micro Kit (Qiagen/Qiacube), followed by treatment with a DNase kit.…”

“…Consequently, the use of intracellular amastigotes may be useful for studies directed to know the transcriptomic changes associated with the parasite’s ability to survive, evade the host defenses, and elude the toxic effects of anti-leishmanial drugs. During a host–pathogen interaction, global changes in the gene expression pattern occur both in the host (macrophages) [ 28 , 29 , 30 , 31 , 32 ] and in the infecting pathogens [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ].…”

“…Recently, based on the dual RNA-seq method, we identified host-specific genes that were modulated after the host macrophages interacted with clinical isolates of L. infantum with different levels of drug susceptibility to the anti-leishmanial drugs associated, or not, with TF [ 28 , 29 ]. In this article, we analyzed the transcriptomic changes in these intracellular amastigotes of L. infantum parasites at 96 h post-infection of THP-1 cells.…”