miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages

Abstract

Visceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-β, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-β, TLR4, IGF-I, chemokine, and HIF1α pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.

Keywords: Leishmania (Leishmania) infantum; THP-1 cells; inflammation; microRNA; pathogenesis; visceral leishmaniasis.

Figure 1

miRNA profiles of L. (L.) infantum infected THP-1-macrophages. Volcano plot of differential expression of miRNAs in L. (L.) infantum promastigote-infected THP-1 macrophages compared to uninfected macrophages at 6h (A) and 24 h (B) post-infection. Each dot represents one miRNA. Red dots indicate up-regulated miRNAs, and blue dots represent downregulated ones (P < 0.05). The horizontal black dotted line corresponds to p=0.05, log 10. The relative up- and down-regulation of miRNAs, expressed as boundaries of 2 or -2 of Fold Regulation, respectively. P-value was determined based on a two-tailed Student’s t-test comparing the 6h p.i. or 24 h p.i. with uninfected macrophages. Significantly expressed miRNAs in different times distributed in four groups (C). Results from three biological replicates developed in independent experiments.

Figure 2

Volcano plot of differential expression of miRNA in plasma from VL patients. Volcano plot showing differential expression of miRNA in plasma samples of active VL patients compared to healthy individuals. Each dot represents one miRNA. Red dots indicate up-regulated miRNAs, and blue dots represent downregulated ones (P < 0.05). The horizontal black dotted line corresponds to p=0.05, log 10. The relative up- and down-regulation of miRNAs, are expressed as boundaries of 2 or -2, of Fold Regulation respectively. P-value was determined based on a two-tailed Student’s t-test. P < 0.05 (Student t-test and Bonferroni correction).

Figure 3

Heatmap of predicted interactions between differentially expressed miRNAs in infected THP-1 cells. The set of differentially expressed microRNAs in L. infantum-infected THP-1 at 6 and 24 h and the biological pathways on which they are suggested to act, according to MiEAA algorithms, are shown in heat map format. Results are shown as -log10 of prediction p-value, and the degree of color saturation corresponds to the -log10 of the p-value.

Figure 4

Heatmap of predicted interactions between differentially expressed miRNAs in VL patient plasma. The set of differentially expressed microRNAs in plasma samples from active VL patients caused by L. infantum and the biological pathways on which they are suggested to act according to MiEAA algorithms are shown in heat map format. Results are shown as -log10 of prediction p-value, and the degree of color saturation corresponds to the -log10 of the p-value.

Figure 5

Parasite load, nitric oxide production, and chemokine levels in L. (L.) infantum promastigote-infected THP-1 cells transiently transfected with miR-548d-3p inhibitor. THP-1 cells were transiently transfected with miR-548d-3p inhibitor (10 nM) or negative control (scrambled miRNA; 10 nM) as described in Methods. After 24h, macrophages were infected with L. (L.) infantum promastigotes and incubated for 6 and 24h post-infection for different evaluations. (A) Parasite load represented as the number of parasites/100 cells. (B) Nitric oxide (NO) production was determined by the accumulation of nitrite in the cell culture supernatants. IP-10/CXCL10 (C), MCP1/CCL2 (D), RANTES/CXCL5 (E), and IL-8/CXCL8 (F) concentrations (pg/mL) were measured by flow cytometry using the CBA kit. * = P < 0.05 (one way ANOVA and Tukey´s test). One representative experiment from three independent assays is shown.

Figure 6

Chemokines in plasma of active VL patients and controls. IP-10/CXCL10, MCP1/CCL2, MIG/CXCL9, RANTES/CXCL5, and IL-8/CXCL8 concentrations were measured in the CBA kit using flow-cytometry. Data are shown as pg/mL, and each symbol corresponds to one subject * = p <0.05 (Mann-Whitney test). Patients (N=8) and controls (N=6).