miR-548d-3p Alters Parasite Growth and Inflammation in Leishmania (Viannia) braziliensis Infection

Abstract

American Tegumentary Leishmaniasis (ATL) is an endemic disease in Latin America, mainly caused in Brazil by Leishmania (Viannia) braziliensis. Clinical manifestations vary from mild, localized cutaneous leishmaniasis (CL) to aggressive mucosal disease. The host immune response strongly determines the outcome of infection and pattern of disease. However, the pathogenesis of ATL is not well understood, and host microRNAs (miRNAs) may have a role in this context. In the present study, miRNAs were quantified using qPCR arrays in human monocytic THP-1 cells infected in vitro with L. (V.) braziliensis promastigotes and in plasma from patients with ATL, focusing on inflammatory response-specific miRNAs. Patients with active or self-healed cutaneous leishmaniasis patients, with confirmed parasitological or immunological diagnosis, were compared with healthy controls. Computational target prediction of significantly-altered miRNAs from in vitro L. (V.) braziliensis-infected THP-1 cells revealed predicted targets involved in diverse pathways, including chemokine signaling, inflammatory, cellular proliferation, and tissue repair processes. In plasma, we observed distinct miRNA expression in patients with self-healed and active lesions compared with healthy controls. Some miRNAs dysregulated during THP-1 in vitro infection were also found in plasma from self-healed patients, including miR-548d-3p, which was upregulated in infected THP-1 cells and in plasma from self-healed patients. As miR-548d-3p was predicted to target the chemokine pathway and inflammation is a central to the pathogenesis of ATL, we evaluated the effect of transient transfection of a miR-548d-3p inhibitor on L. (V.) braziliensis infected-THP-1 cells. Inhibition of miR-548d-3p reduced parasite growth early after infection and increased production of MCP1/CCL2, RANTES/CCL5, and IP10/CXCL10. In plasma of self-healed patients, MCP1/CCL2, RANTES/CCL5, and IL-8/CXCL8 concentrations were significantly decreased and MIG/CXCL9 and IP-10/CXCL10 increased compared to patients with active disease. These data suggest that by modulating miRNAs, L. (V.) braziliensis may interfere with chemokine production and hence the inflammatory processes underpinning lesion resolution. Our data suggest miR-548d-3p could be further evaluated as a prognostic marker for ATL and/or as a host-directed therapeutic target.

Keywords: Leishmania braziliensis; THP-1 cells; active cutaneous leishmaniasis; microRNA; pathogenesis; self-healed cutaneous leishmaniasis.

Figure 1

Cartographical representation of the State of Pernambuco, Northeastern Brazil. Regions in blue indicate Moreno (1), Jaboatão (2), Paudalho (3), and Bezerros (4) municipalities.

Figure 2

miRNA profiles of L. braziliensis infected THP-1-macrophages. Volcano plot of differential expression of miRNA in L. (V.) braziliensis promastigote-infected THP-1 macrophages in relation to non-infected cells at 6 h (A) and 24 h (B) post-infection compared to uninfected-macrophages. Each dot represents one miRNA. Red dots indicate the up-regulated miRNAs, and the blue dots represent the down-regulated miRNAs (P < 0.05). 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 two-tailed Student’s t test. Significantly expressed miRNAs in different times distributed in four groups (C). Experiments were repeated three times.

Figure 3

Volcano plot of differential expression of miRNA in plasma of cutaneous leishmaniasis patients. Volcano plot of differential expression of miRNA in plasma samples of active disease patients (A) and self-healed (B) compared to healthy individuals, and self-healed (C) compared to active disease patients. Each dot represents one miRNA. Red dots indicate the up-regulated miRNAs, and the blue dots represent the down-regulated miRNAs (P < 0.05). 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 two-tailed Student’s t test. P < 0.05 (Student t test and Bonferroni correction).

Figure 4

Predicted interactions between the set of differentially expressed microRNAs in THP-1 cells after 6 and 24 h post infection. The genes on which they are suggested to act in the Leishmaniasis pathway in humans are shown as seen in KEGG. miRNAs were classified into four groups according to their modulation through time: down-regulated in 6 and 24 h (A), up-regulated on 6 h and down-regulated in 24 h (B), down-regulated in 6 h and up-regulated in 24 h (C) and up-regulated in both timepoints (D).

Figure 5

Predicted interactions between the set of differentially expressed microRNAs in THP-1 cells after 6 and 24 h post infection with L. braziliensis (A) and in plasma samples from self-healed patients compared to active disease individuals (B) and the biological pathways related to inflammatory response on which they are suggested to act according to MiEAA algorithms. In (B), pathways in red were predictably targeted by in vitro and ex vivo miRNA sets, while the others in blue were evidenced only in miRNAs significantly quantified in plasma.

Figure 6

Parasite load (number of amastigotes/100 cells) (A) and chemokine levels (pg/ml) (B–E) in L. (V.) braziliensis promastigote-infected THP-1 cells transiently transfected with miR-548d-3p inhibitor at 6 and 24 h post-infection. One experiment was carried out by adding the miR-548d-3p inhibitor (10 nM) or negative control (scrambled miRNA; 10 nM) with the transfection reagent diluted in RPMI medium or only RPMI medium (non-transfected cells) to wells containing 10⁶ THP-1 adherent cells and maintained for 24 h at 37°C (5% CO2) then infected with L. (V.) braziliensis promastigotes. (A) * = P < 0.05 (one way ANOVA and Student t test). MCP1/CCL2 (B), RANTES/CXCL5 (C), IL-8/CXCL8 (D), and IP-10/CXCL10 (E) concentrations were measured by flow-cytometry using the CBA kit. (B–E) * = p <0.05 (Kruskal-Wallis and Bonferroni tests).

Figure 7

Chemokine concentration (pg/ml) in plasma of cutaneous leishmaniasis patients and controls. MCP1/CCL2 (A), RANTES/CXCL5 (B), IL-8/CXCL8 (C), MIG/CXCL9 (D), and IP-10/CXCL10 (E) concentrations were measured by flow-cytometry using the CBA kit. * = p < 0.05 (Kruskal-Wallis and Bonferroni tests). N =5 patients and controls per group.