MicroRNA-21 and microRNA-148a affects PTEN, NO and ROS in canine leishmaniasis

Canine Visceral leishmaniasis (CanL) poses a severe public health threat in several countries. Disease progression depends on the degree of immune response suppression. MicroRNAs (miRs) modulate mRNA translation into proteins and regulate various cellular functions and pathways associated with immune responses. MiR-21 and miR-148a can alter the parasite load and M1 macrophages are the principal cells in dogs’ leishmanicidal activity. A previous study found increased miR-21 and miR-148a in splenic leukocytes (SL) of dogs with CanL using microarray analysis and in silico analysis identified PTEN pathway targets. PTEN is involved in the immune regulation of macrophages. We measured PTEN and the production of reactive oxygen species (ROS) and nitric oxide (NO) before and after transfection SLs of dogs with CanL with mimic and inhibition of miR-21 and miR-148a. PTEN levels increased, NO and ROS decreased in SLs from dogs with CanL. Inhibition of miRNA-21 resulted in PTEN increase; in contrast, PTEN decreased after miR-148a inhibition. Nitrite (NO2) levels increased after transfection with miR-21 inhibitor but were decreased with miR-148a inhibitor. The increase in miR-21 promoted a reduction in ROS and NO levels, but miR-148a inhibition increased NO and reduced ROS. These findings suggest that miR-21 and miR-148a can participate in immune response in CanL, affecting PTEN, NO, and ROS levels

Relationship of peripheral blood mononuclear cells miRNA expression and parasitic load in canine visceral leishmaniasis.

Visceral leishmaniasis (VL) in humans is a chronic and often fatal disease if left untreated. Dogs appear to be the main reservoir host for L. infantum infection, however, in many regions other canids such as jackals, foxes, wolves and other mammals, such as hares or black rats, have been implicated as wild reservoirs. Most dogs cannot form an effective immune response against this infection, and this could be modulated by small non-coding RNAs, called microRNAs, responsible for post-transcriptional control of gene expression. Here, we evaluated the expression of miRNAs in peripheral blood mononuclear cells (PBMC) of symptomatic dogs naturally infected with Leishmania (L.) infantum (n = 10) and compared to those of healthy dogs (n = 5). Microarray analysis revealed that miR-21, miR-424, miR-194 and miR-451 had a 3-fold increase in expression, miR-192, miR-503, and miR-371 had a 2-fold increase in expression, whereas a 2-fold reduction in expression was observed for miR-150 and miR-574. Real-time PCR validated the differential expression of miR-21, miR-150, miR-451, miR-192, miR-194, and miR-371. Parasite load of PBMC was measured by real-time PCR and correlated to the differentially expressed miRNAs, showing a strong positive correlation with expression of miR-194, a regular positive correlation with miR-371 expression, and a moderate negative correlation with miR-150 expression in PBMC. These findings suggest that Leishmania infection interferes with miRNAs expression in PBMC, and their correlation with parasite load may help in the identification of therapeutic targets in Canine Visceral Leishmaniasis (CVL)

Data on differentially expressed miRNAs in dogs infected with Leishmania infantum

This paper contains data on differentially expressed miRNAs in peripheral blood mononuclear cells (PBMC) of dogs naturally infected by Leishmania (L.) infantum compared to healthy dogs. In recent years, studies with miRNAs have shown that these molecules play a critical role in the regulation and function of immune response.Differentially expressed miRNAs were identified by microarray, validated by real time PCR and compared with parasite load in the dogs. Targets and pathways were analyzed using the Ingenuity Pathway Analysis program. Keywords: Dog, Leishmania infantum, miRN

MicroRNA-194 regulates parasitic load and IL-1β-dependent nitric oxide production in the peripheral blood mononuclear cells of dogs with leishmaniasis.

Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host's immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-β in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1β blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1β and NO in PBMCs from diseased dogs, and blockade of IL-1β following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1β. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets

Correlation between miR-194 expression and relative gene expression of TRAF6 in PBMC from dogs with leishmaniasis.

Expression of miR-194 and relative expression gene of TRAF6 in PBMCs from dogs with leishmaniasis (N = 10) was quantified using qPCR. Data represents a negative correlation between miR-194 expression and relative gene expression of TRAF6. Asterisks indicate significant correlation by Pearson correlation test.</p

RFLP analysis of ITS1-PCR fragments amplified from DNA samples of standard isolates using the Hae III enzyme.

M: molecular marker (100 bp); Li: Leishmania infantum. Samples 1 to 28 showed an identical sampling profile to L. infantum. RFLPs were identified on 3% agarose gels stained with gel red and indicated by an arrow. (TIF)</p

Red blood cell parameters.

Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host’s immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-β in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1β blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1β and NO in PBMCs from diseased dogs, and blockade of IL-1β following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1β. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.</div

Quantifying parasite load in PBMCs from dogs with leishmaniasis after transfection with Mimic and miR-194 Inhibitor.

Parasite load was quantified using flow cytometry (N 18) (A) and qPCR (N = 9) (B). Data are expressed as the median and interquartile range (25 and 75). Symbols represent individual data for each animal. Asterisks indicate significant differences (Friedman’s test followed by Dunn’s test, p < 0.05).</p

White blood cells and platelet counts.

Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host’s immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-β in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1β blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1β and NO in PBMCs from diseased dogs, and blockade of IL-1β following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1β. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.</div