Dogs with canine visceral leishmaniasis have a boost of extracellular vesicles and miR‐21‐5p up‐expression

Abstract

This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra‐centrifuged to recover EVs (Can‐EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate‐poli‐acrylammide gel eletroforesis (SDS‐PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum‐EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can‐EVs from dogs with CanVL (7.78 × 1010 Can‐EVs/mL) were higher (p < .0001) than the non‐infected dogs (mean: 1.47 × 1010 Can‐EVs/mL). These results suggested that concentrations of Can‐EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR‐21‐5p, miR‐146a‐5p, miR‐125b‐5p, miR‐144‐3p, miR‐194‐5p, miR‐346, miR‐29c‐3p, miR‐155‐5p, miR‐24‐3p, miR‐181a‐5p, and miR‐9‐5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up‐expressed miR‐21‐5p and miR‐146a‐5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can‐EVs, as well as, the up‐expression of miR‐21‐5p and miR‐146a‐5p in infected dogs.

Extracellular Vesicles from Leishmania (Leishmania) infantum Contribute in Stimulating Immune Response and Immunosuppression in Hosts with Visceral Leishmaniasis

“…The comparison between groups (control versus infected) were statistically different. These data indicate that the concentration of EVs may be able to distinguish infected from uninfected hosts [ 19 ].…”

“…The protocols were prepared as described before [ 19 ]. Promastigotes of L. (L.) infantum (MHOM/BR/1972/LD) were maintained at 25 °C, in 199 medium, pH 7.2 containing fetal bovine serum (FBS) 10%, gentamicin 30 μg/mL, hemin 50 μL/mL, and human urine 50 μL/mL.…”

“…Five healthy dogs were used for negative control. The canine sera have been previously tested for Can-VL through ELISA, Fast test, and real-time PCR [ 19 , 37 ].…”

“…In order to guarantee the invasion in the host cells, pathogens release effector molecules through the cellular membrane, and the via extracellular vesicles (EVs) are transported to host cells. These compounds cause the modulation of cytokines and microRNAs (miRNAs) for preparing the cells for parasite infection [ 16 , 17 , 18 , 19 ]. In vivo studies, analyzing mice and macrophages, complement these findings, reporting that EVs released by Leishmania (Leish-EVs) can modulate the immune-regulating and signaling pathways [ 19 , 20 , 21 , 22 , 23 , 24 ].…”

“…These compounds cause the modulation of cytokines and microRNAs (miRNAs) for preparing the cells for parasite infection [ 16 , 17 , 18 , 19 ]. In vivo studies, analyzing mice and macrophages, complement these findings, reporting that EVs released by Leishmania (Leish-EVs) can modulate the immune-regulating and signaling pathways [ 19 , 20 , 21 , 22 , 23 , 24 ].…”