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

Author(s)

Sidnei Ferro Costa

• Document type: Doctoral Thesis
• Press: Araçatuba. 2023-04-27.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária. Araçatuba
• Defense date: 2023-03-23
• Advisor: Valéria Marçal Felix de Lima

Abstract

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 parasite load. We studied PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis. Basal expression of miR-194 was measured using qPCR. PBMCs were transfected with synthetic miR-194 mimic and inhibitor, and cultured at 37 ºC, 5% CO2 for 48 hours. Expression of possible targets iNOS, NO, T-Bet, GATA3, and FoxP3 were measured using flow cytometry; gene expression of TRAF6 was measured using qPCR, and level 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. Parasite load was measured using cytometry and qPCR. Functional assays followed by IL-1β blockade and assessment of NO production were also performed. Transfection with miR-194 mimic promoted an increase in the 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 IL1β. These results contribute to our understanding of the mechanisms of immune evasion by the parasite and the identification of possible therapeutic targets. (AU)

• FAPESP's process: 19/14894-0 - MiRNA 194 in the regulation of the immunological response of peripheral blood mononuclear cells in Canine Leishmaniasis
• Grantee: Sidnei Ferro Costa
• Support Opportunities: Scholarships in Brazil - Doctorate