MicroRNA-194 regula a carga parasitária e a produção de óxido nítrico dependente de IL-1β em células mononucleares do sangue periférico de cães com leishmaniose
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Data
2023-03-23
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Universidade Estadual Paulista (Unesp)
Resumo
Os cães domésticos são os principais reservatórios urbanos de Leishmania infantum, o agente causador da leishmaniose visceral (LV). Na leishmaniose canina (CanL), a modulação da resposta imune do hospedeiro pode estar associada à expressão de pequenos RNAs não codificantes denominados microRNA (miR). A expressão do miR-194 aumenta em células mononucleares do sangue periférico (CMSP) de cães com leishmaniose com correlação positiva com a carga parasitária. Estudamos CMSP de 5 cães saudáveis e 28 cães com leishmaniose. A expressão basal do miR-194 foi analisada utilzando qPCR. As CMSP foram transfectadas com Mimic e Inibidor sintéticos do miR-194 e cultivadas a 37º C, 5% de CO2 por 48 horas. A expressão de possíveis alvos do miR-194 foi analisada: iNOS, NO, T-Bet, GATA3 e FoxP3 foram analisadas por citometria de fluxo; a expressão gênica de TRAF6 foi analisada por qPCR, e o nível das citocinas IL-1β, IL-4, IL-6, IL-10, TNF-α, IFN-γ e TGF-β no sobrenadantes de cultura celular foi mensurada por Ensaio Imunoenzimático de Captura (ELISA). A carga parasitária foi analisada por citometria e qPCR. Ensaio funcional com Inibitor do miR-194 e bloqueio de IL-1β e avaliação da produção de NO também foram realizados. Transfecção com o mimic do miR-194 promoveu um aumento da carga parasitária. Não houve alterações significativas na expressão de T-bet, GATA3 ou FoxP3 com aumento ou inibição do miR-194. A inibição de miR-194 aumentou IL-1β e NO em CMSP de cães doentes, e o bloqueio de IL-1β após a inibição de miR-194 diminuiu os níveis de NO. Esses achados sugerem que o miR-194 é regulado positivamente em CMSP de cães com leishmaniose e regula positivamente a carga parasitária, possivelmente diminuindo a produção de NO dependente da IL-1β. Esses resultados aumentam nossa compreensão dos mecanismos moleculares de evasão da resposta imune pelo parasita e a identificação de possíveis alvos terapêuticos.
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.
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.
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Leishmaniose visceral, Cães, MicroRNAs, Genes, Imunologia, Visceral leishmaniasis, Dogs, MicroRNA, Immunology