microRNA-148a regula citocinas inflamatórias e atividade microbicida na leishmaniose canina
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Data
2022-04-08
Autores
Rebech, Gabriela Torres
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Universidade Estadual Paulista (Unesp)
Resumo
A leishmaniose canina (LCan) é um grave problema de saúde pública, pois animais infectados facilitam a transmissão do protozoário Leishmania para humanos pela picada do vetor flebotomíneo durante o repasto sanguíneo. A progressão da LCan está relacionada à supressão efetiva da resposta imune, possivelmente associada a pequenos RNAs chamados microRNAs (miR), que podem afetar a tradução do mRNA em proteínas e, consequentemente, regular o funcionamento das células. O aumento de miR-148a em leucócitos esplênicos (LS) de cães com LCan foi observado em estudos anteriores e análises in silico identificaram possíveis vias envolvidas na regulação da resposta imune que são afetadas por este miR. Portanto, através do uso de cultura celular e transfecção, in vitro, avaliamos o envolvimento de miR-148a, na regulação de citocinas inflamatórias como TNF-α, IL-6, IL-12, IL-1β, iNOS, expressão de MHCII, CD80, CD3, dos fatores de transcrição T-bet (Th1) e GATA-3 (Th2) e sua relação com a carga parasitária em LS de cães com LCan. Primeiramente, LS coletados de cães saudáveis e doentes (LCan), foram transfectados com oligonucleotídeos inventariados de inibidor e mimetizador de miR-148a. Após 48 horas, a expressão das proteínas MHCII, CD80, iNOS, CD3, Tbet, GATA-3 foi avaliada por citometria de fluxo e a concentração de TNF-α, IL-12, IL-6 e IL-1β foi medida no sobrenadante de cultura por ELISA de captura. A transfecção de LS com mimetizador de miR-148a diminuiu a concetração de iNOS nas células e de TNF-α, IL-6 e IL-12 no sobrenadante do LS cultivado de cães LCan. Curiosamente, a transfecção com inibidor de miR-148a diminuiu a carga parasitária nas células. Esses resultados sugerem um papel regulador negativo deste miR na resposta imune à infecção por L. infantum. Concluímos que miR-148a pode afetar a resposta imune regulando citocinas inflamatórias durante LCan. Nossos resultados contribuem para o entendimento da complexa interação hospedeiro/parasito na leishmaniose canina e podem auxiliar no desenvolvimento de possíveis tratamentos.
Canine leishmaniasis (CanL) is a serious public health concern, because infected animals facilitate transmission of the Leishmania protozoan to humans by the bite of sandfly vector during the blood meal. Progression of CanL is related to effective suppression of immune response, possibly associated with small RNAs called microRNAs (miR), which can affect mRNA translation into proteins and, consequently, regulate cells function. The increase of miR-148a in splenic leukocytes (SL) of dogs with CanL was observed in previous studies and in silico analysis identified possible pathways involved in immune response regulation that are affected by this miR. Therefore, through the use of, in vitro, cell culturing and transfection, we evaluated the involvement of miR-148a, in regulation of inflammatory cytokines such as TNF-α, IL-6, IL-12, IL-1β, iNOS, MHCII expression, CD80, CD3, of the T-bet (Th1) and GATA-3 (Th2) transcription factors and their relationship with parasite load in SL of dogs with CanL. Primary, SL obtained from healthy and diseased dogs (CanL), were transfectedwith miR-148a mimic and inhibitor inventoried oligonucleotides. After 48 hours, expression of MHCII, CD80, iNOS, CD3, Tbet, GATA-3 proteins was evaluated by flow cytometry and concentration of TNF-α, IL-12, IL-6 and IL-1β was measured in culture supernatant by capture ELISA. Transfection of SL with miR-148a mimic decreased iNOS concentration in the cells and TNF-α, IL-6 and IL-12 in the supernatant of the cultured SL from CanL dogs. Interestingly, transfection with miR-148a inhibitor decreased parasite load in cells. These results suggest a negative regulatory role of this miR in immune response to L. infantum infection. We conclude that miR-148a can affect immune response by regulating inflammatory cytokines during CanL. Our results contribute to the understanding of the complex host/parasite interaction in canine leishmaniasis and could assist the development of possible treatments.
Canine leishmaniasis (CanL) is a serious public health concern, because infected animals facilitate transmission of the Leishmania protozoan to humans by the bite of sandfly vector during the blood meal. Progression of CanL is related to effective suppression of immune response, possibly associated with small RNAs called microRNAs (miR), which can affect mRNA translation into proteins and, consequently, regulate cells function. The increase of miR-148a in splenic leukocytes (SL) of dogs with CanL was observed in previous studies and in silico analysis identified possible pathways involved in immune response regulation that are affected by this miR. Therefore, through the use of, in vitro, cell culturing and transfection, we evaluated the involvement of miR-148a, in regulation of inflammatory cytokines such as TNF-α, IL-6, IL-12, IL-1β, iNOS, MHCII expression, CD80, CD3, of the T-bet (Th1) and GATA-3 (Th2) transcription factors and their relationship with parasite load in SL of dogs with CanL. Primary, SL obtained from healthy and diseased dogs (CanL), were transfectedwith miR-148a mimic and inhibitor inventoried oligonucleotides. After 48 hours, expression of MHCII, CD80, iNOS, CD3, Tbet, GATA-3 proteins was evaluated by flow cytometry and concentration of TNF-α, IL-12, IL-6 and IL-1β was measured in culture supernatant by capture ELISA. Transfection of SL with miR-148a mimic decreased iNOS concentration in the cells and TNF-α, IL-6 and IL-12 in the supernatant of the cultured SL from CanL dogs. Interestingly, transfection with miR-148a inhibitor decreased parasite load in cells. These results suggest a negative regulatory role of this miR in immune response to L. infantum infection. We conclude that miR-148a can affect immune response by regulating inflammatory cytokines during CanL. Our results contribute to the understanding of the complex host/parasite interaction in canine leishmaniasis and could assist the development of possible treatments.
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Palavras-chave
LCan, microRNA-148a, Resposta imune, Fator de transcrição, Citocina, CanL, Immune response, Transcription factor, Cytokine