miR-148a regulation interferes in inflammatory cytokine and parasitic load in canine leishmaniasis

Abstract

Canine leishmaniasis (CanL) is a severe public health threat. Infected animals mediate transmission of the Leishmania protozoan to humans via the sandfly’s bite during a blood meal. CanL progression depends on the degree of suppression of the immune response, possibly associated with microRNAs (miR), which can modulate mRNA translation into proteins and (consequently) regulate cell function. Increased 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, we evaluated the involvement of miR-148a in the regulation of TNF-α, IL-6, IL-12, IL-1β, iNOS, MHCII, CD80, CD3, T-bet, and GATA-3 transcription factors and their relationship with parasite load in SL of dogs with CanL. Splenic leukocytes obtained from healthy and diseased dogs were transfected with miR-148a mimic and inhibitor oligonucleotides. After 48 hours, expression levels of MHCII, CD80, iNOS, CD3, T-bet, and GATA-3 were evaluated by flow cytometry, and concentrations of TNF-α, IL-12, IL-6, and IL-1β were measured in culture supernatants by capture enzyme-linked immunosorbent assays. Transfection of SL with miR-148a mimics decreased iNOS levels in cells and TNF-α, IL-6, and IL-12 in the supernatants of cultured SL from CanL dogs. Interestingly, transfection with miR-148a inhibitor decreased parasite load in SL cells. These results suggest a direct or not regulatory role of this miR in the immune response to Leishmania infantum infection. We conclude that miR-148a can modulate immune responses by regulating inflammatory cytokines during CanL. Our results contribute to understanding the complex host/parasite interaction in CanL and could assist the development of treatments. Author summary The Leishmania infantum is a protozoan parasite that causes visceral leishmaniasis (VL), the most lethal form of the disease in humans. In the domestic environment, dogs are the primary reservoir of this parasite; the infection may present as slimming, extensive skin lesions, excessive nail growth, and others. Treatments for canine leishmaniasis (CanL) are ineffective because treated dogs often re-develop signs and symptoms when the medication is stopped. However, the immune response determines the presentation and cellular immune responses are most effective for parasite replication control. Resistance depends on the inflammatory or microbicidal activity of cytokines. Nevertheless, the suppression of this immune response is evident during CanL and is related to many factors. Recent reports of dogs with CanL found increased small RNAs called microRNAs (miR) that regulate cell function and might be associated with the immune regulation observed during this disease. Therefore, we inhibited and increased levels of a specific miR (miR-148a) in vitro in cells of dogs (Leishmania-infected/sick and uninfected/heathy) and measured immunological markers. We observed a reduction of some essential inflammatory cytokines when the miR was increased and parasite number reduction when the miR was inhibited. These results suggest that this miR influences immunological responses in dogs with CanL related to disease progression. The findings contribute to the understanding of immunological aspects of CanL, facilitating the development of immunotherapeutic approaches.