Visceral Leishmaniasis is a chronic zoonosis and, if left untreated, can be fatal. Infected dogs have decreased cellular immunity (Th1) and develop a potent humoral response (Th2), which is not effective for elimination of the protozoan. Immune response can be modulated by microRNAs (miRNAs), however, characterization of miRNAs and their possible regulatory role in the spleen of infected dogs have not been done. We evaluated miRNA expression in splenic leukocytes (SL) from dogs naturally infected with Leishmania infantum and developing leishmaniasis (CanL; n = 8) compared to healthy dogs (n = 4). Microarray analysis showed increased expression of miR 21, miR 148a, miR 7 and miR 615, and downregulation of miR 150, miR 125a and miR 125b. Real-time PCR validated the differential expression of miR 21, miR 148a and miR 615. Further, decrease of miR 21 in SL, by means of transfection with a miR 21 inhibitor, increased the IL-12 cytokine and the T-bet/GATA-3 ratio, and decreased parasite load on SL of dogs with CanL. Taken together, these findings suggest that L. infantum infection alters splenic expression of miRNAs and that miR 21 interferes in the cellular immune response of L. infantum-infected dogs, placing this miRNA as a possible therapeutic target in CanL.

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.

Visceral leishmaniasis in humans is a chronic and fatal disease if left untreated. Canine leishmaniasis (CanL) is a severe public health problem because infected animals are powerful transmitters of the parasite to humans via phlebotomine vectors. Therefore, dogs are an essential target for control measures. Progression of canine infection is accompanied by failure of cellular immunity with reduction of circulating lymphocytes and increased cytokines that suppress macrophage function. Studies showed that the regulation of the effector function of macrophages and T cells appears to depend on miRNAs; miRNA-21 (miR-21) shows increased expression in splenic leukocytes of dogs with CanL and targets genes related to the immune response. Mimics and inhibitors of miR-21 were used in vitro to transfect splenic leukocytes from dogs with CanL. After transfection, expression levels of the proteins FAS, FASL, CD69, CCR7, TNF-α, IL-17, IFN-γ, and IL-10 were measured. FAS, FASL, CD69, and CCR7 expression levels decreased in splenic leukocytes from dogs with CanL. The miR-21 mimic decreased CD69 expression in splenic leukocytes from CanL and healthy groups. The miR-21 inhibitor decreased IL-10 levels in culture supernatants from splenic leukocytes in the CanL group. These findings suggest that miR-21 alters the immune response in CanL; therefore, miR-21 could be used as a possible therapeutic target for CanL.

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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.

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PGE 2 is a product derived from arachidonic acid, produced by the combination of cyclooxygenases (COX-1 and COX-2). 1 COX-1, expressed constitutively in most cells, is the dominant source of prostanoids that subserve housekeeping functions, such as gastric epithelial cytoprotection and homeostasis. 2 COX-2, induced by inflammatory stimuli, hormones and growth factors, is the most important source of prostanoid formation in inflammation and proliferative diseases, such as cancer. 2 PGE 2 is produced by various cell types. 3 PGE 2 is one of the most abundant PGs produced in the body, is most widely characterized in animal species and exhibits versatile biological activities.Under physiological conditions, PGE 2 is an important mediator of many biological functions, such as regulation of immune responses, blood pressure, gastrointestinal integrity and fertility. AbstractCanine leishmaniasis (CanL) is caused by the intracellular parasite Leishmania infantum. Prostaglandin E2 (PGE 2 ) exerts potent regulatory effects on the immune system in experimental model Leishmania infection, but this influence has not yet been studied in CanL. In this study, PGE 2 and PGE 2 receptor levels and the regulatory effect of PGE 2 on arginase activity, NO 2 , IL-10, IL-17, IFN-γ, TNF-α and parasite load were evaluated in cultures of splenic leucocytes obtained from dogs with CanL in the presence of agonists and inhibitors. Our results showed that splenic leucocytes from dogs with CanL had lower EP2 receptor levels than those of splenic leucocytes from healthy animals. We observed that NO 2 levels decreased when the cells were treated with a PGE 2 receptor agonist (EP1/EP2/EP3) or COX-2 inhibitor (NS-398) and that TNF-α, IL-17 and IFN-γ cytokine levels decreased when the cells were treated with a PGE 2 receptor agonist (EP2) or PGE 2 itself. The parasite load in splenic leucocyte cell cultures from dogs with CanL decreased after stimulation of the cells with PGE 2 . We conclude that Leishmania infection of dogs modulates PGE 2 receptors and speculate that the binding of PGE 2 to its receptors may activate the microbicidal capacity of cells. K E Y W O R D Sdogs, Leishmania spp., prostaglandin E2, receptors

Canine leishmaniasis (CanL) is a chronic disease caused by Leishmania infantum, and the limitations of the current treatments have encouraged new alternatives, such as the use of immunomodulatory nutrients. The objective of this study was to determine the serum levels of vitamin A (retinol), vitamin D (25(OH)VD3), and zinc (Zn) in dogs with CanL and the effect of in vitro supplementation with the respective active forms ATRA, 1,25(OH)2VD3, and SZn on spleen leukocyte cultures. Serum retinol, 25(OH)VD3, and Zn were determined by HPLC, ELISA, and ICP-MS, respectively. Spleen leukocyte cultures were used for the detection of NO and ROS by flow cytometry; the IFN-γ, TNF-α, and IL-10 levels were determined by ELISA; and the parasite load was determined by microscopy. We detected low serum levels of retinol and Zn and high levels of 25(OH)VD3 in the CanL group. The in vitro supplementation of CanL spleen leukocytes with ATRA, 1,25(OH)2VD3, and SZn, in addition to a soluble leishmania antigen (SLA) treatment, increased the NO and ROS levels, while the treatments with only ATRA and SZn increased the TNF-a levels. Increased IL-10 and IFN-g levels were observed with the addition of SLA to the medium, although the addition of the three nutrients led to a reduction of the IL-10 levels, and the addition of 1,25(OH)2VD3 and SZn led to a reduction of IFN-g. A supplementation with 1,25(OH)2VD3 and SZn reduced the parasite load but only in the absence of SLA. We suggest that the nutrients we tested are involved in the leishmanicidal mechanism, showing a potential for investigation in future studies.