RESUMO
In this study, we evaluated the performance of a new enzyme-linked immunosorbent assay (ELISA) variant known as indirect "plasmonic ELISA" (pELISA) for the detection of Leishmania spp. infection. Serum samples from 170 dogs from an area where canine leishmaniosis (CanL) is endemic and from 26 healthy dogs from a nonendemic area were tested by indirect pELISA, and the results were compared to those of an indirect ELISA (both with recombinant antigen rK28) and those of an immunochromatographic test (dual-path platform, TR-DPP®) using real-time PCR on blood samples or conjunctival swabs as the gold standard. The pELISA, indirect rK28 ELISA and the TR-DPP® immunochromatographic test presented sensitivities of 94.7%, 89.5% and 79.0% and specificities of 100%, 92.7% and 91.5%, respectively. The analysis of the results revealed that the specificity of the indirect pELISA was greater than that of the method recommended by the Ministry of Health in Brazil and may increase the feasibility of diagnosis in resource-constrained countries because it does not require sophisticated instruments to read. Thus, this method can be used as an additional tool for the detection of Leishmania spp. infection in these areas.
Assuntos
Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Leishmaniose/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/análise , Brasil , Doenças do Cão/sangue , Cães , Leishmaniose/sangue , Leishmaniose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Testes Sorológicos/métodosRESUMO
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 the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1ß, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-ß in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1ß blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in 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-1ß and NO in PBMCs from diseased dogs, and blockade of IL-1ß 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 IL-1ß. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , MicroRNAs , Animais , Cães , Citocinas/genética , Doenças do Cão/parasitologia , Fatores de Transcrição Forkhead , Leishmania infantum/fisiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares , MicroRNAs/genética , Óxido Nítrico/metabolismo , Carga Parasitária , Fator 6 Associado a Receptor de TNF , Leishmaniose/veterinária , Interleucina-1beta/metabolismoRESUMO
In the Americas, L. infantum (syn. chagasi) is the main cause of human visceral leishmaniasis. The role of neutrophils as part of the innate response to Leishmania spp. infection is dubious and varies according to the species causing the infection. Global expression of coding RNAs, microRNAs and long non-coding RNAs changes as part of the immune response against pathogens. Changes in mRNA and non-coding RNA expression resulting from infection by Leishmania spp. are widely studied in macrophages, but scarce in neutrophils, the first cell to encounter the trypanosomatid, especially following infection by L. infantum. Herein, we aimed to understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum. We isolated neutrophils from whole blood of healthy male donors (n = 5) and split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq and miRNA microarray were performed. A total of 212 genes were differentially expressed in neutrophils following RNA-Seq analysis (log2(FC)±0.58, FDR≤0.05). In vitro infection with L. infantum upregulated the expression of 197 and reduced the expression of 92 miRNAs in human neutrophils (FC±2, FDR≤0.01). Lastly, 5 downregulated genes were classified as lncRNA, and of the 10 upregulated genes, there was only 1 lncRNA. Further bioinformatic analysis indicated that changes in the transcriptome and microtranscriptome of neutrophils, following in vitro infection with L. infantum, may impair phagocytosis, apoptosis and decrease nitric oxide production. Our work sheds light on several mechanisms used by L. infantum to control neutrophil-mediated immune response and identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis.
Assuntos
Leishmania infantum , MicroRNAs , Neutrófilos , RNA Longo não Codificante , RNA Mensageiro , Humanos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Leishmania infantum/genética , Leishmania infantum/imunologia , RNA Longo não Codificante/genética , MicroRNAs/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/genética , Adulto , Perfilação da Expressão GênicaRESUMO
Leishmania infantum causes visceral leishmaniosis, a neglected tropical disease that can modulate the host immune response by altering the expression of small non-coding RNAs called microRNAs (miRNAs). Some miRNAs are differentially expressed in peripheral blood mononuclear cells (PBMCs) of dogs with canine visceral leishmaniosis (CanL), like the down-regulated miR-150. Even though miR-150 is negatively correlated with L. infantum parasitic load, it is unclear if miR-150 directly affects L. infantum parasitic load and (if so) how this miRNA would contribute to infection. Here, we isolated PBMCs from 14 naturally infected dogs (CanL group) and six healthy dogs (Control group) and treated them in vitro with miR-150 mimic or inhibitor. We measured L. infantum parasitic load using qPCR and compared treatments. We also measured miR-150 in silico predicted target protein levels (STAT1, TNF-α, HDAC8, and GZMB) using flow cytometry or enzyme-linked immunosorbent assays. Increasing miR-150 activity diminished L. infantum parasitic load in CanL PBMCs. We also found that inhibition of miR-150 reduced GZMB (granzyme B) levels. These findings demonstrate that miR-150 plays an important role in L. infantum infection in canine PBMCs, and they merit further studies aiming at drug development.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , MicroRNAs , Animais , Cães , Leucócitos Mononucleares , Granzimas , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , MicroRNAs/genética , Doenças do Cão/parasitologiaRESUMO
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-1ß, 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-1ß 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.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , MicroRNAs , Animais , Cães , Citocinas , Doenças do Cão/parasitologia , Interleucina-12/genética , Interleucina-6 , Leishmania infantum/genética , Leishmaniose/veterinária , Leishmaniose Visceral/parasitologia , MicroRNAs/genética , Carga Parasitária , Fator de Necrose Tumoral alfa/genéticaRESUMO
Canine Visceral leishmaniasis (CanL) poses a severe public health threat in several countries. Disease progression depends on the degree of immune response suppression. MicroRNAs (miRs) modulate mRNA translation into proteins and regulate various cellular functions and pathways associated with immune responses. MiR-21 and miR-148a can alter the parasite load and M1 macrophages are the principal cells in dogs' leishmanicidal activity. A previous study found increased miR-21 and miR-148a in splenic leukocytes (SL) of dogs with CanL using microarray analysis and in silico analysis identified PTEN pathway targets. PTEN is involved in the immune regulation of macrophages. We measured PTEN and the production of reactive oxygen species (ROS) and nitric oxide (NO) before and after transfection SLs of dogs with CanL with mimic and inhibition of miR-21 and miR-148a. PTEN levels increased, NO and ROS decreased in SLs from dogs with CanL. Inhibition of miRNA-21 resulted in PTEN increase; in contrast, PTEN decreased after miR-148a inhibition. Nitrite (NO2) levels increased after transfection with miR-21 inhibitor but were decreased with miR-148a inhibitor. The increase in miR-21 promoted a reduction in ROS and NO levels, but miR-148a inhibition increased NO and reduced ROS. These findings suggest that miR-21 and miR-148a can participate in immune response in CanL, affecting PTEN, NO, and ROS levels.
RESUMO
Visceral Leishmaniasis (VL) is a neglected tropical disease, caused by L. infantum in the New World, where dogs are the main reservoir. These parasites can regulate host immune response through miRNA differential expression in the early stages of infection; however such early response has not yet been investigated in the canine model. PBMC from healthy dogs were exposed to L. infantum in vitro and microarray analysis showed an upregulation of miR-206, miR-302d, miR-433, miR-214, miR-493, miR-514, miR-1835, miR-210, miR-539, miR-432, miR-188, miR-345 and downregulation of miR-489 and miR-503 in comparison to non-exposed control cells, at 24 h post-exposure. In silico target prediction showed that the upregulated miRNAs target 1541 genes, which can modulate important pathways involved in the early immune responses, like the "MAPK signaling pathway", one of the most relevant pathways to Leishmania survival inside host cells. These findings shed light on parasite modulation of host immunity following Leishmania infection, which in turn can be explored for drug development.
Assuntos
Doenças do Cão/parasitologia , Leishmania infantum , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares/metabolismo , MicroRNAs/metabolismo , Animais , Células Cultivadas , Doenças do Cão/sangue , Doenças do Cão/genética , Doenças do Cão/imunologia , Cães , Regulação para Baixo , Leishmaniose Visceral/sangue , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Sistema de Sinalização das MAP QuinasesRESUMO
Recently, a novel Enzyme-Linked Immunosorbent Assay (ELISA) strategy has emerged, known as "plasmonic ELISA" (pELISA), which enables the detection of disease biomarkers at low concentrations with the naked eye. For the first time, this research has developed a signal-generation mechanism for the detection of anti-Leishmania sp. IgG antibodies with the naked eye using pELISA. The immunoassay incorporates an indirect ELISA with successive growth of gold nanoparticles to obtain blue or red-colored solutions in the presence or absence of anti-Leishmania sp. IgG antibodies in canine serum, respectively. The technique we developed was successfully tested in canine serum positive and negative for canine leishmaniasis (CanL), and was shown to be an effective method that could be used as an additional tool for CanL diagnosis. It will be particularly useful in resource-constrained countries, because it does not require sophisticated instruments to read the results, increasing the practicality of CanL detection in these areas.