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1.
FASEB J ; 35(9): e21755, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383962

RESUMO

Visceral leishmaniasis (VL) is a debilitating human pathogenesis in which the body's immune functions are severely compromised. Various subsets of T cells, including Th17 cells are important regulators of immune responses observed in various pathologies. The role of Th17 cells and its correlation with immuno-regulatory cytokines are however not well understood in human VL. Herein we studied how IL-17 is associated with the progression of Leishmania donovani infection using murine model of VL. We found induction of a strong IL-17 response at the early phase of infection which progressively reduced to basal level during chronic VL. The mechanistic study of this behavior was found to be linked with the role of regulatory T cells (CD4+ CD25+ T cells) that suppresses the proliferation of the Th17 cell population. Moreover, TGF-ß and IL-35 derived from CD4+ CD25+ T cells are the key mediators for the downregulation of IL-17 during chronic VL. Thus, this study points to an antagonistic effect of Tregs and Th17 cells that can be used for designing better therapeutic and preventive strategies against leishmaniasis.


Assuntos
Interleucinas/imunologia , Leishmaniose Visceral/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Células Cultivadas , Subunidade alfa de Receptor de Interleucina-2/imunologia , Leishmania donovani/parasitologia , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th17/parasitologia
2.
J Immunol ; 207(5): 1322-1332, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34341171

RESUMO

MicroRNA-21 (miR-21) inhibits IL-12 expression and impairs the Th1 response necessary for control of Leishmania infection. Recent studies have shown that Leishmania infection induces miR-21 expression in dendritic cells and macrophages, and inhibition of miR-21 restores IL-12 expression. Because miR-21 is known to be expressed due to inflammatory stimuli in a wide range of hematopoietic cells, we investigated the role of miR-21 in regulating immune responses during visceral leishmaniasis (VL) caused by Leishmania donovani infection. We found that miR-21 expression was significantly elevated in dendritic cells, macrophages, inflammatory monocytes, polymorphonuclear neutrophils, and in the spleen and liver tissues after L. donovani infection, concomitant with an increased expression of disease exacerbating IL-6 and STAT3. Bone marrow dendritic cells from miR-21 knockout (miR-21KO) mice showed increased IL-12 production and decreased production of IL-10. On L. donovani infection, miR-21KO mice exhibited significantly greater numbers of IFN-γ- and TNF-α-producing CD4+ and CD8+ T cells in their organs that was associated with increased production of Th1-associated IFN-γ, TNF-α, and NO from the splenocytes. Finally, miR-21KO mice displayed significantly more developing and mature hepatic granulomas leading to reduction in organ parasitic loads compared with wild type counterparts. Similar results were noted in L. donovani-infected wild type mice after transient miR-21 depletion. These observations indicate that miR-21 plays a critical role in pathogenesis of VL by suppressing IL-12- and Th1-associated IFN-γ and also inducing disease-promoting induction of the IL-6 and STAT-3 signaling pathway. miR-21 could therefore be used as a potential target for developing host-directed treatment for VL.


Assuntos
Células Dendríticas/imunologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , MicroRNAs/genética , Monócitos/imunologia , Neutrófilos/imunologia , Células Th1/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Resistência à Doença , Imunidade Celular , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT3/metabolismo , Regulação para Cima
3.
Life Sci ; 282: 119793, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34242658

RESUMO

Visceral leishmaniasis (VL) is a neglected and highly lethal disease. VL is endemic in South American countries, with Brazil being responsible for 96% of the cases. In this continent, VL is caused by the protozoan Leishmania (Leishmania) infantum (L. infantum), transmitted by the bite of infected female phlebotomine sandflies. Immediately after the inoculation of L.infantum promastigotes into the vertebrate host, the complement, as part of the first line of innate response, becomes activated. L. infantum promastigotes glycocalyx is rich in carbohydrates that can activate the lectin pathway of complement system. In this study, we evaluated whether the lectin pathway collectins [manose binding lectin (MBL) and collectin-11 (CL-11)] and ficolins (-1, -2 and -3) interact with L.infantum promastigotes, using confocal microscopy and flow cytometry. The binding of MBL, CL-11 and ficolins -1 and -3, but not ficolin-2, was observed on the surface of live metacyclic promastigotes after incubation with normal human serum (NHS) or recombinant proteins. C3 and C4 deposition as well as complement mediated lyses was also demonstrated after interaction with NHS. These results highlight a role for collectins and ficolins in the initial immune response to L.infantum.


Assuntos
Proteínas do Sistema Complemento/imunologia , Lectinas/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Ativação do Complemento , Interações Hospedeiro-Parasita , Humanos , Leishmania infantum/fisiologia
4.
Int J Mol Sci ; 22(13)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34281214

RESUMO

Transcriptional analysis of complex biological scenarios has been used extensively, even though sometimes the results of such analysis may prove imprecise or difficult to interpret due to an overwhelming amount of information. In this study, a large-scale real-time qPCR experiment was coupled to multivariate statistical analysis in order to describe the main immunological events underlying the early L. infantum infection in livers of BALB/c mice. High-throughput qPCR was used to evaluate the expression of 223 genes related to immunological response and metabolism 1, 3, 5, and 10 days post infection. This integrative analysis showed strikingly different gene signatures at 1 and 10 days post infection, revealing the progression of infection in the experimental model based on the upregulation of particular immunological response patterns and mediators. The gene signature 1 day post infection was not only characterized by the upregulation of mediators involved in interferon signaling and cell chemotaxis, but also the upregulation of some inhibitory markers. In contrast, at 10 days post infection, the upregulation of many inflammatory and Th1 markers characterized a more defined gene signature with the upregulation of mediators in the IL-12 signaling pathway. Our results reveal a significant connection between the expression of innate immune response and metabolic and inhibitory markers in early L. infantum infection of the liver.


Assuntos
Leishmaniose Visceral/metabolismo , Fígado/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Interleucina-12/metabolismo , Leishmaniose Visceral/imunologia , Metabolismo dos Lipídeos , Camundongos Endogâmicos BALB C , Células Th1/fisiologia
5.
Acta Trop ; 221: 106018, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34157292

RESUMO

Leishmania (Leishmania) amazonensis is an important etiological agent of American cutaneous leishmaniasis (ACL) in Brazil. The species causes a large spectrum of clinical manifestations in humans and dogs, ranging from cutaneous, cutaneous diffuse, mucocutaneous, and visceral involvement, however, the factors that drive the development of different disease forms by the same species are not yet fully known. In the present work, it was systematically reviewed the studies addressing phenotypic and genotypic characteristics of Leishmania (L.) amazonensis isolates causing cutaneous and visceral clinical frames in humans and dogs, comparing the results observed. For this, four research databases were searched for the following keywords: (Leishmania amazonensis AND visceral leishmaniasis) AND (tropism OR virulence OR visceralization OR adaptations OR mutation OR clinical presentation OR resistance OR survival OR wide spectrum). The results revealed that the complexity disease seems to involve the combination of genetic factors of the parasite (as modifications in molecules related to the virulence and metabolism) and also of the host's immune background and status. Nonetheless, the exact mechanism that leads to different clinical manifestations between strains of the same species is still uncertain and future studies must be developed to better elucidate this phenomenon.


Assuntos
Leishmania , Leishmaniose Cutânea , Leishmaniose Mucocutânea , Leishmaniose Visceral , Animais , Cães , Genótipo , Humanos , Leishmania/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/veterinária , Leishmaniose Mucocutânea/imunologia , Leishmaniose Mucocutânea/veterinária , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/veterinária , Fenótipo
6.
Biomed Res Int ; 2021: 8845826, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34095312

RESUMO

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania (L.) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN-γ and TNF-α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection.


Assuntos
Leishmania donovani/genética , Leishmaniose Visceral/terapia , Células Th1/imunologia , Imunidade Adaptativa/imunologia , Animais , Antígenos de Protozoários/imunologia , Quimera , Cricetinae , Citocinas/metabolismo , Feminino , Fatores Imunológicos/metabolismo , Imunoterapia/métodos , Leishmania donovani/imunologia , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Linfócitos/metabolismo , Fosfopiruvato Hidratase/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Triose-Fosfato Isomerase/imunologia , Vacinas/farmacologia
7.
Mol Immunol ; 137: 20-27, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34182228

RESUMO

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.


Assuntos
Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Leucócitos Mononucleares/imunologia , Receptores de Interleucina-10/imunologia , Células Th1/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Células Cultivadas , Cães , Feminino , Interferon gama/imunologia , Leucócitos Mononucleares/parasitologia , Masculino , Células Th1/parasitologia
8.
Exp Parasitol ; 226-227: 108122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115995

RESUMO

Visceral leishmaniasis (VL) is an infectious disease caused by an intracellular protozoan belonging to Leishmania species. Interleukin (IL)-22 plays an important role in inflammatory response, chemotaxis, regulation of cellular proliferation and tissue repair. Considering the role of IL-22 in control of leishmaniasis and the effect of its single nucleotide polymorphisms (SNPs) on respective function and production, this study aimed to investigate the probable association of IL-22 SNPs with VL. The study was carried out on 110 patients with VL, 102 healthy individuals with negative leishmanin skin test (negative control group (NCG)), and 144 healthy individuals with positive leishmanin skin test (LSTPG). Four SNPs in IL-22 including rs2227501, rs2227503, rs2227513 and rs1026786 were analyzed by polymerase chain reaction-restricted fragment length polymorphism (PCR- RFLP) in the study groups. The frequency of A allele and AA genotype at rs1026786 were significantly higher in the LSTPG group than in the patients (P = 0.013 and P = 0.001, respectively). Conversely, the frequency of AG genotype was significantly higher in the patients and the NCG than in the LSTPG group (P = 0.0001 and P = 0.002, respectively). For rs2227503, the frequency of AG genotype was significantly higher in the LSTPG group than in the NCG (P = 0.025). The haplotype TGAA frequency was significantly higher in the NCG, compared to patients and LSTPG group (P = 0.004 and P = 0.023, respectively). The frequencies of haplotypes TAAG and TGAG were significantly higher in the patients than in the LSTPG group (P = 0.046 and P = 0.014, respectively). The TAAA/TAAG frequency was significantly higher in the patients than in the LSTPG group (P = 0.013). Inheritance of rs1026786 A allele and AA genotype of IL-22 could be a possible protective factor against VL, whereas the inheritance of the haplotypes TAAG and TGAG may predispose Iranian population to the disease.


Assuntos
Predisposição Genética para Doença , Interleucinas/genética , Leishmaniose Visceral/imunologia , Alelos , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único
9.
Infect Immun ; 89(7): e0000921, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33875473

RESUMO

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.


Assuntos
Interações Hospedeiro-Parasita , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Monócitos/imunologia , Monócitos/parasitologia , Fagossomos/imunologia , Fagossomos/parasitologia , Glicoesfingolipídeos/metabolismo , Interações Hospedeiro-Parasita/imunologia , Leishmania donovani/metabolismo , Leishmania donovani/patogenicidade , Monócitos/metabolismo , NADPH Oxidases/metabolismo , Virulência , Fatores de Virulência
10.
Infect Immun ; 89(7): e0076420, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33820818

RESUMO

We showed previously that antioxidant enzyme heme oxygenase 1 (HO-1) is critical for Leishmania survival in visceral leishmaniasis. HO-1 inhibits host oxidative burst and inflammatory cytokine production, leading to parasite persistence. In the present study, screening of reported HO-1 transcription factors revealed that infection upregulated (4.1-fold compared to control [P < 0.001]) nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2). Silencing of NRF2 reduced both HO-1 expression and parasite survival. Investigation revealed that infection-induced transient reactive oxygen species (ROS) production dissociated NRF2 from its inhibitor KEAP1 and enabled phosphorylation-dependent nuclear translocation. Both NRF2 and HO-1 silencing in infection increased production of proinflammatory cytokines. But the level was greater in NRF2-silenced cells than in HO-1-silenced ones, suggesting the presence of other targets of NRF2. Another stress responsive transcription factor ATF3 is also induced (4.6-fold compared to control [P < 0.001]) by NRF2 during infection. Silencing of ATF3 reduced parasite survival (59.3% decrease compared to control [P < 0.001]) and increased proinflammatory cytokines. Infection-induced ATF3 recruited HDAC1 into the promoter sites of tumor necrosis factor alpha (TNF-α) and interleukin 12b (IL-12b) genes. Resulting deacetylated histones prevented NF-κB promoter binding, thereby reducing transcription of inflammatory cytokines. Administering the NRF2 inhibitor trigonelline hydrochloride to infected BALB/c mice resulted in reduced HO-1 and ATF3 expression, decreased spleen and liver parasite burdens, and increased proinflammatory cytokine levels. These results suggest that Leishmania upregulates NRF2 to activate both HO-1 and ATF3 for disease progression.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Heme Oxigenase-1/metabolismo , Interações Hospedeiro-Patógeno , Leishmania donovani/fisiologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/microbiologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mediadores da Inflamação/metabolismo , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo
11.
Front Immunol ; 12: 632512, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815385

RESUMO

Visceral leishmaniasis (VL) is a fatal parasitic disease if untreated. Treatment options of VL diminish due to emerging drug resistance. Although the principal host cells for the multiplication of Leishmania are macrophages, neutrophils are the first cells infected with the parasites rapidly after parasite inoculation. Leishmania can survive in neutrophils despite the potent antimicrobial effector functions of neutrophils that can eliminate the parasites. Recently, the growing field of immunometabolism provided strong evidence for the therapeutic potential in targeting metabolic processes as a means of controlling immune effector functions. Therefore, the understanding of the immunometabolic profile of neutrophils during Leishmania infection could provide new promising targets for host-directed therapies against VL. To our knowledge, this is the first study addressing the bioenergetics profile of L. donovani-infected primary human neutrophils. Transcriptome analysis of L. donovani-infected neutrophils revealed an early significant upregulation of several glycolytic enzymes. Extracellular flux analysis showed that glycolysis and glycolytic capacity were upregulated in L. donovani-infected neutrophils at 6 h post infection. An increased glucose uptake and accumulation of glycolytic end products were further signs for an elevated glycolytic metabolism in L. donovani-infected neutrophils. At the same time point, oxidative phosphorylation provided NADPH for the oxidative burst but did not contribute to ATP production. Inhibition of glycolysis with 2-DG significantly reduced the survival of L. donovani promastigotes in neutrophils and in culture. However, this reduction was due to a direct antileishmanial effect of 2-DG and not a consequence of enhanced antileishmanial activity of neutrophils. To further address the impact of glucose metabolism during the first days of infection in vivo, we treated C57BL/6 mice with 2-DG prior to infection with L. donovani and assessed the parasite load one day and seven days post infection. Our results show, that seven days post-infection the parasite load of 2-DG treated animals was significantly higher than in mock treated animals. This data indicates that glycolysis serves as major energy source for antimicrobial effector functions against L. donovani. Inhibition of glycolysis abrogates important neutrophil effector functions that are necessary the initial control of Leishmania infection.


Assuntos
Glucose/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Neutrófilos/imunologia , Animais , Células Cultivadas , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Fosforilação Oxidativa , Carga Parasitária , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória
12.
Med Microbiol Immunol ; 210(2-3): 133-147, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33870453

RESUMO

Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.


Assuntos
Acarbose/farmacologia , Acarbose/uso terapêutico , Imunidade , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Reposicionamento de Medicamentos , Feminino , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Carga Parasitária , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Resultado do Tratamento
13.
Parasitol Res ; 120(5): 1771-1780, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33792813

RESUMO

Leishmaniasis is a vector-borne parasitic disease caused by protozoa of the genus Leishmania. Twenty different species are known to cause disease in humans with varying degrees of pathology. These diseases are transmitted throughout the geographic range of phlebotomine sandflies, found between the latitudes 50°N and 40°S. This study explores antibody dependent enhancement (ADE) as the cause of disease exacerbation in heterologous exposure of L. major primed mice to L. infantum challenge. BALB/c mice received serum from L. major infected or naive mice. All mice were challenged with L. infantum and tissue parasite burdens were recorded. Animals that received anti-L. major serum exhibited significantly higher parasite burdens. Surprisingly, these parasite burdens were higher than those of mice infected with L. major and challenged with L. infantum. In vitro phagocytosis assays were carried out to measure parasite uptake in the presence of naive vs. anti-L. major serum. J774A.1 murine monocytes were cultured with either L. major or L. infantum in the presence of anti-L. major serum, naive serum, or no serum. Significantly higher rates of L. major uptake by J774A.1 cells occurred in the presence of anti-L. major serum, but no measurable increase of L. infantum phagocytosis was seen. Our results suggest that increased disease severity observed in vivo in mice previously exposed to L. major and challenged with L infantum is not a result of extrinsic ADE. We speculate that intrinsic ADE, due to biased memory T cell responses caused by Fcγ signaling, could account for disease exacerbation seen in the animal model.


Assuntos
Leishmania infantum/imunologia , Leishmania major/imunologia , Leishmaniose/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Linhagem Celular , Modelos Animais de Doenças , Imunização Passiva , Memória Imunológica , Leishmaniose/imunologia , Leishmaniose Cutânea/complicações , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Psychodidae , Linfócitos T/imunologia
14.
Acta Trop ; 220: 105922, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33878308

RESUMO

Visceral Leishmaniasis is a major neglected tropical disease with increasing incidences of drug resistance. This has led to the search for a suitable drug target for chemotherapeutic intervention. Potassium channels are a family of membrane proteins which play a vital role in homeostasis and any perturbation in them alters cell survival which makes them an attractive target. To characterize a calcium-activated potassium channel from Leishmania donovani (LdKCa), a putative ion-channel like protein which showed sequence similarity with other Trypanosoma cruzi putative potassium channels was selected. It was cloned and expressed with a histidine tag. MALDI confirmed that it is a potassium channel. Homology model of LdKCa was generated by I-TASSER. It is a transmembrane protein localized in the plasma membrane as predicted by DeepLoc tool. In silico analyses of the protein showed that it is a small conductance calcium activated potassium channel. Point mutation in conserved signature domain 'TXGYGD' affects the protein function as predicted by heat map analysis. The LdKCa model predicted amino acids S207, T208 and M236 as ligand-binding sites. The sequence HSLRGRSARVIQLAWRLRKARKVGPHAPSLKQKVYTLVLSWLLT was the highest scoring B-cell epitope. The highest scoring T-cell epitope was RLYSVIVYL. This study may provide new insights into antigenicity features of leishmanial calcium-activated potassium channels.


Assuntos
Leishmaniose Visceral/imunologia , Canais de Potássio Cálcio-Ativados/imunologia , Animais , Sítios de Ligação , Cálcio/metabolismo , Cálcio/uso terapêutico , Membrana Celular , Simulação por Computador , Humanos , Leishmania donovani , Canais de Potássio Cálcio-Ativados/genética , Mapas de Interação de Proteínas , Linfócitos T/imunologia
15.
BMC Infect Dis ; 21(1): 369, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33874901

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) is severe and potentially fatal. Brazil is one of the countries with the greatest endemicity for the disease in the world. The reduction of CD4+ T lymphocytes, B cells activation and high levels of inflammatory cytokines (IL-6/IL-8/TNF/IL-1ß), plasma LPS, soluble CD14, anti-Leishmania IgG3 and low leptin levels are involved in the immunopathogenesis of VL, most associated with severe VL. Despite relapses occurring in about 4-5% of patients with VL not associated with HIV infection, the factors underlying relapses are little known. Our aim was to identify clinical, laboratory and immunological parameters that may be associated with recurrences in VL. METHODS: Fifteen VL patients recruited from Hospital Eduardo de Menezes (BH-MG) were grouped into relapsing (R-VL, n = 5) and non-relapsing (NR-VL, n = 10) and evaluated during active disease, immediately after treatment (post-treatment) and 6 months post-treatment (6mpt). Clinical and laboratory data obtained from medical records were correlated with CD4+ and CD8+ T cell counts and anti-Leishmania Igs and IL-6 plasma levels and compared to those parameters of ten healthy controls. RESULTS: During the active phase of VL, despite similarity in the clinical symptoms, the rates of thrombocytopenia, elevated transaminases (AST and ALT) and hyperbilirubinemia were higher in the NR-VL group compared to R-VL (p < 0.05), a profile reversed during the post-treatment phase. All patients had low CD4+ T counts in active phase, however, NR-VL patients had a higher gain of this cell type than R-VL in the post-treatment (p < 0.05). There was a significant reduction in IgG3 levels during the follow-up in the NR-VL group compared to the R-VL, especially at 6mpt (p < 0.05). In addition, IgG3 levels were negatively correlated with CD4+ T counts in the R-VL group (r = - 0.52). Elevated levels of IL-6 were observed in active VL and correlated with clinical markers of severity. CONCLUSIONS: During active phase of VL, the NR-VL patients presented more severe laboratorial abnormalities compared to R-VL, probably because the latter had already received previous treatment. On the other hand, R-VL exhibited greater impairment of immune reconstitution and a high degree of B lymphocyte activation, which must be a factor that favored relapses.


Assuntos
Anticorpos Antiprotozoários/sangue , Linfócitos T CD4-Positivos/citologia , Imunoglobulina G/sangue , Leishmania/imunologia , Leishmaniose Visceral/patologia , Adulto , Anfotericina B/uso terapêutico , Brasil , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ácido Desoxicólico/uso terapêutico , Combinação de Medicamentos , Feminino , Infecções por HIV/complicações , Humanos , Interleucina-6/sangue , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Masculino , Pessoa de Meia-Idade , Recidiva
16.
Front Immunol ; 12: 626110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33763070

RESUMO

TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How Leishmania donovani suppresses these pathways is not well studied. We earlier reported, TLR4 is hypersialylated due to reduced membrane-bound neuraminidase (Neu1) on infected-macrophages. We hypothesized that such enhanced sialoglycoconjugates on host cells may modulate the interactions with siglecs- which are the inhibitory receptors. Here, we examined the impact of such sialylation on overall TLR4 activation both in murine cell line J774A.1 and primary bone marrow derived macrophages (BMDM). Supporting this hypothesis, we demonstrated siglec-E engages hypersialylated TLR4 during infection. Such sialic acids-siglec-E interaction enhanced siglec-E phosphorylation that mediated its strong association with SHP1/SHP2 and also upregulated their phosphorylation in both types of macrophages. Pre-treatment of parasites and host cells with neuraminidase reduced SHP1/SHP2 phosphorylation and triggered TLR4 activation respectively through enhanced nuclear translocation of p-65. Moreover, a reciprocal interplay between Neu1 and siglec-E differentially regulates MyD88- and TRIF-pathways through sialic acids on TLR4 as their common substrate during infection. Correspondingly, Neu1 overexpression enhanced MyD88-signaling while still suppressing TRIF-activation. However, silencing siglec-E specifically activated TRIF-signaling. Pro-inflammatory cytokines corresponding to MyD88 and TRIF pathways were also upregulated respectively. Additionally, Neu1 overexpression or siglec-E silencing prevented TLR4 ubiquitination and subsequent degradation by Triad3A. Neu1-overexpression and siglec-E-silencing together followed by infection activated both MyD88 and TRIF-signaling through their enhanced TLR4-association. This elevated the MyD88-specific cytokines and TRIF-mediated IRF3 and IFN-ß genes, thus upregulating the pro-inflammatory cytokines and nitric oxide levels and reduced anti-inflammatory cytokines. All these significantly inhibited parasite survival in macrophages thus demonstrating a previously unidentified dualistic regulation of TLR4signaling pathways activation through sialic acids by interplay of Neu1 and siglec-E during Leishmania infection.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Leishmania donovani/patogenicidade , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Fator 88 de Diferenciação Mieloide/metabolismo , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Imunidade Inata , Mediadores da Inflamação/metabolismo , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Mesocricetus , Camundongos Endogâmicos BALB C , Neuraminidase/genética , Óxido Nítrico/metabolismo , Fosforilação , Transdução de Sinais
17.
Vet Immunol Immunopathol ; 233: 110198, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33548792

RESUMO

Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum, from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (p = 0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (p = 0.0004) and higher IHC scores of IL-1ß (p = 0.0275) and IL-4 (p = 0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions.


Assuntos
Citocinas/imunologia , Doenças do Cão/imunologia , Leishmania infantum , Leishmaniose Visceral/veterinária , Linfonodos/parasitologia , Animais , Brasil/epidemiologia , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Doenças do Cão/patologia , Cães , Feminino , Leishmania infantum/imunologia , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Linfonodos/patologia , Masculino , Carga Parasitária
18.
PLoS Negl Trop Dis ; 15(2): e0009137, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33617528

RESUMO

BACKGROUND: Reports have shown correlations between the immune response to vector saliva and Leishmaniasis outcome. We followed dogs in an endemic area for two years characterizing resistance or susceptibility to canine visceral leishmaniasis (CVL) according to Leishmania infantum diagnosis and clinical development criteria. Then, we aimed to identify a biosignature based on parasite load, serum biological mediators' interactions, and vector exposure intensity associated with CVL resistance and susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: A prospective two-year study was conducted in an area endemic for CVL. Dogs were evaluated at 6-month intervals to determine infection, clinical manifestations, immune profile, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. After two years, 78% of the dogs were infected with L. infantum (53% susceptible and 47% resistant to CVL). Susceptible dogs presented higher splenic parasite load as well as persistence of the parasite during the follow-up, compared to resistant ones. Susceptible dogs also displayed a higher number of correlations among the investigated biological mediators, before and after infection diagnosis. At baseline, anti-saliva antibodies, indicative of exposure to the vector, were detected in 62% of the dogs, reaching 100% in one year. Higher sandfly exposure increased the risk of susceptibility to CVL by 1.6 times (CI: 1.11-2.41). We identified a discriminatory biosignature between the resistant and susceptible dogs assessing splenic parasite load, interaction of biological mediators, PGE2 serum levels and intensity of exposure to sandfly. All these parameters were elevated in susceptible dogs compared to resistant animals. CONCLUSIONS/SIGNIFICANCE: The biosignature identified in our study reinforces the idea that CVL is a complex multifactorial disease that is affected by a set of factors which are correlated and, for a better understanding of CVL, should not be evaluated in an isolated way.


Assuntos
Suscetibilidade a Doenças/veterinária , Doenças do Cão/parasitologia , Leishmaniose Visceral/veterinária , Psychodidae , Animais , Mordeduras e Picadas/veterinária , Brasil , Dinoprostona/sangue , Suscetibilidade a Doenças/parasitologia , Doenças do Cão/imunologia , Cães , Feminino , Insetos Vetores , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/transmissão , Masculino , Carga Parasitária/veterinária , Estudos Prospectivos , Saliva/imunologia , Baço/parasitologia
19.
PLoS Negl Trop Dis ; 15(2): e0009126, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33524030

RESUMO

The increasing use of immunosuppressants in areas where visceral leishmaniasis (VL) is endemic has increased the number of people susceptible to developing more severe forms of the disease. Few studies have examined the quality of the immune response in immunosuppressed patients or experimental animals with VL. The present work characterises the parasite load developed in, and immune response to, Leishmania infantum-induced VL in C57BL/6 mice that, prior to and during infection, received immunosuppressant treatment with methylprednisolone (MPDN), anti-tumour necrosis factor (anti-TNF) antibodies, or methotrexate (MTX). The latter two treatments induced a significant reduction in the number of CD4+ T lymphocytes over the infection period. The anti-TNF treatment was also associated with a higher parasite load in the liver and a lower parasite load in the spleen. This, plus a possibly treatment-induced reduction in the number of cytokine-producing Th1 cells in the spleen, indicates the development of more severe VL. Interestingly, the MPDN and (especially) MTX treatments provoked a greater presence of soluble Leishmania antigen-specific multi-cytokine-producing T cells in the spleen and a lower liver parasite load than in control animals. These results highlight the need to better understand how immunosuppressant treatments might influence the severity of VL in human patients.


Assuntos
Imunidade , Imunossupressores , Leishmaniose Visceral/imunologia , Carga Parasitária , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos , Citocinas/imunologia , Feminino , Humanos , Imunidade Celular , Imunoglobulina G , Leishmania infantum/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Linfócitos T , Inibidores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/imunologia
20.
Cell Immunol ; 361: 104272, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33445051

RESUMO

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.


Assuntos
Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/tratamento farmacológico , Animais , Antígenos de Protozoários/imunologia , Citocinas/imunologia , Imunidade Celular/imunologia , Imunização/métodos , Leishmania donovani/imunologia , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Vacinação/métodos
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