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1.
Int Immunopharmacol ; 110: 108969, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35738089

RESUMO

The protozoan parasite Leishmania donovani, residing and replicating within the cells of the monocyte-macrophage (mono-mac) lineage, causes visceral leishmaniasis (VL) in humans. While, Leishmania infantum, is the main causative agent for zoonotic VL, where dogs are the main reservoirs of the disease. The chemotherapy is a serious problem because of restricted repertoire of drugs, drug-resistant parasites, drug-toxicity and the requirement for parenteral administration, which is a problem in resource-starved countries. Moreover, immunocompromised individuals, particularly HIV-1 infected are at higher risk of VL due to impairment in T-helper cell and regulatory cell responses. Furthermore, HIV-VL co-infected patients report poor response to conventional chemotherapy. Recent efforts are therefore directed towards devising both prophylactic and therapeutic immunomodulation. As far as prophylaxis is concerned, although canine vaccines for the disease caused by Leishmania infantum or Leishmania chagasi are available, no vaccine is available for use in humans till date. Therefore, anti-leishmanial immunotherapy triggering or manipulating the host's immune response is gaining momentum during the last two decades. Immunomodulators comprised of small molecules, anti-leishmanial peptides, complex ligands for host receptors, cytokines or their agonists and antibodies have been given trials both in experimental models and in humans. However, the success of immunotherapy in humans remains a far-off target. We, therefore, propose that devising a successful immunotherapy is an act of balancing enhanced beneficial Leishmania-specific responses and deleterious immune activation/hyperinflammation just as the swings in a trapeze.


Assuntos
Imunoterapia/métodos , Leishmania donovani , Leishmania infantum , Vacinas contra Leishmaniose , Leishmaniose Visceral/terapia , Animais , Reservatórios de Doenças , Cães , Humanos , Hospedeiro Imunocomprometido , Fatores Imunológicos/uso terapêutico , Leishmania donovani/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/prevenção & controle , Leishmaniose Visceral/transmissão , Células Precursoras de Monócitos e Macrófagos/parasitologia
2.
Exp Parasitol ; 239: 108286, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35660529

RESUMO

Leishmania donovani, an obligate intracellular parasite, the causative agent of visceral leishmaniasis is known to subvert the host immune system for its own survival. Although the precise mechanism is still unknown, emerging evidences indicate that L. donovani efficiently suppress MHC I mediated antigen presentation, rendering inadequate CD8+T cell activation and weakening host defense against parasite. The role of transcription factor EB (TFEB) was recognized in modulating antigen presentation besides its role in lysosomal biogenesis and function. Here, we investigated the regulatory role of TFEB in the modulation of presentation of Leishmania antigen in host tissue. Our results showed an increased expression of TFEB after Leishmania infection both in vitro and in vivo and there was a decrease in the expression of Th-1 cytokine IFNγ along with MHC class I and CD8+T cells indicating attenuation of cell mediated immunity and possibly MHC I restricted antigen presentation. Silencing of TFEB resulted in increased expression of IFNγ and MHC I along with increased CD8+T cells population without any significant change in CD4+T cell number. We also observed a decreased parasite burden in TFEB silenced condition which indicates enhanced parasite clearance by alteration of immunological response possibly through induction of presentation of Leishmania antigen through MHC I. The present study explains the role of TFEB silencing in parasite clearance through regulating the antigen presentation of Leishmania antigen thereby promises to formulate a potential therapeutic strategy against visceral leishmaniasis.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Animais , Apresentação de Antígeno , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/imunologia , Controle de Doenças Transmissíveis , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição/imunologia
3.
Front Immunol ; 13: 801182, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154115

RESUMO

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-ß). Here, we show that the gene expression of IFN-ß by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2 -/- mice, while the levels in macrophages from myd88-/- mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2 -/- macrophages completely abolished induction of IFN-ß gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2 -/-) or from protein kinase R (PKR) knock-out mice (pkr -/-), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr -/- macrophages but was fully restored by the addition of exogenous IFN-ß, and parasite burdens were reduced in the spleen of pkr -/- mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development.


Assuntos
Interferon-alfa/metabolismo , Interferon beta/metabolismo , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos Peritoneais/imunologia , Transdução de Sinais/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , eIF-2 Quinase/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Técnicas de Inativação de Genes , Interferon-alfa/genética , Interferon beta/genética , Leishmaniose Visceral/parasitologia , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Receptor 2 Toll-Like/genética , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia , eIF-2 Quinase/genética
5.
Mol Immunol ; 141: 33-42, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34798496

RESUMO

Visceral leishmaniasis (VL) is a chronic tropical disease responsible for devastating epidemics worldwide. Though current treatment relies on drugs, the emergence of resistance, toxic side-effects, and strenuous administration has led to an ineffective remedy. Hence, vaccination remains an alternative and desirable approach for VL control. Though extensive research on anti-leishmanial vaccine candidates has been carried out in past decades, presence of an effective molecule is still missing. In the present study, we have evaluated the immunogenicity and prophylactic potential of a recombinant T-complex protein-1 gamma subunit of L. donovani (rLdTCP1γ), against VL in hamster model. The antigen exhibited in vitro stimulation of lymphoproliferative and NO response in miltefosine and amphotericin B treated hamsters depicting its immunotherapeutic/immunogenic nature. Immunization with rLdTCP1γ revealed a strong protective response against experimental VL as indicated by reduced parasite load in the spleen of immunized group compared to infected control. The immunized animals gained body weight and exhibited significant reduction in the spleen and liver weight as compared to infected controls on days 60, 90, 120 post-challenge. A substantial augmentation of cell-mediated immune response as depicted by an increased lymphocyte proliferation, nitric oxide production, DTH responses and increased levels of IgG2 was observed in rLdTCP1γ immunized hamsters. The Th1 stimulatory potential, imparted by the antigen, was found to be intensified in the presence of adjuvant Bacillus Calmette-Guérin (BCG). The efficacy was further assisted by an upregulated mRNA transcript of Th1 induced cytokines (IL-12, IFN-γ and TNFα) and downregulation of IL-4 and IL-10. The results are thus suggestive of rLdTCP1γ having the potential of a strong vaccine candidate against VL.


Assuntos
Antígenos de Protozoários/imunologia , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Proteínas de Protozoários/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular , Cricetinae , Citocinas/imunologia , Imunização/métodos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Células Th1/imunologia , Vacinação/métodos
6.
Front Immunol ; 12: 747049, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34733282

RESUMO

Although macrophages are considered for host cells for the multiplication of Leishmania, recent studies indicate the important role of neutrophil granulocytes as host cells for these intracellular parasites. Neutrophils have been shown to be massively and rapidly recruited to the site of Leishmania infection where they represent the first cells to encounter the parasites. Exposure to ATP and UTP have been shown to enhance anti-Leishmania activity of macrophages and intralesional injection of UTP led to strongly reduced parasite load in vivo. Since the in vivo anti-leishmanial effect of extracellular UTP correlated with enhanced neutrophil recruitment and enhanced ROS production at the site of Leishmania infection we hypothesized that exposure to extracellular nucleotides can directly enhance the killing of Leishmania by neutrophils. Since purinergic signaling is an essential mechanism of neutrophil activation the aim of the present study was to assess whether purinergic exposure results in the activation of anti-leishmanial neutrophil functions and, therefore, represent an essential component of enhanced anti-leishmanial defense in leishmaniasis. We could show that exposure to ATP and UTP led to activation and enhanced CD11b expression of primary human neutrophils in vitro. Leishmania-induced ROS production was strongly enhanced by extracellular ATP and UTP. Importantly, exposure to ATP and UTP resulted in enhanced killing of Leishmania donovani by neutrophils. In addition, ATP strongly enhanced the secretion of IL-8 and IL-1ß by Leishmania-exposed neutrophils. Our results suggest that signaling via the P2 receptor and phosphorylation of Erk1/2, Akt and p38 are involved in the purinergic enhancement of anti-leishmanial functions of neutrophils.


Assuntos
Trifosfato de Adenosina/imunologia , Leishmania donovani/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Uridina Trifosfato/imunologia , Células Cultivadas , Humanos , Leishmaniose Visceral/imunologia
7.
Immunobiology ; 226(6): 152148, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34773853

RESUMO

Visceral leishmaniasis (VL) or Kala-azar is a vector borne protozoan infection caused by Leishmania donovani in the Indian subcontinent mainly India, Nepal and Bangladesh. It is a major public health problem in these countries mostly affecting the socio-economically poor population. Leishmaniasis ranks the third most important disease after malaria and filariasis but is still considered as one of the neglected tropical diseases of the world. For development of better therapeutic agents and effective vaccine against VL, there is a need to understand host immunological changes that play a vital role during course of infection. Therefore, we investigated the role of Th17 pathway in Balb/c mice during Leishmania donovani infection and treatment with amphotericin B. Mice were divided in four groups i.e. Control, Infected, Uninfected treated and Infected treated. The cytokine levels were estimated in the spleen of Balb/c mice on days 1, 3, 7, 14, 17, 21, 28, 35, 45 and 60 post infection and during course of treatment. The mRNA levels of the Th17 pathway during active Leishmania donovani infection and after treatment were determined by real time polymerase chain reaction (RT-PCR) and protein levels by flow cytometry and ELISA. Results of our study revealed that active infection was associated with low levels of Th17 cytokines IL-17, IL-22 and IL-23 and elevated levels of IL-6, IL-1ß and TGF-ß. Amphotericin B treatment restored production of pro-inflammatory cytokines IL-17 and IL-22. The levels of transcription factor RORγt were found to correlate with the levels of IL-17 during infection and also after chemotherapy whereas STAT3 levels were elevated during infection and vice versa after treatment. The findings of this study suggest that Th17 cytokines IL-17 and IL-22 are associated with protection against VL infection and development of any interventions or chemotherapeutic agents targeting Th17 pathway could be an important approach for VL treatment.


Assuntos
Interações Hospedeiro-Parasita/imunologia , Imunomodulação , Leishmania donovani , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Animais , Biomarcadores , Cricetinae , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Leishmania donovani/imunologia , Leishmaniose Visceral/parasitologia , Ativação Linfocitária/imunologia , Camundongos , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Baço/patologia
8.
Front Immunol ; 12: 748325, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34712235

RESUMO

Leishmaniasis is endemic to the tropical and subtropical regions of the world and is transmitted by the bite of an infected sand fly. The multifaceted interactions between Leishmania, the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development. Leishmania parasites establish a chronic infection by subversion and attenuation of the microbicidal functions of phagocytic innate immune cells such as neutrophils, macrophages and dendritic cells (DCs). Other innate cells such as inflammatory monocytes, mast cells and NK cells, also contribute to resistance and/or susceptibility to Leishmania infection. In addition to the cytokine/chemokine signals from the innate immune cells, recent studies identified the subtle shifts in the metabolic pathways of the innate cells that activate distinct immune signal cascades. The nexus between metabolic pathways, epigenetic reprogramming and the immune signaling cascades that drive the divergent innate immune responses, remains to be fully understood in Leishmania pathogenesis. Further, development of safe and efficacious vaccines against Leishmaniasis requires a broader understanding of the early interactions between the parasites and innate immune cells. In this review we focus on the current understanding of the specific role of innate immune cells, the metabolomic and epigenetic reprogramming and immune regulation that occurs during visceral leishmaniasis, and the strategies used by the parasite to evade and modulate host immunity. We highlight how such pathways could be exploited in the development of safe and efficacious Leishmania vaccines.


Assuntos
Imunidade Inata , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Animais , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Humanos , Evasão da Resposta Imune , Imunogenicidade da Vacina , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Mastócitos/imunologia , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Células T Matadoras Naturais/imunologia , Neutrófilos/imunologia
9.
PLoS Negl Trop Dis ; 15(8): e0009627, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34403413

RESUMO

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.


Assuntos
Antígenos de Protozoários/imunologia , Medula Óssea/parasitologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Fígado/parasitologia , Baço/parasitologia , Imunidade Adaptativa , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/sangue , Medula Óssea/metabolismo , Feminino , Imunização , Interferon gama/metabolismo , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Baço/metabolismo
10.
PLoS Negl Trop Dis ; 15(8): e0009632, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34351903

RESUMO

BACKGROUND: Visceral leishmaniasis is a disease caused by disseminated Leishmania donovani infection which affects almost half a million people annually. Most of the patients are reported from the Indian sub-continent, Eastern Africa and Brazil. In this study, we aimed to determine the levels of antibodies and cytokines in visceral leishmaniasis patients and to examine associations of parasitemia with the clinical states of patients. A prospective study was carried out, enrolling a total of 48 active VL patients who were evaluated before, during different time points and, three months after treatment. Serum cytokine concentrations, antibody levels, parasitemia, laboratory (hematologic and biochemical) measurements, and clinical parameters were assessed. RESULTS: Counts of WBC and platelets, and measurements of hemoglobin (Hb) increased during treatment (P ≤ 0.05). Elevated levels of circulating IL-10, IFN-γ, and TGF-ß1 were measured before treatment. The observed increase in serum IL-10 remarkably declined within 7 days after the start of treatment. Anti-leishmanial antibody index (AI) was high in all VL patients irrespective of spleen aspirate parasite grade before treatment and at different times during treatment. However, a significant (P ≤ 0.05) decrease of AI was observed 120 days post-treatment. IL-2 serum levels were below the detection limit at all sampling points. CONCLUSIONS: The present results suggest that IL-10, IFN-γ, and TGF-ß1 can be used as markers of active visceral leishmaniasis. In addition, measuring circulating cytokines concentrations, particularly IL-10, in combination with other clinical evaluations, could be used as criteria for the cure. The observation that a high serum concentration of IFN-gamma at baseline was associated with low parasitemia deserves further investigations.


Assuntos
Anticorpos Antiprotozoários/sangue , Interferon gama/sangue , Interleucina-10/sangue , Leishmania donovani/imunologia , Leishmaniose Visceral/sangue , Fator de Crescimento Transformador beta1/sangue , Adolescente , Adulto , Criança , Pré-Escolar , Etiópia , Feminino , Hospitais Gerais , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Leishmania donovani/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Parasitemia/sangue , Estudos Prospectivos , Fator de Crescimento Transformador beta1/imunologia , Adulto Jovem
11.
Commun Biol ; 4(1): 929, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330999

RESUMO

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen-/- immunized hamsters. This study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.


Assuntos
Deleção de Genes , Genes de Protozoários , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Leishmania donovani/genética , Leishmaniose Visceral/imunologia , Proteínas de Protozoários , Vacinas Atenuadas/imunologia
12.
Cytokine ; 146: 155623, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34144446

RESUMO

Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.


Assuntos
Citocinas/metabolismo , Eugenol/uso terapêutico , Imunidade , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Óxido Nítrico/biossíntese , Fosforilcolina/análogos & derivados , Administração Oral , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/biossíntese , Interações Medicamentosas , Quimioterapia Combinada , Eugenol/administração & dosagem , Eugenol/farmacologia , Feminino , Imunidade/efeitos dos fármacos , Concentração Inibidora 50 , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Leishmania donovani/ultraestrutura , Leishmaniose Visceral/parasitologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/parasitologia , Macrófagos/ultraestrutura , Masculino , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , Parasitos/efeitos dos fármacos , Parasitos/crescimento & desenvolvimento , Parasitos/imunologia , Parasitos/ultraestrutura , Fosforilação/efeitos dos fármacos , Fosforilcolina/administração & dosagem , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
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
14.
Infect Genet Evol ; 93: 104947, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34052416

RESUMO

Tropical and subtropical areas of the world are affected by leishmaniasis, which is caused by Leishmania spp. It has been categorized as an NTD (neglected tropical disease) because of its negligence. The sand fly of genus Phlebotomus acts as the vector for the transmission of the promastigote form of this protozoan parasite to the mammalian host where it converts to amastigote form in the macrophages. Visceral form of leishmaniasis (VL) is a deadly infection in the endothelial system of the human and other mammals. Only a few chemotherapeutic agents are available for the treatment of this infectious disease whereas no vaccine is available for the control of leishmanial infection. Therefore in the current study, we have tested the effects of gardiquimod (a TLR agonist) as an adjuvant in combination with the formalin-killed antigen of L. donovani as a vaccine. The mice were vaccinated thrice at an interval of 2 weeks and challenged with L. donovani promastigotes after 2 weeks of the last vaccination. We assessed the parasite load, delayed-type hypersensitivity (DTH) responses, humoral and cell-mediated immune response in BALB/c mice before and after challenge infection with L. donovani. Immunized mice were found to have the least parasite load, high DTH response, elevated levels of Th1 cytokines, IgG2a, and nitric oxide than non-immunized and infected control mice. The efficacy of the vaccine was boosted with the use of adjuvant gardiquimod that depicts its potential as an adjuvant in this study. Our study is reporting the adjuvant effects of gardiquimod for the first time. Further studies using other Leishmania species can be performed to signify its role.


Assuntos
Adjuvantes Imunológicos/farmacologia , Aminoquinolinas/farmacologia , Imidazóis/farmacologia , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Receptores Toll-Like/antagonistas & inibidores , Animais , Feminino , Leishmaniose Visceral/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C
15.
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
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.
Parasit Vectors ; 14(1): 111, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597000

RESUMO

BACKGROUND: Asymptomatic Leishmania infections outnumber clinical infections on the Indian subcontinent (ISC), where disease reservoirs are anthroponotic. Diagnostics which detect active asymptomatic infection, which are suitable for monitoring and surveillance, may be of benefit to the visceral leishmaniasis (VL) elimination campaign on the ISC. METHODS: Quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), and the direct agglutination test (DAT) were carried out on blood samples, and the Leishmania antigen ELISA was carried out on urine samples collected from 720 household and neighbouring contacts of 276 VL and post-kala-azar dermal leishmaniasis (PKDL) index cases, with no symptoms or history of VL or PKDL, in endemic regions of Bangladesh between September 2016 and March 2018. RESULTS: Of the 720 contacts of index cases, asymptomatic infection was detected in 69 (9.6%) participants by a combination of qPCR (1.0%), LAMP (2.1%), DAT (3.9%), and Leishmania antigen ELISA (3.3%). Only one (0.1%) participant was detected positive by all four diagnostic tests. Poor agreement between tests was calculated using Cohen's kappa (κ) statistics; however, the Leishmania antigen ELISA and DAT in combination captured all participants as positive by more than one test. We find evidence for a moderately strong association between the index case being a PKDL case (OR 1.94, p = 0.009), specifically macular PKDL (OR 2.12, p = 0.004), and being positive for at least one of the four tests. CONCLUSIONS: Leishmania antigen ELISA on urine detects active asymptomatic infection, requires a non-invasive sample, and therefore may be of benefit for monitoring transmission and surveillance in an elimination setting in combination with serology. Development of an antigen detection test in a rapid diagnostic test (RDT) format would be of benefit to the elimination campaign.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/sangue , Infecções Assintomáticas/epidemiologia , Leishmania donovani/isolamento & purificação , Leishmaniose Cutânea/sangue , Leishmaniose Visceral/sangue , Adolescente , Adulto , Idoso , Bangladesh/epidemiologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Leishmania donovani/genética , Leishmania donovani/imunologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Adulto Jovem
18.
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
19.
Cytokine ; 137: 155319, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002744

RESUMO

Leishmania donovani, a protozoan parasite, inflicts the disease Visceral leishmaniasis (VL) Worldwide. The only orally bioavailable drug miltefosine is toxic, whereas liposomal amphotericin B (AmpB) is expensive. Lupeol, a triterpenoid from Sterculia villosa bark, was exhibited immunomodulatory and anti-leishmanial activity in experimental VL. Herein, we evaluated synergism between sub-optimum dose of AmpB and lupeol in anti-leishmanial and immunomodulatory effects in L. donovani-infected BALB/c mice. We observed that a combination of sub-optimum dose of lupeol and AmpB significantly reduced the hepatic and splenic parasitic burden accompanied by enhanced nitric oxide production, robust induction of Th1 cytokines (IL-12 and IFN-γ) but suppressed Th2 cytokine (IL-10 and TGF- ß) production. The treatment with the lupeol-AmpB combination enhanced p38mitogen-activated protein kinase (p38MAPK), but reduced extracellular signal-related kinase (ERK-1/2), phosphorylation and up-regulated pro-inflammatory response. The present work thus indicates a lupeol-AmpB-mediated immunotherapeutic approach for eliminating the parasite-induced immunosuppression.


Assuntos
Anfotericina B/farmacologia , Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Triterpenos Pentacíclicos/farmacologia , Animais , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Sinergismo Farmacológico , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Immunoblotting , Imunomodulação/efeitos dos fármacos , Imunomodulação/imunologia , Leishmania donovani/imunologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Camundongos Endogâmicos BALB C , Nitritos/imunologia , Nitritos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/parasitologia
20.
Cytokine ; 147: 155322, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33127259

RESUMO

In humans, infection with Leishmania manifests into a spectrum of diseases. The manifestation of the diseases depend on the resultant evasion of the parasite to immune responses namely by macrophages, which is an exclusive host of Leishmania. The B cells valiantly mount antibody responses, however, to no avail as the Leishmania parasites occupy the intracellular niches of the macrophages and subvert the immune response. Extensive studies have been documented on the role of cell-mediated immunity (CMI) in protection and counter survival strategies of the parasites leading to downregulation of CMI. The present review attempts to discuss the cytokines in progression or resolution of visceral form of leishmaniasis or kala-azar, predominantly affecting the Indian subcontinent. The components/cytokine(s) responsible for the regulation of the critical balance of T helper cells and their subsets have been discussed in the perspective. Therefore, any strategy involving the treatment of visceral leishmania (VL) needs to consider the balance and regulation of T cell function.


Assuntos
Citocinas/imunologia , Leishmaniose Visceral/imunologia , Animais , Linfócitos B/imunologia , Humanos , Imunidade Celular/imunologia , Leishmania donovani/imunologia , Macrófagos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia
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