RESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions. METHODS: Hamsters were intradermally infected in the footpad with 10(5) promastigotes of L. (V.) braziliensis and 110 days post-infection skin lesions and popliteal lymph nodes were removed for RNA and DNA extraction, both from the same tissue fragment. Gene expression of IFN-É£, IL-10, TGF-ß TNF, IL-4, IL-6, iNOS and arginase were measured using non-infected animal tissue as a calibrator. Parasite load was quantified from DNA extracted from lesions by qPCR targeting Leishmania kDNA and normalized by hamster GAPDH, using a SYBR Green-based absolute quantification methodology. RESULTS: A relative quantification RT-qPCR assay was standardized for the evaluation of mRNA levels from skin and lymph node samples of golden hamsters, with PCR efficiencies ranging from 92.3 to 116.4 %. In uninfected animals, higher basal mRNA levels in lymph nodes were observed for IFN-É£, TGF-ß, TNF and IL-4 (111.4 ± 92.2; 5.6 ± 1.2; 5.3 ± 1.7; and 60.3 ± 26.8, respectively) in comparison to skin. In golden hamsters infected with L. (V.) braziliensis, an increase in the expression of all immunological markers evaluated was observed, ranging from 2.7 ± 0.2 for TGF-ß to 1018.5 ± 809.0 for iNOS in skin lesions, and 2.4 ± 1.6 for TGF-ß to 600.2 ± 666.4 for iNOS in popliteal lymph nodes. Interestingly, significantly higher levels of IFN-É£, TNF and IL-10 mRNA were observed in skin in comparison to lymph nodes, while a lower significant level of arginase mRNA was observed in skin. In parallel, parasite loads were quantified by qPCR from the skin lesions of infected animals, ranging from 27.0 to 6647.0, with a median of 553.4 (416.7-1504.0) parasites/mg skin equivalents, whereas lesion size varied from 0.3 to 3.1 mm. Despite the tendency of larger lesions to present higher parasite load, the correlation observed was not statistically significant. CONCLUSIONS: In this study, we describe for the first time a sensitive, reproducible and cheaper molecular assay to quantify from the same tissue fragment the gene expression of immunological markers and the parasite load in skin lesions, observing a mixed profile of immune response in the hamster model infected by L. (V.) braziliensis.
Assuntos
Leishmania braziliensis , Leishmaniose Cutânea/imunologia , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Cricetinae , Citocinas/genética , Citocinas/metabolismo , DNA de Protozoário/análise , DNA de Protozoário/genética , Feminino , Regulação da Expressão Gênica , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/parasitologia , Mesocricetus , RNA Mensageiro , RNA de Protozoário , Sensibilidade e Especificidade , Pele/parasitologiaRESUMO
BACKGROUND: Previous results have shown that oral and intranasal administration of particulate Leishmania (Leishmania) amazonensis antigens (LaAg) partially protects mice against L. amazonensis infection. However, vaccination studies on species of the subgenus Viannia, the main causative agent of cutaneous and mucosal leishmaniasis in the Americas, have been hampered by the lack of easy-to-handle bio-models that accurately mimic the human disease. Recently, we demonstrated that the golden hamster is an appropriate model for studying the immunopathogenesis of cutaneous leishmaniasis caused by L. (Viannia) braziliensis. Using the golden hamster model, our current study investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection. METHODOLOGY/PRINCIPAL FINDINGS: Golden hamsters vaccinated with either two intranasal (IN) doses of LaAg (10 µg) or two intramuscular doses of LaAg (20 µg) were challenged 2 weeks post-vaccination with L. braziliensis. The results showed that IN immunisation with LaAg significantly reduced lesion growth and parasitic load as well as serum IgG and IgG2 levels. At the experimental endpoint on day 114 post-infection, IN-immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA levels that returned to uninfected skin levels. In contrast to the nasal route, intramuscular (IM) immunisation failed to provide protection. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time that the nasal route of immunisation can induce cross protection against L. braziliensis infection.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania braziliensis/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/prevenção & controle , Administração Intranasal , Animais , Anticorpos Antiprotozoários , Cricetinae , Imunoglobulina G/sangue , Interferon gama/metabolismo , Vacinas contra Leishmaniose/administração & dosagem , Carga Parasitária , Pele/metabolismo , Pele/parasitologiaRESUMO
Estudos anteriores no modelo murino demonstraram a possibilidade deindução de proteção através da imunização por via mucosa, tanto com antígenosbrutos (lisado total de promastigotas de Leishmania amazonensis - LaAg) comoatravés de vacina de DNA (DNA plasmideal com o gene que codifica a proteínaLACK - LACK DNA), contra a leishmaniose cutânea (LC) causada por L.amazonensis, e a leishmaniose visceral causada por L. infantum. No entanto,estudos de vacinação contra as espécies do subgênero Viannia, principaisresponsáveis pela LC nas Américas, são dificultados devido à falta de modelosexperimentais de fácil manuseio que reproduzam a doença humana. Recentementefoi demonstrado pelo nosso grupo que o hamster dourado é um modelo adequadopara o estudo da imunopatogênese da leishmaniose cutânea causada por L. (V.)braziliensis e que o antígeno LaAg administrado por via intranasal induziu proteçãocontra a infecção por L. (V.) braziliensis no modelo. Entretanto, as abordagensimunológicas e moleculares para os estudos no modelo hamster são limitadas pelapouca disponibilidade de insumos comerciais disponíveis. Nesse trabalho,padronizamos um ensaio por RT-qPCR para avaliação de marcadores molecularesem pele de hamsters infectados por L. braziliensis, pela expressão gênica de IFN-gama,TGF-beta, TNF, IL-10, IL-4, IL-6, iNOS e arginase, e investigamos o efeito protetor daimunização intranasal com a vacina gênica LACK DNA, administrada em protocolosprime-boost homólogo (50mig/dose) e heterólogo (LACK DNA-50mig / LaAg-10mig) nainfecção por L. braziliensis...
Previous studies in murine models demonstrated the possibility to induceprotection by mucosal immunization with crude antigens (total lysate of Leishmaniaamazonensis promastigotes - LaAg) as well as through DNA vaccine (plasmid DNAwith the gene encoding LACK protein - LACK DNA) against cutaneous leishmaniasis(CL) caused by L. amazonensis and visceral leishmaniasis caused by L. infantum.However, vaccination studies against Viannia subgenus (the main cause for CL inAmericas) are hampered due to the lack of experimental models which are easilyexecuted to mimic this disease as it occurs in humans. Recently, it was demonstratedby our group that the golden hamster is an appropriate model to studyimmunopathological aspects of cutaneous leishmaniasis caused by L. (V.)braziliensis. Furthermore, it was also demonstrated that LaAg antigen administeredintranasally induces protection against L. (V.) braziliensis infection in the model.However, immunological and molecular approaches for studies in hamster modelsare limited by the low availability of commercial products. In this study, westandardized an assay by RT-qPCR for assessment of molecular markers in the skinof hamsters infected by L. braziliensis, through the relative gene expressionquantification of IFN-gama, TGF-beta, TNF, IL-10, IL-4, IL-6, iNOS and arginase, and theprotective effect of intranasal immunization with LACK DNA vaccine (administered inhomologous prime-boost protocol 50 mcg/dose) and heterologous prime-boost(LACK DNA 50 mcg in first dose/LaAg-10mg in second dose), against L. braziliensisinfection. The results demonstrated that immunization with LACK DNA homologousprime boost did not induce protection against L. braziliensis infection in hamstermodel since there was no significant difference in lesion size, in parasite load, andIgG and IgG 2 anti-Leishmania levels on day 110 after infection, compared with thecontrol groups (PBS and DNA)...
Assuntos
Cricetinae , Vacinas contra Leishmaniose , Leishmania braziliensis/imunologia , Carga Parasitária , Mesocricetus/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Estudos anteriores no modelo murino demonstraram a possibilidade deindução de proteção através da imunização por via mucosa, tanto com antígenosbrutos (lisado total de promastigotas de Leishmania amazonensis - LaAg) comoatravés de vacina de DNA (DNA plasmideal com o gene que codifica a proteínaLACK - LACK DNA), contra a leishmaniose cutânea (LC) causada por L.amazonensis, e a leishmaniose visceral causada por L. infantum. No entanto,estudos de vacinação contra as espécies do subgênero Viannia, principaisresponsáveis pela LC nas Américas, são dificultados devido à falta de modelosexperimentais de fácil manuseio que reproduzam a doença humana. Recentementefoi demonstrado pelo nosso grupo que o hamster dourado é um modelo adequadopara o estudo da imunopatogênese da leishmaniose cutânea causada por L. (V.)braziliensis e que o antígeno LaAg administrado por via intranasal induziu proteçãocontra a infecção por L. (V.) braziliensis no modelo. Entretanto, as abordagensimunológicas e moleculares para os estudos no modelo hamster são limitadas pelapouca disponibilidade de insumos comerciais disponíveis. Nesse trabalho,padronizamos um ensaio por RT-qPCR para avaliação de marcadores molecularesem pele de hamsters infectados por L. braziliensis, pela expressão gênica de IFN-gama,TGF-beta, TNF, IL-10, IL-4, IL-6, iNOS e arginase, e investigamos o efeito protetor daimunização intranasal com a vacina gênica LACK DNA, administrada em protocolosprime-boost homólogo (50mig/dose) e heterólogo (LACK DNA-50mig / LaAg-10mig) nainfecção por L. braziliensis.
Os resultados demonstraram que a imunização comLACK DNA em prime boost homólogo não induziu proteção contra infecção porL. braziliensis no modelo hamster uma vez que não foi observada diferençasignificativa no tamanho da lesão, na carga parasitária, e nos níveis de IgG e IgG 2anti-Leismania, 110 dias após a infecção, comparados aos grupos controles (PBS eDNA). O protocolo prime-boost heterólogo induziu redução no crescimento da lesão,acompanhado pela redução de IgG anti-Leismania. Os animais consideradosprotegidos (lesão < 0,50mm) apresentaram expressão gênica de IFN-gama, TNF, iNOS,IL-10, TGF-beta, arginase na pata similar a uma pele normal, utilizada como calibradorde reação, enquanto que os animais considerados não protegidos apresentaramníveis de expressão de RNAm destes mesmos alvos comparáveis aos dos animaisdo grupo controle infectado. Demonstramos a aplicabilidade do ensaio de RT-qPCRpara avaliação de fatores relacionados à resposta imune no modelo, permitindoaumentar as abordagens para estudos da LC. Os resultados obtidos com o uso doprotocolo prime-boost heterólogo demostraram que esta estratégia pode serpromissora na busca de uma vacina eficaz contra infecção por L. (V.) braziliensis nomodelo hamster. (AU)
Previous studies in murine models demonstrated the possibility to induceprotection by mucosal immunization with crude antigens (total lysate of Leishmaniaamazonensis promastigotes - LaAg) as well as through DNA vaccine (plasmid DNAwith the gene encoding LACK protein - LACK DNA) against cutaneous leishmaniasis(CL) caused by L. amazonensis and visceral leishmaniasis caused by L. infantum.However, vaccination studies against Viannia subgenus (the main cause for CL inAmericas) are hampered due to the lack of experimental models which are easilyexecuted to mimic this disease as it occurs in humans. Recently, it was demonstratedby our group that the golden hamster is an appropriate model to studyimmunopathological aspects of cutaneous leishmaniasis caused by L. (V.)braziliensis. Furthermore, it was also demonstrated that LaAg antigen administeredintranasally induces protection against L. (V.) braziliensis infection in the model.However, immunological and molecular approaches for studies in hamster modelsare limited by the low availability of commercial products. In this study, westandardized an assay by RT-qPCR for assessment of molecular markers in the skinof hamsters infected by L. braziliensis, through the relative gene expressionquantification of IFN-gama, TGF-beta, TNF, IL-10, IL-4, IL-6, iNOS and arginase, and theprotective effect of intranasal immunization with LACK DNA vaccine (administered inhomologous prime-boost protocol 50 mcg/dose) and heterologous prime-boost(LACK DNA 50 mcg in first dose/LaAg-10mg in second dose), against L. braziliensisinfection. The results demonstrated that immunization with LACK DNA homologousprime boost did not induce protection against L. braziliensis infection in hamstermodel since there was no significant difference in lesion size, in parasite load, andIgG and IgG 2 anti-Leishmania levels on day 110 after infection, compared with thecontrol groups (PBS and DNA).
The heterologous prime-boost protocol inducedreduction in lesion growth, accompanied by reduction in IgG anti-Leishmania levels.The animals considered protected (lesion < 0.50mm) showed IFN-gama, TNF, iNOS, IL-10, TGF-beta, and arginase gene expression levels in footpad similar to normal pawskin (used as reaction calibrator) whereas animals considered not protected showedmRNA expression levels of these same targets comparable to those animals in theinfected control group. We demonstrated the applicability of RT-qPCR assay toevaluate factors related to immune response in this model, contributing to developthe approaches to CL studies. The results obtained with the heterologous primeboostimmunization protocol demonstrated that this may be a promising strategy inthe search for an effective vaccine against infection by L. (V.) braziliensis in hamstermodels. (AU)