Avaliação da capacidade estimulatória de células dendríticas em pacientes com hanseníase
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2022-01-29
Autores
Germano, Giovanna Valle [UNESP]
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Universidade Estadual Paulista (Unesp)
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A hanseníase é considerada ainda um problema de saúde pública com mais de 200.000 novos casos detectados em 2019, sendo 27.863 no Brasil. Causada pelo Mycobacterium leprae, patógeno intracelular obrigatório, a hanseníase quando não tratada pode resultar em incapacidades físicas permanentes devido a tropismo do bacilo por células de Schwann dos nervos periféricos. É uma doença espectral com duas formas polares estáveis e três intermediárias instáveis, sendo o polo tuberculoide caracterizado pelo predomínio de resposta imune celular e doença localizada, enquanto o polo virchowiano apresenta elevada resposta humoral e doença disseminada. As células dendríticas (DCs) ativam a resposta imune específica, portanto a expressão de moléculas coestimulatórias e as citocinas liberadas pelas DCs durante a infecção pelo M. leprae determinam o perfil de resposta imune gerado e podem prevenir ou favorecer sua sobrevivência e disseminação. O objetivo deste estudo foi analisar o perfil de ativação de DCs derivadas de pacientes com hanseníase infectadas por M. leprae viável bem como sua capacidade de ativar linfócitos (LFs) autólogos. DCs foram diferenciadas a partir de monócitos de sangue periférico de controles saudáveis (CS), pacientes tuberculoides (6 tuberculoide-tuberculoides e 2 dimorfo-tuberculoides) e virchowianos (5 virchowiano-virchowianos e 5 dimorfo-virchowianos) empregando GM-CSF e IL-4. As DCs foram estimuladas por (i) M. leprae vivo na multiplicidade de 10:1 (bacilos:DC, MLDCs); (ii) coquetel de maturação padrão (CQM) composto por IL-1β, IL-6, TNF, PGE2 (mDCs); ou (iii) permaneceram não estimuladas (iDCs) por 48 horas. Após o estímulo, as DCs foram co-cultivadas com LFs por cinco dias. O estímulo de DCs com M. leprae viável não induziu expressão de moléculas coestimulatórias e levou à produção de baixos níveos de IL-1β, IL-6, TNF e IL-23 tanto em pacientes quanto em controles. DCs de pacientes virchowianos produziram mais IL-1β frente ao M. leprae do que aquelas de pacientes tuberculoides e maiores quantidades de IL-23 e IL-10 comparado aos controles. Independente da forma clínica da hanseníase, DCs de pacientes produziram menos IL-12p70 em resposta ao M. leprae do que DCs de controles saudáveis. Quanto à ativação dos LFs, verificamos que pacientes virchowianos apresentaram menor proliferação e produziram níveis menores de IL-2 após estímulo com M. leprae. A produção de IFN-, TNF, IL-9 e IL-22 nas culturas de MLDCs e LFs foi semelhante à encontrada quando iDCs foram empregadas e menor do que observado em culturas com mDCs. Nos pacientes virchowianos a frequência de LFs IL-4+ foi maior em paralelo com menor número de LFs expressando IFN- e IL-17A nas culturas estimuladas com MLDCs quando comparados com controles saudáveis. Em conjunto, nossos dados sugerem que o M. leprae é um fraco ativador de DCs, o que compromete a capacidade destas células induzirem a ativação de LFs autólogos. Pacientes com hanseníase virchowiana apresentam menor frequência de LFs produtores de citocinas de perfil Th1 e Th17 após estímulo com M. leprae o que pode contribuir para a persistência do bacilo.
Leprosy is still considered a public health problem with more than 200,000 new cases detected in 2019, being 27,863 registered in Brazil. Caused by Mycobacterium leprae, an obligate intracellular pathogen, leprosy can result in permanent physical disabilities when untreated due to the tropism of the bacillus by Schwann cells from peripheral nerves. It is a spectral disease presenting two polar stable forms and three intermediate unstable forms. Tuberculoid pole is characterized by the predominant cell-mediated immune response and localized disease while lepromatous pole shows prevalent humoral response and disseminated disease. Dendritic cells (DCs) activate specific immune responses, so the pattern of costimulatory molecules expressed, and the cytokines released by DCs during M. leprae infection determine the profile of immune response generated that can prevent or favor its survival and dissemination. This study aimed to analyze the profile of DCs derived from leprosy patients infected with viable M. leprae as well as their ability to activate autologous lymphocytes. DCs were differentiated from peripheral blood monocytes of healthy controls, tuberculoid (6 tuberculoid-tuberculoid and 2 borderline-tuberculoid) and lepromatous (5 lepromatous-lepromatous and 5 borderline-lepromatous) leprosy patients employing GM-CSF and IL-4. DCs were stimulated by (i) live M. leprae at a multiplicity of 10:1 (bacilli:DC, MLDCs); (ii) standard maturation cocktail (CQM) composed by IL-1β, IL-6, TNF, PGE2 (mDCs); or (iii) remained unstimulated (iDCs) for 48 hours. After stimulation, DCs were cultured with lymphocytes for five days. Stimulation of DCs with viable M. leprae did not induce the expression of costimulatory molecules but stimulated the production of low levels of IL-1β, IL-6, TNF and IL-23 in both patients and controls. DCs from lepromatous patients produced more IL-1β against M. leprae than those from tuberculoid patients and greater amounts of IL-23 and IL-10 compared to healthy controls. Regardless of the clinical form of leprosy, DCs from leprosy patients produced fewer IL-12p70 in response to M. leprae than DCs from healthy controls. Considering the activation of lymphocytes, we found that lepromatous patients showed lower proliferation and produced lower levels of IL-2 after stimulation with M. leprae. The production of IFN-, TNF, IL-9 and IL-22 in cultures of MLDCs and lymphocytes was similar to that found when iDCs were used and lower than that observed in cultures with mDCs. In lepromatous patients the frequency of IL-4+ lymphocytes were higher in parallel with a lower number of lymphocytes expressing IFN- and IL-17A in cultures stimulated with MLDCs when compared to healthy controls. Together, our data suggest that M. leprae is a weak activator of DCs, which compromises the ability of these cells to induce the activation of autologous lymphocytes. Lepromatous leprosy patients had a lower frequency of lymphocytes producing Th1 and Th17 cytokines after stimulation with M. leprae, which may contribute to the persistence of the bacillus.
Leprosy is still considered a public health problem with more than 200,000 new cases detected in 2019, being 27,863 registered in Brazil. Caused by Mycobacterium leprae, an obligate intracellular pathogen, leprosy can result in permanent physical disabilities when untreated due to the tropism of the bacillus by Schwann cells from peripheral nerves. It is a spectral disease presenting two polar stable forms and three intermediate unstable forms. Tuberculoid pole is characterized by the predominant cell-mediated immune response and localized disease while lepromatous pole shows prevalent humoral response and disseminated disease. Dendritic cells (DCs) activate specific immune responses, so the pattern of costimulatory molecules expressed, and the cytokines released by DCs during M. leprae infection determine the profile of immune response generated that can prevent or favor its survival and dissemination. This study aimed to analyze the profile of DCs derived from leprosy patients infected with viable M. leprae as well as their ability to activate autologous lymphocytes. DCs were differentiated from peripheral blood monocytes of healthy controls, tuberculoid (6 tuberculoid-tuberculoid and 2 borderline-tuberculoid) and lepromatous (5 lepromatous-lepromatous and 5 borderline-lepromatous) leprosy patients employing GM-CSF and IL-4. DCs were stimulated by (i) live M. leprae at a multiplicity of 10:1 (bacilli:DC, MLDCs); (ii) standard maturation cocktail (CQM) composed by IL-1β, IL-6, TNF, PGE2 (mDCs); or (iii) remained unstimulated (iDCs) for 48 hours. After stimulation, DCs were cultured with lymphocytes for five days. Stimulation of DCs with viable M. leprae did not induce the expression of costimulatory molecules but stimulated the production of low levels of IL-1β, IL-6, TNF and IL-23 in both patients and controls. DCs from lepromatous patients produced more IL-1β against M. leprae than those from tuberculoid patients and greater amounts of IL-23 and IL-10 compared to healthy controls. Regardless of the clinical form of leprosy, DCs from leprosy patients produced fewer IL-12p70 in response to M. leprae than DCs from healthy controls. Considering the activation of lymphocytes, we found that lepromatous patients showed lower proliferation and produced lower levels of IL-2 after stimulation with M. leprae. The production of IFN-, TNF, IL-9 and IL-22 in cultures of MLDCs and lymphocytes was similar to that found when iDCs were used and lower than that observed in cultures with mDCs. In lepromatous patients the frequency of IL-4+ lymphocytes were higher in parallel with a lower number of lymphocytes expressing IFN- and IL-17A in cultures stimulated with MLDCs when compared to healthy controls. Together, our data suggest that M. leprae is a weak activator of DCs, which compromises the ability of these cells to induce the activation of autologous lymphocytes. Lepromatous leprosy patients had a lower frequency of lymphocytes producing Th1 and Th17 cytokines after stimulation with M. leprae, which may contribute to the persistence of the bacillus.
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Palavras-chave
Hanseníase, Mycobacterium leprae, Células dendríticas, Linfócitos, Leprosy, Lymphocytes, Dendritic cells