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RESPOSTA IMUNOPATOLÓGICA EM MACRÓFAGOS RAW 264.7 E CAMUNDONGOS BALB/C INFECTADOS COM LEISHMANIA INFANTUM AO TRATAMENTO COM CXCL10
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Resumen en portugues
A infecção por Leishmania infantum pode ser assintomática ou sintomática
(calazar), podendo neste caso, haver manifestação de formas graves e até óbito,
mesmo em casos adequadamente tratados com drogas antiparasitárias. Na
leishmaniose visceral por L. infantum, o hospedeiro não gera uma resposta
imunológica eficaz. CXCL10 é uma quimiocina indutora da produção de IFN-γ e,
portanto, associada à resposta imunológica celular de perfil Th1, considerada
necessária para o controle da infecção por L. infantum. Com a perspectiva de
contribuir para o desenvolvimento de novas abordagens terapêuticas, o trabalho
objetivou investigar in vitro e in vivo se o tratamento com CXCL10 confereria
proteção contra a infecção. In vitro, macrófagos RAW 264.7 foram infectados com L.
infantum e tratados ou não com 20, 50 e 100 ng/mL de CXCL10 para avaliação da
carga parasitária, produção de NO, IL-4 e IL-10 após 24 e 48 horas do tratamento. In
vivo, camundongos BALB/c foram infectados com L. infantum e tratados ou não com
CXCL10 (5 μg/kg; I.P) após 1, 3 e 7 dias. Após 1, 7, 23 e 45 do início do tratamento
foi verificado o número de parasitos em baço e fígado, a produção das citocinas IFNγ,
IL-4, TGF- β e IL-10 por esplenócitos, o fenótipo e a freqüência das populações de
células Treg Foxp3+ e Foxp3- produtoras de IL-10 e o efeito de CXCL10 na polpa
branca do baço. In vitro, CXCL10 reduziu carga parasitária, não dependente de NO,
e inibiu a liberação das citocinas IL-4 e IL-10. In vivo, CXCL10 foi capaz de reduzir a
carga parasitária tanto no fígado quanto no baço, quatro semanas após a infecção.
Também induziu IFN-γ após 23 dias de tratamento, correlacionando-se com a
redução da carga parasitária, e reduziu IL-10 e TGF-β. Após tratamento, houve
decréscimo de células Treg produtoras de IL-10 intracelular e observou-se também
que CXCL10 foi capaz de controlar a hiperplasia no baço durante a fase aguda da
infecção. Este estudo sugere um papel protetor de CXCL10 contra L. infantum,
mediado por IFN-γ, não dependente de NO, com supressão de células Treg IL-10+ e
preservação da microarquitetura do baço. Esses dados podem fornecer informações
para o desenvolvimento de novas abordagens para futuras intervenções
terapêuticas para a leishmaniose visceral.
Resumen en ingles
Leishmania infantum infection may be asymptomatic or symptomatic (kalazar), in which case there may be severe manifestations and even death, even in cases adequately treated with antiparasitic drugs. In visceral leishmaniasis by L. infantum, the host does not generate an effective immune response. CXCL10 is a chemokine that induces the production of IFN-\03B3 and, therefore, is associated with the Th1 profile cellular immune response considered necessary for the control of L. infantum infection. With the prospect of contributing to the development of new therapeutic approaches, the work aimed to investigate in vitro and in vivo whether treatment with CXCL10 would confer protection against infection. In vitro, RAW 264.7 macrophages were infected with L. infantum and treated with 20, 50 and 100 ng/mL CXCL10 for parasite load, NO, IL-4 and IL-10 production after 24 and 48 hours of treatment. In vivo, BALB/c mice were infected with L. infantum and treated or not with CXCL10 (5 \03BCg/kg; I.P) after 1, 3 and 7 days. After 1, 7, 23 and 45 days of treatment the number of spleen and liver parasites was determined, the production of the cytokines IFN-\03B3, IL-4, TGF-\03B2 and IL-10 by splenocytes, phenotype and frequency of IL-10-producing Foxp3+ and Foxp3- cells and the effect of CXCL10 on the white spleen pulp In vitro, CXCL10 reduced parasite load, non-NO dependent, and inhibited the release of IL-4 and IL-10 cytokines. In vivo, CXCL10 was able to reduce parasite burden in both liver and spleen four weeks post infection, also induced IFN-\03B3 after 23 days of treatment, correlating with the reduction of parasite burden, and reduced IL-10 and TGF-\03B2. After treatment, there was also a decrease in intracellular IL-10 producing Treg cells and it was also observed that CXCL10 was able to control spleen follicular hyperplasia during the acute phase of the infection. This study suggests a protective role of non- NO-dependent IFN-\03B3 mediated CXCL10 against L. infantum with suppression of IL- 10 + Treg cells and preservation of spleen microarchitecture. These data may provide information for the development of new approaches for future therapeutic interventions for visceral leishmaniasis Pneumoconiosis is a pulmonary disease of occupational origin, considered to be highly prevalent among workers exposed to mineral dust, such as silica and asbestos, in the various branches of industrial production. The inhalation of these mineral dust can develop an intense interstitial pulmonary fibrosis, characterized by inflammatory reaction, release of chemotactic factors, production of collagen and consequent formation of granuloma in the lung. Biological indicators may be useful in understanding this inflammatory process of pulmonary disease, in identifying the pre-fibrogenic phase, in monitoring and treating these diseases. It carries out a study of the level of concentration of three cytokines: Tumor Necrosis factor alpha (TNFÓ), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in pulmonary disease caused by exposure to silica and to asbestos, in pneumoconiosis. The specific objective was to determine their concentration in the blood serum of patients exposed to silica and asbestos and to verify the association of cytokines with exposures and pneumoconiosis. It was studied 161 people, of which 107 workers exposed to mineral dust, 85 exposed to silica and 22 exposed to asbestos, and 54 workers not exposed in the comparison group. For the quantitative determination of TNFÎ ±, IL-1β and IL-6, blood was collected from all workers included in the design and the Elisa method was used for the analysis. Significant differences were also found between patients with pneumoconiosis and the group not exposed to the three cytokines at differentiated levels. Analysis of the results did not discriminate the role of cytokines in the pre-fibrotic phase of disease. The results in the subgroup exposed to silica with silicosis did not show elevated levels of IL-6 and TNF, but showed elevated IL-1β levels of cytokines dosed in blood. In the subgroup exposed to asbestosis with asbestosis, there was only an increase in IL-6 levels. In the field of public health, there are still no instruments capable of predicting pathological processes in workers exposed to mineral dust, especially silica and asbestos. The field of study of cytokines and other markers is promising, but so far the only weapon to reduce or eliminate damage from mineral dust in the workplace is the removal of the harmful agent from the environment.
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