Antiparasitic and anti-inflammatory activities of ß-lapachone-derived naphthoimidazoles in experimental acute Trypanosoma cruzi infection.

BACKGROUND: Chagas disease, which is caused by the protozoan Trypanosoma cruzi, is endemic to Latin America and mainly affects low-income populations. Chemotherapy is based on two nitrocompounds, but their reduced efficacy encourages the continuous search for alternative drugs. Our group has characterised the trypanocidal effect of naphthoquinones and their derivatives, with naphthoimidazoles derived from ß-lapachone (N1, N2 and N3) being the most active in vitro. OBJECTIVES: In the present work, the effects of N1, N2 and N3 on acutely infected mice were investigated. METHODS: in vivo activity of the compounds was assessed by parasitological, biochemical, histopathological, immunophenotypical, electrocardiographic (ECG) and behavioral analyses. FINDINGS: Naphthoimidazoles led to a decrease in parasitaemia (8 dpi) by reducing the number of bloodstream trypomastigotes by 25-50% but not by reducing mortality. N1 protected mice from heart injury (15 dpi) by decreasing inflammation. Bradycardia was also partially reversed after treatment with N1 and N2. Furthermore, the three compounds did not reverse hepatic and renal lesions or promote the improvement of other evaluated parameters. MAIN CONCLUSION: N1 showed moderate trypanocidal and promising immunomodulatory activities, and its use in combination with benznidazole and/or anti-arrhythmic drugs as well as the efficacy of its alternative formulations must be investigated in the near future.

Leishmania (V.) braziliensis infection promotes macrophage autophagy by a LC3B-dependent and BECLIN1-independent mechanism.

Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phagocytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimination. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirming BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell.

Leishmania (V.) braziliensis infection promotes macrophage autophagy by a LC3B-dependent and BECLIN1-independent mechanism

Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phago cytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimi nation. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirm ing BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell.

A autofagia do hospedeiro na infecção pelo Trypanosoma cruzi: possíveis mecanismos envolvidos e resposta imune

A doença de Chagas é provocada pelo protozoário Trypanosoma cruzi e dentre suas manifestações clínicas, destaca-se a inflamação cardíaca. Diversas vias da célula hospedeira estão relacionadas ao ciclo do parasito, incluindo a fagocitose e autofagia. Além disso, o desenvolvimento da patogênese depende da resposta imune do hospedeiro. A autofagia é um processo de degradação de componentes citoplasmáticos presente em células eucarióticas associado a sinalização celular, cuja regulação pode afetar processos celulares, incluindo as infecções intracelulares, possibilitando o desenvolvimento da doença. No presente estudo, avaliou-se a participação da autofagia durante a infecção por T. cruzi in vitro e in vivo, analisando: (1) a contribuição desta via durante a infecção em células fagocíticas e não-fagocíticas profissionais; (2) o papel potencial da autofagia na miocardite chagásica aguda, utilizando sua modulação farmacológica pela rapamicina. Primeiro, a análise de autofagia através da avaliação da expressão de LC3 em células cardíacas e macrófagos submetidos à indução por rapamicina ou meio de estresse nutricional aumentou o número de autofagossomos sem a perda de viabilidade. Em culturas infectadas, ambas as células mostraram maior expressão de LC3, principalmente aquelas nas quais o parasito foi internalizado O tratamento com rapamicina compromete a viabilidade do parasito, contudo o estresse nutricional não o afeta, inclusive na infectividade. Assim, as células sob estresse nutricional diminuem a infecção nas duas células hospedeiras, em diferentes momentos do ciclo do parasito. Enquanto macrófagos reduzem a internalização, confirmada pela menor fagocitose de partículas inertes e parasitos mortos, em células não-fagocíticas, a diminuição ocorre pelo bloqueio da proliferação ou impedimento da diferenciação, eliminando o parasito, também corroborado pelas células ATG5-/- submetidas à infecção. In vivo, diferentes cargas parasitárias induziram autofagia, principalmente em células endoteliais e cardiomiócitos, sem associação ao número de parasitos inoculados. O tratamento por 10 dias com rapamicina levou a alterações elétricas, sem induzir danos bioquímicos renais, cardíacos ou hepáticos. Em animais infectados, a rapamicina não afetou a carga parasitária, como previamente observado in vitro. Apesar disso, o fármaco induziu um aumento do número de células T de memória no baço e redução do peso do órgão. Rapamicina ainda apresentou papel protetor no coração dos animais infectados diminuindo o intervalo PR, dano cardíaco por CK-MB e infiltrados inflamatórios, reforçando a via autofágica como benéfica ao hospedeiro durante a infecção pelo T. cruzi, reforçando a possibilidade de intervenções terapêuticas nesta via na modulação da resposta imune