Autophagic Induction Greatly Enhances Leishmania major Intracellular Survival Compared to Leishmania amazonensis in CBA/j-Infected Macrophages

CBA mouse macrophages control Leishmania major infection yet are permissive to Leishmania amazonensis. Few studies have been conducted to assess the role played by autophagy in Leishmania infection. Therefore, we assessed whether the autophagic response of infected macrophages may account for the differential behavior of these two parasite strains. After 24 h of infection, the LC3-II/Act ratio increased in both L. amazonensis- and L. major-infected macrophages compared to uninfected controls, but less than in chloroquine-treated cells. This suggests that L. amazonensis and L. major activate autophagy in infected macrophages, without altering the autophagic flux. Furthermore, L. major-infected cells exhibited higher percentages of DQ-BSA-labeled parasitophorous vacuoles (50%) than those infected by L. amazonensis (25%). However, L. major- and L. amazonensis-induced parasitophorous vacuoles accumulated LysoTracker similarly, indicating that the acidity in both compartment was equivalent. At as early as 30 min, endogenous LC3 was recruited to both L. amazonensis- and L. major-induced parasitophorous vacuoles, while after 24 h a greater percentage of LC3 positive vacuoles was observed in L. amazonensis-infected cells (42.36%) compared to those infected by L. major (18.10%). Noteworthy, principal component analysis (PCA) and an hierarchical cluster analysis completely discriminated L. major-infected macrophages from L. amazonensis-infected cells accordingly to infection intensity and autophagic features of parasite-induced vacuoles. Then, we evaluated whether the modulation of autophagy exerted an influence on parasite infection in macrophages. No significant changes were observed in both infection rate or parasite load in macrophages treated with the autophagic inhibitors wortmannin, chloroquine or VPS34-IN1, as well as with the autophagic inducers rapamycin or physiological starvation, in comparison to untreated control cells. Interestingly, both autophagic inducers enhanced intracellular L. amazonensis and L. major viability, while the pharmacological inhibition of autophagy exerted no effects on intracellular parasite viability. We also demonstrated that autophagy induction reduced NO production by L. amazonensis- and L. major-infected macrophages but not alters arginase activity. These findings provide evidence that although L. amazonensis-induced parasitophorous vacuoles recruit LC3 more markedly, L. amazonensis and L. major similarly activate the autophagic pathway in CBA macrophages. Interestingly, the exogenous induction of autophagy favors L. major intracellular viability to a greater extent than L. amazonensis related to a reduction in the levels of NO

Avaliação do papel do receptor marco na infecção de macrófagos murinos por Leishmania major

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-10-29T14:03:53Z No. of bitstreams: 1 Niara de Jesus Almeida.Avaliação...2014.pdf: 2142198 bytes, checksum: c0f1caa073f88305a34bffb0a94263ff (MD5)Made available in DSpace on 2014-10-29T14:03:53Z (GMT). No. of bitstreams: 1 Niara de Jesus Almeida.Avaliação...2014.pdf: 2142198 bytes, checksum: c0f1caa073f88305a34bffb0a94263ff (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil.Camundongos CBA são resistentes à infecção por Leishmania major e permissivos à infecção por L. amazonensis. Adicionalmente, macrófagos de camundongos CBA controlam à infecção por L. major, mas não por L. mazonensis in vitro. Em estudo comparativo realizado por nosso grupo foi demonstrado que o receptor scavenger MARCO teve expressão aumentada em resposta à infecção por L. major, mas não na infecção por L. amazonensis. Ainda, o bloqueio do receptor com o anticorpo específico reduziu a infecção por L. major em 30%, indicando que esta proteína tem participação no reconhecimento de promastigotas de L. major em macrófagos de CBA. Assim, nossa hipótese é que o receptor MARCO participa do reconhecimento e fagocitose de L. major por macrófagos, direcionando o curso da infecção. O objetivo do presente estudo consistiu em evidenciar o papel do receptor MARCO na infecção de macrófagos por L. major. Inicialmente, células J774 foram transfectadas com os vetores pcDNA3.1-MARCO (J774-MARCO) ou pcDNA3.1 (J774-MOCK). Foi observado que a expressão do gene do MARCO foi cinco vezes maior nas células J774-MARCO em comparação às células J774-MOCK. Ao avaliarmos o efeito da superexpressão sobre o metabolismo de células J774-MARCO foi observado que a atividade metabólica mitocondrial foi maior nos clones J774-MARCO e J774-MOCK, 14% e 39% respectivamente, em comparação com as células J774 controle não transfectadas. Entretanto, a diferença no metabolismo não alterou a viabilidade celular dos clones transfectados. A superexpressão de MARCO não aumentou a ligação de L. major em células J774, mas favoreceu tanto o aumento no percentual de infecção de L. major como o número de parasitos/célula nos tempos iniciais até 24 h após a infecção (p ≤ 0,05). Adicionalmente, foi investigado se MARCO estaria induzindo modificações na membrana celular que favorecessem a entrada de L. major. Foi demonstrado que as células superexpressando MARCO (67%) apresentaram um maior espraiamento da membrana celular, com a formação de lamelipódios e estruturas semelhantes a filopódios, eventos observados em um número reduzido de células J774-MOCK (24%). Ao avaliarmos o efeito da superexpressão de MARCO na produção de quimiocinas e citocinas durante a infecção por L. major foi observado que a adição de L. major induziu níveis significativamente maiores de MCP-1 e TNF-α nos tempos de 24 e 48 h após a infecção em comparação com as células J774-MOCK (p < 0,01). Níveis maiores de IL-6 foram observados após 48 h de infecção por L. major nas células J774-MARCO em comparação às células controle (p < 0,05). Similarmente, em resposta à infecção por L. major, células J774-MARCO produziram maior quantidade de NO nos tempos de 24 (p < 0,001) e 48 h (p < 0,01) após a infecção. Todavia, a superexpressão de MARCO não teve efeito sobre a sobrevivência intracelular do parasito. Em suma, nossos achados sugerem que a superexpressão do receptor scavenger MARCO favorece a entrada de L. major em células J774, além de desencadear uma resposta imune efetora direcionando o curso da infecção por Leishmania.CBA mice are resistant to Leishmania major yet permissive to L. amazonensis infection. In addition, CBA macrophages control L. major, but not L. amazonensis infection in vitro. In a comparative study performed by our group increase in expression of the scavenger receptor MARCO has been detected in response to L. major, but not to L. amazonensis infection. Moreover, ED31 monoclonal antibody against MARCO reduced by 30% L. major infection in CBA macrophages. These findings indicate that MARCO plays a role in L. major recognition by CBA macrophages. We hypothesized that MARCO receptor participates in the recognition and phagocytosis of L. major by macrophages, directing the outcome of infection. In the present study, we aimed to further disclose the role MARCO plays in L. major infection of murine macrophages. First J774 cells were transfected with pcDNA3.1-MARCO vector (MARCO-J774) or pcDNA3.1 vector (MOCK-J774). Expression of MARCO gene shown to be five times higher in MARCO-J774 cells compared to MOCK-J774 cells. Overexpression of MARCO enhanced mitochondrial metabolic activity in 14% and 39% of both MARCO-J774 and MOCK-J774, respectively, when compared to the control non-transfected J774 cells. However, enhancement in mitochondrial metabolic activity did not alter the cell viability of transfected clones. MARCO overexpression did not increase the binding of L. major in J774 cells, but increased both the percentage of infection of L. major and the number of parasites / cell until 24 h after infection (p ≤ 0,05). Additionally, we investigated whether MARCO be inducing changes in cell membrane that would favor L. major uptake by macrophages. We observed that MARCO-J774 cells (67%) showed a pronounced spread of cell membranes, with short microvilli and lamellipodia, events observed in a reduced number of MOCK-J774 (24%). Then, we evaluated production of pro-inflammatory chemokine and cytokine induced by L. major in MARCO-J774 cells. L. major addition to MARCO-J774 cells induced higher significantly levels of MCP-1 and TNF-α compared to MOCK-J774 cells at 24 and 48 h post infection (p < 0,01). Higher levels of IL-6 were also observed at 48 h of L. major infection in MARCO-J774 cells compared to control (p < 0,05). Similarly, NO production induced by L. major was much more higher in MARCO-J774 cells compared to MOCK-J774 at 24 and 48 h post infection (p ≤ 0,01). Although in MARCO-overexpressing cells, enhancement in pro-inflammatory chemokines and cytokines in response to L. major has been observed, no effect on parasite intracellular survival has been detected. In summary, our findings suggest that the scavenger receptor MARCO overexpression favors the entry of L. major in J774 cells, and trigger an effector immune response that directs outcome of Leishmania infection

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

<div><p> BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.</p></div