Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va

Abstract

Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

Figure 1

Effect of latrunculin and nocodazole on the interaction of Leishmania braziliensis with RAW 264.7 macrophages. Adherent macrophages, with or without 17 h previous activation by LPS and IFN-γ, were treated with latrunculin (5 μM) or nocodazole (5 μM) for 10 min and then incubated with promastigotes for 30 min. The association indices were determined by multiplying the percentage of infected macrophages by the mean number of parasites per cell. The bars represent the mean ± standard errors of mean (SEM) of at least three independent experiments performed in triplicate. *P ≤ 0.05 in relation to control inactivated macrophages, ^Δ P ≤ 0.05 in relation to control inactivated macrophages,^◆ P ≤ 0.05 in relation to control activated macrophages.

Figure 2

Effect of latrunculin and nocodazole on the production of NO by RAW 264.7 macrophages after interaction with L. brasiliensis. NO levels were inferred from the level of nitrite measured in the media after various treatments. Adherent macrophages were activated (black bars) for 17 h with LPS (100 ng/mL) + IFN-γ (1 μg/mL) or remained untreated (open bars). For the measurement of NO after interactions with parasites in (a), macrophages were treated with latrunculin (5 μM) or nocodazole (5 μM) for 10 min and then exposed to promastigotes for 30 min. In the absence of parasites (b), activated and nonactivated adherent macrophages were treated with latrunculin (5 μM) or nocodazole (5 μM) for 10 min. The bars represent the mean ± standard errors of mean (SEM) of at least three independent experiments performed in triplicate. *P ≤ 0.05 compared to control activated macrophages in (a) and compared to control inactivated macrophages in (b); ^∇ P ≤ 0.05 compared to control nonactivated macrophages; ^◆ P ≤ 0.05 compared to control activated macrophages.

Figure 3

Effect of latrunculin and nocodazole on cytokine production in RAW 264.7 macrophages. Levels of IL-10 (a and b) and TNF-α (c and d) were measured in the media after various treatments. Adherent macrophages were treated with LPS (100 ng/mL) + IFN-γ (1 μg/mL) for 17 h before treatment with latrunculin (5 μM) or nocodazole (5 μM) for 1 h and then exposed to promastigotes for 30 min (a and c). Adherent macrophages not exposed to parasites were incubated for 17 h in the absence or in the presence of LPS (100 ng/mL) plus IFN-γ (1 μg/mL) and then treated with latrunculin (5 μM) or nocodazole (5 μM) for 1 h (b and d). The bars represent the mean ± standard errors of mean (SEM) of at least three independent experiments performed in triplicate. *P ≤ 0.05 compared to control infected macrophages (a) and compared to control non-activated and uninfected macrophages; ^∇ P ≤ 0.05 compared to control non-activated and uninfected macrophages; ^◆ P ≤ 0.05 compared to control activated uninfected macrophages.

Figure 4

Integrity of the cytoskeleton influences the interaction between L. braziliensis and macrophages. Adherent Raw 264.7 macrophages were incubated for 10 min with (a) fresh DMEM, (b) 5 μM latrunculin A to disrupt actin filaments, or (c) 5 μM nocodazole to disrupt microtubules before exposure for 30 min to parasites that had been previously marked with CFSE. The panels are representative fields of colour-combined fluorescent images displaying parasites in green and F-actin (phalloidin staining) in red. Scale bars indicate imaging for all panels at 63x magnification.

Figure 5

Internalisation of L. braziliensis requires myosin Va. The role of myosin Va was examined by observing the distribution of endogenous myosin Va in relation to parasites and the effect of the expression of a dominant negative tail domain fused to eGFP. Adherent Raw 264.7 macrophages were incubated with fluorescently marked parasites for 30 min before fixation, preparation and imaging by confocal microscopy. (a) shows endogenous myosin Va stained by an antibody (green) and parasites (red). (b) shows the myosin Va tail (green) and parasites (red). (c) shows mock-transfected macrophages by phase microscopy overlaid with a fluorescent image of parasites (red). Scale bars indicate imaging for all panels at 63x magnification.