Inhibition of axenic growth of <i>Leishmania</i> and reduction in parasite load by 17-AAG.

<p>(<b>A</b>) Axenic promastigotes were exposed to several concentrations of 17-AAG (25, 125, 500 nM) for 48 h and the number of viable parasites was assessed as described in Materials and Methods. Data are presented as the percentage inhibition of parasite growth related to untreated controls (4,754×10⁷). Bars represent means ± SD of one representative out of two experiments performed in sextuplicate (one-way ANOVA, Dunnett’s Multiple Comparison Test, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001). (<b>B, C</b>) Drug effects at early times after infection. Following 6 h of incubation with parasites, macrophage cultures were treated for 6, 24 and 48 h with specific concentrations of 17-AAG (25, 125, 500 nM); (<b>D, E</b>) Drug effects at later stages after infection. Following 6 h of incubation with parasites, macrophage cultures were reincubated for additional 48 h then submitted to treatment with specific concentrations of 17-AAG (25, 125, 500 nM). Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001).</p

Alterations suggestive of autophagy in intracellular parasites treated with 17-AAG.

<p>Transmission electron microscopy was used to investigate ultrastructural morphological alterations in intracellular parasites inside 17-AAG-treated macrophages. (<b>A</b>) Control infected macrophages. After 12 h of treatment, several morphological alterations were seen in intracellular parasites, including: (<b>B</b>) numerous small vesicles some containing cytoplasmic material inside <b>(black arrow)</b>, (<b>C</b>) vacuoles larger in size <b>(black arrow-head)</b>, (<b>D</b>) with intravacuolar materials degraded <b>(white arrow)</b>. After 24 h of treatment, the intracellular parasites presented a large number of vesicles occupying most of the cytoplasm containing well-preserved nuclei, mitochondria, and subpellicular microtubules (<b>E–F</b>). After 48 h of treatment, no preserved parasites inside cells were observed, yet empty vesicles, and membrane-bounded structures with an electron-density similar to parasite cytosol in parasitophorous vacuoles were seen (<b>G</b>). At 12 and 24 h after treatment, several alterations were also visible in parasite cytoplasm, including myelin figures <b>(*)</b> (<b>H–I</b>), vesicles with double-layered membranes <b>(white arrow-head)</b> (<b>J–M</b>)<b>,</b> and portions of mitochondria inside membrane-bounded structures (<b>M</b>). The nuclei (<b>N</b>) and mitochondria (<b>M</b>) and kinetoplast (<b>K</b>) remained intact in all groups.</p

Reduced O₂

<p>⁻<b>and NO production in macrophage cultures treated with 17-AAG.</b> (<b>A</b>) O₂⁻ production was measured at early stages of infection using a lucigenin-derived CL method. Points on the graph represent photon emissions per second by macrophage cultures 2 min prior to the addition of <i>L. amazonensis</i> promastigotes, as well as throughout the incubation period of 20 min. Data are derived from one representative experiment out of four performed in uniplicate (Mann Whitney test, p = 0.028); (<b>B</b>) Intracellular O₂⁻ production was assessed by determining cell fluorescence in the presence of hydroethidine (5 µM) and expressed as MFI using a flow cytometer. The histogram overlay depicts the MFI of hydroethidine-labeled cells (solid lines) in comparison to unlabeled control cells (shaded areas). Data are derived from one representative experiment out of three performed in uniplicate (Mann Whitney test, <i>p</i> = 1). (<b>C</b>) NO production was measured by detecting nitrite in the supernatants of 17-AAG-treated cells, as described in Materials and Methods. Bars represent NO production measurement expressed as means ± SD of one representative experiment out of four performed in triplicate or more (Student’s <i>t</i> test, ***<i>p</i><0.0001).</p

Modulation of mediator production by treatment with 17-AAG.

<p>Mediators released by 17-AAG-treated cells were measured in cell supernatants using an inflammatory CBAKit, as described in Materials and Methods: (<b>A</b>) IL-6; (<b>B</b>) IL-10; (<b>C</b>) IL-12; (<b>D</b>) TNF-α; (<b>E</b>) MCP-1. Bars represent means ± SD of a single experiment performed in sextuplicate (Student’s <i>t</i>-test and Mann-Whitney, **<i>p</i><0.001, ***<i>p</i><0.0001).</p

Irreversibility of treatment with 17-AAG on intracellular <i>Leishmania</i>.

<p>To assessment the reversibility of parasite growth inhibition by treatment with 17-AAG parasite load was determined by quantifying the percentage of infected macrophages (<b>A</b>) and the number of parasites per macrophage (<b>B</b>) as described in Materials and Methods. Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, ***<i>p</i><0.0001, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001).</p

Reduction of parasite intracellular viability by 17-AAG.

<p>Treatment’s effect on parasite viability was assessed after 24 h (<b>A</b>) and 48 h (<b>B</b>) of infection, as described in Materials and Methods. Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, ***<i>p</i><0.0001, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001; Mann Whitney test, **<i>p</i><0.001).</p