Chemotherapeutic Potential of 17-AAG against Cutaneous Leishmaniasis Caused by <i>Leishmania (Viannia) braziliensis</i>

<div><p>Background</p><p>Leishmaniasis remains a worldwide public health problem. The limited therapeutic options, drug toxicity and reports of resistance, reinforce the need for the development of new treatment options. Previously, we showed that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a Heat Shock Protein 90 (HSP90)-specific inhibitor, reduces <i>L. (L.) amazonensis</i> infection <i>in vitro</i>. Herein, we expand the current knowledge on the leishmanicidal activity of 17-AAG against cutaneous leishmaniasis, employing an experimental model of infection with <i>L. (V.) braziliensis</i>.</p><p>Methodology/Principal findings</p><p>Exposure of axenic <i>L. (V.) braziliensis</i> promastigotes to 17-AAG resulted in direct dose-dependent parasite killing. These results were extended to <i>L. (V.) braziliensis</i>-infected macrophages, an effect that was dissociated from the production of nitric oxide (NO), superoxide (O⁻²) or inflammatory mediators such as TNF-α, IL-6 and MCP-1. The leishmanicidal effect was then demonstrated <i>in vivo</i>, employing BALB/c mice infected with <i>L. braziliensis</i>. In this model, 17-AAG treatment resulted in smaller skin lesions and parasite counts were also significantly reduced. Lastly, 17-AAG showed a similar effect to amphotericin B regarding the ability to reduce parasite viability.</p><p>Conclusion/Significance</p><p>17-AAG effectively inhibited the growth of <i>L. braziliensis</i>, both <i>in vitro</i> and <i>in vivo</i>. Given the chronicity of <i>L. (V.) braziliensis</i> infection and its association with mucocutaneous leishmaniasis, 17-AAG can be envisaged as a new chemotherapeutic alternative for cutaneous Leishmaniasis.</p></div

Treatment with 17-AAG controls <i>L. (V.) braziliensis</i> replication inside macrophages.

<p><i>L. (V.) braziliensis-</i> infected macrophages were treated with increasing concentrations of 17-AAG or with vehicle alone (DMSO). After 12–72 h, glass coverslips were stained with H&E and assessed for the percentage of infected macrophages (A) and the number of amastigotes per 100 macrophages (B) by light microscopy. Data, shown as mean ±SEM, are shown as the percentage of DMSO -treated cultures, from one of three independent repeats (*<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001).</p

<i>In vivo</i> treatment with 17-AAG decreases <i>L. (V.) braziliensis</i> infection.

<p>Mice were infected with <i>L. (V.) braziliensis</i> and four weeks later, mice were treated with 17-AAG, 3x a week for 3 weeks (boxed area) or with vehicle (DMSO) alone. (A) The course of lesion development was monitored weekly. (B) Disease burden [shown as Area Under the Curves (AUC) depicted in (A)] in mice treated with 17-AAG or injected with DMSO. Parasite load was determined at the infection site (C) and at the dLN (D), 6 weeks later, by limiting dilution analysis. Data, shown as mean ±SEM, are from one of two independent repeats, each performed with 10 mice in each group (**<i>p</i><0.01; *<i>p</i><0.05).</p

17-AAG induces killing of <i>Leishmania (V.) braziliensis</i> promastigotes in a dose-dependent and irreversible manner.

<p><i>L. (V.) braziliensis</i> promastigotes were exposed to increasing concentrations of 17-AAG, to vehicle alone (DMSO) or were left unexposed (Lb) for 48 h. (A) The number of viable parasites was evaluated by direct counting. <i>L.(V) braziliensis</i> promastigotes were treated with 65 nM (IC₅₀) of 17-AAG for 24 h (B) and (C) 48 h. After washing, promastigotes were cultured for additional 48 h and the number of viable parasites was evaluated. Data, shown as mean ±SEM, are from one of two independent repeats (**<i>p</i><0.01 and ***<i>p</i><0.001).</p

Treatment with 17-AAG reduces intracellular <i>L. (V.) braziliensis</i> viability.

<p><i>L. (V.) braziliensis</i>- infected macrophages were treated for 24 h with increasing concentrations of 17-AAG or with vehicle alone (DMSO). The number of viable parasites was evaluated by further culture (5 days) in Schneider medium, free of 17-AAG (A). Infected macrophages were treated with 220 nM (IC₅₀) of 17-AAG for 24–72 h. The number of viable parasites was evaluated by further culture for five days in Schneider medium, free of 17-AAG (B). Data, shown as mean ±SEM, are from one of two independent repeats (**<i>p</i><0.01 and *** <i>p</i><0.001).</p

Treatment with 17-AAG down regulates ROS and cytokine production in <i>L. (V.) braziliensis-infected</i> cells.

<p><i>L. (V.) braziliensis-</i>infected macrophages were treated with 17-AAG (220nM) + IFN-. After 48h, supernatants were assayed for nitrite production (A) and for presence of (B) superoxide, following addition of hydroxylamine. The presence of secreted (C) TNF-α, (D) IL-6 and (E) CCL2 was determined in culture supernatants by Cytometric Bead Array, after 24 h of treatment. Data, shown as mean ± SEM, are from one of two independent repeats (**<i>p</i><0.01; *<i>p</i><0.05).</p

Comparison of the effects of 17-AAG and amphotericin B on the growth of intracellular <i>L. (V.) braziliensis</i>.

<p><i>L. (V.) braziliensis-</i>infected macrophages were treated with 17-AAG or with amphotericin B (AMB) for 48 h. The number of viable parasites was evaluated by further culture in Schneider medium, free of 17-AAG. Data, shown as mean ±SEM, are from one of two independent repeats (***<i>p</i><0.001). (ND, not detected).</p

Vaccination with <em>L. infantum chagasi</em> Nucleosomal Histones Confers Protection against New World Cutaneous Leishmaniasis Caused by <em>Leishmania braziliensis</em>

<div><h3>Background</h3><p>Nucleosomal histones are intracellular proteins that are highly conserved among <em>Leishmania</em> species. After parasite destruction or spontaneous lysis, exposure to these proteins elicits a strong host immune response. In the present study, we analyzed the protective capability of <em>Leishmania infantum chagasi</em> nucleosomal histones against <em>L. braziliensis</em> infection using different immunization strategies.</p> <h3>Methodology/Principal Findings</h3><p>BALB/c mice were immunized with either a plasmid DNA cocktail (DNA) containing four <em>Leishmania</em> nucleosomal histones or with the DNA cocktail followed by the corresponding recombinant proteins plus CpG (DNA/Protein). Mice were later challenged with <em>L. braziliensis,</em> in the presence of sand fly saliva. Lesion development, parasite load and the cellular immune response were analyzed five weeks after challenge. Immunization with either DNA alone or with DNA/Protein was able to inhibit lesion development. This finding was highlighted by the absence of infected macrophages in tissue sections. Further, parasite load at the infection site and in the draining lymph nodes was also significantly lower in vaccinated animals. This outcome was associated with increased expression of IFN-γ and down regulation of IL-4 at the infection site.</p> <h3>Conclusion</h3><p>The data presented here demonstrate the potential use of <em>L. infantum chagasi</em> nucleosomal histones as targets for the development of vaccines against infection with <em>L. braziliensis</em>, as shown by the significant inhibition of disease development following a live challenge.</p> </div

Intracellular cytokine production by CD4+ cells from mice immunized with nucleosomal histones and challenged with <i>L. braziliensis</i> plus sand fly saliva.

<p>BALB/c mice (5 per group) were immunized as described. Two weeks after the last immunization, mice were challenged with <i>L. braziliensis</i>+sand fly saliva. Five weeks later, draining lymph nodes were pooled and cells were preincubated with Brefeldin A for four hours before staining. Data represent the frequency of CD4+ cells positive for IFN-γ (A, B), IL-4 (C, D) and IL-10 (D, E) with signals for the particular cytokine that were greater than the background signals established using isotype controls. Data are presented as the mean+SEM and are from two independent experiments.</p

Intracellular cytokine production by CD8+ cells from mice immunized with nucleosomal histones and challenged with <i>L. braziliensis</i> plus sand fly saliva.

<p>BALB/c mice (5 per group) were immunized as described. Two weeks after the last immunization, mice were challenged with <i>L. braziliensis</i>+sand fly saliva. Five weeks later, draining lymph nodes were pooled and cells were preincubated with Brefeldin A for four hours before staining. Data represent the frequency of CD8+ cells positive for IFN-γ (A, B), IL-4 (C, D) and IL-10 (D, E) with signals for the particular cytokine that were greater than the background signals established using isotype controls. Data are presented as the mean+SEM and are from two independent experiments.</p