Oral treatment with T6-loaded yeast cell wall particles reduces the parasitemia in murine visceral leishmaniasis model

Abstract

Yeast cell wall particles isolated from Saccharomyces cerevisiae (scYCWPs) have a rich constitution of β-glucan derived from the cell wall. After removing intracellular contents, β-glucan molecules are readily recognized by dectin-1 receptors, present on the cytoplasmic membrane surface of the mononuclear phagocytic cells and internalized. Leishmania spp. are obligate intracellular parasites; macrophages are its primary host cells. An experimental murine model of visceral leishmaniasis caused by L. infantum was used to evaluate the antileishmanial activity of oral administration of these particles. A low-water soluble thiophene previously studied in vitro against L. infantum was entrapped into scYCWPs to direct it into the host cell, in order to circumvent the typical pharmacokinetic problems of water-insoluble compounds. We found that scYCWPs + T6 reduced the parasitic burden in the liver and spleen. There was an increase in IFN-γ levels related to nitric oxide production, explaining the reduction of the L. infantum burden in the tissue. Histological analysis did not show signals of tissue inflammation and biochemical analysis from plasma did not indicate signals of cytotoxicity after scYCWPs + T6 treatment. These findings suggested that scYCWPs + T6 administered through oral route reduced the parasitic burden without causing toxic effects, satisfying requirements for development of new strategies to treat leishmaniasis.

Figure 1

Body mass over the course of treatment and organs mass at the last day of treatment of noninfected (a) and L. infantum-infected (b) mice. Animals were treated with empty scYCWPs (scYCWPs), T6-loaded scYCWPs (scYCWPs + T6), T6 alone (T6), and miltefosine was used as standard drug against visceral leishmaniasis. Results were expressed as mean ± standard error (n = 4). Significant differences at *p < 0.05 and **p < 0.01.

Figure 2

L. infantum burden in the spleen (a) and liver (b) determined by qPCR after treatment with empty scYCWPs (scYCWPs), T6-loaded scYCWPs (scYCWPs + T6), and T6 alone (T6). Miltefosine was used as the antileishmanial activity control. Each point represents one animal (n = 4). At least 75% of the mice were considered in the statistical analysis. Significant differences and p values are represented in the graph space.

Figure 3

Cytokine measurements from the spleens of noninfected (a) and L-infantum infected (b) mice after treatment with empty scYCWPs (scYCWPs), T6-loaded scYCWPs (scYCWPs + T6) and T6 alone (T6). Miltefosine was used as the standard drug against visceral leishmaniasis. Data were expressed as cytokine production in pg/ml and shown as mean ± standard error (n = 4). Using the BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit manufacturer’s instruction, 2,500 events were analyzed for each sample. Significant differences at *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Biochemical analysis of plasma from (a) noninfected and (b) L. infantum-infected animals. In both assays, animals were treated with empty scYCWPs (scYCWPs), T6-loaded scYCWPs (scYCWPs + T6), and T6 alone (T6). Miltefosine was used as the standard antileishmanial drug to treat infected mice. Renal, cardiac and hepatic functions were measured through creatinine, CKMB, and alkaline phosphatase, AST and ALT levels, respectively, by UV kinetic determination. All tests were performed in duplicate with three animals per group and the results were shown as mean ± standard error. Significant differences at *p < 0.05, **p < 0.01, ***p < 0.001.