Immunotherapeutic Potential of Eugenol Emulsion in Experimental Visceral Leishmaniasis

The therapy of visceral leishmaniasis (VL) is limited by resistance, toxicity and decreased bioavailability of the existing drugs coupled with dramatic increase in HIV-co-infection, non-availability of vaccines and down regulation of cell-mediated immunity (CMI). Thus, we envisaged combating the problem with plant-derived antileishmanial drug that could concomitantly mitigate the immune suppression of the infected hosts. Several plant-derived compounds have been found to exert leishmanicidal activity via immunomodulation. In this direction, we investigated the antileishmanial activity of eugenol emulsion (EE), complemented with its immunomodulatory and therapeutic efficacy in murine model of VL. Oil-in-water emulsion of eugenol (EE) was prepared and size measured by dynamic light scattering (DLS). EE exhibited significant leishmanicidal activity with 50% inhibitory concentration of 8.43±0.96 μg ml-1 and 5.05±1.72 μg ml─1, respectively against the promastigotes and intracellular amastigotes of Leishmania donovani. For in vivo effectiveness, EE was administered intraperitoneally (25, 50 and 75 mg/kg b.w./day for 10 days) to 8 week-infected BALB/c mice. The cytotoxicity of EE was assessed in RAW 264.7 macrophages as well as in naive mice. EE induced a significant drop in hepatic and splenic parasite burdens as well as diminution in spleen and liver weights 10 days post-treatment, with augmentation of 24h-delayed type hypersensitivity (DTH) response and high IgG2a:IgG1, mirroring induction of CMI. Enhanced IFN-γ and IL-2 levels, with fall in disease-associated Th2 cytokines (IL-4 and IL-10) detected by flow cytometric bead-based array, substantiated the Th1 immune signature. Lymphoproliferation and nitric oxide release were significantly elevated upon antigen revoke in vitro. The immune-stimulatory activity of EE was further corroborated by expansion of IFN-γ producing CD4+ and CD8+ splenic T lymphocytes and up-regulation of CD80 and CD86 on peritoneal macrophages. EE treated groups exhibited induction of CD8+ central memory T cells as evidenced from CD62L and CD44 expression. No biochemical alterations in hepatic and renal enzymes were observed. Our results demonstrate antileishmanial activity of EE, potentiated by Th1 immunostimulation without adverse side effects. The Th1 immune polarizing effect may help to alleviate the depressed CMI and hence complement the leishmanicidal activity.