Dual immunomodulatory and antileishmanial potential of TLR7/8 agonists against <i>Leishmania donovani</i>

Visceral leishmaniasis (VL), caused by <i>Leishmania donovani</i>, is a neglected tropical disease with limited therapeutic options and increasing drug resistance. This study investigates the immunological mechanisms and antiparasitic efficacy of imidazoquinoline-based Toll-like receptor 7/8 (TLR7/8) agonists as host-directed agents in an <i>in vitro</i> VL model. Using RAW 264.7 macrophages and <i>L. donovani</i> promastigotes and amastigotes, we examined macrophage activation, nitric oxide (NO) induction, and cell cycle disruption in parasites. The lead compounds (5 and 10) significantly enhanced NO production in macrophages, both in unstimulated and LPS-stimulated conditions, indicating robust innate immune activation. Additionally, parasite-derived reactive oxygen species (ROS) levels were markedly elevated, suggesting oxidative stress as a mechanism of direct leishmanicidal action. Flow cytometric analysis revealed G₀/G₁ arrest in treated promastigotes, further supporting interference with parasite proliferation. Importantly, these compounds exhibited low cytotoxicity toward host cells and favorable selectivity indices. Notably, this is the first <i>in vitro</i> study to comprehensively demonstrate the ability of TLR7/8 agonists to exert direct parasiticidal effects along with immune modulation in the context of VL. The results underscore the potential of TLR-targeted immunomodulation to enhance host defense mechanisms against intracellular protozoan infections and contribute to the development of novel immunopharmacological interventions for VL.