Notch signaling governs crucial aspects of intercellular communication spanning antigen-presenting cells and T-cells. In this study, we investigate how L. donovani takes advantage of this pathway to quell host immune responses. We report induction of Notch ligand, Jagged1 in L. donovani-infected bone marrow macrophages (BMMфs) and subsequent activation of RBPJκ in T cells, which in turn, upregulates transcription factor GATA3. Activated RBPJκ also associates with histone acetyltransferase, p300, which binds with Bcl2L12 promoter and enhances its expression. Interaction of Bcl2L12 with GATA3 in CD4+ T cells facilitates its binding to IL-10 and IL-4 promoters, thereby increasing the secretion of these cytokines. Silencing Jagged1 hindered these events in a BMMф-T cell co-culture system. Upon further scrutiny, we found that parasite LPG induces the host PI3K/Akt pathway, which activates β-catenin and Egr1, the two transcription factors responsible for driving Jagged1 expression. Vivo morpholino-silencing of Jagged1 suppresses anti-inflammatory cytokine responses and reduces organ parasite burden in L. donovani-infected Balb/c mice, suggesting that L. donovani induced host Jagged1/Notch signaling skews macrophage-T cell crosstalk into disease-promoting Th2 mode in experimental VL.

Objectives Previously, a series of side chain-modified quinolinyl β-enaminones was identified to possess significant activity against chloroquine-sensitive or -resistant Plasmodium falciparum and Brugia malayi microfilariae. The present study evaluates in vitro and in vivo activity of the series against Leishmania donovani and reports their mode of action. Methods The in vitro activity of 15 quinolinyl β-enaminone derivatives against Leishmania promastigotes and amastigotes was assessed by luciferase assay. The reduction of organ parasite burden was assessed by Giemsa staining in L. donovani-infected BALB/c mice and hamsters. Intracellular Ca2+ and ATP level in active derivative (3D)-treated promastigotes were determined by fluorescence and luminescence assays. Flow cytometry was performed to determine loss of mitochondrial membrane potential (MMP) using JC-1 dye, reactive oxygen species (ROS) generation using 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) dye, phosphatidylserine externalization by Annexin V-FITC staining and cell-cycle arrest by propidium iodide (PI) staining. Results Compounds 3A, 3B and 3D showed significant in vitro efficacy against L. donovani with IC50 < 6 µM and mild cytotoxicity (∼75% viability) at 25 µM on J774 macrophages. 3A and 3D at 50 mg/kg and 100 mg/kg reduced parasite burden (>84%) in infected mice and hamsters, respectively, whereas 3D-treated animals demonstrated maximum parasite burden reduction without organ toxicity. Mode-of-action analysis revealed that 3D induced apoptosis by inhibiting mitochondrial complex II, reducing MMP and ATP levels, increasing ROS and Ca2+ levels, ultimately triggering phosphatidylserine externalization and sub-G0/G1 cell-cycle arrest in promastigotes. Conclusions Compound 3D-mediated inhibition of L. donovani mitochondrial complex induces apoptosis, making it a promising therapeutic candidate for visceral leishmaniasis.

A series of uniquely functionalized 2,3,-dihydro-1H-pyyrolo[3,4-b]quinolin-1-one derivatives were synthesized in one to two steps by utlilizing post-Ugi modification strategy and were evaluated for antileishmanial efficacy against visceral leishmaniasis (VL). Among...