Leishmania donovani Isolates with Antimony-Resistant but Not -Sensitive Phenotype Inhibit Sodium Antimony Gluconate-Induced Dendritic Cell Activation

Abstract

The inability of sodium antimony gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb^RLD) and antimony-sensitive (Sb^SLD) was compared in vitro. Unlike Sb^SLD, infection of DCs with Sb^RLD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb^RLD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-κB pathways. In contrast, Sb^SLD failed to block activation of SAG (20 µg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-κB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb^SLD also inhibited SAG (20 µg/ml)-induced activation of PI3K/AKT and NF-κB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 µg/ml. In contrast, Sb^RLD inhibited these SAG-induced events regardless of duration of DC exposure to Sb^RLD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb^SLD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb^RLD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-κB was found to transcriptionally regulate expression of murine γglutamylcysteine synthetase heavy-chain (mγGCS_hc) gene, presumably an important regulator of antimony resistance. Importantly, Sb^RLD but not Sb^SLD blocked SAG-induced mγGCS expression in DCs by preventing NF-κB binding to the mγGCS_hc promoter. Our findings demonstrate that Sb^RLD but not Sb^SLD prevents SAG-induced DC activation by suppressing a PI3K-dependent NF-κB pathway and provide the evidence for differential host-pathogen interaction mediated by Sb^RLD and Sb^SLD. Kala-azar, a life-threatening parasitic disease caused by Leishmania donovani (LD), is widening its base in different parts of the world. Currently, there is no effective vaccine against kala-azar. The antimonial drugs like sodium antimony gluconate (SAG) have been the mainstay of therapy for this disease. Recently, due to the emergence of antimony-resistance in parasites, SAG often fails to cure kala-azar patients, which is compounding the disaster further. It is still unknown how infection with LD exhibiting antimony-resistant phenotype, in contrast to antimony-sensitive phenotype, is handled by the kala-azar patients upon SAG treatment. This demands an understanding of the nature of host immune responses against these two distinct categories of parasites. Accordingly, we compared the impact of infection with LD exhibiting antimony-resistant versus antimony-sensitive phenotype on dendritic cells (DCs). DCs upon activation/maturation initiate anti-leishmanial immunity. We showed that parasites with antimony-resistant but not antimony-sensitive phenotype prevented SAG-induced DC activation/maturation by blocking activation of NF-κB. The latter is a key signaling pathway regulating DC activation/maturation. Our studies for the first time provide both a cellular and molecular basis for differential response of host cells to parasite isolates with antimony-resistant and antimony-sensitive phenotype, which may influence the outcome of the disease.