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Leishmania donovani attachment stimulates PKC-mediated oxidative events in bone marrow-derived macrophages
A K Bhunia1, D Sarkar, P K Das
1Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India.
Abstract:
We investigated activation signaling events in bone marrow-derived macrophages after infection with Leishmania donovani, an intracellular parasite of macrophages. Leishmania donovani infection caused a general suppression of activation parameters like O2- and NO production. However, conditions which allow parasite attachment and prevent entry resulted in triggering of O2- and NO production and stimulation of O2 consumption. Optimal NO and O2- production occurred when bone marrow-derived macrophages and Leishmania ratio was 1:100. The activation signal for O2- production was initiated 15 min after parasite attachment, whereas augmentation of NO production started 6 h after attachment Activation of O2- and NO generation by L. donovani attachment was inhibited by staurosporine as well as by prolonged treatment of phorbol myristate acetate suggesting a protein kinase C-dependent mechanism. Translocation studies showed that protein kinase C activity in cell membrane fraction rapidly and transiently increased following parasite attachment. No such protein kinase C translocation event occurred in L. donovani infected bone marrow-derived macrophages. Phorbol myristate acetate was found to stimulate membrane translocation of protein kinase C in parasite attached cells whereas it was impaired in infected cells. However, both attachment and infection induced a similar shift of phorbol receptors from cytosolic to membrane fraction indicating that in infected cells the translocation of protein kinase C protein was not impaired but the activity of the membrane associated enzyme was somehow inhibited. These results suggest that although internalization of intracellular parasites like L. donovani caused inhibition of nitrite and superoxide release, mere attachment on macrophage surface resulted in an activation of protein kinase C-mediated downstream oxidative events.
Insights
Leishmania donovani infection suppresses macrophage activation. However, parasite attachment alone triggers superoxide and nitric oxide production via protein kinase C activation, despite infection inhibiting enzyme activity.
Area of Science:
- Immunology
- Cell Biology
- Parasitology
Background:
- Macrophages are key immune cells that phagocytose and eliminate intracellular pathogens.
- Leishmania donovani is an intracellular parasite that infects macrophages, modulating host cell functions.
- Macrophage activation involves oxidative burst responses, including superoxide (O2-) and nitric oxide (NO) production.
Purpose of the Study:
- To investigate the signaling events in bone marrow-derived macrophages (BMMs) upon Leishmania donovani infection.
- To determine the role of protein kinase C (PKC) in macrophage activation during Leishmania donovani interaction.
- To differentiate the effects of parasite attachment versus internalization on macrophage activation pathways.
Main Methods:
- Bone marrow-derived macrophages were co-cultured with Leishmania donovani at varying ratios.
- Production of O2- and NO was measured following infection or parasite attachment.
- Protein kinase C activity and translocation were assessed using biochemical assays and cell fractionation.
- Inhibition studies were performed using staurosporine and phorbol myristate acetate (PMA).
Main Results:
- Leishmania donovani infection generally suppressed O2- and NO production in BMMs.
- Parasite attachment alone, without entry, triggered significant O2- and NO production, peaking at a 1:100 BMM to Leishmania ratio.
- PKC activation, evidenced by membrane translocation, was observed upon parasite attachment but inhibited during infection.
- PMA stimulation confirmed that while PKC translocation occurred in attached cells, its activity was impaired in infected cells.
Conclusions:
- Macrophage activation via O2- and NO production is differentially regulated by Leishmania donovani attachment and internalization.
- Parasite attachment initiates PKC-dependent oxidative signaling, whereas internalization leads to PKC-mediated inhibition of these responses.
- Understanding these distinct signaling pathways is crucial for developing strategies against Leishmania infections.