Abstract
One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H2O2 for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H2O2-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H2O2 treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.
Highlights
Leishmania inhibits oxidative burst-mediated apoptosis of macrophages during phagocytosis
L. donovani infection was found to cause 68.8 Ϯ 8.4, 71.6 Ϯ 5.8, 61.6 Ϯ 7.4, and 40.2 Ϯ 3.2% reactive oxygen species (ROS) production in RAW 264.7 macrophages at 5, 10, 15, and 30 min post-infection, respectively (Fig. 1A). To determine whether this initial oxidative outburst could lead to macrophage apoptosis, cells were infected with L. donovani for the indicated time points, washed to remove un-internalized parasites, and incubated overnight at 37 °C, and the percentage of apoptotic cells was measured by annexin V-PI flow cytometric analysis
To determine whether this amount of ROS produced during early hours of L. donovani infection was capable of causing macrophage apoptosis, cells were administered with the indicated concentrations of H2O2 to mimic the initial oxidative burst condition in the case of L. donovani during 5–30 min of infection
Summary
Leishmania inhibits oxidative burst-mediated apoptosis of macrophages during phagocytosis. It was observed that infected cells were capable of ROS production during early hours, there was complete abrogation of the downstream caspase cascade that was found to be mediated by SOCS proteins Silencing of these proteins resulted in reduced thioredoxin levels and increased apoptosis in infected macrophages through de-activation of PTPs. SOCS knockdown cells displayed decreased parasite survival, marking reduction in disease progression. SOCS knockdown cells displayed decreased parasite survival, marking reduction in disease progression Taken together, these results suggest that L. donovani employs differential induction of host SOCS proteins to subvert macrophage apoptotic machinery triggered by parasite internalization-mediated oxidative burst, establishing its replicative niche inside the host
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