C3/CD11b-Mediated Leishmania major Internalization by Neutrophils Induces Intraphagosomal NOX2-Mediated Respiratory Burst but Fails to Eliminate Parasites and Induces a State of Stalled Apoptosis.

Recruited neutrophils are among the first phagocytic cells to interact with the phagosomal pathogen Leishmania following inoculation into the mammalian dermis. Analysis of Leishmania-infected neutrophils has revealed alterations in neutrophil viability, suggesting that the parasite can both induce or inhibit apoptosis. In this study, we demonstrate that entry of Leishmania major into murine neutrophils is dependent on the neutrophil surface receptor CD11b (CR3/Mac-1) and is enhanced by parasite opsonization with C3. Infected neutrophils underwent robust NADPH oxidase isoform 2 (NOX2)-dependent respiratory burst based on detection of reactive oxygen species within the phagolysosome but largely failed to eliminate the metacyclic promastigote life cycle stage of the parasite. Infected neutrophils displayed an "apoptotic" phosphatidylserine (PS)-positive phenotype, which was induced by both live and fixed parasites but not latex beads, suggesting that PS expression was parasite specific but does not require active infection. In addition, neutrophils from parasite/neutrophil coculture had increased viability, decreased caspase 3, 8, and 9 gene expression, and reduced protein levels of both the pro and cleaved forms of the classical apoptosis-inducing executioner caspase, Caspase 3. Our data suggest that CD11b-mediated Leishmania internalization initiates respiratory burst and PS externalization, followed by a reduction in both the production and cleavage of caspase 3, resulting in a phenotypic state of "stalled apoptosis."

NOX2-Derived Reactive Oxygen Species Control Inflammation during Leishmania amazonensis Infection by Mediating Infection-Induced Neutrophil Apoptosis.

Reactive oxygen species (ROS) produced by NADPH phagocyte oxidase isoform (NOX2) are critical for the elimination of intracellular pathogens in many infections. Despite their importance, the role of ROS following infection with the eukaryotic pathogen Leishmania has not been fully elucidated. We addressed the role of ROS in C57BL/6 mice following intradermal infection with Leishmania amazonensis. Despite equivalent parasite loads compared with wild-type (WT) mice, mice deficient in ROS production by NOX2 due to the absence of the gp91 subunit (gp91phox-/-) had significantly more severe pathology in the later stages of infection. Pathology in gp91phox-/- mice was not associated with alterations in CD4+ T cell-mediated immunity but was preceded by enhanced neutrophil accumulation at the dermal infection site. Ex vivo analysis of infected versus uninfected neutrophils revealed a deficiency in infection-driven apoptosis in gp91phox-/- mice versus WT mice. gp91phox-/- mice presented with higher percentages of healthy or necrotic neutrophils but lower percentages of apoptotic neutrophils at early and chronic time points. In vitro infection of gp91phox-/- versus WT neutrophils also revealed reduced apoptosis and CD95 expression but increased necrosis in infected cells at 10 h postinfection. Provision of exogenous ROS in the form of H2O2 reversed the necrotic phenotype and restored CD95 expression on infected gp91phox-/- neutrophils. Although ROS production is typically viewed as a proinflammatory event, our observations identify the importance of ROS in mediating appropriate neutrophil apoptosis and the importance of apoptosis in inflammation and pathology during chronic infection.