Heme Drives Oxidative Stress-Associated Cell Death in Human Neutrophils Infected with Leishmania infantum

Abstract

Free heme is an inflammatory molecule capable of inducing migration and activation of neutrophils. Here, we examine the heme-driven oxidative stress-associated cell death mechanisms in human neutrophils infected with Leishmania infantum, an etiologic agent of visceral leishmaniasis (VL). We first performed exploratory analyses in a population of well characterized treatment-naïve VL patients as well as uninfected controls, who were part of previously reported studies. We noted a positive correlation between serum concentrations of heme with heme oxygenase-1 (HO-1) and lactate deydrogenase, as well as, a negative correlation between heme values and peripheral blood neutrophils counts. Moreover, in vitro infection with L. infantum in the presence of heme enhanced parasite burden in neutrophils, while increasing the production of reactive oxygen species and release of neutrophilic enzymes. Additional experiments demonstrated that treatment of infected neutrophils with ferrous iron (Fe+2), a key component of the heme molecule, resulted in increased parasite survival without affecting neutrophil activation status. Furthermore, stimulation of infected neutrophils with heme triggered substantial increases in HO-1 mRNA expression as well as in superoxide dismutase-1 enzymatic activity. Heme, but not Fe+2, induced oxidative stress-associated cell death. These findings indicate that heme promotes intracellular L. infantum survival via activation of neutrophil function and oxidative stress. This study opens new perspectives for the understanding of immunopathogenic mechanisms involving neutrophils in VL.

Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

“…Heme is an extremely toxic molecule, which can induce significant oxidative stress and cell death ( 33 ). Indeed, we have previously published that heme induces inflammatory cell death in neutrophils ( 34 ) and in macrophages ( 22 ) in the context of Leishmania infection in vitro . Importantly, heme is the canonical substrate of HO-1 ( 35 ), and accumulation of heme in tissues is directly associated with induction of this enzyme ( 36 ).…”

RIPK1–RIPK3–MLKL-Associated Necroptosis Drives Leishmania infantum Killing in Neutrophils

“…Neutrophil can play a positive ( 46 ) or negative role ( 47 ) in the outcome of the infection. We previously demonstrated the sophisticated interplay between innate immune response and different cell death pathways in Leishmania infection ( 28 , 36 , 48 , 49 ). Moreover, neutrophils can undergo necroptosis followed by the ligation of adhesion receptors under inflammatory conditions ( 50 , 51 ).…”

“…Neutrophils, monocytes, and macrophages can control parasites by ROS that are produced by the respiratory burst after phagocytosis ( 66 , 67 ). Moreover, recently our group shows that heme drives oxidative stress-associated cell death in human neutrophils infected by L. infantum ( 49 ). Also, in hemorrhagic shock models, it was demonstrated that exosomes released from macrophages promote neutrophil necroptosis mainly by NADPH oxidase-derived ROS production within neutrophils ( 68 ).…”

Leishmania braziliensis Subverts Necroptosis by Modulating RIPK3 Expression

“…Cells were infected with parasites at early stationary phase [multiplicity of infection (MOI): 10]. After 4 h, cells were washed and further cultured for 24 h. Cell culture medium was then replaced by Schneider medium and the plates were kept at 24°C for 5 days, when proliferating extracellular motile promastigotes were counted in a Neubauer hemocytometer, parasite load is represented as quantity of viable Promastigotes/mL ( Farias Luz et al, 2016 ; Quintela-Carvalho et al, 2017 ). Extraction of total RNA from macrophages was performed with Trizol.…”