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

Abstract

Necroptosis is a pro-inflammatory cell death, which happens in the context of caspase-8 inhibition, allowing activation of the receptor interacting protein kinase 1–receptor interacting protein kinase 3–mixed lineage kinase domain-like (RIPK1–RIPK3–MLKL) axis. Recently, necroptosis has emerged as a key component of resistance against pathogens including infected macrophage by Leishmania infantum, the ethiologic agent of Visceral leishmaniasis (VL). VL is the most severe form of Leishmaniasis, characterized by systemic inflammation and neutropenia. However, the role of neutrophil cell death in VL has not been characterized. Here, we showed that VL patients exhibited increased lactate dehydrogenase levels in the serum, a hallmark of cell death and tissue damage. We investigated the effect of necroptosis in neutrophil infection in vitro. Human neutrophils pretreated with zVAD-fmk (pan-caspase inhibitor) and zIETD-fmk (caspase-8 inhibitor) increased reactive oxygen species (ROS) level in response to Leishmania infection, which is associated with necroptotic cell death. MLKL, an important effector molecule downstream of necroptosis pathway, was also required for Leishmania killing. Moreover, in absence of caspases-8, murine neutrophils displayed loss of membrane integrity, higher levels of ROS, and decreased L. infantum viability. Pharmacological inhibition of RIPK1 or RIPK3 increased parasite survival when caspase-8 was blocked. Electron microscopy assays revealed morphological features associated with necroptotic death in L. infantum infected-neutrophils pretreated with caspase inhibitor, whereas infected cells pretreated with RIPK1 and RIPK3 inhibitors did not show ultra-structural alterations in membrane integrity and presented viable Leishmania within parasitophorous vacuoles. Taken together, these findings suggest that inhibition of caspase-8 contributes to elimination of L. infantum in neutrophils by triggering necroptosis. Thus, targeting necroptosis may represent a new strategy to control Leishmania replication.

Infection of Human Neutrophils With Leishmania infantum or Leishmania major Strains Triggers Activation and Differential Cytokines Release

“…Further studies are necessary to further investigate apoptosis induced by these species and cellular mechanisms involved in death. Notably L. infantum infected neutrophils were shown to undergo necroptosis in presence of specific caspase 8 inhibitor (and so when apoptosis was inhibited) (Barbosa et al, 2018).…”

miR-425 deficiency promotes necroptosis and dopaminergic neurodegeneration in Parkinson’s disease

“…In particular, RIPK1, a death domain-containing Ser/Thr kinase, has an established role in mediating the deleterious mechanisms downstream of type I tumor necrosis factor α receptor (TNFR1) 10 . Activated RIPK1 and RIPK3 form the necrosome complex and then recruit MLKL, leading to necroptosis execution and mitochondrial membrane disintegration 11,12 . The involvement of necroptosis is reported in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer’s disease 6,13,14 .…”

The Caspase Inhibitor Z-VAD-FMK Alleviates Endotoxic Shock via Inducing Macrophages Necroptosis and Promoting MDSCs-Mediated Inhibition of Macrophages Activation

“…To date, the effect of zVAD-mediated necroptosis in the regulation of inflammatory diseases has remained controversial and zVAD may play multiple roles in inflammatory diseases (21). Although many studies have shown that zVAD-mediated necroptosis contributes to the pathogenesis of inflammation, some other studies indicated that necroptosis plays an antiinflammatory role by preventing specific viral and bacterial infections, and could contribute to tissue generation (22,23). For example, zVAD showed positive effect on lung injury in the severe acute pancreatitis model (SAP) in rats, but had a negative effect in severe pneumovirus disease in mice (24,25).…”