RIPK1-RIPK3-MLKL-Associated Necroptosis Drives Killing in Neutrophils.

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 , 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 . Human neutrophils pretreated with zVAD-fmk (pan-caspase inhibitor) and zIETD-fmk (caspase-8 inhibitor) increased reactive oxygen species (ROS) level in response to infection, which is associated with necroptotic cell death. MLKL, an important effector molecule downstream of necroptosis pathway, was also required for killing. Moreover, in absence of caspases-8, murine neutrophils displayed loss of membrane integrity, higher levels of ROS, and decreased 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 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 within parasitophorous vacuoles. Taken together, these findings suggest that inhibition of caspase-8 contributes to elimination of in neutrophils by triggering necroptosis. Thus, targeting necroptosis may represent a new strategy to control replication.