Induced necroptosis limits Toxoplasma gondii replication in a RIPK3/MLKL-dependent manner

Toxoplasma gondii is an obligate intracellular parasite capable of subverting host defenses to establish infection. Necroptosis, a lytic pro-inflammatory form of programed cell death, has emerged as a host defense mechanism against intracellular pathogens. However, its relevance in controlling T. gondii replication remains unclear. Here, we investigated the role of necroptosis in limiting T. gondii replication using bone marrow-derived macrophages (BMDMs) deficient in key necroptotic mediators, RIPK3 and MLKL. We demonstrate that under naïve conditions, T. gondii replication proceeds unimpeded in RIPK3^-/- and MLKL^-/- BMDMs. However, co-treatment with TNF-α and the pan-caspase inhibitor Z-VAD-FMK, conditions that promote necroptosis, significantly reduced parasite replication in wild-type BMDMs but not in those lacking RIPK3 or MLKL. This suppression was dependent on RIPK1 activity, as pharmacological inhibition with Necrostatin-1 abrogated the effect. We further confirmed that TNF-α and Z-VAD-FMK treatment induced necroptotic cell death characterized by loss of plasma membrane integrity, both of which were absent in RIPK3^-/- and MLKL^-/- cells. These findings establish that the activation of necroptosis can effectively limit T. gondii replication in BMDMs and underscore the importance of RIPK1-RIPK3-MLKL signaling in mounting a cell-intrinsic immune defense. Our study provides new insight into the functional capacity of necroptosis in restricting intracellular parasites and highlights its potential as a therapeutic target in toxoplasmosis.