Toxoplasma gondii is an intracellular parasite that scavenges host lipids, including polyunsaturated fatty acids (PUFAs), which are prone to peroxidation by reactive oxygen species, generating membrane-damaging lipid peroxides. As lipid peroxides threaten both host cells and intracellular pathogens, understanding the role of host antioxidant systems during infection is crucial in identifying potential therapeutic targets. A CRISPR knockout (KO) screen for host genes that modulate parasite proliferation identified the lipid peroxide reducing protein glutathione peroxidase 4 (GPX4) as a candidate promoter of T. gondii growth. While GPX4 protects host cells against PUFA-derived lipid oxidation and ferroptotic cell death, its effects on T. gondii remain unknown. We hypothesize that GPX4 promotes T. gondii growth by preventing lipid peroxide-mediated damage. Using tamoxifen-inducible GPX4 KO mouse embryonic fibroblasts, we found that GPX4 depletion decreases T. gondii burden and that the lipid peroxidation inhibitor Ferrostatin-1 (Fer-1) rescues this defect. Confocal microscopy revealed oxidized lipids accumulate in T. gondii upon PUFA addition. Lipidomics showed PUFAs are released from triglycerides during infection and incorporated into diacyl-PUFA phospholipids. PUFA supplementation to infected cells inhibits T. gondii growth, while Fer-1 reversed this defect. Our data show that GPX4 promotes parasite growth and that defects caused by GPX4 depletion and PUFA supplementation result from lipid peroxide accumulation in T. gondii.

Red blood cell (RBC) transfusions are not fully antigen-matched, which can lead to RBC alloimmunization. This immune response may cause hemolytic complications and limit transfusion compatibility. Inflammation can increase alloimmunization risk, and murine models show that type I interferons (IFNs) are necessary for inflammation-induced alloantibody formation, though the responsible subtype remains unclear. To determine if IFNɑ2 is sufficient to promote RBC alloimmunization, recombinant IFNɑ2 was administered with transfusion of RBCs expressing the KEL alloantigen. KEL+ RBCs were transfused into WT recipients, and anti-KEL IgM and IgG were measured by flow cytometry. To evaluate effects on RBC survival, a second fluorescently labeled transfusion was performed and circulating KEL+ RBCs were tracked. WT mice with recombinant IFNɑ2 exhibited significantly higher anti-KEL IgG responses, demonstrating that IFNɑ2 is sufficient to promote RBC alloimmunization. To determine if IFNɑ2 is required, KEL+ RBCs were transfused into WT and IFNɑ2 KO recipients treated with or without (poly I:C), to mimic IFN-mediated inflammation. No significant differences were observed, except (poly I:C) increased IgG to similar levels in WT and IFNɑ2 KO mice. (Poly I:C) treated IFNɑ2 KO mice showed intermediate clearance, suggesting partially improved post-transfusion survival. Our findings indicate that IFNα2 is sufficient but not required to promote inflammation-induced RBC alloimmunization, suggesting compensatory signaling by other type I IFNs.

This abstract has been withheld from publication.

Toxoplasma gondii is an obligate intracellular parasite infecting nearly one-third of the human population. Acute infections consist of a repetitive lytic cycle of invasion, replication, and egress. T. gondii replicates via endodyogeny, a unique process mediated by the centrosomes in which two daughter buds form simultaneously within one maternal parasite. At the beginning of replication, the centrosomes tether to a tubulin-based organelle, the conoid, to serve as a scaffold for daughter bud assembly. The conoid is part of the apical complex, a group of organelles at the parasite’s apex that mediate invasion and egress. While the apical complex forms early in division, components that are specific to developing daughter parasites and how they mediate bud assembly are unknown. We have previously localized a novel T. gondii protein, Daughter Conoid Protein 1 (DCP1), to the conoid of daughter buds. Examination of DCP1 with centrosome and apical complex markers reveal the protein is expressed immediately upon the initiation of endodyogeny and remains in the daughter conoid until the buds reach maturity. Knockout of DCP1 leads to severe defects in parasite growth, morphology, and replication, and examination of the centrosomes in the knockout suggests DCP1 is important for proper division checkpoints. Therefore, this study provides new insight into the regulation of T. gondii endodyogeny and identifies putative targets for the design of novel therapeutics that can specifically target the parasite.

Resident macrophages are innate immune cells that are constantly exposed to pathogens, yet the impact of recurrent endotoxin exposure on human healthspan has not been studied. We hypothesize the long-term impact of a remodeled epigenome establishes a distinct heritable program that drives immunocompromised states in macrophages throughout aging. This study examines whether macrophages can survive repeated endotoxin stimulation in vitro. Additionally, we aim to delineate the molecular mechanisms of endotoxin induced epigenetic remodeling in a macrophage. To test this, primary macrophages derived from the bone marrow were stimulated repeatedly with endotoxin, then assessed for acute cell death, cellular senescence, and the capacity to restore metabolic functions. We found that repeated exposure to endotoxin did not induce macrophage senescence or cell death; DNA damage resolved and macrophages regained proliferative capacity. However, progeny retained a persistent maladaptive memory characterized by diminished effector function. Future work will test whether epigenetic perturbations can restore chromatin accessibility and evaluate infection outcomes using reporter mice, labeled bacteria, and graft infection models to further define the physiological impact of maladaptive macrophage memory. This work will provide an understanding on how repeated inflammatory challenges impair bacterial clearance in aging and identify key mechanisms to reverse the effects of an aged immune system.