(434) Investigating the role of the innate immune system in releasing neutrophil extracellular traps (NETs) in response to acute Toxoplasma gondii infection

Introduction/Rationale: Approximately one-third of the global human population is infected with the parasite Toxoplasma gondii. The immune system depends on neutrophils and one of their key mechanisms, neutrophil extracellular traps (NETs), to trap and neutralize T. gondii. Additionally, NETs help amplify both innate and adaptive responses to this parasite. NETs consist of DNA, myeloperoxidase, citrullinated histone 3, and elastase. Traditionally, it was believed that NET release caused neutrophil cell death. However, vital NETosis, which enables NETs while keeping the cell alive. While T. gondii induces NETs, the question remains whether they are released via vital NETosis. Furthermore, the cellular interactions and signals that trigger vital NETosis are unknown.

Methods: In this study, the promyelocytic cell line HL-60 was differentiated into neutrophil-like cells (NLCs) using retinoic acid and DMSO. NLCs were then infected with type I (RH) and type II (PA7) T. gondii at a multiplicity of infection of 2, in the presence of SYTOX, a dye that binds to extracellular DNA. Fluorescence levels were measured and compared with PMA-stimulated NLCs, which induce suicidal NETosis.

Results: Results showed an increase in extracellular DNA in T. gondii-infected NLCs. Immunofluorescence staining for DNA and citrullinated histone 3 at 4, 6, and 16 hours confirmed NET formation via confocal microscopy. The viability of T. gondii-infected NLCs at late time points (16 hours post-infection) was verified by live/dead viability staining and by measuring extracellular ATP. We observed a higher proportion of NET-releasing neutrophils that maintained membrane integrity, consistent with vital NETosis.

Conclusion: The next phase of this research will involve determining whether neutrophil complement receptors mediate T. gondii-induced vital NETosis. Understanding how neutrophils remain viable after entrapping T. gondii is essential to determine whether they can also deploy other effector mechanisms to prevent uncontrolled parasite replication.