The role of NETosis and ROS production in human neutrophil-mediated host defense against Toxoplasma gondii infection

Toxoplasma gondii has the remarkable ability to infect and replicate in neutrophils. These innate immune cells are rapidly recruited to sites of T. gondii infection and, at first glance, would appear to be an inhospitable option for an intracellular parasite since neutrophils have several antimicrobial effector mechanisms. Nevertheless, studies have shown that, in T. gondii-infected hosts, a high proportion of neutrophils in the intestine and lymph nodes contain replicating parasites. Despite evidence of neutrophil contributions to immunity against T. gondii, the interactions of T. gondii with neutrophils are still poorly understood and have been primarily studied in the mouse. Here, we differentiated the human promyelocytic cell line HL-60 into neutrophil-like cells (NLCs) and focused on characterizing two neutrophil effector functions during acute T. gondii infection: the release of neutrophil extracellular traps (NETs) and the production of reactive oxygen species (ROS). NETs are web-like chromatin structures that can trap and kill pathogens in the extracellular space. We first observed the presence of extracellular DNA in T. gondii-infected neutrophil cultures by using Sytox Orange, a cell-impermeable dye. Next, we confirmed that the increase in Sytox fluorescence signal was due to NETs, as opposed to merely cell death, by staining the cells with an anti-citrullinated histone 3 antibody. We also demonstrated that neutrophils remain viable after undergoing NETosis, as assessed by staining the cells with a live dye and performing a cytotoxicity assay measuring lactate dehydrogenase (LDH) activity. Our current experiments are characterizing the release of DNA through vesicles, as opposed to membrane lysing, and the effectiveness of NETosis in killing T. gondii parasites. In parallel, we were able to observe the production and release of ROS from T. gondii-infected NLCs. Analysis of assembling and activation of the NADPH oxidase suggested that T. gondii-induced ROS in neutrophils is mainly dependent on NOX2 activity. Our current experiments are focused on characterizing the cellular events that lead to ROS production, as well as the contributions of it to other effector functions. Together, these findings expand our understanding of the neutrophil-mediated host defense against T. gondii and contribute to characterizing the very early immune events that affect the progression of the host response and the disease.