Giardia intestinalis trophozoites activate human PMN and induce NET formation but dampen neutrophil ROS production.

INTRODUCTION: Giardia intestinalis is a zoonotic enteric protozoan parasite causing giardiasis in humans, domestic animals and wildlife. More than 300 million human cases of diarrhea due to giardiasis have annually been reported. Despite its high global prevalence, human polymorphonuclear neutrophil (PMN)-mediated early innate immune responses against G. intestinalis remain poorly investigated. This study aimed to evaluate whether vital G. intestinalis trophozoites activate PMN and foster neutrophil metabolic responses, thereby eventually driving NET formation. METHODS: Human PMN were exposed to G. intestinalis trophozoites and Giardia-derived excretory/secretory products (ESPs); stimulation of PMN with PMA served as positive control for both NET induction and neutrophil oxidative (OCR) and glycolytic (PER) responses. RESULTS AND DISCUSSION: NET release was illustrated by scanning electron microscopy (SEM), confirmed and quantified by fluorescence microscopy via the co-localization of histone, neutrophil elastase (NE) and extracellular DNA. PMN activation and metabolic responses were assessed at the level of oxygen consumption rates (OCR), proton efflux rates (PER), and ROS production. Microscopy analyses showed that vital G. intestinalis trophozoites activated PMN, triggering neutrophil phagocytosis and NET-based entrapment of trophozoites. Furthermore, the presence of PMN in trophozoite growth cultures dampened parasite replication efficacy. Trophozoite exposure fostered both OCR and glycolytic PER responses in PMN but failed to drive neutrophil ROS production. To investigate whether the lack of ROS production is a Giardia-mediated immune evasion strategy, the ability of G. intestinalis trophozoites to inhibit PMA-induced ROS generation in neutrophils was assessed. Trophozoites significantly diminished PMA-driven ROS production, impairing a key PMN effector mechanism. To elucidate if these effects were based on parasite-derived products, Giardia-ESPs were tested for their effects on neutrophil metabolic responses and PMA-mediated ROS production. No changes were observed, excluding ESPs-driven effects. In conclusion, our results showed that G. intestinalis trophozoites activate human PMN on an oxidative- and glycolytic level, stimulating them to extrude NETs and/or to engage in phagocytosis, and to impair parasite's binary fission.