Giardia duodenalis trophozoites triggered bovine neutrophil extracellular traps formation dependent on P2X1 receptor and PAD4 in vitro

Abstract

Giardia duodenalis is an important intestinal protozoan parasite, infections of which are frequently seen in cattle and cause intermittent diarrhea and weight loss in young animals around the world. The release of neutrophil extracellular traps (NETs) is an effector mechanism of neutrophils to fight against invading pathogens including parasites. In this present study, we aimed to investigate the effect of Giardia duodenalis trophozoites on bovine NETs formation, and to further examine its basic characteristics and molecular mechanisms. Scanning electron microscopy analyses displayed that Giardia duodenalis trophozoites exposure triggered NET-like filamentary structures released by bovine polymorphonuclear leukocytes (PMNs), and many trophozoites were entrapped within these structures. Immunofluorescence analyses illustrated that these structures were mainly composed of DNA, histones, Myeloperoxidase (MPO) and neutrophil elastase (NE), which confirmed the classical characteristics of NETs. NETs quantification showed that Giardia duodenalis trophozoites significantly increased NETs formation, and it is in a dose-dependent manner from 4:1–1:2 ratio of PMN: trophozoites. Furthermore, pharmacological inhibitory experiment indicated that P2X1 receptor and PAD4 were essential for Giardia duodenalis trophozoites-triggered NETs formation. Additionally, LC3B-based immunostaining analyses revealed that autophagy and NETs formation occurred simultaneously in Giardia duodenalis trophozoites-exposed bovine PMN, imply that autophagy may play a key role in Giardia duodenalis trophozoites-triggered bovine NETs. In summary, these findings suggest that NETs formation might have a crucial role in innate host defense against Giardia duodenalis trophozoites. Hence, we call for future molecular investigations not only on Giardia duodenalis trophozoites-triggered NETs but also on its potential role in giardiasis-related pathology in vivo.