title: NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.
abstract: Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca(++) concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum.
authors.affiliation: Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, Germany. Electronic address: Tamara.Munoz-Caro@vetmed.uni-giessen.de.
authors.forename: Tamara
authors.initials: T
authors.lastname: Muñoz-Caro
authors.affiliation: Institute of Parasitology, University of Leipzig, 04103 Leipzig, Germany. Electronic address: Matthias.Lendner@vetmed.uni-leipzig.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Lendner
authors.affiliation: Institute of Parasitology, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Arwid
authors.initials: A
authors.lastname: Daugschies
authors.affiliation: Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, Germany.
authors.forename: Carlos
authors.initials: C
authors.lastname: Hermosilla
authors.affiliation: Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Taubert
mesh_terms: D000818:Animals; D020013:Calcium Signaling; D002417:Cattle; D045744:Cell Line, Tumor; D016785:Cryptosporidium parvum / Q000276:immunology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D065206:Extracellular Traps / Q000469:parasitology / Q000502:physiology*; D006801:Humans; D007113:Immunity, Innate; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D019272:Leukocyte Elastase / Q000378:metabolism; D008297:Male; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology / Q000469:parasitology*; D009195:Peroxidase / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D034101:Sporozoites / Q000201:enzymology / Q000276:immunology; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D016209:Interleukin-8; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D009195:Peroxidase; D019255:NADPH Oxidases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019272:Leukocyte Elastase
keywords: Apicomplexa; Ca(2+) influx; Cryptosporidium parvum; ETosis; Neutrophil extracellular traps