Cryptosporidium parvum-induced neutrophil extracellular traps in neonatal calves is a stage-independent process

Abstract

Introduction: Infections with the apicomplexan obligate intracellular parasite Cryptosporidium parvum lead to cryptosporidiosis-a worldwide zoonotic infection. C. parvum is one of the most common diarrheal pathogens in young calves, which are the main reservoir of the pathogen. Cryptosporidiosis leads to severe economic losses in the calf industry and being a major contributor to diarrhea morbidity and mortality in children. Polymorphonuclear neutrophils (PMN) are part of the innate immune system. Their effector mechanisms directed against invasive parasites include phagocytosis, production of antimicrobial molecules as well as the formation of so-called neutrophil extracellular traps (NETs). Like other leukocytes of the innate immune system, PMN are thus able to release chromatin fibers enriched with antimicrobial granular molecules extracellularly thereby immobilizing and partially killing invasive bacteria, viruses, fungi and parasites.

Methods: In vitro interactions of neonatal bovine PMN and C. parvum-oocysts and sporozoites were illustrated microscopically via scanning electron microscopy- and live cell imaging 3D holotomographic microscopy analyses. C. parvum-triggered NETosis was quantified via extracellular DNA measurements as well as verified via detection of NET-typical molecules [histones, neutrophil elastase (NE)] through immunofluorescence microscopy analysis. To verify the role of ATP in neonatal-derived NETosis, inhibition experiments were performed with NF449 (purinergic receptor antagonist with high specificity to P2X1 receptor).

Results and discussion: Using immunofluorescence- and SEM-based analyses, we demonstrate here for the first time that neonate bovine PMN are capable of forming NETs against C. parvum-sporozoites and oocysts, thus as a stage-independent cell death process. Our data further showed that C. parvum strongly induces suicidal neonatal NETosis in a P2X1-dependent manner, suggesting anti-cryptosporidial effects not only through firm sporozoite ensnarement and hampered sporozoite excystation, but also via direct exposure to NETs-associated toxic components.

Keywords: Cryptosporidium parvum; NETosis; calves; neonates; polymorphonuclear neutrophils.

Figure 1

C. parvum oocysts induce neonatal PMN F-actin polymerization. In total, 1 x 10⁵ PMN were analyzed by flow cytometry using Alexa Fluor 488 phalloidin to detect F-actin. A shift to the right of the mean fluorescence intensity (MFI) is observed in the C. parvum-confronted condition (A). The analysis of the fluorescence intensity as mean ± SD confirms this observation (B). Statistical significance was defined as p < 0.05 applying a Mann–Whitney test (n = 3).

Figure 2

3D holotomographic microscopy of neonatal bovine PMN confronted with C. parvum. Overall, 1 × 10⁶ neonatal PMN were incubated in RPMI media containing 0.5% BSA and DRAQ5 2μM. Cells were registered using 3D holotomographic microscopy based on RI. (A) The nuclei stained by DRAQ5 and the merge of RI and DRAQ5 fluorescence channels are shown. (B) The digital staining based on the RI and 3D reconstruction is shown in the lower part.

Figure 3

C. parvum-induced NETs in bovine neonatal PMN. Overall, 2 x 10⁵ non-stimulated neonatal PMN (upper panel) or C. parvum oocysts-confronted neonatal PMN (lower panel) were fixed after 180min, and the specimens were analyzed by scanning electron microscopy (SEM).

Figure 4

Stage-independent induction of bovine neonatal NETs by C. parvum oocysts and sporozoites analyzed via confocal microscopy. In total, 2 x 10⁵ neonatal bovine PMN were stimulated with oocysts and sporozoites of C. parvum for 180min and fixed. Shows NET formation by co-localization of DNA, blue, histones, red, and neutrophil elastase, green. Shows the positive control: neonatal bovine PMN with Ca²⁺ ionophore.

Figure 5

C. parvum-induced bovine neonatal NET is dependent on purinergic ATP binding. In total, 2 x 10⁵ neonatal bovine PMN were pre-treated with the inhibitor NF449 (10μM) for 30min, and C. parvum oocysts were added. Furthermore, there were two control groups: non-stimulated neonatal bovine PMN and neonatal bovine PMN, co-cultured with C. parvum oocysts. The DNA release was significantly inhibited when NF449 was used. p-values were calculated using ANOVA, followed by Dunnett's multiple comparison post-hoc test.