Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent

Abstract

Eimeria bovis is an important coccidian parasite that causes high economic losses in the cattle industry. We recently showed that polymorphonuclear neutrophils (PMN) react upon E. bovis sporozoite exposure by neutrophil extracellular trap (NET) formation. We focused here on the molecular mechanisms that are involved in this process. The sporozoite encounter led to an enhanced surface expression of neutrophil CD11b suggesting a potential role of this receptor in E. bovis-mediated NETosis. Antibody-mediated blockage of CD11b confirmed this assumption and led to a significantly decreased sporozoite-triggered NET. In addition, E. bovis-induced NETosis was found to be Ca2+-dependent since the inhibition of store-operated calcium entry (SOCE) significantly diminished NET. Furthermore, NADPH oxidase, neutrophil elastase (NE) and myeloperoxidase (MPO) were confirmed as key molecules in sporozoite-triggered NETosis, as inhibition thereof blocked parasite-triggered NET. PMN degranulation analyses revealed a significant release of matrix metalloprotease-9 containing granules upon sporozoite exposure. We further show a significantly enhanced phosphorylation of ERK1/2 and p38 MAPK in sporozoite-exposed PMN indicating a key role of this signaling pathway in E. bovis-mediated NETosis. Accordingly, ERK 1/2 and p38 MAPK inhibition led to a significant decrease in NET formation. Finally, we demonstrate that sporozoite-induced NETosis is neither a stage-, species-, nor host-specific process.

Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility

“…The cells were then washed by filling up the Falcon tubes with PBS and centrifuging for 10 minutes at 300 Â g. Finally, the supernatant was removed and the cell pellets were resuspended in 2 mL of PBS. PMN were counted with a Neubauer hemocytometer (6).…”

“…NETs are released after PMN cell death process, reported as NETosis, and are primarily situated in the extracellular space. NETosis has been shown to be an NADPH oxidase (NOX)-dependent mechanism, which ultimately leads to the extrusion of a mixture of nuclear as well as cytoplasmic granule contents resulting in the formation of DNA-rich web-like structures that are adorned with histones (H1, H2A/H2B, H3, H4) and granular effector molecules, such as neutrophil elastase (NE), pentraxin, lactoferrin, MPO, and others (5,6). Unlike NOXdependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of ERK activation and a rather moderate level of Akt activation, whereas the activation of p38 is similar in both pathways (7).…”

Suitable in vitro culture of Eimeria bovis meront II stages in bovine colonic epithelial cells and parasite-induced upregulation of CXCL10 and GM-CSF gene transcription

“…This vital staining has been successfully used before in case of E. bovis Muñoz-Caro et al 2015), Eimeria arloingi , and Besnoitia besnoiti (Muñoz-Caro et al 2014a, b). Infections of bovine endothelial cells with CFSE-stained sporozoites resulted in fluorescent macromeronts, which were traceable inside the host cell Fig.…”

“…Taking into account that Eimeria infections are highly hostand host cell-specific and that stage-specific innate immune reactions are a common feature Muñoz-Caro et al 2015), it appears essential that basic research is performed on different developmental stages in the respective host cell type (Hermosilla et al 2012). Nonetheless, fully permissive in vitro systems, representing one important prerequisite for any basic investigation related to specific parasite stages-host cell interactions, allowing for the production of any stage of question are still lacking for E. bovis merozoites I/II or gamonts while they have been successfully established for chicken Eimeria species, e.g., Eimeria tenella (Hofmann and Raether 1990).…”

Beneficial and Deleterious Effects of Neutrophil Extracellular Traps on Infection

“…This parasite stage of E. bovis seems to be a beter inducer of NETs than PMA. NETs have been shown to diminish infection by parasite immobilization and also by parasite killing, although to a lesser extent [38,39].…”