Bovine monocyte extracellular traps (METs) formed against Cryptosporidium parvum under physioxic and hyperoxic conditions

Abstract text

Introduction: Cryptosporidiosis is caused by the zoonotic apicomplexan protozoa Cryptosporidium parvum infecting small intestinal epithelial cells. Referring to early host innate immune reactions during cryptosporidiosis, little is yet known. Nonetheless, previous studies showed that human and bovine NETosis – an important host innate effector mechanism of polymorphonuclear neutrophils (PMN)- is elicited by C. parvum stages (Muñoz-Caro et al., 2015). Conversely to PMN, very little is known on monocyte-derived effector mechanisms against this enteric parasite. Objectives: Aim of the current study was to investigate the release of monocyte extracellular traps (METs) against two C. parvum stages, i. e. oocysts and sporozoites, under physioxia and hyperoxia. Additionally, the role of monocarboxylate transporters (MCTs), Notch pathway signalling and purinergic signalling via the P2X1 receptor for C. parvum-triggered METosis was evaluated. Materials and methods: C. parvum oocysts and sporozoites were exposed to bovine monocytes to investigate METs under hyperoxic (21% O2) and intestinal physioxic (5% O2) conditions. For the detection of C. parvum-triggered METosis, immunofluorescence, confocal, 3D holotomographic and scanning electron microscopic analyses were performed. The role of MCT1, MCT2, P2X1 and Notch pathway in C. parvum-mediated METosis was studied via functional inhibition assays by pretreating monocytes with the inhibitors AR-C141900, ARC155858 and NF449, Compound E and DAPT, respectively. Results: Exposure of monocytes to both parasite stages resulted in suicidal METosis as confirmed by SEM and Live cell 3D holotomographic microscopy. Immunofluorescence and confocal microscopic analyses unveiled that METs were capable to entrap parasites. Pre-treatments of monocytes with MCT inhibitors reduced MET formation after parasite exposure under both oxygen conditions, but not significantly. The same held true for NF449 pre-treatments resulting in the barely significant reduction of C. parvum-triggered METosis. Regarding Notch pathway, reduction was not significant in both oxygen conditions, but it seems that Compound E is more relevant to METosis Conclusions: Exposure of C. parvum sporozoites and oocysts to bovine monocytes leads to suicidal METosis under physioxia and hyperoxia. Overall and in contrast to neutrophil-mediated reactions,P2X1- and MCT-mediated pathways seem of minor importance in C. parvum-induced suicidal METosis.