ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by Cryptosporidium parvum under Physioxia Conditions

Abstract

Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by Cryptosporidium parvum. Neutrophil extracellular traps (NETs), also known as suicidal NETosis, are a powerful and ancient innate effector mechanism by which polymorphonuclear neutrophils (PMN) battle parasitic organisms like protozoa and helminths. Here, C. parvum oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN beneath both 5% O2 (physiological conditions within small intestinal tract) and 21% O2 (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O2) and hyperoxia (21% O2). Besides, C. parvum-induced suicidal NETosis was affirmed by total break of PMN, co-localization of extracellular DNA decorated with pan-histones (H1A, H2A/H2B, H3, H4) and neutrophil elastase (NE) by means of confocal- and immunofluorescence microscopy investigations. C. parvum-triggered NETs entrapped sporozoites and impeded sporozoite egress from oocysts covered by released NETs, according to scanning electron microscopy (SEM) examination. Live cell 3D-holotomographic microscopy analysis visualized early parasite-induced PMN morphological changes, such as the formation of membrane protrusions towards C. parvum while undergoing NETosis. Significant reduction of C. parvum-induced suicidal NETosis was measured after PMN treatments with purinergic receptor P2X1 inhibitor NF449, under both oxygen circumstances, this receptor was found to play a critical role in the induction of NETs, indicating its importance. Similarly, inhibition of PMN glycolysis via 2-deoxy glucose treatments resulted in a reduction of C. parvum-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in C. parvum-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce C. parvum-mediated NET extrusion. Concerning Notch signaling, no significant reduction was detected after PMN treatments with two specific Notch inhibitors, i.e., DAPT and compound E. Overall, we here describe for the first time the pivotal role of ATP purinergic receptor P2X1 in C. parvum-mediated suicidal NETosis under physioxia (5% O2) and its anti-cryptosporidial properties.

MCT-Dependent Cryptosporidium parvum-Induced Bovine Monocyte Extracellular Traps (METs) under Physioxia

“…Moreover, the gut is a distinct niche from other organs with its own physioxia, also described as normoxia, and with it being the largest human organ surface with numerous commensals, micro-and macroorganisms [79][80][81]. Physioxia has been shown to have implications not only in the production of C. parvum-triggered NETs [82] but also in the metabolism of bovine small intestinal explants (BSI) and primary bovine intestinal epithelial cells (BSIECs) infected with C. parvum [52,53]. Knowing this fact, the metabolic profile of suicidal METosis induced by C. parvum was investigated under two different oxygen conditions, namely physioxia (5% O 2 ) and hyperoxia (21% O 2 ).…”

Early immune and host cell responses to Cryptosporidium infection

“…In the same study, polymorphonuclear leukocyte cells (PMN)s isolated from C. parvum -infected mice were shown to play a role in converting resident macrophages to M1 macrophages. Another possible mechanism is through NETosis, an effector mechanism by which extracellular fibers (primarily DNA) from neutrophils bind pathogens ( Hasheminasab et al, 2022 ). In one study, neutrophil extracellular traps (NETs) were found to form when bovine neutrophils were incubated with either sporozoites or oocysts.…”

The Role of Intestinal Flora in Anti-Tumor Antibiotic Therapy

“…1, Ref. [24][25][26][27][28][29][30][31][32]). Microbial barrier, mucus barrier [33], epithelial barrier [33], vascular barrier [34], and immune barrier [33] in the intestinal tract can resist the invasion of harmful substances from the external intestinal environment.…”

“…Intestinal flora disorder is common in patients with CRC. Short-chain fatty acids can regulate intestinal flora [32]. Recent studies have found that SCFAs, such as butyric acid can also inhibit the proliferation of cancer cells or participate in immune regulation, hence achieve anti-tumor effects [44].…”

“…(5) Imbalance of intestinal flora and inflammation can increase vascular barrier permeability [31], allowing some pathogenic bacteria to enter the bloodstream. This will also allow some immune cells and polymorphonuclear (PMN) [32] to enter the vascular barrier to regulate immune response.…”