ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia Conditions
Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by <i>Cryptosporidium parvum.</i> Neutrophil extracellular traps (NETs), also known as suicidal NETosis, are a powerful and ancient innate effector mechanism by which polymorphon...
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MDPI AG
2022-03-01
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| author | Seyed Sajjad Hasheminasab Iván Conejeros Zahady D. Velásquez Tilman Borggrefe Ulrich Gärtner Faustin Kamena Anja Taubert Carlos Hermosilla |
| author_facet | Seyed Sajjad Hasheminasab Iván Conejeros Zahady D. Velásquez Tilman Borggrefe Ulrich Gärtner Faustin Kamena Anja Taubert Carlos Hermosilla |
| author_sort | Seyed Sajjad Hasheminasab |
| collection | DOAJ |
| description | Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by <i>Cryptosporidium parvum.</i> 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, <i>C. parvum</i> oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN under both 5% O<sub>2</sub> (physiological conditions within small intestinal tract) and 21% O<sub>2</sub> (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O<sub>2</sub>) and hyperoxia (21% O<sub>2</sub>). Besides, <i>C. parvum</i>-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. <i>C. parvum</i>-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 <i>C. parvum</i> while undergoing NETosis. Significant reduction of <i>C. parvum</i>-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 <i>C. parvum</i>-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in <i>C. parvum</i>-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce <i>C. parvum</i>-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 <i>C. parvum</i>-mediated suicidal NETosis under physioxia (5% O<sub>2</sub>) and its anti-cryptosporidial properties. |
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| spelling | doaj.art-f093de6125c64eb8a9625fd1dc57c9b42023-11-30T20:52:16ZengMDPI AGBiology2079-77372022-03-0111344210.3390/biology11030442ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia ConditionsSeyed Sajjad Hasheminasab0Iván Conejeros1Zahady D. Velásquez2Tilman Borggrefe3Ulrich Gärtner4Faustin Kamena5Anja Taubert6Carlos Hermosilla7Institute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, GermanyInstitute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, GermanyInstitute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, GermanyInstitute of Biochemistry, Justus Liebig University Giessen, 35392 Giessen, GermanyInstitute of Anatomy and Cell Biology, Justus Liebig University Giessen, 35392 Giessen, GermanyLaboratory for Molecular Parasitology, Department of Microbiology and Parasitology, University of Buea, Buea P.O. Box 63, CameroonInstitute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, GermanyInstitute of Parasitology, Biomedical Research Center Seltersberg (BFS), Justus Liebig University Giessen, 35392 Giessen, GermanyCryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by <i>Cryptosporidium parvum.</i> 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, <i>C. parvum</i> oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN under both 5% O<sub>2</sub> (physiological conditions within small intestinal tract) and 21% O<sub>2</sub> (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O<sub>2</sub>) and hyperoxia (21% O<sub>2</sub>). Besides, <i>C. parvum</i>-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. <i>C. parvum</i>-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 <i>C. parvum</i> while undergoing NETosis. Significant reduction of <i>C. parvum</i>-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 <i>C. parvum</i>-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in <i>C. parvum</i>-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce <i>C. parvum</i>-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 <i>C. parvum</i>-mediated suicidal NETosis under physioxia (5% O<sub>2</sub>) and its anti-cryptosporidial properties.https://www.mdpi.com/2079-7737/11/3/442<i>Cryptosporidium parvum</i>NETosispurinergic receptor P2X1NotchglycolysisPMN |
| spellingShingle | Seyed Sajjad Hasheminasab Iván Conejeros Zahady D. Velásquez Tilman Borggrefe Ulrich Gärtner Faustin Kamena Anja Taubert Carlos Hermosilla ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia Conditions Biology <i>Cryptosporidium parvum</i> NETosis purinergic receptor P2X1 Notch glycolysis PMN |
| title | ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia Conditions |
| title_full | ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia Conditions |
| title_fullStr | ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia Conditions |
| title_full_unstemmed | ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia Conditions |
| title_short | ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by <i>Cryptosporidium parvum</i> under Physioxia Conditions |
| title_sort | atp purinergic receptor p2x1 dependent suicidal netosis induced by i cryptosporidium parvum i under physioxia conditions |
| topic | <i>Cryptosporidium parvum</i> NETosis purinergic receptor P2X1 Notch glycolysis PMN |
| url | https://www.mdpi.com/2079-7737/11/3/442 |
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