Hanna Stracke (Gießen / DE), Dr. Seyed Sajjad Hasheminasab (Gießen / DE), Dr. Iván Conejeros (Gießen / DE), Dr. Zahady Velásquez (Gießen / DE), PD Dr. Ulrich Gärtner (Gießen / DE), Dr. Faustin Kamena (Buea / CM), Prof. Dr. Florian Wagenlehner (Gießen / DE), Prof. Dr. Anja Taubert (Gießen / DE), Prof. Dr. Carlos Hermosilla (Gießen / DE)
Abstract text
Introduction
Cryptosporidium parvum is a zoonotic apicomplexan protozoan parasite causing cryptosporidiosis, a severe enteritis resulting in catharralic diarrhoea, especially in new-borns, toddlers, immunosuppressed patients and calves. Cryptosporidiosis leads to increased toddler mortality worldwide and significant economic losses in the livestock industry. Neutrophil extracellular trap (NET) formation is an important effector mechanism of polymorphonuclear neutrophils (PMN) to fight against viruses, bacteria, fungi and protozoa.
Objectives
The aim was to study the role of distinct signalling pathways (i. e. NF-ƙB, HIF- α, PI-3 kinase), ATP purinergic receptors (PANX1, PX2X1), lactate transporters (MCT1, MCT2) and general metabolic requirements in C. parvum-triggered human NETosis.
Material and Methods
Human PMN were isolated from whole blood by negative immunoselection. C. parvum-PMN interactions were documented via microscopy, live cell imaging and metabolic assays. For the latter, oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of PMN were assessed via Seahorse instrumentation. The role of selected signalling pathways was studied via chemical inhibition using CBX, NF499, YC-1, LY-294002, Bay11-7821, AR-C14990 and AR-C155858 to inhibit PANX1, P2X1, HIF1, PI3-kinase, NF-κB, MCT1 and MCT1/MCT2, respectively.
Results
Upon parasite exposure to PMN, immunofluorescence- and confocal microscopy confirmed the presence of classical NET markers (histones and neutrophil elastase) in DNA-positive NET-like structures. Via live cell 3D-holotomographic microscopy, early morphological changes during the NETotic process such as cell membrane extrusions/protrusions and nuclear area expansion (NAE) was documented. C. parvum-induced human suicidal NETosis was significantly diminished by PMN pre-treatments with PANX1 inhibitor carbenoxolone (CBX), whilst chemical inhibition of MCT1-, MCT2, HIF-α-, NF-ƙB- and PI-3 kinase failed to affect NETosis under physioxia (5% O2). Overall, neither OCR nor ECAR were changed in C. parvum-exposed PMN even though PMN reacted by NET formation.
Conclusion
C. parvum oocysts and sporozoites both induced human suicidal NETosis. We here provide first evidence on a pivotal role of PANX1 in this neutrophil effector mechanism.