Daniela Grob (Gießen / DE), Camilo Larrazabal (Gießen / DE), Tim Vellmer (Würzburg / DE), Christian J. Janzen (Würzburg / DE), PD Dr. Ulrich Gärtner (Gießen / DE), Prof. Dr. Anja Taubert (Gießen / DE), Prof. Dr. Carlos Hermosilla (Gießen / DE), Dr. Iván Conejeros (Gießen / DE)
Abstract text
Introduction
Trypanosoma b. brucei is an euglenozoan parasite that is able to infect a wide range of hosts, including humans and bovines. In cattle, T. b. brucei is one of the pathogenic agents causing Animal African Trypanosomiasis (AAT) or Nagana disease. T. b. brucei life cycle is indirect, with the tsetse flies acting as vectors. In the vertebrate host, T. b. brucei multiplies extracellularly as trypomastigote forms in the host blood, thereby directly being exposed to cells of the innate immune system, such as polymorphonuclear neutrophils (PMN). PMN own several innate effector mechanisms to combat invading pathogens, including neutrophil extracellular trap (NET) formation.
Objectives
To study early interactions between bovine PMN and T. b. brucei trypomastigotes, PMN activation and NET formation. The role of P2X1 and P2Y6 purinergic receptors in trypomastigote-driven NETosis was also evaluated.
Materials & methods
T. b. brucei trypomastigotes were cultured in HMI-79 medium. Bovine PMN were isolated from whole blood with hypotonic lysis of erythrocytes. PMN activation was evaluated on the level of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Percentages of PMN forming NETs were studied by scanning electron, epifluorescence and live cell 3D holotomographic microscopy. For positive controls, PMN were treated with ionomycin or monosodium urate (MSU) crystals. To block P2X1 and P2Y6 purinergic receptors, PMN were treated with the inhibitors NF449 and MRS2578, respectively, before parasite exposure.
Results
The exposure to T. b. brucei typomastigotes induced immediate activation of PMN as indicated by increased OCR and induce a metabolic switch towards glycolysis in PMN as deduced from enhanced ECAR. PMN responded to parasite encounter via NET formation. T. b. brucei rypomastigotes predominantly induced aggNETs. Ionomycin or MSU also resulted in aggNET formation. Furthermore, T. b. brucei typomastigote-driven PMN activation and aggNET formation revealed as dependent on P2X1 and P2Y6 purinergic receptors since PMN pretreatments with NF449 and MRS2578 impaired all three mechanisms finally resulting in decreased OCR, ECAR and aggNET formation.
Conclusion
Exposure of PMN to T. b brucei trypomastigotes leads to PMN activation and triggering an enhancement in OCR/ECAR and aggNET formation. All these neutrophil functions depend on purinergic signaling pathways via P2Y6 and P2X1 receptors.