Neutrophil Extracellular Traps (NETs) in Patients with Congenital Neutropenia
Authors: Happle Christine , Germeshausen Manuela , Zeidler Cornelia , Welte Karl , Skokowa Julia 
Published: October 2019

Abstract
Abstract 15
Activated neutrophils kill microbes by phagocytosis and extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of decondensed chromatin and granular proteins such as neutrophil elastase (NE) and cathepsin G. Enzymatic generation of reactive oxygen species (ROS) by NADPH oxidase and the release of serine proteases such as NE have been shown to be essential factors for NET formation. Patients with chronic granulmatous disease (CGD), who lack a normal generation of ROS, show a defective NET formation.
Since myeloid cells of patients with severe congenital neutropenia (CN) show an aberrant expression pattern of granular proteases such as neutrophil elastase (NE) or myeloperoxidase, we aimed to analyse NET formation in activated neutrophils of these patients. CN is a heterogeneous hematological disorder, characterized by peripheral blood neutrophil counts below 0,2×109/l and a maturation arrest of myelopoiesis at the promyelocytic/myelocytic stage. 60% of CN patients harbor autosomal dominant mutations within the ELA2 gene encoding for NE, but also mutations in other genes (e.g. HAX1, G6PC3, WAS, GFI1, p14) have been found to be disease-causing. Previously, we described severely diminished levels of NE in myeloid cells of CN patients. Here, we aimed to explore NET formation in neutrophils of CN patients. Moreover, we intended to analyze the effects of a reduced ELA2 expression and gene mutations as seen in CN patients on NET formation in vitro.
Granulocytes of CN patients undergoing G-CSF therapy were extracted by density centrifugation, stimulated with phorbol myristate acetate (PMA, 50 nM, up to 240 min) and then tested for NET formation. NETs were stained with an extracellular DNA dye or DAPI. Our analyses showed normal NET formation in peripheral blood granulocytes of two patients with HAX1-related neutropenia, whereas there was a significantly lower amount of NETs in two patients with ELA2 mutations. One further patient out of three CN patients with unknown mutations showed a reduced amount of NETs in bone marrow PMNs.
To further evaluate the possible effect of downregulated ELA2 expression on NET formation, we transduced primary human CD34+ cells with a lentiviral-based shRNA construct downregulating the expression of NE. Subsequently, these cells were differentiated into granulocytes with a cytokine cocktail containing G-CSF and tested for their ability to form NETs. We found an almost completely abolished NET formation in cells transduced with ELA2 shRNA as compared to control cells.
Hitherto, CGD is the only immunodeficiency with a clearly defective NET formation. Our results point to an impaired formation of NETs also in CN patients carrying ELA2 mutations. This supports the recent finding of a central role for NE in NET formation. Two patients with HAX1 related CN showed a normal ability to form NETs. Our further work will aim to better define the subgroup of CN patients defective in NET formation.

Disclosures:
No relevant conflicts of interest to declare.