High-Content Screening Identifies Vanilloids as a Novel Class of Inhibitors of NET Formation.

Elvira Sondo,Nicoletta Pedemonte,Gian Marco Ghiggeri,Emanuela Pesce,Roberta Bertelli

doi:10.3389/fimmu.2019.00963

Elvira Sondo, Nicoletta Pedemonte + Show 3 more

Open Access

https://doi.org/10.3389/fimmu.2019.00963

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Abstract

Neutrophils migrate to sites of infection where they phagocytose, degranulate, and/or, in the presence of appropriate stimuli, release decondensed chromatin strands (called neutrophil extracellular traps, NETs) for trapping and possibly killing microorganisms. NET formation is characterized by marked morphological cell changes, in particular within the nucleus. Lytic NET formation can be observed in neutrophils undergoing cell death, which is referred to as NETosis. Dysregulation of NET production and/or degradation can exert pathogenic effects, contributing to the pathogenesis of various diseases, including cystic fibrosis, autoimmune diseases and inflammatory conditions. By employing a phenotypic assay based on high-content imaging and analysis, we screened a library of biologically active compounds and identified vanilloids as a novel class of chemical compounds able to hinder NETosis induction and NET release. Vanilloids also markedly decrease cytosolic ROS production. The identification of novel vanilloid NET inhibitors, able to stop excessive or aberrant NET production might offer new therapeutic options for those disorders displaying NET overproduction.

Highlights

Neutrophil granulocytes are polymorphonuclear cells that migrate to sites of infection where they phagocytose, degranulate and/or, in the presence of appropriate stimuli, they may undergo marked morphological cell changes, in particular within the nucleus
We first evaluated the feasibility of Opera Phenix System for the detection and quantification of NETs, through the detailed nuclear morphology analysis in neutrophils exposed to phorbol 12-myristate 13-acetate (PMA) or ionomycin, after formalin fixation and Hoechst 33342 staining
NET formation was detected in > 60% of neutrophils exposed to ionomycin (5–10 μM) after a short treatment (45 min); when neutrophils were treated with PMA at the maximal effective dose (100 nM), the same percentage of NETotic cells was observed after 210 min (Figures 1B,C)

Summary

Introduction

Neutrophil granulocytes are polymorphonuclear cells that migrate to sites of infection (as part of the first line of immune defense) where they phagocytose, degranulate and/or, in the presence of appropriate stimuli, they may undergo marked morphological cell changes, in particular within the nucleus. NET production is involved in host defense and NETs have a potentially protective role against infections, their excessive production or ineffective removal can exert pathogenic effects, resulting in tissue/organ damage, and contribute to the pathogenesis of various diseases [3,4,5]. It has been known for over 40 years that cystic fibrosis patients have high levels of cell-free DNA in their airways (correlating with obstructive lung disease), of which NETs have recently emerged as the principal source [6,7,8]. It has been demonstrated that NETs can directly cause epithelial and endothelial cell death [9]

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