Background

Neutrophils are the major innate immune cells in the blood (50-70% of white blood cells in humans) (Mestas and Hughes, 2004). These cells have a diameter of 5-7 µm and are characterized by a segmented nucleus, secretory vesicles in their cytoplasm, and a short lifespan (Borregaard, 2010). Neutrophils are produced in the bone marrow in response to G-CSF secretion under the control of key transcription factors such as PU.1, C/EBPα, GFI-1, and C/EBPϵ (Hidalgo et al., 2019). Neutrophils participate in pathogen destruction by releasing the cytotoxic contents of their secretory vesicles or by phagocytosis (Borregaard, 2010). Recently, several discoveries have shed light on neutrophil biology and function. First, a study using radiolabeling of neutrophils demonstrated an unexpected longer half-life of these cells, which can reach 5.4 days in humans and 18 hours in mice (Pillay et al., 2010). Second, in 2004, Brinkmann and colleagues discovered a novel bactericidal action of neutrophils. Indeed, it was observed that the release of particular structures could trap bacteria and limit their dissemination throughout the organism (Brinkmann et al., 2004). These new structures were named Neutrophil Extracellular Traps or NETs. NETs are web-like structures composed of extracellular free DNA associated with a particular form of citrullinated histone 3 (Cit-H3), in which arginine residues have been replaced by citrulline, and various antimicrobial peptides from secretory vesicles, such as myeloperoxidase (MPO), neutrophil elastase (NE), or cathelicidins (Papayannopoulos, 2018). The release of these structures can be triggered by various stimuli derived from bacteria, viruses, parasites, and fungi, but also by immune complexes, crystals, pro-inflammatory cytokines, and chemokines (Papayannopoulos, 2018). Within neutrophils, the activation of pyruvate kinase C, formation of reactive oxygen species (ROS), and the activation of peptidyl arginase 4 and neutrophil elastase allow chromatin decondensation, nuclear membrane disruption, and NET release (Papayannopoulos, 2018). NETs can immobilize pathogens and prevent their dissemination but are also able to kill them directly (Brinkmann et al., 2004; Papayannopoulos, 2018). Unfortunately, excessive NET release or inappropriate NET accumulation can trigger important tissue damage and lesions (Narasaraju et al., 2011) or inadequate activation of immune cells, thereby promoting various immune-mediated disorders (Hakkim et al., 2010; Radermecker et al., 2019) or coagulation issues (Middleton et al., 2020). Thus, NETs are increasingly implicated in various pathological processes, as evidenced by experimental work in mouse models (Radermecker et al., 2019; Villanueva et al., 2011). The investigation into their potential contributions to human disease is therefore of great interest. NET detection in humans is quite challenging and can be performed either indirectly in physiological fluids or directly in tissue sections. To date, NETs have been mainly measured in physiological fluids such as serum and bronchoalveolar lavage fluid (BALF) (Toussaint et al., 2017; Middleton et al., 2020). NET measurements in fluids rely on the detection of one or two of their components (extracellular DNA, citrullinated histone 3, or complexes of DNA/MPO or DNA/NE). Extracellular free DNA can be detected using DNA quantitation assays on serum or BALF supernatant (Toussaint et al., 2017). Methods for the detection of Cit-H3 have been developed, including western blotting (Liu et al., 2016; Thålin et al., 2017). A more specific technique relies on the detection of complexes formed by DNA and one of the antimicrobial peptides released from secretory vesicles, like MPO or NE, by ELISA (Caudrillier et al., 2012; Caldarone et al., 2019). These techniques are rapid but lack specificity and do not provide any information about the location of NETs in the tissue. Here, we describe a protocol allowing the direct visualization and quantitation of NETs in situ by confocal microscopy. NETs are identified as extracellular structures exhibiting a co-localization of Cit-H3 and MPO, two important components of NETs. This technique is currently considered the gold standard of NET detection. Furthermore, we propose a novel software-based objective method to quantitate NETs in tissues based on the measurement of the volume of structures exhibiting a co-localization of Cit-H3 and MPO outside the cell (Radermecker et al., 2019). This technique is specific, quantitative, and provides information about the location of NETs in tissues (Radermecker et al., 2020). Identification of specific NET-rich areas in human tissues may promote a better understanding of their pathological roles in disease.