Background

Activated neutrophils are rapidly recruited to sites of infection and injury; they contribute to host defense against pathogens and inflammation. About two decades ago, it was observed for the first time that in response to pathogens, a fraction of the neutrophil population can undergo a distinct type of cellular death, different from apoptosis or necrosis. This involves early chromatin decondensation, co-localization of the nuclear and granular compartments and finally, release of this mix into the environment. The extracellular DNA-scaffolded strands anchoring nuclear components (such as histones) or cellular constituents, some with strong anti-pathogen properties like elastase or myeloperoxidase (MPO), are known as “neutrophil extracellular traps” or NETs and have effective antimicrobial activity (Brinkmann et al., 2004). More recent evidence shows that NETs also associate with non-infectious conditions including cancers, systemic lupus erythematosus, sickle cell disease, atherosclerosis, and thrombosis (Jorch and Kubes, 2017). It is yet not understood whether the NETs, or any of their components (cell-free DNA or histones) contribute to the pathology of these disorders as drivers, or whether they are by-products of an unbalanced immune response. NETs might also function as biomarkers in certain diseases and provide information regarding the efficacy of a treatment regimen (Barnado et al., 2016). NETs-associated components such as elastase or MPO are present in the plasma of individuals with autoimmune diseases and also in infections, which complicates the specificity of these molecules as biomarkers. In this light there is a great need for new methodologies for NETs characterization that offers consistent criteria between studies, and are reliable for both mechanistic and clinical applications.

Other NETs detection and quantification methods are being developed in addition to the classical widely used immunofluorescence microscopy technique. These include use of confocal microscopy that provides information on the structure of the NETs (Santos et al., 2018), ELISA-based assay to quantify the citrullinated histone 3 (H3cit) in human plasma (Thalin et al., 2017), high throughput live detection of NETosis- and apoptosis-related nuclear changes using membrane-permeable and -impermeable nuclear dyes in human neutrophils (Gupta et al., 2018), quantification of free chromatin (predominantly associated with elastase) in human whole blood by using microfluidics (Muldur et al., 2018). Several studies monitored NETs components (H3cit, MPO and extracellular DNA) in human and mice by using conventional flow cytometry (Gavillet et al., 2015; Masuda et al., 2017; Lee et al., 2018; Zharkova et al., 2019). While this technique allows high-throughput analysis it also does have noteworthy limitations, regarding the choice of the probes, the preparation of the samples and the way gating impacts final conclusions (Manda-Handzlik et al., 2016; Masuda et al., 2017). On the other hand, while Imaging Flow Cytometry (IFC) does allow high-throughput analysis, it also combines visualization and analysis features from both conventional flow cytometry and fluorescence microscopy (Basiji, 2016). NETs-associated morphological nuclear changes (i.e., chromatin decondensation and DNA trails) and whether and to what degree MPO co-localized with the nuclear compartment were investigated with IFC (Zhao et al., 2015; Ginley et al., 2017; Pelletier et al., 2017). Thus, different markers and different methods are currently employed to detect and quantify NETs in vitro. PAD4-mediated histone citrullination has been long designated as a hallmark of NETs formation, and thus a desirable marker to follow. More recent reports, however, predominantly in the mouse model, suggest that PAD4-mediated citrullination of histone 3 (H3cit) is stimulus-dependent (Neeli and Radic, 2013; Claushuis et al., 2018; Guiducci et al., 2018; Liang et al., 2018) and therefore the utility of this site as a NETs marker might be limited.

Here, we describe in detail a novel IFC protocol that allows specific detection and quantification of citrullination of histone 4 (H4cit3) as a NETs marker in whole neutrophils, prior to the release of the DNA and the cytoplasmic protein strands into the extracellular space. Other analysis parameters that look at nuclear and cellular morphological changes (nuclear decondensation and supercondensation, multi-lobulated nuclei and cell membrane damage) can bring additional information on the behavior of the analyzed neutrophil population. To establish our methodology we determined the responses of healthy human neutrophils treated for 5 different periods of time (between 2 min and 60 min) with NETs agonists: pharmacological inducers, PMA and calcium ionophore (a well-known inducer of histone citrullination, and for our experimental conditions, the positive control), Hemin (an inducer present under hemolytic conditions) and LPS and IL-8 (inducers associated with infectious pathogens). We used in vitro fluorescence microscopy to confirm the formation of DNA-elastase-MPO strands in healthy neutrophils treated with the stimuli used for the IFC tests (Barbu et al., 2020). We further validated this technique in untreated and Hemin-treated neutrophils from healthy donors and patients with sickle cell disease at steady state (Barbu et al., 2019).

The increased sensitivity and objective analysis make this methodology highly suitable for NETs detection in both research and clinical studies, that explore mechanistic answers or possible therapeutic strategies.

This protocol has 4 major parts: one-step neutrophil purification, induction of NETosis, specific staining, IFC acquisition and analysis (as highlighted in Figures 1A and 1C), with the first 3 steps requiring 3 days and up to one week to complete all steps.

Figure 1. Day-by-day description of the major steps for specific detection of NETosis markers by Imaging Flow Cytometry. A. One-step polymorphoponuclear (PMN) cells isolation from whole blood with Polymorphprep medium; with this method neutrophils purity should be consistently above 80%. Total processing time: 90 min. B. An example of correct separation of whole blood by using Polymorphprep gradient medium. C. Detailed description of polymorphonuclear cells treatment and staining procedure. A time recommendation is not included for the neutrophil stimulation step as researchers should choose an appropriate treatment time according to their experimental purpose. Each washing step might need up to 20 min to complete, depending on the number of samples processed. This time should be taken into account while calculating total time required to complete all steps per day. Short final wash followed by samples storage at 4 °C. Total stimulation and staining processing time for Day 1: 4+ h.