WO2025215010A1 - Method for investigating neutrophils - Google Patents

Abstract

The invention relates to methods for analyzing chromatin from a population of neutrophil cells isolated from a whole blood sample. In particular, the method is used to measure the chromatin dynamics of neutrophils during induced NETosis.

Description

METHOD FOR INVESTIGATING NEUTROPHILS

FIELD OF THE INVENTION

The invention relates to methods of detecting and measuring the chromatin dynamics of neutrophils, in particular primary human neutrophils, during induced NETosis. Such methods allow for improved detection and diagnosis of diseases associated with dysregulated extracellular trap formation and also for the screening of potential drug candidates which can treat such diseases.

BACKGROUND OF THE INVENTION

Human neutrophils undergo dramatic nuclear and chromatin changes as part of their response to a myriad of immune challenges. Neutrophils can phagocytose pathogens, release antimicrobial proteins and undergo a process termed NETosis in which neutrophils extrude their chromatin in the form of neutrophil extracellular traps (NETs). NETs trap, degrade and remove pathogens from circulation, but when uncontrolled can become maladaptive, with excess NETosis leading to negative autoimmune responses, thrombosis, and organ damage. Despite its implications, our understanding of the clinically relevant molecular mechanisms underpinning NETosis has remained limited, in part due to the inherent challenges of working with primary neutrophils and other neutrophil model systems. For example, the intricacies of the chromatin landscape in these short-lived and delicate immune cells have posed formidable technical obstacles. In many cases, without fixation, chromatin mapping of primed or activated neutrophils is not possible due to rapid destabilization of cellular membranes and neutrophil responses to environmental perturbations including centrifugation and temperature changes.

White blood cells are an important component of the innate immune system. They are recruited to the site of an infection as part of the immune response where they protect against a broad range of pathogens by mechanisms including degranulation, phagocytosis and intracellular degradation of pathogens as well as by the formation of extracellular traps (ETs). Neutrophils make up some 60% of human white blood cells and can form neutrophil extracellular traps (NETs). NETs are the best known and most studied ETs. ETs and NETs are web-like decondensed chromatin structures ejected extracellularly by white blood cells, particularly activated neutrophils, as a natural mechanism to trap pathogens such as bacteria, viruses, fungal spores and parasites. ETs and NETs help to prevent infection by trapping and degrading invading pathogens locally and also help prevent their spread throughout the body.

Huge numbers of neutrophils are generated daily in the bone marrow from myeloid progenitor cells. The myeloid cells progress through a number of neutrophil precursor cells, including myeloblasts, promyelocytes, myelocytes, metamyelocytes and band cells before becoming mature neutrophils. Under normal conditions, neutrophils are thought to live less than 24 hours after entering the circulation. During this time neutrophils take on various phenotypes, including activated phenotypes, as they migrate out of the vasculature into the tissues where they complete their functions before being eliminated by macrophages. However, some neutrophils may persist for longer times including some extravasated neutrophils.

Neutrophil biology is not well understood and remains a subject of study. Neutrophils are highly plastic and heterogeneous in phenotype and function. The neutrophil phenotype and activation state may change with age at different points in the neutrophil life-history. Neutrophils are abundant in the blood but also occur in many different tissues through extravasation from the vascular system, for example into the lungs or spleen. Neutrophils located in different tissues may have different phenotypes representing different neutrophil sub-populations. Different neutrophil phenotypes and different activation states may occur in different disease states, for example in infection or inflammation. Neutrophils are also thought to have a variety of functions not limited to immunity.

Neutrophils migrate to the site of an injury, infection, inflammation, or other insult to the body as a response to chemotaxis. Thus, neutrophils may be found in many different tissues and in many body fluids if recruited there in response to an infection or injury. Neutrophils have been observed in bronchoalveolar lavage fluid (BALF), cerebrospinal fluid (CSF), sputum, saliva, urine and stool.

The ejection of decondensed chromatin to produce NETs by neutrophils involves a process known as NETosis. NETosis of a neutrophil may be stimulated or induced by many different factors including by the presence of a bacterial, fungal or viral pathogen, as well as by many chemical activators including, without limitation, heparin, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), reactive oxygen species such as hydrogen peroxide or moieties that generate reactive oxygen species (for example, glucose oxidase), ionomycin or calcium ionophores (Cal) (Fuchs et al, 2007, Neubert ef al, 2019).

NETosis may also be induced by the complement system and coagulation of blood. Coagulation or clotting of blood at an open wound stimulates NETosis providing a scaffold on which platelets and blood cells may aggregate during clot formation to close the wound, whilst also providing protection against infection by trapping and degrading any pathogens that may enter the body locally and thus preventing the spread of infection to other parts of the body. The process of NETosis is well known in the art and described in reviews (Fousert et al, 2020, Thalin et al, 2019, Sollberger et al, 2018, Snoderly et al, 2019, Niedzwiedzka-Ryswej et al, 2019, Papayannopoulos, 2018, Delgado-Rizo et al, 2017). NETosis involves decondensation of tightly coiled chromatin in the nucleus of a neutrophil to produce unwound strings of chromatin with a concomitant increase in nuclear size in response to intracellular mediators that include reactive oxygen species, produced by NADPH oxidase or mitochondria, which activate myeloperoxidase (MPO), neutrophil elastase (NE) and protein-arginine deiminase type 4 (PAD4). The decondensed chromatin is ejected extracellularly to form NETs. Pathogens are trapped and subjected to degradation by histone and DNA components of NETs which have antimicrobial properties. NETs also contain additional antimicrobial moieties to neutralize captured pathogens including for example myeloperoxidase and proteolytic enzymes.

Once produced by NETosis, NETs are reported to persist for some 24 hours in the vasculature (Kolaczkowska et al, 2015), but are eventually degraded by mechanisms thought to include digestion of DNA by DNase enzymes secreted into the circulation and by phagocytosis (Farrera and Fadeel, 2013).

Activated neutrophils may undergo NETosis if induced to do so, for example by the presence of a pathogen. Additionally, neutrophils secrete a variety of pro-inflammatory cytokines and surface molecules (major histocompatibility complex II) leading to altered membrane composition and increased activity in the cytoplasm (for example to produce cytokines). Excessive cytokine release may lead to further neutrophil activation, NETosis and, in turn, further cytokine release in a classic “cytokine storm” positive feedback loop, for example as has been associated with severe sepsis and COVID-19.

Neutrophils in circulation also release harmful intracellular granule contents when recruited, primed, and activated following encounters with chemokines, cytokines or pathogens. However, neutrophils can also become primed or activated during immune dysregulated conditions such as sepsis where primed neutrophils exhibit a 10 to 20-fold increase in their response if stimulated to do so. Septic patients have been described to contain a primed population of neutrophils. Similarly, acute respiratory distress syndrome (ARDS) patients are reported to have high plasma levels of tumor necrosis factor-a (TN Fa) which primes neutrophils resulting in hyper-responsiveness and lung injury. Overactivation of neutrophils may result in dysfunction and cause tissue damage. For example, while neutrophil recruitment in response to lung injury is an appropriate response, excessive recruitment of activated neutrophils into the pulmonary vasculature may lead to ARDS in patients with severe trauma or hemorrhagic shock.

Whilst NETs are very effective in protection against infection by trapping invading pathogens, excessive NETosis is a major cause of pathology and is involved in a large number of disease processes including, without limitation, sepsis, septic shock, organ failure, metabolic diseases, cancer, obesity, most or all autoimmune conditions, most or all inflammatory conditions, Alzheimer’s disease, atherosclerosis, bacterial infection, cystic fibrosis, pancreatitis, viral infection, type I and type II diabetes, cancer, vasculitis, thrombosis, pneumonia, respiratory infections, gout, rheumatoid arthritis (RA), psoriasis, systemic lupus erythematosus, atherosclerosis, stroke and sickle cell disease (Sollberger et al, 2018, Thalin et al, 2019, Neubert et al, 2019). The list of diseases in which NETs plays a pathological role is a growing list as workers discover NETs association in more diseases.

Inappropriate production of NETs is not only associated with these diseases but is a causative factor in disease mechanism, disease progression or disease severity. The prolonged presence of NETs may cause tissue damage and development of an autoimmune reaction against NETs components leading to inflammatory, autoimmune, and vascular diseases. Cytotoxic proteases and histones in NETs may cause endothelial damage in sepsis and small vessel vasculitis. In severe influenza, the alveolar-capillary surfaces of the lungs may become embroiled with NETs and damaged by cytotoxic NETs-associated proteins including histones and MPO (Moorthy et al, 2013). Highly elevated plasma NETs levels have been observed in patients with COVID-19 and sepsis. In hospitalised COVID-19 and sepsis patients, plasma NETs levels are higher in patients with severe disease (with organ failure), than in patients with less severe disease. Plasma NETs levels also correlate with disease severity as assessed by Sequential Organ Failure Assessment (SOFA) Score and are predictive of mortality (Morimont et al, 2022, Rea et al, 2021). NETs measurements in bronchoalveolar lavage fluid samples taken from patients with pneumonia (Maruchi et al, 2018) and in serum samples taken from patients with COVID-19 infections (Zuo etal, 2020) have shown that NETs levels were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. NETs levels therefore predict which patients are in need of high levels of respiratory support.

NETs are implicated as a cause of cancer related thrombosis and as a facilitator of metastatic stage IV cancer disease progression by a variety of mechanisms including through the entrapment of tumour cells in NETs, facilitating the spread of NET-bound tumour cells around the body and by assisting in the establishment of metastatic cell growth at new locations (Teijeira et al, 2020, Rayes et al, 2019, Cools-Lartigue et al, 2013, Niedzwiedzka-Ryswej et al, 2019).

Rayes et al, 2019 reported that the level of circulating NETs measured in heparin plasma samples is elevated in late stage cancer and correlates with disease stage. They also reported that metastatic disease progression is inhibited by inhibition of NETs formation and that the level of circulating NETs is a better predictor of tumor progression than neutrophil count or neutrophil to lymphocyte ratio.

NETs and NETosis are subject to a great deal of research interest and are clearly of clinical relevance in many disease areas. It is clear that NETs and NETosis are associated with and/or a causative factor in a wide variety of disease processes. The propensity of neutrophils to NETosis is an extremely useful monitor of the health or state of the immune system of a subject. The measurement of the propensity for NETosis, particularly a propensity or predisposition to dysregulated or excessive NETosis, has a very wide array of applications including, for example without limitation, diagnostic and prognostic applications for the risk of severity of a broad range of diseases from rheumatoid arthritis to cancer to sepsis, and to determine and monitor the efficacy of immune related or other therapies and/or their effect on the immune system. Despite this, no functional tests for neutrophil activation are used in patient care. This is because such tests are either performed in cell culture or do not perform well.

A reliable, functional test for the propensity of white blood cells to undergo NETosis would be useful for the diagnosis and management of many patients in many disease areas, in particular to identify patients at high risk for excessive or dysregulated NETosis or with a high capacity or propensity for NETosis.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a method for analyzing chromatin, comprising:

(a) treating chromatin from a population of neutrophil cells isolated from a whole blood sample with an insertional enzyme complex to produce tagged fragments of genomic DNA, and wherein the neutrophil cells are fixed before isolation from the whole blood sample;

(b) sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and

(c) making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region. According to a further aspect of the invention, there is provided a method for comparing two samples comprising:

(a) analyzing a first population of cells using the method defined herein to produce a first epigenetic map; and

(b) analyzing a second population of cells using the method defined herein to produce a second epigenetic map; and

(c) comparing the first epigenetic map to the second epigenetic map.

According to a further aspect of the invention, there is provided a method for comparing two samples containing neutrophils derived from whole blood comprising:

(a) analyzing a first population of cells using the method defined herein to produce a first epigenetic map wherein the first population of cells is challenged with a candidate modulator of NETosis or a modulator of NETosis before isolation of the neutrophils from a whole blood sample; and

(b) analyzing a second population of cells using the method defined herein to produce a second epigenetic map wherein the second population of cells is challenged with a candidate modulator of NETosis or a modulator of NETosis after isolation of the neutrophils from a whole blood sample.

According to a further aspect of the invention, there is provided a diagnostic method, comprising: analyzing chromatin from a patient using the method defined herein, to produce an epigenetic map; and providing a diagnosis or prognosis based on the epigenetic map.

According to a further aspect of the invention, there is provided a method for treatment selection, comprising: analyzing chromatin from a patient using the method defined herein, to produce an epigenetic map; and selecting a treatment for the patient based on the epigenetic map.

According to a further aspect of the invention, there is provided a method for identifying the effect of a candidate modulator of NETosis, comprising: analyzing chromatin from a patient treated with the candidate modulator of NETosis using the method defined herein, to produce an epigenetic map; and identifying whether the candidate modifier of NETosis is a modifier of NETosis based on the epigenetic map.

According to a further aspect of the invention, there is provided a kit comprising:

(a) a modulator of NETosis;

(b) reagents for isolating neutrophils from whole blood cells;

(c) reagents for isolating nuclei from a population of cells;

(b) an insertional enzyme complex, and

(c) transposase reaction buffer, wherein the components of the kit are configured such that, combining the reaction buffer, transposon tags and adaptors with nuclei in vitro results in both lysis of the nuclei to release chromatin and production of tagged fragments of genomic DNA.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 is a schematic overview of the process of chromatin decondensation which is investigated by the method of the invention.

FIGURE 2 is a schematic overview of the convention process of ATAC-seq.

FIGURE 3 is a schematic overview of a process of an embodiment of the invention.

FIGURE 4 is a schematic overview of FACS sorting after fixation and isolation and shows the difference between whole blood fixation according to the invention and fixation following neutrophil isolation.

FIGURE 5 shows that chromatin accessibility patterns are different during NETosis induction in whole blood and isolated neutrophils. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed following fixation of neutrophils and whole blood at different time points following PMA treatment versus treatment with a DMSO control, revealing that distinct chromatin accessibility changes.

FIGURE 6 shows that chromatin accessibility patterns are different during NETosis induction in whole blood and isolated neutrophils, including at different time points.

FIGURE 7 is a heat map which separates out PMA treated and DMSO treated samples and shows examples of the most different regions across the whole genome. It illustrates that there are differences between the two groups and that there are also differences across time. FIGURE 8 shows the results of HOMER Motif analysis and illustrates that the present invention can be used to identify enrichments of DNA-binding motifs. It also illustrates that the method of the invention can be used to identify transcription factor binding sites and gene regulatory networks involved in NETosis.

FIGURE 9: Neutrophils fixed in whole blood prior to isolation shows consistency across donors. (A) Merged replicate tracks visualized using the IGV Genome Browser with untreated healthy donors (n=6). Neutrophils isolated prior to fixation (top) and whole blood fixed prior to isolation (donors D48, D78, D79, D81 , D83, D85) are shown below. Top bars (grey) indicate loci specific to different immune cells: Housekeeping (all) - ACTB; B-cell (PAX5 and CD19), T-cell (CD8A and CD3E), monocytes (ITGAM, CSF1 R, and CD14), and neutrophils (accessible regions - CD14, CLEC7A, and HCAR; inaccessible regions - AZU1 and MPO). (B) The location of peaks (MACS2 peaks (q < 0.01)) across genome structures was generated using nf-core/atacseq. Peaks annotations are plotted as a percentage of all peaks found within a sample (Transcription start site (TSS), Transcript termination sites (TTS)).

FIGURE 10: PMA stimulation drives a stable chromatin response in whole blood fixed neutrophils. (A) Principal Component Analysis (PCA) based on unbiased clustering of dimethyl sulfoxide (DMSO) versus phorbol 480 12-myristate 13-acetate (PMA) using the merged replicate data. Treatment is indicated by squares (DMSO) or circles (PMA). PC1 (x-axis) represents 65.2% of the total variance and PC2 (y-axis) represents 15.5% of variance. Donor numbers are indicated by the number adjacent to the datapoint. (B) Merged donor tracks visualized using IGV genome browser. T30 DMSO, T30 PMA, T60 PMA, T90 PMA, T120 PMA at various loci. Housekeeping gene TBP, increased accessibility at the CXCL2, 3, 5 locus (T60-T120), bimodal response shown at ACTG1 , and decreased accessibility at CXCR2. (C) Heatmap showing the total number of differential accessible regions (DARs) between all pairwise comparisons (each timepoint for each treatment condition) (DESeq2 p.adj > 0.01 and Iog2(fold change) less than -1.5 or greater than 1.5). (D) HOMER motifs within DARs that gain accessibility in T60-T1120 PMA compared to T30 DMSO are plotted by by-log10(p. value) on the y-axis and the HOMER rank on the x-axis. The top 50 known motifs were graphed, and the top 15 known motifs based on p. value are annotated in green. (E) Similar to (D), but DARs that have more accessibility in T30 DMSO compared to T60-T120 PMA are shown.

FIGURE 11 : Comparison of chromatin accessibility changes between isolated and whole blood fixed neutrophils. (A) Heatmap showing the overlap of DARs at each timepoint and condition. This indicates whether the DAR was also found at other timepoints between Isolated and whole blood (WB). The diagonal represents 100% of DARs called at each row, and subsequent values within the row represent the percentage of those DARs found. (B) GO pathway analysis of all DARs in Isolated and Whole blood. Each group includes Overlapping DARs. (C) Similar to (B) but Reactome pathway analysis was used.

FIGURE 12: Whole blood PMA induction leads to a more complex immune response compared to the isolated system. (A) HOMER motifs graphed by -log 10(p. value) on the y-axis and HOMER rank on the x-axis for DARs which gained accessibility in Isolated (left), Whole blood (middle), and Overlapped (right). The top 50 known motifs were graphed, and the top 15 known motifs based on p. value are annotated. (B) Similar to (A) but with downregulated DARs that lose accessibility. (E) Reactome Pathway heatmap showing the -Log10(P. value) for each pathway: Isolated (left), Whole blood (middle), and Overlapped (right) DARs.

FIGURE 13: Principal Component Analysis (PCA) based on unbiased clustering of control formulations (untreated, DMSO or vehicle) versus natural factors (NF).

FIGURE 14: Zscore heatmap showing the total number of differential accessible regions (DARs) between all the tested natural factors, calcium ionophore (Cl), PMA and the control formulations (untreated, DMSO or vehicle).

FIGURE 15: HOMER motifs graphed by -Iog10(p. value) on the y-axis and HOMER rank on the x-axis for transcription factor motifs that are upregulated (left) or downregulated (right) following natural factor stimulation.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "NET" or "neutrophil extracellular trap" refers to an extracellular complex of nucleosomes and proteins. Upon activation, neutrophils and other cells undergo a cell death program termed "NETosis" and release portions of nuclear DNA in the form of nucleosomes in complex with various proteins having antimicrobial activity, thereby forming Neutrophil Extracellular Traps (NETs). NETosis is a critical part of the innate immune system by which pathogens are inactivated, degraded and removed from circulation. NETosis culminates in the extrusion of chromatin to form NETs that can span dozens of nanometers in diameter. As a result, when dysregulated, NETs can be pathophysiological, leading to thrombosis and organ damage. Furthermore, nucleosomes that make up chromatin are complexes containing positively charged histone proteins coupled with negatively charged DNA. These opposing forces make nucleosomes very stable, however when decoupled can cause damage to surrounding cells. Much of our understanding of NET formation is based on studies of isolated neutrophils or immortalized neutrophil-like cells, and while these models have helped understand the basic biology of NETosis, they do so in a non-physiologically relevant context and often with non-biological stimuli like Phorbol 12-myristate 13-acetate (PMA), which can bypass regulatory mechanisms. Here we measured NETosis induction and the associated changes in chromatin structure in response to physiologically relevant molecules that are implicated in NETosis related diseases in isolated neutrophils and intact whole blood. Interestingly, we find that while these factors are able to induce NETosis in whole blood, they are unable to induce NETosis in isolated neutrophils, suggesting that a critical molecule or cell type may be present in circulation that supports NETosis induction with a minimal pool of physiological factors. Furthermore, we found distinct chromatin accessibility changes preceding NET formation following PMA induction in whole blood compared to isolated neutrophils, suggesting that chromatin decondensation and extrusion signaling mechanism(s) may be different in whole blood compared to isolated neutrophils. These findings open the possibility that using more physiologically relevant NETosis models may lead to a better understanding of the regulatory mechanisms governing NETosis in vivo and the potential for new therapeutic interventions.

In particular, we studied chromatin accessibility changes using ATAC-seq in both unstimulated primary human neutrophils and neutrophils undergoing NETosis. blood. To assess the impact of the factors present in whole blood, we compared chromatin changes when NETosis was induced in isolated neutrophils with NETosis induction in whole blood. To enable this study, we fixed neutrophils in whole blood with and without treatment with PMA prior to isolation. PMA is a known activator of NETosis through protein kinase C (PKC) activation leading to many downstream transcription factors to be activated, including NF-kB, NFAT, and others.

We performed a time course of PMA induced NETosis, across 180 minutes in both isolated and whole blood systems and the results suggest an organized series of chromatin remodeling changes being triggered within 20 minutes of PMA stimulation, which was the earliest time point we measured. We see both localized changes occurring at specific chromatin locations (<1 kb) as well as broader changes affecting >10 kb. These insights underscore a rapid and dynamic response to PMA induced PKC activation. Notably, at around the 45-minute mark, we observed a stabilization of the accessible chromatin, suggesting a potential conclusion of the initial phase of PMA-induced chromatin remodeling, which precedes extracellular changes.

These findings collectively underscore the presence of a finely tuned, ordered, and reproducible response among neutrophils stimulated with PMA. The findings in this study highlight the complex process of NETosis and provides a methodology to study the chromatin dynamics in primary human neutrophils in a biologically relevant context. By shedding light on the intricate epigenetic machinery underpinning NETosis, this research endeavors to pave the way for innovative strategies aimed at deciphering the complexities of immune responses and pathologies in the future and potential therapeutic intervention. Furthermore, this study lays the groundwork for studying NETosis induction using physiologically relevant induction methodologies both in an isolated and whole blood setting.

The invention relates to the analysis of ETs or NETs production using a whole blood sample outside of the body by induction of NETosis in the sample.

The method of the invention involves analyzing chromatin, comprising:

(a) treating chromatin from a population of blood cells, such as neutrophil cells, isolated from a whole blood sample with an insertional enzyme complex to produce tagged fragments of genomic DNA;

(b) sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and

(c) making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region.

Reference to a population of “blood cells” will be understood to include any cell found in circulating blood, such as white blood cells, red blood cells or platelets. White blood cells include granulocytes (such as neutrophils, eosinophils and basophils), lymphocytes (such as B lymphocytes and T lymphocytes) and monocytes. Methods of the invention are particularly focused on treating chromatin obtained from white bloods cells, in particular neutrophils. Therefore, in one embodiment, the population comprises white bloods cells. In a further embodiment, the population comprises neutrophil cells.

In one embodiment, the neutrophil cells are fixed before isolation from the whole blood sample. In an alternative embodiment, the neutrophil cells are fixed after isolation from the whole blood sample.

A preferred method of the invention involves analyzing chromatin, comprising:

(a) treating chromatin from a population of neutrophil cells isolated from a whole blood sample with an insertional enzyme complex to produce tagged fragments of genomic DNA, and wherein the neutrophil cells are fixed before isolation from the whole blood sample; (b) sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and

(c) making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region.

A method of the invention may comprise:

(a) fixing a population of neutrophil cells present in a whole blood sample, e.g. by applying a fixation buffer;

(b) isolating the population of neutrophil cells from the whole blood sample;

(c) treating chromatin from the isolated neutrophil cells with an insertional enzyme complex to produce tagged fragments of genomic DNA;

(d) sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and

(e) making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region.

Optionally, the whole blood sample is challenged before the neutrophil cells are fixed with at least one candidate modulator of NETosis or at least one modulator of NETosis. For example, a stimulant of NETosis may be applied to the whole blood sample, prior to step (a).

Alternatively, the method may comprise:

(a) isolating a population of neutrophil cells from a whole blood sample;

(b) optionally challenging neutrophil cells with at least one candidate modulator of NETosis or at least one modulator of NETosis;

(c) fixing the isolated neutrophil cells, e.g. by applying a fixation buffer;

(d) treating chromatin from the isolated neutrophil cells with an insertional enzyme complex to produce tagged fragments of genomic DNA;

(e) sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and

(f) making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region.

Extracellular traps (ETs) may be formed by a variety of cell types including neutrophils. Therefore, in one embodiment of the methods described herein, the ETs are Neutrophil extracellular traps (NETs). NETs are extracellular traps formed by neutrophils. The terms ETs and NETs are used interchangeably herein. Similarly, the term NETosis as used herein is intended to encompass the process of the extracellular trap production by any cell. Neutrophils occur predominantly in blood and haematopoietic tissues. However, many other body fluids contain neutrophils due to migration of neutrophils to the site of an injury, infection or inflammation. Thus, neutrophils may be found in multiple body fluids including blood, cerebrospinal fluid (CSF), sputum, saliva, urine and stool.

One aspect underlying the present invention is to measure the propensity of neutrophils in a body fluid sample taken from a subject to undergo NETosis as an indicator of an increased propensity or risk of the subject to suffer a NETs or NETosis related condition or a NETs or NETosis related complication of a disease or a NETs or NETosis related disease flare or crisis of a disease. Such measurements predict the risk of many disease complications including, without limitation, prediction of the development, or the risk of developing, a dysregulated immune response to an infection (for example leading to a severe COVID-19 infection or sepsis), cancer disease progression, inflammatory or autoimmune disorders, vascular or microvascular disease, thrombosis, microthrombi, disseminated intravascular coagulation (DIC), vasculitis, venous thromboembolism, poor wound healing, diabetic complications including for example vascular complications and diabetic foot complications and many others.

Many drugs affect NETosis. For example, treatment of patients with RA or radiographic axial spondyloarthritis treated with anti-TNF-a drugs (e.g. Infliximab) or anti-interleukin-6 receptor (anti-l L6R) drugs (e.g. Tocilizumab) targeted at immunological interventions results in reduced NETs levels. Drug treatment of neutrophils isolated from healthy subjects in vitro also results in reduced NETs production (Ruiz-Limon et al, 2020)

The present invention makes use of the technique called ATAC-seq to investigate chromatin opening. In general terms, ATAC-Seq stands for Assay for Transposase- Accessible Chromatin with high-throughput sequencing. The ATAC-Seq method relies on next-generation sequencing (NGS) library construction using the hyperactive transposase Tn5. NGS adapters are loaded onto the transposase, which allows simultaneous fragmentation of chromatin and integration of those adapters into open chromatin regions. The library that is generated can be sequenced by NGS and the regions of the genome with open or accessible chromatin are analyzed using bioinformatics.

The ATAC-Seq method was first published in 2013 in the journal Nature Methods by lead researcher Jason Buenrostro in the labs of Howard Chang and William Greenleaf at Stanford University (Buenrostro et al, 2013). An overview of the process is shown in FIGURE 3.

The present invention may also make use of the ATAC-see protocol to investigate chromatin. ATAC-see, or transposase-accessible chromatin with visualization, was described in Chen et al, 2016. The technique can be used to provide information on the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion (NETosis). In this embodiment, the insertional enzyme complex comprises a transposase (e.g. Tn5) with fluorophore-conjugated adaptors. ATAC-see fluorescently labels accessible loci so that the molecular accessibility of chromatin can be identified as points of light. After imaging, inserted adaptors allow deep sequencing to map open chromatin sites on the same sample in a manner identical to that of ATAC-seq.

In the present invention the whole blood sample is isolated from an individual.

The neutrophils are fixed before isolation from the whole blood sample using any known method such as the use of formaldehyde or paraformaldehyde (PFA). Other fixers such as chemical cross-linkers, e.g. methanol and DSG (disuccinimidyl glutarate) and also nonchemical cross linkers such as UV light may be used.

The whole blood sample may be fixed at any suitable time after collection of the blood sample, such as up to 2, 3, 4, 12, 13, 25, 60, 90, 120, or 180 minutes after collection of the blood sample, or after challenge. Preferably, the whole blood sample is fixed between 30 and 180 minutes, in particular between 60 and 120 minutes after challenge.

The method comprises (a) treating chromatin from a population of neutrophil cells isolated from a whole blood sample with an insertional enzyme complex to produce tagged fragments of genomic DNA, and wherein the neutrophil cells are fixed before isolation from the whole blood sample.

In one embodiment, the tagged fragments are produced by simultaneous fragmentation of the chromatin and addition/ligation of a tag (e.g. inserting a sequence into the DNA). This process is also known as “tagmentation”. Thus, the chromatin is tagmented, i.e. cleaved and tagged in the same reaction, using an insertional enzyme in the insertional enzyme complex.

The present invention makes use of an insertional enzyme complex. This refers to a complex comprising an insertional enzyme and two adapter molecules (the “transposon tags”) that are combined with polynucleotides to fragment and add adapters to the polynucleotides. The term “insertional enzyme” or “tagging enzyme” as used herein, refers to any enzyme capable of addition of nucleotide sequences (e.g. transposon tags) to the target DNA molecule. Thus, in one embodiment the insertional enzyme complex comprises a ligase. T4 DNA ligase is widely employed for joining two double DNA strands between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. In a further example the insertional enzyme is a recombinase enzyme. Examples of such recombinase enzymes include retroviral integrase and transposase enzymes.

In some cases, the insertional enzyme can insert the nucleic acid sequence into the polynucleotide in a substantially sequence-independent manner. The insertional enzyme can be prokaryotic or eukaryotic. Examples of insertional enzymes include, but are not limited to, transposases, HERMES, and HIV integrase. The transposase can be a Tn transposase (e.g. Tn3, Tn5, Tn7, Tn10, Tn552, Tn903), a MuA transposase, a Vibhar transposase (e.g. from Vibrio harveyi ), Ac-Ds, Ascot-1 , Bs1 , Cin4, Copia, En/Spm, F element, hobo, Hsmarl , Hsmar2, IN (HIV), IS1 , IS2, IS3, IS4, IS5, IS6, IS10, IS21 , IS30, IS50, IS51 , IS150, IS256, IS407, IS427, IS630, IS903, IS911 , IS982, IS1031 , ISL2, L1 , Mariner, P element, Tam3, Tc1 , Tc3, Te1 , THE-1 , Tn/O, TnA, Tn3, Tn5, Tn7, Tn10, Tn552, Tn903, Toll , Tol2, Tyl, any prokaryotic transposase, or any transposase related to and/or derived from those listed above.

In one embodiment the transposase is derived from Tn5 transposase.

In another embodiment the transposase is derived from MuA transposase.

In one embodiment, the insertional enzyme complex comprises a tag sequence for attaching an identifiable tag to the target DNA. Such tag sequences may comprise adapter sequences and/or labelled nucleotides. References herein to “adapter sequences” (or “adaptor sequences”) refer to short, chemically synthesized, oligonucleotides that can be ligated to the ends of other DNA or RNA molecules.

The molecular tags can comprise sequencing adaptors, locked nucleic acids (LNAs), zip nucleic acids (ZNAs), RNAs, affinity reactive molecules (e.g. biotin, dig), self-complementary molecules, phosphorothioate modifications, azide or alkyne groups. In some cases, the sequencing adaptors can further comprise a barcode label. Further, the barcode labels can comprises a unique sequence. The unique sequences can be used to identify the individual insertion events. Any of the tags can further comprise fluorescence tags (e.g. fluorescein, rhodamine, Cy3, Cy5, thiazole orange, etc.). In some embodiments, the sequencing adapters comprise a barcode sequence. Such barcode sequences may allow for the identification of a particular nucleic acid composition in subsequent analysis and processing and allow for tagged DNA molecules from multiple samples to be combined in subsequent steps, whilst retaining the ability to identify and analyse individual DNA molecules of interest. The adapter sequences, such as adapter sequences comprising a barcode sequence, may also be used to identify and/or enrich/isolate the tagged DNA onto which they have been ligated, e.g. they may be used to selectively purify the tagged DNA. For example, the adapter sequences, optionally comprising a barcode sequence and/or Unique Molecular Identifier, may be used to amplify the tagged DNA by PCR. Thus, in one embodiment enrichment and/or isolation is by PCR amplification. In a further embodiment, the adapter sequences allow or enable subsequent library preparation and sequencing of the tagged DNA.

In another embodiment, the insertional enzyme attaches or comprises labelled nucleotides and the DNA is tagged with said labelled nucleotides. In some embodiments, the labelled nucleotides are biotinylated nucleotides or fluorescently labelled nucleotides, such as biotinylated or fluorescently labelled NTPs. Such labelled nucleotides may be used to identify and/or enrich/isolate the tagged DNA into which they are incorporated or onto which they have been ligated, e.g. they may be used to selectively purify the tagged DNA.

Additionally, the insertional enzyme can further comprise an affinity tag. In some cases, the affinity tag can be an antibody. The antibody can bind to, for example, a transcription factor, a modified nucleosome or a modified nucleic acid. Examples of modified nucleic acids include, but are not limited to, methylated or hydroxymethylated DNA. In other cases, the affinity tag can be a single-stranded nucleic acid (e.g. ssDNA, ssRNA). In some examples, the singlestranded nucleic acid can bind to a target nucleic acid. In further cases, the insertional enzyme can further comprise a nuclear localization signal.

After the chromatin has been fragmented and tagged to produce tagged fragments of genomic DNA, at least some of the adaptor tagged fragments are sequenced to produce a plurality of sequence reads. The fragments may be sequenced using any convenient method. For example, the fragments may be sequenced using Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' sequencing by ligation (the SOLiD platform) or Life Technologies' Ion Torrent platform. Examples of such methods are described in the following references: Margulies et al (Nature 2005 437: 376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9); Shendure et al (Science 2005 309: 1728-32); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox et al (Methods Mol Biol. 2009; 553:79- 108); Appleby et al (Methods Mol Biol. 2009; 513:19-39) and Morozova et al (Genomics. 2008 92:255-64), which are incorporated by reference for the general descriptions of the methods and the particular steps of the methods, including all starting products, methods for library preparation, reagents, and final products for each of the steps. As would be apparent, forward and reverse sequencing primer sites that are compatible with a selected next generation sequencing platform can be added to the ends of the fragments during the amplification step. In certain embodiments, the fragments may be amplified using PCR primers that hybridize to the tags that have been added to the fragments, where the primer used for PCR have 5' tails that are compatible with a particular sequencing platform. In certain cases, the primers used may contain a molecular barcode (an “index”) so that different pools can be pooled together before sequencing, and the sequence reads can be traced to a particular sample using the barcode sequence.

In another aspect, the present disclosure provides a method for determining accessibility of a polynucleotide at a site, wherein the polynucleotide is from a cell sample, said method comprising: inserting a plurality of molecular tags with an insertional enzyme into the polynucleotide and using the molecular tags to determine accessibility at the site. The cell sample can be from a primary source. The cell sample may consist of a single cell. The cell sample may consist of a finite number of cells (e.g. less than about 500,000 cells).

The method can further comprise using the determined accessibility to identify one or more proteins that are bound to the polynucleotide at the site. In some instances, at least one of the proteins is a transcription factor. Additionally, the method can comprise using the molecular tags to generate an accessibility map of the polynucleotide.

The polynucleotide may be fragmented into a plurality of fragments during the insertion of the molecular tags. In some cases, the fragments may be amplified. In some cases, the fragments can be sequenced to generate a plurality of sequencing reads. This may be used to determine the accessibility of the polynucleotide at any given site. The fragments may be sequenced using a high-throughput sequencing technique. In some cases, the sequencing reads can be normalized based on the sequence insertion preference of the insertional enzyme. The length of the sequenced reads can be used to determine a chromatin state annotation.

Embodiments of the methods involve making an epigenetic map of a region of the genome of the cells. This step may be done by mapping information obtained from the sequence reads to the region. In these embodiments, the sequence reads are analyzed computationally to produce a number of numerical outputs that are mapped to a representation (e.g., a graphical representation) of a region of interest.

In one embodiment, the information is obtained using the nucleotide sequence(s) at the beginning (start) and, optionally, the end of a sequence read.

As will be explained in greater detail below, many types of information may be mapped, including, but not limited to: (i) cleavage sites for the transposase; (ii) the sizes of the fragments produced in step a); (iii) fragment length; (iii) the positions of sequence reads of a defined range in length; and (iv) sequence read abundance. In a further embodiment, the fragments of a defined size range are nucleosome-free fragments.

The resultant epigenetic map can provide an analysis of the chromatin in the region of interest. For example, depending on which information is mapped, the map can show one or more of the following: a profile of chromatin accessibility along the region; DNA binding protein (e.g., transcription factor) occupancy for a site in the region; nucleosome-free DNA in the region; positioning of nucleosomes along the region; and a profile of chromatin states along the region. In some embodiments, the method may further comprise measuring global occupancy of a binding site for the DNA binding protein by, e.g., aggregating data for one DNA binding protein over a plurality of sites to which that protein binds. In certain instances, the map can also be annotated with sequence information, and information about the sequence (e.g., the positions of promoters, introns, exons, known enhancers, transcriptional start sites, untranslated regions, terminators, etc.) so that the epigenetic information can be viewed in context with the annotation.

In certain embodiments, the epigenetic map can provide information regarding active regulatory regions and/or the transcription factors that are bound to the regulatory regions. For example, nucleosome positions can be inferred from the lengths of sequencing reads generated. Alternatively, transcription factor binding sites can be inferred from the size, distribution and/or position of the sequencing reads generated. In some cases, novel transcription factor binding sites can be inferred from sequencing reads generated. In other cases, novel transcription factors can be inferred from sequencing reads generated.

Thus the present invention can be used to identify changes in gene promoters, changes in enhancers and/or changes in non-coding regulatory regions. In particular the changes that occur during NETosis. The present invention also allows the identification of clusters or a combination of changes which may occur during NETosis. The present invention allows one of these changes to be targeted and this may prevent a deleterious combination of events occurring. For example, with reference to Figure 8 which shows that inter alia JUN is involved in the process, targeting regions around JUN could prevent the induction of deleterious NETosis.

The cells that are analyzed are neutrophil cells. These are isolated from a whole blood sample. References to “isolation” or “selection” will be understood to include both positive and negative isolation methods, i.e. positive isolation methods where the cell of interest (e.g. neutrophil cells) is actively bound and removed from a whole blood sample, compared to negative selection methods where cells that are not wanted in the whole blood sample are removed and the cell of interest is left in the remaining sample.

The population of cells may be selected by EasySep™, MACS or FACS from a heterogeneous population of cells in whole blood by known methods using labeled antibodies to cells surface markers. In one embodiment the neutrophils are isolated from the whole blood using EasySep™. When in another embodiment the method involves use of isolated neutrophils which isolated from whole blood before fixing, it is preferable that these cells are isolated using MACS.

In one embodiment the whole blood sample is challenged before the neutrophil cells are fixed with at least one candidate modulator of NETosis or at least one modulator of NETosis. For ease of reference candidate modulator and modulator may be used interchangeably.

The modulator or candidate modulator of NETosis may be a stimulator of NETosis or an inhibitor of NETosis. In one embodiment the modulator of NETosis is PMA. However, others are known in the art or the invention may involve investigating the role of a potential modulator of NETosis.

Stimulators of NETosis

Many chemical activators, stimulants or inducers of NETosis are known in the art and any such stimulant of NETosis may be used for methods of the invention to induce neutrophils to undergo NETosis.

Therefore, in a further aspect of the invention, NETosis of neutrophils is stimulated by addition of a substance or moiety that stimulates or induces NETs production in a sample. Such stimulants are known in the art and include, without limitation, any bacterial, fungal or viral pathogen or component thereof (living or dead, active or inactive), or any chemical activator of NETosis including, without limitation, heparin, PMA, lipopolysaccharides (LPS), reactive oxygen species or moieties that generate reactive oxygen species (for example, hydrogen peroxide or glucose oxidase), or calcium ionophores (Cal or Cl).

In one embodiment the stimulant of NETosis added is selected from: PMA, LPS, Cal or a bacterial, fungal or viral pathogen or a component thereof. In one embodiment the whole blood sample, to which a stimulant of NETosis is added, is collected in a plasma sample collection tube such as an EDTA, heparin, citrate or other plasma sample collection tube. In some embodiments the stimulant of NETosis added to the whole blood sample stimulates a rapid NETosis.

In one embodiment, the stimulant of NETosis is a natural factor. References herein to a “natural factor” is understood to refer to a naturally occurring modulator of NETosis, such as naturally occurring cytokines and chemokines at physiologically relevant concentrations. Naturally occurring molecules associated with neutrophils or NETosis activation are known in the art, such as interleukins: ll-1 b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 along with other molecules TNF- a, LT-a, IFN-y, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1 , CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, ferritin, HMGB1 , C5a and LPS.

In one embodiment, the natural factor is selected from TNF-alpha (TNF-a), LT-alpha (LT-a), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Complement component 5a (C5a), N-formyl-methionyl-leucyl-phenylalanine (fMLP), lnterleukin-1 B (IL-1 B), Interferon gamma (IFN-y), lnterleukin-8 (IL-8), Leukotriene B4 (LTB4), ferritin and lipopolysaccharides (LPS).

It will be understood that more than one (i.e. a combination) of NETosis stimulants may be used. Therefore, in one embodiment, the whole blood sample is challenged with a composition comprising one or more (e.g. at least two or at least three) stimulants of NETosis. It will be understood that the stimulants may be added individually, in combination or at staggered time points (i.e. prior to fixation).

In one embodiment, the stimulant of NETosis is selected from TNF-a, LT-a, C5a and/or fMLP. In a further embodiment, the stimulant of NETosis is selected from TNF-a and/or LT-a.

In one embodiment, the whole blood sample is challenged with a composition comprising or consisting of at least three inducers of NETosis of which at least one is TNF-a or LT a. In a further embodiment, the composition comprises or consists of at least three inducers of NETosis including TNF-a or LT-a in combination with one or more of IFN-y, GM-CSF, LTB4, C5a, ferritin, LPS or fMLP.

Inhibitors of NETosis

Any moiety that inhibits NETosis in a body fluid sample may be useful in the current invention. Many NETosis inhibitors have been described in the literature and the number is increasing rapidly as the interest in NETosis has increased greatly (partially due to the recent outbreak of COVID-19). Any of these NETosis inhibitors may be used for the invention as described herein. Some examples of NETosis inhibitors described in the literature include, without limitation, anti-citrullinated protein antibodies (Chirivi et at, 2016), GSK484 (an inhibitor of the Protein Arginine Deiminase 4 NETosis pathway), high concentrations of heparin (heparin is reported to be an activator of NETosis at low concentration and an inhibitor of NETosis at high concentrations), anthracycline drugs, including without limitation epirubicin, daunorubicin, doxorubicin and idarubicin, blockers or antibodies to CD32, blockers or antibodies to CD62p and blockers or antibodies to CD162 (Perdomo et a/; 2019), indolylmaleimide moieties (Dodo et a , 2019), leukocyte elastase inhibitor, diisopropyl fluorophosphate, diphenylene iodonium, phenylmethylsulfonyl fluoride, aminoethylbenzene sulfonylfluorid (Farley et a/; 2012) and BMS-P5 (Li et at, 2020). Sondo et at, 2019 identified 70 putative inhibitors of NETosis, 22 of which fully inhibited NETosis onset. These included kinase inhibitors, vanilloids (for example, capsaicin and dihydrocapsaicin) and tetrahydroisoquinolines. Similarly, a large number of NETosis inhibitors are disclosed in WO2016/127255. It will be understood that any of these NETosis inhibitors, as well as any yet to be described, may be used in the present invention as described herein.

It will be understood that NETosis is inhibited in a whole blood sample by the addition of an inhibitor of coagulation. Thus, NETosis may be inhibited indirectly by preventing or inhibiting induction of NETosis by coagulation. Many inhibitors of coagulation are known in the art including heparin, citrate and EDTA. Thus EDTA, or other anti-coagulant, may be added to a whole blood sample (for example in a serum blood collection tube) after leaving the whole blood sample for NETosis to occur to prevent further coagulation and indirectly inhibit NETosis. Therefore, in one embodiment the inhibitor of NETosis is an anticoagulant.

Moieties such as azide that induce rapid cell death without cell lysis may also prevent further NETosis and be useful for the termination of NETosis in methods of the invention. Inhibitors of NETosis that fully inhibit or prevent NETosis are particularly useful for preventing NETosis in blood or other body fluid samples for use in the present invention.

Analysis methods

The whole blood sample may be challenged or assayed at any suitable time after collection of the blood sample, such as up to 2, 3, 4, 12, 13, 25, 60, 120, or 180 minutes after collection of the blood sample, or after challenge.

In one embodiment, the cells are lysed prior to treating with an insertional enzyme complex. The chromatin used in the method may be made by any suitable method, for example using a suitable lysing buffer.

In one embodiment, wherein the treating step (a) comprises: isolating nuclei from the population of cells; and combining the isolated nuclei with said insertional enzyme complex, wherein the combining results in both lysis of the nuclei to release said chromatin and production of said tagged fragments of genomic DNA.

The population of cells used in the assay may be any suitable number of cells, such as 1 to 100,000 cells.

In one embodiment, the whole blood sample is collected in an EDTA tube.

In some embodiments, the method can be used to compare two samples. In these embodiments, the method may comprise analyzing a first population of cells using the abovedescribed method to produce a first epigenetic map; and analyzing a second population of cells using the above-described method to produce a second epigenetic map; and comparing the first epigenetic map to the second epigenetic map, e.g., to see if there are any changes in chromatin openness or transcription factor occupancy, for example.

Thus in one embodiment there is provided a method for comparing two samples comprising:

(a) analyzing a first population of cells using the method of the invention to produce a first epigenetic map; and

(b) analyzing a second population of cells using the method of the invention to produce a second epigenetic map; and

(c) comparing the first epigenetic map to the second epigenetic map. In some embodiments the first population of cells has been challenged with at least one candidate modulator of NETosis or at least one modulator of NETosis. In some embodiments the second population of cells has not been challenged with a candidate modulator of NETosis or a modulator of NETosis.

In one embodiment there is provided a method for comparing two samples containing neutrophils derived from whole blood comprising: analyzing a first population of cells using the method of the invention to produce a first epigenetic map wherein the first population of cells is challenged with a candidate modulator of NETosis or a modulator of NETosis before isolation of the neutrophils from a whole blood sample; and analyzing a second population of cells using the method of the invention to produce a second epigenetic map wherein the second population of cells is challenged with a candidate modulator of NETosis or a modulator of NETosis after isolation of the neutrophils from a whole blood sample.

In some embodiments, the first population of cells and the second population of cells are collected from the same individual at different times. In other embodiments, the first population of cells and the second population of cells are different populations of cells collected from different individuals.

The method may be used to identify the effect of a test agent, e.g., a drug, or to determine if there are differences in the effect of two or more different test agents. In these embodiments, two or more identical populations of cells may be prepared and, depending on how the experiment is to be performed, one or more of the populations of cells may be incubated with the test agent for a defined period of time. After incubation with the test agent, the chromatin of the populations of cells can be analyzed using the methods set forth above, and the results can be compared. In a particular embodiment, the cells may be blood cells, and the cells can be incubated with the test agent ex vivo. These methods can be used to determine the mode of action of a test agent, to identify changes in chromatin structure or transcription factor occupancy in response to the drug, for example.

According to another aspect of the invention, there is provided a method for identifying the effect of a candidate modulator of NETosis, comprising: analyzing chromatin from a patient treated with the candidate modulator of NETosis using the method described herein, to produce an epigenetic map; and identifying whether the candidate modifier of NETosis is a modifier of NETosis based on the epigenetic map.

Diagnostic and treatment methods

The method described above may also be used as a diagnostic (which term is intended to include methods that provide a diagnosis as well as methods that provide a prognosis). These methods may comprise, e.g., analyzing chromatin from a patient using the method described above to produce an epigenetic map; and providing a diagnosis or prognosis based on the epigenetic map.

The method set forth herein may be used to provide a reliable diagnostic to any condition associated with altered chromatin or DNA binding protein occupancy. The method can be applied to the characterization, classification, differentiation, grading, staging, diagnosis, or prognosis of a condition characterized by an epigenetic pattern (e.g., a pattern of chromatin accessibility or DNA binding protein occupancy). For example, the method can be used to determine whether the epigenetic map of a sample from an individual suspected of being affected by a disease or condition is the same or different compared to a sample that is considered “normal” with respect to the disease or condition. In particular embodiments, the method can be directed to diagnosing an individual with a condition that is characterized by an epigenetic pattern at a particular locus in a test sample, where the pattern is correlated with the condition. The methods can also be used for predicting the susceptibility of an individual to a condition.

The method can also be used to determining a proper course of treatment for a patient having a disease or condition, e.g., a patient that has cancer. A course of treatment refers to the therapeutic measures taken for a patient after diagnosis or after treatment. Therefore, according to one aspect, there is provided method for treatment selection, comprising: analyzing chromatin from a patient using the method described herein, to produce an epigenetic map; and selecting a treatment for the patient based on the epigenetic map.

Kits

In another aspect the present invention provides a kit comprising:

(a) a modulator of NETosis;

(b) reagents for isolating neutrophils from whole blood cells;

(c) reagents for isolating nuclei from a population of cells;

(b) an insertional enzyme complex, and

(c) transposase reaction buffer, wherein the components of the kit are configured such that, combining the reaction buffer, transposon tags and adaptors with nuclei in vitro results in both lysis of the nuclei to release chromatin and production of tagged fragments of genomic DNA.

The kit may optionally contain other components, for example: PCR primers, PCR reagents such as polymerase, buffer, nucleotides etc., as described above. The various components of the kit may be present in separate containers or certain compatible components may be recombined into a single container, as desired.

In addition to above-mentioned components, the subject kits may further include instructions for using the components of the kit to practice the subject methods, i.e., instructions for sample analysis.

It will be understood that the embodiments described herein may be applied to all aspects of the invention, i.e. the embodiment described for the uses may equally apply to the claimed methods and so forth.

The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLES

EXAMPLE 1

Fresh whole blood is collected in K2 EDTA tubes and processed within one hour of the blood draw. The blood is treated with 250 nM PMA or a dimethylsulfoxide (DMSO) vehicle control and then aliquoted into 2ml tubes. The tubes are then rotated end over end at 37°C over a time course of minutes up to 180 minutes. After the appropriate amount of incubation time, the aliquot is removed and a fixation buffer is added (final concentration of 1% formaldehyde) and rotated for 10 minutes at room temperature. Afterwards, the reaction is stopped with the addition of 1 :20 (vokvol) 2.5M glycine and cell pellets are subsequently washed with phosphate buffered saline (PBS).

Neutrophils are then isolated with the EasySep Neutrophil Isolation kit in line with the manufacturer’s instructions. Pure neutrophils are snap frozen in liquid nitrogen and stored at -80°C. ATAC-see is then performed in line with the published protocol (Chen et al. Nat. Methods 2016 Dec; 13(12): 1013-1020; Note that “see” is not a typo, the method was called ATAC-see and is similar to the standard ATAC-seq).

EXAMPLE 2: Chromatin Changes Associated with Neutrophil Extracellular Trap (NET)

Formation in Whole Blood Reflect Complex Immune Signaling

Materials and methods

Whole Blood Collection

Whole blood was obtained from healthy donors in K2-EDTA tubes (PrecisionMed, San Diego). Research was approved under WCG IRB Protocol number 20161665, and all participants provided written informed consent. Each subject was healthy, aged 18-50, with BMI < 30, and not taking Non-steroidal anti-inflammatory drugs (NSAIDs).

Whole Blood Treatment

Whole blood was pooled and 20 mL aliquots distributed into two 50 mL tubes. Untreated samples were collected as 2 mL aliquots in 5 mL tubes and fixed as described below. The remaining 18 mL was treated with either 250 nM PMA or DMSO. Subsequently, 2 mL aliquots were placed in 5 mL tubes for each time point (30 min, 60 min, 90 min, and 120 min) and maintained at 37°C until further processing.

The cells were fixed with a 10x solution of formaldehyde (Formaldehyde 11%, 1M NaCI, 0.1 mM EDTA, 0.5mM HEPES), added at 1 :10 volume to whole blood aliquots. After incubation at room temperature for 10 minutes, fixation was quenched with a 1 :20 volume of glycine (2.5M). Neutrophils were then isolated from the fixed whole blood using the MACSxpress Whole Blood Neutrophil Isolation Kit for humans (Miltenyl Biotec). Briefly, the bead mixture was added to the whole blood sample, followed by incubation on a rotator. Magnetic separation yielded isolated neutrophils, which were washed in 1x PBS and subjected to red blood cell (RBC) lysis to eliminate contaminants. Cells were washed in ice-cold 1x PBS and pelleted to retain only intact neutrophils, removing potential NET fragments or other DNA. 125,000 cells per condition/time point/replicate were flash frozen in liquid nitrogen and stored at -80°C until further processing. Tn5 Assembly

Recombinant Tn5 transposase protein (Active Motif) was assembled with custom oligos mosaic end (ME) ME_Rev, ME_A, and ME_B (IDT), and activity was tested as previously described (Buenrostro et al, 2013).

ATAC-Seq Protocol

The fixed ATAC-Seq protocol was performed as previously reported (Atteberry et al, 2024). Briefly, adapted from Chen et al. (2016), nuclei were transposed in a reaction mixture containing assembled Tn5 transposase, followed by cross-link reversal, DNA purification, library preparation, and sequencing.

ATAC-Seq Processing and Peak Calling

ATAC-Seq processing and alignment were conducted using an ATAC-Seq Nextflow pipeline (https://nf-co.re/atacseqZ2.1.2) with the nf-core framework as previously described (Atteberry et al, 2024). Samples were aligned to the hg38 reference genome, with the fragment size parameter set to 200. Peak calling was performed using MACS2 (Zhang et al, 2008), identifying narrow peaks at a false discovery rate (FDR) of 0.01 . This pipeline followed current ENCODE sequencing standards (Landt et al, 2012).

Untreated ATAC-Seq Analysis

Consensus peaks (.featureCounts.txt) and annotated peaks (.annotatePeaks.txt) were generated using the nf-core pipeline for subsequent analysis. Read counts were normalized across samples using scaling factors from bigwig normalization (https://cran.r- project.org/web/packages/scales/index.html) to balance total reads. For untreated samples, normalized peak counts were combined with annotated peaks to create a consensus peak dataset. UpSetR plots (https://cran.r-project.org/web/packages/UpSetR/index.html) were generated using the .boolean.annotatePeaks.txt files. Additional metrics, including read counts, peak annotations, bigwigs, fraction of reads in peaks (FRiP) scores, insert sizes, and alignment metrics, were generated using the nf-core ATAC-Seq pipeline.

Treated ATAC-Seq Analysis

Treated samples were analyzed similarly to untreated samples, with the following modifications. Samples were normalized and divided into six groups based on time course (T30, T60, T90, and T120 minutes) and treatment (DMSO vs. PMA). Samples with less than 10 million reads or a FRiP score below 5 were excluded. Peaks were retained if at least 2/3 of donors at a given timepoint/treatment had a peak within that interval. For DESeq2 (Love et al, 2014) analysis, normalized counts of consensus peaks were filtered to include only those with a baseMean > 50, and subsequent analyses were performed on this dataset. Principal Component Analysis (PCA) was conducted using prcomp, and pairwise comparisons were performed with DESeq(), summarizing results with the results() function. Significant regions (padj < 0.01) were z-score normalized based on normalized peak counts, and heatmaps were generated using pheatmap (https://cran.r-project.org/web/packages/pheatmap/index.html) with row clustering. Volcano plots were created using DESeq2's results() function, defining significant regions by -loglO(padj) > 4. For all-vs.-all comparisons, significant regions from each grouping (treatment and timepoint) with padj < 0.01 were included. Pheatmap (https://cran.r-project.org/web/packages/pheatmap/index.html) was used with both row and column clustering. Overlaps between T30 DMSO vs. PMA T30-T120 were analyzed using innerjoin (tidyverse) (29) and visualized with eulerr version 7.0.2 for Venn diagrams in a custom R script.

HOMER Analysis

HOMER was used to determine motif enrichment in differentially accessible regions (DARs) after PMA stimulation, as previously described (Atteberry et al, 2024), with the following modifications. Significant regions were sorted by padj, and a background set of DARs was randomly selected from the total consensus peaks generated in Nextflow for the dataset (.featureCounts.txt) for motif enrichment. Next, findMotifsGenome.pl was used with hg38 and standard settings and results were visualized using a custom R script.

Isolated Data Processing

Isolated neutrophil data was obtained from the Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra, BioProject: PRJNA1120432). This data was processed using the same pipeline and settings as untreated and treated ATAC-Seq analysis, including alignment, normalization, peak calling with MACS2, and DESeq2 analysis. Replicate-merged datasets for isolated samples were used in all comparisons.

Pathway Analysis

DAR gene lists from ATAC-Seq were analyzed for pathway enrichment using the following bioinformatics tools: DAVID (https://david.ncifcrf.gov) was used to identify functional domains and motifs. Gene lists were divided into whole blood and isolated neutrophil datasets, and functional annotation was performed using Reactome, KEGG, and GO databases to identify pathways relevant to NET formation. Metascape (https://metascape.org) was also used to analyze the same gene lists. Enrichment analysis was conducted through Metascape's express analysis mode, followed by custom downstream analysis using Python3. Results were processed using the Pandas library (v2.2.0) (Patel et al, 2023), focusing on the top 20 p-values per condition, with missing values replaced by 0. NumPy (v1.26.4) (Patel et al, 2023) handled missing values, and heatmaps were generated using Seaborn (vO.13.2) (Waskom, 2021) with Matplotlib (v3.8.3) (Hunter, 2007) for customization. These heatmaps visualized enrichment scores (-loglO(pvalue)) with average clustering and significance annotations.

Results

Whole Blood Fixation Followed by Neutrophil Isolation Shows Stable Chromatin Structure Across Donors

Previous work has assessed chromatin changes associated with NET formation in isolated neutrophils. To expand upon this and understand the interplay of the whole blood matrix and potential immune interactions, we induced NET formation in whole blood, followed by formaldehyde fixation, neutrophil isolation, and ATAC-Seq. Whole blood was either untreated (n=6), PMA stimulated (n=5), or DMSO stimulated (n=5), and compared to neutrophil ATAC- Seq data where cells were isolated prior to fixation. To minimize the impact of stress during isolation, we fixed whole blood with formaldehyde prior to neutrophil isolation, aiming to maintain neutrophils close to their circulating state. This approach yielded highly pure neutrophils (-90%), with monocytes as the main contaminant.

Untreated whole blood samples fixed prior to isolation (donors D48, D76, D79, D81 , D83, and D85) showed high correlation across donors and with post-isolation samples (Isolated), with minimal presence of accessible chromatin which would be expected from contamination of other common immune cells (Figure 9A). We examined gene loci expected to have accessible chromatin in neutrophils (CD14, ACTB, CLEC7A, and HCAR) and regions expected to lack accessibility (AZU1 and MPO). Despite lower purity in whole blood fixed samples, loci specific to B cells (PAX5 and CD19), T cells (CD8A and CD3E), and monocytes (ITGAM, CSF1 R) showed no chromatin accessibility, as expected. This suggests that chromatin structure in the whole blood model is consistent across donors and with what is observed in neutrophils that are isolated prior to fixation.

Called peaks were reproducible across donors and showed limited variability in gene annotations (Figure 9B). Specifically, over 4,500 peaks were shared among all six donors, indicating a set of commonly accessible chromatin regions in whole blood neutrophils. Although certain donors (D48 and D81) had more unique peaks, the majority of peaks were shared across multiple donors, demonstrating considerable overlap. Notably, over 24,000 peaks were found in at least two donors, while nearly 16,500 peaks were common across three donors. This consistency highlights the robustness of chromatin accessibility profiles in whole blood neutrophils across donors, despite individual variability.

PMA Stimulation Drives a Dynamic Chromatin Response Over Time Compared to DMSO Controls

To assess chromatin accessibility changes during PMA-induced NET formation in whole blood, we treated whole blood from five donors (D48, D76, D79, D81 , and D85) with 250 nM PMA or DMSO and selected time points prior to NET release. Samples were fixed at 30, 60, 90, and 120 minutes, followed by neutrophil isolation and ATAC-Seq. Samples with fewer than 10 million reads and a FRiP score below 5 were excluded. Called peaks (MACs, q < 0.01) and their annotations showed limited changes based on treatment conditions, with differences mainly due to donor variability.

To determine whether PMA treatment drove global changes in chromatin accessibility, we performed Principal Component Analysis (PCA) and observed separation between PMA and DMSO groups (Figure 10A). Comparing chromatin accessibility at various loci showed stability of TBP (housekeeping gene) and increased accessibility at CXCL2, CXCL3, CXCL5, and CD69 loci in PMA-treated samples at 60 minutes. A bimodal response at ACTG1 indicated rapid opening followed by decreased accessibility by 90 minutes consistent with previous findings. There were also regions with decreased accessibility, such as the CXCR2 locus (Figure 10B). These results indicate a gain and loss of chromatin accessibility with PMA induction.

These results support the concept that PMA treatment induces chromatin changes preceding NET release. DESeq2 analysis revealed few significant DARs in the DMSO group between 30 and 120 minutes, indicating chromatin stability across the time course. However, comparing DMSO at 30 minutes to PMA at all timepoints showed numerous significant DARs. Pairwise comparison of the number of DARs (q < 0.01) revealed that DARs were consistent and reproducible across the dataset, with limited chromatin changes at 30 minutes in PMA treatment, but significant changes at 60, 90, and 120 minutes compared to DMSO at 30 minutes (Figure 10C).

To gain a better understanding of the pattern of chromatin accessibility changes following PMA treatment, we determined the number of DARS across different time points. Consistent with the results described above, we found limited chromatin accessibility changes at 30 minutes, with only 105 DARs identified. Of these, 64 were present across all four time points (30-, 60-, 90-, and 120 minutes), and the majority of the changes occurred after 30 minutes. Specifically, 2,189 DARs were shared across the 60, 90 and 120 minutes of PMA. Thus, the 30-minute PMA-treated timepoint was excluded from further analyses. The remaining PMA treated DARs (60, 90, 120 minutes) were combined to generate a comprehensive list and using DMSO at 30 minutes as a baseline, we assessed chromatin accessibility progression over time in response to PMA treatment. DARs were sorted by adjusted p-values, and the top 1 ,000 were z-score normalized (data not shown). We next determined whether certain transcription factor (TF) binding sites were enriched in regions that were gaining or losing accessibility and found that regions gaining accessibility had TF motifs associated with NET formation, including the AP-1 complex (JUN, FOS, ATF) and FRA1/2 (Figure 10D). In contrast, regions losing accessibility were linked to differentiation and proliferation pathways, including the ELF family and PU.1 (Figure 10E).

PMA-induced Chromatin Accessibility Changes in Whole Blood and Isolated Neutrophils Are Similar but Distinct

Comparing NET induction in isolated neutrophils and whole blood, we first assessed if the order of isolation and fixation affected consensus peaks (defined as peaks shared by at least 50% of donors in each group). Approximately 57% (14,420) peaks were shared between conditions, with 8% (2,073 peaks) unique to whole blood fixed samples and 35% (8,813 peaks) unique to neutrophils fixed after isolation. Overlapping peaks indicate a strong consensus between datasets and numerous peaks that occur only when fixation occurs after neutrophil isolation.

To determine whether NET associated chromatin changes are different when NETs are formed in isolated neutrophils or in the context of whole blood, we analyzed DARs following PMA induction in both systems. We identified 6,282 significant DARs, with approximately 20% (1 ,114/6,282) overlapping between systems, 30% (1 ,888/6,282) unique to isolated neutrophils, and over 50% (3,280/6,282) unique to the whole blood system. PMA induced DARs were then assessed as to whether they were found in the consensus peaks from untreated isolated or whole blood neutrophils. About 46% of PMA induced DARs were outside consensus peak sets, indicating PMA-induced new chromatin accessibility. Interestingly, 70% of overlapping PMA induced DARs were found within original consensus peaks, compared to 60% in isolated and 54% of whole blood DARs.

Temporal analysis of PMA-induced DARs showed consistent patterns across induction systems (Figure 11 A). Despite only around 20% of the PMA induced DARs overlapping, samples generally clustered by treatment (DMSO vs. PMA). The DARs present in both isolated and whole systems exhibited stronger fold changes in whole blood compared to isolated samples, with few divergent changes, indicating a robust PMA response across systems.

Gene Ontology (GO) analysis of DAR-associated genes revealed stronger enrichment in immune response pathways in whole blood compared to isolated samples (Figure 11 B). Reactome pathway analysis showed immune-specific pathways, including cytokine signaling and innate immune responses, were more prominent in whole blood (Figure 11C), whereas signaling cascades downstream from cell surface receptors and genes involved in response to starvation were more prominent in isolated neutrophils. These results suggest NET formation in whole blood involves more complex cell signaling pathways during PMA stimulation, reflecting interactions with other immune cells or factors present in whole blood.

Whole blood PMA induction leads to a more complex immune response than isolated neutrophil PMA induction

We next sought to further understand the role of the extracellular environment in NET formation by assessing changes between T30 DMSO and PMA treatments in both systems by z-score normalizing the DAR counts (n = 6,282) and mapping them over time. DARs were next categorized based on changes in accessibility with PMA induction into isolated neutrophils only (n=944), whole blood only (n=1 ,640), or in both (i.e., overlapping; n= 557) and the top half were retained (based on adjusted p-value) in each category for z-score normalization. We observed that the isolated-only group had fewer DARs with increased accessibility, whereas whole blood and overlapping groups showed more balance of increasing and decreasing accessibility with PMA treatment.

To better understand the underlying biology of NET formation in whole blood and isolated neutrophil environments, we separated DARs into increasing/decreasing chromatin accessibility and identified enriched TF binding motifs. We next identified overrepresented TF binding site sequences and found regions with increased accessibility in overlapping and whole blood groups were enriched for TFs associated with NET formation or inflammation, such as the AP-1 complex (42-44), and MAF/NFE2L2 (NRF2) (Figure 12A). In the isolated group, the most identified motifs were not correlated with a NET response, except for HIF-1a and STAT3, which are known to play roles in neutrophil and inflammatory responses (Figure 12B). These motifs were also present in whole blood and overlapping groups but were ranked below the top 20 based on p-value. Overall, DARs with decreased accessibility showed limited correlation to TFs directly involved in NET formation across all categories (Figure 12B). We examined the genes associated with altered chromatin accessibility to identify affected pathways in response to PMA stimulation. Using Metascape’s Reactome pathway analysis (Milacic et al, 2024 and Fagerberg et al, 2014), we identified significant pathways in the three groups, finding many related to immune system regulation, including cytokine signaling, interleukins, and neutrophil degranulation. Interestingly, the top categories varied among whole blood, isolated, and overlapping groups. Immune pathways were less significant in the isolated group, likely because the main neutrophil response originated from overlapping regions in isolated samples (Figure 12C). In contrast, whole blood DARs showed much higher significance in interleukin and cytokine signaling compared to isolated or overlapping DARs (Figure 12C). Taken together, these data demonstrate dynamic changes in chromatin regions involved in NET formation, suggesting that similar yet distinct pathways govern NET formation in whole blood and isolated environments.

EXAMPLE 3: Chromatin Changes Associated with NET Formation following treatment with natural factors.

The experiment outlined in Example 2 was performed again but using natural NETosis- inducing factors instead of PMA. In brief, we induced NET formation in whole blood, followed by formaldehyde fixation, neutrophil isolation, and ATAC-Seq. Natural factors were added individually, in combination or at staggered time points prior to fixation. A summary of the combination/staggered additions is provided in Table 1 , below.

Table 1

Principal component analysis, Zscore heatmaps and HOMER analysis as described for Example 2, were obtained for neutrophils stimulated with natural factors. The results are shown in Figures 13-15. PCA analysis showed a general grouping of the untreated group (i.e. untreated, DMSO or vehicle formulations) and that TNFR stimulation drives combination therapy (Figure 13). Zscore heatmap analysis identified 1171 significant DARs (p < 0.01) and also showed that TNFR stimulation generally groups together (Figure 14). Interestingly, the natural factors I L-1 p, IFNy and IL-8 had a distinct response compared to other natural factors (three right-hand columns of the heatmap in Figure 14). HOMER analysis showed high upregulation of NF-KB following stimulation with natural factors, which is different compared to the other transcription factors which were also shown to be up-regulated with PMA-induced NET formation (Figure 15). Taken together, these data demonstrate that the method can also be used to analyse the dynamic changes in chromatin regions involved in NET formation following stimulation with natural factors.

Discussion

Neutrophils are critical to the innate immune response, with NET formation being essential for responding to infections and modulating immune responses. However, maladaptive NET formation can lead to autoimmune diseases and a dysregulated host immune response such as seen in sepsis. Given the complex blood milieu, understanding neutrophil induction and NET formation in the context of immune cell crosstalk is crucial. Chromatin accessibility is key in gene regulation, and investigating neutrophil responses under pathophysiological conditions can provide insights into maladaptive NET formation.

This study investigated differential chromatin accessibility in neutrophils within whole blood and compared them to isolated neutrophils post-PMA stimulation. Neutrophil isolation methods, whether through Ficoll gradients or commercial kits like MACSxpress, can significantly impact neutrophils with the procedures potentially inducing stress responses and biasing experimental outcomes. The impact of the cell isolation procedures was shown through GO analysis which showed enrichment of genes associated with biotic stimulus response, and Reactome pathway analysis identified cell starvation, underscoring how isolation could alter neutrophil quiescence. Thus, studying neutrophils in a physiologically relevant environment is essential. Comparing whole blood to isolated neutrophils revealed NET formation is slower and requires higher PMA concentrations in whole blood. While isolated neutrophils showed dramatic chromatin changes at 30 minutes, whole blood samples exhibited minimal changes at the same timepoint, suggesting plasma components and cellular interactions buffer or inhibit early NET formation. Despite timing differences, ATAC-Seq revealed dynamic chromatin accessibility changes, including increased, decreased, and bimodal responses. Motif analysis identified TFs consistent with NET formation in both the whole blood and overlapping DARs, such as the AP-1 complex, but not in the isolated only DARs. This indicates the whole blood ex vivo system effectively models and potentially exhibits a stronger induction of NET formation.

Fold change revealed a stronger chromatin response in whole blood samples as compared to isolated neutrophils. The upregulation of cytokine/interleukin signaling pathways in whole blood suggests immune cell crosstalk, whereas isolated DARs showed limited activation of immune-specific pathways. Enriched TF motifs in whole blood and overlapping categories were more NET-related, while isolated categories showed motifs unrelated to NET formation, except for HIF-1a and STAT3, which are involved in neutrophil and inflammatory responses. Furthermore, across all groups, motifs found in less accessible regions upon stimulation were associated primarily with TFs in the ELF family, Pll.1 , and ERG which are known to be involved in autoimmune disease. This suggests overlapped regions represent baseline neutrophil responses to PMA, while whole blood samples exhibit additional responses driven by the blood environment.

Our study demonstrates that examining NET formation within a human whole blood environment captures layered complexities of neutrophil responses by revealing additional chromatin accessibility changes and enriched immune-specific pathways not observed in isolated neutrophils. These changes likely reflect significant crosstalk among immune cells influencing neutrophil behavior during NET formation, pivotal in NET-associated pathogenesis such as immunothrombosis and sepsis.

Given the complex nature of human blood and immune responses — with diverse cellular components and myriad cytokines — studying NET formation within this intricate environment is critical for translating scientific findings into clinical applications. A deeper understanding of the NETosis pathway in humans, considering these complexities and chromatin dynamics, is crucial for developing targeted therapies to modulate NETosis in inflammatory and autoimmune conditions.

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Claims

1 . A method for analyzing chromatin, comprising:

(a) treating chromatin from a population of neutrophil cells isolated from a whole blood sample with an insertional enzyme complex to produce tagged fragments of genomic DNA, and wherein the neutrophil cells are fixed before isolation from the whole blood sample;

(b) sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and

(c) making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region.

2. The method of claim 1 , wherein the whole blood sample is isolated from an individual.

3. The method of any of claims 1-2, wherein the neutrophils are fixed using formaldehyde or paraformaldehyde (PFA).

4. The method of any of claims 1-3, wherein the whole blood sample is challenged before the neutrophil cells are fixed with at least one candidate modulator of NETosis or at least one modulator of NETosis.

5. The method of claim 4, wherein the modulator of NETosis is phorbol 12-myristate 13-acetate (PM A).

6. The method of any of claims 1-5, any wherein the neutrophils are isolated using MACs or EasySep.

7. The method of any of claims 1-6, wherein the cells are lysed prior to treating with the insertional enzyme complex.

8. The method of any of claims 1-7, wherein the information is obtained using the nucleotide sequences at the beginning and, optionally, the end of a sequence read.

9. The method of any of claims 1-8, wherein the information mapped in (c) is selected from one or more of:

(i) cleavage sites for the transposase;

(ii) the sizes of the fragments produced in step (a);

(iii) sequence read length; (iii) the positions of sequence reads of a defined range in length; and

(iv) sequence read abundance.

10. The method of claim 9, wherein the fragments of a defined size range are nucleosome- free fragments.

11. The method of any of claims 1-10, wherein the epigenetic map shows one or more of:

(i) a profile of chromatin accessibility along the region;

(ii) DNA binding protein occupancy for a binding site in the region;

(iii) nucleosome-free DNA in the region;

(iv) positioning of nucleosomes along the region; and/or

(v) chromatin states.

12. The method of claim 11 , further comprising measuring global occupancy of a binding site for said DNA binding protein.

13. The method of claim 11, wherein the DNA binding protein is a transcription factor.

14. The method of any of claims 1-13, wherein the population of neutrophil cells is composed of 1 to 100,000 cells.

15. The method of any of claims 1-14, wherein the treating step (a) comprises: isolating nuclei from the population of cells; and combining the isolated nuclei with said insertional enzyme complex, wherein the combining results in both lysis of the nuclei to release said chromatin and production of said tagged fragments of genomic DNA.

16. The method of any of claims 1-15, wherein the transposase is derived from Tn5 transposase.

17. The method of any of claims 1-15, wherein the transposase is derived from MuA transposase.

18. The method of any of claims 4 to 17, wherein the whole blood sample is challenged up to 180 minutes after collection of the blood sample.

19. The method of any of claims 1 to 18, wherein the whole blood sample is collected in an EDTA tube.

20. A method for comparing two samples comprising:

(a) analyzing a first population of cells using the method of any of claims 1-19 to produce a first epigenetic map; and

(b) analyzing a second population of cells using the method of any of claims 1-19 to produce a second epigenetic map; and

(c) comparing the first epigenetic map to the second epigenetic map.

21. The method of claim 20, wherein the first population of cells has been challenged with at least one candidate modulator of NETosis or at least one modulator of NETosis.

22. The method of claim 20, wherein the second population of cells has not been challenged with a candidate modulator of NETosis or a modulator of NETosis.

23. A method for comparing two samples containing neutrophils derived from whole blood comprising:

(a) analyzing a first population of cells using the method of any of claims 1-19 to produce a first epigenetic map wherein the first population of cells is challenged with a candidate modulator of NETosis or a modulator of NETosis before isolation of the neutrophils from a whole blood sample; and

(b) analyzing a second population of cells using the method of any of claims 1-19 to produce a second epigenetic map wherein the second population of cells is challenged with a candidate modulator of NETosis or a modulator of NETosis after isolation of the neutrophils from a whole blood sample.

24. The method of any of claims 20, 21 or 23, wherein the second population of cells has been challenged with a different candidate modulator of NETosis or modulator or NETosis, or a different combination of candidate modulators of NETosis or a different combination of modulators of NETosis, to that used to challenge the first population of cells.

25. The method of any of claims 20-24, wherein the first population of cells and the second population of cells are collected from the same individual at different times.

26. The method of any of claims 20-25, wherein (a) and (b) involve a challenge at the same or a different time point following collection of the whole blood, such as up to 180 minutes after collection.

27. The method of any of claims 20-26, wherein the first population of cells and the second population of cells are different populations of cells collected from different individuals.

28. A diagnostic method, comprising: analyzing chromatin from a patient using the method of any of claims 1-19, to produce an epigenetic map; and providing a diagnosis or prognosis based on the epigenetic map.

29. A method for treatment selection, comprising: analyzing chromatin from a patient using the method of any of claims 1-19, to produce an epigenetic map; and selecting a treatment for the patient based on the epigenetic map.

30. A method for identifying the effect of a candidate modulator of NETosis, comprising: analyzing chromatin from a patient treated with the candidate modulator of NETosis using the method of any of claims 1-27, to produce an epigenetic map; and identifying whether the candidate modifier of NETosis is a modifier of NETosis based on the epigenetic map.

31. A kit comprising:

(a) a modulator of NETosis;

(b) reagents for isolating neutrophils from whole blood cells;

(c) reagents for isolating nuclei from a population of cells;

(b) an insertional enzyme complex, and

(c) transposase reaction buffer, wherein the components of the kit are configured such that, combining the reaction buffer, transposon tags and adaptors with nuclei in vitro results in both lysis of the nuclei to release chromatin and production of tagged fragments of genomic DNA.