Novel rapid high-throughput method of netosis induction and inhibition with physiologically relevant molecules

Abstract

The invention relates to methods of screening candidate substances as regulators of NETosis using a NETosis model in whole blood. The invention also relates to compositions which find use in the NETosis model.

Description

[0001] NOVEL RAPID HIGH-THROUGHPUT METHOD OF NETOSIS INDUCTION AND INHIBITION WITH PHYSIOLOGICALLY RELEVANT MOLECULES

[0002] FIELD OF THE INVENTION

[0003] The invention provides a NETosis model in whole blood. The model may be used in methods of screening candidate substances as regulators of NETosis. The invention also relates to compositions and kits which find use in such screening methods.

[0004] BACKGROUND OF THE INVENTION

[0005] Sepsis results from a dysregulated host response to infection and affects over 1 .7 million adults annually in the US1 2. Rapid recognition and intervention is critical to avoid progression to septic shock and death. Despite advances in critical care, sepsis remains a major global health burden. New strategies are urgently needed to unravel the intricacies of sepsis pathophysiology, in order to understand individual patient susceptibility and develop more effective diagnostic tools and treatments.

[0006] Multiple sepsis etiologies, rapid evolution and systemic impact, have limited progress on novel treatments. Neutrophils represent 50-70% of white blood cells in humans and play a pivotal role in the initial response to infection. Whilst Neutrophils can phagocytose pathogens, they also release extracellular traps to rapidly immobilize pathogens and prevent dissemination. NETosis, first described by Brinkmann et al in 20043, involves the formation of Neutrophil Extracellular Trap (NETs) through rapid decondensation of nuclear chromatin, driven by changes to histone post translational modifications, followed by externalization of web like NETs containing long strands of chromatin and include associated antimicrobial granular enzymes (neutrophil elastase (NE) and myeloperoxidase (MPO)) (Figure 1A)3. While NETs serve as a vital defense mechanism, mounting evidence suggests that dysregulation and excessive formation can contribute to pathogenesis in sepsis and other inflammatory disorders through host directed bystander effects, initiation of a hyperinflammatory feedback loop and Disseminated Intravascular Coagulation4. Elevated nucleosome levels, a component of NETs, have been described in several studies of NETosis related conditions including COVID-19 and sepsis, and are negatively correlated with survival5-8.

[0007] NETosis research has largely relied on mouse models, in-vitro models using isolated primary neutrophils or neutrophil-like cells induced from immortalized cell lines9-11. While these models have provided valuable insights into NETosis, they have significant limitations. Murine immune responses, though informative, can differ substantially from human responses in part due to the significantly lower proportion of neutrophils, their maturation profile and half-life12'14. Immortalized HL-60 cells are highly dependent on culture conditions and do not recapitulate neutrophil fragility. NETosis induction in isolated primary neutrophils overcomes many of these limitations providing important insights into neutrophil activation and regulation15'17. However, neutrophils are very fragile and the method of isolation impacts their response to various stimuli18. To bridge the gap between existing models and the clinical reality of human sepsis, relevant ex vivo human models that enable rapid processing and require minimal handling are essential14. Various synthetic, as well as physiologically relevant factors induce NETosis. In-vivo, Staphylococcus aureus and Lipopolysaccharides (LPS) are commonly used NETosis inducers, while in-vitro models typically use LPS or Calcium Ionophore (Cl) and, the most commonly reported inducer, phorbol 12-myristate 13-acetate (PMA)19. PMA is an extremely powerful inducer of NETosis but is not physiologically relevant as it can bypass natural regulatory pathways governing NET production, thus preventing the ability to understand full regulatory feedback loops, limiting the clinical relevance of findings from PMA induced studies20.

[0008] An ex-vivo human NETosis model using physiologically relevant triggers in the presence of other blood cells (e.g. macrophages, platelets) and circulating proteins offers several distinct advantages. Primarily, investigation of the dynamics of NET formation, regulation and function in a clinical context would better translate insights to patient settings. Additionally, human cell models enable exploration of patient-specific factors including genetic predisposition and influence of pre-existing conditions, which can significantly impact sepsis outcomes. By dissecting the molecular mechanisms underlying NETosis using human cells, we can potentially identify novel therapeutic targets and develop personalized sepsis treatment strategies.

[0009] Here, we developed a human primary cell-based “Synthetic-Sepsis” model using intact whole blood to study NETosis induction with physiologically relevant molecules. We show NETosis induction using panels of pro-inflammatory molecules varied in both time-scale and magnitude of NET release compared to non-physiological PMA induction. Furthermore, we show differential NETosis profiles based on specific combinations of molecules, which we hypothesize could distinguish between beneficial and pathogenic NETosis. Moreover, we show that TNF-a or LT-a was necessary but not sufficient for NETosis induction and that in the presence of C5a, rapid onset of NETosis occurred within two hours of exposure. A minimal combination of LT- a, C5a and fMLP was able to consistently induce NETosis in multiple donors. We believe our novel model could delineate underlying complexities of NETosis, potentially leading to the development of innovative diagnostic tools and targeted interventions for sepsis and other NETosis related pathologies.

[0010] SUMMARY OF THE INVENTION

[0011] According to one aspect of the present invention there is provided a method for screening a candidate substance as a regulator of NETosis comprising: a) providing a multiwell plate; b) distributing a candidate regulator across the multiwell plate; c) distributing a fluorescing intercalating dye across the multiwell plate; d) distributing a sample of whole blood across the multiwell plate; e) allowing erythrocytes in the whole blood sample to sediment leaving an upper plasma layer and a buffy coat containing neutrophils at the interface of the layers; f) measuring NET formation in the buffy coat using the level of fluorescence of the intercalating dye wherein the level of NET formation can be used to determine whether the candidate substance is a regulator of NETosis; wherein steps b), c) and d) can occur simultaneously or in any order. According to a further aspect of the present invention there is provided a method of identifying a candidate compound capable of inhibiting NETosis comprising: a) providing a whole blood sample; b) adding at least three inducers of NETosis to the whole blood sample wherein at least one of the inducers is TNF-a or LT-a; c) adding a candidate compound to the whole blood sample; and d) determining whether the level of NETosis is reduced as a result of adding the candidate compound; wherein steps a) to c) are conducted in any order.

[0012] According to a further aspect of the present invention there is provided a composition comprising at least three inducers of NETosis of which at least one is TNF-a or LT-a.

[0013] According to a further aspect of the present invention there is provided a kit for regulating NETosis wherein the kit comprises a composition as defined herein.

[0014] According to a further aspect of the present invention there is provided a use of the composition as described herein or the kit as described herein, in a method for screening for a candidate inhibitor of NETosis wherein the method is as defined herein and the composition is distributed across the multiwell plate before step e).

[0015] According to a further aspect of the present invention there is provided a use of the composition as described herein or the kit as described herein in a method for screening a candidate compound for toxicity.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] Figure 1 : Induction of NETosis in a whole-blood system with H3.1 Nu.Q® and Incucyte Systems: (A) Diagram of how we measure NETosis using our “Synthetic Sepsis”, ex-vivo, or high-throughput screening methods. (B) PMA titration (•) and a DMSO control (grey •) on primary isolated neutrophils using Cytotox Green dye (Sartorius #4633) on an S3 Incucyte imaging system from Sartorius. (C) Similar to Fig 1 B, but isolated neutrophils were first incubated with MPO inhibitor (ABAH) (•), a DMSO control (grey •), and 100 nM PMA (black •). (D) Representative images from the Incucyte system for Fig 1C. These were taken at the start of the assay (left panels) and after six hours (right panels). The scale bars indicate 400 pm. (E) Similar to Fig 1 B, but isolated neutrophils were treated with various concentrations of Cl ( ’ ) and compared to a DMSO control (grey •) and 100 nM PMA (black •). (F) Similar to Fig 1 B and Fig 1E, but isolated neutrophils were treated with various concentrations of LPS (■) and compared to a DMSO control (grey •) and 100 nM PMA (black •). (G) Whole blood from two donors was treated with the indicated concentrations of a vehicle control, PMA, Cl, or LPS for four hours and measured with H3.1 Nu.Q®. (H) Whole blood was treated with either 800 nM PMA or DMSO (grey) and measured for H3.1 Nu.Q® over six hours indicating the ability to capture NETosis.

[0018] Figure 2: High-throughput ex-vivo method of screening NETosis in whole-blood: (A) Workflow for the ex-vivo system. Briefly, reagents excluding whole-blood are dispensed into a 384-well plate using the MANTIS system (Formulatrix). Whole blood is mixed with Sytox Green (ThermoFisher #S7020) and then dispensed into the same plate. The plate is mixed and spun down to separate plasma and erythrocytes. Finally, top read fluorescence is completed. (B) The assay used 250 nM PMA to activate NETosis and the time of NETosis (tNE-r) was measured to show consistency across the assay plate. (C) PMA titration ( *■ , ’ , •, ♦) and a DMSO control (grey •) was completed over six hours. (D) The high-throughput method was compared to H3.1 Nu.Q®, where Sytox signal can be seen on the left axis and Nu.Q signal on the right axis. A vehicle control (grey ♦) and 800 nM PMA indicate Sytox Green RFU from the high-throughput model. A corresponding vehicle control (grey ■) and 800 nM PMA indicate measurements from H3.1 Nu.Q® readings (ng / mL). (E) Similar to Fig 20, but 250 nM PMA (black •) was compared to treatment with 4 pM Cl ( *■ ), and a DMSO control (grey •). (F) Similar to Fig 2E, but 250nM PMA (black •) was compared to treatment with 12 pg / mL LPS ( * ), and a DMSO control (grey •). (G) Similar to Fig 2E and Fig 2F, but 250 nM PMA (black •) was compared to treatment with 250 nM PMA with 500 pM ABAH (■). Also included was a DMSO control ( * ) and a DMSO control with 500 pM ABAH ( ’ ). The whole blood was incubated for 45 minutes with or without the ABAH added prior to addition of PMA.

[0019] Figure 3: Selection of naturally occurring chemokines and cytokines using sequential pooling: (A) Design of the multi-factor screening process. Briefly, individual factors were selected and then pooled. The pooled factors were then tested using the high-throughput screening method and Sytox Green (ThermoFisher #S7020) signal was measured. The contribution of each factor was determined and factors which did not increase NETosis were removed, and the new pools were designed. (B) There were four distinct signal profiles for the various pools. The distinct signals were compared to a DMSO control (■) and 250 nM PMA (•). Example A (dark ♦) showed delayed response with continued gradual increase, Example B ( *■ ) initial response with secondary response with continued increase, Example C ( ’ ) showed an initial response with early plateau, and Example D (light ♦) showed no response. (C) Analysis workflow. Raw signal was averaged over 30-minute windows followed by the first derivative. Afterwards, multivariate regression modeling was completed to determine factor contributions in each pool. (D) An example of the multivariate regression (Fig 3C), highlighting four common factors: I L-1 p (•) showed less NETosis signal, IL-5 (■) had limited response, C5a ( ’ ) and TNF-a ( * ) showed a high amount of contribution to the NETosis signal. (E) The full list of factors tested and their responses. Two unique donors were tested in each screen and the final pool was indicated with an X.

[0020] Figure 4: Understanding of the 8-factor pool and determining minimal factor pool needed for NETosis: (A) Example of Boolean (binary) representation of the first derivative NETosis signal. If signal was above the threshold (3.3 x Standard Deviation of Vehicle Signal - dashed line), it was marked as positive and converted to a 1 , otherwise it was marked as negative and converted to a 0. (B) Boolean representation of NETosis signal over six healthy donors. The factors were pooled in a full factorial manner, such that all possible combinations were present and indicated by the number of factors in each pool. (C) Similar to Fig 4B, but donors were broken up between male and female. (D) Similar to Fig 4B and Fig 4C but removing either LT-a and / or TNF-a (top) or C5a and / or LPS (bottom). The overlap of the donors showed limited NETosis without LT-a and / or TNF-a. (E) Using a limited pool of 5 factors (left panel), the change in NETosis signal over background signal is shown (right panel). Sequential removal of one factor is shown in each panel on the right, where each row is an individual donor. For each panel (right), the donors are kept in the same order.

[0021] Figure 5: Determination of minimal factors required to induce NETosis in the ex-vivo system: (A) A male and female donor were tested over three time points (over 30 days) with a full factorial of the five-factor pool from Fig 4E (LT-a, GM-CSF, C5a, LPS, and fMLP). Factors are indicated on the left panel where the shaded box denotes the factor was present in that row. Decreasing p-value is indicated by increasing circle size and raw change in the NETosis signal (RFU) after 4 hours is shown. (B) Various concentrations of the five-factor were tested on the same male and female donors were completed to determine optimal concentrations required for induction of NETosis. Delta RFU after 4 hours is shown on the y- axis and log concentration of the factors (ng / mL) is shown on the x-axis. 95% confidence is shown as dashed lines. The factors are ordered as for Fig 5A (From top to bottom: LT-a, GM-CSF, C5a, LPS, and fMLP). (C) EC50 concentrations were determined after removing the two factors which showed limited response (GM-CSF and LPS). 95% confidence is shown in the dashed line. The calculated EC50 values indicated by a vertical dashed line were: LT-a: -0.6 ± 0.8 ng / mL, C5a: 2.2 ± 0.4 ng / mL, and fMLP: 1.8 ± 0.5 ng / mL. The factors from left to right are: LT-a, C5a and fMLP). (D) Using the pool of three factors at the optimized EC50 concentrations, inhibitors targeting the following were compared to a Null treatment: 280 pM MPO (ABAH - Sigma #A41909-10G), 65 pM NE (NEi II - Sigma #324744- 5MG), 350 pM pan-PAD (Cl-Amidine - Sigma #506282-10MG), 25 pM PAD4 (GSK484 - Sigma #SML1658-5MG), 40 pM Receptor-interacting protein kinase 1 (NecroStatin-1 - Sigma #), and 15 pM Caspase 3 / 7 (Sigma # 218826-1MG). * indicates p > 0.05, ** indicates p > 0.005, and **** indicates p > 5e-5.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] According to one aspect of the present invention there is provided a method for screening a candidate substance as a regulator of NETosis comprising: a) providing a multiwell plate; b) distributing a candidate regulator across the multiwell plate; c) distributing a fluorescing intercalating dye across the multiwell plate; d) distributing a sample of whole blood across the multiwell plate; e) allowing erythrocytes in the whole blood sample to sediment leaving an upper plasma layer and a buffy coat containing neutrophils at the interface of the layers; f) measuring NET formation in the buffy coat using the level of fluorescence of the intercalating dye wherein the level of NET formation can be used to determine whether the candidate substance is a regulator of NETosis; wherein steps b), c) and d) can occur simultaneously or in any order.

[0024] By “distributing” we include dispensing into all or a portion of the multiwell plate.

[0025] Extracellular traps (ETs) may be formed by a variety of cell types including neutrophils. Neutrophil extracellular traps (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. NETs are composed of decondensed or unwound chromatin consisting primarily of strings of nucleosomes with a beads on a string type of structure decorated or granulated with myeloperoxidase (MPO), neutrophil elastase (NE) and other proteins. Each nucleosome consists of a protein complex of eight highly conserved core histones (comprising of a pair of each of the histones H2A, H2B, H3, and H4). Around this complex are wrapped approximately 145 base pairs (bp) of DNA. Further DNA, that is often referred to as “linker DNA”, connects each nucleosome in a “string” to the next, i.e. the DNA connecting one nucleosome to another in chromosomes. Another histone, H1 , which may be located on the nucleosome outside of the core histones, binds to linker DNA and this may also be present in some nucleosome strings.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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).

[0030] 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. There is thus a need to provide a method of screening for regulators of NETs and regulators which can be used in such screens. The present invention addresses this need.

[0031] The present invention employs fluorescent intercalating DNA dyes.

[0032] An intercalating dye refers to a dye whose fluorescence is enhanced by intercalation into a double stranded nucleic acid. DNA intercalators, which bind between adjacent base pairs of dsDNA are used as fluorescent probes to visualize DNA. Intercalating dyes specific for double-stranded nucleic acid (dsDNA) show an increased fluorescence upon intercalating with the dsDNA. As a consequence, the more dsDNA are generated during NETosis, the higher gets the increase in fluorescence. Thus, by using intercalating dyes real-time detection of the production of NETs is enabled, and this method allows the degree of NETosis to be determined with reference to a standard sample. Accordingly, a “change in the fluorescence” shall mean either an increase or decrease of fluorescence intensity of the reporter.

[0033] Thus, the present invention provides detecting the presence or absence of quantifying the amount of NETosis by measuring fluorescence of said intercalating dye; and determining, based on the fluorescence of intercalating dye the effectiveness of a candidate NETs regulator.

[0034] According to a one embodiment of the method according to the invention, the intercalating dye can be selected from, but not limited to, Cytotox Green, Sytox Green, SYBR® Green (N',N'-dimethyl-N-[4-[(E)-(3-methyl-1 ,3-benzothiazol-2-ylidene)methyl]-1- phenylquinolin-1-ium-2-yl]-N-propylpropane-1 ,3-diamine), PICOGREEN® (2-(n-bis-(3- dimethylaminopropyl)-amino)-4-(2,3-dihydro-3-methyl-(benzo-1 ,3-thiazol-2-yl)-methylidene)- 1-phenyl-quinolinium), YOYO-1 , ethidium bromide.

[0035] In one embodiment the intercalating dye is Cytotox Green or Sytox Green.

[0036] Fluorescence is measurable by fluorometers. A fluorometer is an instrument designed to measure the various parameters of fluorescence, including its intensity and wavelength distribution of the emission after excitation. There are many terms that refer to instruments used to measure the fluorescence of a sample: fluorometer, spectrofluorometer, fluorescence spectrophotometer, fluorescence spectrometer, fluorimeter, etc. In general, they use a light source to excite the sample at a certain wavelength and measure the emitted fluorescence at a second wavelength. The sample may be held in many formats, including cuvettes, capillaries and petri dishes. ‘Fluorescence microplate reader’ refers to an instrument that can measure the fluorescence of samples in a microplate, most often a 96- or 384-well plate. Microplates offer higher throughput and are useful for screening or other applications where many samples must be analyzed.

[0037] Thus, DNA intercalating dyes provide a method to image DNA topology by means of fluorescence microscopy.

[0038] The level may be measured with reference to a “baseline” level, which may also be referred to as the “first”, “initial” or “background” level. The baseline level refers to the level of NETs measured before the addition of the candidate regulator or with reference to a control that does not contain the candidate regulator.

[0039] In one embodiment the time of NETosis, or the time at which the level of fluorescence is measured, is determined as the time at which the level of fluorescence exceeds 3.3 times the standard deviation of a control.

[0040] The difference between two measurements provides a measure of the level of NETs generated ex vivo in the whole blood sample and can be used as a measure of the level of neutrophils (or other white cells) present in the whole blood sample to produce NETs if stimulated to do so by a candidate regulator. In this case of NETs being produced the candidate regulator will be a inducer of NETs. On the other hand a reduction in the level of NETs being produced when the whole blood is in contact with a candidate regulator compared to a control in which a stimulator of NETs is present will be an inhibitor of NETs. However, in this scenario the addition of a NETosis activator is optional because NETosis can be induced spontaneously by coagulation (for example using a serum blood collection tube).

[0041] For inhibition studies, candidate inhibitors are mixed with whole blood and also mixed with an inducer of NETs. These components may be added in any order.

[0042] Methods of the invention enable the rate of NETosis to be corrected for background by subtraction of the baseline level of circulating nucleosomes present in the samples of the same subjects.

[0043] The multiwell plate may be of any suitable dimension. Examples of conventional multiwell plates are 24, 96, 384 and 1536 well formats. In one embodiment a 384 multiwell plate is used. A multiwell plate is also known as a microplate or a microtiter plate and is a flat plat with multiple wells used as small test tubes.

[0044] In one embodiment, the sample of whole blood is ethylenediamine tetraacetic acid (EDTA) whole blood.

[0045] In one embodiment the whole blood sample is processed by centrifugation, and the sample is then assayed for NETs. Therefore, in one embodiment the multiwell plate is centrifuged to sediment erythrocytes leaving the upper plasma layer and the buffy coat.

[0046] In one embodiment the whole blood sample is left for at least 15 minutes (such as at least 30 minutes, 45 minutes, 1 hour, 3, 4, 8, 12 or 24 hours) following addition of the candidate regulator for the development of any NETosis.

[0047] In one embodiment, NET formation is measured through the upper plasma layer, such as from above.

[0048] In one embodiment the method comprises comparing the level of NETs detected in the whole blood sample to the level of NETs detected in a reference sample.

[0049] In one embodiment the whole blood sample is oxygenated. Described herein is a method of investigating the kinetics and mechanisms of NETosis. The methods are designed to be carried out on a whole blood sample in vitro or ex vivo. The whole blood sample is obtained from a subject. References to acts carried out on a body fluid sample “obtained” from a subject are intended to encompass acts carrier out on a body fluid sample already obtained or “obtainable” from a subject.

[0050] References to “subject” , “individual” or “patient” are used interchangeably herein. The subject may be a human or an animal subject. In one embodiment, the subject is a human. In one embodiment, the subject is a (non-human) animal. In some embodiments the invention encompasses animal subjects (wild or domesticated). In some embodiments, the invention relates to veterinary uses including for livestock and companion animals such as cats, dogs, horses, donkeys, rats, rabbits, mice, guinea pigs, sheep, goats, pigs, deer, llamas, cows and cattle. In one embodiment, the subject is a non-human mammal, such as a dog, mouse, rat or horse, in particular a dog.

[0051] NET formation has been shown to occur via two distinct pathways: dependent or independent of NADPH oxidase (NOX) (Schoen et al. 2022; Khan et al. 2017). Phorbol 12- myristate 13-acetate (PMA) stimulation has been shown to be NOX dependent (Damascena et al. 2022), whereas other activators such as calcium ionophore (Cl) are NOX independent (Parker et al. 2012). Both the NOX-dependent and -independent NET formation pathways likely contribute to host defense against pathogens, and dysregulation of either pathway can exacerbate inflammatory conditions. For instance, excessive reactive oxygen species (ROS) production in the NOX pathway can lead to tissue damage and inflammation, and the aberrant activation of protein kinases in the non-oxidative pathway can contribute to autoimmune responses and tissue injury (Nguyen et al. 2017). Understanding the intricate regulation of these pathways is crucial for developing targeted therapeutic strategies to modulate NET formation and mitigate inflammatory diseases. It is also desirable to identify NETosis regulators that impact both pathways.

[0052] The present invention provides a method of identifying candidate inducers of NETosis. Many chemical activators, stimulants or inducers of NETosis are known in the art; however the present invention provides a means of screening for inducers of NETosis which are physiologically relevant, i.e. which induce NETosis in whole blood. We have identified novel combinations of NETosis inducers. These compositions preferably are natural products, i.e. trigger NETosis in an in vivo natural situation.

[0053] As demonstrated herein, whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: 11-1 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. We used combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations. Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and / or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude; however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors can be used to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-a or LT-a was required to cause a NETosis response, underlining the potential significant roles these factors play in inflammatory disease.

[0054] In one aspect of the present invention there is provided a composition comprising at least three inducers of NETosis of which at least one is TNF-alpha (TNF-a) or LT-alpha (LT-a).

[0055] In one embodiment the composition comprises TNF-a and LT-a.

[0056] In one embodiment the composition, comprises at least four inducers of NETosis.

[0057] In one 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 Interferon gamma (IFN- y), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Leukotriene B4 (LTB4), Complement component 5a (C5a), ferritin, lipopolysaccharides (LPS), N-formyl-methionyl- leucyl-phenylalanine (fMLP).

[0058] In one embodiment the inducer of NETosis is not phorbol 12-myristate 13-acetate (PMA).

[0059] In one embodiment the inducer of NETosis is not LPS or calcium ionophore (Cl).

[0060] In one embodiment the composition comprises or consists of at least one of TNF-a or LT-a in combination with one or more of IFN-y, GM-CSF, LTB4, C5a and ferritin.

[0061] In one embodiment the composition comprises or consists of a combination of LT-a and GM- CSF, C5a, LPS, and fMLP.

[0062] In one embodiment the composition comprises or consists of LT-a, C5a, and fMLP.

[0063] In one embodiment the composition comprises or consists of TNF-a or LT-a and C5a.

[0064] According to a further aspect of the invention, there is provided a method of identifying a candidate compound capable of inhibiting NETosis comprising: a) providing a whole blood sample; b) adding at least three inducers of NETosis to the whole blood sample wherein at least one of the inducers is TNF-a or LT-a; c) adding a candidate compound to the whole blood sample; and d) determining whether the level of NETosis is reduced as a result of adding the candidate compound; and wherein steps a) to c) are conducted in any order.

[0065] In one embodiment the level of NETosis is measured by detecting or measuring the level of cell free nucleosomes or a component thereof, MPO, NE or cfDNA.

[0066] Measurements of the level of NETs produced in a biological sample may be carried out in principle by measuring any protein or nucleic acid component of NETs. Components measured may include specific NETs component proteins, for example myeloperoxidase (MPO) and neutrophil elastase (NE). Similarly, measurements of any chromatin components may be used including without limitation cfDNA, cell free nucleosomes, cell free nucleosomes containing particular histone isoforms or histone post-translational modifications (PTMs), for example citrullinated cell free nucleosomes, cell free MPO-DNA complexes, cell free NE-DNA complexes and many other components. Histone post- translational modifications and their measurement are described, for example, in W02005 / 019826 which is herein incorporated by reference. Histone isoforms and their measurement are described, for example, in WO2013 / 030579 and WO2016 / 067029 which are herein incorporated by reference. Protein-nucleosome adducts and their measurement are described, for example, in WO2013 / 084002 which is herein incorporated by reference. Many further protein NETs components are known in the art and any may be used for methods of the invention including for example those listed by Bruschi et al, 2019.

[0067] In one embodiment the cell free nucleosomes comprise histone H3.1.

[0068] The detection or measurement may comprise an immunoassay, immunochemical, mass spectrometry, chromatographic, chromatin immunoprecipitation or biosensor method. In one embodiment the detection and / or measurement may comprise an immunoassay such as a homogeneous immunoassay (HIA). In a preferred embodiment, detection and / or measurement may comprise a 2-site immunoassay method for nucleosome moieties. A 2- site immunoassay method for the measurement of nucleosomes may employ any antibodies that bind to any part of a nucleosome or moiety adducted to a nucleosome. Such a method is preferred for the measurement of nucleosomes or nucleosome incorporated epigenetic features in situ employing two anti-nucleosome binding agents or an anti-nucleosome binding agent in combination with an anti-histone modification or anti-histone variant or anti- DNA or anti-adducted protein detection binding agent. Also, detection and / or measurement may comprise a 2-site immunoassay employing a labelled anti-nucleosome detection binding agent in combination with an immobilized anti-histone modification or anti-histone variant or anti-adducted protein binding agent.

[0069] The inventors herein used a 2-site immunoassay for detecting H3.1 -nucleosomes by employing an immobilized anti-histone H3.1 antibody directed to bind to an epitope around amino acids 30-33 of the histone H3.1 protein to capture clipped and non-clipped nucleosomes, together with a labelled anti-nucleosome antibody directed to bind to an epitope present in intact nucleosomes but not present on isolated (free) histone or DNA nucleosome components. This type of epitope may be referred to as a “conformational nucleosome epitope” herein because it requires the native three-dimensional configuration of the target nucleosome to be intact. In one embodiment, the method of detection or measurement comprises contacting the body fluid sample with a solid phase comprising a binding agent that detects cell free nucleosomes or a component thereof, and detecting binding to said binding agent.

[0070] In one embodiment, the method of detection or measurement comprises: (a) contacting the sample with a first binding agent which binds to an epigenetic feature of a cell free nucleosome; (b) contacting the sample bound by the first binding agent in step (a) with a second binding agent which binds to cell free nucleosomes; and (c) detecting or quantifying the binding of the second binding agent in the sample.

[0071] In another embodiment, the method of detection or measurement comprises: (a) contacting the sample with a first binding agent which binds to cell free nucleosomes; (b) contacting the sample bound by the first binding agent in step (a) with a second binding agent which binds to an epigenetic feature of the cell free nucleosome; and (c) detecting or quantifying the binding of the second binding agent in the sample.

[0072] In a particular embodiment, the method of detection or measurement comprises: (i) contacting the sample with a first binding agent which binds to an epigenetic feature of a cell free nucleosome, wherein the epigenetic feature is histone H3.1 ; (ii) contacting the sample bound by the first binding agent in step (i) with a second binding agent which binds to cell free nucleosomes; and (iii) detecting or quantifying the binding of the second binding agent in the sample.

[0073] As demonstrated herein, levels of nucleosomes comprising H3.1 increased with NETosis induction, consistent with DNA release as measured by Sytox green. NETosis was detected 1.5 hours after PMA induction using H3.1 Nu.Q® and Sytox assays. DNA Methylation analysis confirmed neutrophil cell of origin post induction. MPO inhibition resulted delayed onset of NETosis, supporting NET release rather than other forms of cell death. NETosis visualization using microscopy confirmed NETosis. Importantly, we found that although PMA successfully induced NETosis in both contexts, Cl failed to elicit a response in the ex vivo whole blood system, highlighting the importance of a full blood model for unbiased NETosis profiling.

[0074] Our findings demonstrate that H3.1 nucleosome levels are elevated in response to NETosis- inducing treatments, with a time course that is consistent with NETosis and correlates with other established NETosis markers, validating its utility as a reliable indicator of NETosis. The successful induction of NETosis in an ex vivo whole blood model provides a valuable tool for studying disease-associated NETosis within a biologically relevant context, which will likely shed additional insight which is not possible using isolated neutrophils. Utilizing these approaches provides a platform to scrutinize a wide array of both synthetic and naturally occurring NETosis regulators. It facilitates insight into the signalling mechanisms at the core of NETosis and the influence of chromatin-associated changes within an in vivo like microenvironment of whole blood. By unravelling the intricate dynamics of neutrophil biology and its interplay with the microenvironment, we can develop tailored interventions to modulate NETosis in sepsis and other inflammatory conditions.

[0075] The compositions of the present invention may be provided in the form of kits. In one embodiment the composition or kits of the invention are used in methods to identify or screen for inhibitors of NETosis. In one such method there is provided a method for screening for the potential of a substance to inhibit NETosis, wherein said method comprises the steps of:

[0076] (i) obtaining a whole blood sample from the subject;

[0077] (ii) adding a stimulant of NETosis to the sample;

[0078] (iii) adding the potential inhibitor of NETosis to the sample;

[0079] (iv) leaving the sample for sufficient time to generate any NETs;

[0080] (v) measuring the level of any NETs in the sample; and using the level of NETs measured as an indicator of the potential of the potential inhibit to prevent or reduce the production of NETs in a blood sample, optionally including a correction for baseline levels of NETs present in the whole blood sample.

[0081] The present invention provides a method of identifying candidate inducers of NETosis. Many chemical activators, stimulants or inducers of NETosis are known in the art; however the present invention provides a means of screening for inducers of NETosis which are physiologically relevant, i.e. which induce NETosis in whole blood.

[0082] According to a further aspect of the invention, there is provided a use of the composition or the kit as described herein, in a method for screening for a candidate inhibitor of NETosis. In particular, the method is as defined herein and the composition is distributed across the multiwell plate before step e).

[0083] The present invention is directed to measuring the impact of a candidate substance on NETosis which can be an indicator of the impact of the substance on the innate immune system. Therefore, methods of the invention may be used in screening a candidate compound (also referred to as a candidate substance or a test substance) for toxicity, in particular immune-related toxicity.

[0084] In one embodiment, the method comprises contacting the whole blood sample with a candidate compound and observing a change (e.g., an effect) in the level of NETosis induced by the candidate compound. “Toxicity” refers to an adverse toxicological property of the candidate compound.

[0085] According to a further aspect of the invention, there is provided a use of the or the kit as defined herein, in a method for screening a candidate compound for toxicity.

[0086] 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.

[0087] The invention will now be illustrated with reference to the following non-limiting examples.

[0088] EXAMPLES

[0089] INTRODUCTION

[0090] Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil Extracellular trap (NET) formation in response to pathogens is increasingly recognized as an essential rapid innate immune response, but when dysregulated contributes to pathogenesis of sepsis and autoimmune disease. Current models of NETosis are limited, routinely employing non-physiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines, limiting the complex biology underlying neutrophil activation and NETosis, that occurs in a whole-blood environment. Here we describe a novel, high- throughput ex-vivo whole blood induced NETosis model using combinatorial pooling of native NETosis inducing factors in a more biologically relevant “Synthetic-Sepsis” model. We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and / or magnitude of NETosis. Despite inter-donor variability, similar sets of pro-inflammatory molecules induced consistent responses across donors. We found at least three biological triggers, were necessary to induce NETosis in our system requiring either TNF-a or LT-a. To our knowledge, we report the first human ex-vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis. These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially, providing novel strategies for disease intervention and treatment.

[0091] Our findings demonstrate:

[0092] • Stimuli to induce NETosis differs between isolated neutrophils and whole blood, reflecting the need for physiologically relevant models.

[0093] • NETosis can be induced in whole blood using a minimum of three physiological factors, enabling mechanistic and regulatory studies.

[0094] METHODS

[0095] Neutrophil isolation & Imaging

[0096] Anonymous healthy donor K2-EDTA whole blood was obtained from PrecisionMed (San Diego). Research was approved under WCG IRB Protocol number 20181025 and all human participants gave written informed consent. Blood was stored at room temperature, and processed within one-hour post-draw. Neutrophils were isolated using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi, 130-104-434) and resuspended in RPMI (Gibco, 11-875-119) containing 250nM Cytotox Green (Sartorious, 4633) to 2.0 x 10A5 cells / mL and seeded at 100pL / well in a 96-well Incucyte ImageLock plates (Sartrious, 4806) coated with 10pg / mL Fibronectin (Sigma-Aldrich, F1141). The plate was centrifuged at 120g for two minutes to seat the neutrophils at the bottom of the plate. NETosis stimuli and inhibitors were diluted in RPMI containing Cytotox Green then added to the neutrophils to the indicated final concentration. Inhibitors were incubated with neutrophils at 37°C for one hour before adding stimuli. NETosis was analyzed using Incucyte’s S3 Live-Cell Analysis System (Sartorious). The plate was imaged every 20 minutes using phase contrast and green fluorescent exposure channels and a 10* objective. Neutrophils were identified by phase contrast, objects smaller than 30 pm2were excluded. Positive NETosis (Green) signal was optimized for measuring total area with Top-Hat for background correction and Edge Split Off. NETosis Induction in Whole Blood

[0097] Anonymous healthy donor K2-EDTA whole blood was obtained from PrecisionMed (San Diego, USA), with samples stored at room temperature and processed within one-hour postdraw. Blood was treated with 0.625pL / mL of CaCh solution (5mM in ddH2O) and mixed. Pooled blood was treated with either PMA (Sigma Aldrich, P1585), or vehicle (DMSO) followed by inversion mixing. Each condition was aliquoted into 2mL low protein binding tubes (Cat#90410, Thermo Scientific) and incubated at 37°C under rotation at 10 RPM. Plasma was isolated from treated blood at designated time points by centrifugation in a swinging bucket rotor for 10 minutes at 1300 x g with no brake at room temperature. Plasma fractions were transferred and nucleosomes measured in duplicate using the H3.1 Nu.Q® NETs immunoassay (Belgian Volition).

[0098] Screening

[0099] Whole blood screening candidates were selected based on literature reports of an association with NETosis (Table 2). Concentrations were equivalent to or in excess of commonly used for in-vitro stimulation. Recombinant lyophilized proteins were resuspended using 0.1% (w / v) human serum albumin (HSA) (A9731 , Sigma-Aldrich) in sterile water, with further dilutions of reagents made using 0.1% HSA in PBS. Reagents were dispensed in plates (3575, Corning) using the Mantis V3 Liquid Handler (Formulatrix) with Silicon LV or PFE LV chips (233581 or 233129, Formulatrix). Wells were backfilled with appropriate solvents (Ethanol, DMSO, 0.1% HSA in PBS), such that the concentration and volume of vehicle was consistent across wells. Plates contained 6 wells of each the following controls: untreated, PMA (50, 250, and 500 nM) randomly distributed across the plate to assess assay performance. The factors contained within remaining wells were determined by the optimal DOE screening design computed using JMP V17.1 (JMP Statistical Discovery).

[0100] Fluorescent Plate Assay

[0101] 50 pL of oxygenated whole blood was dispensed across a prepared 384-well fluorescent assay microplate using the Mantis Continuous Flow Silicon Chip (233127, Formulatrix). After dispensing, the plate was sealed (235307, Thermo Scientific) and mixed (orbital shaking) at 1400RPM for 10 seconds. Plates were then centrifuged at 1300 x g for 10 minutes at room temperature using a swinging bucket rotor, without braking. Centrifuged plates were preheated to 37°C on a dry-heat block (25-038A, Lonza) for 15 minutes prior to reading.

[0102] Kinetic fluorescent measurements were obtained using a SpectraMax i D5 Multi-Mode MicroPlate Reader (Excitation = 510nm, Emission = 550nm), top read, at two minute sampling intervals for 24 hours. For inhibition studies, inhibitors, vehicle, and backfill were added to the wells followed by oxygenated whole blood and incubated at room temperature for 45-60 minutes after which PMA or physiological molecules were dispensed, the plate was sealed, and the assay continued as described above.

[0103] Screening Design and Statistical Analysis

[0104] Optimal screening design was computed using JMP V17.1 (JMP Statistical Discovery) custom screening design, with all main effects and fourth degree interactions included, but assigned estimatibility to “if possible”. The number of runs specified for each plate was set to 360 with no additional center points or replicate runs. RFU signal was averaged into 30- minute intervals, followed by calculating the change in the smoothened RFU values at 30- minute intervals. These values were used for input into multivariate regression modeling using JMP V17.1 , with the concentration of each molecule in each treatment being the dependent variable, and each change in RFU value at 30 minute intervals being the independent variable. The effect of each factor was assessed by t-test to determine if the predicted coefficient at any given period was non-zero, and if the factor was acting to increase or decrease signal at any given time. Factors with a significant non-zero effect on the signal and acted to increase signal over time (P < 0.1) in one or both tested donors for that screen, were selected to move forward for additional screening.

[0105] Dose-Response profiles for the three-factor combination were characterized by space-filling DOE (JMP V17.1). The selected design was sphere-packing optimal with 88 different concentration combinations of LT-a, C5a, and fMLP and tested in triplicate. The system’s dose-response surface was generated using gaussian process regression and gaussian correlation structure, with nugget parameter estimation. Each individual factor’s EC50 was obtained by determining the log concentration at the signal half-max for each factor, given that the two other factors are at their maximal concentrations.

[0106] Data Sharing

[0107] Raw data or additional information will be provided upon emails to the corresponding author.

[0108] RESULTS

[0109] NETosis Induction and real-time monitoring in whole blood and isolated neutrophils

[0110] We set out to develop a more biologically relevant NETosis model than isolated neutrophils. Our approach allows the investigation of NETosis neutrophils in whole blood either in a low throughput or high-throughput screening approach (Figure 1A). We first validated our NET quantification approach, using extracellular DNA intercalation and fluorescence, in a classical isolated neutrophil model of NETosis. A dose dependent increase in DNA release measured by Cytotox Green was seen in response to PMA in an Incucyte S3 incubated imaging platform (Sartorius), with signal onset after 2 hours of treatment (Figure 1B). Treatment with the MPO inhibitor, ABAH, substantially delayed the onset of DNA release and reduced the overall level of release, consistent with NETosis inhibition (Figure 1C). The results were confirmed by fluorescent microscopy at 6 hours post PMA treatment (Figure 1D). Cl and LPS, established NETosis inducers, also induced NETosis in isolated neutrophils in a dose dependent manner (Figure 1E and Figure 1F).

[0111] To study NETs in a more biologically relevant system, (i.e. in the presence of other blood proteins and cell types) we incubated whole blood in EDTA tubes from two healthy donors with PMA, Cl or LPS. NETosis was quantified by nucleosome release and H3.1 nucleosome levels started to increase three hours after treatment with PMA (Figure 1H). Differential nucleosome elevation was seen in both donors over the time course. Only PMA induced NETosis in whole blood, unlike in isolated neutrophils, where all three molecules induced NETosis (Figure 1G). This unexpected finding suggests that isolated neutrophils respond differently than whole blood. High-throughput model for NETosis induction

[0112] To identify physiologically relevant NETosis activators we adapted the model for high- throughput screening. As neutrophils are fragile, we designed a rapid screening format with limited handling utilizing a MANTIS liquid dispenser (Formulatrix) to distribute molecules across a 384-well plate followed by 50 pl aliquots of EDTA whole blood containing Sytox Green per well (Figure 2A). The plate was centrifuged to sediment erythrocytes leaving an upper plasma layer and buffy coat containing neutrophils at the interface (Figure 2A). NET formation was measured from the top through the plasma via intercalation and fluorescence of extracellular DNA using a SpectraMax plate reader.

[0113] To evaluate consistency across the plate and limit potential artifacts (edge effects, temperature variation and oxygenation), a series of optimizations were performed across an entire 384 plate using 250 nM of PMA to induce NETosis (Figure 2B). Experimental parameters were optimized to ensure that small changes in fluorescent intensity at the onset of NETosis could be reliably detected. The time of NETosis (tNEi), was calculated as the time at which the fluorescent signal exceeded 3.3 x the standard deviation of a vehicle treated control group. Assay optimization focused on reducing the standard deviation in tNET to + / - 5 minutes across the plate. We performed a randomized PMA titration across the entire plate and showed a dose response curve of NETosis induction (Figure 2C). Nucleosome release was measured in plasma isolated from treated whole blood in parallel using the H3.1 Nu.Q® NETs assay (Volition) and showed the time course of nucleosome release was consistent with the Sytox Green signal (Figure 2D). In this high-throughput, induced NETosis model we found that both Cl and LPS failed to induce NETosis (Figure 2E and Figure 2F), reflecting the results seen in whole blood (Figure 1G). MPO inhibition (ABAH) delayed the onset of NETosis (Figure 2F) consistent with the inhibition of NETosis, as noted in isolated neutrophils.

[0114] Variability of NETosis Profiles

[0115] The high-throughput NETosis model was used to screen candidate NETosis regulators selected based on reported association with NETosis or neutrophil biology (Table 2). Design of Experiments (DOE) was performed to combine selected factors in an iterative screen to identify the minimal candidate pool required to induce DNA release (Figure 3A). Initially, 6- 19 factors were combined into individual wells with each factor represented in approximately half of the wells. Four distinct fluorescence patterns were observed following treatment with the various combinations: no response; initial response with early plateau; delayed response with continued gradual increase and initial response with secondary response with continued increase (Figure 3B). To assess the relative contribution of each factor to the fluorescence signal at each time point, we averaged the raw signal over 30-minute windows, calculated the first derivative, and performed multivariate regression modeling (Figure 3C). The contribution of each factor across each of the wells was used to determine the potential extent the specific factor contributed to the signal. Figure 3d shows examples of four observed patterns: C5a and TNF-a contributed to a rapid onset of DNA release, with TNF-a having a secondary protracted effect on the system. Interleukin-5 (IL-5) did not appear to have a significant impact on fluorescence signal and lnterleukin-1 p (IL-1 ) appeared to reduce the fluorescence signal, indicating potential inhibition or buffering capacity. We performed two rounds of screening in blood from two different healthy donors (n=4) and found that TNF-a, LT-a, IFN-y, GM-CSF, LTB4, C5a, and Ferritin consistently contributed to increased Sytox Green signal across donors whereas LPS and fMLP only contributed in one of the two screens (Figure 3E). Molecules with negative or no effect on signal were removed for subsequent NETosis inducer screens. Ferritin was observed to consistently contribute to an increase in signal but was removed from subsequent screens as it was horse derived.

[0116] Biological Relevant NETosis Induction Consistency Across Donors

[0117] Levels of endogenous cytokines vary across individuals and in response to environmental stimuli contributing to natural variability in innate immune response. Inter and intra donor variability of NETosis induction was evaluated using the eight selected factors. To normalize the effect each combination of factors had across donors: we performed a Boolean transformation of the first derivative change in RFU over the time course with a cutoff at 3.3 SD x of the background signal (Figure 4A). We tested combinations of the selected 8 factors (TNF-a, LT-a, IFN-y, GM-CSF LTB4, C5a, LPS, and fMLP) in six donors and found signal consistently increased across donors with more factors (Figure 4B). Four factors increased NETosis in most of the six donors tested with two distinct patterns: early onset between 30 minutes and two hours, followed by a later increase after three hours. Female donors were generally less responsive than males, especially with earlier onset NETosis (Figure 4C). Either TNF-a or LT-a was required for consistent increase in signal across donors. In their absence, the remaining six compounds failed to induce consistent signal (Figure 4D). Interestingly, C5a or LPS appeared to be critical for the early onset signal (30 minutes to 2 hours) (Figure 4D). To investigate the relative role of each factor, we selected a pool of five factors (LT-a, GM-CSF, C5a, LPS, and f LP) which induced early DNA release in all donors tested but not the later release, hypothesizing that the secondary response could reflect mechanisms other than NETosis. The impact of each factor in combination with the other factors across the six donors is shown in Figure 4E. These results highlighted differences between male and female donors, for example, it appeared GM-CSF and LPS were less important for the early response in males compared to females and that male donors were more likely to undergo NETosis with less stimulation compared to the female donors with the five factor pool.

[0118] Minimal factors required for NETosis induction

[0119] To determine whether individual donors responded to the same combination of compounds over time we had two donors undergo multiple blood draws over a month period and found general consistency for NETosis initiation with the five factor pool (LT-a, GM-CSF, C5a, LPS, and fMLP) across and within donors, with a pool of LT-a, C5a and fMLP giving the most consistent result with the fewest number of factors (Figure 5A). Variability was reduced as more factors were utilized, however we observed a possible set of three factors (LT-a, C5a, and fMLP) which had consistency and similar results as those pools which included either GM-CSF and or LPS. Due to this, space-filling DOE and Gaussian Process Regression was used to evaluate the concentration dependence of each factor in blood samples from two healthy donors. We found that GM-CSF did not play a significant role in either donor, while LPS appeared to decrease the overall observed signal in a dose-dependent manner (Figure 5B). Consequently, we removed GM-CSF and LPS from the pool to generate the minimal combination of factors required to induce an increase in NETosis. All three remaining factors were required to generate an increase in Sytox Green signal (Figure 50), with the EC50 of each being consistent with published values for neutrophil activation (Table 1). To confirm the signal we were seeing upon treatment with these natural triggers was a result of NETosis, we treated whole blood with the optimized concentrations of LT-a, C5a, and fMLP, along with a variety of inhibitors. We showed a significant decrease in signal when inhibitors targeting Neutrophil Elastase II, protein arginine deiminase (PAD), and PAD4 were included (Figure 5D). However, there was no change in signal when an apoptosis inhibitor (Caspase 3 / 7 inhibitor I) or necroptosis inhibitor (necrostatin-1) were included. This evidence suggests the increased signal from Sytox Green was a result of NETosis and not an alternative form of cell death. We did see a marginal, yet not significant decrease in DNA release upon treatment the MPO inhibitor (ABAH), suggesting that NETosis induction using these natural triggers may be different than what is seen with PMA.

[0120] DISCUSSION

[0121] Sepsis is a major global disease. There are an estimated 48.9 million cases annually and 11 million sepsis related deaths21. Additionally, patients who recover from sepsis are at greater risk of readmission to hospital and have a greater risk of death when compared to age and sex-match controls22. A key driver for pathogenesis and potentiator of sepsis is excessive NET formation23. Physiologically relevant models mimicking endogenous NETosis enable mechanistic investigation of the complex signaling cascades underlying NETosis activation. Our representative ex-vivo model of these cascades offers the opportunity for screening therapeutic interventions under disease mimetic conditions which reflect underlying conditions which may predispose patients to adverse outcomes. To our knowledge, we show for the first time the ex-vivo induction and real time kinetic read out of NETosis using naturally occurring molecules in a whole blood system. We found that activation with TNF-a or LT-a was required for rapid onset NETosis, highlighting the importance of these molecules in sepsis. Indeed, TNF-a has been shown to be elevated in sepsis and is a putative therapeutic target24.

[0122] Interestingly, we found that Cl, and LPS showed a differential ability to induce NETosis in whole blood compared to isolated neutrophils, which could be due to the presence of an additional cell type or factor present in whole blood, and reflects the complexity of the whole blood model compared to isolated neutrophils. Mol et al, previously reported that treating neutrophils with pairs of factors similar to what we identified (GM-CSF, fMLF, TNF, and LPS), resulting in neutrophils displaying a variety of neutrophil associated behaviors (e.g. ROS production, degranulation, phagocytosis) but they were not able to induce NETosis17. Our ability to induce NETosis in our ex-vivo model using no less than three different factors, demonstrates the complex signaling needed for NETosis to occur, and suggests that there is something present in whole blood that support NETosis. Whole blood has a variety of proteins and other cells types (e.g. platelets and macrophages) which are known to induce NETosis and additional work is necessary to determine whether these cells play a role in the NETosis we observe in our ex vivo model.

[0123] NETs play a critical role as part of the innate immune response to infection, immobilizing pathogens to prevent dissemination and clearing them from circulation23. However, excessive NETosis can be pathogenic and lead to host directed bystander effects and thrombosis25. Thus, NETosis can be both beneficial and detrimental25. The differential time courses and magnitudes of NETosis seen in our ex-vivo whole blood model in response to specific combinations of pro-inflammatory compounds may be correlated to beneficial vs pathogenic NETosis (Figure 3B). It is possible that the compound combinations in which the amount of NETosis was minimal represents the beneficial NETosis, whereas conditions that led to higher release may reflect pathogenic NETosis. Furthermore, some combinations trigger a biphasic release pattern with a moderate early minimal response followed by a secondary exacerbated response, which could be reflective of a positive feedback loop in which nucleosomes released from the early NETs further stimulate additional NETosis26. These patterns provide an opportunity for biomarker identification as well as therapeutic intervention for future study.

[0124] In our initial screens we found that pools with molecules that interact with similar cell-surface markers / signaling cascades induced consistent responses across donors, which suggested a potential convergence of required signaling pathways. At least three naturally occurring factors in combination (LT-a, fMLP and C5a) were necessary to consistently induce NETosis in our system indicating a potential requirement of the activation of TNF Receptor 1 , TNF Receptor 2, or LT~p receptors for NETosis to occur27. NETosis induction did not occur in the absence of TNF-a or LT-a, underlying the potentially significant roles these factors play in inflammatory disease, and suggests an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.

[0125] These findings emphasize the importance of expanding the understanding of neutrophil physiology in a biologically relevant context. Physiological triggers of NETosis could be used to better understand NET associated disease pathology, risk factors, and potential therapeutic targets, providing novel strategies for disease intervention and treatment. Our novel ex-vivo NETosis approach has the potential to be used for screening drug candidates as inhibitors and inadvertent activators of NETosis and through a comprehensive exploration of human cell-based NETosis, we can take significant strides toward mitigating the devastating impact of sepsis on global healthcare.

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[0170] Table 1 : Summary of EC50 values reported in the literature and identified in the current study. component 5a; fMLP, N-formyl-Met-Leu-Phe; ROS, reactive oxygen species

[0171] Table 2: List of compounds assessed for NETosis regulation activity. All compounds were resuspended according to manufacturer’s recommendation.

Claims

CLAIMS1 . A method for screening a candidate substance as a regulator of NETosis comprising: a) providing a multiwell plate; b) distributing a candidate regulator across the multiwell plate; c) distributing a fluorescing intercalating dye across the multiwell plate; d) distributing a sample of whole blood across the multiwell plate; e) allowing erythrocytes in the whole blood sample to sediment leaving an upper plasma layer and a buffy coat containing neutrophils at the interface of the layers; f) measuring NET formation in the buffy coat using the level of fluorescence of the intercalating dye wherein the level of NET formation can be used to determine whether the candidate substance is a regulator of NETosis; wherein steps b), c) and d) can occur simultaneously or in any order.

2. The method according to claim 1 , wherein the multiwell plate is centrifuged to sediment erythrocytes leaving the upper plasma layer and the buffy coat.

3. The method according to claim 1 or claim 2, wherein the multi-well plate is a 384-well plate.

4. The method of any preceding claim, wherein the sample of whole blood is EDTA whole blood.

5. The method of any preceding claim, wherein the candidate substance is a candidate inducer of NETosis.

6. The method of any one of claims 1 to 4, wherein the candidate substance is a candidate inhibitor of NETosis and the method includes the step of inducing NETosis, for example by adding at least one substance which is an inducer of NETosis across the multiwell plate or by allowing coagulation to occur.

7. The method of claim 6, wherein the at least one substance which is an inducer of NETosis is a composition comprising at least one of TNF-alpha (TNF-a) or LT-alpha (LT-a).

8. The method of any preceding claim, wherein the time of NETosis is determined as the time at which the level of fluorescence exceeds 3.3 times the standard deviation of a control.

9. The method of any preceding claim, wherein the fluorescing intercalating dye is Cytotox Green or Sytox Green.

10. The method of any preceding claim, wherein NET formation is measured through the upper plasma layer, such as from above.

11. A method of identifying a candidate compound capable of inhibiting NETosis comprising: a) providing a whole blood sample; b) adding at least three inducers of NETosis to the whole blood sample wherein at least one of the inducers is TNF-a or LT-a; c) adding a candidate compound to the whole blood sample; and d) determining whether the level of NETosis is reduced as a result of adding the candidate compound; and wherein steps a) to c) are conducted in any order.

12. The method of claim 11 , wherein the level of NETosis is measured by detecting or measuring the level of cell free nucleosomes or a component thereof, myeloperoxidase (MPO), neutrophil elastase (NE) or cell free DNA (cfDNA).

13. The method of claim 12, wherein the cell free nucleosomes comprise histone H3.1.

14. The method of any preceding claim, wherein the method is used in screening the candidate compound for toxicity.

15. A composition comprising at least three inducers of NETosis of which at least one is TNF-a or LT-a.

16. The composition of claim 15, comprising TNF-a and LT-a.

17. The composition of claim 15 or claim 16, comprising at least four inducers of NETosis.

18. The composition of any one of claims 15 to 17, comprising or consisting 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, lipopolysaccharides (LPS), fMLP19. The composition of any one of claims 15 to 18, wherein the inducer of NETosis is not phorbol 12-myristate 13-acetate (PM A).

20. The composition of any one of claims 15 to 19, wherein the inducer of NETosis is not LPS or calcium ionophore (Cl).21 . The composition of any one of claims 15 to 20, comprising or consisting of at least one of TNF-a or LT-a in combination with one or more of IFN-y, GM-CSF, LTB4, C5a and ferritin.

22. The composition of any one of claims 15 to 21 , comprising or consisting of a combination of LT-a and GM-CSF, C5a, LPS, and fMLP.

23. The composition of any one of claims 15 to 22, comprising or consisting of LT-a, C5a, and fMLP.

24. The composition of any one of claims 15 to 23, comprising or consisting of TNF-a or LT-a and C5a.

25. A kit for regulating NETosis wherein the kit comprises a composition as defined in any one of claims 15 to 24.

26. Use of the composition of any one of claims 15 to 24 or the kit of claim 25 in a method for screening for a candidate inhibitor of NETosis wherein the method is as defined in any one of claims 1 to 10 and the composition is distributed across the multiwell plate before step e).

27. Use of the composition of any one of claims 15 to 24 or the kit of claim 25 in a method for screening a candidate compound for toxicity.

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