WO2017055604A1 - Assay for etosis and extracellular traps - Google Patents

Abstract

Disclosed herein is an improved method for the detection, quantification and/or analysis of extracellular traps (ETs) and processes generally known and/or classed as types or forms of, ETosis as well as NET-inducing capacity. The methods disclosed herein may be (semi)- automated, high throughput and reproducible methods. In particular, the methods of this disclosure may be applied to the detection, analysis and/or quantification of ETs and ETosis as might be produced by or occurring in, a number of different cell types. For example, the methods may be applied to the detection, quantification and/or analysis of such processes and structures in neutrophils - the structures in neutrophils being termed neutrophil extracellular traps (NETs) and the process in neutrophils being known as "NETosis".

Description

ASSAY FOR ETOSIS AND EXTRACELLULAR TRAPS

FIELD OF THE INVENTION

The present disclosure provides methods for the detection, quantification and/or analysis of extracellular traps formed by a process known generally as, ETosis. For example, the methods of this invention may be exploited in the detection, quantification and analysis of NETosis and NET-like structures. The disclosure provides methods which may also find application in the diagnosis of certain diseases (or a predisposition/susceptibility thereto) and in assays for testing agents for an effect on NETosis. The disclosure further relates to kits for use in such methods. BACKGROUND OF THE INVENTION

NETosis is an alternative death pathway of neutrophils characterised by the release of neutrophil extracellular traps (NETs). A number of studies have postulated an important role for NETs in the inductions and/or perpetuation of autoimmune diseases (AIDs) such as systemic lupus erythematosus (SLE), Anti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis and rheumatoid arthritis (RA). The exact role of NETs in the pathogenesis of AIDs remains to be elucidated. One of the limitations is the lack of a well-defined and reliable assay to directly quantify NET formation.

There are essentially two current ways of quantifying in vitro NETs. The first method involves immunohistochemistry (IHC). Methods based only on IHC have several issues: first, when assessing NETosis by IHC 5-10 high power fields (HPFs) are selected for quantification. This selection is prone to operator subjectivity as they tend to be biased towards the NET-like structures. Secondly, only a small number of neutrophils can be assessed in 5-10 high power fields, covering 0,5-1 % of all cultured neutrophils introducing a high probability of sampling errors when quantifying NETosis or NET-like structures. Thirdly, NET(-like) structures are expulsed from neutrophils and lay on top on neutrophils. When using IHC, the focus of a high power field is directed towards neutrophils introducing the problem that the majority of NET(-like) structures are not into view, hence not quantified. As such, they are unable to provide a true and reliable assessment of the level of NETosis within a given sample. The second method requires quantification of extracellular DNA - most usually performed using some form of immunological technique such as ELISA. Methods based solely on the quantification of nucleic acid (for example by ELISA) do not take into account the origin of the nucleic acid. While some of the extracellular nucleic acid may result from the process of NETosis, it may also be present as a consequence of non- NETosis type of death pathways - for example apoptosis or necrosis. The present disclosure provides methods which avoid problems associated with prior art method and facilitate the improved analysis, detection and/or quantification of NETosis

SUMMARY OF THE INVENTION

Disclosed herein is an improved and sensitive method for the detection, quantification and/or analysis of extracellular traps (ETs) and processes generally known and/or classed as types or forms of, ETosis as well as NET-inducing capacity (of, for example, sera - including sera from patients suffering from or susceptible/predisposed to a disease, condition or autoimmune disease/condition). The methods disclosed herein may be (semi-)automated, high throughput and reproducible methods. In particular, the methods of this disclosure may be applied to the detection, analysis and/or quantification of ETs and ETosis as might be produced by or occurring in, a number of different cell types. For example, the methods may be applied to the detection, quantification and/or analysis of such processes and structures in neutrophils - the structures in neutrophils being termed neutrophil extracellular traps (NETs) and the process in neutrophils being known as "NETosis".

It should be noted that throughout this specification reference will be made to "ETosis" and this should be taken to embrace any innate immune process resulting in the formation of extracellular traps. Further, the disclosure refers to extracellular traps (ETs) and this term may embrace structures produced by cells and comprising nucleic acid (decondensed chromatin-DNA) and perhaps antimicrobial enzymes and peptides. Without being bound by theory, it is suggested that the function of these ETs is to trap and kill pathogens (including microorganisms). It should be understood that the terms "ETosis" and "ETs/ET like structures" embrace processes occurring in neutrophils (referred to as "NETosis") and neutrophil extracellular traps (referred to as "NETs"). The terms may also refer to similar processes occurring in other cell types such as other cells of the innate and acquired immune system, mast cells, eosinophils, basophils macrophages and the like.

The methods described herein exploit cell staining and nucleic acid staining procedures in order to provide methods which avoid problems associated with prior art ETosis quantification methods, which methods are (among other problems) prone to user/operator subjectivity (the user/operator being biased towards cells which exhibit ET-like structures), sampling errors, use of too few high power fields (HPFs) and erroneous focussing. Problems of this type are most often associated with immunohistochemistry based ETosis quantification techniques. Further the methods described herein avoid problems associated with the non-discriminatory detection of extracellular nucleic acid. Problems of this type are most often associated with ELISA based ETosis quantification techniques. The methods of this invention may be used to directly visualise and/or quantify a level or amount of ETosis in response to any given stimulus.

In a first aspect, disclosed herein is a method for quantifying, analysing or detecting ETosis and/or ET-like structures, said method comprising: providing cells; contacting the cells with a cell membrane stain and subjecting the cells to conditions which promote or induce ETosis; contacting the cells with a nucleic acid stain; and determining the ratio of cells positively labelled for nucleic acid with the number of labelled cells.

It should be understood that throughout this specification, the term "comprising" is used to denote that aspects and embodiments of this disclosure/invention "comprise" a particular feature or features. It should be understood that the term "comprising" may also encompass aspects and/or embodiments which "consist essentially of" or "consist of" the relevant feature or features.

The conditions which promote or induce ETosis may include, for example, conditions which comprise protocols for "stimulating" or "affecting" ETosis in cells. Where the cells are neutrophils, the conditions used may induce NETosis, a process characterised by the release (from neutrophils) of neutrophil extracellular traps (NETs). As such, conditions suitable to promote or induce NETosis are any conditions which bring about the phenomenon or process of NETosis in neutrophils. One of skill in this field will be familiar with those techniques which can be used to promote or induce ETosis in cells (for example neutrophils) and any suitable technique may be applied here. For example, cells may be subject to conditions which include contact with one or more ETosis "inducers". For example an ETosis inducer may include one or more selected from the group consisting of plasma, serum, immunoglobulins, immune complexes (ICxs), cell culture medium, and/or Phorbol 12- myristate 13-acetate (PMA) - all of which are known to induce ETosis -including NETosis. Thus, the conditions which promote or induce ETosis may comprise contacting cells with one or more ETosis inducers selected from the group consisting of plasma, serum, immunoglobulins, cell culture medium, and/or Phorbol 12-myristate 13-acetate (PMA). The method may exploit any cell or populations of cells capable of undergoing or affecting the process of ETosis. For example the cells may be (or comprise) cells of the innate and/or acquired immune system. The cells may comprise white blood cells. The cells may comprise, for example, a neutrophil, a mast cell, a basophil, an eosinophil or any similar cell. The cell may be a neutrophil. The cells may be a homogenous population of cells or a heterogeneous population of cells.

The cells may be obtained from or provided by healthy subjects - that is, subjects not known to be suffering from (or susceptible or predisposed to) diseases and/or conditions likely to give rise to the phenomenon of ETosis. Additionally or alternatively, cells for use in the methods disclosed herein may be obtained from or provided by subjects suffering from, for example, autoimmune diseases. The term autoimmune diseases may include, for example SLE, ANCA and RA.

Cells (for example neutrophils) for use in a method of this invention may be obtained by any suitable method. For example, the cells may be obtained from blood provided by healthy donors and/or (as described above) those suffering from or predisposed to some form of disease associated with ETosis (for example an autoimmune disease). Techniques such as density gradient centrifugation and erythrocyte lysis may be used to isolate and purify certain cell types (for example neutrophils) for use.

The cells may then be maintained in any suitable vessel. For example, the cells may be counted, diluted if required and seeded into multi-well vessels, for example 96-well culture plates. Once seeded, the cells may be maintained in culture. The cells may be maintained under conditions suitable to promote growth and/or viability of the cells.

Prior to, during or after seeding or maintaining the cells, may be subjected to a protocol or compound which stains cell membranes. One of skill will be familiar with staining compounds that can be used to mark, label or stain a cell membrane and any such compounds may be used here. In particular, the methods of this disclosure may exploit compounds which label cell membranes in an optically detectable manner. For example, the methods of this disclosure may exploit a fluorescent cell membrane label. As such, the methods described herein may comprise a step in which the cells are contacted with a labelling compound so as to yield labelled cells. Labelled cells for use in the methods disclosed herein may be referred to as "membrane labelled". Thus, where the cells are neutrophils, a method described herein may require that the neutrophils are "membrane labelled" using some form of labelling compound. Suitable cell membrane labelling compounds include, for example, PKH fluorescent cell dyes. The methods of this invention may exploit PKH26.

The cells for use in the methods disclosed herein may be maintained or incubated under conditions which promote or induce ETosis for a specific predetermined time. The length of time may depend on the precise method used to promote or induce ETosis. As such, the period of time may be construed as any time sufficient (or required) to induce or promote ETosis. The conditions which induce ETosis may further include exploitation of specific incubation temperatures and pH.

By way of example, when subjecting the cells to conditions which promote or induce ETosis, the cells may be maintained under those conditions for anywhere between about a few minutes to a few hours. For Example the cells may be maintained under such conditions for about 10, 20, 30, 40 or 50 minutes to about 1 , 2, 3, 4, 5 hours or more.

After a suitable period of contact between the cells and the conditions which promote or induce ETosis, the cells may be contacted with a compound capable of labelling cell membranes.

The methods disclosed herein further comprise a step wherein the (seeded, membrane labelled and ETosis induced) cells are contacted with a nucleic acid stain or label. Again, suitable stains or labels will be known to those skilled in this field. Advantageously, the methods may exploit live-cell impermeable nucleic acid stains. The nucleic acid staining step may occur during or after the step in which the cells are subjected to conditions which promote or induce ETosis. For example, sometime (perhaps minutes, for example 5, 10, 15 or 20 minutes) prior to completion of the step in which the cells are induced or promoted to undergo ETosis, the cells may be additionally contacted with a nucleic acid stain. The nucleic acid stain may comprise a live-cell impermeant fluorescent stain. The colour (or fluorescence) of the nucleic acid stain may be different from the colour or fluorescence of the cell (membrane) label. For example, the nucleic acid stain may comprise Sytox^® Green nucleic acid stain.

The nucleic acid stain may be brought into contact with the cells for a time sufficient to affect the staining (or labelling) of any extracellular traps (which comprise nucleic acid). For example the nucleic acid stain may be brought into contact with the neutrophils for at least about 5, 10, 15 or 20 minutes. After completion of the nucleic acid stain procedure, the cells may be fixed. Cell fixing procedures are well known to the skilled person and may include, for example, the use of paraformaldehyde (for example 4% paraformaldehyde solutions). Thus, the cells may be fixed by contact with paraformaldehyde. After fixing, the cells may be subjected to an imaging protocol configured to capture images of the stained cells. The imaging protocol may capture images using a filter for visualisation of the stained cells and images using a filter for visualisation of the nucleic acid stain. For example, where the cells are stained or labelled with a red fluorescent label (for example PKH26), images may be obtained using a Cy3 filter. Where the cells are stained or labelled using a green fluorescent (live-cell) impermeant nucleic acid stain, the images may be captured using an alexa488 filter.

The images may be captured using any suitable imaging system.. A protocol for imaging cells (and ultimately for determining the ratio of cells positively labelled for nucleic acid with the number of labelled cells) may comprise imaging or capturing one or more high power fields (HPFs). Where the cells have been seeded into a multi-well vessel, the one or more high power fields may be taken or captured across surface of the seeded well.

The imaging protocol and/or the process of determining the ratio of cells positively labelled for nucleic acid with the number of labelled cells, may require the imaging or capturing of (in any given vessel or well thereof) anywhere between about 2 and about 250 (or more) individual (discrete or separate) HPFs. For example, the process may exploit 5, 10, 15, 25, 30 or 35 HPFs. The process may exploit, for example 25 HPFs.

Each high power field may comprise one image or two or more images which may be stacked. Where the HPF comprises two or more (stacked) images, each image may be separated by a z-distance (a distance in the "z" axis). For example, the z-distance may be a predetermined and/or fixed distance such that each image of the HPF is separated by the same amount. The z-distance may be variable such that the distance between each image of any given HPF can be varied. The z-distance may be a distance of about 0.1 m to about 100pm or any distance therebetween. The precise distance may not be important but suitable z-distances may include, for example, anything from about 0.5 pm to about 20 pm, about 1-10 pm or about 1.2 pm, about 1.4 pm, about 1.6 pm, about 1.8 pm, about 2 pm. The z-distance may be 1.4 pm. The z-distance and total number of stacked images in each HPF may be decided with consideration given to the diameter and/or height of the cell and/or position/location of the extracellular DNA (stained by the nucleic acid stain). The inventors have noted that the staining of the ETs (i.e. the extracellular DNA) occurs mostly on top of the cells - for example on top of neutrophils and as such it is advantageous to have the z- distance and number of images span at least the whole height of the cell. This avoids problems associated with focus plane sampling bias where (in prior art methods) a single focus plane focuses only on the cells (or a single plane thereof) and misses a major part of the nucleic acid staining (which may lie above (or below) the selected focus plane). The methods disclosed herein avoid this problem by selecting a number of images (for example 12) and a z-distance (for example 1.4pm) which ensures that the image stacks cover at least the complete height/diameter of the cell (and perhaps more). For example, the inventors may begin by imaging an HPF some 2-3 or more images below the focus plane and then progress imaging upwards (by predetermined z-distance increments) to ensure that the whole cell diameter/height is imaged and considered in the analysis. In this way, the method avoids false negative results where, due to the position of the selected focus planne, a degree of nucleic acid staining on any given cell is missed.

Each HPF may comprise 2-50 stacked images (the precise number of images may not be crucial and thus could be higher than 50 if desired). For example, each HPF may comprise 10-15 stacked images, for example 12 stacked images.

As stated, the HPFs (each HPF comprising one or more images) may be captured using any suitable imaging device as it scans the surface of the cell seeded vessel. The HPFs may be evenly spread across the relevant surface and taken en-route through some predetermined transit across the surface of the vessel (or a well thereof). For example, the HPFs may be evenly spread across the surface of the vessel (or a well thereof) by a standardised "zig-zag" pattern. The imaging device may translocate or move across the surface along an x-axis direction of travel and a y-axis direction of travel. Each HPF may be separated from another by an x distance and a y distance. In this way, the HPFs are distributed as an array across the vessel surface. For example, each HPF may be separated from another by an x-axis distance of about 50 pm - about 800 m, for example about 100 pm - about 700 pm, about 200 pm - about 600 pm, about 300 pm - about 500 pm. Each HPF may be separated by an x-distance of about 400 pm. Each HPF may be separated from another by a y-axis distance of about 100 pm - about 1000 pm, for example about 200 pm - about 800 pm, about 300 pm - about 700 pm, about 400 pm - about 600 pm. Each HPF may be separated by a y-axis distance of about 500 pm.

The images may be captured at any suitable magnification. It will be appreciated that the level of magnification used may depend on the imaging system. For example the images may be obtained using a 20x objective.

Each image may comprise one image taken using a filter for visualisation of the labelled cells and another image taken using a filter for visualisation of the nucleic acid label. In this way the imaged surface of the vessel or well is imaged not only for visualisation of the cells per se (via visualisation of the fluorescently stained or labelled membranes) but also for any stained or labelled nucleic acid (via visualisation of the fluorescent live-cell impermeable nucleic acid stain). The imaging system may be exploited or set to automatically capture images of the labelled cells.

Image exposure times for each of the cell label and nucleic acid label images may be determined using the appropriate controls. For example the exposure time for the nucleic acid stained images may be determined using PMA stimulated neutrophils which are known to undergo maximal NETosis. The exposure time for cell-membrane stained cells may be determined using a negative control comprising un-stimulated and viable neutrophils. Once an exposure time is determined, the same exposure time may be used for each similar image.

Depending on the number of HPFs captured and the number of images comprised within each HPF, the HPFs may represent anything from about 1 % to about 100% of the total surface of the vessel or a well thereof. For example, the HPFs may represent at least about 20%-30%, for example 25% of the total surface of the vessel or a well thereof.

It should be noted that prior art methods may only achieve consistent sampling of about 0.5-1 % of the requisite well. This can introduce some considerable sampling error. The methods of this invention routinely take account of the number of cells/amount of NETosis in a greater area and thus sampling errors are reduced. Each HPF may comprise 12 stacked images, the images being separated by a z-distance of about 1.4μιη. Each HPF may be separated by a x-axis distance of about 400 μηι and a y-axis distance of about 500 μιη. The method of this invention may require the capturing of about 25 HPFs in total. After capture, the images are analysed in order to determine (a) the number of cells and (b) the amount of nucleic acid staining. It should be noted that the present invention represents an improvement over prior art methods as not only does it use two methods (cell staining an nucleic acid staining) to address the deficiencies associated with the individual immunohistochemistry (IHC) and ELISA based techniques currently used, but it avoids the user being influenced and biased towards cells exhibiting ET-like structures. By imaging cells by visualisation of a fluorescent cell membrane label, the assessment of the number of cells is done without regard for the presence of any ET-like structures or ETosis. As such, when a user counts the number of fluorescently labelled cells in an image - this is a reflection of the true number of stained cells. A further advantage of the methods described herein is that the nucleic acid staining protocol stains only extracellular nucleic acid. Additionally, by combining the nucleic acid staining images with the cell images, it is possible to ignore images which show nucleic acid stained as a consequence of some process other than ETosis - for example through cell death or the like. It should be understood that prior art ELISA based nucleic acid detection procedures are unable to discriminate between extracellular nucleic acid occurring as a consequence of ETosis and the presence of extracellular nucleic acid occurring as a consequence of some non-ETosis based process. In effect, the methods of this invention allow the user to properly correct the amount of stained nucleic acid (an indication of the level of ETosis) for the number of cells visualised.

Another advantage of the methods described herein is that the number of cells analysed is increased largely, as well as the percentage area of the well covered, reducing sampling errors.

Additionally, the fact that the method (image acquisition and quantification) can be automated is an advantage over prior art methods.

It is known that the prior art methods for quantifying ETs and ETosis are subject to a great deal of variability - not least because, as stated above, prior art techniques fail to prevent user/operator bias towards cells exhibiting ETosis. This also leads to user/operator variability as some users/operators are affected to a greater extent by the presence of ETosis when counting cells. Thus in any given sample, the level of ETosis quantification reported by one user/operator might vary considerably from the level reported by another user/operator. Surprisingly, the methods described herein reduce user/operator effect and hence the variability of results reported from different users/operators. Indeed in one experiment it was found that two operators quantifying ETosis in a sample processed according to the methods described herein, reported substantially the same result - in other words the user/operator effect was minimal.

Image analysis may be completed using an automated image processing software.

The images may be analysed for cell-membrane staining. For example, where the cell-membrane staining is affected using a red fluorescent label such as PKH26, the images may be analysed for PKH26 staining - this being a(n unbiased) measure of the total number of cells imaged. The images may also be analysed for nucleic acid staining. For example, where the nucleic-acid stain is affected using a green fluorescent label such as Sytox® Green, the images may be analysed for Sytox® Green staining. The analysis may aim to determine the percentage of the area imaged that is positively stained for cells and percentage of the same area that is positively stained for nucleic acid.

In order to quantify ETosis or the number of ET-like structures, the percentage area of positive nucleic acid staining (via Sytox® Green) may be divided by the percentage area of positive cell staining (via PKH26). In addition to facilitating ETosis quantification, this corrects the level of ETosis for the number of cell counted.

One of skill will appreciate that images with a higher ratio are indicative of a greater amount of extracellular DNA present. This in turn is a marker of ETosis. The methods of this disclosure may not use antibody stains.

The methods described herein represent a further advantage over prior art methods in that there are limited pipetting steps

The methods described herein may be used or exploited in method of diagnosing disease. For example, the methods may be used to facilitate or aid the diagnosis of autoimmune diseases.

Without wishing to be bound by theory, a serum factor, for example immunoglobulin (for example IgG), derived, provided or obtained from patients suffering from or predisposed to autoimmune diseases may induce or promote more ETosis than immunoglobulin (IgG) from healthy patients (i.e., patients not suffering from or predisposed/susceptible to autoimmune diseases). As such, by inducing or promoting ETosis in cells (for example neutrophils) using immunoglobulin (IgG) obtained from or provided by subjects to be tested (those subjects perhaps being suspected of suffering from or being predisposed/susceptible to an autoimmune disease), a diagnosis of possible autoimmune disease (or susceptibility/predisposition thereto) may be based on the level of ETosis observed using a method described herein.

The methods of this invention may be further used to quantify NET-inducing capacity in sera from patients with certain diseases and/or conditions including, for example, autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). As stated, a level of ETosis may be associated with autoimmune disease, for example, immune complexes (ICxs) are considered as pathogenic compounds in some human autoimmune conditions and are capable of inducing NETs. Further, neutrophil ETosis may be sensitive to the presence of immunoglobulin (IgG) from patients with (or predisposed/susceptible to) autoimmune disease. As such, where a method of this invention exploits a patient source of immunoglobulin as a means to induce or promote ETosis, a read out from the method that reveals a high level of ETosis may be an indication that the patient source of immunoglobulin has been provided by or obtained from a patient suffering from an autoimmune diseases or predisposed or susceptible to an autoimmune disease.

Thus, this invention provides a method of diagnosing an autoimmune disease or a predisposition or susceptibility thereto, said method comprising executing a method according to the first aspect of this invention, wherein the step of inducing or promoting ETosis exploits an immunoglobulin sample obtained from or provided by a patient to be tested. As stated the patient to be tested may be a subject suspected of suffering from an autoimmune disease or a subject suspected of being predisposed or susceptible thereto.

This invention provides a method of predicting or determining the outcome or prognosis for a patient diagnosed with an autoimmune disease or a predisposition and/or susceptibility thereto, said method comprising executing a method according to the first aspect of this invention, wherein the step of inducing or promoting ETosis exploits an immunoglobulin sample obtained from or provided by a patient to be tested. As stated the patient to be tested may be a subject diagnosed with an autoimmune disease, a subjected suspected of suffering from an autoimmune disease or a subject suspected of being predisposed or susceptible thereto.

An advantage of the methods described herein, including the methods of diagnosis of this invention, is that the present methods are highly sensitive - that is they can be used to detect low levels of NETs. As such, the methods may be used to properly discriminate between subjects that still have functionally active antibodies circulating from those with circulating autoantibody but no detectable signs of ETosis. This is important as many autoimmune diseases are diagnosed on the basis of observed symptoms. Further the response to any given treatment is also assessed on the basis of an associated improvement or relief of symptoms (in other words, successful treatments often relive certain symptoms). While the presence of circulating autoantibodies can be useful in diagnosis of autoimmune diseases, they are not of use when assessing the effectiveness of a treatment - this is because, even though the treatment may be effective in relieving symptoms, autoantibodies may still be produced and detectable in the circulation. Thus the assays of this invention may be used in order to test the ability of autoantibodies to induce ETosis.

The methods described herein may be used to detect NET release as induced by RA and SLE serum samples. Further applications of the methods of this disclosure may include, for example, use of the methods to investigate the ability of circulating factors to induce NET release in autoantibody-mediated diseases, such as ANCA-associated vasculitis, SLE, rheumatoid arthritis, antiphospholipid syndrome and cryoglobulinemic vasculitis.

The methods described herein may find further application in the assessment of the NET-inducing capability of sera derived from patients having or suffering from (metastatic) cancer and thrombotic (cardio-)vascular diseases. Without wishing to be bound by theory, it is suggested that NETs may play an important role in the metastasis of cancers and thrombosis (such as myocardial infarction and stroke).

The methods of this invention may also be exploited in drug testing assays. For example an agent being tested for possible use in the modulation of ETosis may be contacted with a method of this invention. Any increase or decrease in the level of ETosis (as compared to the level of ETosis determined in a method which does not involve contact with the test agent) will indicate whether or not the test agent is a modulator of ETosis.

As such, a third aspect of this invention provides a method for detecting modulators of ETosis, said method comprising inducing ETosis as described above and in the presence of a test agent and performing the ETosis quantification method according to the first aspect of this invention. As stated, the results of a method according to the third aspect of this invention may be compared to the results of a method in which ETosis is induced in the absence of the test agent. This will allow the user to determine whether or not the test agent is a modulator of ETosis. It should be understood that a ETosis modulator may increase or decrease a level of ETosis.

A fourth aspect of this invention may provide a kit for use in quantifying ETosis, said kit comprising a cell membrane stain and a nucleic acid stain (as described above). The kit may further comprise equipment, buffers, vessels and the like necessary to obtain or process neutrophils for use. The kit may further comprise vessels and other equipment for use in executing a method according to the first aspect of this invention. For example the kit may comprise cell culture vessels and multi-well plates. The kit may comprise instructions for use.

The present invention will now be described in detail with reference to the following Figures which show:

DETAILED DESCRIPTION

Figure 1 : SLE derived neutrophils release more DNA in the supernatant in the absence of a stimulus Figure 2: DNA measurements with Picogreen in response to PMA stimulation and fluorescence immunostaining do not correlate. In this example, images of healthy neutrophils after 4 hours of stimulation with PMA are shown (A). Cells are labelled with Calceingreen and DNA is stained with Hoechst 33258. More NET formation would be expected in the condition after stimulation with 0,4 nM PMA since 50% of the PMA response is at this point as shown in the Picogreen assay (B).

Figure 3: Automated fluorescence confocal laser scanning microscopy can specifically detect neutrophil extracellular traps. A. Schematic representation of the automated image acquisition: in each well 12 z-stacked images at 25 high power fields (HPFs) are captured by confocal laser scanning microscopy (CLSM), Each HPF has an horizontal interval of 400um and a vertical interval of 500um. As such 70% of the well is covered imaging 1 1 % of the total area; B. Using automated, digital image analysis, area of PKH-26 is highly correlated to absolute neutrophil number; C. Mean area PKH-26 area of 12 z-stacked images is highly correlated with absolute neutrophil numbers; D. Representative confocal images at 20x magnification are shown. Extracellular DNA staining (Hoechst) stains positive for citrillunated histones (aCitH3) and neutrophil elastase (NE) which are specific markers for NETs.

Figure 4: Automated digital analysis of stacked CLSM images results in a high-sensitivity quantification of neutrophil extracellular traps. Representative stack of 6 montages of 25 HPFs at 20x magnification showing the layered topology of neutrophils and NETs; B. Z-axis 2D-reconstruction of stacked images illustrating the topological position of neutrophils and NETs demonstrating the sampling bias of the single focus plane

Figure 5 Automatization of 3D CSLM image acquisition. The yield of (potentially netting) neutrophils is augmented by increasing the area per well imaged. (A) At a 40* magnification, 3D CSLM is capable of automatically imaging up to 2.8% of each well area. (B) At a 20xmagnification, 3DCSLMis capable of automatically imaging up to 11.1 % of each well area. (C)Depicted is the total amount of imaged neutrophils correlated to the imaged well area. Mean± SEM are shown. (D) Thus, optimal yield was achieved by automatically capturing a fixed pattern 25 high power fields (HPFs) images for each well. (E) For image analysis, 25 HPF images are merged to one montage resulting in a total of 12montages for each well suitable for digital image analysis. (F) When capturing high numbers of neutrophils, the percentage area of PKH26 correlates significantly with the amount of imaged neutrophils (unstimulated). (G) This correlation is preserved even after ICx-induced NET release (ICx 12.5 pg/ml). Seventy-two montages were analysed. (H, I) Relative in vitro neutrophil cell loss after ICx-induced NET release is lower compared to PMA-induced NETosis. The amount of neutrophil was quantified by percentage area of PKH26 positivity. Depicted is the relative reduction in percentage area.

Materials and methods

Preparation of neutrophils Twenty millilitres of whole blood was collected in EDTA-coated tubes. Neutrophils were isolated by density gradient centrifugation with Ficoll-amidotrizoaat (LUMC, Leiden The Netherlands) followed by erythrocyte lysation. Cells were counted using trypan blue, labelled with PKH26 (2 uM, Sigma-Aldrich, Saint-Louis, MO, USA) and then 1x10⁵ neutrophils were seeded per well into a 96-well culture plate in phenol red-free RPMI 1640 medium (Life Technologies, The Netherlands) supplemented with 2% fetal calf serum (FCS). Neutrophils were stimulated during 3 ¾ hours with either medium (negative control), intravenous immunoglobulins (IVIG) (Nanogam 50mg/mL; Sanquin, Amsterdam, The Netherlands), purified IgG, heat-aggregated immune complexes or 25 nM PMA (Sigma-Aldrich). Hereafter, an impermeable DNA dye 0,5 uM Sytox green (Life Technologies) was added for 15 minutes after which neutrophils were fixated with 4% paraformaldehyde (PFA; LUMC).

Visualisation of neutrophil extracellular traps

Within 24 hours, neutrophil extracellular traps (NETs) were visualized by confocal laser scanning microscopy (CLSM) using the automated BD Pathway 855 (BD Biosciences, San Jose, CA, USA). Briefly, 12 z-stacked images of 25 predefined high power fields (HPFs) at a 20x magnification were captured. The HPFs were evenly spread throughout the well by a standardized 5x5 zig-zag pattern with 400um (length) and 500um (width) spaced between each high HPF. Hereby, the 25 HPFs covered 41 % of each well of which 10% of the total well area is captured in 12 z-stacked images.

By way of a non-limiting example, the well area covered by the above-described microscopic imaging was calculated as follows: 1) the area of a HPF at a 20* magnification was calculated at 3.3 * 10⁶ μιτι² (length 417.39 μηι x width 318.01 μιη); 2) the area of a HPF at a 40 x magnification was calculated at 0.8 * 10⁶ μιη² (length 210.49 μηη ^χ width 160.38 μιτι); 3) total area covered was calculated by multiplying the number of HPFs by the HPF area at a given magnification. One of skill will appreciate that the precise detail of the calculation will depend on the well area being assessed.

The microscope was programmed to automatically focus on PKH26 membrane staining. Then, for each image PKH26 (Cy3) and Sytox green (alexa488) was visualized. The CLSM exposure time for Sytox green was set on the positive control and for PKH26 on the negative control. The same exposure time was applied to all images in the same experiment.

Automatic digital image analysis for the quantification of NET formation

Acquired images were automatically analysed by ImageJ image analysis software (NIH, Bethesda, MD, USA). Extracellular DNA of NETs was quantified as the cumulative area of positive Sytox green. To correct for the number of neutrophils, the area of positive PKH26 staining was quantified. Thus, the ratio of both area is calculated representing the amount of NETs in each sample. A higher ratio indicates a higher extracellular DNA present.

Fluorescence immunohistochemistry of NETs Neutrophils were seeded onto polylysine-coated coverslips and NETs were induced according to the abovementioned protocol. Fixated neutrophils were blocked with 1 % BSA in PBS and stained with polyclonal rabbit anti-human citrullinated histon3 (1 ug/ml, Abeam), myeloperoxidase (MPO) (Hycult) and neutrophil elastase (NE) (Abeam) in PBS and incubated for two hours. Then, neutrophils were washed and incubated with a 1 /750 dilution secondary anti-rabbit Alexa647 antibody (Dako, Agilent Technologies, Denmark). After 30 minutes incubation, neutrophils were washed and stained with Hoechst 33258 1 ug/ml or Sytox green (both from Life Technologies). Images were acquired with the Leica DMI6000 inverted microscope using a 20x magnification.

Results NETs are induced at lower levels by human immune complexes as compared to PMA

Immune complexes are considered as pathogenic compounds in human autoimmune diseases and capable of inducing NETs. We first confirmed that human heat-aggregated IgG ICx indeed induced NET formation. Extracellular DNA was detected by immunofluorescence and we confirmed that the extracellular DNA was positive for citrullinated histones (citH3) and neutrophil elastase (NE). Visually, ICx induced NET release at much lower levels compared to PMA. We were unable to pick up low levels of ICx-induced NET release compared to medium control, which was indeed the case for PMA-induced NET release. Of note, similar results were obtained when using Picogreen and Sytox green fluorescence measurements on supernatants of these conditions. In-depth exploration to improve visualization of NETs, we noted that NETs were topological^ superimposed on the neutrophils: Using stacked imaging, neutrophils were found attached to the bottom of a well while released NETs were predominantly observed on top of the neutrophils. Comparing NET quantification, we demonstrated a significant higher result for ICx-induced NET release in three dimensional (3D) imaging (mean NET area ± SEM: 1.03 ± 0.1 1) as compared to 2D imaging (mean NET area ± SEM: 0.04 ± 0.01 , p = 0.01). Altogether, sensitivity to quantify low levels of NETs upon ICx stimulation was increased by visualizing NET release 3- dimensionally (3D). Automatization of 3D CSLM image acquisition

Because ICx induced NETs in fewer neutrophils as compared to PMA, we aimed to further augment the sensitivity of the NET quantification assay by increasing the total imaged surface to yield a higher number of neutrophils. By varying the fully automated image acquisition technique we showed that increasing number of HPFs and reducing the magnification led to larger image areas, correlating with a 10-fold increase of imaged neutrophils (see Fig. 5A, B).When the 40* magnification was used, the area of the well captured is 2.8% when 25 HPFs were imaged, representing 194 ± 66 neutrophils (mean + SEM). For optimal acquisition, 1 1.1 % area of the well was captured when 25 HPFs were imaged with the 20* magnification, thereby analysing 1500 ± 247 neutrophils (mean + SEM).We confirmed that imaging a larger well area by increasing the number of HPFs, resulted in a higher yield of neutrophils (Fig. 5C). To exclude observer subjectivity, 25 HPFs were captured in a constant pattern throughout each well, as illustrated in Fig. 5D. Captured images from 25 HPFs were stitched together to form one montage and 12 montages per well were mounted to form a 3D-image, which was used for digital image analysis (Fig. 5E). When performing image analysis, the amount of NET release is calculated by the total area of Sytox green (%), corrected for the amount of imaged cells calculated by the total area of PKH26 (%). The NET area per amount of imaged neutrophil is thus calculated by the total area of NETs (Sytox positive) divided by the amount of neutrophils, represented by the mean area of PKH26. Regardless of the induction of NETosis, we demonstrated that the total area of PKH26 (%) correlated significantly with neutrophil counts in unstimulated conditions (r = 0.99, p b 0.0001 ) and ICx-stimulated conditions (r = 0.95, p b 0.0001) (Fig. 5F, G). PKH staining is drastically decreased upon PMA stimulation (75% with 12.5 nM PMA) compared to ICx stimulation (34% with 12.5 g/ml ICx) (Fig. 2H, I), suggesting increased neutrophil death and/or cell loss with PMA compared to ICx-induced NETosis.

High sensitivity quantification of ICx-induced NET release shows a ROS-independent process

Using the above-described high-sensitivity NET quantification assay, we confirmed its applicability by further investigating ICx-induced NET release. Low levels of ICx induced NET release (mean NET area per neutrophil ± SEM: 1.1 ± 0.5) were not significantly different when diphenyleneiodonium (DPI) was added (mean NET area per neutrophil ± SEM: 0.8±0.2, p N 0.05). PMA induced large amounts of NET release (mean NET area per neutrophil ± SEM: 107 ± 5.8) which was almost completely inhibited by DPI (mean NET area per neutrophil ± SEM: 2.9±0.0, p b 0.005). These findings were confirmed in cross-sectional overviews of 3-dimensional images of each condition with and without DPI. Overall, these findings indicate that human ICx induce NETs in a ROS-independent manner.

RA and SLE serum samples show NET-inducing capability compared to normal human serum To further confirm applicability of this assay in the analysis of NET release in ICx-mediated autoimmune diseases, we next used this assay to quantify NET release induced by serum of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The mean NET area ± SEM per imaged neutrophil upon induction with RA serum was 0.46 ± 0.08, 0.69 ± 0.18 for SLE and 0.12 ±0.06 for the NHS control (Fig. 6A). For RA, 18 out of 27 serum samples (67%) showed increased NET-inducing capacity compared to the NHS control and in SLE, 17 out of 20 samples (85%) showed increased NET-inducing capacity (Fig. 6B).

Claims

Claims

1. A method for quantifying, analysing or detecting NET inducing capacity, ETosis and/or ET-like structures, said method comprising: providing cells; contacting the cells with a cell membrane stain and subjecting the cells to conditions which promote or induce ETosis; contacting the cells with a nucleic acid stain; and determining the ratio of cells positively labelled for nucleic acid with the number of labelled cells.

2. The method of claim 1 , wherein the conditions which promote or induce ETosis include conditions which comprise protocols for stimulating or affecting ETosis in cells.

3. The method of any one of claims 1 or 2, wherein the cells are subject to conditions which include contact with one or more ETosis inducers.

4. The method of claim 4, wherein the ETosis inducer is one or more selected from the group consisting of plasma, serum, immunoglobulins, immune complexes, cell culture medium, and/or Phorbol 12-myristate 13-acetate (PMA).

5. The method of claim 4, wherein the serum is convalescent serum, serum from a patient suffering from an autoimmune disease, ANCA-associated vasculitis, SLE, rheumatoid arthritis, antiphospholipid syndrome and/or cryoglobulinemic vasculitis.

6. The method of any preceding claim, wherein the cells are any cells capable of undergoing or affecting the process of ETosis.

7. The method of any preceding claim, wherein the cells are cells of the innate and/or acquired immune system.

8. The method of any preceding claim, wherein the cells comprise white blood cells and/or cells selected from the group consisting of neutrophils; a mast cells; basophils; and eosinophils.

9. The method of any preceding claim, wherein the cell membrane stain is a PKH fluorescent cell dye.

10. The method of any preceding claim, wherein the cell membrane stain is PKH26.

11. The method of any preceding claim, wherein the nucleic acid stain is a live-cell impermeant fluorescent nucleic acid stain.

12. The method of any preceding claims, wherein the nucleic acid stain is Sytox^® Green nucleic acid stain.

13. The method of any preceding claim, wherein determining the ratio of cells positively labelled for nucleic acid with the number of labelled cells comprises capturing a plurality of high power field (HPF) images, each HPF image comprising between 2-50 stacked images.

14. The method of claim 13, wherein each image to be stacked is separated from another image by a pre-determined z-distance.

15. The method of claim 13, wherein the z-distance is between about 0.5μιη and about 20pm.

16. A method of diagnosing an autoimmune disease or a predisposition or susceptibility thereto, said method comprising executing a method according to any one of claims 1-14, wherein the step of inducing or promoting ETosis exploits an immunoglobulin sample obtained from or provided by a subject to be tested.

17. The method of claim 16, wherein the subject to be tested is a subject suspected of suffering from an autoimmune disease or a subject suspected of being predisposed or susceptible to an autoimmune disease.

18. The method of claims 16 or 17, wherein the autoimmune disease is one or more selected from the group consisting of ANCA-associated vasculitis, SLE, rheumatoid arthritis, antiphospholipid syndrome and cryoglobulinemic vasculitis.