Name: supervised machine learning for semi-quantification of extracellular dna in glomerulonephritis
Uploaded: 2020-06-18T14:00:00
Description: Watch this Scientific Journal Video about Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis at JoVE.com

We acknowledge Monash Micro Imaging for the use of Nikon C1 upright confocal laser scanning microscope and the Monash Histology Platform for the processing of kidney tissue.

This method enables detection of pan ecDNA from all forms of cell death. The same method and antibodies are used for human kidney biopsy tissue (from step 4). All animal and human subjects had Ethics approval from Monash University, and Monash Health, Clayton, Victoria, Australia.
1. Staining for ecDNA with DAPI and &#946;-Actin
<ol>
	<li>Induce a 20 day model of anti-myeloperoxidase glomerulonephritis (anti-MPO-GN) in 8-10 week old C57/Bl6 mice, with controls9.
	<ol>...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-neutrophil+cytoplasmic+antibodies+and+effector+CD4++cells+play+nonredundant+roles+in+anti-myeloperoxidase+crescentic+glomerulonephritis.">Anti-neutrophil cytoplasmic antibodies and effector CD4+ cells play nonredundant roles in anti-myeloperoxidase crescentic glomerulonephritis.</a> Journal of the American Society of Nephrology. 17 (7), 1940-1949 (2006).</li><li>Nagler, M., Insam, H., Pietramellara, G., Ascher-Jenull, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+DNA+in+natural+environments:+features,+relevance+and+applications.">Extracellular DNA in natural environments: features, relevance and applications.</a> Applied Microbiology and Biotechnology. 102 (15), 6343-6356 (2018).</li><li>Burnham, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+cell-free+DNA+is+a+versatile+analyte+for+monitoring+infections+of+the+urinary+tract.">Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract.</a> Nature Communications. 9 (1), 2412 (2018).</li><li>Gobe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+apoptosis+in+kidney+tissue+sections.">Identification of apoptosis in kidney tissue sections.</a> Methods in Molecular Biology. 466, 175-192 (2009).</li><li>Olander, M., Handin, N., Artursson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-Based+Quantification+of+Cell+Debris+as+a+Measure+of+Apoptosis.">Image-Based Quantification of Cell Debris as a Measure of Apoptosis.</a> Analytical Chemistry. 91 (9), 5548-5552 (2019).</li><li>Arganda-Carreras, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trainable+Weka+Segmentation:+a+machine+learning+tool+for+microscopy+pixel+classification.">Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification.</a> Bioinformatics. 33 (15), 2424-2426 (2017).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Apel, F., Zychlinsky, A., Kenny, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neutrophil+extracellular+traps+in+rheumatic+diseases.">The role of neutrophil extracellular traps in rheumatic diseases.</a> Nature Reviews Rheumatology. 14 (8), 467-475 (2018).</li><li>Chapman, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caught+in+a+Trap?+Proteomic+Analysis+of+Neutrophil+Extracellular+Traps+in+Rheumatoid+Arthritis+and+Systemic+Lupus+Erythematosus.">Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus.</a> Frontiers in Immunology. 10, 423 (2019).</li><li>Oliveira, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sudan+Black+B+treatment+reduces+autofluorescence+and+improves+resolution+of+in+situ+hybridization+specific+fluorescent+signals+of+brain+sections.">Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections.</a> Histology &amp; Histopathology. 25 (8), 1017-1024 (2010).</li><li>Kessenbrock, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netting+neutrophils+in+autoimmune+small-vessel+vasculitis.">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nature Medicine. 15 (6), 623-625 (2009).</li><li>Schreiber, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+controls+NET+generation+and+mediates+complement+activation,+endothelial+damage,+and+autoimmune+vasculitis.">Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (45), E9618-E9625 (2017).</li><li>Antonelou, M., Perea Ortega, L., Harvey, J., Salama, A. 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Multiple protocols exist that measure proinflammatory markers in the serum and urine of both patients and mouse models of glomerulonephritis. This described protocol allows analysis of the products of cell death (ecDNA, NETs and ecMPO) within the glomerulus directly. The most crucial steps in this protocol is the tissue preparation and imaging. The major restricting element of using a fluorescent staining method for analysis is the tissue autofluorescence. Formalin fixed paraffin tissue is subject to autofluorescence tha...

Myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV) is an autoimmune disease that results in renal failure from pathological glomerular injury with considerable cell death and release of deoxyribonucleic acid (DNA)1,2. DNA can activate the immune system by acting as a danger signal. Under normal healthy conditions, the nuclear location of DNA offers protection from exposure to the immune system. Self-DNA that is released extracellularly during either pathogenic processes or autoimmunity is seen by the immune system as a potent proinflammatory damage associated molecular self-protein (DAMP)3. Extra cellular DNA (ecDNA) is released from dying cells through several distinct mechanisms that are governed by distinct biochemical pathways, such as apoptosis, necroptosis neutrophil extracellular trap formation (NETs), necrosis or pyroptosis4,5,6,7,8.
We describe herein methods to stain and measure ecDNA released from dying cells in sections of formalin fixed paraffin embedded (FFPE) kidneys from experimental anti-MPO GN and kidney biopsies from patients with MPO-AAV9,10. Multiple methods exist for the detection of circulating double stranded DNA (dsDNA) and DNA complexes from both serum and urine and from in vitro assays11,12. These methods, although accurate in determining the amount of ecDNA, do not determine where the ecDNA is released anatomically. There are methods that describe specific measurement of ecDNA such as tunel for apoptosis and measurement of cell debris13,14. There is no method that describes measuring ecDNA culminated from all forms of cell death in FFPE kidneys where the pathological damage occurs. This is important to determine if experimental therapeutic treatments are clearing the ecDNA from the sites of pathological injury in the actual target organ.
The acquisition of multiple images from kidney samples creates a high volume of data that is analyzed commonly by one single user. This is labor intensive, time consuming and can be subject to unreliable reproducibility by other users, due to user bias. Trainable Weka segmentation is an open-source software plugin for ImageJ that uses cutting edge bioinformatic tools to classify pixels using machine learning algorithms15,16. This method is &#34;trainable&#34; whereby it learns from the user&#39;s classification of segments of pixels and applies the new learnt classification to other images. This method relies on common analysis tools within the ImageJ program that are used to &#34;classify&#34; each pixel in a segment as belonging to a specific &#34;class&#34;. Once the program learns the &#34;classifiers&#34;, they can be used to identify other similar classified segments within the same image. This model is then saved and applied to other sets of images within the same experiment.
Current obstacles to determining ecDNA in situ in kidney sections is the endogenous autofluorescence from fixation in formalin and the labor-intensive analysis of the images. We describe here how to quench this autofluorescence, detect ecDNA, and use supervised machine learning for high throughput measurement of ecDNA. We have previously published the measurement of NETs and extracellular MPO (ecMPO) using a macro in ImageJ, we now demonstrate semi automation of these methods using supervised machine learning1. We demonstrate the adaptability of the machine learning tool, to classify an alternative stain for NETs and ecMPO within the same image. These staining methods described here for detecting ecDNA, NETs and ecMPO can be translated to other solid organs and diseases where ecDNA, NETS and ecMPO plays a role in perpetuating disease such as rheumatoid arthritis and lupus17,18.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A2153</td>
			<td>5% and 1% solutions are made up in PBS, can be made in bulk and frozen- discard once thawed.</td>
		</tr>
		<tr>
			<td>Chicken anti Goat IgG (H+L) cross absorbed antibody Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21468</td>
			<td>Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail</td>
		</tr>
		<tr>
			<td>Chicken anti mouse IgG (H+L) cross absorbed antibody, Alex Fluor 647</td>
			<td>ThermoFisher Scientific</td>
			<td>A-121468</td>
			<td>Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail</td>
		</tr>
		<tr>
			<td>Chicken anti rabbit IgG (H+L) Cross absorbed antibody Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21441</td>
			<td>Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail</td>
		</tr>
		<tr>
			<td>Chicken sera</td>
			<td>SIGMA</td>
			<td>C5405</td>
			<td>Made up in 1%BSA/PBS</td>
		</tr>
		<tr>
			<td>Coverslips 24 x60 mm</td>
			<td>Azerscientific</td>
			<td>ES0107222</td>
			<td>#1.5 This is not standard thickness- designed for use in confocal microscopy</td>
		</tr>
		<tr>
			<td>EDTA 10mM</td>
			<td>SIGMA</td>
			<td>E6758</td>
			<td>Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x Solution</td>
		</tr>
		<tr>
			<td>Ethanol 30%, 70% and 100%</td>
			<td>Chem Supply</td>
			<td>UN1170</td>
			<td>Supplied as 100% undenatured ethanol- dilute to 30% and 70% using distilled water</td>
		</tr>
		<tr>
			<td>Formaldehyde, 4% (10% Neutral buffered Formalin)</td>
			<td>TRAJAN</td>
			<td>NBF-500</td>
			<td>Kidney is put into a 5ml tube containing 3ml of formalin for 16 hours at RT, formalin should be used in a fume hood</td>
		</tr>
		<tr>
			<td>Glass histology slides- Ultra Super Frost, Menzel Glaze, 25x75 x1.0mm</td>
			<td>TRAJAN</td>
			<td>J3800AM4</td>
			<td>Using positive charged coated slides is essential. We do not recommend using poly-L-lysine for coating slides as tissue dislodges from slides during the antigen retrieval step</td>
		</tr>
		<tr>
			<td>Goat anti human/mouse MPO antibody</td>
			<td>R&#38;D</td>
			<td>AF3667</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Histosol</td>
			<td>Clini Pure</td>
			<td>CPL HISTOSOL 08</td>
			<td>Used neat, in 200ml staining rack containers, use in a fume hood</td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>VECTOR Labs</td>
			<td>H-400</td>
			<td>Use to draw circle around kidney tissue</td>
		</tr>
		<tr>
			<td>Mouse anti human/mouse Peptidyl arginase 4 (PAD4)</td>
			<td>ABCAM</td>
			<td>ab128086</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Nikon C1 confocal scanning laser head attached to Nikon Ti-E inverted Microscope</td>
			<td>Coherent Scientific</td>
			<td></td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>SIGMA</td>
			<td>P38135</td>
			<td>0.01M PB/0.09% NaCl Make up 5L at a time</td>
		</tr>
		<tr>
			<td>Pressure Cooker 6L Tefal secure 5 Neo stainless</td>
			<td>Tefal</td>
			<td>GSA-P2530738</td>
			<td>Purchased at local homeware store</td>
		</tr>
		<tr>
			<td>Prolong Gold DAPI</td>
			<td>Life Technologies</td>
			<td>P36962</td>
			<td>Apply drops directly to coverslip</td>
		</tr>
		<tr>
			<td>Rabbit anti human/mouse Beta Actin antibody</td>
			<td>ABCAM</td>
			<td>ab8227</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Rabbit anti human/mouse H3Cit antibody</td>
			<td>ABCAM</td>
			<td>ab5103</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Staining rack 24 slides</td>
			<td>ProScitech</td>
			<td>H4465</td>
			<td>Staining rack chosen has to be able to withstand boiling under pressure and incubation in 60 degree oven</td>
		</tr>
		<tr>
			<td>Sudan Black B</td>
			<td>SIGMA</td>
			<td>199664</td>
			<td>0.3% Add 3g to a 1L bottle in 70% Ethanol, filter and protect from the light- stable for 6 months at room temperature</td>
		</tr>
		<tr>
			<td>Tris 10mM</td>
			<td>SIGMA</td>
			<td>T4661</td>
			<td>Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x solution</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Trajan</td>
			<td>XL005/20</td>
			<td>Must be use used in a fume hood</td>
		</tr>
	</tbody>
</table>

supervised machine learning for semi-quantification of extracellular dna in glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

These images represent the multiple steps required to successfully use trainable Weka segmentation to minimize the labor-intensive manual measurement of ecDNA in fluorescently stained FFPE kidney tissue from a mouse with induced anti-MPO GN. These steps are summarized in Figure 1 and Figure 2 with images taken directly from the Weka segmentation program, outlining every step in the analysis process. Measurements from this analysi...

Watch this Scientific Journal Video about Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis at JoVE.com

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

Glomerulonephritis causes pathological glomerular injury with considerable cell death and extracellular DNA release. This protocol enables the visualization of extracellular DNA at the site of release during pathological injury. The advantage of this technique is that it measures extracellular DNA from cell death in the kidney.As opposed to serum or urine, extracellular DNA measurement is a surrogate marker for pathological damage. The significance of this method is that both the extracellular DNA staining and image analysis are easily transferrable to other autoimmune diseases such as rheumatoid arthritis and lupus. This method requires a basic knowledge of kidney histology.To enable the accurate detection of glomerular extracellular DNA, the researcher must be able to correctly identify glomeruli. For extracellular DNA staining, first heat the samples of interest in a slide rack in 60 degrees Celsius for 60 minutes before immersing the slides in two 40-minute changes of solvent in a fume hood. After the second immersion, rehydrate the samples two times in 100%ethanol and one time in 70%ethanol for five minutes per treatment.After the last treatment, use tongs to place the slides into boiling antigen retrieval solution in a pressure cooker on high for 10 minutes. After unmasking the antigen epitopes, transfer the pressure cooker from heat into a sink and immediately run cold tap water over the lid. Allow the slides to equilibrate for 20 minutes in the antigen retrieval solution before washing the samples two times in 0.01 molar PBS on an orbital shaker for five minutes per wash.After the second wash, use a hydrophobic pen to draw circles around the kidney tissue samples and block any nonspecific staining with 60 microliters of 10%chicken serum in 5%bovine serum albumin for 30 minutes at room temperature. At the end of the incubation, carefully replace the blocking solution with 60 microliters of the primary antibody of interest in a humidity chamber overnight at four degrees Celsius. The next morning, wash the slides on an orbital shaker in PBS for two minutes per wash before adding 60 microliters of an appropriate fluorophore conjugated secondary antibody to each slide for a 40-minute incubation at room temperature.At the end of the incubation, wash the slides in PBS as demonstrated, followed by treatment in 0.3%Sudan black in 70%ethanol to quench any potential formalin-induced autofluorescence. After 30 minutes, wash the slides in tap water to remove any precipitate and immerse the slides in PBS for 10 minutes to prevent any further Sudan black precipitate formation. At the end of the incubation, mount the slides onto confocal glass coverslips with three 60 microliter drops of mounting solution supplemented with DAPI and seal the coverslips with nail polish.Allow the slides to cure for 24 hours at room temperature. Store them at four degrees Celsius protected from light until imaging by confocal laser scanning microscopy according to standard protocols. Be sure to seal the coverslips with nail polish to prevent any movement during imaging and to clean the coverslips of ethanol to enhance the imaging resolution.After imaging 20 glomeruli per slide, open the trainable Weka Segmentation plugin in ImageJ and drop the first image onto the ImageJ toolbar. Click image, color, and split channels. An image showing the primary antibody staining will appear.To create a merged file of the single images, click color and merge channels. A popup box will appear asking for a color to be assigned to each channel. Check the create composite and keep source images boxes and click OK.A merged composite image will appear.Using the primary antibody staining as a guide, draw a region of interest around the glomerular tuft. To perform a Gaussian blur on the single blue DAPI image, click process, filters, and Gaussian blur. Set the blur at one to two.The image will become a little blurry, but the nuclei will appear smooth, the background will be softened. Next, click plugins, segmentation, and trainable Weka Segmentation, and use the free hand tool to trace the intact nuclei. When all of the nuclei of interest have been added, the add class one box, use the line tool to delineate the areas of background to the class two box.Click create new class and use the line tool to outline the DAPI-stained extranuclear DNA. Click train classifier and get probability. Toggle the mouse to obtain a black and white image of all the components with the object of selection highlighted in white.To duplicate this image, click image and duplicate. Under image, adjust and threshold. Use the mouse button to adjust the threshold until only the extracellular DNA is observed.After thresholding, click process, binary, and make binary, and copy the previously generated glomerular region of interest. Press Control Shift E to select edit, selections, and restore to restore the selection. To measure only the extracellular DNA particles within the glomerulus, click analyze particles and OK.A result sheet showing the number of particles, area, average pixels, and percentage of glomeruli containing extracellular DNA will be generated.To save the classifier as an extracellular DNA classifier, click save classifier and save the data. To reapply the model to subsequent images, repeat the initial image processing as demonstrated before selecting load classifier and the saved classifier file. The log menu will pop up and run the model.Once the model has run, click load data and select the extracellularDNA.arff. Then click create result. If the resulting image has failed to pick up all the nuclei, background, or extracellular DNA, click retrain classifier and add more classifiers as necessary.Here, single channel images showing representative DNA and beta actin staining are shown. Merging the two images allows a region of interest to be drawn around the glomerular area for measurement of the extracellular DNA within the mouse kidney glomeruli using trainable Weka Segmentation as demonstrated. This model can be adopted to identify extracellular DNA in kidney biopsy specimens from a control patient or comparison to that of a kidney biopsy from a patient with myeloperoxidase ANCA-associated vasculitis.In addition, this program can be translated to other stains within kidney tissue samples, for example, to stain for neutrophil extracellular traps and the deposition of extracellular myeloperoxidase in ANCA-associated vasculitis. Importantly, no significant differences are observed when the same data is analyzed by multiple users. The most critical step of the analysis is providing enough examples of background, nuclei, and extracellular DNA with glomeruli as classifiers for the Weka Segmentation program to learn from.This method is easily transferrable. For example, further analysis of the different types of cell death can be investigated through staining for specific antibodies against markers for apoptosis or necrosis. Our technique can be used with other inflammatory processes such as assessing TLR expression in autoimmune kidney disease or extracellular myeloperoxidase expression in human kidney biopsies.

supervised-machine-learning-for-semi-quantification-extracellular-dna

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...