Name: in vitro stimulation and visualization of extracellular trap release in differentiated human monocyte-derived macrophages
Uploaded: 2019-11-01T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages at JoVE.com

This work was supported by a Perpetual IMPACT Grant (IPAP201601422) and Novo Nordisk Foundation Biomedical Project Grant (NNF17OC0028990). YZ also acknowledges the receipt of an Australian Postgraduate Award from the University of Sydney. We would like to thank Mr. Pat Pisansarakit and Ms. Morgan Jones for assistance with the monocyte isolation and tissue culture.

The HMDM were isolated from human buffy coat preparations supplied by the blood bank with ethics approval from the Sydney Local Health District.
1. HMDM Culture
<ol>
	<li>Isolate the monocytes from buffy coat preparations prepared from the peripheral blood of healthy human donors using a commercially available preparation to isolate lymphocytes, followed by countercurrent centrifugal elutriation19,20.</li>
	<li>Confirm the presence o...

<ol>
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S., 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=Cytokines+induced+neutrophil+extracellular+traps+formation:+implication+for+the+inflammatory+disease+condition.">Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition.</a> PLoS One. 7 (10), 48111 (2012).</li><li>Rayner, B. 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S., 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=Peptidylarginine+deiminase+inhibition+reduces+vascular+damage+and+modulates+innate+immune+responses+in+murine+models+of+atherosclerosis.">Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis.</a> Circulation Research. 114 (6), 947-956 (2014).</li><li>Megens, R. T., 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=Presence+of+luminal+neutrophil+extracellular+traps+in+atherosclerosis.">Presence of luminal neutrophil extracellular traps in atherosclerosis.</a> Thrombosis and Haemostasis. 107 (3), 597-598 (2012).</li><li>Doring, Y., Weber, C., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Footprints+of+neutrophil+extracellular+traps+as+predictors+of+cardiovascular+risk.">Footprints of neutrophil extracellular traps as predictors of cardiovascular risk.</a> Arteriosclerosis Thrombosis and Vascular Biology. 33 (8), 1735-1736 (2013).</li><li>Goldmann, O., Medina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+world+of+extracellular+traps:+not+only+neutrophils+but+much+more.">The expanding world of extracellular traps: not only neutrophils but much more.</a> Frontiers in Immunology. 3 (420), 1-10 (2012).</li><li>Boe, D. M., Curtis, B. J., Chen, M. M., Ippolito, J. A., Kovacs, E. 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+traps+and+macrophages:+new+roles+for+the+versatile+phagocyte.">Extracellular traps and macrophages: new roles for the versatile phagocyte.</a> Journal of Leukocyte Biology. 97 (6), 1023-1035 (2015).</li><li>Pertiwi, K. R., 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=Extracellular+traps+derived+from+macrophages,+mast+cells,+eosinophils+and+neutrophils+are+generated+in+a+time-dependent+manner+during+atherothrombosis.">Extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time-dependent manner during atherothrombosis.</a> Journal of Pathology. 247 (4), 505-512 (2019).</li><li>Okubo, 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=Macrophage+extracellular+trap+formation+promoted+by+platelet+activation+is+a+key+mediator+of+rhabdomyolysis-induced+acute+kidney+injury.">Macrophage extracellular trap formation promoted by platelet activation is a key mediator of rhabdomyolysis-induced acute kidney injury.</a> Nature Medicine. 24 (2), 232-238 (2018).</li><li>Boeltz, S., 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=To+NET+or+not+to+NET:current+opinions+and+state+of+the+science+regarding+the+formation+of+neutrophil+extracellular+traps.">To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.</a> Cell Death and Differentiation. 26 (3), 395-408 (2019).</li><li>Doster, R. S., Rogers, L. M., Gaddy, J. A., Aronoff, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Extracellular+Traps:+A+Scoping+Review.">Macrophage Extracellular Traps: A Scoping Review.</a> Journal of Innate Immunology. 10 (1), 3-13 (2018).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K., Dockrell, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+markers+of+macrophage+differentiation+in+PMA-stimulated+THP-1+cells+and+monocyte-derived+macrophages.">The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.</a> PLoS One. 5 (1), 8668 (2010).</li><li>Mestas, J., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> Journal of Immunology. 172 (5), 2731-2738 (2004).</li><li>Brown, B. E., Rashid, I., van Reyk, D. M., Davies, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+of+low-density+lipoprotein+results+in+the+time-dependent+accumulation+of+cholesteryl+esters+and+apolipoprotein+B-100+protein+in+primary+human+monocyte-derived+macrophages.">Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophages.</a> FEBS Journal. 274, 1530-1541 (2007).</li><li>Garner, B., Dean, R. T., Jessup, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+macrophage-mediated+oxidation+of+low-density+lipoprotein+is+delayed+and+independant+of+superoxide+production.">Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independant of superoxide production.</a> Biochemical Journal. 301, 421-428 (1994).</li><li>Morris, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acid+ionization+constant+of+HOCl+from+5+&#176;C+to+35+&#176;C.">The acid ionization constant of HOCl from 5 &#176;C to 35 &#176;C.</a> Journal of Phyical Chemistry. 70, 3798-3805 (1966).</li><li>Pan, G. J., Rayner, B. S., Zhang, Y., van Reyk, D. M., Hawkins, C. 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The generation and visualization of MET formation using M1 differentiated HMDMs represents a new in vitro model that may be useful for investigating the potential pathological role of these macrophage structures, particularly under chronic inflammatory conditions. It provides a robust protocol for the stimulation of primary human macrophages to release METs, which can also be utilized in related studies with human monocyte or murine macrophage cell lines. The successful implementation of this protocol for the stimulation...

The release of ETs from neutrophils was first identified as an innate immune response triggered by bacterial infection1. They consist of a DNA backbone to which various granule proteins with anti-bacterial properties are bound, including neutrophil elastase and myeloperoxidase2. The primary role of neutrophil ETs (NETs) is to capture pathogens and facilitate their elimination3. However, in addition to the protective role of ETs in immune defense, an increasing number of studies have also discovered a role in disease pathogenesis, particularly during the development of inflammation-driven diseases (i.e., rheumatoid arthritis and atherosclerosis4). The release of ETs can be triggered by various pro-inflammatory cytokines including interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF&#945;)5,6, and the localized accumulation of ETs can increase tissue damage and evoke a pro-inflammatory response7. For example, ETs have been implicated as playing a causal role in the development of atherosclerosis8, promoting thrombosis9, and predicting cardiovascular risk10.
It is now recognized that in addition to neutrophils, other immune cells (i.e., mast cells, eosinophils, and macrophages) can also release ETs on exposure to the microbial or pro- inflammatory stimulation11,12. This may be particularly significant in the case of macrophages, considering their key role in the development, regulation, and resolution of chronic inflammatory diseases. Therefore, it is important to gain a greater understanding of the potential relationship between ET release from macrophages and inflammation-related disease development. Recent studies have shown the presence of METs and NETs in intact human atherosclerotic plaques and organized thrombi13. Similarly, METs have been implicated in driving kidney injury through the regulation of inflammatory responses14. However, in contrast to neutrophils, there are limited data on the mechanisms of MET formation from macrophages. Recent studies using human in vitro models of MET formation show some differences in the pathways involved in each cell type (i.e., regarding the absence of histone citrullination with macrophages)6. However, some have shown that NET release can also occur in the absence of histone citrullination15.
The overall goal of this protocol is to provide a simple and direct method to assess MET release in a clinically relevant macrophage model. There are a number of different in vitro macrophage cell models that have been used to study METs (i.e., the THP-1 human monocyte cell line and various murine macrophage cell lines)16. There are some limitations associated with these models. For example, the differentiation of THP-1 monocytes to macrophages usually requires a priming step, such as the addition of phorbol myristate acetate (PMA), which itself activates protein kinase C (PKC)-dependent pathways. This process is known to trigger ET release4 and results in a low basal MET release from THP-1 cells. Other studies have highlighted some differences in bioactivity and inflammatory responses mounted by macrophages in vivo compared to PMA-treated THP-1 cells17.
Similarly, the behavior and inflammatory responses of different murine macrophage-like cell lines do not completely represent the response spectrum of primary human macrophages18. Therefore, for the purpose of investigating macrophage ET formation in the clinical setting, primary human monocyte-derived macrophages (HMDMs) are believed to be a more relevant model rather than monocytic or murine macrophage-like cell lines.
ET release from M1 polarized HMDMs has been demonstrated following exposure of these cells to a number of different inflammatory stimuli, including the myeloperoxidase-derived oxidant hypochlorous acid (HOCl), PMA, TNF&#945;, and IL-86. Described here is a protocol to polarize HMDMs to the M1 phenotype and visualize subsequent MET release upon exposure to these inflammatory stimuli. PMA is used as a stimulus of MET release to facilitate comparisons to previous studies that have used neutrophils. Importantly, HOCl, IL-8, and TNF&#945; are also used to stimulate MET release, which are believed to be better models of the inflammatory environment in vivo. The microscopic method for visualization of ET release involves staining the extracellular DNA in live cell cultures using SYTOX green, an impermeable fluorescent green nucleic acid stain that has been successfully applied in previous neutrophil studies. This method allows for rapid and qualitative assessment of ET release, but it is not appropriate as a stand-alone method for the quantification of ET release extent. Alternative methodology should be used if quantification is required to compare the extent of ET release resulting from different treatment conditions or interventions.

<table>
	<tbody>
		<tr>
			<td>120Q broad spectrum fluorescent light source</td>
			<td>EXFO Photonic Solutions, Toronto, Canada</td>
			<td></td>
			<td>x-cite series</td>
		</tr>
		<tr>
			<td>Corning CellBIND Multiple Well Plate (12 wells)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3336</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Differential Quik Stain Kit (Modified Giemsa)</td>
			<td>Polysciences Inc.</td>
			<td>24606</td>
			<td>Characterisation of monocytes</td>
		</tr>
		<tr>
			<td>Hanks balanced salt solution (HBSS)</td>
			<td>Thermo-Fisher</td>
			<td>14025050</td>
			<td>For washing steps and HOCl treatment</td>
		</tr>
		<tr>
			<td>Hypochlorous acid (HOCl)</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>Interferon gamma</td>
			<td>Thermo-Fisher</td>
			<td>PMC4031</td>
			<td>For M1 priming</td>
		</tr>
		<tr>
			<td>Interleukin 4</td>
			<td>Integrated Sciences</td>
			<td>rhil-4</td>
			<td>For M2 priming</td>
		</tr>
		<tr>
			<td>Interleukin 8</td>
			<td>Miltenyl Biotec</td>
			<td>130-093-943</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>59202C</td>
			<td>Added to culture media</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide</td>
			<td>Integrated Sciences</td>
			<td>tlrl-eblps</td>
			<td>For M1 priming</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Axis-Shield PoC AS</td>
			<td>1114544</td>
			<td>For isolation of monocytes</td>
		</tr>
		<tr>
			<td>Olympus IX71 inverted microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12- myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td>For MET stimulation</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>For washing steps</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>SYTOX green</td>
			<td>Life Technologies</td>
			<td>S7020</td>
			<td>For MET visulaization</td>
		</tr>
		<tr>
			<td>TH4-200 brightfield light source</td>
			<td>Olympus, Tokyo, Japan</td>
			<td></td>
			<td>x-cite series</td>
		</tr>
		<tr>
			<td>Tumor necrosis factor alpha</td>
			<td>Lonza</td>
			<td>300-01A-50</td>
			<td>For MET stimulation</td>
		</tr>
	</tbody>
</table>

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).

Brightfield images showing the morphological changes of HMDM in response to stimuli for cell differentiation are shown in Figure 1. M1 polarized macrophages from experiments with HMDM exposed to IFN&#947; and LPS showed an elongated and spindle-like cell shape, as indicated by the black arrows in Figure 1 (middle panel). For comparison, the morphology of the M2 polarized macrophages after exposure of HMDM to IL-4 for 48 h were typically round and flat, as indica...

Watch this Scientific Journal Video about In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages at JoVE.com

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

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 ...

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