How to Take Out a Virus: Spotlight on NETosis
As COVID-19 lingers on, many are searching for new therapeutic strategies to “take out” this new virus. One approach is to manipulate one of the immune system’s cell death mechanisms for removing infected cells: NETosis. We talked with both Dr. Marta Rodriguez-Garcia (formerly at Dartmouth, now at Tufts) and Dr. Jos Raats (ModiQuest) about using a live-cell imaging technique to study the role of NETosis in HIV and COVID-19, respectively.
This article is posted on our Science Snippets Blog
Quantifying NETosis
In NETosis, neutrophils corner and destroy the invading pathogen by forming neutrophil extracellular traps (NETs). These tangled, extracellular, web-like formations contain DNA from neutrophils, nuclear chromatin, proteases and other antimicrobial molecules. NETs are thought to provide a physical barrier, trapping pathogens in their spider-like webs to prevent further infection. This also exposes them to more concentrated antimicrobial proteins near the site of infection.
Quantifying NETosis in real time can provide important insights on how this response can be an effective, first-line defense against viral pathogens. However, studying NETosis can be challenging due to its similarities with other cell death mechanisms, like apoptosis and necrosis, and technically laborious by standard fluorescence microscopy methods. The assays are also typically performed as endpoint assays, thereby missing important kinetic information.
Interview with Dr. Marta Rodriguez-Garcia: NETosis and HIV
Dr. Rodriguez-Garcia and colleagues used a high-throughput live-cell imaging technique developed by another group to study the impact of NET formation by neutrophils at mucosal sites during HIV infection. This assay, which was developed on the Incucyte® System, not only quantified NETosis in real time, but also distinguished it from other cell death mechanisms through signature kinetic patterns that arose with known NET-inducing stimuli.1 The assay was faster and more sensitive compared to fluorescence microscopy, and generated unbiased, reproducible kinetic data.
Incucyte® live-cell imaging and analysis was used to capture the in vitro dynamics of GFP-tagged HIV-VLP (HIV viral-like particles) entrapped by human genital neutrophils, which had been cultured in cell impermeant DNA-dye (red). When the neutrophils released DNA as part of the NET, this was visualized as a red signal, while HIV-VLP had a green signal. HIV-VLP entrapment by the NETs was visualized as co-localization of red (NET) and green (HIV-VLP) signals. They found that NETs were released within minutes of viral exposure.2
Sartorius: How did live-cell imaging with Incucyte® help you capture and understand the unique biology of human genital neutrophils?
Dr. Rodriguez-Garcia: Using the Incucyte® platform allowed for the characterization of the very early events of NET formation following viral exposure. Given that the number of neutrophils isolated from human genital tissues can be limiting, the ability to perform time-lapse imaging in a 96-well format was key to understanding the dynamics of NET formation following HIV exposure, and also, for the simultaneous comparison of different genital tissues and treatments to understand the underlying mechanisms of NET formation in the genital tract.
Sartorius: This study highlights the different responses of neutrophil populations within the body. Is it possible that other mucosal areas might have similar NET alterations?
Dr. Rodriguez-Garcia: It is very possible. There is still much to learn about neutrophils in mucosal surfaces and how the tissue environment modifies their functions.
Neutrophils isolated from the female genital tract were cultured in media with impermeant DNA dye (red) and stimulated with GFP-labeled HIV viral-like particles (green) to induce NET formation. Left panel shows the GFP-labeled viral-like particles entrapped in NETs. Right panel shows the merged image of phase contrast and fluorescent signals, with HIV trapped in the NETs appearing as green/yellow. Image is courtesy of Dr. Marta Rodriguez-Garcia, Tufts University School of Medicine.
Interview with Dr. Jos Raats: NETosis and COVID-19
As with any immune reaction, excessive NETosis can have undesirable and even serious consequences. Most recently, Zuo et al. showed that cell-free DNA, myeloperoxidase DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3) were present in serum samples of COVID-19 patients.3 MP0-DNA and Cit-H3 are markers for NET formation, and cell-free DNA correlated clinically with other markers of acute phase disease.
Drs. Chirivi, Raats, and colleagues recently reported that NETs are released into the extracellular environment during inflammation.4 They tested the efficacy of engineered therapeutic anti-citrullinated protein antibodies (tACPA) on NET disease pathology in mouse inflammatory disease models of arthritis, pulmonary fibrosis, colitis, and sepsis. They used Incucyte® live-cell analysis to capture images of NET release from neutrophils in the presence of an engineered therapeutic anti-citrullinated protein antibody (tACPA), which bound to citrulline at position3 (Cit3) in histone 2A (citH2A) and 4 (citH4). The antibody showed both therapeutic and prophylactic potential for inflammatory disease associated with NET pathology.
Sartorius: How did the use of live-cell imaging add to your understanding of the kinetic effects of the tACPA antibody on NET binding and release?
Dr. Raats: A good example of this was provided by the study towards the role of Fab2 and full-size antibodies in NET inhibition. Both s and Full-size antibody inhibited NET formation. The different staining capacities of the live imaging allowed real-time visualization of the NET formation and the subsequent inhibition thereof. This made clear on what point in the NET formation tACPA antibodies bind to the neutrophil and inhibit the NET formation.
Sartorius: Could this type of antibody have a possible application for infectious diseases such as viral infections?
Dr. Raats: This antibody could potentially have utility in the treatment of secondary consequences of virus infections. The antibody interferes with NET formation and blocks the effect and promotes clearance of toxic histones that cause tissue damage. Therefore, it may be useful to inhibit or prevent the tissue damaging and thrombus inducing effects of NETs induced by viral infections. However, this is currently only a hypothesis and further testing by Citryll B.V. may shed further light on this.
Tools for COVID-19 Research and Beyond
Understanding and exploiting the immune system’s “hit-man” can help expand our toolkit for combatting diverse diseases, including our latest viral nemesis. Live-cell imaging and analysis equips scientists with real-time visual and kinetic information about key events, like NETosis, that can inform therapeutic targeting and management. In addition to instrument technologies and virology assays, Sartorius supports COVID-19 research with cell and protein analysis tools for vaccine research, equipment and consumables for diagnostic testing and solutions for environmental monitoring, including air and wastewater.
COVID-19 won’t be our last pandemic. Are you ready to take out the next virus?
References:
- Gupta S, Chan DW, Zaal KJ, Kaplan MJ. A High-Throughput Real-Time Imaging Technique To Quantify NETosis and Distinguish Mechanisms of Cell Death in Human Neutrophils. J Immunol. 2018;200(2):869-879. doi:10.4049/jimmunol.1700905
- Barr FD, Ochsenbauer C, Wira CR, Rodriguez-Garcia M. Neutrophil extracellular traps prevent HIV infection in the female genital tract. Mucosal Immunol. 2018;11(5):1420-1428. doi:10.1038/s41385-018-0045-0
- Zuo Y, Yalavarthi S, Shi H, et al. Neutrophil extracellular traps in COVID-19. JCI Insight. 2020;5(11):138999. Published 2020 Jun 4. doi:10.1172/jci.insight.138999
- Chirivi, R.G.S., van Rosmalen, J.W.G., van der Linden, M. et al. Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases. Cell Mol Immunol (2020). doi.org/10.1038/s41423-020-0381-3