A Novel Real-Time Imaging Technique to Quantify Neutrophil Extracellular Traps and Distinguish Mechanisms of Cell Death in Neutrophils

Sarthak Gupta¹, Diana Chan², Kristien Zaal³, Evelyn Ralston⁴ and Mariana J. Kaplan², ¹Systemic Autoimmunity Branch, National Institue of Arthritis, and Musculoskelatal and Skin Diseases/NIH, Bethesda, MD, ²Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases/NIH, Bethesda, MD, ³Light Imaging Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, ⁴Light Imaging Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases/NIH, Bethesda, MD

Meeting: 2017 ACR/ARHP Annual Meeting

Date of first publication: September 18, 2017

Keywords: NETosis and Neutrophil Extracellular Traps

Session Information

Date: Sunday, November 5, 2017

Title: Innate Immunity and Rheumatic Disease Poster I

Session Type: ACR Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose: Neutrophils play a key role in host defenses and have recently been implicated in the pathogenesis of autoimmune diseases by several mechanisms including formation of neutrophil extracellular traps (NETs) through a distinct form of programmed cell death called NETosis. Techniques to assess and quantitate NETosis in vitro in an unbiased, reproducible and efficient way are lacking, considerably limiting the advancement of this field. We optimized and validated a new method to automatically quantify the percentage of neutrophils undergoing NETosis in real time using IncuCyte ZOOM™, a two-color, live-content imaging platform, and the membrane permeability properties of two different DNA dyes. We also evaluated whether this technology would allow for the differentiation of various forms of neutrophil cell death.

Methods: Neutrophils were isolated from healthy controls and their nuclei were stained with the membrane permeable DNA binding NUCLEAR-ID red dye to count all cells. They were then seeded on a 96-well plate and incubated with various stimuli or inhibitors and a membrane-impermeable DNA dye, Sytox Green, that helped to assess cell death. Images were taken every 10minutes and a processing definition was devised to automatically count all neutrophils at baseline and neutrophils undergoing cell death based on fluorescence intensity and stained nuclear area size.

Results: This imaging platform enabled efficient, real-time imaging and quantification of cells undergoing NETosis. Findings were confirmed with established method of immunofluorescence microscopy and the percentage counts of cells undergoing NETosis correlated well. The platform’s ability to rapidly measure NETosis and effects of drugs to modulate it was used to test various concentrations of an inhibitor of NETosis (Akt-inhibitor XI) which showed a dose dependent effect. This method was also able to distinguish between distinct neutrophil cell deaths. Neutrophils undergoing NETosis induced by phorbol-myristate acetate, bacterial toxin nigericin, calcium ionophore A23187 or platelet activating factor, exhibited a loss of multi-lobulated nuclei, nuclear decondensation and eventual membrane compromise. Necrosis induced by freeze-thaw resulted in instantaneous damage to membrane integrity with minimal change to nuclear morphology. In contrast, apoptotic cells induced by staurosporine showed nuclear condensation and cytoplasmic blebbing.

Conclusion: The IncuCyte ZOOM platform is a novel assay that quantifies NETosis in a rapid, automated and reproducible way, while retaining the ability to distinguish between different types of neutrophil cell death, and offers a significant advancement in the study of neutrophils. It is a powerful tool to assess neutrophil physiology and to swiftly develop novel neutrophil targets in autoimmune diseases.

Disclosure: S. Gupta, None; D. Chan, None; K. Zaal, None; E. Ralston, None; M. J. Kaplan, None.

To cite this abstract in AMA style:

Gupta S, Chan D, Zaal K, Ralston E, Kaplan MJ. A Novel Real-Time Imaging Technique to Quantify Neutrophil Extracellular Traps and Distinguish Mechanisms of Cell Death in Neutrophils [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/a-novel-real-time-imaging-technique-to-quantify-neutrophil-extracellular-traps-and-distinguish-mechanisms-of-cell-death-in-neutrophils/. Accessed .

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