Customer Spotlight

Cytation and Lionheart Help to Identify cFLIPL as a Key Regulator of Cell Death and Inflammation

Tufts University

21-April-20

At Tufts University’s Graduate School of Biomedical Sciences, Dr. Alexander Poltorak seeks to understand how the host immune system responds when facing a pathogen. If the response is too strong or too weak, the results can be devastating. “If we look at the SARS-CoV-2 virus impacting the world right now, many deaths aren’t a direct result of the coronavirus itself,” he says. “Instead, it’s a complication of the infection; a violent, unbalanced response from the immune system resulting in many different pathological processes or attacks taking place in the lungs.”

On the other end of the unbalanced response spectrum, he cites the Black Death, or bubonic plague, that killed millions of people in the Middle Ages. During that pandemic, Y. pestis impaired the host immune response in order to bolster its own unrestricted proliferation in the body.

Dr. Poltorak’s most recent research findings are published in Science and titled, “cFLIPL protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation” (DOI: 10.1126/science.aay3878). Graduate student Hayley Muendlein serves as first author on the paper. By monitoring infected macrophages and their death rates over time, the collaborators identified the long isoform of cellular FLICE-like inhibitory protein (cFLIPL) as a key regulator of the balance between cell death and inflammation.

The Cytation™ 3 Cell Imaging Multi-Mode Reader and Lionheart™ FX Automated Microscope were extensively used to produce data in this project. He points to features that enable kinetic monitoring of cell death over time, saying, “These instruments were eye opening for us; we could literally see things that we were blind to before.”

Using data from the Cytation, curves were created that unveiled real-time dynamics of cell death. The team could see even subtle differences in cell death kinetics between activated wild-type and cFLIPL-deficient macrophages; information that could not be gleaned from end-point data.

The Lionheart was also instrumental in the visualization and quantification of inflammasome activation as measured by formation of the apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD) also known as ASC, in wild-type and cFLIPL-deficient macrophages. Inflammasome activation is a hallmark of pyroptotic cell death. However, since ASC-speck formation is fleeting and only occurs in a fraction of the cell population, it can be difficult to accurately quantify. Using the Lionheart 60x imaging capacity, they were able to visualize ASC-speck formation in real-time, providing us with a more accurate quantification of inflammasome activation.

The data indicates that if cFLIPL levels are sufficiently high, then the macrophage is committed to producing cytokines and the inflammatory response. At very high levels, this response may even induce the violent immune response known as a cytokine storm. Conversely, if cFLIPL levels are low, the cells engage pyroptotic machinery, preventing the pathogen from replicating further.

A representative 60x image taken using the Lionheart FX Automated Microscope of cFLIPL-deficient macrophages
A representative 60x image taken using the Lionheart FX Automated Microscope of cFLIPL-deficient macrophages stimulated with lipopolysaccharide. DAPI nuclear staining (blue) was used to quantify total cells, bright perinuclear ASC staining (green) indicates inflammasome activation in a particular cell, and nuclear propidium iodide incorporation (red) marks dead cells.

 

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To learn more about Tufts University, visit their web site.
Thanks to Dr. Alexander Poltorak for sharing his BioTek experience.



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