InflamCellDeath
Behind pyroptotic cell death
Mechanism and function of gasdermininduced inflammatory cell death Project Objectives
The objectives of InflamCellDeath is to characterize pyroptosis, a regulated form of necrotic and highly pro-inflammatory cell death that is induced in response to infections or cell stress. The aim is to understand how gasdermin family members, the executors of pyroptosis, are activated and regulated, and how pyroptotic cell death contributes to pathogen clearance and immune defense
A pro-inflammatory form of programmed cell death, pyroptosis causes cells to expand and eventually burst, releasing content that then causes inflammation. We spoke to Professor Petr Broz about the work of the InflamCellDeath project in investigating the basis of pyroptotic cell death and its wider impact. A form of
programmed cell death first reported around 20 years ago, pyroptosis differs significantly from the more wellknown process of apoptosis. While apoptosis is a non-inflammatory, immunologically silent form of cell death, pyroptosis is proinflammatory. “A pyroptotic cell enlarges and balloons until it explodes. This releases cytokines and so-called danger signals that cause inflammation in the tissue,” explains Professor Petr Broz. As the Principal Investigator of the InflamCellDeath project, Professor Broz is analysing the underlying basis of pyroptotic cell death. “We know that pyroptosis is caused by a type of protein called gasdermin D, which is a pore-forming protein,” he outlines. “This protein is activated by proteolytic cleavage, by proteases. It’s believed that the N terminal part of the protein is cleaved from the C-terminal part, which is the regulatory, inhibitory domain.”
Membrane pores The N-terminus then targets the plasma membrane, where it forms huge pores. These pores allow the uncontrolled influx of water into the cell, which eventually leads to the death of the cell. “This influx of water results in the swelling and rupture of the cell, and eventually the release of inflammatory content,” says Professor Broz.
This form of programmed cell death normally occurs during infection. “Pyroptosis is the outcome of a signalling cascade known as the inflammasome pathway. A pattern recognition receptor recognises pathogenderived molecules, and assembles a signalling platform known as the inflammasome,” explains Professor Broz. “A protein called caspase 1 activates gasdermin D to kill off the cell, so that the pathogen can’t replicate anymore. It will also cause inflammation, so that other immune cells converge on the site of infection.”
says there is a lot of interest in the idea from the pharmaceutical sector. “Could we develop an inhibitor that would prevent the pore from forming and prevent the inflammation?” he asks. “This is a very interesting area for pharmaceutical companies.” There is a lot of interest in particular in the NLRP3 inflammasome, which is an important component of the innate immune system. Activation of the NLRP3 inflammasome is associated with several inflammatory disorders, and gasdermin D has been identified as an important target for therapeutic
A pyroptotic cell enlarges and balloons until it explodes. This releases cytokines that cause inflammation in the tissue. This can be very beneficial in terms of clearing a pathogen, yet an excessive inflammatory response in itself can damage healthy tissue. In some cases the inflammasome complex assembles even without an infection, due for example to a certain mutation, which may then be a factor in the development of auto-inflammatory disease. “Where the inflammasome pathway activates, you get caspase 1 activation and inflammation,” outlines Professor Broz. Blocking the gasdermin D protein could be an effective therapeutic strategy in this respect, and Professor Broz
development. “By better understanding the gasdermin D protein, we hope to eventually help develop inhibitors and assess whether, by blocking the gasdermin D protein, we can prevent inflammation in those types of diseases,” says Professor Broz. The project is largely exploratory at this stage however, with researchers aiming to define, in vivo, the role of gasdermin D in the inflammasomedependent bacterial host defence. “We hope to provide more insights into the gasdermin D protein, and to pinpoint where it is important,” continues Professor Broz.
Pyroptosis
Apoptosis
Time-lapse microscopy images showing a cell dying by apoptosis (upper row) and pyroptosis (lower row).
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EU Research
Project Funding
Funded by an European Research Council Consolidator Grant.
Contact Details
Atomic force microscopy (AFM) images of Gasdermin D pores.
Gasdermin D pore formation A further strand of the project’s research centres around investigating how the gasdermin D protein is regulated and activated. This work is central to understanding how pores are formed in a cell membrane during pyroptosis. “We have found that there are two phases of gasdermin pore formation. There is an initial phase where the gasdermin pores are formed in the plasma membrane, but the cell doesn’t die. Still, the gasdermin proteins function to release cytokines through the pores – so the pores themselves serve as a release mechanism of cytokines. It is only later on, once too many pores have accumulated around the plasma membrane, that the cell eventually dies,” says Professor Broz. “Not all cells die after the activation of caspase 1.” The gasdermin D protein is the focus of a lot of attention in research, yet it is just one of a family of six, and Professor Broz is keen to learn more about the other members as well. These proteins are among the most cytotoxic substances that cells are known to make, yet relatively little is known about their function and how they are regulated. “We don’t really know much about the conditions under which these highly efficient cell killers are activated. What’s their function? In which diseases would we need to inhibit them?” says Professor Broz. These types of questions will form an important part of Professor Broz’s research agenda in future. “We are trying to identify the function of the other gasdermin family members,” he continues. “We know that
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all of these gasdermins have the ability to cause pyroptotic cell death.” Research suggests that gasdermin E plays an important role in the innate immune defence against cancer, as a proinflammatory form of cell death could result in an immune reaction against the disease. However, less is currently known about the other family members. “We don’t know whether they function in infection, or what their biological function is,” says Professor Broz. It has been established that these proteins are specifically expressed in the skin and the gut, but there is much that remains unclear. “It’s known that if these proteins get activated they can cause very strong inflammation, but currently we don’t know the signal,” explains Professor Broz. “Mutations have been identified that cause the activation of these proteins, so they can get activated without a signal. This causes very strong inflammation in the skin, leading to alopecia.” This research into other members of the wider gasdermin family will be combined with continued investigation into gasdermin D. With the project set to enter the third year of its funding term, Professor Broz and his colleagues are working towards some ambitious objectives. “By the end of the project we hope to have identified how the gasdermin D protein is regulated on a posttranslational level, and how pore formation is regulated,” he outlines. “We also aim to identify novel functions for gasdermin D in inflammatory diseases in general, and to identify an inhibitor of the protein.”
Project Coordinator, Petr Broz, Ph.D., Associate Professor Department of Biochemistry University of Lausanne Chemin des Boveresses 155 CH-1066 Epalinges Switzerland T: +41 (0)21 692 5656 E: petr.broz@unil.ch W: https://cordis.europa.eu/project/ id/770988 Rühl S., Shkarina K., Demarco B., Heilig R., Santos J.C. and Broz P. ESCRT-dependent membrane repair negatively regulates pyroptosis downstream of GSDMD activation. Science, 362 (6417) pp. 956-960 (2018) Santos J.C., Dick M.S., Lagrange B., Degrandi D., Pfeffer K., Yamamoto M., Meunier E., Pelczar P., Henry T. and Broz P. LPS targets host guanylate-binding proteins to the bacterial outer membrane for non-canonical inflammasome activation. The EMBO journal, 37 (6) pp. 1-19 (2018) Chen K.W., Demarco B., Heilig R., Shkarina K., Boettcher A., Farady C.J., Pelczar P. and Broz P. Extrinsic and intrinsic apoptosis activate pannexin-1 to drive NLRP3 inflammasome assembly. The EMBO Journal, 38 (10) (2019). Broz P., Pelegrín P. and Shao F. The gasdermins, a protein family executing cell death and inflammation. Nature Reviews Immunology 2019 Nov 5.
Professor Petr Broz
Professor Petr Broz is an Associate Professor in the Department of Biochemistry at the University of Lausanne, Switzerland. He has previously worked in Europe and the United States, including post-doctoral training at Stanford University. His current research focuses on host defense mechanisms, inflammasomes and the induction of pyroptosis, a lytic inflammatory cell death.
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