By Steven Shen

Graphic design by Yu-Wen Jan

Did you know that over one million cells die every second throughout the human body?¹ Fortunately, the immune system helps maintain balance by clearing away these dead cells while also defending against infections and harmful invaders like bacteria and viruses. A critical piece of this puzzle is understanding the molecular mechanisms of cell death and their regulatory effects on inflammation; as well as, uncovering how our body and brain may respond to injuries. Cell death occurs in various ways, which include: 1–apoptosis, an essential and programmed form of cell death; 2–necrosis, an uncontrolled form of cell death, often triggered by injury; and 3–pyroptosis, a highly inflammatory form of cell death that releases damage-associated molecular patterns (DAMPs) to amplify inflammatory responses.² DAMPs are molecules released by damaged cells to help with repair and to assist in restoring homeostasis. However, DAMPs can also often contribute to inflammation and disease. Specifically, pyroptosis is of interest in the field of cell biology, as the release of DAMPs constitutes a normal physiological response that not only enhances repair but also contributes to disease pathogenesis.²

Given its role in both tissue repair and disease progression, pyroptosis has become a key focus in medical research. Dr. Benjamin Steinberg, an Institute of Medical Science (IMS) alumnus and faculty member, is a pediatric anesthesiologist and neuroscientist at The Hospital for Sick Children (SickKids). He studies the molecular mechanisms of cell death to develop new therapeutics and improve patient outcomes for inflammatory diseases such as sepsis and pulmonary arterial hypertension. He completed his MD/PhD at the University of Toronto (U of T) from 2003 to 2011, followed by a residency in Anesthesiology at U of T. In 2013, he pursued additional fellowship training as a resident in Neuroscience and Immunology at the Feinstein Institute for Medical Research in New York under the supervision of Dr. Kevin Tracy. In 2018, he joined SickKids as a staff anesthesiologist and clinician scientist, focusing on the role of cell death in inflammation and disease.

In recent years, Dr. Steinberg’s research has been increasingly focused on pyroptosis, or what he often refers to as “cell rupture.” He explains, “historically, the field at large assumed that [the] cell rupture component was just a passive process—if a cell cannot sustain [the] energy-requiring process for repair and maintenance, it is effectively metabolically dead and will break apart.” Extending beyond this initial understanding from the field—what has truly fascinated Dr. Steinberg and has guided his research—is understanding the precise terminal events that trigger this lytic cell death process, thereby sparking his interest in this space. In fact, his research group and collaborators recently made a groundbreaking discovery, identifying the molecular mechanism by which a membrane protein called Ninjurin-1 (NINJ1) executes the final step of cell rupture.³

The discovery of NINJ1 in cell rupture, as Dr. Steinberg describes, helps “fill the void in our understanding of how the cell death process release[s] all those molecules that we study”, namely DAMPs and proinflammatory cytokines. His research group and others have found that NINJ1 functions through a polymerization event where it forms large protein complexes that cause the cell to break apart or rupture. Still, the question remained: what triggers the polymerization process of NINJ1? Normally, the body is well-equipped to manage cells that undergo programmed cell death by employing pathways like phagocytosis (the removal of dead cells and foreign substances by phagocytes). In contrast, our body is less equipped to handle ruptured cells and the pro-inflammatory damaging molecules they release. This is what makes the study of NINJ1 polymerization of special interest. Dr. Steinberg’s translational objective would be to find a way to hold the cell together, thus preventing the release of pro-inflammatory molecules from ruptured cells.

With the goal of preventing cell rupture and the excessive release of pro-inflammatory molecules, Dr. Steinberg and his team began searching for factors that could regulate NINJ1’s polymerization. Their investigations led them to an intriguing candidate: glycine, one of the simplest amino acids, that appears to play a key role in stabilizing the cell membrane and inhibiting NINJ1-induced cell rupture.⁴ The discovery of glycine as a cytoprotective agent better positioned his research group towards more translational projects, aiming to preserve cellular integrity in treating various diseases such as acute liver injury.⁵ In pre-clinical models, glycine is a valuable tool used to preserve cellular integrity.⁴ One of the biggest issues with the clinical application of glycine, however, is its poor pharmacokinetics—it may require a very high dosage to achieve the desired effect at a specific target organ. This limitation prompted Dr. Steinberg and his team to explore other potential therapeutic targets.

One avenue of alternative therapeutic targets Dr. Steinberg has investigated is an anti-NINJ1 monoclonal antibody that specifically blocks NINJ1 polymerization, preventing cell rupture. Through collaborations with researchers including Dr. Balyne Sayed—a liver transplant surgeon at SickKids—they demonstrated that anti-NINJ1 antibodies could reduce inflammation associated with liver injury, as demonstrated by a reduction in neutrophil infiltration in their mouse model.⁵ This breakthrough discovery offers a promising strategy to mitigate acute inflammatory liver injury in transplantations, particularly those caused by ischemia (impaired blood flow) and subsequent reperfusion (restoration of blood flow to an organ or tissue).  

Despite these significant advancements in uncovering new mechanisms of cell death, Dr. Steinberg emphasizes the need to further explore the translational potential of anti-NINJ1 therapies, particularly in treating chronic injuries such as pulmonary vascular disease. Combining anti-NINJ1 treatments with other interventions may help address the multifaceted nature of chronic diseases. As Dr. Steinberg notes, “cell death is more nuanced than ever appreciated, and the consequences can be severe.” His research offers a beacon of hope, and a path toward new treatments that could prevent cell-death-related inflammation and ultimately improve the lives of his patients in the future.

References

1. Campisi L, Cummings RJ, Blander Margarian J. Death-Defining Immune Response After Apoptosis. Am J Transplant. 2014 Jun;14(7):1488–1498.
2. Wallach D, Kang TB, Kovalenko A. Concepts of tissue injury and cell death in inflammation: a historical perspective. Nat Rev Immunol. 2014 Jan;14(1):51–9. 
3. David L, Borges JP, Hollingsworth LR, et al. NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks. Cell. 2024 Apr 25;187(9):2224-2235.e16. 
4. Borges JP, Sætra RS, Volchuk A, et al. Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death. eLife. 11:e78609. 
5. Kayagaki N, Stowe IB, Alegre K, et al. Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury. Nat. 2023 May;618:1072–1077.