A new study on mechanisms underlying pyroptosis sheds light on inflammatory processes and may help improve treatment of autoimmune and autoinflammatory diseases.

The study, entitled “Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death,” was published on Sep. 17 in Nature as an advanced online publication. Professor SHAO Feng, an investigator at the National Institute of Biological Sciences (NIBS) in Beijing and a guest investigator at the Institute of Biophysics of the Chinese Academy of Sciences, led the research.

Inflammatory cell death or pyroptosis plays a crucial role in immune defense again microbial infection. Pyroptosis is a kind of programmed necrosis featuring pore formation and rupture of the plasma membrane, resulting in massive leakage of cytosolic contents and strong inflammation. However, excessive pyroptosis causes many autoinflammatory and autoimmune diseases. Recent studies have also suggested a critical role for pyroptosis in HIV infection.

Pyroptosis is induced by two types of inflammatory caspases, caspase-1 and caspase-4/5/11. Caspase-1 was the first caspase identified and was shown to cause cell death in the early 1990s (misclassified as apoptosis then). Caspase-1 is activated by the inflammasome complex upon sensing microbial infection, representing a critical cytosolic innate defense. Human caspase-4/5 and mouse caspase-11 are activated by direct binding to cytosolic LPS (also known as endotoxin), playing a key role in bacteria-induced septic shock.

The study in question reveals the molecular mechanism for inflammatory caspases-mediated pyroptosis.

In this research, scientists performed genome-wide CRISPR-Cas9 genetic screens on caspase-1 and caspase-11-mediated pyroptosis in mouse bone marrow macrophages. Both screens hit the same gene, which encodes the unknown-function protein GSDMD. By generating GSDMD-deficient mouse macrophages and human HeLa cells, they confirmed that GSDMD is required and sufficient for pyroptosis mediated by all known inflammasome complexes (caspase-1) as well as cytosolic LPS (caspase-11). GSDMD deficiency does not affect caspase-1 activation and its processing of interleukin (IL)-1, but blocks the release of mature IL-1. This suggests that IL-1 secretion requires pyroptosis and further indicates that GSDMD acts downstream of inflammatory caspases.

Through biochemical analyses, the researchers further discovered that GSDMD is a generic and specific substrate of caspase-1/4/5/11, which cleave the link between the N- and C-terminal domains of GSDMD. A cleavage-resistant mutant of GSDMD cannot restore defective pyroptosis in GSDMD-deficient cells, suggesting that cleavage is required for pyroptosis.

The researchers also found that the cleaved N-terminal domain by itself is sufficient to drive cell pyroptosis. In the absence of infection, GSDMD is kept in an autoinhibited inactive state due to the interaction between the N- and C-terminal domains. An engineered GSDMD protein with another protease or caspase-3/7 cleavage site between the two domains can cause pyroptosis in the presence of the cognate protease or can even switch apoptotic cell death into pyroptosis.

GSDMD belongs to the gasdermin family, which is functionally uncharacterized; the family also contains GSDMA, GSDMB, GSDMC, DFNA5 and DFNB59. The SHAO study discovered that the N-terminal domains of most gasdermins also possess the capacity to induce pyroptosis.Like GSDMD, they are kept in the inactive state by intramolecular autoinhibitory interaction in the absence of infection.

Genetic mutations in human DFNA5 and mouse Gsdma3 that produce fragments capable of inducing pyroptosis are known to cause human nonsyndromic hearing impairment, and mouse alopecia and skin inflammation, respectively. These diseases are likely caused by gasdermins-mediated abnormal pyroptosis. Interestingly, other gasdermins are not cleaved by inflammatory caspases, suggesting that they employ other mechanisms to respond to microbial infection and trigger pyroptosis-mediated immune defenses.

This study identifies for the first time a generic substrate (GSDMD) for all inflammatory caspases whose cleavage determines pyroptosis. The study also reveals the molecular mechanism for pyroptosis and inflammatory cell death, and provides a new attractive drug target for therapeutics to treat inflammatory caspases-associated autoinflammatory/autoimmune diseases as well as endotoxin-induced septic shock. Moreover, the study identifies for the first time the pyroptosis function of the large gasdermin family, which not only redefines the concept of pyroptosis but also suggests a new paradigm for understanding programmed necrosis.

This research was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, the 973 National High-Technology Projects Program, the Beijing Scholar Program of the Beijing Municipal Government, the China National Science Foundation and the Howard Hughes Medical Institute in the United States.