Dr. Feng Shao's lab reveals the key molecular mechanism for inflammatory cell death (pyroptosis).

    On Sept. 16, 2015, Studies from Dr. Feng Shao's laboratory at National Institute of Biological Sciences (NIBS), Beijing, reveal the key molecular mechanism for inflammatory cell death (pyroptosis). The work titled "Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death" is published as an "article" in the journal Nature as Advance Online Publication.

    Inflammatory cell death or pyroptosis plays a crucial role in immune defenses 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. Excessive pyroptosis causes many autoinflammatory and autoimmune diseases; recent studies have also suggested a critical role of pyroptosis in HIV infection. Pyroptosis is induced by two types of inflammatory caspases, caspase-1 and caspase-4/5/11. The caspase family contains more than a dozen members, most of which function in apoptosis. Caspase-1 and caspase-4/5/11 belong to the inflammatory caspase group. Caspase-1 is indeed the first caspase identified and was shown to cause cell death in early 1990s (misclassified as apoptosis then). Caspase-1 is activated by the inflammasome complex upon sensing microbial infection, representing a critical cytosolic innate defense. In their previous studies, Dr. Feng Shao's laboratory has identified several receptors for cytosolic bacteria that mediate inflammasome assembly and downstream caspase-1 activation (Zhao et al., Nature 2011; Xu et al., Nature 2014).     In a recent study (Shi et al., Nature 2014), they also show that human caspase-4/5 and mouse caspase-11 are innate receptors for cytosolic LPS (also known as endotoxin); LPS binding induces oligomerization and activation of caspase-4/5/11, triggering cell pyroptosis, which plays a key role in bacteria-induced septic shock. However, the mechanism for how inflammatory caspases induce pyroptosis has long been unknown.

    In this new study, Dr. Feng Shao and his team performed genome-wide CRISPR-Cas9 genetic screens on caspase-1 and caspase-11-mediated pyroptosis in mouse bone marrow macrophages. Both screens hit a same gene encoding an 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 extracellular release of the mature IL-1. This suggests that IL-1 secretion requires pyroptosis and further indicates that GSDMD functions 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 that cleave a linker region between the N-and C-terminal domains of GSDMD. A cleavage-resistant mutant of GSDMD can not rescue the defective pyroptosis in GSDMD-deficient cells, suggesting that the cleavage is required for pyroptosis. The researchers further found that the released N-terminal domain of GSDMD by itself is sufficient to drive cell pyroptosis; in the absence of infection or stimulation, GSDMD is kept in an autoinhibite inactive state due to the strong interaction between the N- and C-terminal domains. An engineered GSDMD protein with other protease or caspase-3/7 cleavage site between the two domains can render cell pyroptosis in the presence of the cognate protease or even can switch apoptotic cell death into pyroptosis, respectively. These results confirm the pyroptosis-inducing activity in the N-terminal domain of GSDMD.

    GSDMD belongs to the gasdermin family that 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 harbor the pyroptosis-inducing activity; like GSDMD, they are kept in the inactive state by the 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 for the first time identifies a generic substrate (GSDMD) for all inflammatory caspases whose cleavage is required and sufficient for inducing pyroptosis. The study also reveals the molecular mechanism underlying pyroptosis and inflammatory cell death, and provides a new attractive drug target for therapeutics of inflammatory caspases-associated autoinflammatory/autoimmune diseases as well as endotoxin-induced septic shock. Moreover, the study for the first time identifies 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.

    PhD student Jianjin Shi from the PTN graduate program and postdoc fellow Yue Zhao from the Shao laboratory are co-first authors of this paper; PhD student Kun Wang and undergraduate thesis student Xuyan Shi also made some contributions; other contributors include Yue Wang, Yinghua Zhuang and Dr. Fengchao Wang at NIBS transgenic facility as well as Huanwei Huang and Tao Cai at NIBS sequencing facility. Dr. Feng Shao is the corresponding authors. The research was supported by the 973 National High-Tech. Projects, the Beijing Municipal Government, China National Science Foundation, the Strategic Priority Research Program of the Chinese Academy of Sciences and Howard Hughes Medical Institute in the States, and carried out at National Institute of Biological Sciences, Beijing.

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature15514.html