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Mechanism and function of gasdermin-induced inflammatory cell death

Periodic Reporting for period 3 - InflamCellDeath (Mechanism and function of gasdermin-induced inflammatory cell death)

Reporting period: 2021-03-01 to 2022-08-31

Pyroptosis is a lytic pro-inflammatory type of programmed cell death that is observed in pathogen-infected cells. This type of cell death is part of the innate immune response of mammals and serve to kill the infected cell and thereby restrict pathogen replication. On a molecular level, pyroptosis is initiated by proteases of the caspase family. These proteases are activated within so-called inflammasomes, cytosolic signalling platforms that are assembled by pattern recognition receptors upon the detection of pathogen- or host-derived danger signals. Pyroptosis is essential for antimicrobial host defense, but also promotes the concomitant release of inflammatory danger signals and leaderless cytokines that is detrimental during chronic inflammatory disease.

Recently it was found that pyroptosis is caused by the caspase-driven cleavage of a single caspase substrate called gasdermin-D. This cleavage generates a cytotoxic N-terminal fragment of gasdermin-D that targets the plasma membrane, where it forms large pores and thus causes pyroptotic cell death. Gasdermin- D is only one member of the larger gasdermin protein family, an emerging group of cell death effectors that share its pore-forming cytotoxic activity and that appear to be major regulators of inflammatory necrotic cell death.

The main goal of this projectis to comprehensively characterize the function of gasdermins in host defense, to investigate the consequences of gasdermin-D pore formation to the host cell and to elucidate the pathways that regulate gasdermin activation. The objectives are:
1) to define the role of gasdermin-D in inflammasome-dependent anti-bacterial host defense
2) to study the role of membrane repair in restricting gasdermin-D-induced membrane
3) to characterize the function and regulation of other gasdermin family members during infection

By characterizing the mechanism and function of gasdermin-induced cell death in host-defense and inflammation this project may contribute to the development of novel therapies for infectious as well as inflammatory diseases.
The work so far focused mainly on two aspects of pyroptotic cell death and gasdermins: 1) the mechanism and regulation of gasdermin activation and 2) the control and execution of pyroptotic cell death.
On the first aspect, we were able to show that besides inflammatory caspases (e.g. Caspase-1, -4, -5 and -11), also apoptotic caspases cleave gasdermin-D, the executor of pyroptosis. Interestingly, caspase-3 inactivates GSDMD by cleaving in the pore-forming N-terminal domain, while caspase-8 activates GSDMD, by removing the C-terminal regulatory domain. This raised an interesting conundrum, that during extrinsic apoptosis, GSDMD can be both activated and inactivated by apoptotic caspases. To explore this surprising results we investigated different pathway of extrinsic apoptosis and different animal models. Our results show that indeed, caspase-8 activity can in most setting activate GSDMD, and that this activation converts apoptosis (an immunologically silent cell death) into pyroptosis, which is pro-inflammatory. We believe that GSDMD-driven cell death accounts for part of the inflammation observed after administration of apoptosis-inducing chemotherapeutic drugs. Thus development of GSDMD inhibitors could have a potential to reduce side effects associated with such treatments.
On the second aspect, we have found that pyroptotic cell death is strongly controlled, and that it proceeds via 2 phases, a non-lytic phase in which GSDMD pores are formed, but the cells lives, and a lytic phase in which pyroptotic cells burst due to GSDMD pore formation. The progress from the first to the second phase is controlled by host membrane repair mechanisms, in particular the ESCRT machinery. ESCRT proteins target the parts of the plasma membrane permeabilised by GSDMD pores, and result in its removal in vesicles that are shed from cells. Furthermore, during the non-lytic phase GSDMD pores serve as conduits fro the release of cytokines, like IL-1b and IL-18, revealing that GSDMD pore formation is the mechanism of unconventional proteins secretion.
In the remaining period, we plan to focus on additional gasdermin family members and their function in host cell death and host-defence. In particular GSDME and GSDMC have now been proposed to be activated by apoptotic caspases, but their physiological function in anti-bacterial defense remains unstudied.
Series of images showing a cell undergoing apoptosis (upper row) and pyroptosis (lower row)