Periodic Reporting for period 4 - InflamCellDeath (Mechanism and function of gasdermin-induced inflammatory cell death)
Période du rapport: 2022-09-01 au 2024-02-29
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, such as caspase-1, -11, -4 and -5. These proteases are activated within so-called inflammasomes, cytosolic signaling 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 that involve the activation of inflammasome complexes, such as gout, pseudogout, asbestosis, or during autoinflammatory diseases caused by inflammasome deregulation.
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 (GSDMD) is only one member of the larger gasdermin protein family, consisting of GSDMA, GSDMB, GSDMC, GSDMD, GSDME and GSDMF in humans, 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 project is 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.
Conclusions of the action:
Our results have shown that gasdermins play an important role in vivo by mediating host defense and immunity against bacterial infections. Interestingly, different gasdermins acting in different cell types are needed to provide an anti-bacterial immune response.
Furthermore, we find that gasdermin activation does not only induce cell death but can also lead to a sub-lethal permeabilization of the plasma membrane that allows the release of proteins via gasdermin pores. Important in this context are membrane repair mechanisms that can remove gasdermin pores from the membrane and thus prevent cell death.
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 (GSDMD) is only one member of the larger gasdermin protein family, consisting of GSDMA, GSDMB, GSDMC, GSDMD, GSDME and GSDMF in humans, 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 project is 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.
Conclusions of the action:
Our results have shown that gasdermins play an important role in vivo by mediating host defense and immunity against bacterial infections. Interestingly, different gasdermins acting in different cell types are needed to provide an anti-bacterial immune response.
Furthermore, we find that gasdermin activation does not only induce cell death but can also lead to a sub-lethal permeabilization of the plasma membrane that allows the release of proteins via gasdermin pores. Important in this context are membrane repair mechanisms that can remove gasdermin pores from the membrane and thus prevent cell death.
Our project uncovered new insights into the mechanism and regulation of gasdermin activation, the control and execution of pyroptotic cell death and the role of gasdermins in vivo.
1) Gasdermin pore formation is regulated even after gasdermin cleavage and pore formation, on the level of membrane repair. Specifically, we found that GSDMD pore formation induced Calcium influx into the cell, which resulted in a recruitment of ESCRT-III complex components towards the membrane region containing gasdermin pores that drive the formation vesicles containing GSDMD pores that were shed from cells. Finally, we found that inactivation of ESCRT-III reduced survival of inflammasome-activated cells.
Ref: Rühl et al. Science 2018
2) We showed that apoptotic caspases-8 cleaves and activates gasdermin-D, thereby switching apoptosis to pyroptosis, and that this plays an important physiological role in in vivo model of TNF-driven cytokine storm and host defense against infection with Yersinia bacteria. Interestingly, we also found that GSDMD and GSDME play cell-type-specific roles in host defense against Yersinia infections.
Ref: Chen et al. EMBO J 2019, Dimarco et al. Sci Adv 2020, Chen et al. PNAS 2021
3) We also explored the effects of GSDMD deficiency in inflammasome-activated cells, and found that GSDMD-deficiency did not completely abrogate cell death. We found that in absence of GSDMD, caspase-1 cleaved BID to induce extrinsic apoptosis and rapid necrotic death. This finding revealed an unexpected redundancy in cellular cell death pathways, and new connections between inflammatory caspases and induction of apoptosis.
Ref: Heilig et al. LSA 2021
4) Using super-resolution microscopy and Cryo-EM microscopy we determined the atomic structure of NINJ1 filaments that form downstream of GSDMD activation and permeabilize the plasma membrane.
Ref: Degen et al. Nature 2023
5) To extend our findings from pyroptosis to other forms of cell death, we investigated plasma membrane rupture during ferroptosis, a novel form of death dependent on iron-mediated ROS production and subsequent lipid peroxidation, and showed that NINJ1 acts as a driver of membrane rupture in ferroptotic cells.
Ref: Ramos et al. EMBO J 2024
Exploitation and dissemination:
The results from this project were published in high impact journals and presented at over 20+ conferences.
Furthermore, we promoted their disseminations through press releases and on twitter.
Articles for the lay audience were also published in EU research in Spring 2020, and on the CORDIS website (upcoming).
The project also contributed to technical advances in the field of cell death: As part of the project we developed a novel toolset for the optogenetic induction of different forms of cell death, among them pyroptosis, apoptosis and necroptosis. Optogenetics allows the manipulation of proteins using blue light illumination and our toolset can be used to kill individual cells in a multicellular tissue to subsequently analyze the response of neighboring cells, or precise timing of cell death induction. The toolset was published and made available to other researchers via Addgene.org.
Ref: Shkarina et al. JBC 2022.
1) Gasdermin pore formation is regulated even after gasdermin cleavage and pore formation, on the level of membrane repair. Specifically, we found that GSDMD pore formation induced Calcium influx into the cell, which resulted in a recruitment of ESCRT-III complex components towards the membrane region containing gasdermin pores that drive the formation vesicles containing GSDMD pores that were shed from cells. Finally, we found that inactivation of ESCRT-III reduced survival of inflammasome-activated cells.
Ref: Rühl et al. Science 2018
2) We showed that apoptotic caspases-8 cleaves and activates gasdermin-D, thereby switching apoptosis to pyroptosis, and that this plays an important physiological role in in vivo model of TNF-driven cytokine storm and host defense against infection with Yersinia bacteria. Interestingly, we also found that GSDMD and GSDME play cell-type-specific roles in host defense against Yersinia infections.
Ref: Chen et al. EMBO J 2019, Dimarco et al. Sci Adv 2020, Chen et al. PNAS 2021
3) We also explored the effects of GSDMD deficiency in inflammasome-activated cells, and found that GSDMD-deficiency did not completely abrogate cell death. We found that in absence of GSDMD, caspase-1 cleaved BID to induce extrinsic apoptosis and rapid necrotic death. This finding revealed an unexpected redundancy in cellular cell death pathways, and new connections between inflammatory caspases and induction of apoptosis.
Ref: Heilig et al. LSA 2021
4) Using super-resolution microscopy and Cryo-EM microscopy we determined the atomic structure of NINJ1 filaments that form downstream of GSDMD activation and permeabilize the plasma membrane.
Ref: Degen et al. Nature 2023
5) To extend our findings from pyroptosis to other forms of cell death, we investigated plasma membrane rupture during ferroptosis, a novel form of death dependent on iron-mediated ROS production and subsequent lipid peroxidation, and showed that NINJ1 acts as a driver of membrane rupture in ferroptotic cells.
Ref: Ramos et al. EMBO J 2024
Exploitation and dissemination:
The results from this project were published in high impact journals and presented at over 20+ conferences.
Furthermore, we promoted their disseminations through press releases and on twitter.
Articles for the lay audience were also published in EU research in Spring 2020, and on the CORDIS website (upcoming).
The project also contributed to technical advances in the field of cell death: As part of the project we developed a novel toolset for the optogenetic induction of different forms of cell death, among them pyroptosis, apoptosis and necroptosis. Optogenetics allows the manipulation of proteins using blue light illumination and our toolset can be used to kill individual cells in a multicellular tissue to subsequently analyze the response of neighboring cells, or precise timing of cell death induction. The toolset was published and made available to other researchers via Addgene.org.
Ref: Shkarina et al. JBC 2022.
The InflamCellDeath project has contributed to a worldwide effort of characterizing the role of gasdermins, pore-forming cell death executioners, and pyroptosis.
The gasdermins have been identified in 2015 and at the start of the project, it was known that gasdermins can form pores, and that their activation is controlled by proteases. Over the last years, the field progressed beyond these initial findings, and gasdermin are now known to be evolutionary conserved cell death executioners that are found in bacteria, fungi and animals. The mechanism of gasdermin activation has been characterized, and we now know many proteases that can activate gasdermins in a cell-type and context-dependent manner. Finally, the gasdermins have been shown to play an important role in vivo, in processes ranging from host defense against pathogens, autoinflammatory disease as well as cancer immunity.
Our project contributed key findings to this effort, such as by showing that cell can repair membranes permeabilized by gasdermin pores, identifying caspase-8 as an activator of GSDMD, defining the roles of GSDDM and GSDME in host defense against bacterial infections, and finally in structurally characterizing NINJ1, the effector of plasma membrane permeabilization downstream of gasdermin pores.
The gasdermins have been identified in 2015 and at the start of the project, it was known that gasdermins can form pores, and that their activation is controlled by proteases. Over the last years, the field progressed beyond these initial findings, and gasdermin are now known to be evolutionary conserved cell death executioners that are found in bacteria, fungi and animals. The mechanism of gasdermin activation has been characterized, and we now know many proteases that can activate gasdermins in a cell-type and context-dependent manner. Finally, the gasdermins have been shown to play an important role in vivo, in processes ranging from host defense against pathogens, autoinflammatory disease as well as cancer immunity.
Our project contributed key findings to this effort, such as by showing that cell can repair membranes permeabilized by gasdermin pores, identifying caspase-8 as an activator of GSDMD, defining the roles of GSDDM and GSDME in host defense against bacterial infections, and finally in structurally characterizing NINJ1, the effector of plasma membrane permeabilization downstream of gasdermin pores.