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Mechanisms and regulation of inflammasome-associated programmed cell death

Periodic Reporting for period 4 - PyroPop (Mechanisms and regulation of inflammasome-associated programmed cell death)

Reporting period: 2020-01-01 to 2021-12-31

Cell death plays a central role in both homeostasis and human pathology. While apoptotic cells are orderly packed in ‘apoptotic bodies’ for uptake by neighboring cells and professional phagocytes, pyroptosis is a non-physiological lytic programmed cell death mode that results in spilling of the intracellular content in the extracellular environment. This lytic programmed cell death mode is increasingly associated with differential pathophysiological outcomes in (acute) infectious and (chronic) inflammatory diseases, respectively. Pyroptosis has been implicated in host defense against bacterial pathogens such as Francisella tularensis, Salmonella Typhimurium, Escherichia coli, Legionella pneumophila and Burkholderia thailandensis. Under conditions of chronic inflammation, however, pyroptosis may be detrimental to the host. This is best illustrated by the observation that genetic deletion of caspase-1 is significantly more effective in controlling early perinatal lethality and inflammatory pathology of Muckle Wells Syndrome-associated Nlrp3 knock-in mice relative to preventing downstream signaling through its inflammatory cytokine substrates interleukin (IL)-1β and IL-18. In addition, recent studies suggest that pyroptosis may be linked to macrophage activation syndrome in autoinflammatory patients with Nlrc4 mutations. A likely cause is that in addition to IL-1β and IL-18, pyroptotic cells release an amount of inflammatory mediators and danger signals (HMGB1, IL-1α, S100A8 and S100A9, heat-shock proteins, etc.) that may contribute importantly to destructive inflammatory responses in the context of chronic inflammatory disease. Once released into the extracellular space, these effectors can enhance inflammatory, cell survival and repair responses through activation of cell surface receptors such as the IL-1 and IL-18 receptors and the receptor for advanced glycation endproducts (RAGE). However, the cellular and biochemical mechanisms by which inflammatory caspases and the pore-forming protein GSDMD drive pyroptosis largely remain to be identified. Additionally, the in vivo role of pyroptosis in driving immune-related pathology and disease outcomes has not been established and it is unclear how inflammasomes can steer alternative cell death modes. Here, we aim to characterize the cell biological and molecular requirements of different inflammasome-induced cell death types, and to explore the therapeutic potential of inflammasome-driven cell death switching in chronic inflammatory diseases with high unmet medical need.
Project PyroPOP’s central aim is to better understand the molecular mechanisms by which inflammasomes regulate cell death of innate immune cells and how inflammasome-induced cell death contributes to inflammatory diseases. The project has led to several primary research publications in leading scientific journals. We have been able to achieve validation of the central hypothesis of the project that inflammasomes are cytosolic platforms that couple pathogen sensing to multiple programmed cell death modes. More specifically, we have demonstrated that inflammasomes induce ASC- and caspase-8-mediated apoptosis signalling when caspase-1 activation is blocked (Van Opdenbosch et al. (2017) Cell Reports 21(12):3427-3444); we have defined the subcellular dynamics of pyroptosis at the single-cell level by high-resolution time-lapse microscopy (de Vasconcelos et al (2019) Cell Death Differ 26(1):146-161). Moreover, we have shown that members of the dipeptidyl protease (DPP) 8 and 9 negatively regulate pyroptosis and canonical activation of the NLRP1b inflammasome in mouse macrophages (de Vasconcelos et al (2019) Life Science Alliance 2:pii:e201900313), and that significant crosstalk exists between pyroptosis and apoptosis pathways in inflammasome-activated cells (Van Opdenbosch et al Cell Reports (2020) 32(4):107959). We have also provided in vivo evidence for the project’s central hypothesis that pyroptosis induction is a critical mechanism that drives inflammasome- and interleukin-1β-induced inflammatory pathology in a mouse model of Familial Mediterranean Fever (Kanneganti et al. (2018) Journal of Experimental Medicine 215(6):1519-1529), and that sensing of Clostridium difficile infection differentially activates the Pyrin inflammasome in myeloid cells, and the mitochondrial apoptosis pathway in intestinal epithelial cells (IEC) in vivo and in IEC organoid systems (Saavedra et al (2018) Nature Communications 9:4846). Furthermore, we recently established that Bacillus anthracis infection results in TNF-mediated cell death induction and activation of the NLRP3 inflammasome as a new virulence pathway in lethal anthrax (Van Hauwermeiren et al (2022) PNAS 119(2):e2116415119). We have also characterized the role of neutrophil inflammasome activation and neutrophil pyroptosis in driving cytokine secretion and inflammasome-associated cell death in a mouse model of CAPS disease. The resulting publication has recently been submitted for publication. We also showed the mechanism of action of small molecule NLRP3 inflammasome inhibitor MCC950/CRID3 and demonstrated that it is significantly less potent in inhibiting CAPS disease-associated mutant NLRP3 (Vande Walle et al (2019) PLoS Biology 17(10):e3000529). In translational studies, we built on new mechanistic understanding of Pyrin inflammasome signaling in FMF monocytes to establish the first functional diagnostic assay for autoinflammatory diseases and validated it’s robustness as a blood-based test for diagnosis and functional subtyping of FMF (Van Gorp et al (2020) Annals of the Rheumatic Diseases 79(7):960-968). In other experiments that have been completed by the end of the project’s lifetime, we have used innovative single-cell technology platforms to re-evaluate a decades old question of how LPS-induced interleukin-1beta secretion is secreted from human monocytes. This has led to interesting new insights of how LPS promotes interleukin-1beta secretion in humans, and the corresponding manuscript is currently being prepared for publication (Vande Walle et al, in preparation). Overall, the results summarized above provide mechanistic understanding of cell death regulation in inflammasome-activated macrophages, epithelial cells and neutrophils as a scientific foundation for potential future exploitation in the development of novel anti-inflammatory therapies.
As summarized above, the project has led to several breakthrough studies that have substantially increased our understanding of inflammasome-induced cell death pathways and its role in inflammatory and infectious diseases. Currently still unpublished work from the project focused on characterizing the role of canonical inflammasome activation in neutrophils. The resulting publication has recently been submitted for publication. Additionally,In other experiments that have been completed by the end of the project’s lifetime, we have used innovative single-cell technology platforms to re-evaluate a decades old question of how LPS-induced interleukin-1beta secretion is secreted from human monocytes. This has led to interesting new insights of how LPS promotes interleukin-1beta secretion in humans, and the corresponding manuscript is currently being prepared for publication (Vande Walle et al, in preparation).
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