Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - PyroPop (Mechanisms and regulation of inflammasome-associated programmed cell death)

Teaser

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...

Summary

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.

Work performed

During the first reporting period, we have made several important breakthroughs and 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. The work that has been performed so far has resulted in 4 peer-reviewed publications, 3 of which reporting primary results and 1 review manuscript, and 2 additional manuscripts that are currently being revised for resubmission. 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) (Objective 3); we have defined the subcellular dynamics of pyroptosis at the single-cell level by high-resolution time-lapse microscopy (de Vasconcelos et al (2018) Cell Death Differ in press) (Objective 1); and we showed that inflammasome-induced pyroptosis is critical for in vivo IL-1beta-mediated immune pathology in a genetic mouse model of Familial Mediterranean Fever (Kanneganti et al. (2018) JEM 215(6):1519-1529) (Objective 4).

Final results

Ongoing studies are focused on characterizing the in vivo role of Pyrin inflammasome-mediated pyroptosis versus other cell death modes in driving inflammatory intestinal pathology in a mouse model of Clostridium difficile-induced pseudomembranous colitis; and communication mechanisms between the NLRP3 inflammasome and death receptors of the TNF family in B. anthracis-infected macrophages. Additionally, we are establishing a single-cell phenotyping platform to characterize at the single-cell level the relationship between pyroptosis and inflammasome-induced cytokine release; and exploring the role of pyroptosis executor GSDMD in IL-1beta-mediated pathology in a mouse model of CAPS disease and HA20 pathology. Studies towards CRISPR-Cas9 screening of the NLRP1 and Pyrin pathways are also ongoing. Overall, the team has achieved remarkable progress during the first reporting period, and progress towards the project goals is moving as planned.