Medical Biology - Theses
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ItemAnalysing the impact of the absence of CARD containing caspases on different forms of cell death( 2018)Cell death is an important process during embryogenesis as well as tissue homeostasis in the adult. Apoptosis, pyroptosis and necroptosis are three of the major programmed cell death pathways. Dysregulation of either of these cell death pathways can promote the development of a variety of diseases, such as cancer or autoimmune pathologies. Cysteine-dependent aspartate-specific proteases, known as caspases, exert key functions in all of these cell death pathways. Of note, certain caspases have been shown to play a role in more than one cell death pathway. This thesis presents the functional analysis of different caspases, in particular caspase activation and recruitment domain (CARD) containing caspases and their contributions to the pyroptotic, apoptotic and other cell death pathways. We have generated a novel triple knockout mouse strain deficient for the CARD containing caspases-1, -11 and -12. We initially used this strain to improve our understanding on the contributions of caspases-1, -11 and-12 to sepsis and different forms of cell death. Previous studies have suggested a role for caspase-12 in endoplasmic reticulum (ER) stress-induced cell death. However, we were not able to attribute a role of caspase-12 to sepsis or ER stress-induced apoptosis in vitro and in vivo. In Chapter 4 we present a study on the roles of different caspases as well as RipK3 during Salmonella infection in vitro and in vivo. There is evidence for a substantial functional overlap between different cell death pathways in the cellular response to pathogens, such as Salmonella. We examined this functional overlap of different cell death processes in the organismal and cellular response to infection by generating mice deficient for multiple caspases and also RipK3, an essential mediator of necroptotic cell death. Upon infection with S. Typhimurium SL1344 strain, primary myeloid cells from caspase-1/11/12/8 RipK3-/- mice showed marked resistance to cell death and survived even at high bacterial loads for up to 24 hours. When infecting the caspase-1/11/12/8 RipK3-/- mice with the vaccine Salmonella Typhimurium strain, they were not able to clear the bacteria from primary organs. Collectively, these findings provide evidence that there is substantial functional overlap between the different cell death pathways and hence the caspases involved in these processes in the cellular as well as organismal response to infection with S. Typhimurium and possibly other pathogens. Lastly, I generated mice lacking all murine CARD containing caspases, i.e. caspase-1, -11, -12, -2 and -9. These preliminary analyses revealed no major defects when comparing the embryonic development of mice lacking caspases-1, -11, -12, -2 and -9 to wildtype. Furthermore, we isolated haematopoietic stem and progenitor cells (HSPCs) from foetal livers derived from caspase-1/11/12/2/9 deficient mice and reconstituted lethally irradiated wildtype mice. Surprisingly, we did not find notable defects in the lymphoid and myeloid compartments in the caspase-1/11/12/2/9 deficient mice at steady state. In thymocyte cell death assays, cells from the quintuple caspase knockout mice still could undergo cell death, induced by the cytotoxic agent ionomycin, albeit at a delayed rate.
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ItemA CRISPR/Cas9-based investigation of inflammasomes in infectious disease and autoinflammation( 2017)Inflammasomes are a family of innate immune signalling platforms that are activated in response to tissue damage or infection. Inflammasome stimulation results in activation of the inflammatory protease caspase-1, which induces a lytic cell death program known as pyroptosis, and maturation and release of the pro-inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. The potent inflammatory cascade triggered through activation of the inflammasomes is protective against many bacterial pathogens that either invade host cells or produce toxins that deregulate key homeostatic mechanisms within innate immune cells such as monocytes and macrophages. De-regulation of inflammasome signalling, such as gain-of-function mutations in inflammasome components, can result in autoinflammatory pathology. In order to investigate the function and regulation of inflammasomes, Clustered, Regularly Interspersed, Short, Palindromic Repeats (CRISPR)/Cas9 gene editing technology has been utilised to delete various inflammasome components from human myeloid cell lines or from mice. The alternative inflammatory caspases, caspase-11 in mice and caspases-4 and -5 in humans are activated directly by cytoplasmic lipopolysaccharide (LPS), a key component of the cell wall of gram-negative bacteria. These caspases are able to induce pyroptosis independently of caspase-1, but are only able to trigger IL-1β and IL-18 release in a caspase-1-dependent manner. In this thesis, the roles of caspase-4 and caspase-5 in the response to cytoplasmic lipopolysaccharide (LPS) and invasive gram-negative bacteria have been investigated in a human monocytic cell line. While both caspases responded to infection with live gram-negative bacteria, free LPS that was transfected into the cytoplasm activated only caspase-4. This suggests that caspases-4 and -5 may be activated by distinct stimuli or through different mechanisms. This work also interrogates the role of the inflammasome-forming receptor pyrin, in both autoinflammatory disease and the anti-bacterial immune response. A serine to arginine mutation in pyrin at amino acid position 242 results in a newly described autoinflammatory condition known as Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND). A monocytic cell line expressing the S242R mutant of pyrin has been created and it was demonstrated that this mutation results in spontaneous inflammasome activity. Under homeostatic conditions, serine 242 is phosphorylated and interacts with the 14-3-3 family of adapter proteins to keep pyrin inactive. Deletion of specific 14-3-3 isoforms also resulted in spontaneous production of mature IL-1β. Finally, the expression of pyrin in various myeloid compartments and its role in in vivo models of bacterial infection have been investigated using a pyrin-deficient mouse line. Two isoforms of pyrin were detected that were differentially expressed among myeloid populations. Additionally, no role for the pyrin inflammasome was observed in a Dextran Sodium Sulfate (DSS)-induced colitis model, or Citrobacter rodentium, Salmonella Typhimurium or Mycobacterium tuberculosis infection models.