Medical Biology - Theses
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ItemThe role of RIPK3 ubiquitylation and MLKL signalling during cell death and autophagy( 2021)Receptor Interacting Serine/Threonine Kinase-3 (RIPK3) is essential for necroptosis, an inflammatory form of programmed cell death pathway implicated in innate immunity, kidney ischemia reperfusion injury, and systemic inflammatory response syndrome. In the classical model, cells committed to necroptosis phosphorylate RIPK1, which in turn drives RIPK3 phosphorylation and oligomerisation. Active RIPK3 oligomers subsequently phosphorylate mixed lineage kinase domain-like protein (MLKL) pseudokinase which induces its translocation to the plasma membrane. The necroptosis pathway culminates in MLKL perforating the plasma membrane as a prelude to cellular rupture and release of inflammatory cytokines and damage-associated molecular patterns to the extracellular milieu. In addition to being a pro-necroptotic kinase, RIPK3 is also capable of triggering apoptosis when its kinase activity is restrained. Moreover, numerous death-independent roles of RIPK3 have been described in the context of inflammation such as arthritis, viral infection, or colitis whereby RIPK3 either promotes or dampens the secretion of pro-inflammatory cytokines. Understanding the molecular regulation of RIPK3 will thereby facilitate the ongoing pre-clinical development of RIPK3 inhibitors. Like most proteins, post-translational modification (PTM) is a critical fine tuner of RIPK3 activities. Ubiquitylation, in particular, has recently garnered attention in the cell death field as loss of this PTM may result in hyperactive RIPK3 which consequently accelerates death and inflammation. However, the post-translational control of RIPK3 signalling is not fully understood. Using mass-spectrometry, I identified a novel ubiquitylation site on murine RIPK3 on lysine 469 (K469). Complementation of RIPK3-deficient cells with a RIPK3-K469R mutant demonstrated that the decoration of RIPK3 K469 by ubiquitin limits both RIPK3-mediated caspase-8 activation and apoptotic killing, in addition to RIPK3 autophosphorylation and MLKL-mediated necroptosis. Unexpectedly, the overall ubiquitylation of mutant RIPK3-K469R was enhanced, which largely resulted from additional RIPK3 ubiquitylation upstream on lysine 359 (K359). Loss of RIPK3-K359 ubiquitylation reduced RIPK3-K469R hyper-ubiquitylation and also RIPK3-K469R killing. Collectively, I therefore propose that ubiquitylation of RIPK3 on K469 functions to prevent RIPK3 hyper-ubiquitylation on alternate lysine residues, which otherwise promote RIPK3 oligomerisation and consequent cell death signalling. I further investigated the consequence of abolishing RIPK3 K469 ubiquitylation by generating Ripk3K469R/K469R mice. In agreement with in vitro findings, primary fibroblasts with mutant RIPK3-K469R enhanced apoptosis, and in vivo studies demonstrate that RIPK3-K469 ubiquitylation contributes to pathogen clearance. Specifically, when Ripk3K469R/K469R mice were challenged with Salmonella enterica serovar Typhimurium, bacterial loads in the spleen and liver were significantly increased relative to wildtype control animals. The increased bacterial burden in the mutant mice was consistent with reduced IFNg produced in the serum, while the elevated MCP-1 cytokine upon infection might be indicative of heightened immune infiltrates. Although necroptosis signalling clearly triggers cell death, how it might impact other cellular responses remains unclear. Therefore, to further delineate the functional outcomes of necroptotic activity I examined how its signalling impacts autophagy. The autophagy pathway is triggered when cells are deprived of nutrients. Although regarded as a pro-survival pathway which acts to recycle and remove damaged organelles, studies have recognised that autophagic pathways can impact cell death processes. In apoptosis, for instance, autophagy acts to limit pro-inflammatory IFN-b secretion, thus decreasing apoptotic immunogenicity. Nonetheless, little is known about the status of autophagy during necroptosis. I demonstrate through various genetic, imaging, and pharmacological approaches that active MLKL translocates to autophagic membranes during necroptosis. However, contrary to previous findings which reported the activation of autophagy upon necroptotic activity based on increased lipidated LC3B, a commonly used marker of autophagy induction, I challenged this conclusion by demonstrating that the accumulation of active LC3B during necroptosis is a consequence of reduced autophagic flux. Therefore, unlike apoptosis which proceeds in tandem with autophagy, the induction of necroptosis negates autophagy in an MLKL-dependent manner. While the function of MLKL-mediated autophagy inhibition warrants further investigation, I propose that attenuating autophagy during necroptosis contributes to the immunogenicity of this cell death modality by limiting the ability of the cell to clear damaged organelles and immunogenic molecules. Overall, my research has helped in outlining how a key necroptotic molecule RIPK3 is regulated post-translationally and how this is relevant in the context of microbial defence. I have also defined novel functional roles for necroptosis signalling in the regulation of autophagic responses. Understanding the molecular regulation of necroptosis signalling and how this cell death pathway is linked to other cellular responses, such as autophagy, is important for the accurate design of new therapeutics to target these pathways in pathological settings.
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ItemDefining programs of cell death that can be harnessed to impact on outcomes of chronic viral infection( 2019)Pathogens causing chronic infections have successfully evolved mechanisms to subvert host immunity. Excessive and inappropriate inflammation together with attrition of repeatedly overstimulated high affinity T cells leads to abrogated immunity and persistence of pathogens such as HIV and HBV. T cell exhaustion has been touted as a prelude to T cell deletion during these infections, however, studies indicate that high affinity T cell clones are deleted at the onset of infection. The T cells that remain have lower affinity for pathogen epitopes and hence their response is weaker and more easily antagonised by inhibitory networks, including T-regulatory (Treg) cells. The killing of immune effector cells during chronic overwhelming infections is juxtaposed to the pathogen’s attempts to promote survival of infected target cells. Keeping infected cells alive is imperative for the maintenance of a microbial replicative niche. In this body of work, I dissected the role of host cell molecules and how they contribute to the death and survival of immune and infected cells. Necroptosis did not contribute to the loss of highly functional virus-specific CD8+ T cells during the course of infection. In contrast, when I interfered with death receptor signalling there was a modest rescue of functional CD8+ T cells. This gain in immune function, however, did not translate to improved viral control. The same mechanism I used to promote the survival of T cells made infected target cells refractory to death receptor mediated killing and therefore, offset any gain in immune function. Whilst examining the role of necroptosis in chronic infection, I made the discovery that the necroptotic inducer molecule, RIPK3, has additional non-necroptotic roles. Ripk3-/- mice cleared LCMV with enhanced kinetics compared to wild-type mice and mice that lacked the necroptotic executioner MLKL. I found that in the absence of RIPK3, chronically infected mice had impaired IFNβ responses. Excessive and prolonged IFNβ production is known to impair immunity. This may partially explain why mice lacking RIPK3 had enhanced numbers of granzyme B expressing T cells and controlled infection better than WT animals. The host-viral dynamics that favour displacement of highly functional cells with poorly activated cells makes the immune system highly vulnerable to inhibition through the activity of Treg cells. I next investigated the role of Treg cells in immune dysfunction during chronic infections and I was particularly interested in the cell death and cell survival pathways that contributed to the turnover and accumulation of these cells. I utilised mice with a Treg-specific deletion of Casp8. These mice had twice as many Treg cells as wild-type mice at steady state. Surprisingly, when these mice were infected with chronic LCMV, only 25% of the animals survived to 145 days post infection. Moribund animals succumbed to overt T cell activation and autoimmunity due to a precipitous drop in Treg cell numbers. Survivors, intriguingly, eliminated LCMV in most organs consistent with a massive gain in immune function. The death of the Treg cells was due to necroptosis. When I ablated the necroptotic pathway, through the deletion of Mlkl, I completely prevented the loss of Treg cells and the fatal immune pathology in Treg conditional caspase-8 deficient mice. I found that differential expression of RIPK3 and MLKL in Treg cells made them highly susceptible to necroptosis during chronic infection compared to Tconv cells. This was also the case for human Tregs and I was able to preferentially kill these cells, over Tconv cells, in vitro by driving necroptosis with a clinical stage caspase-8 antagonist called emricasan. Necroptosis is a lytic form of cell death that promotes inflammation and it has been implicated in chronic liver disease. I initially investigated if necroptosis in the liver contributed to the control of chronic LCMV, HBV or the malaria parasite Plasmodium berghei. Ablation of necroptosis had no impact on liver-pathogen dynamics and no impact on general liver function and architecture. In many cell types caspase-8 inhibits death receptor induced necroptosis. So, I reasoned that this molecule must be inhibiting induction of necroptosis in the liver of infected animals. I examined this by infecting mice that had a conditional loss of caspase-8 within hepatocytes. Despite abundant, infection driven, death ligands I observed no necroptosis in the liver. Even drug induced ablation of NF-ĸb survival signalling, downstream of TNF, failed to promote liver necroptosis in the aforementioned scenarios. The liver’s inability to undergo necroptosis was confirmed in mice with a human chimeric liver. I showed this refractoriness was due to liver repression of RIPK3 in humans and mice. The work conducted in this thesis provides important insights into the cell death pathways that are engaged in diverse cell types during chronic viral infections and I provide evidence that antagonising them therapeutically may lead to better clinical outcomes.
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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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ItemDissecting the role of TNF signalling in Mycobacterium tuberculosis disease pathogenesis to identify novel therapeutic targets( 2018)Mycobacterium tuberculosis (Mtb) is a formidable public health challenge, with a global epidemic, fuelled partly by rampant antibiotic resistance, that has the medical community grappling with more infected individuals than at any other time in history. Mtb is remarkable in its ability to efficiently disarm its primary host cell, the macrophage. One of our most crucial immunological defences against this highly skilled pathogen is the cytokine tumour necrosis factor (TNF), which can promote either cell survival or programmed cell death via apoptosis or necroptosis, depending on the cellular context. Given this essential role, TNF and its downstream pathways represent attractive therapeutic targets for tuberculosis (TB). Despite decades of research, however, fundamental insights into the means by which TNF mediates host protection remain elusive and have been hampered by reports of a pathological role of this cytokine in TB. The aim of this thesis is to systematically dissect the various components of TNF signalling and their impact on Mtb disease outcomes in order to identify aspects of the pathway that may be amenable to therapeutic intervention. This is achieved using a cutting-edge genetic approach and physiologically-relevant animal models of TB. Recent work suggested that TNF induces programmed forms of necrosis in Mtb-infected macrophages, thus promoting Mtb pathogenesis by facilitating mycobacterial escape and dissemination. In Chapters 3 and 4, I show that neither necroptosis, dependent on mixed lineage kinase domain-like (MLKL), nor a previously-undescribed death modality dependent on receptor-interacting protein kinase 3 (RIPK3) and B cell lymphoma-extra large (BCL-XL), are responsible for macrophage death during Mtb infection, and do not contribute to disease progression. This is in spite of the observation that the former pathway is strongly primed upon infection, suggesting that necroptosis is favoured by Mtb but ultimately restricted by the host. In contrast to lytic death, apoptosis of infected cells is considered beneficial to the host as the process is intrinsically microbicidal. In Chapter 5, I show that TNF is the primary death ligand driving the extrinsic apoptotic death pathway in infected macrophages during Mtb infection. Furthermore, I demonstrate that this pathway is beneficial in terms of eliminating intracellular bacilli and promoting the activation of adaptive immunity. Having established that apoptosis is protective, I postulate in Chapter 6 that the ability to pharmacologically modulate this process presents a potential therapeutic opportunity. Inhibitor of apoptosis (IAP) protein antagonists promote programmed cell death upon death ligand stimulation. I show that clinical-stage IAP antagonists selectively promote the apoptotic death of Mtb-infected macrophages in mice, and that this promotes the clearance of Mtb. I also extend these findings to infections caused by Burkholderia pseudomallei, in which a single dose of IAP antagonists completely eliminated the pathogen from the lungs. In summary, this thesis demonstrates that host TNF overwhelmingly promotes signalling pathways that are protective against Mtb. This refutes prior work suggesting that regulated necrosis is induced by TNF, and that advocated for the use of inhibitors of these pathways for the treatment of TB. The insights gained from this work have, however, led to the identification of a viable therapeutic strategy for Mtb and other intracellular pathogens, based on the finding that TNF-driven apoptosis of infected cells is beneficial to the host and can be harnessed with clinical-stage pharmaceuticals.
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ItemInvestigation of cell death pathways in response to TNF and IFNγ( 2017)During my PhD I investigated the regulation of the TNF and IFNγ signalling pathways and their ability to induce cell death. IFNγ is a critical cytokine in the immune response against viral and intracellular bacterial infections. It has also been associated with auto-inflammatory and auto-immune disorders (Pollard et al., 2013; Zhang, 2007), where it was found upregulated together with other pro-inflammatory cytokines like TNF (Ohmori et al., 1997). TNF signalling and the mechanism of cell death induction downstream of the TNF receptor complex has been investigated in detail over the past 4 decades. Although IFNγ was first described 50 years ago, and before TNF, significantly fewer IFNγ signalling components have been discovered compared to the TNF signalling complex. Nevertheless, both cytokines induce equally potent and potentially dangerous systemic responses at low concentrations and must be tightly regulated. I therefore hypothesised that additional IFNγ signalling regulators must exist. In order to discover such novel regulators of the IFNγ signalling pathway I enriched for the IFNγ receptor and identified binding partners using mass spectrometry. Using this approach I identified SPTLC1 and 2, which are two subunits forming the serine palmitoyltransferase, directly interacting with the IFNγ receptor chain 2 (IFNGR2) constitutively. Weak interaction between SPTLC1/2 and IFNGR1, however, was only detected upon IFNGR complex formation induced by IFNγ stimulation suggesting that IFNGR1 interacts with SPTLC1/2 indirectly via IFNGR2. SPTLC2 deficient single cell mouse dermal fibroblast showed either normal or increased phosphorylation of STAT1 upon IFNγ stimulation and lack of SPTLC2 had no impact on transcription of classical IFN target genes. Secondly, I investigated the mechanism of cell death induced by IFN in combination with Smac-mimetics, a group of small molecule inhibitors of the inhibitor of apoptosis proteins (IAPs), which have been heavily investigated in context of TNF signalling. Previous studies revealed that inhibition of IAPs renders cells sensitive to TNF induced cell death, which is primarily apoptosis mediated by caspase-8. However, inhibition of caspase-8 by caspase inhibitors triggers an alternative cell death pathway called necroptosis. Here I found that the combination of IFN/Smac-mimetic had a similar impact on survival and, more precisely, induced RIPK3 dependent caspase-8 mediated apoptosis in mouse dermal fibroblasts. Surprisingly, IFN/Smac-mimetic induced killing in HT29 cells was not blocked by deleting caspase-8 and effectors of the necroptotic pathway like RIPK3 and MLKL. In contrast, deficiency of RIPK1 largely protected cells from IFN induced cell death, indicating that a novel form of RIPK1 dependent cell death was being induced. In trying to discover the mechanism we observed that caspase-10 was significantly upregulated by IFN and activated by IFN/Smac-mimetic treatment. HT29 cells deficient for caspase-10, caspase-8 and either MLKL or RIPK3 were completely resistant to IFN/Smac-mimetic revealing an important role for caspase-10 in IFN/Smac-mimetic induced killing. Thirdly I focused on the activation and function of MLKL, the most downstream member of the necroptotic pathway known. Necroptosis has been best studied downstream of the TNF signalling complex, upon IAP and caspase inhibition. We and others propose a model where phosphorylation of the MLKL pseudokinase domain by RIPK3 triggers a molecular switch, leading to exposure of MLKL’s N-terminal four-helix bundle domain, its oligomerisation, membrane translocation, and ultimately cell death. We additionally identified novel phosphorylation sites S158, S228, S248. By mutating these sites and overexpressing phosphomimetic and -ablating MLKL mutants in Mlkl-/- or Ripk3-/-/Mlkl-/- deficient murine fibroblasts I demonstrated that these sites influence MLKL activity and discovered a potential inhibitory effect of RIPK3 on cell death induced by MLKL. Finally, I examined the evolutionarily conservation of the necroptosis inducing activity of MLKL by analysing the function of MLKL orthologs. While the intrinsic ability to lyse membranes, which was tested in liposome assays, is highly conserved, several MLKL orthologs including human MLKL failed to induce cell death when expressed in murine fibroblasts. This suggests the presence of additional poorly conserved, species-specific factors that inhibit or activate MLKL.
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ItemSHARPIN and RIPK1 are key regulators of cell death and inflammation in vivo( 2015)Therapeutic tumour necrosis factor (TNF) inhibition has been remarkably successful in the treatment of inflammatory and autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. Receptor interacting serine/threonine protein kinase 1 (RIPK1) and SHANK-associated RH domain interacting protein (SHARPIN) are proteins that regulate TNF and other immune signalling pathways. In mice, RIPK1 deficiency causes perinatal lethality whilst SHARPIN deficiency due to the inactivating chronic proliferative dermatitis gene mutation results in multi-organ disease including severe dermatitis. In both cases the underlying mechanism for the pathology has been unknown. TNF is best known for upregulating pro-survival and inflammatory transcriptional pathways although cell death in the form of caspase-8-dependent apoptosis or Receptor interacting serine/threonine protein kinase 3- (RIPK3) and Mixed lineage kinase domain-like- (MLKL) dependent programmed necrosis can also result. Whilst apoptosis is generally viewed as being immunologically inert and non-inflammatory, programmed necrosis, known as necroptosis, results in the release of cellular contents into the extracellular matrix and this can drive inflammation. In vitro, SHARPIN and RIPK1 regulate the transcriptional arm of TNF signalling and RIPK1 is also required for TNF-induced necroptosis. Both SHARPIN and RIPK1 also regulate transcriptional and cell death signalling from multiple other immune receptors. We therefore hypothesised that deregulated cell death was causative for the phenotypes caused by RIPK1 and SHARPIN deficiency. This work shows that SHARPIN deficient keratinocytes and dermal fibroblasts are sensitive to TNF-induced cell death. The SHARPIN deficient skin phenotype was completely prevented by deletion of the gene encoding TNF or its death receptor Tumour necrosis factor receptor 1 (TNFR1). SHARPIN deficient mice with caspase-8 heterozygosity and RIPK3 deficiency were almost fully protected from multi-organ inflammation and dermatitis, whilst unexpectedly, combined SHARPIN, RIPK3 and caspase-8 deficiency resulted in lethality. We also show that RIPK1 deficient mice on a C57BL/6 background die perinatally due to TNFR1-independent RIPK3- and MLKL-dependent systemic inflammatory disease, providing strong evidence that RIPK1 is both dispensable for, but required to inhibit, necroptosis. Mice doubly deficient for RIPK1 and RIPK3 or RIPK1 and MLKL were overtly normal at birth but after three to five days became runted and appeared to succumb to an intestinal phenotype marked by excessive apoptosis. Caspase-8 deficiency did not prevent the RIPK1 deficient perinatal lethality but prevented the intestinal phenotype. Finally, analogous to the deleterious effects of caspase-8 and RIPK3 in SHARPIN deficient mice, mice triply deficient for RIPK1, RIPK3 and caspase-8 were viable well into adulthood, and fertile, but eventually developed severe lymphoproliferative disease. These data show that in mice, RIPK1 or SHARPIN deficiency results in widespread inflammation and pathology due to unrestrained caspase-8-dependent apoptosis and RIPK3- and MLKL-dependent necroptosis, in a tissue specific manner. The results highlight that excessive apoptosis can be inflammatory, and that TNF can cause inflammation indirectly by causing excessive cell death and not only by upregulating inflammatory cytokines. RIPK1 activates necroptosis and has been successfully therapeutically targeted in pre-clinical trials. This research shows that RIPK1 also inhibits necroptosis in some tissue types, indicating a protective function for RIPK1 that must be preserved when therapeutically targeting it. These findings may have implications for the treatment of human disease states including stroke, heart attack and inflammatory bowel disease thought to involve excessive and pathological cell death.