Browsing by Author "Miao, Edward A"

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. Although pyroptosis and apoptosis have distinct signaling pathways, when a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we also now engineer S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. In both engineered strains, apoptosis occurred somewhat slower than pyroptosis. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche, but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. In order to clear an infection, distinct cell types may have specific tasks that they must complete before they die. In some cells, either apoptotic or pyroptotic signaling may initiate the same tasks, whereas in other cell types these modes of cell death may lead to different tasks that may not be identical in defense against infection. We recently suggested that such diverse tasks can be considered as different cellular “bucket lists” to be accomplished before a cell dies. As demonstrated here, engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We engineer S. Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. However, after 48 hours the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFNg-deficient mice this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell’s physiology that make host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

The inflammasomes are a group of pattern recognition receptors (PRRs) with unique characteristics and critical to innate immunity by translating microbial and damage signals into inflammation. While early investigation into inflammasomes focused on their ability to respond to invading pathogens, now inflammasomes are known to collectively respond to a broad range of sterile damage signals. Indeed, several inflammasomes, and most notably the NLRP3 inflammasome, are known to promote pathogenesis of several autoinflammatory conditions, including the autoimmune neuroinflammation which is modeled in the mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). While this association of inflammasomes with EAE is well established, the timing, tissue localization, and cell-specificity of inflammasome activation in the central nervous system (CNS) remains poorly understood during disease. Thus, this dissertation details our investigation into the specific sites and timing of inflammasome activation during EAE, as well as determining which inflammasome is activated in the CNS during disease. The interrogation of inflammasome activation in vivo during disease states required our use of genetically modified mice with fluorescent-protein tagged inflammasome adaptor ASC (ASC-Citrine), which serves as a reporter of active inflammasomes. We identified in situ inflammasome activation in specific CNS cell types of these mice using antibody-based immunofluorescent staining techniques and confocal microscopy, and confirmed the result by genetically modified mice, which allowed cell-specific expression of ASC-Citrine. Further, we used mice genetically deficient in inflammasome components ASC and AIM2 to investigate the involvement of these proteins in disease development. Our study concluded with several insights. Firstly, inflammasome activation occurs in the antigen draining lymph nodes prior to symptom onset during EAE, but inflammasome activation occurs more significantly in the spinal cord at 30 days post induction (dpi) of EAE. Spinal cord inflammasome activation during EAE occurs selectively in radioresistent cells, mainly in astrocytes. Further, this astrocyte inflammasome is dependent on AIM2, and does not result in outcomes, which are canonically observed in myeloid cells, such as release of the inflammatory cytokine, IL-1β. Indeed, AIM2 limits EAE development. Lastly, astrocyte inflammasomes differ morphologically from the traditional structure (known as the “ASC speck”) as seen in macrophages. This dissertation thus expands our understanding of inflammasome activation during EAE, and introduces new areas of investigation into the AIM2 inflammasome during EAE and inflammasome activation in astrocytes.