Viral infection stimulates the immune response to produce many cytokines and chemokines, the proteins imperative to fight a brewing infection. This response begins through recognition of pathogen-associated molecular patterns (PAMPs) from the virus, or from other signatures characteristic of tissue damage, damage-associated molecular patterns (DAMPs), by pattern recognition receptors (PRRs) that in turn stimulate pro-inflammatory signaling cascades. The results of these signaling pathways include the release of cytokines and chemokines that work to further upregulate immune responses and attract immune cells to the site of infection, respectively. In the case of SARS-CoV-2 infection, these responses can become problematic if they go unmitigated or unresolved, resulting in the severe COVID-19 manifestation of the 'cytokine storm,' or multisystem inflammatory syndrome in children (MIS-C). One classically increased protein in cytokine storm of COVID-19 patients is C-X-C motif chemokine 10 (CXCL10), which has been explored as a prognostic marker as it is shown to be predictive of disease outcome in hospitalized patients. To prevent severe outcomes like cytokine storm, a delicate balance must be struck, to ensure that this inflammation does not result in high levels of diffuse tissue damage. To achieve this, anti-inflammatory pathways exist within the immune system and help dampen the signals being induced. One such unique anti-inflammatory protein is a pattern recognition receptor known as NLRX1 (Nucleotide binding oligomerization domain, leucine rich repeat containing X1), that can interact with two main pathways involved with anti-viral immunity, the NFB and interferon pathways, downregulating them to keep off-target tissue damage at bay. NLRX1 is also involved in several other cellular processes, including modulating cell death processes and cellular metabolism which can also impact viral replication and clearance indirectly. In this work, we investigated both the pro- and anti-inflammatory arms of the anti-SARS-CoV-2 response focusing on two key proteins – pro-inflammatory chemokine CXCL10 and immunoregulatory PRR NLRX1. The roles of these two proteins were explored utilizing transcriptomic analysis of both human and mouse RNA samples, immortalized cell culture work, humanized mouse models of SARS-CoV-2 infection, and mouse-adapted virus models to be able to utilize deficient mouse models. In this work we better characterize the immune response to SARS-CoV-2 and its related immune-driven pathobiology of disease. The data presented in this work continues to elucidate CXCL10's role as an important driver of viral clearance of SARS-CoV-2, translating data from human patient nasal swabs to the animal model of disease, exploring differential inflammation and immune responses in the absence of CXCL10. Additionally, the work shown here provides further understanding of NLRX1 and its role in antiviral immunity with the context of SARS-CoV-2 infection. The interactions between this protein and the virus remains to be fully characterized, however, it appears they have some degree of mutual inhibition as determined by animal and cell culture models. The culmination of work here emphasizes the importance for both the pro- and anti-inflammatory responses in SARS-CoV-2 infection and offers insight into two possible related targets for future drug development. / Doctor of Philosophy / When a virus invades the body, the immune system kicks off many signaling cascades to keep the virus from replicating, clear virus already established in cells, and clean up the tissues surrounding the infected area of the remnants of cells that already succumbed to the virus. While this immune response is important to fight off the virus that has made its way into the body, overactive immune responses can result in hospital stays requiring supportive care to aid recovery from possible off-target tissue damage. One such case of this happening is when SARS-CoV-2 induces such a strong response, the immune system becomes overzealous and results in overproduction of pro-inflammatory cytokines and chemokines, signaling proteins in the immune system, which can lead to the characteristic 'cytokine storm' of severe COVID-19 disease. One of the proteins most often overproduced is the chemokine CXCL10, and this protein has been used as a biomarker in clinical practice to successfully predict severe disease outcomes in COVID-19 patients. To help combat severe disease outcomes and high levels of tissue damage, the immune system has inborn checks and balances to ensure that proteins like CXCL10 do not reach the level of overproduction as in the cytokine storm of COVID-19. One of these natural checkpoints is a protein called NLRX1, which interacts with two of the main pathways that can lead to the overproduction of cytokines seen overproduced in the case of cytokine storm. NLRX1 also has other roles in other interesting facets important for viral infections, including the metabolism of the cell and cellular death processes. The culmination of these roles could offer up NLRX1 as a possible target for treatments in the future. The work put together here explores both sides of the immune response, turning it 'on' with pro-inflammatory signaling, and turning it 'off' with anti-inflammatory signaling, trying to find just the right amount of inflammation to clear a viral invader while also impeding off target and diffuse tissue damage as the body fights the virus. This work focuses on two key proteins, CXCL10 to represent pro-inflammatory responses, and NLRX1 to represent the anti-inflammatory signaling. Understanding both arms of the immune response to SARS-CoV-2 infection is crucial to being able to identify potential targets for future treatments to help combat severe outcomes of SARS-CoV-2 infection. Using multiple levels across the translational spectrum, including cell culture, animal models, and human patient RNA from COVID test swabs, we explore both facets of SARS-CoV-2 immunity, focusing on these two proteins. Utilizing mouse models bearing deletions of the genes required to make these proteins and a mouse-adapted strain of SARS-CoV-2, this work characterizes how important these individual proteins are in the immune response to SARS-CoV-2, and work as proxies to understand the broader impacts of either the positive or negative regulation of immune signaling. Because of the work culminated here, these two tangentially related proteins are also offered up as possible future drug targets for the development of treatments in severe COVID-19 disease with cytokine storm presentation.

Wirtszellen verfügen über bisher unzureichend verstandene zellautonome Immunmechanismen zur Abwehr von intrazellulären Bakterien. In dieser Arbeit wurden zwei Abwehrmechanismen charakterisiert, die in Endothelzellen und Makrophagen Infektionen durch C. pneumoniae bekämpfen. Es konnte gezeigt werden, dass C. pneumoniae über einen MAVS-abhängigen Signalweg in humanen Endothelzellen erkannt wird. Diese Erkennung aktiviert die Transkriptionsfaktoren IRF3 und IRF7 und nachfolgend eine IRF3/7-abhängige Typ I-IFN-Produktion. Typ I-IFN bewirken auto- und parakrin eine Kontrolle der intrazellulären Infektion mit C. pneumoniae. Zum anderen wurde gezeigt, dass das mitochondriale Molekül NLRX1 eine zellautonome Abwehr gegen C. pneumoniae in Endothelzellen und in Makrophagen vermittelt. Diese NLRX1-abhängige intrazelluläre Abwehr ist unabhängig von verschiedenen, bisher mit NLRX1 in Verbindung gebrachten Signalwegen. Die Ergebnisse zeigen somit zum ersten Mal, dass NLRX1 eine zellautonome Abwehr gegen intrazelluläre Bakterien vermittelt. Daraus gewonnene Erkenntnisse sowie die Ergebnisse zukünftiger Arbeiten zur Klärung der NLRX1- und MAVS-aktivierenden chlamydialen Moleküle und den durch Typ-I-IFN-abhängigen intrazellulären Abwehrmechanismen könnten bei der Erforschung neuartiger antibakterieller Therapien hilfreich sein. Diese ist angesichts der weltweiten signifikanten Zunahme von mehrfach-resistenten Infektionserregern, unbedingt notwendig. / The cell autonomous defense mechanisms against intracellular bacteria in host cells are so far insufficiently understood. In the present work two defense mechanisms involved in the elimination of C. pneumoniae in endothelial cells and in macrophages were characterized. It could be shown that C. pneumoniae is recognized by a MAVS-dependent signaling pathway in human endothelial cells. This recognition activates the transcription factors IRF3 and IRF7 and subsequently an IRF3/7-dependent type I-IFN production. Type-I-IFNs induce an auto- and paracrine control mechanism against the intracellular infection with C. pneumoniae. Additionally it could be shown for the first time that the mitochondrial NLR molecule NLRX1 mediates a cell autonomous defense mechanism against C. pneumoniae and most likely other intracellular bacteria in human endothelial cells and murine macrophages. This NLRX1-dependent intracellular defense mechanism is independent of the different mechanisms which were so far linked to NLRX1. The outcome of this work and future studies to identify chlamydial molecules responsible for the activation of the MAVS- and NLRX1-dependent signaling pathways as well as the effector mechanisms responsible for Type-I-IFN-dependent control of intracellular chlamydial replication could be very helpful in the development of novel antibacterial therapies.

Résumé : La mort cellulaire neuronale est un phénomène qui se produit pendant le développement du cerveau, mais aussi dans les conditions pathologiques. Selon l’environnement où la cellule se retrouve; l’apoptose ou la nécrose peuvent contribuer à cette mort neuronale. La nécrose produit un environnement qui promeut l’inflammation ainsi que la cytotoxicité. L’apoptose est un processus hautement organisé qui permet l’homéostasie tissulaire. Un récepteur NOD récemment découvert, Nlrx1, jouerait un rôle dans la régulation de l’inflammation et de la mort cellulaire pendant les infections. Par conséquent, notre hypothèse suppose que Nlrx1 joue un rôle neuroprotecteur en contrôlant la mort neuronale. Afin de déterminer le mécanisme protecteur de Nlrx1 in vitro, un Knock-Down, un Knock-In et un témoin Scrambled de Nlrx1 dans les cellules N2a ont été générés. Des essais LDH de mort cellulaire avec la staurosporine ou le stress oxydatif comme la roténone, le MPP+ ou le H[indice inférieur 2]O[indice inférieur 2] ont été exécutés. Suite au traitement de 24 heures à la staurosporine, les cellules N2a Knock-In subissent plus de mort cellulaire que les cellules N2a Knock-Down et les cellules Scrambled. Quand ces cellules sont traitées à la roténone ou au H[indice inférieur 2]O[indice inférieur 2], les cellules Knock-In subissent moins de mort cellulaire que les cellules Scrambled. Les cellules N2a Knock-Down ont plus de mort cellulaire que les cellules Scrambled quand elles sont traitées à la roténone ou au MPP+. Les analyses par immunobuvardage de type Western des protéines HSP90 et HMGB1 ainsi que par cytométrie en flux ont montré que les cellules Knock-In ont moins de cellules nécrotiques lorsque traitées à la roténone comparé aux cellules contrôles Scrambled. Le ratio des cellules nécrotiques/cellules apoptotiques était aussi plus élevé dans les cellules Knock-Down comparé aux cellules Scrambled. Par microscopie électronique, il a été possible d’observer que les cellules N2a Knock-In contiennent plus de mitochondries que les cellules Knock-Down et Scrambled en conditions témoins. Ces résultats ont aussi été confirmés par marquage au mitotracker en cytométrie de flux L’immunobuvardage de type Western a montré que dans les cellules Knock-In, il y avait une augmentation de la protéine phosphorylée-DRP1 active, une protéine impliquée dans la fission mitochondriale. Ces résultats pourraient expliquer le nombre augmenté de mitochondries observé dans les cellules Knock-In. Des expériences d’immunoprécipitation ont montré une association entre Nlrx1 et DRP1, ainsi qu’avec la forme active phosphorylée de DRP1. En ajoutant le Mdivi, un inhibiteur de la fission mitochondriale, aux traitements de roténone ou H[indice inférieur 2]O[indice inférieur 2], la mort cellulaire était augmentée dans les cellules Knock-In comparé aux cellules Scrambled. Également, la nécrose était augmentée dans les cellules Knock-In à des niveaux semblables à ceux retrouvés chez les cellules Scrambled et Knock-Down. Ces résultats suggèrent que Nlrx1 serait impliquée dans la régulation de l’équilibre entre la nécrose et l’apoptose, en favorisant la survie cellulaire. Nlrx1 pourrait alors servir de molécule neuroprotectrice dans les maladies médiées par le stress oxydatif. / Abstract : Neuronal cell death is a phenomenon that occurs during brain development as well as in pathological diseases. Depending on the environment in which the cells are; a poptosis or necrosis can contribute to neuronal cell death. Necrosis produces an environment that promotes inflammation and cytotoxicity and apoptosis is a highly organized process that maintains tissue homeostasis. A recently discovered NOD receptor, Nlrx1, is thought to play a role in regulation of inflammation and cell death during infection. Therefore, we hypothesize that Nlrx1 plays a neuroprotective role by controlling cell death in neurons. To determine the protective mechanism of Nlrx1 in vitro, a Knock-Down, a Knock-In and a Scrambled control of Nlrx1 in N2a cells was generated. LDH assays for cell death detection with staurosporine or oxidative stress, such as rotenone, MPP+ or H[subscript 2]O[subscript 2], have been done. After 24h treatment of staurosporine, N2a Knock-In cells showed higher cell death than N2a Knock-Down and Scrambled. When cells were treated with rotenone or H[subscript 2]O[subscript 2], N2a Knock-In cells had less cell death than Scrambled cells. N2a Knock-Down cells resulted in more cell death than Scrambled cells when treated with rotenone or MPP+.Western Blotting of HSP90 and HMGB1 as well as flow cytometry of cell death demonstrated N2a Knock-In cells to have less necrotic cells when treated with rotenone compared to Scrambled. The ratio of necrotic cells on apoptotic cells was also higher in N2a Knock-Down cells compared to Scrambled cells. Electron microscopy of control cells showed that Knock-In cells contains more mitochondria than Knock-Down and Scrambled cells. These results were confirmed by mitotracker staining by flow cytometry. Western blotting showed that there was an increased in Knock-In cells of active phosphorylated-DRP1 protein, a protein implicated in mitochondrial fission. Thus, it could explain the increased number of mitochondria seen in Knock-In cells. Immunoprecipitation showed that Nlrx1 protein interacts with DRP1 as well as active phosphorylated-DRP1. Adding Mdivi, a mitochondrial fission inhibitor, to rotenone or H[subscript 2]O[subscript 2] treatments, cell death was increased in Knock-In cells compared to Scrambled. Also, necrosis was also augmented in Knock-In cells to levels comparable to Scramble and Knoc k-Down cells. These results suggest an implication for Nlrx1 in regulating the balance of necrosis to apoptosis, permitting cells to survive. Nlrx1 could serve as a neuroprotective molecule in diseases mediated by oxidative stress.