Although induction of innate immune responses during viral infection is essential, it can cause acute inflammation and lead to devastating results. The deleterious effect of innate immune responses has been demonstrated in gene therapy where administration of a replication deficient adenoviral vector (Ad) caused fatality during a clinical trial. Despite recent advances in our understanding of the innate immunity, there is a lack of understanding on how different cell types interact to mount inflammatory responses, which may play an important role in regulating immune responses in vivo. In this study, we investigated the interaction between macrophages and epithelial cells, the two major cell types capable of sensing and responding to viral infection in the airway, in induction of inflammatory responses against replication deficient Ads. We show in Chapter 2 that Ad infection of the macrophage-epithelial cell co-culture resulted in synergistic induction of inflammatory responses. Ad infection of the co-culture compared to macrophages alone resulted in higher cytotoxicity and induction of significantly higher levels of inflammatory mediators including pro-inflammatory cytokines, chemokines, nitric oxide, and reactive oxygen species. We found that these synergistic responses require macrophages and epithelial cells to be in close proximity suggesting that a novel mechanism regulates the inflammatory responses. In Chapter 3, we studied whether ATP plays a role in regulating inflammatory responses during acute Ad infection. Using the co-culture system, we found that ATP signaling through P2X7 receptor (P2X7R) is critical as inhibition or deficiency of P2X7R resulted in reduced inflammatory responses. We demonstrate that ATP-P2X7R signaling regulates inflammasome activation and IL-1β secretion. Furthermore, intranasal administration of Ad resulted in high mortality in mice but inhibition of ATP-P2X7R signaling enhanced survival and reduced inflammatory responses. These results suggest that ATP released by the infected cells plays an important role in regulating inflammatory responses during acute viral infection.

Alcohol abuse is an enormous problem causing death and disability to over 43 million people worldwide each year (WHO). Chronic alcohol consumption also contributes to abnormal brain morphology and significant brain volume loss indicative of neurodegeneration. Until there are effective treatments to alter maladaptive behavioral patterns in alcohol abuse, more research is needed to prevent alcohol-induced toxicity and degeneration. We used C. elegans as a model system to identify genetic modulators of alcohol toxicity and explored whether prolonged alcohol exposure damages the nervous system. In our study, we exposed L4-larval stage worms to varying concentrations of ethanol for three days and found a dose-dependent deficit in crawling. Furthermore, we evaluated degeneration by assessing the health of neurons using fluorescent reporters. Compared to the untreated group, we found that ethanol-exposed worms had a significant neurodegeneration. Previous findings using C. elegans have suggested that the innate immune pathway may protect against neurodegeneration caused by drug toxicity (Schreiber & McIntire, 2012). We find that deletion of either the innate immune gene nsy-1 (orthologous to the mammalian ASK-1 MAPKKK) or pmk-1 (orthologous to the mammalian p38 MAPK) caused hypersensitivity to ethanol toxicity. Conversely, boosting innate immune signaling via gain-of-function mutation in nsy-1 produced resistance to ethanol toxicity and ameliorated ethanol-induced cholinergic degeneration. Our findings indicate that prolonged exposure to ethanol leads to both behavioral impairments and neuronal degeneration in C. elegans and that the ASK1/p38 MAP kinase pathway may play a role in ethanol-induced damage to the nervous system. / text

Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.

Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.

Vaccinia virus E3 protein is known to bind double-stranded RNA and mediate interferon resistance. Alanine scanning mutagenesis was performed on its dsRNA binding domain, sufficient for wild-type tropism and immune suppression in vitro, and dsRNA binding and host range function assayed. Residues involved in dsRNA binding were required for host range function; however, seven dsRNA binding mutants were unable to rescue ΔE3L replication. Utilizing recombinant viruses, non-rescue mutants were unable to inhibit protein Kinase R phosphorylation despite dsRNA binding. Furthermore, host range was found to correlate with cytokine suppression and replication in IFN stimulated Huh7R cells. Additionally, no direct association was found between dsRNA binding and PKR interaction, refining the suppression model. Novel protein-protein interactions were discovered between E3 and cellular proteins via differential gel electrophoresis. This study represents the first full mapping of E3 residues involved in dsRNA binding and tropism, forming a basis for future study.

Vaccinia virus E3 protein is known to bind double-stranded RNA and mediate interferon resistance. Alanine scanning mutagenesis was performed on its dsRNA binding domain, sufficient for wild-type tropism and immune suppression in vitro, and dsRNA binding and host range function assayed. Residues involved in dsRNA binding were required for host range function; however, seven dsRNA binding mutants were unable to rescue ΔE3L replication. Utilizing recombinant viruses, non-rescue mutants were unable to inhibit protein Kinase R phosphorylation despite dsRNA binding. Furthermore, host range was found to correlate with cytokine suppression and replication in IFN stimulated Huh7R cells. Additionally, no direct association was found between dsRNA binding and PKR interaction, refining the suppression model. Novel protein-protein interactions were discovered between E3 and cellular proteins via differential gel electrophoresis. This study represents the first full mapping of E3 residues involved in dsRNA binding and tropism, forming a basis for future study.

Although induction of innate immune responses during viral infection is essential, it can cause acute inflammation and lead to devastating results. The deleterious effect of innate immune responses has been demonstrated in gene therapy where administration of a replication deficient adenoviral vector (Ad) caused fatality during a clinical trial. Despite recent advances in our understanding of the innate immunity, there is a lack of understanding on how different cell types interact to mount inflammatory responses, which may play an important role in regulating immune responses in vivo. In this study, we investigated the interaction between macrophages and epithelial cells, the two major cell types capable of sensing and responding to viral infection in the airway, in induction of inflammatory responses against replication deficient Ads. We show in Chapter 2 that Ad infection of the macrophage-epithelial cell co-culture resulted in synergistic induction of inflammatory responses. Ad infection of the co-culture compared to macrophages alone resulted in higher cytotoxicity and induction of significantly higher levels of inflammatory mediators including pro-inflammatory cytokines, chemokines, nitric oxide, and reactive oxygen species. We found that these synergistic responses require macrophages and epithelial cells to be in close proximity suggesting that a novel mechanism regulates the inflammatory responses. In Chapter 3, we studied whether ATP plays a role in regulating inflammatory responses during acute Ad infection. Using the co-culture system, we found that ATP signaling through P2X7 receptor (P2X7R) is critical as inhibition or deficiency of P2X7R resulted in reduced inflammatory responses. We demonstrate that ATP-P2X7R signaling regulates inflammasome activation and IL-1β secretion. Furthermore, intranasal administration of Ad resulted in high mortality in mice but inhibition of ATP-P2X7R signaling enhanced survival and reduced inflammatory responses. These results suggest that ATP released by the infected cells plays an important role in regulating inflammatory responses during acute viral infection.

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Tribbles (Trib) protein was first described in Drosophila as a regulator of proliferation, later being implicated as a G2/M modulator. In mammalian systems, three Trib gene family members have been identified, which share a conserved motif similar to the catalytic domain of serine/threonine kinases. However, they lack several conserved residues in the ATP-binding pocket and the core motif of the catalytic domain necessary for catalytic function. Tribbles 1 (Trib1) is involved in inflammation through its ability to regulate MAPK, NF-κB and the CCAAT Enhancer Binding Protein (C/EBP). Moreover, Trib1 is associated with human disease, such as atherosclerosis and acute myeloid leukaemia. In this thesis, I investigated the functional role of Trib1 in Toll-like Receptor (TLR)-induced inflammatory responses together with pro- or anti-inflammatory cytokines. The RAW264.7 myeloid cell line was stimulated with TLR2/9 ligands in the presence or absence of IFN-γ or IL-10. I observed a high level of Trib1 expression in the presence of IFN-γ and TLR2 ligands, but weak Trib1 expression following treatment with IL-10 and TLR9 ligands. In gene knock-down experiments using small interfering RNAs (siRNA) to reduce Trib1 expression, C/EBPβ was up-regulated in both stimulated (by IFN-γ and TLR2 ligands) and resting macrophage populations. TNF-α production was increased following Trib1 knockdown after treatment with IFN-γ and/or TLR2 ligands but IL-6 secretion remained unchanged. Furthermore, ERK1/2 expression was reduced in Trib1 siRNA-treated cells and failed to induce chemokinesis in macrophages. Finally, Trib1 was demonstrated to act as a modulator of cell cycle (G2/M) transition and displays a delayed apoptotic phenotype. The work in this thesis demonstrates that mammalian Trib1 contributes to the pro-inflammatory response and functions as a regulator of the ERK1/2 and C/EBPβ pathways following TLR ligand-mediated activation. Its novel functions include acting as a modulator of G2/M arrest and suppressing macrophage migration.