The innate immune system houses cellular signaling proteins called pattern recognition receptors (PRRs) that are responsible for recognizing highly-conserved molecular patterns associated with pathogens or damage to elicit an immune response. However, NLRX1 is a unique PRR in the NOD-like receptor (NLR) family that instead functions to attenuate pro-inflammatory pathways that are activated by other PRRs, such as NF-κB and type-1 interferon signaling which both have implications in cancer. NLRX1 can regulate additional cancer-associated pathways, such as MAPK and AKT, and cancer-associated functions like metabolism and reactive oxygen species (ROS) production. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a murine mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden in vivo when NLRX1 is expressed in healthy host cells. NLRX1 exerts its protection through limiting the recruitment of eosinophils to the tumor, suppressing epithelial-mesenchymal transition (EMT), and attenuating the formation of the metastatic niche. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes cancer-associated characteristics in vitro and disease burden in vivo by promoting EMT. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context. Conversely, in murine pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics in vitro, and that this is likely driven by NF-κB, MAPK, AKT, and inflammasome signaling with a potential to also limit immune evasion. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type and identifies the underlying mechanisms through which NLRX1 functions in these two cancer models. This is critical information for drug development initiatives so therapies can be developed that target NLRX1 in the appropriate cell type and in the appropriate disease. / Doctor of Philosophy / Inflammation, which is characterized by redness, heat, pain, swelling, and sometimes loss of function, is a critical way in which our bodies fight infections and repair tissue damage. However, chronic inflammation occurs when our bodies are unable to turn inflammation off and can result in cancerous mutations. Therefore, the successful resolution of inflammation is critical to maintaining inflammatory balance and has previously been dubbed the "Goldilocks Conundrum". The immune system houses a class of cellular signaling proteins called pattern recognition receptors (PRRs), which often function to turn inflammation on. However, a unique PRR in the NOD-like receptor (NLR) family called "NLRX1" functions to turn inflammation off and therefore plays an important role in preventing damaging chronic inflammation. NLRX1 has historically been studied in the context of infectious diseases, but because NLRX1 is involved in inflammation and because inflammation is a critical factor of cancer, its role as a tumor suppressor or tumor promoter has recently become an area of interest. NLRX1 has also been found to regulate biological pathways beyond inflammation that are also important for cancer initiation and progression. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a mouse mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden when NLRX1 is expressed in healthy, non-tumor cells. NLRX1 exerts its protection through impacting the immune cells recruited to the tumor, limiting the ability of the tumor cells to leave the original tumor and spread throughout the body in the process known as metastasis, and suppressing the formation of a favorable tumor metastasis environment in the lung. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes disease burden by helping tumor cells leave the original tumor and spread throughout the body. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context, including if the cell is healthy or cancerous. Conversely, in mouse pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type. This is critical information for drug development initiatives so therapies can be developed that turn NLRX1 on or off in the appropriate cell type and in the appropriate disease.

Macrophage migration, an essential component of many biological processes and pathologic conditions, is mediated by integrated cellular signaling processes and cytoskeleton rearrangement. Recent advances indicate that the innate immunity signaling process plays a key role in the regulation of macrophage migration. Furthermore, our lab has provided evidence demonstrating the involvement of a key innate immunity signaling kinase, IRAK-1, as a critical modulator of murine macrophage migration. Macrophage migration induced by a potent PKC activator, phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS) was significantly decreased in IRAK-1-/- murine macrophages compared with wild type cells. Mechanistically, we first demonstrated that IRAK-1 works downstream of PKCε and directly binds to VASP, a cytoskeleton regulatory protein, to regulate PMA-induced macrophage migration. Secondly, we proved that IRAK-1 is required for LPS-induced macrophage migration and expression of MCP-1, a chemotactic cytokine for macrophages, via transcription factor C/EBPδ instead of NFκB. IRAK-1 binds directly to IKKε and inhibition or knock-down of IKKε results in a significant decrease in C/EBPδ expression and MCP-1 mRNA expression. Lastly, we identified the direct association between IRAK-1 and Rac1, a member of the Rac subfamily in the Rho family of GTPases. These finding further confirmed the essential role of IRAK-1 during macrophage migration. Our research provides a novel facet regarding the molecular signaling processes regulating macrophage migration. / Ph. D.

Host defense peptides (HDPs) are a large group of small positively charged peptides that play an important role in innate immunity. Their role is more critical at early ages when other components of the immune system have not fully developed. There are three classes of avian HDPs: avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). The objective was to compare expression of HDPs in male turkey poults at day of hatch (D0), D7, D14, D21 and D28 from the thymus, spleen, bursa, duodenum, jejunum and ileum. The expression of AvBD1, AvBD2, AvBD8, AvBD9, AvBD10, AvBD13, Cath2, Cath3 and LEAP-2 was measured using qPCR (n=6 birds/tissue/age). Data were analyzed by one-way ANOVA and Tukey's test, and significance considered at P ≤ 0.05. AvBDs and Caths exhibited greater expression in immune organs than intestinal tissues, with the greatest expression of AvBDs observed in the spleen. The intestinal tissues showed very low expression of AvBDs except for AvBD10 at D0. Similar to AvBDs, Caths expression in the immune organs was greater than the intestinal tissues with the spleen having the greatest expression among immune organs. Conversely, LEAP-2 showed greater expression in the intestinal tissues than in the immune tissues, which showed very low LEAP-2 expression unlike other HDPs. Understanding the differential expression of HDPs could reveal the innate immune status of poults, and may subsequently allow improvement of their health through appropriate mitigation strategies. / Master of Science

Avian paramyxoviruses (APMVs) consist of twelve distinct serotypes (APMV-1 to -12) isolated from a wide variety of domestic and wild birds. APMV-1/Newcastle disease virus (NDV) is the most characterized and globally important avian pathogen, because of the huge economic loss associated with the disease. However, very little information is known about the pathogenicity of APMV 2-12. APMV expresses six structural and two accessory proteins. The functions of APMV accessory proteins (V and W) are not fully established. Only the function of V protein in NDV is studied so far. V protein was found to be an IFN antgonist and a major virulent determinant of NDV. In this study, we tested for the potential functions of W protein in NDV and fuctions of V protein in other APMV serotypes. Vaccination failure is a major cause for NDV outbreak in developing and tropical countries, because of thermolabile nature of vaccine strains. Thermostable and thermolabile NDV strains exhibit difference in W protein length. In the first part of our study, we mutated the genome of a thermolabile NDV strain to express W protein of different lengths, rescued recombinant viruses by reverse genetics system and tested for thermostability. Our results showed that W protein does not confer thermostability to NDV. In the second part of study, we constructed plasmids expressing APMV -2, -3 and -6V proteins and tested for IFN antagonism by a dual luciferase reporter assay. Our results showed that APMV-3V acts as IFN antagonist by blocking IFN induction and thereby may play an important role in the evasion of innate immunity. / Master of Science

Human airway epithelial cells (HAECs) play a pivotal role in creating a mechanical barrier to prevent environmental insults from entering deeper into the lung tissue and in facilitating host defence against pathogens and allergens by producing immune mediators and recruiting inflammatory cells. ABCF1, is a unique member of the ABC transporter family that it is highly expressed in the airway epithelium, however, its function in HAECs is currently not known.In this thesis, we explored the role of ABCF1 as a dsDNA viral sensor in HAECs. Our findings demonstrated that while ABCF1 is required for an immune response to a double-stranded DNA (dsDNA) viral mimic, VACV-70, our transcriptomic analysis suggested a role in pro-inflammatory responses downstream of toll-like receptors (TLR) 3 and 4 signalling pathways. We followed this outcome by investigating ABCF1 in mediating pro-inflammatory responses to TNF-α and Poly(I:C) through A20, NF-κB and IRF-3 regulated signalling pathways. Our study demonstrated that Poly(I:C) and TNF-α induced IL-8 are regulated by ABCF1 through pathways independent of NF-κB, and IRF-3 activation, although the exact mechanism remains unclear. The next approach was to run a hypothesis-free in silico investigation of the ABCF1 protein-protein interaction (PPI) network using publicly available databases and Gene Ontology (GO) term enrichment analysis. Following our in silico results of ABCF1 protein interactors, we validated a novel interaction of ABCF1 and SYK in human airway epithelial cells following Poly(I:C) stimulation. We have demonstrated that silencing ABCF1 under stimulation by VACV-70, TNF-α and Poly(I:C) in HAECs affects the induction of immune mediators, and a candidate protein interaction partner, SYK, is involved in immune signalling, however its exact mechanism is not defined. We propose that further insights into the functions of ABCF1 may aid in understanding how HAECs maintain mucosal immune homeostasis. / Thesis / Doctor of Philosophy (Medical Science) / The human lungs are exposed to over 10,000 litres of air each day from normal respiration, and it is the first point of contact between the outside environment and the internal anatomy. The cells on the surface of the air passages in the lungs are called human airway epithelial cells (HAECs), and they represent a critical defence against inhaled foreign objects that may include air pollution, allergens, bacteria, and viruses. HAECs have a group of proteins called ABC transporters, that are capable of different activities that are essential for maintaining normal lung health. One unique ABC transporter called ABCF1 was found to regulate defences against viral and bacterial infections in non-lung cells. It is unclear whether ABCF1 has the same function and protective capacity in HAECs. In this Ph.D. thesis, we investigated how ABCF1 functions in HAECs to detect and respond to respiratory infections. By understanding how ABCF1 is involved in protecting the lungs against these infections, novel treatments can be developed to minimize morbidity and mortality in both healthy and vulnerable individuals. In our studies, we evaluated the changes in the response of HAECs that had normal or absent levels of the ABCF1 gene under conditions that resemble an infection. We used computational tools to help us investigate the proteins that interact with ABCF1 and predict their potential function. Our studies have concluded that ABCF1 does have a protective capacity in HAECs, however, we have yet to elucidate how and what other proteins are involved to help it function.

Innate leukocytes manifest dynamic and distinct inflammatory responses upon challenges with rising dosages of pathogen associated molecular pattern molecules (PAMPs) such as lipopolysaccharide (LPS). To differentiate signal strengths, innate leukocytes may utilize distinct intra-cellular signaling circuitries modulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules in Toll-like receptor 4 (TLR4) signaling and potentially playing key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip may serve as a negative regulator of NFkB signaling pathway in cells challenged with higher dosages of LPS, it acts as a positive regulator for low-grade chronic inflammation in leukocytes programmed by subclinical low-dosages of LPS. We aim to show recent progress in our understanding of complex innate leukocyte dynamics and its relevance in the pathogenesis of resolving versus non-resolving chronic inflammatory diseases. / Ph. D. / White blood cells, or leukocytes, have a dynamic inflammatory response to rising doses of bacterial cell wall components. Lipopolysaccharide (LPS) is a ubiquitous component of gram negative bacteria that is recognized by Toll-like receptor 4 (TLR4) and can shed into the blood stream, causing low-grade non-resolving inflammation. In order to differentiate between varying signal strengths of LPS, leukocytes utilize signaling within the cell, which is often regulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules in TLR4 signaling and potentially plays key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip serves to inhibit the pro-inflammatory NFκB signaling in cells challenged with higher dosages of LPS, it acts to increase low-grade chronic inflammation in leukocytes programmed by low-dosages of LPS. In these studies we show recent progress in elucidating the mechanism for Tollip involvement in low-grade non-resolving inflammation in mouse fibroblast cells.

Doctor of Philosophy / Department of Anatomy and Physiology / Elizabeth G. Davis / Activation of innate immunity through Toll-like receptor (TLR) signaling can also enhance antigen-specific adaptive immunity. TLR9 is an endosomal receptor for unmethylated bacterial and viral cytosine-phosphate-guanine DNA (CpG-DNA). West Nile virus (WNV) infection may result in meningitis and encephalitis in humans and horses, especially aged and immunocompromised individuals. Using flow cytometric analyses and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated equine cell-mediated immunity (CMI) to an inactivated West Nile virus vaccine in healthy yearling and adult horses. We also studied the potential of enhancing equine adaptive immunity to viruses and other pathogens by activation of innate immunity though TLR9 signaling pathway. We found vaccination with inactivated WNV vaccine induced strong WNV-specific T helper type 1 (Th1) and Th2 CMI with a Th1 bias, also effectively induced WNV-specific CTLs in yearling horses. In adult horses, the pre-existing Th1 CMI bias against WNV was enhanced following booster vaccination with inactivated WNV vaccine. Molecular characterization and flow cytometric analysis of TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in neutrophils (PMNs), CD4[superscript]+ and CD8[superscript]+ T cells and other leukocytes. Conservation of equine TLR9 and a high expression profile among leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity. Unmethylated CpG-DNA can significantly activate equine PMNs. It also induces expression of interferon (IFN)-[Alpha], IFN-[Beta], IFN-[Gamma], and interleukin (IL)-12p35 in PBMCs, as well as IFN-[alpha] and IFN-[gamma] in monocyte-derived DCs. Enhanced expression of IFNs in immune cells by CpG-DNA is not only crucial for host viral clearance, but also important in mediating host immune responses due to IFNs' anti-inflammatory effects. Compared to the relatively weaker activation of equine innate immunity by inactivated WNV, the tested CpG-DNA species showed potential as vaccine adjuvants for enhancement of CTLs and Th1 CMI against intracellular pathogens, characterized by significant induction of type I IFNs and Th1-specific cytokines such as IL-12p35 and IFN-γ. These data provide a basis for further investigation of these CpG-DNA species as potentially effective vaccine adjuvants in horses.

The innate immune system is at the interface between the host's immune system and the initial contact with HIV. Understanding the correlates of innate immune protection against Human Immunodeficiency Virus is an important goal for development of effective anti-HIV therapies or vaccines. Not all exposures to HIV end in infection. The innate immune system has been linked to the reduced susceptibility of HIV-exposed seronegative (HESN) female commercial sex workers in Kenya by a number of studies. This thesis is a comparison of Toll-like receptor (TLR) responses in different immune cells in peripheral blood mononuclear cells (PBMCs) from HESN and HIV negative (susceptible) female commercial sex workers (CSWs). This study tested the hypothesis that higher TLR8 responsiveness in PBMCs of HESN to ssRNA analogous to HIV's genetic material, would result in higher effector responses capable of making HIV target cells more refractory in vitro, compared to susceptible controls. The results showed that PBMCs of HESN were often hypo-responsive to TLR4 and TLR7 stimulations evidenced by often reduced cytokine responses to the corresponding ligands, but hyper-responsive to TLR8 following stimulation with ssRNA analogous to HIV's genetic material. The 'dichotomy' in TLR responsiveness of HESN PBMCs was associated with differential expression of cognate TLRs in PBMCs, and altered activation of TLR signalling pathways. The opposing pattern of TLR7 and TLR8 responsiveness corresponded to the ability of HIV to infect target cells in vitro; where pre-treatment of PBMCs with TLR7 enhanced HIV replication whereas TLR8 stimulation inhibited HIV replication. The differences in outcomes of the HIV infection assays were associated with distinct cytokine profiles, where TLR7 stimulation induced robust type I IFNs responses without proinflammatory TNF-α and IL-12 cytokine responses,while TLR8 stimulations produced type II IFN responses accompanied by robust proinflammatory responses in both groups. The cytokine milieu of HESN PBMCs prior to and following TLR4 and TLR8 stimulations was more tightly regulated, but was associated with higher activation of CD8+, NK cells, monocytes but not blood DCs. These results demonstrate that the lower activation or 'quiescent' state of HESN PBMCs did not limit the ability of their cells to recognize ssRNA analogous to HIV derived genetic material and mount potent responses capable of limiting HIV infection in vitro, supporting the overall hypothesis tested. This thesis contributes to the growing knowledge on the dichotomous outcomes between TLR7 and TLR8 treatments with respect to HIV infection that could be instrumental in the design of novel HIV inventions such as vaccines or microbicides. / May 2016

Swine influenza viruses (SIV) are a common and an important cause of respiratory disease in pigs. Pigs can serve as mixing vessels for the evolution of reassortment viruses containing both avian and human signatures, which have the potential to cause pandemics. NS1 protein of influenza A viruses is a major antagonist of host defence and it regulates multiple functions during infection by interacting with a variety of host proteins. Therefore, it is important to study swine viruses and NS1-interacting host factors in order to understand the mechanisms by which NS1 regulates virus replication and exerts its host defense functions. Influenza A viruses enter the host through the respiratory tract and infect epithelial cells in the respiratory tract, which form the primary sites of virus replication in the host. Thus, studying SIV infection in primary swine respiratory epithelial cells (SRECs) would resemble conditions similar to natural infection. The objectives of this study were to identify NS1-interacting host factors in the virus-infected SRECs and to understand the physiological role of at least one of the factors in influenza virus infection. The approaches to meet this objective were to generate a recombinant SIV carrying a Strep-tag in the NS1 protein, infect SRECs with the Strep-tag virus, purify NS1-interacting host protein complex from the infected cells by pull-down using strep-tactin resin and then study the physiological role of one of the NS1-interacting partners during influenza infection. Using a reverse-genetics strategy, a recombinant virus carrying the Strep-tag NS1 was successfully rescued and the SRECs were infected with this recombinant virus. The Strep-tag in the NS1 protein facilitated the isolation of an intact NS1-interacting protein complex and the proteins present in the complex were identified by liquid chromatography-tandem mass spectrometry. The identified proteins were grouped to enrich for different functions using bioinformatics. This gave an insight into the different functions that NS1 may regulate during infection and the potential host partners involved in these functions. Among the host proteins identified as potential interaction partners, RNA helicases were particularly of interest to study. Influenza being an RNA virus, RNA helicases could have important functions in the virus life cycle. Among the identified RNA helicases, DDX3 has been shown to regulate IFNβ induction and affect the life cycle of a number of viruses. However, its function in influenza A virus life cycle has not been studied. Hence, this study explored whether DDX3 has any role in the influenza A virus life cycle. Immunoprecipitation studies revealed viral proteins NP and NS1 as direct interaction partners with DDX3. DDX3 is a known component of stress granules (SGs) and influenza A virus lacking the NS1 gene is reported to induce SG formation. Therefore, the role of DDX3 in SG formation, induced by PR8 influenza A virus lacking NS1 (PR8 del NS1) was explored. The results from this study showed that DDX3 co-localized with NP in SGs indicating that DDX3 may interact with NP in the SGs. NS1 protein was found to inhibit virus-induced SGs and DDX3 downregulation impaired virus-induced SG formation. The contribution of the different domains of DDX3 to viral protein interaction and virus-induced SG formation was also studied. While DDX3 helicase domain did not interact with NS1 and NP, it was essential for DDX3 localization in virus induced SGs. Moreover, DDX3 downregulation resulted in the increased replication of PR8 del NS1virus, accompanied by an impairment of SG induction in infected cells. Since DDX3 is reported to regulate IFNβ induction, the role of DDX3 in influenza A virus induced IFNβ induction was also examined. Using small molecule inhibitors and siRNA-mediated gene knockdown, the RIG-I pathway was identified as the major contributor of influenza induced IFNβ induction in newborn porcine tracheal epithelial (NPTr) cells. DDX3 downregulation and overexpression also showed that DDX3 has an inhibitory effect on IFNβ expression induced by both influenza infection and low molecular weight (LMW) poly I:C treatment, which is also a RIG-I ligand. RNA competition assay to identify the mechanism of DDX3-mediated inhibition, showed that RIG-I binding affinity to its ligands LMW poly I:C and influenza viral RNA (vRNA) is much higher than that of DDX3. Furthermore, DDX3 downregulation enhanced titers of the PR8 del NS1 virus, while it did not affect the titers of the wild-type strains of PR8 and SIV/SK viruses. Overall, the results show that DDX3 has an antiviral role and the SG regulatory function of DDX3 has a profound effect on virus replication than the IFNβ regulatory function.

Fractures of bone are very common, affecting 2% of the population per annum. Whilst the majority heal uneventfully, 10-15% exhibit delayed or non-union. These complications tend to occur in patients who have sustained high-energy open fractures, which are limb-threatening injuries, or low-energy osteoporotic fractures, which are associated with high morbidity and mortality rates. Enhancement or acceleration of fracture repair would confer significant benefit to these patients as well as reduce the public health burden. Inflammation represents the earliest response following trauma and initiates a cascade of downstream events crucial for wound healing. However, the mechanism by which this occurs remains poorly defined. A detailed understanding of how these upstream events initiate fracture healing is a necessary step in the development of therapeutics to enhance this process. Our group previously reported that addition of low dose recombinant human TNF (rhTNF) at the fracture site accelerated fracture repair in a murine tibial fracture model. Here I show that local rhTNF treatment is only effective when administered within 24 hours of injury, when neutrophils represent the major inflammatory cell infiltrate. Endogenous TNF was expressed at the fracture site initially by neutrophils and after 3 days by monocytes/macrophages. Systemic administration of anti-TNF resulted in impaired fracture healing. The addition of rhTNF to the fracture environment in an air pouch model enhanced neutrophil recruitment, and promoted the recruitment of monocytes through CCL2 production. Conversely, inhibition of either neutrophils or the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility fractures represent a major unmet medical need and they are associated with permanent disability and premature death. Using a murine model of fragility fractures, rhTNF treatment improved fracture healing during the early phase of repair. Translated clinically, accelerated healing would permit earlier load bearing and reduce the morbidity and mortality associated with delayed patient mobilisation.