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Host cell pattern recognition receptors (PRRs) are a first line of defence against pathogens and function to generate a productive innate immune response. PRRs sense pathogen-associated molecular patterns (PAMPs), such as viral genomic DNA, which is a major PAMP during DNA virus infection. Viral DNA sensing leads to the activation of the STING-TBK1-IRF3 signalling axis and the production of type I interferon. Previously, our work identified the non-homologous end-joining protein DNA-PKcs, part of the DNA-dependent protein kinase (DNA-PK) complex, as an intracellular PRR for cytoplasmic viral DNA in murine cells. After screening several human cell lines, we established a robust system to dissect the DNA sensing pathway in human fibroblasts. In these human cells DNA-PKcs was found to be essential for the production of type I interferon via the STING pathway in response to DNA and DNA virus infection and we found that the kinase activity of DNA-PKcs was not required for this response. Many DNA viruses evade the immune response by inhibiting the pathway. We make use of attenuated Herpes simplex virus 1 (HSV- 1) and Vaccinia virus (VACV) that lack immunomodulatory proteins and drive type I interferon production. DNA-PKcs-/- cells have a defective immune response after infection with attenuated HSV and VACV. Furthermore, primary patient fibroblasts harbouring a mutation in DNA-PKcs showed a gain-of-function effect and an enhanced immune signaling to DNA and DNA virus infection. DNA-PKcs has also been linked to cell death during retrovirus integration although this has not been studied extensively. We carried out some preliminary work in this study, showing that DNA-PKcs-/- cells are more prone to cell death during HSV-1 infection and have reduced yields of virion production. This work demonstrates the role of DNA-PKcs as a viral DNA sensor in human cells and adds to the knowledge of the DNA sensing processes that are essential for anti-viral innate immunity.

In the dental pulp, an acute inflammatory response is crucial for initiating healing and repair. The dental pulp is capable of mounting such a response by expressing sophisticated immune response components. Many studies investigated the role of different immune response components in dental pulp inflammation and repair, but the emerging role of Absent in melanoma 2 (AIM2) and Nod-like receptor protein 3 (NLRP3) inflammasomes is yet to be elucidated. In this thesis, the role of AIM2 and NLPR3 receptors was explored, both in bacterial and sterile inflammation context. Furthermore, the potential of modulating the inflammatory responses via these receptors to mediate repair. The findings of the thesis confirmed the importance of inflammation in dental pulp healing and repair following injury and demonstrated novel roles for NLRP3 inflammasomes and DNA sensors in this process.

Microbiota colonisation of the intestinal tract makes it difficult for pattern recognition receptors (PRR) to discriminate between beneficial microbes and harmful pathogens. We aim to define the roles of cytosolic Nod-like receptors (NLR) in intestinal immunity and homeostasis. Upon activation, some NLR form inflammasomes that mediate the release of inflammatory cytokines and pyroptosis, an inflammatory form of cell death. NLR activation in the non-hematopoietic compartment was shown to be protective during acute intestinal infection. To identify the cell type responsible for this protection, we generated transgenic mice in which the key inflammasome adaptor molecule Asc is selectively ablated in intestinal epithelial cells (IEC) (AscΔVC) and observed that inflammasomes are important for controlling Citrobacter rodentium clearance in these mice. To further dissect the importance of pathogen clearance by IEC inflammasome, ex vivo cultures of primary IEC organoids were established. Thus far this system has revealed profound differences in inflammasome regulation between IEC organoids and bone marrow-derived macrophages (BMDM). This research will inform our understanding of cell type-specific regulation of inflammasomes.

The adenosine deaminase acting on RNA (ADAR) enzymes catalyse the hydrolytic deamination of adenosine (A) to inosine (I) in double stranded (ds) RNA. Mutations in ADAR1 underlie the autoimmune disorder Aicardi Goutiѐres syndrome (AGS). Patients with AGS display heightened levels of type I interferon (IFN) and IFN stimulated genes (ISGs). The first aim of my thesis was to determine whether the mutations found in the human ADAR1 gene affected RNA editing. I found that the ADAR1 mutants identified in the AGS patients have reduced editing activity. Interestingly, the mutations have a greater effect on the IFN-inducible cytoplasmic isoform, ADAR1p150 than on the constitutive ADAR1p110 isoform. These results imply that A-to-I editing plays a role in regulating the type I IFN response. The Adar1 null mouse dies by E12.5 with a type I IFN signature similar to that observed in the AGS patients. The second aim of my thesis was to characterize the immune signalling pathway aberrantly activated in the absence of Adar1. A colleague in our research group rescued the Adar1 null mouse to birth by blocking the cellular response to cytoplasmic dsRNA by generating a double mutant with the mitochondrial antiviral signalling adaptor, Mavs. In the Adar1-/-; Mavs-/- mutant I found that the aberrant immune response is rescued at E11.5. This indicates that MAVS is the downstream adaptor in the aberrant immune response that underlies the embryonic lethality in the Adar1-/- mouse. The third aim of my thesis was to determine if the lack of inosine modification within cellular RNA was triggering the aberrant immune response in the Adar1-/- mouse. To study this, Adar1-/-; p53 -/- mouse embryonic fibroblasts (MEFs) were generated. By reintroducing various ADAR isoforms into the Adar1-/-; p53 -/- MEFs I found that to rescue the aberrant immune response requires both catalytic activity and the location of an ADAR protein within the cytoplasm. Moreover, I demonstrated that transfecting inosine-containing dsRNA oligonucleotides into Adar1-/-; p53 -/- MEFs suppresses the aberrant immune response. Overall my results suggest that A-to-I editing by ADAR1 is an essential RNA modification that is required by the cell to distinguish between ‘self’ and ‘non-self’ RNA. Editing of cellular RNAs prevents an autoimmune response whereas editing of viral RNA may act to suppress a heightened antiviral immune response and prevent long-term damage to the cell.

Urinary tract infections (UTIs), including cystitis and pyelonephritis, are one of the most common infections across species. Approximately 80% of these infections are caused by uropathogenic Escherichia coli (UPEC). The host’s innate immune response is paramount in the defense against UPEC and is initiated by urinary tract epithelial cells with subsequent recruitment of leukocytes. Despite a robust immune response, UPEC can persist in the urinary tract, can cause recurrent infections, and can be resistant to antibiotic therapy. There is a growing and imminent risk for antibiotic resistant UTIs, thus necessitating investigations into alternative therapeutic options. One possibility is utilizing mechanisms to enhance existing host innate immune defenses; however, our understanding of innate immunity against UPEC, especially in the kidney, is limited. In this work, we aimed to characterize the host immune response to UPEC in cell culture and in various important mouse models of UTI to identify potential targets for augmenting host defense against UPEC.The first portion of this thesis investigates the in vitro pathogenesis of UPEC in one of the host’s most important UTI renal defenders, the kidney collecting duct intercalated cell. Previous work from our lab and others have shown that intercalated cells are required for protection against UPEC due to direct binding to these cells, their role in immune cell recruitment, and secretion of bactericidal antimicrobial peptides. We found that upon UPEC infection, intercalated cells activate multiple innate immune pathways including those associated with pattern recognition receptor (PRR) signaling. We identified that nucleotide oligomerization domain (NOD) 2 and Toll-like receptor (TLR) 4 are two intercalated cell PRRs that, upon initial activation, can protect against subsequent UPEC infection. Additionally, NOD2 activation induces upregulation of multiple innate immune pathway genes along with genes associated with various antimicrobial peptides, cytokines and chemokines. This work expanded on UPEC pathogenesis in intercalated cells and discovered a novel intercalated cell PRR, NOD2, utilized in host defense against UPEC.The second portion of this research focuses on the in vivo pathogenesis of UPEC and histopathologic characterization of three commonly used mouse models of cystitis and pyelonephritis, the C57BL/6J, C3H/HeN, and C3H/HeOuJ strains. There have been previous studies acknowledging the bacterial burden differences between mouse strains but in-depth assessments correlating urinary tract organ pathology and host innate immune responses to bacterial burden are limited. Additionally, current UTI histopathologic scoring systems are subjective and may not adequately reflect true observable pathologic differences. We developed an updated scoring system, identified stark differences in strain susceptibility to UPEC UTI, and correlated bacterial burden to organ pathology and urinary biomarker expression. Upon UPEC infection, C57BL/6J mice had rapid clearance of urine bacterial burden and cystitis without pyelonephritis. In contrast, the C3H/HeN and C3H/HeOuJ had more persistent bacterial burden but have differential susceptibility to cystitis and pyelonephritis. The C3H/HeOuJ mouse had delayed but persistent cystitis and severe pyelonephritis while the C3H/HeN mouse did not demonstrate significant pyelonephritis. Finally, urine bacterial burden and histopathology differences between strains corresponded with varying expression of urinary pro-inflammatory proteins, such as interleukin (IL)-1β, IL-6, KC/GRO (CXC ligand 1), and tissue necrosis factor (TNF)-α expression. This research demonstrates the utility of a novel UTI scoring system and highlights key strain specific bacterial burden, pathologic, and immune response differences in UPEC pathogenesis, which can impact in vivo studies aimed at treating UTIs. Lastly, given the observable protective effects of NOD2 signaling in vitro, we assessed impact of NOD2 loss in vivo and in cell culture. Upon infection with UPEC, NOD2-/- C57BL/6J mice exhibited increased urinary burden and bladder pathology with no significant differences in pyelonephritis susceptibility but had rare formations of renal urothelial intracellular bacterial communities. Unexpectedly, loss of NOD2 or disruption of NOD2 signaling in vitro did not impact UPEC attachment and invasion in intercalated cells. This work demonstrates that although NOD2 is protective, it may not be required for intercalated cell defense. In addition, given the observed pathology differences in the lower urinary tract NOD2 may have a role elsewhere in the urinary tract. In summary, this thesis addresses, in part, the complex pathogenesis of UTIs and innate immune response in both in vitro and in vivo modeling systems. We have identified a novel intercalated cell innate immune signaling pathway, NOD2, that can be activated during UPEC UTIs. We have also highlighted the spectrum of mouse model pathology during UTI and developed a novel histopathologic scoring system which can be utilized for thorough assessment of pre-clinical therapeutic efficacy.

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.

Abstract: The constant exposure of the intestine to antigens forces the mucosal immune system to be tolerant to avoid a constitutively inflamed state. Unfortunately, immune tolerance can allow colorectal cancers (CRCs) to escape detection. The majority of CRCs are chromosomally instable (CIN) and display the features of an immune ignorant tumour, including the exclusion of cytotoxic T cells (CD8+). Nonetheless, approximately 15% of all CRCs are microsatellite instable (MSI) and have high infiltrating CD8+ T cells and a more favourable prognosis as a result. A prevailing hypothesis to explain MSI CRC’s enhanced immunogenicity stems from hypermutability driving the accumulation of tumour associated antigens that stimulate an adaptive immune response. Although this is agreed to be a contributing factor, it is likely not the only influence because not all hypermutable tumours have enhanced anti-tumour immunity. One alternative explanation is that different defective DNA repair pathways may influence the growth of specific intestinal bacteria, driving immunogenicity. A major way intestinal epithelial cells communicate with microbes is through pattern recognition receptors (PRRs), including NLRP3 and STING, which have higher expression in MSI compared to other CRCs. We suspect that the activity of such receptors is aiding in MSI CRC’s better prognosis, either by recruiting cytotoxic immune cells, or by inducing programed tumour cell death. We sought to determine if MSI CRCs have altered bacteria recognition when compared to CRCs with other types of genetic instability, and if this could impact the anti-tumour immune response. We discovered that cooperation between NLRP3 and STING signaling aids in MSI CRC’s enhanced prognosis, thereby identifying new possible therapeutic targets to enhance anti-tumour immunity and immunotherapy responsiveness in the more aggressive CIN CRCs.

Pattern recognition receptors are innate immune sensors that recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) with crucial roles in host defense against microbial infection, autoimmune diseases and cancer. Cytosolic nucleic acids including DNA and RNA originate from pathogens or self-cells, which form major groups of PAMPs and DAMPs. A range of nucleic acid sensors have evolved to sense various types of nucleic acids. How different DNA-sensing pathways regulate microbial infection and cancer is the focus of this dissertation. Stimulator of IFN genes (STING) is a cytosolic innate immune sensor for cyclic dinucleotides that also serves a dual role as an adaptor molecule for a number of intracellular DNA receptors. A physiological role for STING in cancer was previously unknown. We showed that STING-deficient mice were highly susceptible to colitis-associated colorectal cancer. Colons of STING-deficient mice exhibited significant intestinal damage and overt proliferation with increased levels of pro-inflammatory cytokines during early stages of tumorigenesis, uncovering an unexpected and important role for STING in mediating protection against colorectal tumorigenesis. Absent in melanoma 2 (AIM2) forms an inflammasome with ASC and caspase-1 upon recognition of double-stranded DNA (dsDNA) in the cytosol leading to caspase-1 activation and caspase-1-dependent pyroptosis and release of cytokines IL-1 and IL-18. Mutations in AIM2 are frequently identified in patients with colorectal cancer, but how AIM2 modulates colonic tumorigenesis is unknown. We found that AIM2-deficient mice were hypersusceptible to colonic tumor development. While production of inflammasome-associated cytokines and other inflammatory mediators was largely intact in AIM2-deficient mice, intestinal stem cells lacking AIM2 were prone to uncontrolled proliferation. Aberrant Wnt signaling expanded a population of tumor-initiating stem cells in the absence of AIM2 driving the tumor development in AIM2-deficient mice. In addition to its role in cancer, AIM2 recognizes bacteria including Francisella tularensis subspecies novicida (F. novicida) and induces inflammasome responses. Type I interferon (IFN) signaling drives activation of AIM2 inflammasome in F. novicida-infected macrophages; however, the relative contribution of IFNs and inflammasome responses in host defense against F. novicida infection is less understood. We found intact AIM2 inflammasome responses in mice lacking type I IFN signaling during infection with F. novicida. Lack of type I IFN signaling conferred protection to F. novicida infection in contrast to the increased susceptibility in AIM2-deficient mice. Interestingly, mice lacking both AIM2 and IFNAR2 were protected against the infection indicating a dominant role for type I IFNs in mediating detrimental responses despite the protective AIM2 inflammasome responses. Gasdermin D (GSDMD) is activated by caspase-1 to generate pores on the plasma membrane to induce pyroptosis downstream of the AIM2 inflammasome. We also demonstrated that mice lacking GSDMD were highly susceptible to F. novicida infection. Interestingly, GSDMD is required for optimal caspase-1 activation during F. novicida infection, providing protection to the host during the infection. In addition, we identified differential mechanisms regulating expression of inflammasome-associated cytokines IL-1β and IL-18. IL-1β is only induced in response to inflammatory stimuli and its expression is not sustained during chronic treatment, while IL-18 is constitutively expressed and further induced after the stimulation in a type I IFN signaling-dependent manner. Overall, this dissertation addresses protective roles for cytosolic nucleic acid-sensing molecules, STING and AIM2, in colon cancer, uncovers an interplay between the AIM2 inflammasome and type IFN signaling during F. novicida infection, and demonstrates a novel function for GSDMD in regulating AIM2 inflammasome. The dissertation finally describes distinctive mechanisms governing inflammasome-associated cytokines IL-1β and IL-18.

OBJECTIVES: Bacterial infections in the normally sterile environment of the middle ear cavity usually trigger host immune response, whereby the innate immune system plays a dominant role as the host’s first line of defense. In this study we evaluated the expression levels of Toll-like receptors (TLRs) -2, -4, -5, -9, and nucleotidebinding oligomerization domain-containing proteins (NODs) -1 and -2, all of which are related to bacterial infection in pediatric patients with otitis media with effusion (OME). METHODS: The study sample consisted of 46 pediatric patients with OME, all of whom had ventilation tubes inserted. The expression levels of TLR-2, -4, -5, -9, NOD-1 and -2 mRNA in middle ear effusion were assessed by polymerase chain reaction. RESULTS: All effusion fluid samples collected from patients with OME showed expression of TLR-2, -4, -5, -9, NOD-1, and -2 mRNA. However, we found no correlations among expression levels of pattern recognition receptors (PRRs) in relation to characteristics of exudates, presence of bacteria, or frequencies of ventilation tube insertion (p>0.05). CONCLUSION: Our findings suggest that exudates of OME patients show PRR expressions that are related to the innate immune response regardless of the characteristics of effusion fluid, presence of bacteria in exudates, or frequency of ventilation tube insertion.

Type I interferon (IFN) regulates innate and adaptive immunity. It protects host cells against viral infection. Many microorganisms have evolved successful mechanisms to inhibit host type I IFN response. Excessive type I IFN is associated with autoimmune diseases such as systemic lupus erythematosus (SLE). Studying the negative regulation of type I IFN will help understand the pathogenesis of infectious diseases and potential therapeutic control of type I IFN in SLE. In murine bone marrow derived dendritic cells (DCs), we found two mechanisms that can inhibit type I IFN production in DCs. First, CpG-B ODNs, previously known as weak IFN inducers, globally inhibit type I IFN production by multiple Toll-like receptor (TLR)-dependent and –independent pathways. It involves transcriptional regulation of first-wave type I IFN induction downstream of multiple receptors by inhibiting binding of IRF1 and chromatin remodeling at the IFN-β and IFN-α4 promoters. Second, M. tuberculosis and TLR2 agonists specifically inhibit MyD88-dependent (TLR7/TLR9-induced) type I IFN induction. The inhibition is mediated through a new protein synthesis –independent direct intracellular mechanism that is correlated with rapid depletion of IRAK1. Overall, further investigation of these mechanisms may help us understand the role of type I IFN in infections and vaccines when multiple TLR agonists are present and may provide therapeutic targets for controlling type I IFN production in vivo.

Infections with rhinovirus (RV), a single-stranded Picornavirus, are the most frequent cause of upper respiratory tract infections (“common colds”). RV also causes exacerbations of chronic lower airways diseases such as asthma, a major source of morbidity and health care expenditure. Little is known about how RV causes exacerbations of lower respiratory tract disease. This thesis examines mechanisms underlying the innate immune response to RV infection, focusing on the pattern recognition receptors required for interferon (IFN) responses. Viral double-stranded RNA produced during replication may be recognized by either Toll-like receptor (TLR)-3, retinoic acid-inducible gene (RIG)-I or melanoma-differentiation-associated gene (MDA)-5. We examined the requirements of RIG-I, MDA5 and IFN response factor (IRF)-3 for RV-induced gene expression in human bronchial epithelial cells. RV-1B induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFNs and IFN-stimulated genes. siRNA against IRF3, MDA5 and TRIF, but not RIG-I, decreased RV1B-induced gene expression and IRF3 dimerization. These results suggest that, in cultured epithelial cells, TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA, and that TLR3 and MDA5 signal through IRF3. The overall airway response to viral infection represents an integration of antiviral and pro-inflammatory pathways. Also, other cells besides airway epithelial cells may contribute to the RV immune response. Therefore, we examined the responses of TLR3- and MDA5-deficient mice to RV1B infection. TLR3-/- mice showed normal IFN responses and viral clearance. MDA5-/- mice showed delayed type I and attenuated type III IFN responses, leading to a transient defect in viral clearance. RV-infected TLR3-/- and MDA5-/- mice showed reduced airway inflammation and responsiveness. Finally, RV-infected MDA5-/- mice with allergic airways disease showed enhanced viral clearance despite deficient IFN responses, as well as decreased inflammatory and contractile responses. Together, these results suggest that MDA5 is the major receptor required for sensing RV dsRNA and initiating innate immune responses. Also, TLR3 and MDA5 initiate pro-inflammatory signaling pathways leading to airways inflammation. Thus, in the context of RV infection, TLR3 and MDA5 signaling is maladaptive. Therapies against TLR3 and MDA5 may be useful in the treatment or prevention of RV-induced asthma exacerbations.