Coinfection with bacteria and viruses is an understudied area of microbiology, despite its potential to modulate pathogen abundance and host survival. We investigated the effect of bacteria on virus infection and developed an in vitro system to study the first step: viral internalization. Our studies show that multiple bacterial species promote the entry of a diverse panel of viruses into lung and gut epithelial cells. Bacteria expressing the toll-like receptor (TLR)5 agonist, flagellin, are most efficient at inducing viral uptake and studies using recombinant flagellin or aflagellate bacterial strains confirm that flagellin has pro-viral activity. Flagellin promotes epithelial cells to support virus entry via TLR5-dependent activation of NF-KB. To extend these observations and study the role of flagellin in the complete viral replicative lifecycle, we studied human immunodeficiency virus (HIV)-1 replication in T cells. Flagellin augments HIV-1 entry and promoter activity and increases the production of extracellular virus. The data presented in this thesis highlight a new role for bacterial flagellin to promote diverse virus infection of epithelial barriers and enhance the spread of HIV-1. This has significant implications for understanding how exposure to multiple pathogens can alter susceptibility to infection and its associated pathogenesis.

Outer membrane vesicles (OMVs) are nanosized proteoliposomes ubiquitously released from the outer membrane of Gram negative bacteria, and are known to contribute to immune priming and disease pathogenesis. However, the current understanding of their interactions with host cells is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery. This work has developed a highly sensitive method to study vesicle entry into host cells in real-time using a genetically encoded, vesicle targeted probe. Using this approach, it was found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of O polysaccharide in lipopolysaccharide creates a bias towards non-receptor mediated endocytosis, which enhances both the rate and efficiency of entry into host cells. This work indicates that the composition of the bacterial cell wall influences the behaviour of OMVs, and is therefore implicated in secretion-system independent delivery of bacterial virulence factors during Gram negative infection.

Hepatocytes can capture dead cells. This phenomenon is called efferocytosis. Furthermore, our lab previously observed live CD4+ T cells captured by hepatocytes. This was reminiscent of entosis. This project aimed to further the understanding of the mechanisms and consequences of these processes. In vitro experimentation showed that efferocytosis could be modulated through cytokine treatment and using macropinocytosis inhibitors. Captured cells were also shown to associate with the uncharacterised receptor, SCARF2. Furthermore, efferocytosis was shown to cause multinucleation in hepatocytes. This was demonstrated in vitro, in vivo using mouse models of acute injury, and ex vivo with cauterised donor human tissue. Increased multinucleation was also associated with hepatocellular carcinoma and vascular invasion. Live CD4+ T cell capture occurred at a lower frequency than efferocytosis and required alternative membrane rearrangements. This process also did not share defining characteristics of entosis, such as E-cadherin association or susceptibility to Rho-kinase inhibitors. Furthermore, anti-inflammatory T-regulatory cells were more likely to enter acidic compartments within their captors in vitro. This project has unearthed novel aspects regarding the regulation and molecular processes of hepatocyte cell-in-cell structure formation. Further understanding into the mechanisms of these processes may provide future targets for therapeutic intervention of inflammatory disease and cancer.

Invariant natural killer T (iNKT) cells are endowed with features of both innate and adaptive immunity. They are activated by the recognition of glycolipid agonists presented by CD1d which makes them excellent candidates for cellular therapies. In order to investigate the ability of iNKT cells to suppress experimental acute Graft versus host disease (GVHD), iNKT cells were first expanded in vitro, which is likely to be required prior to their use as a cellular therapeutic. Interestingly, the expanded cells showed increased frequencies of IL-10, IL-13 and IL-17 producing cells and were found to robustly suppress alloreactive T cell proliferation in vitro compared to freshly isolated cells. However, in a model of acute GVHD induced by alloantigen-reactive TCR-transgenic T cells neither freshly isolated nor expanded iNKT cells suppressed GVHD, although some survival benefit was seen following the activation of host iNKT cells. These data indicate that iNKT cells can be expanded ex vivo, that they can acquire different functional properties and that such cells robustly suppress alloreactive T cell proliferation in vitro. Therefore, further investigation into the suppressive behaviour of these cells is warranted despite a failure to suppress acute GVHD in the current study.

TNF receptor/ligand superfamily members signal through pathways giving rise to proteins that regulate lymphocyte proliferation, activation, differentiation and survival. Absence of TNF ligands OX40L and CD30L impairs survival of GC T cells and affinity maturation of antibody responses. Direct effects of these molecules on B cells in antibody responses are not characterised. I dissected roles of OX40 and CD30 for B cells using T-independent type II (TI-II) antigen NP-Ficoll. Humoral immunity is impaired in OX40 deficiency. Defects in class switched and non-class switched antibody production are due to reduced development of antigen-specific switched and non-switched plasma cells. CD30 has an opposing role, deficiency results in similar or higher switched and non-switched antibody titres and higher numbers of antigen-specific plasma cells that develop rapidly. This may explain why in OX40/CD30 double deficiency, there is a less pronounced defect than in OX40 single deficiency. B cell intrinsic roles are revealed for OX40 and CD30 that suggest OX40 on B cells is critical for TI-II plasmablast differentiation or survival and B cell CD30 inhibits onset of plasmablast differentiation.

Innate immunity is a branch of the immune system that is responsible for controlling the early events of pathogen infection. One of the key components of the innate immune systems arsenal are the interferon (IFN) cytokines. IFNs are small signalling proteins that are released by cells in response to invading pathogens, and viruses in particular. They are named for their ability to interfere with viral replication. The result of IFN signalling is the up-regulation of a diverse collection of genes termed interferon-stimulated genes (ISGs). These genes act in synchrony to limit the replication of viruses. The protein products of ISGs are involved in a multitude of cellular pathways that limit replication and additionally intercept viral proteins and nucleic acid directly. Some of these ISGs are mediators of an important cell-death response, apoptosis. Apoptosis is a vital component of innate immune signalling and controls viral replication by sacrificing the infected cell to limit further infection of neighbouring cells. The function of specific ISGs in mediating this response is poorly understood.

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Introduction Autophagy is a cellular process whereby elements within cytoplasm become engulfed within membrane vesicles and trafficked to fuse with lysosomes. This is a common cellular response to starvation, allowing non-essential cytoplasmic contents to be recycled in times of energy deprivation. However, autophagy also plays an important role in immunity and inflammation, where it promotes host defence and down-regulates inflammation. A specialised bacterial virulence mechanism, the type III secretion system (T3SS) in Pseudomonas aeruginosa (PA), an extracellular bacterium, is responsible for the activation of the inflammasome and IL-1β production, a key cytokine in host defence. The relationship between inflammasome activation and induction of autophagy is not clear. Hypothesis and aims The central hypothesis is that induction of autophagy occurs following PA infection and that this process will influence inflammasome activation in macrophages. Our aims were to determine the role of the T3SS in the induction of autophagy in macrophages following infection with PA, and to investigate the effects of autophagy on inflammasome activation and other pro-inflammatory pathways following infection with these bacteria. Materials and methods Primary mouse bone marrow macrophages BMDMs were infected with PA, in vitro. Induction of autophagy was determined using five different methods: - electron microscopy, immunostaining of the autophagocytic marker LC3, FACS, RT-PCR assays for autophagy genes, and post-translational conjugation of phosphatidylethanoloamine (PE) to LC3 using Western blot. Inflammasome activation was measured by secretion of active IL-1β and caspase-1 using ELISA and Western blot. Functional requirements of proteins were determined using knockout animals or SiRNA mediated knockdown. Result and Conclusions PA induced autophagy that was not dependent on a functional T3SS but was dependent on TLR4 and the signaling molecule TRIF. PA infection also strongly induced activation of the inflammasome which was absolutely dependent on a functional T3SS. We found that inhibition of inflammasome activation increased autophagy, suggesting that the inflammasome normally inhibits this process. Further experiments showed that this inhibitory effect was due to the proteolytic action of caspase-1 on the signaling molecule TRIF. Using a construct of TRIF with a mutation in the proteolytic cleavage site, prevented caspase-1 cleavage and increased autophagy. TRIF is also involved in the production of interferon-β following infection. We also found that caspase-1 cleavage of TRIF down-regulated this pathway as well. Caspase-1 mediated inhibition of TRIF-mediated signaling is a novel pathway in the inflammatory response to infection. It is potentially amenable to therapeutic intervention. Recognition of a pathogen infection is a key function of the innate immune system that allows an appropriate defensive response to be initiated. One of the most important innate immune defences is provided by a multi-subunit cytoplasmic platform termed the inflammasome that results in production of the cytokine IL-1β. The human pathogen Pseudomonas aeruginosa activates the inflammasome following infection in a process that is dependent on a specialized bacterial virulence apparatus, the type III secretory system (T3SS). Here, we report the novel finding that this infection results in mitochondrial damage and release of mitochondrial DNA into the cytoplasm. This initiates activation of an inflammasome based on the protein NLRC4. Autophagy induced during infection removes damaged mitochondria and acts to down-regulate NLRC4 activation following infection. Our results highlight a new pathway in innate immune activation following infection with a pathogenic bacterium that could be exploited to improve outcomes following infection.

Much is known about the disease pathology related to schizophrenia, however, little is known with regards to its aetiology. Recent evidences suggest a neurodevelopmental hypothesis for schizophrenia where environmental factors including: infection, stress and malnutrition, can adversely affect the pregnant mother thereby elevating the risk for schizophrenia developing in the offspring during adulthood (Meyer et al., 2008d; Meyer and Feldon, 2009; 2012; Forrest et al., 2012; Meyer, 2013). Since a variety of viral and bacterial infections in animal models have demonstrated to increase the risk in schizophrenia, it is proposed that factors common to the immune response may mediate this link. While many laboratories have reported several behavioural abnormalities following maternal immune activation, we sought to examine molecular changes following poly(I:C) exposure, a synthetic viral mimetic, in the pregnant mother and assessed a range of protein markers with known developmental roles, since an appreciable understanding of the molecular alterations taking place would permit suitable therapies to follow. Interestingly, poly(I:C) was able to induce a range of changes resembling those observed during schizophrenia, where the major NMDA receptor subunit GluN1 and α-Synuclein was reduced in postnatal day 21 animals born to mothers treated with poly(I:C) during gestation days 14, 16 and 18. Furthermore, these changes suggest a mechanism by which maternal immune activation may lead to the subsequent emergence of schizophrenia. Another aspect of this work examined the role of the kynurenine pathway on brain development. There is increasing evidence suggesting the involvement of the kynurenine pathway, a biochemical pathway responsible for the oxidative metabolism of tryptophan, in the disease pathology of schizophrenia, including neurodegenerative disorders such as Parkinson’s, Alzheimer’s and Huntington’s disease (Giorgini et al., 2005; Ting et al., 2009; Bonda et al., 2010). Since immune activation induces the activation of the kynurenine pathway, it was hypothesised that alterations in central kynurenine concentrations during development may be involved in mediating the subsequent increased risk for schizophrenia (Forrest et al., 2013, Khalil et al., 2013, 2014). As very little is known about the physiological activity of the kynurenine pathway during development, we sought to examine the potential consequence of disrupting this pathway and examining its effects upon brain development. Therefore, a kynurenine monooxygenase inhibitor, Ro61-8048, was administered to pregnant rats during gestation day 14, 16, and 18, that would inhibit the synthesis of the neurotoxic metabolite quinolinic acid, while redirecting the pathway to increase the neuroprotectant kynurenic acid. Brain development was assessed by examining changes in protein expression of markers intimately involved in synaptic transmitter release machinery, neurogenesis and many aspects of neuronal development. Interestingly, we found the kynurenine pathway is highly active during brain development, and induces a variety of changes in protein markers that may be involved in precipitating a range of neuronal and cognitive deficits. While Ro61-8048 induced no changes in the embryo brains at 5 and 24 h following treatment, delayed changes were seen in postnatal day 21 animals displaying a decrease in RhoB expression as examined in the western blots. Since the full blow symptoms of schizophrenia become apparent during early adulthood, we sought to examine any changes in protein expression in postnatal day 60 animals in regions of the cortex, hippocampus, midbrain and cerebellum. Interestingly, profound alterations were seen in doublecortin and the netrin receptors responsible for axonal guidance. Perhaps the most striking protein change in the postnatal day 60 animals is the significant alteration induced in the expression of disrupted in schizophrenia (DISC)-1, a protein strongly linked with schizophrenia. Glutamate function was assessed as indicated by the density of glutamate transporters, VGLUT-1 and VGLUT-2, in the CA1 region of the hippocampus of postnatal day 60 animals using immunocytochemistry. While the relative density of glutamate transporters were substantially increased, there were no changes in the GABA transporters, indicating that while GABA transmission remained the same, glutamate function may have increased in the absence of an increase in synaptic connections. Spine densities of pyramidal neurons in the hippocampus were also examined, using the golgi-impregnation method, to reveal a significant loss in spines of the apical and basal dendrites, consistent with reports in schizophrenia. To conclude, the kynurenine pathway is highly active during development, and alterations in central kynurenines during pregnancy, as induced by environmental factors such as stress and infection, may be involved in the subsequent emergence of neurodevelopmental disorders.