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Flagellin is a highly immunogenic, bacterial protein considered to be abundant in the intestinal lumen. It has been reported to be an immunodominant antigen in patients with inflammatory bowel disease (IBD). In the work presented in this thesis several complementary murine models of IBD were employed to elucidate the role of innate immune sensing of flagellin in the development of intestinal inflammation. Pattern recognition receptors (PRRs) enable mammals to discriminate self from non-self through the recognition of microbial signatures, such as bacterial flagellin. Flagellin is detected by at least two distinct PRRs, NOD-like receptor 4 (NLRC4) and Toll-like receptor 5 (TLR5). Our experiments revealed that NLRC4 promoted protective effects during acute intestinal inflammation mediated by infection with Citrobacter rodentium, a close relative of enteropathogenic E. coli. Following infection with C. rodentium, Nlrc4^-/- mice developed more severe weight loss, increased bacterial colonisation levels and exacerbated intestinal inflammation compared to WT counterparts. Bone marrow chimera experiments revealed that NLRC4 expression in non-hematopoietic cells provided protection and intestinal epithelial cells expressed high NLRC4 mRNA levels. These results suggest that NLRC4 inflammasome activation in the intestinal epithelium provides potent, protective effects during infection with a mucosal pathogen. In contrast, TLR5 was shown to promote protective effects during chronic, T-cell mediated intestinal inflammation driven by adoptive transfer of naïve CD4+ T-cells into lymphopenic Rag^-/- hosts. The absence of TLR5 in Rag^-/- recipients resulted in accelerated and exacerbated IBD. Furthermore, chronic T-cell dependent colitis driven by Helicobacter hepaticus, a flagellated, enteric bacterium, was more severe in mice deficient in TLR5. Finally, construction of a H.hepaticus Type 6 secretion system deletion mutant revealed delayed pathogenicity in an innate model of intestinal inflammation, most likely due to reduced initial colonisation of mutant H.hepaticus.

Probiotics confer health benefits through many mechanisms including modulation of the gut immune system. Gut mucosal macrophages play a pivotal role in driving mucosal immune responses. The local environment and macrophage subset determine immune response: tolerance, associated with an M2-like, regulatory macrophage phenotype and inflammatory activation with an M1-like phenotype. The aims of this study were firstly to investigate the immunomodulatory effects of a panel of heat-killed (HK) probiotic bacteria and their secreted proteins (SP) of Bifidobacterium breve (BB), Lactobacillus rhamnosus GG (LR), L. salivarius (LS), L. plantarum (LP), L. ferrmentum (LF), and L. casei strain Shirota (LcS) on cytokine production and TLR expression in monocultures of monocytes, macrophage subsets, and intestinal epithelial cells. Normally, mucosal gut macrophages resemble the M2 subset and fail to express CD14, a co-receptor for LPS signalling. Therefore, probiotic modulation of LPS-induced NF-kB activity and cytokine expression was investigated using a THP-1 monocyte-derived reporter cell line, model of CD14 hi / lo M1 and M2 macrophages. Secondly, a transwell co-culture system was developed to investigate probiotic modulation of macrophage-influenced epithelial barrier function. Parameters investigated included cytokine, TLR and hBD-2 expression, TEER and IHC staining of the tight junction protein, ZO-1. Probiotics selectively modulated monocyte and macrophage subset cytokine expression. Probiotics (HK and SP) suppress CD14 lo , augment CD14 hi M1, and differentially regulated TNF-α production in M2s. M2 macrophage IL-6 production was suppressed by both HK and SPs, and differentially regulated in CD14 lo and CD14 hi M1s. NF-κB activation failed to parallel probiotic regulation of TNF-α and IL-6. Probiotics (HK-LF and HK-LcS) selectively modulated both endogenous and exogenous TNF-α and IL-10, as well as their induction of epithelial cell expression of TLR and hBD-2. Epithelial expression of TEER, ZO-1 and the endogenous TLR signal regulator, Tollip, were suppressed upon co-culture with pro-inflammatory M1 macrophages paralleled by a suppression of IL-10 and up-regulation of TNF-α and IL-8. In the presence of LPS, HK-LF enhanced TEER, ZO-1 and partially rescued Tollip expression, whereas HK-LcS had no effect on TEER and ZO-1 and displayed a weaker rescue effect on Tollip compared with LF. In the M2/epithelial cell co-culture, both probiotics enhanced TEER and ZO-1 in the presence of LPS, whilst displaying a differential modulation of Tollip, dependant on the format of probiotic (HK or SP). In conclusion, probiotic strains can differentially exert immune activatory or suppressive functions and immunomodulation is determined by strain, inflammatory environment, and mucosal macrophage effector phenotype. Future probiotic development must consider prophylactic use in healthy individuals or therapeutic treatment of defined pathological conditions, strain-specific effects, gut mucosal integrity, and immune phenotype of mucosal macrophages.

The HIV/AIDS pandemic continues to be one of the most devastating global health pandemics in history. With women accounting for approximately 60% of all new HIV infections, preventative strategies that provide women with the ability to protect themselves is imperative. To this end, identifying natural factors expressed in the female genital tract (FGT) capable of inhibiting HIV may prove to be novel candidates for female-controlled microbicide preventative strategies. The work in this thesis examined the differences in CVL (cervicovaginal lavage) composition between HESN (HIV-exposed seronegative) women and HIV-susceptible women. Distinct differences in the female genital tract proteomes, and HIV inhibitory activity exhibited by CVL, were observed among women highly exposed to HIV compared to women at lower exposure. Furthermore, while HESN women as a group did not demonstrate stronger inhibitory effects compared to susceptible women from the Pumwani cohort, it was apparent that CVL from individual women was capable of inhibiting HIV consistently over longitudinal analysis. From the antiproteases that were identified as over-expressed within the CVL of HESN women, Serpin B1 exhibited the strongest and most consistent HIV inhibitory activity. The mechanism for this activity does not appear to be directly against HIV but rather through effects exerted on HIV target cells. Specifically, Serpin B1 alters the proliferative capacity and induces early apoptotic markers on these cells. Proteomic pathway analysis of the proteins over-expressed following treatment, suggests that Serpin B1 may up-regulate the expression of proteins associated with inhibition of the mTOR pathway. This inhibition may be caused by induction of increased production of ROS (reactive oxygen species) by macrophages or through Granzyme A activity, and subsequent dysfunction of the mitochondria, potentially inducing an autophagic state. However, this would need to be confirmed with further molecular studies. These results defined a potential mechanism of HIV inhibition for Serpin B1. Hence, the overabundance of Serpin B1 in the CVL of HESN women may, in fact, be contributing to their protective phenotype against HIV infection. These findings suggest that Serpin B1 could be considered as a candidate in future microbicides. However, these findings must be validated in in vivo models. / February 2017

The intestinal tissue is charged with a delicate immunological task. The intestinal immune system needs to be tolerant towards nutrients and microbiota present in the intestinal lumen, while simultaneously detecting and responding to dangers such as pathogens. A single-cell layer of intestinal epithelial cells (IECs) acts as a first line of defence. There is a T cell population located between the IECs that have been named intraepithelial lymphocytes (IELs). As the main lymphoid population within the intestinal barrier, IELs are thought to have an important role in intestinal homeostasis maintenance, as well as a role in intestinal inflammatory and autoimmune diseases such as inflammatory bowel disease and celiac disease. Despite extensive research on IEL biology, there are still questions remaining in terms of the development, maintenance and activation of IELs. Furthermore, IELs survive poorly in vitro, which hinders mechanistic insights. In this thesis, a co-culture system between IELs and intestinal organoids, "mini-guts", provides an in vitro model for IELs. With this IEL-organoid co-culture system, IELs associated with the organoids survive for at least 4 days. Additional findings suggest that IELs are kept in a poised state of activation due to differences in their mitochondria compared to other T cells found in spleen, lung and skin. Upon activation or intestinal inflammation, the mitochondrial mass in IELs increases. This increase correlates with effector functions such as cytokine production and proliferation. In addition, the composition of the mitochondria-specific lipid, cardiolipins, alters drastically in IELs after activation. These data support a model of mitochondria-dependent activation of IELs. The mitochondria-dependent activation in IELs appears to have at least two pathways: one T cell receptor-dependent and one microbiota-dependent. The latter pathway suggests a model in which IELs can become activated regardless of the cause of intestinal epithelial barrier damage.

Efficient delivery of vaccines and therapeutic agents in vivo is a critical aspect for ensuring a desired immunological or biological response is achieved. This work focuses on developing effective delivery systems for vaccines and therapeutics to achieve biological potency while maintaining patient friendly administration. Traditional vaccines are administered via parenteral injection, which requires skilled personnel for administration and does not elicit strong mucosal immune responses. An alternative approach is to develop an oral vaccine; however, this requires antigens to be protected during transit through the gastrointestinal tract and be transported across specialized intestinal sampling cells called M cells. These M cells are extremely rare, making them an important target for oral vaccines. The protein invasin, from Yersinia psuedotuberculosis, naturally binds [mathematical symbols] integrin, a receptor found exclusively on M cells within the gastrointestinal tract. Therefore, we generated combinatorial libraries of invasin, followed by a directed evolution and high-throughput screening strategy to identify invasin variants with increased affinity towards [mathematical symbols] integrin. This process led to the creation of an invasin variant exhibiting a nine-fold decrease in EC₅₀, which could be used for targeted oral vaccine systems. In order to test for increased vaccine efficacy due to the engineered invasin ligand, we developed a polymeric microparticle delivery system. These microparticles were formulated to encapsulate the model antigen ovalbumin and be decorated with the invasin targeting ligands. To measure physiological trafficking and intestinal retention, novel fluorescent nanocrystals were loaded into particles conjugated to invasin. These nanocrystals served as a contrast agent for in vivo imaging in mice. While these particles were unsuccessful in generating an antibody response toward ovalbumin when administered to mice, a response directed to the targeting ligand itself was observed. These findings provide insights for further optimizing a delivery system for oral vaccination. In addition to developing an oral vaccine delivery system, we created a high concentration therapeutic protein formulation, suitable for low-volume subcutaneous administration. By adding crowding agents, we were able to generate reversible protein nanoclusters with low viscosity. These nanoclusters were found to revert to monomer upon dilution and pharmacokinetic profiles similar to solutions. / text