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Mononuclear phagocytes play a key role in tissue homeostasis and in host defense against pathogen invasion. However, the phenotypic identities and functional properties of this heterogeneous population within the peritoneal cavity remains poorly elucidated. In the context of peritoneal dialysis, it is hypothesized that the modification of macrophage/dendritic cell biology by the dialysis process would alter tissue homeostasis, host susceptibility to infection and local immunity, compromising long-term outcomes of the patients. The research work carried out in this thesis has tested this hypothesis by examination of the immunobiology (mainly phenotype and function) of human and murine peritoneal macrophage and dendritic cell subsets. Moreover, the potential link between the altered immunobiology of peritoneal mononuclear phagocytes and clinical outcomes of the dialysis patients has been investigated. Several key findings were generated during these studies: 1) multiple discrete peritoneal macrophage and dendritic cell subsets have been phenotypically identified in humans (from different clinical settings of peritoneal dialysis) and in mice (from naïve and inflamed conditions); 2) in humans, the phenotypes and the activation/maturation status of peritoneal macrophages and dendritic cells are modified throughout the dialysis course and also during acute peritonitis; some characteristic alterations are associated with adverse patient outcomes; 3) in mice, the distinctive kinetics of the respective peritoneal macrophage and dendritic cell subsets in clinically relevant acute peritonitis models have been characterized; 4) individual peritoneal macrophage and dendritic cell subsets (human and mice) display differences in their phagocytic capacity and the ability to process and present antigen.

The strategy of the adaptive immune system in eliminating viruses from infected tissues is the activation of CD8+ T cells with specific T cell receptor in the LN draining the site of virus entry and subsequent migration of these cells to the sites of the viral infection. L-selectin, a well characterized LN homing receptor, is variably expressed on virus peptide activated CD8+ T cells, regulated through two separate mechanisms of early ectodomain shedding and late gene silencing. The role of L-selectin in homing of activated CD8+ T cells to sites of virus infection is not studies in detail. Here we show that despite being primed normally in the draining LN, there were a hierarchy in homing ability of adoptively transferred CD8+ T cells expressing mutant L-selectin(which resist shedding and gene silencing upon T cell activation), wildtype Lselectin and deficient in L-selectin (Ko) to the site of virus infection. The Lselectin specific recruitment was confirmed by using antibody blockade strategy and short-term competitive homing experiments. Furthermore, Lselectin dependent homing of virus specific CD8+ T cells rather than hyperfunctional or hyperproliferative T cells conferred anti-viral immunity against two evolutionarily distinct viruses, vaccinia and influenza viruses which infect mucosal and visceral organs, respectively.

Human Vγ9Vδ2 T cells constitute a novel type of APC (γδ T-APCs) capable of stimulating CD4+ T cell responses. The outcome of γδ T-APC induced CD4+ T cell responses in terms of cytokine profiles, and the physiological implications for infection and autoimmunity, remain unknown. This study demonstrates that γδ T cells are able to act as potent APCs, inducing proliferation in naive and memory CD4+ T cell populations. Resulting cytokine responses triggered in naive CD4+ T cells included production of IFN-γ and IL-22. Of note, γδ T cells had a greater capacity to promote production of IL-22 in naive and memory T cells than monocytes and monocyte-derived DCs in identical experiments. The microenvironment of γδ T-APCs played a major role in the subsequent polarisation of CD4+ T cell responses, with APCs induced in the presence of IL-15 being superior in promoting IL-22 responses in naive CD4+ T cells compared to γδ T-APCs generated in the presence of other cytokines. Unexpectedly, the IL-22 induction in CD4+ T cells was IL-6 independent, but instead involved TNF-α and ICOS-L, both expressed by the γδ T-APCs. In addition, γδ T-APCs induced in the presence of IL-21 favoured increased induction of IL-10 in CD4+ T cells. The observation that γδ T-APCs are able to drive IL-22 responses in naive and memory T-cell populations presents a novel function for these APCs, with implications for a multitude of infection/disease scenarios. One such scenario is Inflammatory Bowel Disease (IBD), where IL-22 and γδ T-cells have previously been shown to play significant roles in disease pathogenesis and progression. Indeed, γδ T cells derived from intestinal biopsies are able to act as fully functional APCs. In summary, γδ T-APCs may be involved in the pathogenesis or maintenance of autoimmune inflammation in the intestine and other peripheral sites.

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.

Despite advances in treatment, peritoneal dialysis (PD)-associated peritonitis remains a major cause of morbidity and mortality in PD patients. Given that peritonitis can be the proximate cause of technique failure and cause ultrafiltration failure at a later time, it is important to understand the peritoneal immune response, microbiology and outcomes of these infections. Data presented in this thesis have shown that leukocytes are recruited to the peritoneal cavity, starting with a rapid accumulation of neutrophils, which are later replaced by a population of mononuclear cells, including monocytes/macrophages and T cells during acute peritonitis. Of note, Vγ9/Vδ2 T cells are also recruited to the peritoneal cavity in the early stage, which implies a significant role of Vγ9/Vδ2 T cells as early responders in acute peritonitis. In patients with acute peritonitis, the capacity of the causative pathogen to produce (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), together with the infiltration of activated Vγ9/Vδ2 T cells are important risk factors and possible predictors of patient outcomes from infection. By performing a detailed immunological and microbiological analysis in PD patients on the first day of peritonitis, our findings provide proof of concept that acute bacterial infections indeed leave characteristic disease-specific ‘immune fingerprints’ of diagnostic and prognostic value. Local fingerprints not only discriminated between episodes of culture-negative and culture-positive PD-associated peritonitis but also predicted infections caused by Gram− or Gram+ bacteria. HPMC play an important role in maintaining homeostasis of the peritoneal immunity. Our data revealed the regulation of Vγ9/Vδ2 T cells by HPMC and demonstrated that resting HPMC were potent suppressors of Vγ9/Vδ2 T-cell cytokine production and proliferation in the presence of HMB-PP. Collectively, these findings improve our insight into the complex cellular interactions in PD-associated peritonitis and peritoneal homeostasis, identify novel biomarkers of possible diagnostic and predictive value and highlight new avenues for therapeutic intervention.

The recruitment of peripheral blood lymphocytes (PBL) to sites of inflammation and their subsequent traffic into the lymphatic circulation is important in host defense. However, surprisingly little is known about their recruitment from the blood vasculature into inflamed tissue, and almost nothing about their egress from inflamed tissue via the lymphatic circulation. We showed that both human macrovascular and microvascular endothelial cells stimulated by TNF\(\alpha\) and IFN\(\gamma\), preferentially recruited memory T-lymphocytes (CD45RO positive cells) from a mixed pool of PBL. T-cells that had migrated across vascular endothelial cells subsequently utilised a combination of \(\beta\)1 and \(\beta\)2 integrins to traverse cytokine activated lymphatic endothelium. In addition we provide evidence that PGD2 was critical for the transmigration of lymphocytes through vascular endothelium. The process of trans-lymphatic migration was also significantly retarded in the presence of a function neutralising antibody against CCR7. Most importantly, we observed that memory T-cells showed a markedly enhanced capacity to migrate across lymphatic endothelium if they had first traversed a vascular endothelial cell barrier. We have shown that addition of exogenous PGD2 to isolated lymphocytes is able to restore the enhanced migration capacity of lymphocytes that have previously migrated through a vascular monolayer. The nature of the priming signal delivered by the process of migration across blood vessel endothelium remains to be fully identified, but is likely to be important in regulating the dynamics of an inflammatory response.

Multidrug resistance (MDR) efflux pumps and biofilm formation are two mechanisms by which bacteria can evade the action of many antimicrobials. MDR efflux pumps confer low level multidrug resistance and are over-expressed in MDR clinical isolates. Biofilms are three dimensional, complex communities of bacteria encased in a self produced extra cellular matrix. Biofilms protect the bacteria within them both physically by acting as a barrier to any external threats, and metabolically by containing high proportions of persister cells. This thesis explores the link between MDR efflux and biofilm formation in S. Typhimurium and shows that genetic inactivation of any one of the nine MDR efflux systems results in a biofilm defect. We found that the transcriptional repression of curli, an essential component of the Salmonella matrix, is the reason for the efflux mutants' inability to form a biofilm. The biofilm defect in a tolC mutant was rescued by inactivation of a gene encoding an osmolarity sensor, envZ, suggesting that membrane stress response is a possible link between efflux and biofilm formation. Chemical efflux inhibitors (EIs) were found to impart curli repression and cause subsequent biofilm defect. This finding is clinically important as biofilms are a major cause of infection EIs could be a potential novel anti-biofilm therapy.