Members of the Mycobacterium tuberculosis complex are the causative agents of tuberculosis, a major global health threat to human populations. The majority of infected individuals harbour a latent infection where bacteria persist in a non-replicating, dormant state. Resuscitation promoting factors (Rpfs) are secreted proteins whose peptidoglycan hydrolyzing abilities have been correlated with the reactivation of dormant bacteria; however, the precise molecular mechanisms underlying this remain poorly understood. The individual roles, localisation and expression patterns of Mycobacterium marinum Rpf homologues during macrophage infection were investigated by immunofluorescence using custom Rpf-specific polyclonal antibodies and confocal microscopy. We found Rpfs to associate to the polar ends of bacteria, along the lengths of the bacterial surface and free in the cytoplasm of infected macrophages suggesting a role for these proteins in apical extension and peptidoglycan biosynthesis. No distinct patterns of Rpf localisation were observed in relation to phagosomal mycobacteria. Microscopy based semi-quantification of Rpf expression levels during macrophage infection, and analysis of in vitro grown bacteria from logarithmic phase by flow cytometry identified that only a subset of mycobacteria produce Rpf at detectable levels. Significant protein sequence homology between RpfA and Listeria monocytogenes ActA suggested that RpfA could play a similar role in actin polymerisation and bacterial motility in the macrophage cytoplasm as ActA. However, under the conditions tested, ΔrpfA M. marinum did not show any differences to wildtype during infection of murine macrophages. Recombinant ActA was demonstrated to have muralytic activity, a previously uncharacterised property of this protein suggesting a function beyond the polymerisation of host cell actin.

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Antigen presenting cells such as macrophages and dendritic cells play an important role between the interface of the innate and adaptive immune responses. One property shared by dendritic cells and macrophages is that upon activation they secrete either pro- or anti-inflammatory cytokines dependent on the pathogen derived product and the T cell derived signal they encounter. The panel of cytokines they produce determines the class of the adaptive immune response. Tpl-2 was originally described as a proto-oncogene. It was later found to function as a Map-3 kinase leading to phosphorylation of MEK and ERK. Upon stimulation of dendritic cells and macrophages with Toll like receptor ligands (TLR) such as LPS and CpG, Tpl-2 phosphorylates MEK, which in turn phosphorylates ERK, leading to production of cytokines. In the Tpl-2 knockout mice, it has been shown that TNF production is decreased. We have confirmed this finding and shown that this is both at the transcriptional and post-transcription level. In the complete absence of Tpl-2 we have shown that production of the suppressive cytokine IL-10 is reduced in response to TLR stimulation in macrophages and dendritic cells. On the other hand, when macrophages and dendritic cells are stimulated with LPS or CpG in the absence of Tpl-2, production of IL-12 is increased. IFN-b is also upregulated in absence of Tpl-2. Tpl-2 can regulate IL-12 either directly via IL-10, but also independently of IL-10 via ERK. IL-10 is an important cytokine in regulating the immune response in order to inhibit immune pathology. We crossed the Tpl-2 knockout with an IL-10 knockout mouse in order to investigate whether Tpl-2 regulates IL-12 and IFN-b in the complete absence of IL-10. These studies are currently in progress. Preliminary results however, show that the Tpl-2/IL-10 double knockout develop diarrhoea and colitis around 8 weeks of age, leading to minimal weight gain and even weight loss compared to littermates. This is in contrast to the Tpl-2 knockout, which does not develop colitis and the IL-10 knockout, which only develops colitis at 4 months of age. These findings have important implications for treatment of autoimmune diseases with drugs that inhibit ERK, to inhibit TNF mediated pathology. The aim of this thesis was to investigate to role ERK has in regulating cytokine production in DC and macrophages. We used two different strategies to do this. Firstly we pharmacologically blocked ERK phosphorylation with an inhibitor. Secondly we used genetically modified mice lacking Tpl-2.

Neural Crest (NC) derived mesenchyme has previously been shown to play an important role in the early development of the foetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26eYfp reporter mice, NC derived mesenchymal cells were revealed in the adult murine thymus. It is reported here that NC derived cells infiltrate the thymus before E13.5, and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at three months of age, NC derived perivascular cells continue to be associated with the vasculature providing structural support to the blood vessels and possibly regulating endothelial cell function. Thymus organogenesis requires co-ordinated interactions of multiple cell types including NC cells that orchestrate the formation, separation and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear, however NC derived mesenchyme has been shown to be important. Here, it is shown that the separation process of the thymus from the pouch is independent of ephrin-B2 expression on thymic NC derived mesenchyme, however in its absence the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analyses of individual NC derived thymic mesenchymal cells shows that the absence of ephrin-B2 impairs their polarisation, and thus motility, as a result of defective EphB receptor signalling. This implies a NC derived cell specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.

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