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Tuberculosis predominantly manifests in the form of a pulmonary infection, but may spread out into other parts of the body and is then referred to as extrapulmonary tuberculosis (EPTB). One form of EPTB is an infection of the central nervous system (brain & spinal cord), CNS-TB. Although CNS-TB is relatively rare, accounting for about 5% of EPTB, it is characterised by high morbidity and mortality, particularly for children and immunosuppressed individuals. To examine the effects of a Mycobacterium tuberculosis infection of neural tissue, researchers have hitherto relied on two animal models namely, in vivo intracranial infections or in vitro culturing with dissociated neural cells. Both models have yielded crucial insights concerning CNS-TB but each have limitations e.g. lack of access to the brain during infection in vivo and absence of the 3D organizational tissue structure in vitro. This study investigated the effect of the vaccine strain for tuberculosis, Bacille Calmette-Guerin (BCG) on neural tissue using the model of organotypic hippocampal slice cultures; an in vitro model that overcomes the previously mentioned obstacles. The study sought to expound on immunological and electrophysiological responses to the infection. A viable and moderate BCG infection was established in the hippocampal slice cultures, confirmed by colony forming units enumeration and immunohistochemistry. However, immunological analysis using ELISA found that BCG infection did not change the production levels of cytokines and elicit a distinguishable immune response. To examine the neuronal function during BCG infection, whole-cell patch clamp technique was applied to the hippocampal slice cultures. The neuronal intrinsic properties were not significantly different between infected and non-infected slices. However, tuberculin PPD (M. tuberculosis extract) moderately and transiently had a depolarizing effect when 'puffed' directly onto neurons. In conclusion, organotypic slice cultures are suitable for the investigation of cellular interactions and neural functions in CNS-TB, and the neuronal impact of PPD warrants further investigation.

Tuberculosis of the central nervous system (CNS TB) is the severest form of tuberculosis. It is classified as extra-pulmonary tuberculosis due to dissemination of Mycobacterium tuberculosis (Mtb) bacilli from the lung to the brain. It affects mostly children and immune suppressed individuals and high incidents of death occur as a result of missed diagnosis and delayed treatment. Therefore, the is a need for improved therapeutic strategy and a better understanding of the CNS immunity; investigate cells targeted for infection, their respective response to infection and interaction with different cell types to the overall protection of the CNS - as accumulating evidence indicates a dynamic neuronal lymphocyte interplay that defines outcomes of diseases. A novel observation was previously made, that neurons are infected by Mtb during in vitro and in vivo infection. The aim of this study was to further characterize neural responses induced by mycobacteria using hippocampal primary neuron cultures, infected with H37RV and BCG. Secondly, this study investigated the importance of interaction between neurons and immune cells in immunity against mycobacterial challenge using an optimised neuron T cell co-culture model. Investigation included identifying the production levels of neuronal cell surface markers and cytokines induced by Mtb. In flowcytometry and ELISA analyses, infection exhibited a robust inflammatory response with increased neuronal production of cytokines such as IL1β, IL6, TNF and regulatory cytokine IL10 in vitro and in vivo. Neuronal MHC class I expression was upregulated by infection, suggesting possible antigen dependent interactions between neuron and CD8+ T cells. In co-cultures, neurons induced expression of Tbet, RorγT and Gata3 T cell transcription factors through direct contact with T cells. These data highlighted the likelihood of neurons activating T cells upon mycobacterial stimulations. It may potentially be utilised to broaden the understanding of CNS immunity under pathological conditions and possibly lead to identification of novel immunomodulatory targets that could be exploited for new rapid sensitive diagnostics and early opportune intervention against CNS TB – reducing morbidity and mortality associated with the disease.

Includes bibliographical references., Neurotuberculosis is the most severe form of extra-pulmonary tuberculosis, characterised by the formation of rich foci a brain form of granulomas, and tuberculous meningitis. Granulomas contain mycobacteria by recruitment of immune cells that surround the bacteria. The cytokine tumour necrosis factor has been found to be involved in the recruitment of the immune cells and structure maintenance of granulomas. Tumour necrosis factor is a multifunctional proinflammatory cytokine which play a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) infection. This cytokine is synthesised by several cell types of hematopoetic origin, such as microglia/macrophages, neutrophils, dendritic cells and lymphocytes, and non-hematopoetic origin such as astrocytes and neurons. During neurotuberculosis, excess of tumour necrosis factor has been implicated in persisting hyperinflammation, however, deficiency of tumour necrosis factor lead to uncontrolled bacterial growth; both phenomena causing necrotic lysis. Thus, a need exists to investigate the contribution of tumour necrosis factor by specific cell types in the control of cerebral M. tuberculosis infection and its protective immune response. In this study, we investigated the role of tumour necrosis factor derived from neurons, microglia/macrophages, neutrophils, CD4+ and CD8+ T cells in host immunity against M. tuberculosis; using an experimental murine model with cell type-specific gene targeting. We found that mice deficient for tumour necrosis factor in neurons (NsTNF-/-), as well as from microglia/macrophages and neutrophils (M-TNF-/-) are not susceptible to M. tuberculosis infection with a phenotype similar to wild type mice. Interestingly, mice with ablation of tumour necrosis factor in myeloid (microglia/macrophages, neutrophils) and CD4+ and CD8+ T cells (MTTNF-/-) were highly susceptible to M. tuberculosis infection with a phenotype similar to that of complete deficient tumour necrosis factor (TNF-/-) mice, which succumbed by 21 days postinfection. Thus, it seems that the resistance observed in M-TNF-/- mice may be caused by the compensation of T cell-derived TNF whose function appeared as non-redundant. Impaired protective immunity observed in MT-TNF-/- and TNF-/- mice were related to alteration of cytokines and chemokines, and also to reduce antigen response and B cells IgM secretion. Our data suggest that neurons as well as microglia/macrophages and neutrophils derived tumor necrosis factor have a very limited role in protective cerebral immune responses. In MT-TNF-/- mice, TNF mediated protective immunity against cerebral M. tuberculosis infection requires primarily T cell-derived TNF as opposed to macrophage/neutrophil derived TNF. These findings may inform the development of immunomodulatory therapy strategies against neurotuberculosis.

The importance of TNF-TNFR signaling in immunity against M. tuberculosis has been established. The aims of this study were to characterize the functions of membrane-bound TNF (Tm-TNF) and soluble TNF (solTNF) and to investigate the role of membrane-bound p55TNFR signaling as well as the in vivo significance of TNFR shedding in host immune responses during infection with M. tuberculosis H37Rv. To address this, mice expressing only the membrane-bound TNF or membrane-bound p55TNFR were exposed to a low dose of M. tuberculosis H37Rv by aerosol inhalation infection. The results presented in this dissertation illustrate that Tm-TNF mice were able to control acute M. tuberculosis infection but succumbed to chronic exposure to M. tuberculosis with pneumonia. We demonstrate that Tm-TNF mice displayed heightened pulmonary macrophage activation reflected by enhanced cell surface expression of MHC-II, CD80 and CD86 as well as enlargement of granulomas. Furthermore, our results show that solTNF has a regulatory function that modulates the magnitude of Th1 immune responses during acute and chronic stages of the infection. The evaluation of the functions of Tm-TNF and solTNF in host immune function in the presence of an established mycobacteria-specific immune response was carried out using a 'drug-based' M. tuberculosis reactivation model. Here, mice that were challenged with a low dose of M. tuberculosis were exposed to INH-RIF treatment for six weeks in drinking water, after which therapy was withdrawn and immune responses during reactivation were analyzed. Our results demonstrate that complete absence of TNF resulted in host susceptibility to recrudescence tuberculosis in the presence of a mycobacteria-specific immune response. TNF deficient mice were unable to suppress bacilli growth and formed diffused granulomas and succumbed early to reappearing tuberculosis compared to WT mice. By contrast, we show that Tm-TNF was sufficient for containment of reappearing mycobacterial growth and sustaining immune pressure in a manner comparable to WT control mice. xii Lastly, the analysis of host immune responses in mice expressing a non-sheddable p55TNFR revealed that persistent p55TNFR cell surface expression does not afford better protection to low dose M. tuberculosis infection. However, we observed a transient elevation in the frequency of pulmonary CD11b+/MHC-II+ cells in mice expressing a non-sheddable p55TNFR relative to WT mice as well as reduced cell surface expression of CD44 on CD4+ T cells. We also found that pulmonary IL-12p70 and TNF concentrations were elevated whereas IFNγ levels were reduced in mice expressing a non-sheddable p55TNFR relative to WT mice. Furthermore, data presented here describe the in vivo functional significance of p75TNFR shedding. We demonstrate using a double mutant mouse strain that in the absence of p75TNFR, mice expressing a non-sheddable p55TNFR display enhanced ability to control M. tuberculosis infection.

Includes abstract., Includes bibliographical references (leaves 85-97)., Tumor necrosis factor alpha (TNFα) is critical for host protective immunity against Mycobacterium tuberculosis infection. TNFRp55 and TNFRp75 can both bind TNFα and conduct signaling, however the respective roles, in particular that of TNFRp75 in an M. tuberculosis aerosol inhalation infection was poorly defined. In this study the role of signaling through TNFRp55 and TNFRp75 was investigated using TNFR deficient mice in an aerosol inhalation M. tuberculosis infection model.

Includes bibliographical references (leaves 109-122 )., This study investigated four indigenous South African medicinal plants that are commonly used in traditional medicine for their bioactivity against Mycobacterium tuberculosis. Crude plant extracts were prepared and characterized using HPLC analysis. The crude rhizome extracts of Agapanthus praecox, leaf extracts of Olea europaea subsp.

Includes bibliographical references (leaves 131-141)., Tuberculosis remains an important public health problem worldwide. There has been increase in the development of drug resistance towards INH and RIF, two of the frontline antimycobacterial drugs currently used in therapeutic regimes. As an attempt to address drug resistance, the World Health Organization has implemented the DOTS strategy in 182 countries. Moreover, new chemical libraries of potential antituberculosis drugs have been designed and synthesised. We therefore assessed 121 derivatives from the acetolactate synthase inhibitors, cysteine protease inhibitors, thiosemicarbazones, and thiolactomycins classes of compounds for in vitro efficacy against M. tuberculosis using the resazurin microtitre plate assay afterwhich active compounds were assessed for cytotoxicity in vitro against elicited peritoneal macrophages using the MTT assay. Of the 38 acetolactate synthase inhibitors tested, 2 derivatives namely RKG162A and RKG1541 were bactericidal against M. tuberculosis. Both derivatives were mildly cytotoxic against macrophages. For cysteine protease inhibitors, 35 derivatives were tested. Four derivatives namely AXE1, AXE4, AXE5, and AXE29 were bactericidal whereas AXE2, AXE3, AXE35, and NAT47 were bacteriostatic.

Bibliography: leaves 75-90., Absence of the TH2 inducing cytokine IL-4 has been shown not to increase the TH1 response and resistance to mycobacterial infection. This study asked whether the combined absence of IL-4 and IL3, as compared to IL-4 deficiency only, would increase resistance to an aerogenic Mycobacterium tuberculosis infection. By using IL-4 gene-deficient mice, it was confirmed that endogenous IL-4 does not reduce host immunity. In contrast, IL-4Rα genedeficient mice, which lack both IL-4 and IL-13 signalling, had reduced bacterial burden in the lungs and other organs, increased survival and cellular immunity with macrophage activation. Therefore IL-4Rα gene-deficient mice have increased resistance to Mycobacterium tuberculosis infection.