Browsing by Author "Jacobs, Muazzam"
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- ItemOpen AccessAstrocyte-mediated immune modulation during mycobacterial infection(2023) Geyer, Sohair; Jacobs, MuazzamCentral nervous system tuberculosis (CNS-TB) is the severest clinical extra-pulmonary manifestation of tuberculosis (TB) disease and constitutes approximately 1% of global cases. Little is known about the cells that regulate immune responses during CNS-TB infection. Microglia are the prime resident immune-effector cells in the CNS; astrocytes however also regulate innate and adaptive immunity in CNS disease and injury. Astrocytes play a progressive role in maintaining the structural and functional integrity of the CNS while supporting neuronal function and participating in host protection during infection of the CNS. These cells exist as distinct populations with complex morphological identities and functional modifications suited to their micro-environment. The principal aim of this study was to elucidate the genomic profile and cellular immune responses of astrocytes to characterise their immunomodulatory potential during CNS-TB infection. The ability of astrocytes to internalise mycobacteria was evaluated by immunocytochemistry and immunohistochemistry. The immune regulatory role of astrocytes was assessed through transcriptomic profiling, flow cytometry and Luminex analysis. Outcomes were validated in mouse models, through flow cytometric analysis of infected brain cells. The novel findings presented here collectively demonstrate the sophistication and complexity of astrocyte activity and behaviour during host immunity. For the first time, astrocytes showed internalisation of M. bovis BCG and M. tuberculosis bacilli, demonstrating that astrocytes are target cells for non-virulent and virulent mycobacterial strains. Extensive transcriptomic analysis of astrocytes revealed elevated expression of multiple pathways in infected cells, particularly those involved in inflammation and immune regulation, and emphasised the innate immune component. Notably, various pro-inflammatory cytokines essential to host defence during CNS-TB infection, such as IL-1b and TNF, were upregulated by astrocytes following the mycobacterial challenge. The data suggests that astrocytes may modulate host immune responses by regulating blood-brain barrier permeability and facilitating immune cell recruitment and activation at the site of infection. This potential was demonstrated by their enhanced expression and production of well-described pro- and anti-inflammatory factors as well as chemotactic factors following mycobacterial infection. Furthermore, increased expression of neurotrophic factors by astrocytes was observed, which can assist in the recovery and repair of the CNS during CNS-TB. By supporting the survival and function of neurons and modulating immune cell activity, astrocyte-derived neurotrophic factors can help to limit infection-induced damage and promote the resolution of inflammation. The dichotomous behaviour of astrocytes during CNS-TB was demonstrated, as they can contribute to the maintenance and protection of CNS function and host immune responses while potentially enhancing pathology during infection. This study explored astrocyte contributions to host immune responses during CNS-TB infection by examining their regulation of CNS inflammation in the presence of mycobacterial challenges, and highlighted the intricate interplay of cytokines/chemokines that need careful modulation to achieve optimal outcomes. These findings demonstrated that astrocytes are crucial regulators of host immunity during mycobacterial infection and play a progressive role in maintaining the structural and functional integrity of the CNS
- ItemOpen AccessCharacterising the potential health risks associated with coal dust(2023) Kamanzi, Kankindi Conchita; von Holdt, Johanna; Jacobs, Muazzam; Broadhurst, Jennifer; Becker MeganCoal dust is inextricably linked to the development of dust diseases. To date, the role of mineral matter in coal has been investigated for its links to pulmonary damage; however, no consensus has been reached on which characteristics are relevant to pulmonary toxicity. This study hypothesises that the toxic potential of inhalable coal dust can be attributed to reactive mineralogy and the specific surface area for interaction between the particles and primary phagocytes such as macrophages. To test this hypothesis, the study developed an advanced understanding of the relationship between the physicochemical and mineralogical characteristics of coal particles and pulmonary toxicity. Three objectives were constructed to achieve this aim. Objective 1 developed a detailed particle characterisation dataset on coal particle samples utilising both routine (X-ray diffraction and X-ray fluorescence) and advanced methods of coal analysis (automated scanning electron microscopy systems). Objective 2 elucidated multivariant relationships between the particle characteristics and the immunological responses from exposed macrophage cells in vitro using advanced statistical methods. Lastly, objective 3 developed a protocol to empirically characterise the relative risk of coal dust-related damage on a cellular level. In developing a detailed characterisation dataset on the coal samples, both routine and automated analysis tools were used to define general, chemical, mineralogical, and mineral specific characteristics. An auto-SEM-EDS-XRD (Automated scanning electron microscope coupled with Energy Dispersive X-ray Spectroscopy and analyses generated by X-ray Diffraction) protocol was developed to obtain a broad spectrum of particle data by mineralogically mapping each particle. This protocol involved the rigorous analysis of uncertainty in the data using comparative datasets generated from XRD and XRF (X-ray Fluorescence) analyses. In summary, the study demonstrated that the combined use of both routine and advanced particle analysis tools allowed for the classification of chemical and mineralogical distributions as well as a discrimination between general and mineral specific particle characteristics. Generally, these results suggested that features relating to general particle characteristics (size, shape, roughness, and surface area) are more strongly a function of mechanical breakage and deformation than compositional variation. To assess the multivariant relationships between the numerous characteristics defined and response measures of cellular toxicity, a PLSR (partial least squares regression) was applied in a novel approach to attempt a single model comparison of such relationships. This model was chosen for its ability to relate response and explanatory variables based on a new set of variables which have undergone dimensionality reduction whilst maximising the covariance. The results from the relationship analysis showed that physical characteristics (particle shape in particular) displayed a greater influence on cytotoxicity and lipid peroxidation over mineral and chemical-based characteristics. Relating this observation to previous research it was suggested that the influence of shape and roughness on phagocytosis may have strong implications for magnitude of direct and indirect cellular harm and the predominance of either intracellular or extracellular damage. The results also showed that, apart from the influence of particle shape, radical-induced stress and cytotoxicity displayed a strong dependency on (1) the chemical and mineralogical reactivity Ca hosted in gypsum, (2) the release/inhibition of Fe from pyrite and Fe-sulfates, and (3) the surface activity of quartz based on its crystallite size. However, the relationships defined in the context of cytotoxicity displayed a more nuanced dependency with the silicate mineral content and their associated properties compared to lipid peroxidation. From this it was suggested that non-radical related pathways to cytotoxicity could also occur from coal dust exposure. Ultimately, the study demonstrates the first analysis which assesses relative impact and magnitude of multiple particle characteristics on cytotoxicity and cellular stress. Finally, to provide a more easily interpretable format for the analysis of the PLSR relationships, a protocol was developed to screen variables based on: (1) their level of importance to the defined relationship and (2) the rank of importance for each influential variable represented on a unified scale. Elements which explained the variability within the sample characteristics and the responses were clustered using the k-means algorithm to determine classes of samples which display similar characteristics or levels of toxicity. The comparison of the classes grouping samples with similar properties versus samples groups with similar toxicity levels showed that even though samples may share similar properties, their reported level of toxicity may differ. This confirms the observations from previous studies which have shown that the relative toxicity of coal dust cannot be explained on the basis of isolated properties. Rather the set of ‘influential variables' showed that a combination of general, chemical, mineralogical and mineral specific data are needed to determine the differences between levels of toxicity. Ultimately, the application of this protocol on 17 different dust-sized coal samples demonstrated the key differences between samples and their influence on levels of cytotoxicity and lipid peroxidation, which until this study have not been demonstrated by a single regression. As an outcome of such results, this study provides a robust analysis strategy for elucidating particle cell relations which can further advance the understanding of coal dust induced disease pathology. Additionally, the protocol has demonstrated the usefulness of disseminating the complex data structures to more easily interpretable data formats such that a generalisable analysis of risk factors related to coal dust-based cellular damage can be utilised by stakeholders in data-based decision making. Ultimately, the results of this study propose that the toxic potential of coal dust is primarily a function of the reactive mineralogical and chemical components within the particles, however, the magnitude of this intrinsic reactivity is subject to the mitigative factors which can either neutralise of supress the anticipated reactivity.
- ItemOpen AccessFatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88(2004) Fremond, Cecile M; Yeremeev, Vladimir; Nicolle, Delphine M; Jacobs, Muazzam; Quesniaux,Valerie F; Ryffel, BernhardToll-like receptors (TLRs) such as TLR2 and TLR4 have been implicated in host response to mycobacterial infection. Here, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with Mycobacterium tuberculosis (MTB). While primary MyD88–/– macrophages and DCs are defective in TNF, IL-12, and NO production in response to mycobacterial stimulation, the upregulation of costimulatory molecules CD40 and CD86 is unaffected. Aerogenic infection of MyD88–/– mice with MTB is lethal within 4 weeks with 2 log10 higher CFU in the lung; high pulmonary levels of cytokines and chemokines; and acute, necrotic pneumonia, despite a normal T cell response with IFN-γ production to mycobacterial antigens upon ex vivo restimulation. Vaccination with Mycobacterium bovis bacillus Calmette-Guérin conferred a substantial protection in MyD88–/– mice from acute MTB infection. These data demonstrate that MyD88 signaling is dispensable to raise an acquired immune response to MTB. Nonetheless, this acquired immune response is not sufficient to compensate for the profound innate immune defect and the inability of MyD88–/– mice to control MTB infection.
- ItemOpen AccessInvestigating transmembrane TNF and transmembrane p55TNFR mediated signaling in host immune function during Mycobacterium tuberculosis infection(2010) Dambuza, Ivy M; Jacobs, MuazzamThe 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.
- ItemOpen AccessThe investigation of indigenous South African medicinal plants for activity against Mycobacterium tuberculosis(2006) Mokgethi, Thabang; Jacobs, MuazzamThis 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.
- ItemOpen AccessInvestigation of neuron T cell interaction in central nervous system tuberculosis(2017) Choshi, Phuti Sophia; Jacobs, Muazzam; Hsu, Nai-JenTuberculosis 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.
- ItemOpen AccessInvestigation of the efficacy of identified acetolactate synthase inhibitors, peptidyl cysteine protease inhibitors, thiolactomycins, and thiosemicarbazone compounds against Mycobacterium tuberculosis(2006) Sebesho, Biopelo Felicity; Jacobs, MuazzamTuberculosis 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.
- ItemOpen AccessMembrane TNF confers protection to acute mycobacterial infection(BioMed Central Ltd, 2005) Fremond, Cecile; Allie, Nasiema; Dambuza, Ivy M; Grivennikov, Sergei; Yeremeev, Vladimir; Quesniaux, Valerie; Jacobs, Muazzam; Ryffel, BernhardBACKGROUND:Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF). METHODS: C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice. RESULTS: While TNF-KO mice succumbed to infection within 4-5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice. CONCLUSION: Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.
- ItemOpen AccessModelling neuroimmune interactions using organotypic slice cultures(2017) Mbobo, Buchule; Jacobs, MuazzamTuberculosis 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.
- ItemOpen AccessReactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice(Public Library of Science, 2011) Dambuza, Ivy; Keeton, Roanne; Allie, Nasiema; Hsu, Nai-Jen; Randall, Philippa; Sebesho, Boipelo; Fick, Lizette; Quesniaux, Valerie J F; Jacobs, MuazzamOf those individuals who are infected with M. tuberculosis , 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.
- ItemOpen AccessThe role of cell type-specific tumour necrosis factor in protective immunity against neurotuberculosis(2013) Francisco, Ngiambudulu Mbandu; Jacobs, MuazzamNeurotuberculosis 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.
- ItemOpen AccessThe role of IL-4 and IL-13 in pulmonary tuberculosis using gene-deficient mice and protective efficacy of the purine-deficient auxotroph of Mycobacterium tuberculosis(2002) Brown, Najmeeyah; Jacobs, MuazzamAbsence 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.
- ItemOpen AccessThe role of TNFRp55 and TNFRp75 in the host immune response to Mycobacterium tuberculosis(2009) Keeton, Roanne Shay; Jacobs, MuazzamTumor 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.
- ItemOpen AccessThe role of Tumour Necrosis Factor, Lymphotoxin alpha and Interleukin-10 in the host's protective immune response against mycobacterial infection(2002) Jacobs, Muazzam; Ryffel, BernardBibliography: leaves 174-198.
- ItemOpen AccessThe characterisation of dendritic cell, microglial, macrophage and T cell responses during mycobacterial infection of the central nervous system(2021) Kgoadi, Khanyisile; Jacobs, Muazzam; Keeton, RoanneBackground: Tuberculosis (TB) remains a global health challenge and a quarter of the global population is infected with latent TB. It is a single infection that causes most deaths and was the number one cause of death in South Africa in 2017. Bacille Calmette-Guerin (BCG) remains the only licensed vaccine for protection against TB. Although TB primarily occurs as a pulmonary infection after inhalation of Mycobacterium tuberculosis (M. tuberculosis) bacilli, it can disseminate to other organs causing extra-pulmonary TB (EPTB). Approximately 5-15% of EPTB cases are attributed to central nervous system tuberculosis (CNS-TB) which commonly manifests as TB meningitis. CNS-TB is a severe form of TB associated with high morbidity and about 50% mortality due to inconclusive diagnosis and treatment challenges. Children and immunocompromised adults like those coinfected with HIV/AIDS are higher risk groups for the development of CNS-TB. Pathogenesis of CNS-TB occurs as a secondary infection during haematogenous dissemination of pulmonary TB to the brain parenchyma and meninges where inflammation occurs after rupture of rich foci into the subarachnoid space. Mechanisms by which M. tuberculosis infects the CNS and specific cell types targeted are not fully characterized. Little is understood of the cells that regulate CNS-TB, their respective functions, their cellular interactions, and contributions to the overall protection of the CNS. Most studies have focussed on microglia and macrophages as the preferential targeted antigen-presenting cells (APCs) by M. tuberculosis and neglected dendritic cells (DCs) to an extent because no consensus had been reached regarding the presence of DCs in a healthy CNS. Both myeloid (APCs) and T cells contribute to protection against CNS-TB. This study characterized the dendritic cell, microglial, macrophage, and T cell responses during mycobacterial infection of the CNS. We also investigated the modulation of T cells by DCs during CNS-TB. Methodology and Results: Wild-type female C57BL/6J mice were intracerebrally (i.c.) infected with M. tuberculosis H37Rv or Mycobacterium bovis BCG while control animals were saline inoculated and naive mice. Mice were euthanized at weeks 2, 4, 6, and organs harvested for experimental analysis. Histology results detected acid-fast bacilli using Ziehl-Neelsen (ZN) stain in the brains of M. tuberculosis and BCG i.c. infected mice, respectively. This was accompanied by a high degree of inflammatory responses in the brain ventricles and meninges of infected mice as compared to saline control mice shown by Hematoxylin and Eosin (H & E) staining. Although controlled brain bacterial burdens were demonstrated from homogenates of M. tuberculosis or BCG infected mice, dissemination to the spleen and lungs occurred. The histopathological results showed the successful reproduction of the murine CNS-TB infection model. For immunophenotyping, flow cytometry analysis of single-cell suspensions generated from brains and cervical lymph nodes were characterized for phenotypic and functional profiles. We detected the recruitment of macrophages and DCs to the brain from the periphery and an expansion of brain APCs (microglia, brain infiltrating macrophages, and DCs) during mycobacterial infection of the CNS. Brain APCs from infected animals displayed highly activated and mature phenotypes as shown by increased numbers of these cells expressing MHCII, co-stimulatory CD86 molecule, pro-inflammatory cytokines (IFNg, TNFa, IL-1b, IL-6, IL-12) and an anti-inflammatory cytokine (IL-10) in comparison to saline control mice. We also demonstrated preferential recruitment of mature conventional DCs (CD11c+, MHCII+) that express chemokine receptor-7 (CCR7) to the brain and cervical lymph nodes (CLNs), a phenomenon that may have contributed to the recruitment and expansion of predominantly effector CD4+ T cells than CD8+ T cells (CD44+CD62L-) to the brain and CLNs during mycobacterial infection of the CNS. Increased numbers of recruited CD4+ T cells and CD8+ T cells expressed T-bet [T-helper (Th1) transcription factor) in the brain and CLNs post-infection. At week 4 post intracerebral infection, increased numbers of these T cells expressed both T-bet and FoxP3 (regulatory transcription factor) during CNS-TB and identified a higher frequency of polyfunctional IFNg+TGF-b+CD4+ T cells than IFNg+TGF-b+IL-10+CD4+ T cells. M. tuberculosis-infected DCs from CLNs of CNS-TB mice were cocultured with naïve CD3+ T cells to generate a DC-T cell coculture, cells were sorted using fluorescence-activated cell sorting (FACS). DC-T cell coculture demonstrated increased percentage expression of IFNg, IL-4, IL-10 and TGF-b responses by CD4+ T cells and CD8+ T cells during CNS-TB. Our in vitro coculture findings validated in vivo findings of recruited brain CD4+ T cell cytokine responses that showed a combination of Th1 and regulatory T cell immune responses. Conclusion: We successfully reproduced the CNS-TB murine model, which proved valuable in studying immune responses. The functional mature phenotypes of detected brain APCs (microglia, brain infiltrating macrophages, DCs) suggest their capabilities of inducing antigen-specific T cell responses that contributed to initiating and mediating immunity during mycobacterial infection of the CNS. Our study findings suggest protection against mycobacterial infection of the CNS was achieved by characterized cells based on reduced brain bacterial burdens and 100% animal survival rate. Detrimental disease outcome was prevented by the balance achieved between proinflammatory and anti-inflammatory responses. The novel mechanism employed by conventional DCs during CNS-TB is modulating CD4+ and CD8+ T cell cytokine responses to Th1 and Treg polarization that achieved M. tuberculosis control in the brain. We demonstrated that DCs can be targeted for strategic therapeutic intervention against CNS-TB. Therefore; we support ongoing research that focuses on DCs for the development of tuberculosis vaccines and host-directed therapy. This study provided new knowledge on immune mechanisms and pathogenesis experienced during TBM, thus adding to the current gap of advancing basic and translational TBM research that will inform clinical interventions. These new insights have the potential to help reduce the high death and disability associated with CNS-TB.
- ItemOpen AccessThe impact of malaria on the immunogenicity and efficacy of mycobacterium bovis BCG vaccination against mycobacterium tuberculosis in mice(2022) Tangie, Emily Nchangnwie; Jacobs, Muazzam; Keeton, RoanneBackground Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine for use against tuberculosis (TB), however, it is poorly efficacious against pulmonary TB in adults. The poor efficacy has been attributed in part to coinfections with many other unrelated pathogens that overlap geographically with Mycobacterium tuberculosis (M. tuberculosis). In this study, we used a murine model to investigate the effect of Plasmodium species virulence and the timing of infection on BCG-induced immune responses and efficacy against M. tuberculosis both in vivo by aerosol challenge and ex vivo by Mycobacterial Growth Inhibition Assay (MGIA). Methods and Results To assess the impact of malaria parasite virulence on BCG, wild-type C57BL/6 mice were vaccinated intraperitoneally with BCG Pasteur and 6 weeks later, they were infected with either virulent Plasmodium berghei (P. berghei) or less-virulent Plasmodium chabaudi chabaudi AS (P. chabaudi) infected red blood cells with appropriate controls. The mice were euthanased at 7, 10, 17, 26 days after malaria infection for multiparameter flow cytometry analysis. We compared P. berghei and P. chabaudi, their effects on B cells, effector and memory T cells and the outcome on BCG-induced protective efficacy against M. tuberculosis H37Rv infection by subsequent aerosol challenge. P. berghei induced a significant decrease in the frequency of central memory T cell (CD44hiCD62Lhi), marginal zone B cell (B220+AA4.1-CD1dlo), and follicular B cell (B220+AA4.1-CD1dhi ) populations. In contrast, infection with the less virulent P. chabaudi induced depletion in the marginal zone B cells but not the follicular B cells or the central memory T cells. A strong effector T cell/effector memory T cell (CD44hiCD62Lo) response enhanced by BCG vaccination was observed in both species. The reduction in the central memory T cells observed during P. berghei infection was attributed to T cell apoptosis. It should be noted that the observed changes in T cell and B cell populations described above are relative proportions of these cells among splenocytes. Surprisingly, BCG-mediated protection against M. tuberculosis H37Rv by aerosol challenge was retained in both the virulent P. berghei and less virulent P. chabaudi species despite modulations in the immune responses. We also investigated the effect of malaria infection post BCG vaccination and malaria infection prior to BCG vaccination on the cytokine responses and the efficacy of BCG vaccine both in vivo and ex vivo. Splenocytes from wild type C57BL/6 mice infected with P. yoelii 17XNL 4 weeks post BCG vaccination were analysed 13 days after P. yoelii 17XNL infection. We compared the cytokine responses and growth inhibition by MGIA in four groups of 6 mice each: naïve, BCG, BCG-malaria, and malaria control. BCG Pasteur Aeras was used as a surrogate for M. tuberculosis in the MGIA. Restimulation with PPD-T induced a significant increase in both the proportion and absolute cell numbers of CD4+ IFNγ+ and CD4+ TNF+ T cells in the BCG vaccinated compared to the naïve control. No significant differences in the proportion and cell numbers of CD4+ IFNγ+ T cells were observed between the BCG and BCGmalaria groups restimulated with PPD-T. This indicates that the presence of malaria did not significantly hamper the IFNγ response to BCG. In contrast, a significant (p<0.05) reduction was detected in the proportion of CD4+TNF+ T cells in the BCG-malaria group compared to the BCG group following restimulation with PPD-T. P. yoelii 17XNL infection also led to significant production of IL-4 by CD4+ T cells in the groups that were infected with malaria with or without BCG vaccination when restimulated with PPD-T or P. yoelii 17XNL peptide pool. Minimal production of CD4+ IL-17 T cells and CD8+ T cells were observed when restimulated with PPD-T. MGIA revealed a significant decrease in net bacterial growth (0.2 log 10) in the BCG and BCG-malaria splenocyte cultures compared to the naïve and malaria control splenocyte cultures, indicating that BCG immunized mice were able to control the growth of mycobacteria in the presence of malaria. For P. yoelii 17XNL infection prior to BCG vaccination, wild type C57BL/6 mice (n=6-11) were infected with either P. yoelii 17XNL or P. chabaudi and after 13 (acute malaria) and 21 (cleared malaria) days or 7 (acute malaria) and 28 (cleared malaria) days respectively, mice were vaccinated with BCG. They were either sacrificed 6 weeks after BCG vaccination for cytokine analysis and MGIA or challenged with M. tuberculosis H37Rv by aerosol for bacterial growth determination. BCG vaccination caused a significant increase in the proportion of CD4+ IFN-γ + and CD4+TNF+ T cells compared to the naïve control when restimulated with PPD-T. However, we observed a significant reduction in the proportion of CD4+TNF+ but not CD4+ IFN-γ + T cell responses in the acute malaria-BCG group compared to the BCG vaccinated mice following in vitro restimulation with PPD-T. Interestingly, these cytokine levels were significantly elevated upon clearance of the P. yoelii 17XNL parasites. Therefore, acute malaria infection at the time of BCG vaccination induces a transient decrease in Th1 response which is restored once the parasite is cleared. An ex vivo MGIA assay on splenocytes showed a significant decrease (0.2 log reduction) in net bacterial growth in all BCG vaccinated groups except the acute malaria-BCG group, compared to the unvaccinated groups with or without malaria. Interestingly, a significant increase in net bacterial growth was observed in the acute malaria group compared to the BCG vaccinated group, indicating that acute P. yoelii 17XNL infection prior to BCG vaccination decreases the protective efficacy of BCG in the MGIA. In an in vivo aerosol challenge with M. tuberculosis, a significant decrease was observed in bacterial burden in the spleen (0.6 log reduction) and lungs (1 log reduction) of mice that were vaccinated during acute P. chabaudi infection, vaccinated when malaria had cleared or vaccinated in the absence of malarial infection. This was further confirmed by higher numbers of acid-fast bacilli observed in all unvaccinated groups compared to all vaccinated groups with or without malaria, implying BCG vaccine efficacy is maintained in an acute or cleared P. chabaudi infection prior to BCG vaccination. This discrepancy between the in vivo aerosol challenge and ex vivo growth inhibition assay may be attributed to the different Plasmodium species with different clinical characteristics used in the study or the different kinetics in the two assays used. Conclusions Therefore, malaria parasite virulence does not inhibit the ability of BCG to control the growth of M. tuberculosis in vivo despite alterations in the immune responses. Additionally, neither Plasmodium infection prior to BCG vaccination nor Plasmodium infection post BCG vaccination abolishes the efficacy of BCG against M. tuberculosis although a decrease in CD4+ Th1 cytokine responses and concomitant increase in bacilli burdens are observed in the group of acute Plasmodium infection prior to BCG vaccination.
- ItemOpen AccessTNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis(BioMed Central Ltd, 2015) Francisco, Ngiambudulu; Hsu, Nai-Jen; Keeton, Roanne; Randall, Philippa; Sebesho, Boipelo; Allie, Nasiema; Govender, Dhirendra; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, MuazzamBACKGROUND: Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. METHODS: We generated neuron-specific TNF-deficient (NsTNF / ) mice and compared outcomes of disease against TNF f/f control and global TNF / mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracerebral M. tuberculosis infection of TNF / mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF / mice were resistant to infection and presented with a phenotype similar to that in TNF f/f control mice. Impaired immunity in TNF / mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. CONCLUSION: Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB.