Browsing by Author "Keeton, Roanne"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    Open Access
    Investigating cytokine responses to different strains of Mycobacterium tuberculosis
    (2021) Booi, Zandile; Burgers, Wendy A; Keeton, Roanne
    Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the world's most prevalent fatal pathogens, infecting one third of the global population. The highly specialised pathogen that infects humans today evolved from a group of bacteria, known as the Mycobacterium tuberculosis complex (MTBC). It is divided into different lineages, based on phylogeography and genetic structure. It is well established that infection with different strains can lead to different types of innate immune responses. Recent studies indicate differences in the adaptive immune response (IL-22 and IL-17) induced by various Mtb strains, however these differences have not been clearly defined. The aim of this study was to investigate CD4+ T cell adaptive cytokine responses (IFN-γ, IL-22 and IL-17) in blood from human volunteers with prior exposure to Mtb, when stimulated with different strains within the MTBC, using a whole blood assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA). My results indicate that the frequency of IFN-γ and IL-22-producing CD4+ T cells was similar for the two strains, as no significant differences were observed. In contrast, IL-17 responses for HN878 was almost 2-fold higher than H37Rv (p=0.04), consistent with published observations. Moreover, three additional Mtb strains (Indo-oceanic, East African-Indian and the Euro-American outbreak strain CDC1551) were tested for their ability to detect cytokine responses. While all five strains enabled the detection of Mtb-specific CD4+ T cell IFN-γ, IL22 and IL-17, no significant differences were observed when comparing the responses between strains. The highest responses were observed for IFN-γ, followed byIL-22 and IL17. In addition to flow cytometry, soluble cytokine concentrations of IFN-γ and IL-22 were examined in plasma in whole blood stimulated with Mtb lysate derived from H37Rv and HN878, using an ELISA. Responses were tested at both 24 and 48 hours of stimulation, and while median responses were higher at the later time point, they were not significantly different. IFN-γ showed no difference between H37Rv and HN878 at 24 hours in the ELISA. In contrast, HN878 IFN-γ responses were lower than H37Rv at 48 hours (p=0.04). The amount of secreted IL-22 did not differ significantly between the twos trains at either time point. Further work is required to confirm these findings in a larger cohort to understand differences in adaptive responses to different Mtb strains. In conclusion, understanding Mtb strain modulation of host immune function is of major importance, as it may provide better insights into human TB immunity and could assist in the development of an effective TB vaccine.
  • No Thumbnail Available
    Item
    Open Access
    Investigating SARS-CoV-2 immune responses in children
    (2025) Benede, Ntombi; Keeton, Roanne; Burgers Wendy
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infec
  • Thumbnail Image
    Item
    Open Access
    NK-, NKT-and CD8-derived IFNγ drives myeloid cell activation and erythrophagocytosis, resulting in Trypanosomosis-associated acute anemia
    (Public Library of Science, 2015) Cnops, Jennifer; Trez, Carl De; Stijlemans, Benoit; Keirsse, Jiri; Kauffmann, Florence; Barkhuizen, Mark; Keeton, Roanne; Boon, Louis; Brombacher, Frank; Magez, Stefan
    African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia.
  • Thumbnail Image
    Item
    Open Access
    Reactivation 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, Muazzam
    Of 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.
  • Thumbnail Image
    Item
    Open Access
    The characterisation of dendritic cell, microglial, macrophage and T cell responses during mycobacterial infection of the central nervous system
    (2021) Kgoadi, Khanyisile; Jacobs, Muazzam; Keeton, Roanne
    Background: 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.
  • Thumbnail Image
    Item
    Open Access
    The 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, Roanne
    Background 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.
  • Thumbnail Image
    Item
    Open Access
    TNF-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, Muazzam
    BACKGROUND: 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.