Browsing by Subject "Clinical Science and Immunology"
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- ItemOpen AccessAlterations in preconception, antenatal, and postnatal maternal gut microbiota influence offspring intestinal microbiota and immunity(2017) Nyangahu, Donald D; Jaspan, Heather B; Horsnell, WilliamMaternal microbiota during pregnancy, as well as maternal disease state, may impact offspring gut bacterial colonisation. Here, we explore the impact of maternal antibiotics during gestation and/or nursing on offspring gut microbiota. Further, we investigate the effect of preconception helminth infections on maternal and infant gut microbiota. For maternal antibiotic experiments, dams were fed vancomycin, polymyxin B, or both, in drinking water during gestation, nursing or gestation plus nursing, and their offspring microbiota analysed at 14 days of life, alongside immunity in the spleens. Offspring born to vancomycin treated mothers had significantly higher relative abundance of Proteobacteria and Tenericutes while maternal oral polymyxin B led to significantly lower abundance of Proteobacteria and Deferribacteres in infants. Maternal oral vancomycin led to significant reduction in proportions of infant central memory CD4+ T cells (CD4+CD44hiCD62Lhi) regardless of antibiotic timing. Effector memory CD4+ T cells were significantly lower in pups born to dams treated with polymyxin B while nursing while proportions of central memory CD4 T cells were significantly increased in gestation only or gestation plus nursing pups. In addition, oral vancomycin in dams during nursing resulted in significantly reduced proportions of both total and follicular B cells in offspring born to antibiotic treated dams. Pups born to Vancomycin treated mothers had a significant delay in growth when infected with Respiratory Syncytial Virus (RSV). On the other hand, pups born to mothers treated with Polymyxin B during gestation or gestation plus nursing were susceptible to Nippostrongylus brasiliensis (Nb) infection. In the second study, we infected female BALB/c mice with 500Nb L3 three weeks prior to mating and examined the effect of preconception helminth infection on offspring microbiota and immunity. Preconception Nb infections led to alterations of maternal gut microbiota during pregnancy. In addition, we observed dramatic differences in offspring microbiota in pups born to previously helminth infected dams. Coriobacteriaceae were predominant in pups born to previously Nb infected dams when compared to uninfected dams. Overall, manipulation of maternal microbiota during gestation or lactation profoundly impacts offspring growth, intestinal microbiota and immunity to RSV and helminths.
- ItemOpen AccessBystander influence of nematode exposure on subsequent herpesvirus infections in vivo(2019) Chetty, Alisha; Horsnell, William; Dewals, BenjaminParasitic worms have the ability to modulate the hosts immune response to promote host control of the infection and also parasite survival in the host. Helminth infections classically induce a potent Th2-biased and regulatory immune imprint. This immune response also influences unrelated inflammatory processes in the host. Studies have shown helminth infections have bystander influences on unrelated conditions such as allergy and autoimmunity. Additionally, helminth infections can alter susceptibility to other infections. In this thesis, we investigate the systemic influences of murine nematode Nippostrongylus brasiliensis infection on host immunity in colonized and non-colonized tissues, and the implications of these effects on susceptibility to subsequent herpesvirus infections in vivo. We show that prior N. brasiliensis infection enhanced control of acute respiratory murid gammaherpesvirus (MuHV-4) infection, with an increase in viral-specific CD8+ T cells in colonized lung tissue. Enhanced effector cytokine responses by cytotoxic T cells were also observed with prior helminth exposure. Conversely, despite enhanced primary control, prior helminth exposure was associated with earlier and heightened genital reactivation of MuHV4. This demonstrates differences in local bystander and systemic effects of helminth exposure on the host, and on unrelated viral infections. We also show that N. brasiliensis infection, which transits the respiratory and gastrointestinal tracts, also systemically influences immunity in the female genital tract (FGT) in vivo. Here, helminth infection induced Th2-type immunity in the FGT, namely increased tissue IL-4, IL-5 and long-lasting eosinophilia. We further demonstrated that systemic influences of N. brasiliensis infection results in exacerbated genital pathology and inflammation, following subsequent intravaginal herpes simplex virus type II (HSV-2) infection. Increased HSV-2 pathology with prior helminth exposure was associated with diminished innate anti-viral immunity, increased IL-33, ILC2 and IL-5 responses, as well as significant eosinophilia. Interestingly, abolition of canonical Th2 immune signalling by the lack of IL-4Rα expression, enhanced innate anti-viral defences and provided protection from HSV-2 pathology. However, N. brasiliensis-induced exacerbation of HSV-2 illness was IL-4Rαindependent, associated with significant genital eosinophilia. Furthermore, antibody-depletion of eosinophils ameliorated nematode-exacerbated HSV-2 pathology, suggesting that nematode-induced genital eosinophilia mediates increased HSV-2 pathology in coinfected mice. We have therefore shown that helminth infections can induce local and systemic bystander immunity to lymphoid and myeloid immune compartments, which alters susceptibility to subsequent herpesvirus infections.
- ItemOpen AccessCancer cell behaviour following parasite exposure(2018) Jacobs, Brittany-Amber; Smith, Katherine; Prince, SharonInfectious diseases, including helminthiases, are estimated to cause 16.1% of global cancer cases. While certain helminths are conclusive causes of cancer, others have been shown to reduce the disease. It is currently unknown why differing helminth infections promote or prevent cancer development and progression, or which cellular mechanisms are altered following exposure. Using several in vitro and in vivo techniques, this study aimed to determine the effect that certain helminths have on the progression of cervical and colorectal cancer. The results revealed that antigen from the hookworm Nippostrongylus brasiliensis significantly reduced cervical cancer cell migration and the expression of two markers of metastasis: vimentin and N-cadherin. Importantly, N. brasiliensis antigen significantly lowered the expression of cell-surface vimentin, while decreasing Human Papillomavirus type16 pseudovirion internalization. In vivo infection with N. brasiliensis significantly decreased vimentin expression within the female genital tract, confirming the relevance of these in vitro findings. Furthermore, exposure to antigen from the gastrointestinal nematode Heligmosomoides polygyrus decreased the in vitro proliferation of human and mouse colorectal cancer cells and simultaneously increased the expression of cell cycle regulator proteins, p53 and p21. Surprisingly, while antigen from H. polygyrus inhibited human colorectal cancer cell migration, it had the opposite effect on mouse colorectal cancer cells, suggesting that its impact on colorectal cancer migration may be, at the very least, species dependent. Using a syngeneic tumour model, the excretory-secretory product from H. polygyrus was shown to significantly increase tumour growth and the expansion of regulatory T cells and neutrophils in the tumour. Similarly, in a model of colitis-associated colorectal cancer this antigen significantly worsened pathology in a TGF-β dependent manner. Undoubtedly, the knowledge gained from this study will contribute to the limited understanding about helminths and the effect that these parasites have on cancer progression.
- ItemOpen AccessCharacterization of CD8 T cell responses in Mycobacterium Tuberculosis infection(2011) Moshi, Noell Dominika; Day, Cheryl; Hanekom, Willem AThe aim of this project was to compare the breadth and magnitude of CFP10 and ESAT6-specific CD8 T cell responses in individuals with latent Mycobacterium tuberculosis (MTB) infection (LTBI) and active TB disease, and further define MTB-specific CD8 T cell phenotypes associated with latent infection and active disease. Ex vivo IFN? Elispots and proliferation assays were used to identify immunodominant ESAT6 and CFP10 15mer peptides targeted by CD8 T cells in LTBI and TB donors. A multiparameter flow cytometry panel was designed and optimized to assess turnover, susceptibility to apoptosis and terminal differentiation/senescence in CD8 T cells from TB and LTBI donors. Bcl-2, Ki67,CD95, CD57, CD127 and IFNγ were thus measured in each group.
- ItemOpen AccessDevelopment of an Autologous Human Dendritic Cell Vaccine against Mycobacterium tuberculosis in Patients with Extensively Drug-Resistant Tuberculosis(2022) Londt, Rolanda Sabrina; Dheda, Keertan; Tomasicchio, MicheleIntroduction: Extensively drug-resistant tuberculosis (XDR-TB), and resistance beyond XDR-TB (often untreatable), is an increasing public health concern globally. Drug resistance has outpaced the drug development pipeline. Therefore, alternative immunotherapeutic approaches are urgently needed. In this proof-of-concept study, and based on precedents in breast and prostate cancer, we aimed to develop an autologous therapeutic dendritic cell (DC) vaccine by evaluating two TB antigen-specific multi-peptide pools in combination with different adjuvants (such a cellular vaccine would require ex-vivo manipulation in a clean room and reinfusion back into the patient). Vaccine efficacy was evaluated using an in vitro mycobacterial containment model. Methods: DCs were derived from monocytes isolated from the peripheral blood of patients with XDR-TB (n=30) and participants with presumed latent TB infection (LTBI; n=15). DCs were matured with a differential combination of cytokines and pattern-recognition receptor agonists (maturation cocktail) together with different TB antigen combinations. The complete cocktail contained interferon-, interferon-, CD40L, IL-1 and TLR-3, TLR-7 and TLR-8 agonists, whilst in the limited cocktail the TLR agonists were absent. Two M.tb-specific multi peptide pools suited to GMP-grade vaccine manufacture were evaluated: (i) an immunodominant peptide pool (ESAT6 + CFP10 + Ag85B + TB10.4; referred to as ECAT) and (ii) a PE/PPE peptide pool. PPD and the lysate of a clinical M.tb strain (HN878), though not amenable to GMP-grade vaccine development, served as controls representing the entire antigenic repertoire of M.tb. Thus, there were four major comparator groups (unstimulated DCs, limited cocktail-only stimulated DCs, antigen-only stimulated DCs, and antigen + complete cocktail stimulated DCs). As the two latter groups were interrogated using the two multi-peptide pools and the two broad-spectrum antigen controls, a total of 10 different experimental groups were generated (see overview figure 3.2). DCs were assessed for the expression of key maturation markers using flow cytometry and the secretion of Th1-polarising cytokines by ELISA. The ability of DC-primed peripheral blood mononuclear cells (PBMCs) to restrict the growth of M.tb-infected monocyte derived-macrophages was evaluated using an in vitro validated mycobacterial containment assay. Results: In patients with XDR-TB, DCs matured with any M.tb-antigen + complete cocktail, compared to DCs matured with M.tb-antigen only, showed significantly higher upregulation of CD80, CD83, CD86, and CCR7 (p< 0.001 for all comparisons), and higher secreted levels of IL12p70 (0.67 versus 0.01 ng/mL per 106 cells; p< 0.001). A similar pattern was seen in the containment experiments: mycobacterial stasis within the XDR-TB group was significantly better with antigen + complete cocktail versus antigen alone (p≤0.0002 for PE/PPE and PPD), and the limited cocktail did not show this effect. Furthermore, PE/PPE + complete cocktail matured DCs achieved a higher magnitude of mycobacterial containment compared to ECAT + complete cocktail-matured DCs (50%, IQR:39-75, versus 46%, IQR: 15-62, p= 0.02). Using PPD and the HN878 lysate did not improve the containment effect. Furthermore, the improved containment effect of the PE/PPE + complete cocktail, versus ECAT + complete cocktail, was only seen in the XDR-TB and not in the LTBI group. Conclusion: In patients with XDR-TB, an effector response primed by PE/PPE antigen and complete cocktail-matured DCs was able to better restrict the growth of M.tb in vitro. These data indicate proof-of-concept feasibility to generate a DC-based immunotherapeutic intervention for therapeutically destitute patients with DR-TB. Further mechanistic studies and future phase 1 and 2 human clinical studies are warranted.
- ItemOpen AccessThe impact of myeloid derived suppressor cells on vaccine immunogenicity in South African HIV-infected and uninfected mothers and their infants(2016) Kidzeru, Elvis Banboye; Jaspan, Heather BBACKGROUND: Each year over 4 million infants die from infections, of which many are vaccinepreventable. Young infants respond poorly to vaccines, but the basis of reduced immunity is controversial. We hypothesized that myeloid-derived suppressor cells (MDSC) that might be induced during gestation, would persist at birth leading to active suppression of infant-immune responses. OBJECTIVE: We evaluated the ontogeny of MDSC and the effect of MDSC on vaccine immunogenicity during early life in South African infants and mothers, and in HIVexposed uninfected (HEU) infants and HIV+ mothers. METHODS: HIV-infected and uninfected mothers and their infants were recruited from Khayelitsha, Cape Town and followed-up for one year. In whole PBMC and after MDSC (CD15+) depleted, we measured BCG, Hepatitis B, Tetanus toxoid and Bordetella pertussis vaccine-specific CD4+ T cell proliferation by CFSE and IFN-γ responses using ELISpot assay.
- ItemOpen AccessAn in vitro investigation of the effect of Khellin and UVA (KUVA) on normal and transformed human melanocytes(1999) Carlie, Gadija; Kidson, Susan; Hulley, PhillipaThe problematic and numerous side effects of PUVA (psoralen and UVA) and other treatments currently in use for vitiligo, have justified the search for an alternative treatment. In this study, the use of khellin, a naturally occurring furochromone, which is similar in structure to psoralens, is explored as an alternative treatment for vitiligo. When khellin is combined with UVA (KUVA), it is reported to repigment vitiligo skin as effectively as PUVA photochemotherapy, but without the adverse effects reported with PUVA. The exact mechanism for khellin-induced repigmentation is still to be determined and no cell biological studies have yet been done to elucidate its mechanism of action. The specific aim of this project was to set up an in vitro tissue culture system to determine the direct effects of khellin, UVA and KUVA on melanocyte proliferation and pigmentation. Studies were carried out on cultures of a human melanoma cell line (Mel-1), normal human melanocytes and 3T3 mouse fibroblasts. Cell proliferation assays revealed that the proliferation of melanoma cells and melanocytes were increased after exposure to khellin for four days at concentrations ranging from 1nM to 0.5mM. A peak of proliferation was obtained at 0.01mM khellin, which stimulated proliferation of melanoma cells and melanocytes by 2.3 5-fold and 2.1- fold, respectively. In contrast, khellin decreased proliferation of fibroblasts over the entire concentration range tested. At concentrations of 0.5mM and above, khellin was cytotoxic to both melanocytic cells and fibroblasts. Cytotoxic assays revealed that 1mM khellin was equally cytotoxic to both melanoma cells and fibroblasts. In addition, these assays revealed that the proliferative response observed with 0.01mM and 0.1mM khellin, did not mask an underlying cytotoxic effect. Exposure to single doses of UVA between 150-280mJ/cm², increased proliferation of melanoma cells with maximal proliferation at 250mJ/cm², while the proliferation of normal melanocytes and fibroblasts were unaffected by this UVA dose. More significantly than khellin or UVA alone, the treatment with the combination of khellin and UVA (KUVA) stimulated proliferation of the melanocytic cells. The combination of 0.01mM khellin plus a single dose of UVA at 250mJ/cm² was the most effective treatment. KUVA combination treatments were found to be more cytotoxic to the fibroblasts than khellin or UVA alone. To test the effect of khellin, UVA and KUVA on melanogenesis, standard radiometric assays were carried out. The combination of khellin and UVA enhanced melanogenesis of the melanocytic cells more significantly than khellin alone or UVA alone. The dose of 0.01mM khellin plus 250mJ/cm² UVA (maximal proliferative dose) increased melanogenesis of the melanocytes by 290% above the untreated control melanocytes. To determine whether khellin and KUVA act by increasing levels of melanogenic proteins, western blot analyses were carried out. The results revealed that there were no differences in the amounts of TRP-2 and tyrosinase in the melanocytic cells treated with khellin alone. In contrast, those treated with KUVA (or UVA alone) had increased levels of the glycosylated form of the enzymes and also possibly had increased levels of the de nova form of the enzymes. These results suggest that UVA might be enhancing glycosylation of melanogenic enzymes and provides a novel insight into the possible mechanism for UVA-induced melanogenesis. The results also revealed that 3T3 fibroblasts expressed the non-glycosylated form of TRP-2, as described by others. In conclusion, this study suggests a model in which khellin acts directly on melanocytic cells by acting as a mitogen and a melanogen at non-toxic concentrations. Khellin possibly increases melanogenesis by acting post-translationally or antagonizing or removing an inhibitor of the melanogenic pathway. The melanocyte-specific effect of khellin and even more so KUVA, seems to suggest that khellin acts along the signal transduction pathways which increases both proliferation and melanogenesis in melanocytes, possibly via endothelin-1 pathways.
- ItemOpen AccessPeripheral inflammatory and regulatory immune changes in HIV positive to HIV positive renal transplant recipients(2018) Rautenbach, Stefan; Gray, CliveINTRODUCTION Since 2008, 43 renal transplants have been performed from HIV positive deceased donors to HIV positive recipients with renal failure predominantly due to HIV-associated Nephropathy (HIVAN). Recipients received Anti-thymocyte globulin (ATG) induction therapy and maintenance immunosuppression. Despite transplantation across Human Leukocyte Antigen (HLA) mismatches, there were few rejection events in the first year post-transplant (PT). Recipient CD4 counts did not decrease and HIV viral load also remained undetectable at one year PT. To gain insight into immune homeostatic mechanisms after ATG induction, immunosuppression and transplantation, a subset of 10 transplant recipients were investigated. This dissertation examined levels of peripheral inflammatory and regulatory cytokines. A polychromatic flow cytometry panel was also developed to measure the phenotypic T cell proportions of T regulatory cells (Tregs) in the blood circulation. METHODOLOGY A multiplexed Luminex assay was used to measure the concentrations of 67 inflammatory and regulatory plasma cytokines immediately pre-transplant, at 1, 3, 6 and 12 weeks PT. Two separate manufacturers of Luminex panels were used and a series of statistical analyses were employed to identify intra- and interplate variation. Firstly, data was cleaned up by excluding analytes for which >90% of measured values were outside of the observable range of the standard curve. Secondly, the measurable values were assessed for differences between replicates (intra-plate variation). A Bland-Altman plot was used to identify and exclude highly divergent replicates of the same sample. Thirdly, a Paired Ttest/Wilcoxon Signed Rank Test was used to investigate differences between inter-plate controls (inter-plate variation). Fold change from baseline was calculated for all values to correct for inter-plate variability. After correcting for variability, fold change trends in all included analytes were examined for each recipient. Trends in recipients with rejection events (rejectors) and recipients without rejection events (non-rejectors) were also compared. Fold change from baseline was assessed to identify single analytes that differed over time using a Paired T-test/Wilcoxon Signed Rank Test. A hierarchical clustering analysis (HCA) was used to identify groups of analytes for which fold change may have been significantly influenced by age, sex, baseline CD4 count at transplantation (TP), number of HLA matches, or time. A mixed effect generalised linear model (MEGLM) was constructed to calculate differences between participants for each cytokine. A polychromatic flow cytometry panel was devised to measure Treg CD4+ T cells consisting of the following antibodies: CD3-BV650, CD4-PE/Cy5.5, CD8- HorizonV500, CD27-PE/Cy5, CD45RA-PerCP/Cy5.5, CD25-BV421, CD127- PE/CF594, and FoxP3-Alexa647. Antibody concentrations in the panel were optimised by titrating each marker on resting, or Phytohaemagglutinin (PHA) stimulated, peripheral blood mononuclear cells (PBMCs). The median fluorescent intensity (MFI) of the positive and negative populations for each marker was used to calculate the signal:noise ratio for all titrating volumes. The optimal volumes were determined by the highest signal:noise ratio for each marker. RESULTS In all recipients, when compared to baseline, IL-2, IFN-α2 and IFN-γ were significantly decreased at 12-weeks PT. IL-35 was significantly decreased at weeks 1, 3, 6 and 12 PT, whilst IL-10 increased significantly at 1-week PT in 5 recipients. Hierarchical clustering showed no association with analyte fold changes due to age, sex, CD4 count, or number of HLA matches. It did show a decrease over time in IL-35, IFN-γ, IL-20, IL-28A, and IL-11 for all 10 recipients. A mixed-effects generalized model (MEGLM) was used to identify analytes with variable concentrations between recipients. It showed that the concentrations of IL-28A, IL-6Rα, IL-6Rβ, sTNFR1, Pentraxin-3 and IFN-γ varied the most between recipients. IL-35 and TNFSF12 were shown to vary the least between recipients. These data suggest heterogeneity in the highly variable analytes, none of which were shown to differ over time. The heterogeneity is likely due to genetic diversity, history of opportunistic infections and relevant prophylaxis. The concentrations of IL-35 did not vary between recipients, but it was shown to decline over time in all recipients. This data suggests a consistent decline in the concentration of IL-35 in all recipients over the first 12-weeks PT. An 8-colour Regulatory T cell (Treg) flow cytometry panel was designed based on the Luminex results and optimised to phenotype peripheral T-cell subsets. It distinguished between different T-cell phenotypes, namely naïve and memory, CD4 or CD8, and activated and regulatory T cells. Antibody titrations identified the optimal volume of each marker to use in a combination cocktail. Due to time constraints, the panel was not used on patient material, but the panel and its optimisation has been described in detail. CONCLUSIONS Statistical investigations into the Luminex results identified variability between replicates and between plates. These differences needed to be accounted for before combining data between plates and kits to arrive at biological conclusions. By using stringent analysis, this dissertation shows that multiplex data is highly variable and a series of statistical approaches should be employed to avoid including erroneous data. A decrease in both inflammatory and regulatory proteins was shown in the 12 weeks after transplantation and ATG induction. The transient increase in IL-10 suggested the induction of effector T-cells to become IL-10 producing Tregs (Tr1), known to occur in response to ATG induction. Combined with the consistent decline in IL-35 in all recipients, these results suggested that there was preferential secretion of IL-10 over IL-35 in some patients early after transplantation.
- ItemOpen AccessRemodelling of Mycobacterial Peptidoglycan During Cell Division and the Epigenetics of Macrophages during M. tuberculosis infection(2021) Kieswetter, Nathan Scott; Guler, Reto; Ozturk, Mumin; Brombacher, FrankTuberculosis (TB) has emerged as the world’s most deleterious infectious disease. The etiological agent of TB, Mycobacterium tuberculosis (Mtb), has evolved the ability to evade the host immune system using several mechanisms; emphasising the need for novel treatment strategies. Peptidoglycan (PG) is an important immunomodulatory heteropolysaccharide structure that can be shed during mycobacterial infection with immunological consequences and as such, changes in PG structure are expected to have important implications on disease progression and host responses. Mycobacterial amidases have been shown to have important roles in the remodelling of PG during cell division in M. smegmatis and are implicated in sensitivity to antibiotic treatment. However, their roles in modulating host immunity remain unknown. Herein, we assess the immune responses to Mtb mutants defective for either one of two amidases, Ami1 and Ami4, in bone marrow-derived macrophages (BMDM) and the C57BL/6 murine models of tuberculosis. Both Ami1 and Ami4 deletion resulted in increased pro-inflammatory response in BMDM. Infection with the Mtb Δami1 mutant in mice resulted in differential induction of proinflammatory cytokines and certain chemokines during the acute phase of the infection, an eff ect that was abrogated in chronic phase infection. The Δami1mutant was found to be susceptible to antibiotics in liquid growth culture but this sensitivity was negated in macrophages and reversed to a tolerant phenotype in mice. The Δami4 mutant, by contrast, did not display differential antibiotic susceptibility and did not significantly alter cytokine and chemokine responses relative to the wildtype control in mice. These findings suggest that Ami1 and Ami4 in Mtb play a nonoverlapping role in antibiotic sensitivity and modulating host immunity during tuberculosis. Additionally, the specific epigenetic alterations which occur during host-Mtb infection that contribute to immune evasion remain unknown. Here, we propose a method to elucidate transcriptomic changes in both human primary monocyte-derived macrophages (MDM) and the Mtb bacillus with which they were infected. In this study, we exhibit a dual-RNA-seq proof-of-concept methodology where, from a single donor, we successfully sequence host RNA from infected MDMs as well as Mtb RNA enriched from those same infected MDMs. Utilizing this optimised methodology, we aim to discover and model epigenetic and transcriptional alterations as well as their effector proteins in primary human macrophages following Mtb infection. Further, we aim to identify novel and annotated ncRNAs which are correlated with these epigenetic modifications.
- ItemOpen AccessThe role of IL-4 receptor alpha in chronic allergic airway disease (AAD)(2013) Jayakumar, Jaisubash; Brombacher, Frank; Nieuwenhuizen, Natalie
- ItemOpen AccessThe association between the oral and vaginal microbiome of young South African women(2019) Esra, Rachel; Jaspan, Heather; Balle, ChristinaBacterial vaginosis (BV) and periodontal disease (PD) are conditions characterised by reduction of healthy bacterial communities in the vaginal and oral microbiomes respectively. Both BV and PD are associated with an increased risk of preterm labour and negative birth outcomes, yet it is unknown whether PD and BV are independent risk factors or may be interrelated. Understanding the health risks associated with pregnancies in young women is critical for developing new preventative interventions and for informing guidelines. Current knowledge of what constitutes a healthy microbiome is largely based on North American studies and may not be applicable to the South African population. This study characterises the oral and vaginal microbiome of South African female adolescents and investigates the association between alterations in oral bacterial diversity and BV in young South African women. DNA was extracted from matched lateral vaginal wall, saliva and periodontal samples and V4 16S sequencing was performed using MiSeq technology. The composition of the core oral microbiome of South African female adolescents was found to be similar to descriptive studies published in other populations. We additionally report a description the vaginal microbiome that is in agreement with previous studies in the South African population. PD-associated bacterial species were enriched in the oral microbiome of women with clinically diagnosed BV and in those with Lactobacillus iners dominant vaginal community types (VCTs) compared to asymptomatic women and those with L. crispatus dominated VCTs respectively. While this data provides evidence in support of a relationship between oral and vaginal dysbiosis, it unclear in which compartment bacterial dysbiosis would originate, should the association holds true.
- 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 role of Cysteinyl leukotriene receptor-1 during experimental helminth infections in murine model(2021) Mosala, Paballo Pertunia; Brombacher, Frank; Ndlovu, HlumaniCysteinyl leukotrienes (cysLTs) are potent inflammatory lipid mediators that play a major role in the pathophysiology of inflammatory diseases. They signal primarily through cysteinyl leukotriene receptor-1 (cysLTR1) and have been reported to drive Th2 immune responses. Initiation and amplification of robust Th2 immune responses is crucial for conferring protective immunity to helminth (Schistosoma mansoni and Nippostrongylus brasiliensis) infection in mice. The role played by cysLTs in the development of protective immune responses to helminth infections is not well documented. Hence in the present study, we investigated the role of cysLTs during helminth infection using cysteinyl leukotriene receptor-1 deficient (cysLTR1-/- ) mice. Under steady state conditions, young naïve cysLTR1-/- mice did not reveal any significant alteration of the cellular, tissue and phenotypic profile although we did observe expansion of central memory T cells (Tcm) in secondary lymphoid organs in cysLTR1-/- as compared to wildtype mice. Primary infection with N. brasiliensis indicated increased worm burden in cysLTR1-/- mice at day 7 post infection and a delay in the resolution of infection by day 9 post infection when compared to wild type mice. Furthermore, we observed reduced Th2 immune responses as well as impaired contractility of the small intestine, which are key features required for protective immunity to N. brasiliensis infection. Furthermore, recall of memory responses to N. brasiliensis was abrogated in cysLTR1 -/- mice, with higher numbers of adult worms recovered at day 5 post re-infection in cysLTR1-/- mice comparison with wild type mice. Additionally, cysLTR1-/- mice exhibited impaired production of IL-13 in the lungs and draining lymph nodes compared with wildtype mice. Finally, there was reduced recruitment of effector CD4+ T cells and central memory CD4+ T cells in the lungs of cysLTR1 deficient mice compared to control mice. Taken together, these data demonstrated an essential role played by cysLTR1 in clearance and resolution of N. brasiliensis infection. CysLTR1-/- mice survived acute S. mansoni infection similarly to wildtype mice. In addition, cysLTR1-/- mice displayed reduced granulomatous inflammation and reduced cellular responses in the liver compared with wildtype mice. Further analysis revealed reduced gut fibrosis but cytokine production, immune cell recruitment in the gut and both type 1 and type 2 antibodies were found to be comparable between wildtype and knockout mice, demonstrating that cysLTs signaling through cysLTR1 contribute to granuloma formation in the liver. Similar to acute schistosomiasis, cysLTR1-/- mice were not susceptible to chronic schistosomiasis and indicated by prolonged host survival. This increased host survival observed in cysLTR1-/- mice was associated with reduced granulomatous inflammation, reduced fibrosis and hepatocellular damage, impaired production of IL-4 in the liver, and reduced intracellular secretion of IL4 by CD4+ T cells and ILC2s in cysLTR1-/- mice compared with wildtype mice. Furthermore, we observed reduced granulomatous inflammation in the lungs of chronically infected cysLTR1-/- mice despite the heightened Th2 immune response in the lungs. Collectively, these data revealed that disruption of cysLTR1 leads to reduced granulomatous inflammation and reduced production of IL-4 in the liver during chronic schistosomiasis. In conclusion, the current study demonstrated both positive and negative roles for cysLTs signaling through cysLTR1 during different helminth infection models. Absence of cysLTR1 during N. brasiliensis leads to delayed expulsion of adult worms and impaired recall of memory responses, indicating that cysteinyl leukotriene signaling via cysLTR1 is essential for orchestrating host protective responses. On the other hand, signaling via cysLTR1 appears to be dispensable for the development of host protective responses during acute schistosomiasis in mice. However, mice deficient of cysLTR1 had reduced liver pathology during chronic schistosomiasis, suggesting that inhibition of this receptor could be a potential therapy for reducing granulomatous liver pathology.
- ItemOpen AccessThe role of Cysteinyl leukotriene type 1 receptor (CysLTR1) during Listeria monocytogenes infection in mice(2020) Poswayo, Sibongiseni Kwakho Luntukazi; Parihar, Suraj; Ozturk, Mumin; Brombacher, FrankSouth Africa recently experienced a Listeriosis outbreak, which was responsible for over 180 deaths, caused by an intracellular, rod-shaped bacilli called Listeria monocytogenes (LM). LM can infect both phagocytic and non-phagocytic cell types and induces its uptake by expressing internalin A and B, then secretes listeriolysin O (LLO), a virulence factor forming pores on the phagosome membrane to escape into the cytosol. Macrophages can phagocytose invading pathogens and induce innate inflammatory responses. Production of cytokines and eicosanoids by antigen presenting cells activates the adaptive immunity. Eicosanoids (epoxyeicosatreinoic acids, prostanoids and leukotrienes) are generated from metabolites of 20-carbon chained polyunsaturated fatty acids and arachidonic acid. Leukotrienes (LTs) are generated from 5- lipoxygenase-metabolism of arachidonic acid to LTB4 and cysteinyl LTs (cysLTs). CysLTs are pro-inflammatory lipids that have pathobiological functions in asthma. CysLTs function through three G-protein coupled receptors (CysLTR1, CysLTR2 and GPR99). The CysLTR1 and its ligands function has been well elucidated in asthmatic and allergic responses however, its role in bacterial infections is unknown. The aim of our study was to elucidate the role of CysLTR1 on disease progression in mice and macrophages infected with LM. In this study, we showed that CysLTR1 mRNA expression is upregulated by LM infection in WT macrophages and mice. Mice deficient of CysLTR1 had no defects at homeostasis. During time kinetic experiments with LM, CysLTR1 knockout mice displayed increased neutrophil recruitment and decreased lymphocyte cells at 3dpi, however, bacterial burdens were comparable to wild-type mice. In addition, macrophages deficient of CysLTR1 have no effect on the intracellular growth of LM. In conclusion, CysLTR1 signalling plays a role in lymphoid cell activation and neutrophilic recruitment during early LM infection, however, further studies are required to better understand the role of CysLTR1 during inflammatory responses.
- ItemOpen AccessThe role of Interleukin-4 induced gene 1 (IL-4i1) in allergic asthma and atopic dermatitis(2022) Ngomti, Amkele; Hadebe, Sabelo; Brombacher, FrankAllergies are described as an unnecessary immune response to non-harmful substances known as allergens. Both allergic asthma and atopic dermatitis (AD) are said to be induced by elevated levels of immunoglobulin E (IgE) and T helper 2 (Th2) immune cells and inflammatory associated cells such as eosinophils, mast, and basophils. Globally, asthma is affecting more than 300 million people and is characterized by chronic airway inflammation, reversible airflow limitation, and airway hyperreactivity. AD is affecting approximately 15%-20% of the pediatric population and 7%-10% of adults in the world and is characterized by dysregulation of skin barrier and immunity, eczematous lesions, dry and itchy skin. Dysfunctional tolerogenic immune response to these innocuous allergens has been described as a leading cause of allergic disease pathogenesis. Interleukin-4 induced gene 1 (IL-4i1) is a secreted L-amino acid oxidase enzyme mainly expressed by antigen-presenting cells (APCs) and upon activation by IL-4 and CD40, can be induced in B lymphocytes. IL-4i1 converts phenylalanine into phenylpyruvate, ammonia, and hydrogen peroxide which can induce effector T cells suppression by inhibiting their activation, proliferation, and cytokine production while promoting a regulatory T cell (Tregs) arm. The contribution of IL-4i1 and its immunoregulatory potential has not yet been explored in allergic asthma and atopic dermatitis. Thus, we proposed to investigate the role of IL-4i1 during allergic asthma and atopic dermatitis using acute mouse models. Female mice of 8-12 weeks old either sufficient (IL-4i1+/+) or deficient of IL4i1 (IL-4i1-/- ) backcrossed to BALB/c genetic background were used in this study. For induction of allergic asthma, a high dose (100µg/per mouse) of house dust mite (HDM) was used in sensitizing mice intratracheally at day 0 and challenged at day 7 to 11 intranasally under anaesthesia. To assess the development of asthma features, we measured lung function on day 14 and collected blood for ELISAs, mediastinal lymph nodes, and lung tissues for FACS and RNA. For induction of AD, a skin irritant vitamin D3 analog (MC903) was used to topically sensitize shaved mice (IL-4i1+/+ or IL-4i1-/- ) for 9 consecutive days. We assessed disease score and skin inflammation at day 10 and collected blood, inguinal lymph nodes, and skin for ELISAs, FACS, RNA, and histology analysis. In both disease models, we saw a significant reduction in total IgE in IL-4i1- deficient mice compared to IL-4i1+/+ littermate controls. A significant upregulation of Th2 cytokines and increased eosinophilia was seen in IL-4i1 deficient mice in the allergic asthma model with no changes in airway hyperresponsiveness. In AD model, we observed a protective effect in the absence of IL-4i1, which was demonstrated by no changes in body weight a reduced skin epidermal thickness, and reduced systemic type 2 cytokines, TSLP, IL-5, and IL-13 producing CD8 T cells. Furthermore, type 2 alarmin, TSLP was reduced at disease site. These results suggest a dichotomy of IL4i1 in regulation of type 2 immune responses depending on disease site. This data further suggests that IL4i1 may be a potential target for therapy against these diseases. Studies are currently underway to understand how IL-4i1 is induced and how it regulates downstream effector molecules and how these target molecules can be inhibited.
- ItemOpen AccessThe role of platelets in the pathogenesis of and immunity to helminth infections(2022) Pollock, Jonathan; Horsnell, William; Darby, MatthewBackground: Platelets are small, anucleate cells which circulate in blood and are often the first to respond to tissue damage and vascular inflammation caused by pathogens. Here they not only maintain tissue integrity and prevent bleeding, but also initiate and regulate a vast variety of immunologic responses. Little is known on the role of platelets in helminth infections, despite our understanding that many helminth species cause significant vascular pathology as they transfer from the circulatory system to diverse tissues as part of their life cycles. Based on previous studies showing tight association between platelets and innate immune responses during infection with other pathogens, we hypothesized that platelets significantly contribute toward acute (vascular) immunity to helminth infection. Objectives: This project aimed to investigate the role of platelets in regulating acute innate immune responses following infection with the murine gastro-intestinal nematode N. brasiliensis (Nb), commonly used to model human helminthiases. Specifically, it aimed to characterized plateletregulated responses involved in acute innate immunity during the pulmonary stage of infection, in which larvae exit the pulmonary vasculature and invade host lung tissue. Methods: C57BL/6mice were infected with 500 L3 Nb larvae, and the association of platelets with acute innate immune responses in the circulation and the lung were established by flow cytometry and immunohistochemistry. In further experiments, mice were depleted of their platelets using antibodies prior to infection with Nb and the effect of this on pulmonary pathology and innate immune responses was inferred from flow cytometric and histologic analyses of pulmonary tissues. Lastly, antibodies were used to interfere with platelet receptors during Nb infection to gain mechanistic insight into platelet regulation of neutrophil responses. Results: Infection with N. brasiliensis was associated with significant changes in the activation of platelets, their localisation into lung tissue and their interaction with innate immune cells. Additionally, platelet -immune cell interaction was associated with changes in the expression of factors known to play a role in driving the early immune response to Nb, including IFN-γ and RELMα. Furthermore, mice depleted of their platelets prior to infection had significantly enhanced pulmonary pathology and rapidly succumbed to infection. This was associated with significant changes in neutrophil responses, and depletion of neutrophils together with platelets significantly protected against enhanced pathology. Finally, direct and indirect targeting of the platelet receptors CD62P and CLEC-2 did not result in significantly enhanced pulmonary pathology but was associated with altered platelet and neutrophil responses. Conclusion: Herein, we have provided evidence that platelets tightly associate with protective host responses during acute N. brasiliensis infection and that their absence correlates with a dysregulated neutrophil response and enhanced helminth – associated pulmonary pathology. These data therefore collectively show that platelets play notable roles in the acute innate immune response to N. brasiliensis and that future investigations into the immunological functioning of platelets during helminth infection are warranted.
- ItemOpen AccessThe role of Surfacant Protein D in the control of human helminth infections(2020) Baker, Zoe; Horsnell, WilliamLung produced surfactant protein D (SP-D) is essential for both homeostasis and as an innate immune opsonin. In the project presented here, we aimed to translate data recently published by our group, which demonstrated that SP-D contributes to protection against murine parasitic nematode infections, to human work. In the first part of this study, we determined whether individuals exposed to helminths have altered serum SP-D in comparison to unexposed individuals, through analysis (ELISA and Western Blot) of bio banked samples in 2 clinical cohorts from South Africa. Secondly, we aimed to identify if SP-D influences the magnitude of anti-nematode responses in human immune cells (type 2 innate lymphoid cells, monocytes and macrophages) through in vitro cell work and flow cytometry. Our findings indicated an association between serum SP-D and exposure to helminths that have a lung migration stage as part of their life cycle (Ascaris spp and Toxocara spp). Furthermore, in vitro analysis demonstrated that human immune cells primed with SP-D might have an altered response to helminth antigen. These findings point toward the need for further investigation into the novel role of SP-D in the control of human helminth infections in the context of immune physiology, as a biomarker and eventually treatment option.