Browsing by Subject "Clinical Sciences and Immunology"
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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 AccessCharacterisation of mucosal tissue in the foreskin after voluntary medical male circumcision(2016) Harryparsad, Rushil; Gray, Clive M; Olivier, Abraham JacobusBackground: Medical Male Circumcision (MMC) reduces the risk of HIV-1 acquisition by up to 60% as shown in a number of randomized controlled trials in Uganda, Kenya and South Africa. MMC has also been shown to reduce the prevalence of other sexually transmitted infections (STIs) like Herpes Simplex Virus (HSV) -2 and Human Papillomavirus (HPV) by 25% and 35% respectively. Asymptomatic STIs may elevate the risk of HIV-1 acquisition by recruiting HIV-1 target cells to the foreskin. The higher permeability of the inner foreskin may play a role in HIV-1 acquisition as well as the number of target cells present in the foreskin. The more inflamed inner foreskin may be increasing the risk of a productive HIV-1 infection. The aims of this dissertation was to a) examine the levels of keratinisation in the inner and outer foreskins after MMC; b) investigate the number of Langerhans, Ki67+ and CD4+ T cells in the inner and outer foreskin and c) identify the impact of asymptomatic STIs on the numbers and proliferative capacity of foreskin-resident Langerhans and CD4+ T cells.
- ItemOpen AccessEffects of hormonal contraceptives on the female genital tract microbiota in South African adolescents(2018) Balle, Christina; Jaspan, Heather; Passmore, Jo-Ann; Lennard, KatieBackground Young women in sub-Saharan Africa are disproportionally affected by HIV and often rely on injectable hormonal contraception (HC) to prevent unintended pregnancies. However, HC might affect HIV-1 risk through changes in the female genital tract (FGT) microbiota. We examined the impact of three different HC methods on the adolescent female genital tract microbiota and related cytokine and HIV target cell levels at the cervical mucosa in a randomized, crossover trial. Study design and methods 131 adolescent females aged 15 to 19 from Cape Town were enrolled into a randomized, crossover study. The participants were randomized into three study arms: 1. progestin-only injectable norethisterone enanthate (Net-En), 2. combined oral contraceptive pills (COCPs) or 3. combined contraceptive vaginal ring (CCVR) for 16 weeks. Participants then switched to one of the other HC options for a final four months. Vaginal samples were collected at baseline, crossover and exit. STI testing and Nugent scoring were performed at all study visits. Vaginal microbiota was characterized by 16S rRNA gene amplicon sequencing, cytokine concentrations were measured by Luminex and CD4+ T cells analysed by flow cytometry. Results Using fuzzy clustering, three major female genital tract bacterial community types were identified. Two of these were dominated by Lactobacillus species (L. crispatus and L. iners, respectively) and the third was comprised of a diverse group of anaerobic bacteria associated with bacterial vaginosis (BV). In an intention-to-treat analysis at crossover, participants randomized to COCP had a significantly less diverse vaginal microbiota compared to participants randomized to either Net-En or CCVR. The same was observed in an according to protocol analysis at crossover. Using differential abundance testing and random forest analyses, we found that species associated with BV and risk of HIV were significantly more abundant in, and predictive of, participants on Net-En (e.g. Prevotella, Sneathia and Dialister) or CCVR (e.g. Prevotella, Mycoplasma and Parvimonas) compared to COCP while L. iners was more common in the COCP group. Cytokine concentrations were positively associated with a diverse vaginal community and with specific bacterial taxa associated with BV and increased risk of HIV including species enriched in participants on Net-En and NuvaRing. In contrast, there were no association of the frequencies of CD4+ T cells expressing CCR5+ with the vaginal community or BV status. There was likewise no significant association with BV or diversity with Th17 cell frequency, yet BVassociated bacteria were more abundant in participants with higher frequencies of Th17 cells. Conclusions Our data generated from a randomized study suggests that COCPs use may exert a positive influence on genital health through an increase in lactobacilli and a decrease in BV-associated bacterial taxa with an accompanying decrease in overall bacterial diversity, vaginal pH and cytokine levels. In contrast, the vaginal microbiota of participants on Net-En and NuvaRing have increased levels of bacteria associated with BV and HIV risk and increased cytokine levels. We did not observe any association of the frequencies of CD4+ T cells expressing CCR5 or Th17-like cells with the vaginal community, BV status or HC use.
- ItemOpen AccessInvestigating the role of IL-4/IL-13 signalling through the IL-4 receptor alpha (IL-4Rα) on keratinocytes in murine models of Leishmania major and Schistosoma mansoni(2017) Govender, Melissa; Brombacher, Frank; Hurdayal, Ramona; Guler, RetoKeratinocytes represent the major cell type in the skin. During cutaneous leishmaniasis (CL) and schistosomiasis, the skin is important during the parasite life cycle. While Th1 immunity is required to control CL, protection during schistosomiasis requires Th2 immunity. Paradoxically, Th2 characteristic IL-4 secreted early during L. major infection in mice, can drive a Th1 response by instructing dendritic cells to produce IL-12. Additionally, keratinocytes at the site of L. major infection in C57BL/6 mice, were postulated to be the source of the IL-4. We investigated if IL-4/IL-13 signalling via the IL-4Rα on keratinocytes contributed to early immunity during CL and schistosomiasis. Keratinocyte-specific IL-4Rα deficient (KRT14creIL-4Rα-/lox) BALB/c and C57BL/6 mice were generated by gene targeting and site-specific recombination (cre/loxP) under control of the KRT14 locus. In the L. major footpad model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, and cytokine and antibody secretion similar to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice had decreased footpad swelling, low parasite burdens, a dominant Th1 cytokine response, and low type 1 and 2 antibody titres, similar to littermate control and resistant C57BL/6. In the L major ear model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, Th1 and Th2 cytokines, and high antibody titres, similar to littermate controls. L. major LV39-infected KRT14creIL-4Rα-/lox BALB/c mice showed significantly decreased parasite burdens in the ear, compared to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice in the ear model, had decreased swelling, low parasite burdens, a dominant Th1 immune response, and low type 1 and 2 antibody titres, similar to littermate control and C57BL/6 mice. In the Schistosoma model, survival of S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice was similar to littermate controls during mortality studies. During acute infection, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice showed gut pathology, hepatosplenomegaly, cytokine production, low type 1 and high type 2 antibodies, similar to littermate controls. In comparison to littermate controls, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice had smaller granulomas. Collectively, our results indicate that IL-4/IL-13 signalling through the IL-4Rα on keratinocytes is not required for control during CL or acute schistosomiasis, but does contribute to efficient granuloma formation during acute schistosomiasis.
- ItemOpen AccessProtein Kinase C - delta (PKC-δ): a critical hub for immunomodulatory functions in macrophages during Mycobacterium tuberculosis infection(2023) Hazra, Rudranil; Parihar, SurajTuberculosis (TB) has reached epidemic levels and emerged as the second deadliest infectious disease globally after CoVID-19. By evolving the ability to evade host defense via intrinsic mechanisms, Mycobacterium tuberculosis (Mtb), the etiological agent of TB has been deleterious to human health and has necessitated novel therapeutic interventions, the primary notion to combat Mtb infection. Hence, the identification of host-modulating candidate genes involved in immune evasion and putative pathogen-killing pathways during Mtb infection is crucial. Additionally, macrophages are the first line of defense against Mtb infection through activating effector genes, which lead to pathogen killing and acquiring long-lasting immunity. One such candidate gene with potential novel therapeutic intervention, Protein Kinase C – δ (PKCδ) has been recognized as a critical marker with clinical and experimental evidence in recent years. An experimental mouse model of global PKCδ knockout (PKCδ-/- ) revealed mechanistic alterations enhancing the susceptibility to various infectious diseases including Mtb infection, suggesting a protective phenotype of PKCδ against invading pathogens. However, the macrophage-specific role of PKCδ during Mtb infection remains unknown and has not been delineated yet. Because the pulmonary microenvironment during Mtb infection is majorly governed by macrophages, initiating innate and skewing adaptive immune response, we have exploited the role of PKCδ in macrophages using the macrophage-specific PKCδ knockout mice (LysMcrePKCδflox/flox). Our success in characterizing this experimental murine strain has resulted in the establishment of an immunologically comparable PKCδ functional study platform, which has been adopted herein to investigate the immunomodulatory effects of Mtb infection in the ablation of PKCδ in macrophages. An early lymphocytic immune response increased neutrophil turnover, and reduced inflammatory macrophages are all accompanied by PKCδ deficiency in macrophages, which was abolished in the chronic stage of infection. Bonemarrow-derived macrophages from LysMcrePKCδflox/flox murine model further showed that the disease susceptibility is a consequence of an array of cellular intrinsic mechanisms and dysregulated proteome which are modulated by PKCδ. Furthermore, increased expression in bronchoalveolar lavage (BAL) samples from active TB patients and increased bacterial burden in PKCδ silenced human monocyte-derived macrophages with decreased pro-inflammatory cytokine response strongly signify PKCδ as a key hub for immunomodulatory functions during Mtb infection and a potential host-directed therapeutic (HDT) target against TB.
- ItemOpen AccessThe Role of IL-4 Receptor Alpha signalling on Foxp3 T Regulatory cells in Listeriosis and Tuberculosis(2020) Chia, Julius Ebua; Brombacher, Frank; Parihar, SurajT regulatory cells are critical in the maintenance of self-tolerance, immune homeostasis and regulation of the immune system. Cytokine signalling is a dominant component of environmental signals which controls the function of Forkhead box P3 (Foxp3) regulatory T cells. This thesis addressed the hypothesis that interleukin-4 receptor alpha (IL-4Rα) signalling on T regulatory cells (T reg) play a role in the stability of T reg cells. Loss of IL-4Rα signalling on T reg cells may shift the immune balance from a Foxp3+ T reg to a Th1 effector function essential for Th1 disease outcome. Regulatory cells have a major function to dampen cytokine production; however, this role can be detrimental for host-protective immune responses in diseases such as tuberculosis. Here, we used two Th1 models of intracellular pathogens Listeria monocytogenes (Lm) and Mycobacterium tuberculosis (Mtb), to understand the role of IL-4Rα signalling on Foxp3+ T regulatory cells. Infection studies with L. monocytogenes demonstrated an impairment of T reg responses, with a decreased bacterial burden and diminished pathology both in the liver and spleen at 7 days post-infection, ultimately translated in better survival. Mechanistically, enhanced Th1 signature with the characteristic T-bet transcriptional factor and increased effector T cells producing IFN-γ, IL-2 following ex-vivo stimulation with PMA/Ionomycin, and heat-killed Lm (HKLM) were observed in Foxp3creIL-4Rα-/lox mice. Furthermore, CD8+ T cells of Foxp3creIL-4Rα-/lox mice showed increased cytotoxicity (Granzyme-B secretion) with higher proliferation capacity (Ki-67), better survival (Bcl-2) and decreased apoptosis (activated caspase3), suggesting contribution towards the observed protection against listeriosis. Subsequently, we investigated the role of IL-4Rα on Foxp3 T reg cells in Mycobacterium tuberculosis infection. To our surprise, in contrast to Lm infection, survival Survival of Mtb-infected Foxp3creIL-4Rα-/lox mice was similar to littermate control following infection with an intermediate dose of Mtb (H37Rv). We observed no differences in acute and chronic stages of infection in bacterial burden and histopathological scores in Foxp3creIL-4Rα-/lox mice when compared to littermate control animals in acute and chronic stages of infection. Importantly, Mtb infected FoxP3creIL-4Rα-/lox mice, exhibited significantly enhanced CD4+ T effector functions with increased pro-inflammatory cytokine secretion upon stimulation ex-vivo.