Electronic Theses and Dissertations (PhDs)

Permanent URI for this collection

https://hdl.handle.net/10539/38222

Browse

Browsing Electronic Theses and Dissertations (PhDs) by School "Pathology"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Exploring the interplay of chemokine receptors ccr5 and cxcr6 in mechanisms of natural control in HIV-1- infected black South Africans
    (2024) Koor, Gemma Whitney
    In sub-Saharan Africa, HIV-1 is a significant cause of morbidity and mortality. However, research remains primarily focused on North American and European population groups, who have remarkably different genetic backgrounds to individuals from sub-Saharan Africa. HIV1 controllers represent a model of HIV-1 functional cure, with some individuals able to control viral replication, and some able to sustain immune function in the presence of high viral loads, both in the absence of antiretroviral therapy (ART). The chemokine receptors CCR5 and CXCR4 are the major coreceptors HIV-1 utilises to enter cells. The use of alternative coreceptors, such as the CXCR6 coreceptor, is thought to contribute to the lower pathogenicity exhibited by the HIV-2 and SIVsmm strains. Building on previous work conducted in our research unit on these two coreceptors in South African populations, this thesis firstly describes CCR5 genetic variants that associate with HIV-1 control or risk of progressive infection in black South Africans, and then explores constitutive expression levels of CCR5 and CXCR6 on various peripheral blood immune cell subsets in the absence of HIV-1 infection in ethnically divergent population groups. The effect of sex, age, and select CCR5 and CXCR6 single nucleotide polymorphisms (SNPs) on expression levels of these two receptors was also investigated. The CCR5 5’UTR and 3’UTR regions were PCR-amplified and sequenced from genomic DNA extracted from 145 ART-naive black South African individuals living with HIV-1 (71 HIV-1 controllers – 23 elite controllers, 37 viraemic controllers, 11 high viral load long-term nonprogressors and 74 progressors). Findings confirmed results from other studies in showing that the CCR5 HHE haplotype is deleterious for HIV-1 disease progression, and the HHA haplotype and HHA/HHC genotype associated with protection from HIV-1 disease progression. Novel haplotypes were characterised, both in the 3’UTR and spanning the CCR5 5’UTR and 3’UTR. Overall, findings suggest that two CCR5 promoter SNPs (-2459 G>A and -2135 T>C) and one CCR5 3’UTR SNP (+2919 T>G) may be key functional variants with regards to HIV-1 control in black South Africans. To gain further insight into the constitutive expression of CCR5 and CXCR6 on peripheral blood immune cells and explore the relationship between select genetic variants and expression, immunophenotyping by flow cytometry was conducted using whole blood from age- and sex-matched ethnically distinct South African HIV-uninfected individuals (17 black, 21 white). Expression levels of CCR5 and CXCR6 were assessed on CD4+ and CD8+ T cells, B cells, monocytes and NK cells, and their respective subsets. The effects of age and sex on expression levels of these two receptors was also investigated. Population-specific differences with regards to CCR5 expression on all cell types, except for B cells, were evident. Generally, black South Africans exhibited a lower expression level of CCR5 compared to white South Africans. CXCR6 expression only differed with regards to percentage of CXCR6-expressing cells, not CXCR6 density (numbers of cell surface receptors). Black individuals had a lower percentage of CXCR6-expressing CD8+ T cell subsets (naïve and effector memory) and a higher percentage of CXCR6-expressing CD14+CD16+ monocytes compared to white individuals. Overall, we found significant population-specific differences in expression levels of both CCR5 and CXCR6, multiple associations with cell activation (as measured by HLADR expression) and CCR5 and CXCR6 expression, and CCR5 and CXCR6 expression was positively significantly correlated on multiple cell subsets. Furthermore, both sex and age influenced CCR5 and CXCR6 expression, however results varied widely across the two population groups studied. Sex differences were only evident in white individuals; predominantly CXCR6 expression was increased in males compared to females. Age associations with CCR5 and CXCR6 expression were also primarily found in white individuals. Four CCR5-related SNPs that are associated with HIV-1 control in this or other studies (rs553615728 -4223 C>T SNP, rs1799987 −2459 G>A SNP, rs746492 +2919 T>G SNP and rs1015164 G>A SNP) were assessed for their potential association with CCR5 expression levels. The +2919 TG genotype significantly associated with a higher percentage of CCR5- expressing total CD8+ T cells, transitional memory and terminally differentiated CD8+ T cells compared to the GG genotype. The +2919 GG genotype associated with a lower percentage of CCR5-expressing B cells compared to the TT and TG+TT genotypes, however, only in white South Africans. The +2919 TG and TG+TT genotypes associated with significantly higher CCR5 density on all CD8+ T cell subsets, except for naïve CD8+ T cells, when compared to the GG genotype. When evaluating two CXCR6 genetic variants previously associated with HIV-1 viraemic control (rs2234355 G>A and rs2234358 G>T) in relation to CXCR6 expression, possession of the rs2234355 SNP GA genotype associated with lower CXCR6 expression on select CD4+ and CD8+ T cell subsets as well as on B cells, while possession of the rs2234358 SNP TT genotype associated with higher CXCR6 expression on multiple cell types, primarily in white South Africans. Possession of the -358TT/+355GA genotype combination associated with lower CXCR6 expression on select subsets of CD4+ T cells and monocytes. In summary, this study provides information on genetic variation in the CCR5 gene in a South African context, describes genetic variants associating with HIV-1 control in black South Africans, adds novel insight into constitutive CCR5 and CXCR6 expression levels on CD4+ and CD8+ T cells, B cells, monocytes and NK cells in HIV-1-uninfected black and white South Africans, and describes the potential associations of select genetic variants and expression. Black and white individuals differed in their baseline expression levels of CCR5 or CXCR6, which was partly driven by host genetic factors that were explored. This work highlights the importance of considering effects of ethnicity, age, and sex in any studies addressing any immune molecules in relation to differential HIV-1 outcomes of infection susceptibility/protection, disease progression, or HIV-1 virological control on antiretroviral therapy. Although conducted on small numbers of individuals, these variables clearly influenced constitutive expression of CCR5 and CXCR6, and further population-specific studies are warranted to gain further insights. Findings from this study have implications for risk of acquisition of HIV-1 infection and for disease progression in people living with HIV-1. Understanding the role of these molecules is important for informing strategies for both HIV1 prevention and HIV cure.
  • Thumbnail Image
    Item
    Variation in the LBP-CD14-TLR4-LY96 gene complex and consequences of microbial translocation in HIV-1 infected black South Africans
    (2024) Mncube, Sizanani
    Persistent immune activation and inflammation in people living with HIV-1 (PLWH) has been associated with higher morbidity and mortality, even in individuals on antiretroviral therapy (ART). Microbial translocation, among other factors, has been identified as a major driver of persistent immune activation. A subgroup of PLWH collectively known as HIV-1 controllers can naturally control the HIV-1 infection without the use of ART. Little is known about the extent and the role of microbial translocation/immune activation in African HIV-1 controllers. Translocated lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls elicits innate immune responses through the activation of the toll-like receptor 4 (TLR4) in a complex pathway, which requires the use of cluster of differentiation 14 (CD14), LPS binding protein, and Lymphocyte antigen 96 (LY96) also known as Myeloid differentiation factor 2 (MD-2). Although numerous studies have reported associations of expression levels of the LPS recognition and signalling molecules as well as variants in the genes encoding for these molecules, with the risk and severity of various inflammatory, autoimmune, and infectious diseases, such studies are limited in African populations. Given the large genetic diversity in African populations, characterisation of both the constitutive expression levels and genetic variation in these molecules is essential to understanding HIV-1 infection in the populations most affected by the AIDS epidemic. We quantified constitutive expression of cell surface TLR4 and CD14 (mCD14) on monocytes and neutrophils using flow cytometry and quantified plasma levels of soluble CD14, LBP, and MD-2 using commercially available ELISA kits in two ethnically divergent South African populations [healthy HIV-1 uninfected black (n=17) and white (n=21) individuals]. Furthermore, the influence of sex and age on the expression levels of these molecules was also investigated. We found higher LBP plasma levels in black South Africans compared to white South Africans (p<0.0001), however these two populations did not differ significantly in expression levels of CD14 (mCD14 and sCD14), TLR4, or MD-2. Sex differences in the TLR4 expression levels, with higher TLR4 on total monocytes (p=0.016) and CD14+ 1CD16- (p=0.009) and CD14+CD16+ (p=0.009) subsets of monocytes in females compared to males were observed in the white South African population but not in the black South African population. Significant population and sex-specific negative correlations between age and CD14 expression on monocytes, monocyte subsets and neutrophils, and TLR4 expression on neutrophils were observed. In addition, we found that there is differential regulation of TLR4 expression on monocytes and neutrophils between black and white South Africans post stimulation with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Together, thesefindings suggest that population differences in plasma levels of LBP, and population-specific sex differences in TLR4 expression, are likely to differentially impact TLR4 functionality. Using whole genome sequencing data (WGS), we next sought to fully describe the genetic variation and linkage disequilibrium (LD) patterns in the LBP, CD14, TLR4, and LY96 genes in HIV-1 uninfected black South Africans (n=87, SA controls), and compared the representation of the variants to select populations from the 1000 Genomes Project. Our results revealed that the representation of genetic variants and LD patterns across these genes in the SA black population more closely mirrored those of representative African subpopulations (Yoruba in Ibadan, Nigeria, and Luhya from Webuye, Kenya) than the European and Asian populations. These findings emphasize that there are vast genetic differences in African populations compared to non-African populations, which could differentially affect gene regulation and associations with various diseases. Several novel variants and putative haplotypes were identified in the SA black population which, upon verification in future studies, will serve to add to understanding the genetic diversity in this population group. Using WGS data, we also assessed the representation of the LBP, CD14, TLR4 and LY96 gene variants in a cohort of black South African ART-naïve HIV-1 controllers (n=39) comprised of elite controllers (n=21), viraemic controllers (n=6), and high viral load long-term nonprogressors (n=12), relative to the SA controls. Only one CD14 5’ flanking region SNP (rs186291587) showed a significant difference in minor allele frequency (MAF) representation in elite controllers when compared to SA controls (p=0.024; OR=13.3, CI: 1.3 – 131.4). The representation of several TLR4 variants showed significant differences when HIV-1 controllers were compared to SA controls and the most significant differences were predominantly found in comparison to the HVL LTNPs - the most significant difference observed was overrepresentation of two SNPs in complete LD (r2=1), a newly identified intronic variant (TLR4 NI-2), and a 3’ flanking region SNP (rs113017335) in HVL LTNPs compared to SA controls (p=0.006; OR=24.71, CI: 2.46-248.51). The representation of several LBP variants also differed between HIV-1 controllers and SA controls, here predominantly when viraemic controllers were compared to SA controls. Minor allele frequency overrepresentation of the LBP intronic SNP (rs1250247980) in the total group of HIV-1 controllers (p=0.003), and viraemic controllers (p=0.0002), relative to the SA controls, was the most significant difference observed. Furthermore, differences in the representation of LY96 variants were observed when the total group of HIV-1 controllers, elite controllers and HVL LTNPs were compared to SA controls - the most significant difference observed was the MAF and heterozygosity overrepresentation of an intronic SNP (rs149605245) in elite controllers compared to SA controls (MAF: p=0.007; heterozygosity: p=0.007). These results suggest a potential role of the LPS recognition and signalling molecules in natural HIV-1 control. Lastly, in ART-naïve black South African elite controllers (n=44), HVL LTNPs (n=12), progressors (24), and in HIV-1 uninfected controls (HUCs, n=17), we measured and compared plasma levels of select innate immune molecules that are considered markers of microbial translocation and gut damage (LBP, sCD14, REG3α), or are important in interacting with TLR4 (MD-2). We found no differences between groups in plasma levels of LBP and MD-2. However, sCD14 was significantly higher in progressors compared to all groups (HUCs, p=0.0001; ECs, p0.05). Marked sexspecific differences in REG3α levels were evident, with females having significantly higher levels compared to males in all groups (HUCs and ECs, p=0.0001; ECs, p<0.0001; HVL LTNPs, p=0.0005), with no differences between HIV-1 uninfected controls, elite controllers and HVL LTNPs. Plasma levels of REG3α were unexpectedly significantly lower in progressors compared to elite controllers (p=0.007) and HVL LTNPs (p=0.018), however similar to HIV-1 uninfected controls (p>0.05). Marked sexspecific differences in REG3α levels were evident, with females having significantly higher levels compared to males in all groups (HUCs and ECs, p<0.0001; HVL LTNPs, p=0.036; progressors, p=0.005). Our data suggests that in black South Africans, REG3α plasma levels are not a reliable marker of gut damage, and that increased levels in elite controllers and HVL LTNPs might contribute to protection from excessive systemic activation in the presence of microbial translocation, consistent with reduced monocyte activation in these groups. Progressors, on the other hand, appear to have an inability to produce REG3α while having substantial monocyte activation. Our findings highlight the importance of sex differences, and that studies conducted in populations of different ethnic backgrounds are often not directly comparable Overall, findings presented in this thesis contribute to the understanding of the baseline expression levels and the genetic diversity in the LBP, CD14, TLR4, and LY96 gene complex in the black South African population, and the representation of these variants in black South African HIV-1 controllers. This thesis also highlights the importance of taking ethnicity, sex, and age into consideration when exploring measures that quantify biological parameters. Understanding of the molecules important in the TLR4 signalling pathway can help elucidate approaches that could contribute to curbing immune activation in the context of HIV-1 infection, as well as other diseases.