Genetic variability within both the HIV-1 coreceptor, CCR5, and its ligand, CCL3L, has been shown to contribute towards differences between individuals in their susceptibility to HIV-1 infection and rate of disease progression. In this study we investigated the extent of genetic variation within the CCR5 gene as well as CCL3L, CCL3La and CCL3Lb gene copy number distribution in two healthy HIV-1 uninfected South African populations, South African Africans (SAA) and South African Caucasians (SAC). The impact of variations within these genes on the expression of CCR5 and CCL3 was subsequently assessed. Furthermore, CCR5 genetic variability, CCL3L gene copy number distribution and the expression of CCR5 and CCL3, was assessed in a similar way in a small cohort of HIV-1 infected long term nonprogressors (LTNPs).
Genotyping of the CCR5 gene in SAA (n=41) and SAC (n=46) HIV-1 uninfected individuals revealed a high degree of genetic variation between the two population groups, both in terms of single nucleotide polymorphism (SNP) profiles and CCR5 haplotype distribution. Seven complex putative haplotypes spanning the length of the sequenced region were identified with only one of the identified haplotypes, SAA/C-HHC, common to both study populations. The effect of genetic variability on promoter activity of four different CCR5 promoter regions for three CCR5 haplotypes,SAA-HHA, SAA/C-HHC and SAC-HHE, were evaluated. Results showed variability in (i) promoter activity between different promoter regions tested, (ii) results obtained with different cells used for analysis, and (iii) the haplotype being analysed, thereby highlighting that both the cellular
environment as well as genetic variability within the promoter region, have the capacity to influence the efficiency of a promoter and consequently CCR5 expression levels. Haplotype-specific promoter analysis demonstrated the SAA-HHA haplotype to have the strongest promoter activity in THP-1 and K562 cells for both P1A (downstream) and P2 (upstream) promoter regions, while in the other cell lines tested (Jurkat and U937), HHA demonstrated intermediate promoter strength.Differences seen between the haplotypes tested in this study and other published studies may be attributable to additional SNPs being tested in the promoter constructs used in this study.
The two population groups differed significantly with regards to cell activation levels, as measured by HLA-DR expression, in CD4+ T cell (P=0.002) and CD56+ NK cell subsets (P<0.001). CCR5 expression, determined both as the number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, was found to differ between SAA and SAC individuals across all peripheral blood cell types. SAA individuals had larger proportions of CCR5-expressing natural killer (NK) cell subsets (P<0.01) but lower CCR5 molecules per cell density on CCR5+CD8+ T cell and CCR5+ NK cell subsets (CD56+, CD16+CD56+ and CD56dim) (all P<0.05) compared to SAC individuals. These differences were maintained even after CCR3D32 heterozygous SAC individuals were included in the analyses. Furthermore, the previously described haplotypes, HHA and HHC, associated with differences in CCR5 expression on different cell subsets between individuals within the same population group. SAA individuals with the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells compared to SAA
individuals that lacked the haplotype (P=0.001). SAC individuals with the HHC haplotype had significantly higher density on NK (CD56+) and CD16+CD56+ NK cell subsets (P=0.030 and P=0.024, respectively) compared to SAC individuals without this haplotype. The latter observation suggests that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals, thus highlighting the potential role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression.
Despite significant differences in CCL3La (CCL3L chemokine coding) and CCL3Lb (nonchemokine coding) copy number between SAA and SAC populations, no difference in CCL3 production by peripheral blood mononuclear cells (PBMCs) was noted between the two study populations. Assuming equal contribution of CCL3 and each copy of CCL3La to CCL3 production,we found that SAC individuals produced higher levels of CCL3 per functional copy of CCL3La compared to SAA individuals (P<0.001). Although, when SAA and SAC individuals with comparable CCL3La and CCL3Lb gene copy numbers were compared, there was no difference in production per functional copy between the two groups (P=0.974). We also determined CCL3La and CCL3Lb gene copy number for a previously established cohort of HIV-1 intrapartum-infected (IP) and exposed uninfected (EU) infants and found that differences previously seen in cord blood
mononuclear cell (CBMC) CCL3 production between IP and EU infants with comparable CCL3L copy numbers could not be attributed to differences in CCL3Lb copy number.
The potential role of differences in CCR5 genotype, CCR5 expression, CCL3 genotypes and CCL3 production levels in the control of HIV-1 infection was then examined by comparing a small group (10 SAA and 4 SAC) of LTNPs to the respective background population. No polymorphisms in the CCR5 open reading frame were detected in these LTNP individuals. However, the HHA haplotype frequency was significantly higher in SAC LTNP individuals compared to SAC control individuals (P=0.010). Interestingly, CCR5 density on CD4+ T cells and monocytes was significantly lower in SAA LTNP individuals (P=0.025 and P=0.022, respectively) with a trend towards a similar relationship in CD8+ T cells (P=0.058), while the proportions of CCR5-expressing CD8+ T cells
were elevated compared to SAA controls (P=0.043). This latter finding reflects the increased immune activation in these individuals compared to uninfected individuals, as evidenced by increased proportions of HLA-DR-expressing T cells (CD8+ and CD4+, P<0.0001). In addition,PHA-induced CCL3 production by PBMCs was significantly lower in LTNP (SAA and SAC combined) compared to control individuals (P=0.004). SAA LTNP individuals had higher proportions of CD8+ T cells (P<0.0001) and lower proportions of natural killer cells (CD56+,P=0.002) compared to control SAA individuals. Thus, CCL3 production differences may be partially explained by differences in the distribution of immune cell subsets between the two study groups.Furthermore, PBMCs of LTNP individuals with low viral loads (<400 copies/ml) produced CCL3 at lower levels than those from individuals with higher viral loads, irrespective of whether or not the
cells were stimulated (P=0.005 and P=0.035, respectively).
In summary, this study demonstrates that: (i) two ethnically divergent populations show marked differences in CCR5 genetic variability, cell activation and CCR5 expression which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression, (ii) both CCR5 genotypic differences and differences in baseline cellular activation levels appear to be contributing towards the observed differences in CCR5 protein expression, and (iii) the two study populations do not differ with respect to CCL3 production by PBMC cultures which suggests that either the two copy per diploid genome gene, CCL3, may play a significant role in CCL3 production and/or that as yet undefined mechanisms regulate production of CCL3 from variable CCL3L copy
number. In addition, a pilot study conducted in a small group of LTNP individuals demonstrates that two major determinants of HIV-1 disease progression, CCR5 and CCL3, are both expressed at lower levels in LTNPs individuals compared to healthy uninfected controls and has identified CCR5 haplotypes which are potentially associated with disease progression.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/14571 |
| Date | 23 April 2014 |
| Creators | Picton, Anabela Correia Pereira |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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