Interleukin-8 genetic diversity, haplotype structure and production differ in two ethnically distinct South African populations.

A single nucleotide polymorphism (SNP), 251 bases upstream from the IL-8 transcription start (-251A>T, rs4073), has been extensively investigated in cancers and inflammatory and infectious diseases in predominantly European and Asian populations. We sequenced the IL-8 gene of 109 black and 32 white South African (SA) individuals and conducted detailed characterization of gene variation and haplotype structure. IL-8 production in phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) of a subset (black: N = 22; white: N = 32) of these individuals was measured using ELISA. Select variants were genotyped for additional black individuals (N = 141), and data from the 1000 Genomes Project were used for haplotype analysis and comparative purposes. In white individuals, the -251A>T SNP formed part of a prevalent six-variant haplotype [haplotype frequency (HF): 61%], Hap-1C, involving the following variants: -251A>T; +394T>G (rs2227307); +780C>T (rs2227306); +1240->A (rs2227541); +1635C>T (rs2227543) and +2770A>T (rs2227543). Hap-1C (-251T+394T+780C+1240+A+1635C+2770A) was composed of two three-variant sub-haplotypes [Hap-1Ca: -251T+394T+1240+A; Hap-1Cb: +780C+1635C+2770A) sharing similarities with haplotypes identified in the black population. Hap-1C was found to be present in European, East and South Asian populations. Four haplotypes were identified in the black population with the two prevalent haplotypes each comprised of two variants: Hap-1B [-251A>T and +1240->A; -251T+1240+A; HF: 14%] and Hap-2B [-743T>C (rs2227532) and +2452A>C (rs2227545); -743C+2452C; HF: 13%]. Populations did not differ in unstimulated PBMC IL-8 production. Upon PHA stimulation, PBMCs from white individuals produced more IL-8 (P = 0.04), suggesting the -251T allele is responsible for higher production, however further analysis revealed that Hap-1C (and constituent sub-haplotypes), did not associate with IL-8 production. Populations did however differ in monocyte number with the white population having significantly more monocytes compared to the black population (P = 0.025), and furthermore monocyte number strongly correlated with IL-8 production in both population groups (black: p = 0.0002, r = 0.71; white: P = 0.0005, r = 0.59). Hap-1B, Hap-2B, and a SNP located one base pair upstream of the IL-8 ATG start codon, +100C>T SNP (rs2227538), all associated with higher IL-8 production in the black population - individuals harbouring at least one of these haplotypes/variant associated with higher IL-8 production (P = 0.003) compared to individuals without. The black population was enriched for individuals harbouring Hap-1B and/or Hap-2B compared to the 1000 Genomes project sub-Saharan African population (P = 0.006), suggesting that SA black individuals may be high IL-8 producers. Given the paucity of IL-8-related studies that have been conducted in populations from sub-Saharan Africa, this study has significantly increased our understanding of this important chemokine in the South African population.

Reduced CCR5 Expression and Immune Quiescence in Black South African HIV-1 Controllers.

Unique Individuals who exhibit either suppressive HIV-1 control, or the ability to maintain low viral load set-points and preserve their CD4+ T cell counts for extended time periods in the absence of antiretroviral therapy, are broadly termed HIV-1 controllers. We assessed the extent to which black South African controllers (n=9), differ from uninfected healthy controls (HCs, n=22) in terms of lymphocyte and monocyte CCR5 expression (density and frequency of CCR5-expressing cells), immune activation as well as peripheral blood mononuclear cell (PBMC) mitogen-induced chemokine/cytokine production. In addition, relative CD4+ T cell CCR5 mRNA expression was assessed in a larger group of controllers (n=20) compared to HCs (n=10) and HIV-1 progressors (n=12). Despite controllers having significantly higher frequencies of activated CD4+ and CD8+ T cells (HLA-DR+) compared to HCs, CCR5 density was significantly lower in these T cell populations (P=0.039 and P=0.064, respectively). This lower CCR5 density was largely attributable to controllers with higher VLs (>400 RNA copies/ml). Significantly lower CD4+ T cell CCR5 density in controllers was maintained (P=0.036) when HCs (n=12) and controllers (n=9) were matched for age. CD4+ T cell CCR5 mRNA expression was significantly less in controllers compared to HCs (P=0.007) and progressors (P=0.002), whereas HCs and progressors were similar (P=0.223). The levels of soluble CD14 in plasma did not differ between controllers and HCs, suggesting no demonstrable monocyte activation. While controllers had lower monocyte CCR5 density compared to the HCs (P=0.02), significance was lost when groups were age-matched (P=0.804). However, when groups were matched for both CCR5 promoter haplotype and age (n=6 for both) reduced CCR5 density on monocytes in controllers relative to HCs was highly significant (P=0.009). Phytohemagglutinin-stimulated PBMCs from the controllers produced significantly less CCL3 (P=0.029), CCL4 (P=0.008) and IL-10 (P=0.028) compared to the HCs, which was largely attributable to the controllers with lower VLs (<400 RNA copies/ml). Our findings support a hypothesis of an inherent (genetic) predisposition to lower CCR5 expression in individuals who naturally control HIV-1, as has been suggested for Caucasian controllers, and thus, likely involves a mechanism shared between ethnically divergent population groups.

Cis-regulatory genetic variants in the CCR5 gene and natural HIV-1 control in black South Africans.

Studies have investigated CCR5 haplotypes (HHA, HHB, HHC, HHD, HHE, HHF*1, HHF*2, HHG*1, HHG*2), defined by seven 5'UTR single nucleotide polymorphisms (SNPs), CCR2-V64I and CCR5Δ32, in HIV-1 disease. CCR5 cis-regulatory regions were sequenced, CCR2-V64I and CCR5Δ32 genotyped, and compared in HIV-1-infected black South Africans: 71 HIV-1 controllers (23 elite controllers, 37 viraemic controllers (VCs), 11 high viral load long-term non-progressors) and 74 progressors. The HHE haplotype and 3'UTR +2919 T > G SNP heterozygosity were underrepresented in total controllers and VCs vs. progressors (p = .004; p = .007 and p = .002, pbonferroni = 0.032; p = .004, respectively). Possession of the +2919 T > G SNP (dominant mode) was associated with HIV-1 progression (controllers vs. progressors: p = .001, pbonferroni = 0.016). The +2919 T > G SNP is in linkage disequilibrium (LD; r2 = 0.73) with two 5'UTR SNPs (-2459G > A and -2135 T > C; r2 = 1: 5'UTR-2SNP-hap). The 5'UTR-2SNP-hap was lower in total controllers and VCs vs. progressors (p = .003, pbonferroni = 0.048; p = .01, respectively). Results suggest -2459G > A, -2135 T > C, and + 2919 T > G as key CCR5 variants in HIV-1 control.

CXCR6 gene characterization in two ethnically distinct South African populations and association with viraemic disease control in HIV-1-infected black South African individuals.

CXCR6 genetic variation was described for HIV-1-uninfected black (n=41) and Caucasian (n=40) South Africans. We also investigated the CXCR6 rs2234358 and rs2234355 single nucleotide polymorphisms in HIV-1 disease control in 124 HIV-1-infected drug-naïve black individuals [elite controllers (n=11), viraemic controllers (VCs, n=30), high viral load long-term nonprogressors (HVL LTNPs, n=11) and progressors (n=72)] compared to healthy controls (HCs; n=232). The rs2234358-T allele was underrepresented in VCs (40.0%) compared to HCs (59%, P=0.006), HVL LTNPs (72.7%, P=0.012) and progressors (59%, P=0.014). The rs2234358-TT genotype was underrepresented in VCs (7%) compared to progressors (32%; OR=6.57, P=0.006) and HCs (35%; OR=7.18, P=0.001, Pbonferroni=0.034). The rs2234355-GA genotype was overrepresented in VCs (80%) compared to HCs (50.4%; OR=0.25, P=0.003) and progressors (29.17%; OR=0.10, P=3.8×10-5, Pbonferroni=0.001). The combination of rs2234355-GA in the absence of rs2234358-TT was overrepresented in VCs (80%) compared to HCs (32.6%, OR=0.12, P=1×10-6, Pbonferroni=3.4×10-5) and to progressors (16.7%; OR=0.05, P<1×10-8, Pbonferroni<1×10-7).

Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes.

The controversy surrounding the findings that copy number variation, of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. This study evaluated the standard method of quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n=23) and Caucasian (n=32) population groups using qPCR and ddPCR. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r=0.99, p<0.0001) compared to qPCR (r=0.87, p<0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course.

Differences are evident within the CXCR4-CXCL12 axis between ethnically divergent South African populations.

The G-protein-coupled receptor, CXCR4, is highly expressed on a number of cell types, and together with its ligand, CXCL12, plays an important role in immune development and trafficking of cells. CXCR4 promotes tumor growth, angiogenesis and metastasis, and is a prognostic marker in a number of different types of tumors. Additionally, CXCR4 is utilized, together with CD4, for entry of T-tropic HIV viruses. Ethnic differences in incidence and mortality of various cancers, and in the response to highly active antiretroviral treatment (HAART) of HIV-1 infected individuals have been reported. The aim of this study was to establish if differences in the CXCR4-CXCL12 axis exist between ethnically divergent uninfected South Africans. CXCR4 density was significantly higher on CD4(+) and CD8(+) T cells, B cells and CD56(dim) NK cells, and CXCL12 levels lower in Black compared with Caucasian individuals. Furthermore, an inverse correlation was observed between CXCR4 density on CD56(+) and CD3(+) cells and age, only in Black individuals. CXCL12-3'A heterozygosity (AG) found in 28% of Caucasians did not explain the higher plasma levels of CXCL12 compared to Black individuals who were all GG genotypes, suggesting that other factors influence homeostatic levels of CXCL12. In conclusion, this study demonstrates that ethnically divergent populations show clear differences in both CXCR4 density and CXCL12 plasma levels which may influence the course of cancer and HIV-1 infection.

Contribution of variable CCL3L copy number to CCL3 protein production in two ethnically divergent South African populations.

When accounting for the specific population, CCL3L copy number, a measure of the sum of chemokine- and non-chemokine-producing genes (CCL3La and CCL3Lb, respectively), has been reported to associate with risk of HIV-1 infection. In this study, we have described the distribution of CCL3La and CCL3Lb copy number variation in two populations, South African Africans (SAA) and South African Caucasians (SAC), and investigated the impact of these variations upon CCL3 protein production. Despite significant differences in CCL3La and CCL3Lb copy number, no differences in CCL3 production were noted between the two 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). However, when individuals with comparable CCL3La and CCL3Lb gene copy numbers were compared, no difference in production per functional copy between SAA and SAC individuals was noted. Furthermore, we demonstrate that differences noted in cord blood mononuclear cell CCL3 production between HIV-1 intrapartum-infected (IP) and exposed uninfected (EU) infants with comparable CCL3L copy numbers could not be attributed to differences in CCL3Lb copy number. Collectively, our findings suggest that either the CCL3 gene may play a significant role in CCL3 production and/or that as yet undefined mechanisms regulate production of CCL3 from variable CCL3L copy number.

CCR5 promoter haplotypes differentially influence CCR5 expression on natural killer and T cell subsets in ethnically divergent HIV-1 uninfected South African populations.

CCR5 plays a critical and central role in HIV-1 infection and, to date, a number of genetic mutations and haplotypes within the gene have been found to positively or negatively influence an individual's susceptibility and rate of disease progression. In this study, we have evaluated the influence of CCR5 haplotypes, HHA, HHC, HHD, and HHE, on CCR5 expression in healthy HIV-1 uninfected individuals from two populations, South African Africans (SAA, n = 22) and South African Caucasians (SAC, n = 31). CCR5 haplotypes were determined through sequencing and real time polymerase chain reaction. Flow cytometry was used to quantitate CCR5 surface expression, as both CCR5 density and percentage of CCR5-expressing cells, on B, T, natural killer (NK) cells and monocytes. SAA individuals positive for the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells in comparison to individuals without HHA (P = 0.001). HHC+ SAC individuals had significantly higher CCR5 molecules per cell (density) on NK (CD56+) and CD16+ CD56+ NK cell subsets (P = 0.030 and P = 0.024, respectively) compared to HHC- SAC individuals. Haplotypes HHD and HHE had no impact on CCR5 expression. Overall, our data highlight 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. Findings raise the question as to the role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression.

Marked differences in CCR5 expression and activation levels in two South African populations.

The chemokine receptor CCR5 is pivotal in determining an individual's susceptibility to HIV-1 infection and rate of disease progression. To establish whether population-based differences exist in cell surface expression of CCR5 we evaluated the extent of CCR5 expression across all peripheral blood cell types in individuals from two populations, South African Africans (SAA) and South African Caucasians (SAC). Significant differences in CCR5 expression, both in number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, were observed between the two study groups, within all cell subsets. Most notably, the percentage of all CCR5(+) cell subsets was significantly lower in SAC compared with SAA individuals (P < 0·01) among natural killer (NK) -cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) whereas CCR5 density was significantly higher in SAC compared with SAA individuals in CCR5(+) CD8(+) T-cell subsets and CCR5(+) NK-cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) (all P < 0·05). These relationships were maintained after exclusion of CCR5Δ32 heterozygous individuals (n = 7) from the SAC dataset. The SAA individuals exhibited significantly higher cell activation levels, as measured by HLA-DR expression, than SAC individuals in CD4(+) T-cell subsets (P = 0·002) and CD56(+) NK-cell subsets (P < 0·001). This study serves to demonstrate that ethnically divergent populations show marked differences in both 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.

Genetic variation within the gene encoding the HIV-1 CCR5 coreceptor in two South African populations.

Polymorphisms within the open reading frame as well as the promoter and regulatory regions can influence the amount of CCR5 expressed on the cell surface and hence an individual's susceptibility to HIV-1. In this study we characterize CCR5 genes within the South African African (SAA) and Caucasian (SAC) populations by sequencing a 9.2kb continuous region encompassing the CCR5 open reading frame (ORF), its two promoters and the 3' untranslated region. Full length CCR5 sequences were obtained for 70 individuals (35 SAA and 35 SAC) and sequences were analyzed for the presence of single-nucleotide polymorphisms (SNPs), indels and intragenic haplotypes. A novel SNP (+258G/C) within the ORF leading to a non-synonomous amino acid (Trp-->Cys) change was detected in one Caucasian individual. Results demonstrate a high degree of genetic variation: 68 SNP positions, four indels, as well as the Delta32 deletion mutant, were detected. Seven complex putative haplotypes spanning the length of the sequenced region have been identified. These haplotypes appear to be extensions of haplotypes previously described within CCR5. Two haplotypes, SAA-HHE and SAC-HHE were found in high frequency in the SAA and SAC population groups studied (20.0% and 18.6%, respectively) and share four SNP positions suggesting an evolutionary link between the two haplotypes. Only one of the identified haplotypes, SAA/C-HHC, is common to both study populations but the haplotype frequency differs markedly between the two groups (8.6% in SAA and 52.9% in SAC). The two population groups show differences in both haplotype arrangement as well as SNP profile.