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. 2017 Mar 22;7(1):232.
doi: 10.1038/s41598-017-00192-x.

CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis

Anjali Joshi  1 , Erin B Punke  1 , Melina Sedano  1 , Bethany Beauchamp  2 , Rima Patel  2 , Cassady Hossenlopp  2 , Ogechika K Alozie  3 , Jayanta Gupta  4   5 , Debabrata Mukherjee  3 , Himanshu Garg  6
Affiliations

CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis

Anjali Joshi et al. Sci Rep. .

Abstract

CCR5 is the major co-receptor for HIV and polymorphisms in the CCR5 gene as well as promoter region that alter cell surface expression have been associated with disease progression. We determined the relationship between CCR5 promoter polymorphisms and CD4 decline and other immunopathological features like immune activation and CD4+ T cell apoptosis in HIV patients. CCR5 promoter haplotype HHC was significantly associated with higher CD4 counts in patients. The relative promoter activity (RPA) of each haplotype was determined in vitro and combined promoter activity based on both alleles (CRPA) was assigned to each patients. Interestingly, CCR5 CRPA correlated inversely with CD4 counts and CD4:CD8 ratio specifically in viremic patients. In normal individuals, the CRPA correlated with the number of CCR5+ CD4+ T cells in the peripheral blood suggesting an effect on CCR5 expression. In a subset of high viremic patients harboring R5 tropic HIV, there was a strong correlation between CCR5 CRPA and both CD4 counts and CD4 T cell apoptosis. Our study demonstrates that, CCR5 promoter polymorphisms correlate with CD4 T cell loss possibly by regulating CD4 T cell apoptosis in HIV patients. Furthermore, assigning CRPAs to each patient is a new method of translating genotype to phenotype.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Association of SNPs in the CCR5 promoter with CD4 counts in HIV infected patients. Different SNPs in the CCR5 promoter region were determined by PCR amplification followed by sequencing using specific primers. Association of specific SNPs (A) 58755A (B) 58934G, (C) 59029A, (D) 59353C, (E) 59402A and (F) 59653C with CD4 counts in HIV infected individuals is shown. Two tailed student's t-test was used for statistical analysis.
Figure 2
Figure 2
HHC haplotype is associated with higher CD4 counts in HIV infected patients. Based on CCR5 promoter haplotype determination, the HIV patient population was divided into HHC group, where at least one allele was HHC, or non HHC, where neither allele was HHC. CD4 counts in HHC versus non HHC groups in (A) all HIV+ and (B) viremic patients (>100 copies/ml viral RNA). CD4:CD8 ratio in HHC versus non HHC CCR5 groups in (C) all HIV+ and (D) viremic patients. CD4 apoptosis (active Caspase+) in HHC versus non HHC groups in (E) all HIV+ and (F) viremic patients. Immune activation defined by the presence of CD8+CD38+ HLADR+ cells in HHC versus non HHC CCR5 groups in (G) all HIV+ and (H) viremic patients. Differences in log viremia between HHC and non HHC groups in (I) all HIV+ and (J) viremic patients. One tailed student's t-test was used for statistical analysis.
Figure 3
Figure 3
Transcriptional activity of different CCR5 promoter haplotypes. (A) 293 T cells were transfected with plasmid DNA containing CCR5 gene promoter haplotype (HHA, HHB, HHC, HHD, HHE, HHF and HHG) upstream of the luciferase gene. Promoter activity was determined 48 h later by measuring luciferase activity in cell lysates. Data was normalized to HHA, the ancestral haplotype. Bars represent mean ± SD of triplicate observations from 4 independent experiments. (B) Relative promoter activity (RPA) of each CCR5 promoter haplotype after normalizing to the ancestral allele HHA.
Figure 4
Figure 4
Combined CCR5 relative promoter activity (CRPA) of both alleles in HIV patients correlates with CD4 counts. Each patient was assigned a numerical CRPA score based on the RPA of both alleles. The patient population was then divided into two groups of CRPA > 229 or CRPA < 229. CD4 counts in CRPA > 229 vs CRPA < 229 in (A) all HIV+ patients (B) viremic patients. CD4:CD8 ratio in CRPA > 229 vs CRPA < 229 in (C) all HIV+ patients (D) viremic patients. CD8 immune activation in CRPA > 229 vs CRPA < 229 in (E) all HIV+ patients (F) viremic patients. CD4 apoptosis in CRPA > 229 vs CRPA < 229 in (G) all HIV+ patients (H) viremic patients. (I) CD4 apoptosis in CRPA > 229 vs CRPA < 229 no HHG2 (after removing one data point from an HHG2 patient). Log viremia in CRPA > 229 vs CRPA < 229 in (J) all HIV+ or (K) viremic patients. One tailed student's t-test was used for statistical analysis.
Figure 5
Figure 5
Correlation of CCR5 CRPA with CD4 decline. Correlation between CD4 counts and CCR5 CRPA in (A) all HIV+ or (B) viremic patients. Correlation of CCR5 CRPA with CD4:CD8 ratios in (C) all HIV+ or (D) viremic patients. (E) CRPA in patients with CD4 counts less than or more that 200/μl of blood. Spearman's correlation with linear regression was used for correlation determination. One tailed student's t-test was used for comparative statistics.
Figure 6
Figure 6
CCR5 CRPA positively correlates with number of CD4+ CCR5+ cells in the peripheral blood. (A) Correlation analysis of CRPA with % CCR5+ CD4+ T cells in peripheral blood of healthy controls. (B) Analysis of % CCR5+ CD4+ T cells in peripheral blood healthy controls after dividing the population into CRPA > 229 and CRPA < 229. Spearman's correlation with linear regression was used for correlation determination. One tailed student's t-test was used for comparative statistics.
Figure 7
Figure 7
Correlation between CCR5 CRPA, CD4 apoptosis and CD4 decline in CCR5 tropic HIV infected viremic patients. Full length functional Env glycoproteins cloned from 11 viremic patients in the HIV patient cohort were previously found to be CCR5 tropic. Correlation analysis of CCR5 CRPA with (A) CD4 counts (B) CD4:CD8 ratio (C) in vivo CD4 apoptosis in these R5 virus harboring patients. Spearman's correlation with linear regression was used for all correlation determination.
Figure 8
Figure 8
Model of HIV pathogenesis proposing a role of Env glycoprotein phenotype (AIP), viremia, immune activation and CCR5 levels in collectively determining CD4 bystander apoptosis. The model incorporates CCR5 levels at the center as a major factor which may regulate both plasma viral load and apoptosis mediated by Env glycoprotein leading to CD4 decline.
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