Host genetic variants regulates CCR5 expression on immune cells: a study in people living with HIV and healthy controls

C-C chemokine receptor 5 (CCR5) is the main HIV co-receptor affecting susceptibility and disease course. Quantitative trait loci (QTL) mapping analysis was performed to assess genetic variants associated with CCR5 expression on circulating immune cells in 209 PLHIV using ART and 304 healthy controls, all of Western European ancestry. The proportions of CCR5 positive cells and CCR5 mean fluorescence intensity (MFI) were assessed by flow cytometry in monocytes and CD4+ and CD8+ T cell subsets using flow cytometry. We identified the rs60939770, which is an intergenic variant in cis-region to CCR5 gene not in linkage disequilibrium with CCR5d32, related to the proportion of CCR5+ memory T regulatory cells, both in PLHIV and healthy controls. Two genome-wide significant loci, in linkage equilibrium with CCR5d32, were found to be associated with CCR5 MFI of multiple subsets of mostly differentiated memory T cells in both groups. The expression of nearby chemokines receptors (CCR1, CCR2, CCR3, CCRL2), existing in the same the same topologically associating domain, were also influenced by these genetic variants. Furthermore, we show the genetic variants which modulate CCR5 surface expression affect the production of other inflammatory mediators, including monocyte- and lymphocyte-derived cytokines as well as CCL4 and IL-8. Our data indicate that the genetic regulation of CCR5 expression is cell-specific and affects the production of various inflammatory mediators. Author Summary CCR5 plays a important role in the acquisition of HIV and it is associated to immune activation in people living with HIV (PLHIV). Using samples of cohorts composed of healthy individuals and PLHIV, we sought to map genomic regions that influence CCR5 expression on monocytes and subsets of CD4+ and CD8+ cells. We identified distinct genetic variants that are associated with CCR5 cell proportions or mean fluorescence intensity in subpopulations of T cells with memory functions in both healthy and PLHIV. The genetic variants also influenced the expression of other nearby chemokine receptors and the production of inflammatory mediators. Thus, we demonstrated that the genetic regulation of CCR5 expression is cell-type specific and may impact HIV susceptibility and disease progression.


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In this study we aimed to conduct a in a genome-wide association study (GWAS)

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After testing the association between common variants (MAF > 0.1) and CCR5 MFI 164 or cell proportions using a linear regression model with age and sex corrected, we identified 165 five independent genome-wide significant loci (p < 5 × 10 -8 ) associated with CCR5 MFI or 166 cell proportions in PLHIV (S1 Table). CCR5 MFI and cell proportions showed associations 167 with three independent variants in the cis-region to the CCR5 locus (

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To validate these genetic associations and verify their specificity to PLHIV, we 174 performed a QTL mapping analysis of the same measurements of the relevant immune cell 175 subsets in an independent cohort of healthy controls (HC). We identified a common genetic 176 loci that was associated with the proportions of CCR5 + CD4 + mTregs for both PLHIV and HC 177 (Fig. 1C). The genetic variant associated with CCR5 MFI in PLHIV was also identified in HC 178 in the same subsets of CD4 + and CD8 + T cells, except for CD8 + central memory T cells in 179 which the association was found in PLHIV only (Fig. 1D). Two additional genetic variants in  Table). Besides the validation of the common genetic associations in two 183 independent cohorts, our results using both cohorts of PLHIV and HC allowed the 184 identification of genetic variants that are important for CCR5 surface expression in specific 185 immune cells subpopulations of PLHIV (Fig. 2). As CCR5 is the major co-receptor of HIV  The strongest association (cis-SNP rs60939770, chromosome 3, P-value = 4.29 × 191 10 -16 ) was identified for the proportion of CCR5 + CD4 + mTreg cells in PLHIV (Fig. 3A). Of 192 importance, these effects were replicated in the HC cohort at genome-wide significance (P-193 value = 3.18 × 10 -10 ). PLHIV carrying at least one rs60939770-G allele had a significantly 194 higher proportion of CCR5 + CD4 + mTreg cells than subjects homozygous for the A allele 195 (Fig. 3B). The same genetic effect was also observed in the HC (Fig. 3C). The rs60939770 196 SNP is an intergenic variant in cis-region to the CCR5 gene and it is in a linkage 197 disequilibrium (LD) with the rs1015164 SNP (R 2 = 0.6823, D' = 0.8594, P-value < 0.0001). (Table S1). Our results reveal that the rs1015164 is strongly correlated to the the 199 percentages of CD4 + mTreg cells expressing CCR5 in PLHIV and HC (P-value < 5 × 10 -8 ) (

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The rs12467868 SNP lies in the intron 3 of the RPS27 gene, a coding ribosomal protein  For CCR5 surface expression (measured as MFI), we identified a genetic 222 association with rs11574435 SNP (P-value < 5 × 10 -8 ) in the majority of both CD4 + and CD8 + 223 T cell subsets of both PLHIV and HC (Fig. 2, S1 Table). In PLHIV, the rs11574435-CC 224 genotype was associated with higher CCR5 MFI expression than individuals with TC 225 genotypes in the subpopulations of CD4 + and CD8 + T cells (Fig. 4). Within the same locus, 226 the rs71327064 SNP (P-value < 6.22 × 10 -9 ) was identified to be associated with CCR5 MFI 227 expression in CD4 + TEMRA cells (Fig. 2, S1 Table). Furthermore, these findings of both 228 SNPs were replicated in the HC cohort with the same allelic direction (P-value < 0.05).

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The CCR5d32 is a well-known causal variant affecting CCR5 expression. It is a

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As the eQTL analysis of candidate genes performed in whole blood of healthy 295 individuals revealed that the rs60939770 is associated with the genes located within the 296 CCR5 TAD (Fig. 2), we next assessed the effects of rs60939770 in modulating the  . We found that the rs60939770 is 319 associated with the production of the innate immune cells-derived soluble mediators, IL-1b, 320 IL6, TNF and monocyte chemoattractant protein-1 (MCP-1) in PLHIV (nominal P-value < 321 0.05). Of note, despite the fact of rs1015164 being in LD with rs60939770, we demonstrated 322 that in terms of functions these two SNPs differ, as rs1015164 was associated with the 323 production of the TNF and IFNg (Fig. 6A). When we further assessed the SNPs identified to 324 be associated with CCR5 MFI levels, we observed that the rs71327064 was associated with 325 the production of both IL-6 and IFNg. Moreover, the rs11574435 SNP was significantly 326 associated with the production of both innate and adaptive inflammatory mediators of 327 PBMCs of PLHIV, including MCP-1, IL-1b, TNF, IL8, IL6 and IFNg, IL17, respectively (Fig.   328   6A). In the HC cohort, rs11574435 SNP was also associated with the levels of IFNg by 329 PBMCs (Fig. 6B). Of importance, CCR5delta32 was also shown to modulate the production

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In the present study, we identified three novel common genetic loci that were

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Live and single cells were selected first. Leukocytes were identified using CD45.

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Lymphocytes and monocytes were identified by granularity and size. Lymphocytes were 921 further characterized using CD4, CD8, CD45RA and CCR7 to identify CD4+ cells and CD8+