Naturally-occurring genetic variants in human DC-SIGN increase HIV-1 capture, cell-transfer and risk of mother-to-child transmission

Abstract

Background: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Dendritic cell-specific ICAM-3 grabbing-nonintegrin (DC-SIGN, also known as CD209) is an HIV-1 receptor that enhances its transmission to T cells and is expressed on placental macrophages.

Methods and findings: We have investigated the association between DC-SIGN genetic variants and risk of MTCT of HIV-1 among Zimbabwean infants and characterized the impact of the associated mutations on DC-SIGN expression and interaction with HIV-1. DC-SIGN promoter (p-336C and p-201A) and exon 4 (198Q and 242V) variants were all significantly associated with increased risk of intrauterine (IU) HIV-1 infection. Promoter variants decreased DC-SIGN expression both in vitro and in placental CD163(+) macrophages (Hofbauer cells) of HIV-1 unexposed infants but not of HIV-1 exposed infants. The exon 4 protein-modifying mutations increased HIV-1 capture and transmission to T cells in vitro.

Conclusion: This study provides compelling evidence to support an important role of DC-SIGN in IU HIV-1 infection.

Figure 1. Inferred haplotypes for DC-SIGN in Zimbabwean population.

(A) Schematic representation of the location of the 20 single nucleotide polymorphisms (SNPs) selected in the DC-SIGN gene. Minor allele frequency (MAF) >5%. (B) DC-SIGN inferred haplotypes. Frequencies of minor alleles are indicated. Dots referred to the major allele. The 10 haplotype-tagged SNPs are shaded. * and ζ referred to SNPs in complete linkage disequilibrium (D’ = 1).

Figure 2. DC-SIGN promoter variants reduced transcriptional activity in vitro and reduced DC-SIGN expression in placental macrophages.

(A, B) Transcriptional activity in vitro (A) Schematic representation of reporter gene constructs corresponding to the DC-SIGN promoter region from positions −507 to −1 with or without promoter variants −336C and −201A. (B) Relative luciferase expression from pGL2-Basic, the parental vector without a promoter. Expression of the DC-SIGN promoter constructs was calculated relative to this value. Results are mean ± S.E.M. values of three independent experiments performed in triplicates and differences in relative luciferase expression between variants and wild-type were examined with Student’s t test. (C) Hofbauer-like cells were analysed by flow cytometry to measure DC-SIGN expression in infants bearing or not promoter variants. Dead cells and Lin⁺ (CD3; CD19; CD56) cells were excluded and subsets were identified for their side scatter (SSC-A) properties and their level of CD14 expression. Placental macrophages were selected for high granularity and CD14 expression (CD14⁺ subset). DC-SIGN was expressed on CD163⁺ and CD163⁻ subsets. Dot plots and flow cytometry histograms are representative experiments of all patients. Mean fluorescence intensity (MFI) of DC-SIGN, HLA-DR and CD68 was compared between both subsets for infants bearing or not promoter variants and born from HIV-1-negative mothers (p-336T/p-201C group n = 4; p-336C or p-336C/p-201A group n = 11). (D) DC-SIGN, HLA-DR and CD68 expression was compared in CD163+ and CD163− subsets from infants bearing or not promoter variants and born from HIV-1-negative mothers (HIV-1 Unexposed; p-336T/p-201C group n = 4; p-336C or p-336C/p-201A group n = 11) or from HIV-1-positive mothers (HIV-1 Exposed; p-336T/p-201C group n = 3; p-336C or p-336C/p-201A group n = 6). Results in C and D are mean ± S.E.M. values of MFI and difference between subsets or variants was calculated with Student’s t test.

Figure 3. DC-SIGN neck variants enhance HIV-1 capture and transmission.

(A) Schematic representation of DC-SIGN constructs representing DC-SIGN neck variants stably expressed in the Raji cell line. (TM; transmembrane domain, Cyt; cytoplasmic domain). (B) Raji-transfectants were selected for similar DC-SIGN cell-surface expression by flow cytometry. Cells stained with anti-DC-SIGN (DCN46) (filled grey histogram) or isotypic control (dashed grey line) are shown. Parental Raji cells are represented by the black line. Antibody titration was achieved at the same dilution for all cell lines using two DC-SIGN monoclonal antibodies (clones DCN46 and 5D7) that recognized different epitopes. (C) HIV-1 capture by DC-SIGN variants. Raji-transfectants were incubated with HIV-1_HXBru-ADA (ADA) or HIV-1_JRCSF (JRCSF) for 2 h at 37°C, extensively washed and lysed. Cell-associated p24 Ag was measured by ELISA. Where indicated, cells were pre-incubated with anti-DC-SIGN (AZN-D1) or with mannan to inhibit DC-SIGN interaction with HIV-1. HIV-1 capture is shown relative to WT (WT = 100%). (D) HIV-1 transfer to T lymphocytes by DC-SIGN variants. Raji-transfectants were pulsed as in (C) and subsequently co-cultivated with activated human primary CD4⁺T lymphocytes from two donors for 5 days. Virus release into the supernatant was measured by ELISA p24. Where indicated, cells were pre-incubated with AZN-D1. HIV-1 transmission is shown relative to WT (WT = 100%). Results are mean ± SD of duplicates for each donor (D) or three independent experiments (C). Student’s t test was used to calculate differences in % capture and transmission among Raji DC-SIGN transfectants L242V, R198Q and WT.