DC-SIGN (CD209) Promoter -336 A/G polymorphism is associated with dengue hemorrhagic fever and correlated to DC-SIGN expression and immune augmentation

Abstract

Background: the C-type lectin DC-SIGN (CD209) is known to be the major dengue receptor on human dendritic cells, and a single nucleotide polymorphism (SNP) in the promoter region of CD209 (-336 A/G; rs4804803) is susceptible to many infectious diseases. We reason that variations in the DC-SIGN gene might have a broad influence on viral replication and host immune responses.

Methods and findings: we studied whether the rs4804803 SNP was associated with a susceptibility to dengue fever (DF) and/or dengue hemorrhagic fever (DHF) through genotyping analysis in a Taiwanese cohort. We generated monocyte-derived dendritic cells (MDDCs) from individuals with AA or AG genotype of rs4804803 to study the viral replication and immune responses for functional validation. A total of 574 DNA samples were genotyped, including 176 DF, 135 DHF, 143 other non-dengue febrile illnesses (OFI) and 120 population controls. A strong association between GG/AG genotypes of rs4804803 and risk of DHF was found when compared among DF, OFI and controls (p = 0.004, 3×10(-5) and 0.001, respectively). The AA genotype was associated with protection against dengue infection compared with OFI and controls (p = 0.002 and 0.020, respectively). Moreover, MDDCs from individuals with AG genotype with a higher cell surface DC-SIGN expression had a significantly higher TNFα, IL-12p40, and IP-10 production than those with AA genotype in response to dengue infection. However, the viral replication in MDDCs with AG genotype was significantly lower than those with AA genotype. With both genotypes, MDDCs revealed an increase in viral replication following the addition of anti-IP-10 neutralizing antibody.

Conclusions/significance: the rs4804803 SNP in the CD209 promoter contributed to susceptibility to dengue infection and complication of DHF. This SNP with AG genotype affects the cell surface DC-SIGN expression related to immune augmentation and less viral replication.

Figure 1. CD209 mRNA and cell surface expression on MDDCs with AA or AG genotype of rs4804803.

(A) Quantitative RT-PCR analysis of CD209 mRNA in MDDCs was run parallel to ß2-microglobulin (B2MG) mRNA expression (control) on the real-time RT-PCR tracing and the gel view of RT-PCR products. (B) Subjects with rs4804803 AG genotype had higher CD209 mRNA expression in MDDCs than those with AA genotype (p = 0.032). (C) Flow cytometric analysis of CD209 surface expression on MDDCs from individuals with the rs4804803 AG genotype (blue line; MFI = 34.1) and with the AA genotype (black line; MFI = 30.4). (D) MDDCs from individuals with AG genotype had significantly higher CD209 cell surface expression than those with AA genotype (p = 0.029). The results are shown as mean ± s.e.m. from subjects with AA (n = 10) or AG (n = 10) genotype in three independent experiments. Statistical values were determined by Mann-Whitney U test. Asterisk (*) indicates a significant difference (p<0.05).

Figure 2. Kinetic surface CD209 expression and viral replication from DEN-infected MDDCs with AA or AG genotype.

(A) Kinetic CD209 surface expression on MDDCs from individuals with AA or AG genotype of rs4804803 by flow cytometry. The surface CD209 expression declined along with infection in subjects with both genotypes. (B) Kinetic changes of DEN-2 replication (virus copies per 10⁵ cells) were assessed after infection at MOI of 5 for 24 to 72 h. Viral replication was significantly higher in MDDCs from individuals with rs4804803 AA genotype than those with AG genotype at 48 hr and 72 hr post-infection (p = 0.006 and 0.003, respectively) The results are shown as mean ± s.e.m. from subjects with AA (n = 8) or AG (n = 8) genotype in three independent experiments. (C) Changes in the yield of DEN-2 replication (virus copies per 10⁵ cells) were assessed at MOIs of 1, 5, and 10 at 72 h post-infection. Viral replication was significantly higher in MDDCs from individuals with AA genotype than those with AG genotype at MOIs of 5 and 10 (p<0.001 and 0.002, respectively). The results are shown as mean ± s.e.m. from subjects with AA (n = 10) or AG (n = 10) genotype in three independent experiments. Statistical values were determined by Mann-Whitney U test. Asterisk (*) indicates a significant difference (p<0.05). ** indicates p<0.01; *** p<0.001.

Figure 3. Kinetic cytokine/chemokine production by DEN-infected MDDCs from individuals with AA or AG genotype of rs4804803.

The supernatant of TNFα, IL-12p40, IP-10 and MCP-1 levels were measured using ELISA after DEN infection at MOI of 5 for 24 to 72 h. The production of TNFα (A), IL-12p40 (B), and IP-10 (C) levels after DEN-2 infection was significantly higher in MDDCs from individuals with AG genotype than those with AA genotype. The production of MCP-1 (D) after DEN-2 infection was not significantly different between the two groups. The results are shown as mean ± s.e.m. from subjects with AA (n = 8) or AG (n = 8) genotype in three independent experiments. Statistical values were determined by Mann-Whitney U test. Asterisk (*) indicates a significant difference (p<0.05). ** indicates p<0.01; *** p<0.001.

Figure 4. IP-10 production by MDDCs involved in the viral replication of DEN infection.

MDDCs were mock-infected or DEN infected (MOI = 5) after preincubation, with and without anti-IP-10 neutralizing antibody (10 µg/mL) for 30 min. Total RNA was extracted from MDDCs and the supernatants were collected 24 h post-infection. The percentage of viral replication (A) and the production of IP-10 levels (B) following DEN-2 infection were significantly higher by MDDCs from individuals with AG genotype than from individuals with AA genotype. Following DEN-2 infection, the production of IP-10 by MDDCs from individuals with AA genotype was not significantly different between those with and without anti-IP-10 neutralizing antibody. The results are shown as mean ± SEM from three independent experiments (n = 7). Statistical values were determined by Wilcoxon signed-rank test. Asterisk (*) indicates a significant difference (p<0.05).