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Variations of dendritic cell-specific intercellualar adhesion molecule-3-grabing nonintegrin neck region in HIV infected individuals

XU, Li-jun; YAO, Hang-ping; LI, Dan; WANG, Zhi-gang; CHEN, Liang; WU, Nan-ping

Section Editor(s): SUN, Jing

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Edited by

Institute of Infectious Diseases, First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, China (Xu LJ, Yao HP and Wu NP)

Shanghai Public Health Clinical Center, Shanghai 201508, China (Li D)

Zhejiang Center for Disease Prevention and Control, Hangzhou, Zhejiang 310004, China (Wang ZG)

Department of Infectious Diseases, Jishuitan Hospital, Beijing 100035, China (Chen L)

Correspondence to: Dr. WU Nan-ping, Institute of Infectious Diseases, First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, China (flwnp@yahoo.com.cn or flwnp@zju.edu.cn)

This study was supported by a grant from the National Natural Science Foundation of China (No. 30471538).

(Received May 27, 2007)

Dendritic cells (DCs) play a critical role in initiating the immune response by virtue of their ability to capture and present antigens to T cells.1 Although the precise mechanism by which DCs acquire human immunodeficiency virus (HIV-1) is not completely understood, migration of DCs from the periphery to the draining lymph nodes may enable CD4+ T cells to become infected.2 DC-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN, CD209), a mannose specific C-type lectin receptor on DCs, plays a vital role in this process by binding HIV-gp120 and helping DCs transport HIV from the infection site to the secondary lymph nodes.3 DC-SIGN related lectin (DC-SIGNR, or L-SIGN, CD209R) shares 77% amino acid identity with DC-SIGN, and is expressed on endothelial cells in the liver, lymph nodes and placental capillaries.4 Both DC-SIGN and DC-SIGNR are HIV receptors.5

Peptide sequence and structural analyses indicate that DC-SIGN consists of four regions: carbohydrate recognition domain (CRD), consisting of 110 to 140 amino acid residues; neck region (also named repeat region), normally including seven 23-amino acid residue tandem repeat alleles; transmembrane region; and an endocellular domain. The CRD and neck regions have the greatest influence on pathogen infection. CRD is necessary for binding and transmission of HIV-1 and other pathogens. The neck region forms a tetramer which stabilizes the DC-SIGN structure. So the number changes of repeat alleles may have a potential impact on the function of CRD.6,7

Recently, associations between host gene polymorphisms and HIV infection have been investigated. A study of 1716 American individuals suggests that heterozygous 7/5R DC-SIGNR tend to decrease the risk of HIV infection;8 however this conclusion is not supported by the investigation of a German population conducted by Lichterfels et al,9 whose research indicates the polymorphisms in the DC-SIGNR neck region does not effect HIV infection and HIV/AIDS progress. Usually, polymorphisms of host genes are related with ethnic background and heredity. In fact, the distribution of DC-SIGN differs widely in populations from industrialized and developing countries as well as between different ethnic populations. The reasons for this diversity are presumably related to geographically determined selection pressures and long-term selected pressures of pathogens.10,11

Little is known about DC-SIGN variations and HIV infection in Chinese populations. For the purpose of exploring this association in our present study we investigate a potential correlation between variations of the DC-SIGN neck region and susceptibility to HIV in Chinese Han population.

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METHODS

Samples

Samples from one hundred and nineteen HIV-1 seropositive Chinese patients were collected during 2004 to 2006 from Chinese Center for Disease Control and Prevention (Beijing), Hangzhou Infectious Hospital, Qingchun Hospital (Hangzhou) and the First Affiliated Hospital of Zhejiang University. All HIV-1 infected subjects were HIV-1 primary infected individuals with an average age of (42±10) years old and did not receive any anti-virus therapies. One hundred and twenty HIV-1 seronegative individuals were sampled as controls. They were outpatients in the First Affiliated Hospital of Zhejiang University for routine health examination and were unrelated to the HIV infected individuals. The average age of the controls was (40±15) years old. All the subjects were males from the Han population except for one female. With the subjects' authorization, five milliliter of blood was taken from a vein and preserved in EDTA-anticoagulant tubes.

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Isolation of DC-SIGN neck region

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples by density gradient centrifugation and washed twice with PBS. PBMC was treated with Trizol (Invitrogen, USA) for total RNA extraction according to standard protocol. The total RNA was resuspended in 50 μl DEPC-treated deionized water, then preserved at -80°C for further use. The DC-SIGN repeat region was analyzed by RT-PCR. We amplified the DC-SIGN repeat region with a sense primer 5′-AACAATCCAGGCAAGACG-3 and an antisense primer 5′-TGCTCAGGCAGGGTCAGT-3′. PCR products were analyzed by 2% agarose gel electrophoresis. The products were then purified with a PCR purification kit (Takara, Japan), cloned into a PMD-18T vector (Takara, Japan) by a T-A reaction, followed by a two-way sequencing. For the heterozygous DC-SIGN, every DNA strip in the gel was separated by gel purification (Takara, Japan) and sequenced. The numbers of repeat allele within DC-SIGN neck region were determined based on the results of agarose gel electrophoresis and nucleotide sequences.

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Statistical analysis

The two-sided Pearson χ2 test was used to determine whether genotypes or repeat alleles of DC-SIGN had differential discrepancies between HIV infected individuals and the controls. Heterozygous and homozygous DC-SIGN were categorized into two groups respectively for analysis. A 2×2 contingency table was used to examine whether the distributions of homozygous/heterozygous genotypes were independent. Yates' continuity correction was used when necessary. A P value less than 0.05 was considered statistically significant. All analysis was performed using the software SPSS11.5.

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RESULTS

Variations of DC-SIGN neck region

The numbers and frequencies of DC-SIGN variations among HIV infected individuals and controls are summarized in Table 1. The percentage of 7/7 repeat allele (7/7R) genotype was 91.41% in HIV infected Chinese, which was significantly lower than the frequencies among control individuals (91.41% vs 97.50%, P=0.038). The dominance of the 7/7R genotype among the HIV-1 seropositive and control patients suggested that the 7/7R genotype was the wild type gene in the population. In order to analyze whether heterozygous DC-SIGN reduced the susceptibility to HIV infection or not, homozygous genotypes (8/8R, 7/7R, 6/6R, 4/R and 3/3R) and heterozygous genotypes (8/7R, 8/6R, 7/6R and 7/5R) present in the Chinese population were categorized into two groups as in Table 2. There were no significant discrepancies of genotypes for DC-SIGN between the two groups (P=0.561).

Table 1

Table 1

Table 2

Table 2

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Gene sketch map for DC-SIGN neck region

Though homozygous 7/7R was the dominant genotype in our research subjects although many different DC-SIGN allelic combinations were found in our research. In order to illustrate the missing fragments of each repeat region, and to identify the genotype of repeat alleles within the neck region, we draw a sketch map of the DC-SIGN gene, as described previously.6,7 The map shows that the first, second, and eighth repeat alleles were conserved among all samples and were rarely absent (Figure).

Figure.

Figure.

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DISCUSSION

The associations of D-SIGN/R variation and susceptibility to HIV infection have been studied in recent years. These studies showed that the variations in the DC-SIGN promoter region had potential effects on pathogen infection. For example, individuals with the -336C variant are more susceptible to HIV infection than individuals with the -336T variant.12

A study including 316 HIV-1-seropositive and 425 HIV-1-seronegative individuals suggest that heterozygous DC-SIGN reduces the risk of HIV-1 infection.13 This conclusion is draw from the fact that the occurrence of heterozygous DC-SIGN is 0 in HIV-seropositive populations, but 3.2% in the HIV-1 seronegative individuals.

Another important factor is that DC-SIGN translated from mRNA rather than from DNA may have great significance on HIV infection. Different DC-SIGN structure from different recombination of DC-SIGN mRNA may directly interact with HIV and could have a potential effect on HIV infection. In our present study, we determined variants of the DC-SIGN neck region from HIV-1 infected and uninfected individuals, respectively. We show that variations within DC-SIGN are significantly more common in the HIV infected cohort than in the control group. Differing from previous report that heterozygous DC-SIGN is not available in HIV-1(+) individuals,13 our date suggest heterozygous DC-SIGN in the Chinese Han population is detectable and heterozygous DC-SIGN does not have an impact on HIV infection.

The reasons for the variations of DC-SIGN detected in HIV-1(+) individuals might be as follows. First, interleukin-4 (IL-4) is a pivotal regulator for DC-SIGN expression in in vitro experiments, and is responsible for allelic DC-SIGN mRNA scission, recombination and rearrangement.7,14 One character of HIV infection is gradual T lymphocyte subpopulation switching from a Th1 population to a Th2 population. In vivo the Th1 population mainly secrets interferon γ (INF-γ) not IL-4, whereas, Th2 population secret mainly IL-4 but not INF-γ. So variation of DC-SIGN in HIV infected people maybe result from a variety of levels of IL-4 secreted by Th2 cells at different stages of HIV infection. Second, mutations of IL-4 receptor (IL-4R) are closely associated with susceptibility to HIV-1 infection and its progression to AIDS.20 So the mutation of the IL-4R maybe also be a factor associated with DC-SIGN variations. Third, because the genotype of DC-SIGN does not impact HIV infection the variations of DC-SIGN that appeared in HIV infected persons is the result from HIV infection, not the result of HIV infection of individuals with different DC-SIGN.

It should be pointed that further research is urgently required to explore potential the significance for the presence of these mutations in the HIV-1 seropositive populations. For example, whether variations of DC-SIGN are related to HIV/AIDS progress or not will require a detailed study. Our results provide a starting point to understanding why mutation rates in DC-SIGN are increased in HIV infected individuals.

In summary, our data reveal that there are more genotypes of DC-SIGN in HIV-infected individuals than in non-infected people, and heterozygous DC-SIGN does not reduce the risk to HIV infection in Chinese Han population.

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REFERENCES

1. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392: 245-252.
2. Geijtenbeek TB, Engering A, Van Kooyk Y. DC-SIGN, a C-type lectin on dendritic cells that unveils many aspects of dendritic cell biology. J Leukoc Bio 2002; l71: 921-931.
3. Geijtenbeek TB, van Duijnhoven GC, van Vliet SJ, Krieger E, Vriend G, Figdor CG, et al. Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesionmolecule 3 and HIV-1. J Biol Chem 2002; 227: 11314-11320.
4. Pohlmann S, Soilleux EJ, Baribaud F, Leslie GJ, Morris LS, Trowsdale J, et al. DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans. Proc Natl Acad Sci U S A 2001; 98: 2670-2675.
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13. Liu H, Hwangbo Y, Holte S, Lee J, Wang C, Kaupp N, et al. Analysis of genetic polymorphisms in CCR5,CCR2, stromal cell-derived factor-1, rantes, and dendritic cell-specific intercellualar adhesion molecule-3-grabing nonintegrin in seronegative individuals repeatedly exposed to HIV-1. J Infect Dis 2004; 190: 1055-1058.
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Keywords:

dendritic cells; specific intercellualar adhesion molecule-3-grabing nonintegrin; HIV; susceptibility

© 2008 Chinese Medical Association