The association of HIV-1 virions with cell-surface factors on macrophages and dendritic cells (DC) has been shown to protect the virus from degradation and promote viral dissemination (Lekkerkerker et al., 2006
; Levy, 2002
), but the full repertoire of these factors remains unknown. Here we demonstrate that HIV-1 adsorbs to DARC on the surface of RBCs and transfers virus to target cells, indicating that RBCs might act as carriers of infectious HIV-1 particles to susceptible cells such as CD4+
T lymphocytes. Although this process was most marked for X4-tropic strains, alteration of gp120 conformation of R5 virus by soluble CD4+
significantly increased the binding of HIV to DARC. HIV-1 did not infect CD4+
cells transfected with DARC. Collectively, these data suggest that DARC might act like DC-SIGN on DCs (Lekkerkerker et al., 2006
) in serving as a tether rather than as a functional entry receptor, although subsequent sequestration and formation of immunological synapses presumably do not occur with RBCs.
To determine whether the in vitro findings have in vivo relevance, we studied the impact of a polymorphism in DARC
in a well-characterized cohort of HIV-infected individuals in which confounding factors (e.g., unequal access to health care and unequal socioeconomic status) have been minimized (Dolan et al., 2007
; Gonzalez et al., 2005
). Contrary to the CCR5-Δ32/Δ32
genotype, which abrogates CCR5 expression and confers protection against HIV transmission in Europeans, we find that the African American-specific DARC
genotype that abrogates DARC surface expression on RBCs is associated with a 40% increase in the odds of HIV acquisition. Whereas, paradoxically, once infection occurs, the DARC
genotype confers a survival advantage to African Americans that is comparable in magnitude to the survival advantage conferred by the CCR5-Δ32
genotype to European Americans. Whether the DARC
genotype also increases the risk of infection and conveys a survival advantage to African populations outside the US remains unknown but would appear likely. If similar effects of the DARC
genotype on HIV acquisition were to be conveyed in Africans, ~11% of the HIV burden in these populations may be attributable to this genotype. To validate this hypothesis conclusively, one would need to study subjects in Africa in whom the DARC
genotype has not attained fixation and in whom other confounding factors, including exposure risk, had been accounted for. Unfortunately, such populations are difficult to identify in Africa because in sub-Saharan black Africans, the −46C/C
genotype is almost universal.
The sum of the in vitro and genetic epidemiologic findings demonstrates that HIV might exploit DARC to its advantage: binding of HIV to DARC, a molecule that is expressed on one of the most abundant cell types (RBCs), might afford a unique biological niche that favors viral survival and persistence. Underscoring this possibility is the observation by Hess et al., (2002)
, who detected RBC-bound HIV in the absence of detectable plasma virus. Thus, it is likely that the net effect of the relationship between DARC and chemokines on HIV disease in vivo is likely to be much more complex, as it seems that DARC regulates the biological effects of chemokines in three distinct ways—either through scavenging, retention, or transportation (Comerford et al., 2007
; Rot, 2005
). Hence, based on the known functions of DARC as well as the results presented herein, we hypothesize that the ultimate impact of DARC on HIV-AIDS pathogenesis is a complex interplay between levels of DARC, circulating chemokines, and HIV virions.
The few studies that have examined the in vivo impact of DARC genotype on free and erythrocyte-bound chemokine levels paint a very complicated picture. The most definitive work to date is by Jilma and colleagues (Mayr et al., 2008
), who determined plasma- and RBC-associated chemokines in those who do (DARC+) or do not (DARC−) express DARC on RBCs, before and after administration of LPS. They found that at baseline (pre-LPS), compared to DARC+ subjects, DARC− individuals had lower plasma- and RBC-associated MCP-1 levels (Mayr et al., 2008
). Post-LPS, plasma MCP-1 levels were 2-fold lower in DARC− compared to DARC+ individuals. Consequently, DARC+ individuals—by virtue of having higher chemokine levels during endotoxemia—have a transient proinflammatory state. Additionally, Fukuma et al., (2003)
show that DARC
genotype influences clearance mechanisms for soluble chemokines as DARC-knockout mice have a more rapid clearance of chemokines injected intravenously with chemokines than their wild-type counterparts. These findings underscore that DARC substantially alters chemokine concentrations in blood with the RBC DARC-phenotype favoring a low chemokine status. These results invoke that one has to consider the complexity of how the amount of plasma chemokine levels and chemokines bound to erythrocyte DARC will impact on competitive binding and displacement of HIV to DARC as well as to CD4+
T cells. Hence, based on our results and these human and murine investigations and knowledge that HIV cell entry invokes a vigorous inflammatory response with release of LPS (Douek, 2007
), we propose the following model (), whose confirmation will require additional studies.
Although it is tempting to speculate that akin to its sink function for chemokines (Comerford and Nibbs, 2005
), DARC might also serve as a sink for the initial inoculum of HIV, we found that DARC binds X4 virus more efficiently than R5 virus. Due to the low initial inoculum and the preferential primary infection of R5 viruses compared to dual-tropic or X4 viruses, it is less likely that the HIV adsorption/trans
-infection function of DARC contributes to the reduced risk of infection in DARC+ subjects. Instead, we suggest this is dominantly attributed by the impact of DARC on chemokine levels. The data of Mayr et al., (2008)
indicate that in addition to the DARC ligands CCL2 (MCP-1) and CXCL1 (GROα), DARC+ subjects are also likely to have higher levels of the circulating and RBC-associated HIV-suppressive chemokine CCL5 compared to DARC–individuals (B. Jilma, personal communication). Hence, at the time of initial viral exposure, when a small inoculum of founder R5 virus is attempting to establish infection and disseminate, greater amounts of RBC-associated HIV-suppressive chemokine might provide a relative shield for binding of HIV to RBCs and subsequent transfer to HIV target cells (). Additionally, the higher plasma levels of CCL5 in DARC+ individuals might convey a protective effect against transmission ().
However, once infection occurs, two events might come into play that together confer an accelerated rate of disease progression in DARC+ individuals. First, during established infection, the inflammatory insult conveyed by HIV (Douek, 2007
) might be greater in DARC+ than DARC− individuals. This is because DARC binds many more proinflammatory than HIV-suppressive chemokines (Gardner et al., 2004
; Rot, 2005
), including those that increase viral replication (e.g., CCL2; Monteiro de Almeida et al., 2005
). Consequently, the chemokine-driven proinflammatory state (e.g., high levels of CCL2, CCL5, and CXCL1) in DARC+ subjects may far outweigh the HIV-suppressive effects of high CCL5 levels, the only major HIV-suppressive chemokine that binds to DARC. Second, by contrast to what might be observed in the setting of the initial exposure of a host to a small viral inoculum, productive clinical HIV infection is characterized by a high plasma viral burden, which might promote improved virus and target-cell interactions. Consequently, after infection is established, there is a greater probability of HIV displacing DARC-bound chemokines, and in turn, DARC erythrocyte-bound HIV making physical contact with CD4-bearing target cells, improving the efficiency of infection. Accordingly, other studies have found that a virus bound to uninfected cells is more stable and infectious than a similar amount of cell-free virus (Levy, 2002
). Hence, after systemic infection has been established, HIV bound to DARC-expressing RBCs might serve as a reservoir for HIV and transport to other cellular targets. The aforementioned two scenarios might explain why HIV-positive subjects who express DARC have a faster rate of disease progression (). Conversely, DARC− HIV-positive subjects might have both a smaller reservoir of HIV-loaded RBCs and a reduced inflammatory state, and this could explain in part the association between the DARC
genotype and a slower disease course.
Substantiating the aforementioned model, as well the contrasting effects of DARC
genotype on HIV acquisition and disease progression, we found that the genotype-phenotype relationships of DARC+ status is remarkably similar to those of a CCL2
) that we showed previously was associated with increased CCL2 expression (Gonzalez et al., 2002
). Thus, it is striking that both DARC+ status as well as the CCL2
genotype track higher expression levels of CCL2. They are also both associated with contrasting effects on HIV acquisition and disease progression, i.e., a reduced risk of HIV acquisition, but increased rate of disease progression to AIDS and HAD (this study and Gonzalez et al., 2002
). It is also noteworthy that in our previous work we only detected these associations for the CCL2
genotype in European Americans but not African Americans HIV+ subjects (Gonzalez et al., 2002
). However, akin to the genotype-phenotype relationships that we describe for the CCL5
genotype before and after stratification for DARC
genotype (), we found that a disease-accelerating effect for the CCL2
genotype was evident only in DARC+ but not DARC− African-American subjects (data not shown).
As there is extensive linkage disequilibrium (LD) around the DARC
locus (Lautenberger et al., 2000
), we cannot exclude with certainty the possibility that the effects ascribed to the −46C/C
genotype might be attributable to some other polymorphism(s)/gene(s) in LD near DARC
. However, based on the in vitro () and in vivo () data described herein, as well as previous findings implicating a role for DARC in HIV-AIDS, we suggest that the genotype-phenotype relationships described herein are most likely directly attributable to DARC. Nevertheless, similar studies in other cohorts are warranted to confirm these results. Although the precise mechanisms by which DARC imparts its effects on HIV-AIDS pathogenesis are unclear, our findings indicate that the triangular relationship between this chemokine-binding protein, its natural ligands (chemokines), and pathogen (HIV) is likely to lead to biologically complex and seemingly contrasting outcomes.
In summary, the results of our proof-of-concept genetic-epidemiologic studies support a conceptual model that the interplay between DARC and chemokines might influence the amount of free versus DARC-bound HIV available for eventual transfer from RBCs to target cells and also chemokine levels that promote inflammation (e.g., CCL2, CCL5, CXCL1, and CXCL8) and compete for the binding of HIV (e.g., CCL5) to CD4+
T cells during HIV infection. By extension, the results of our studies indicate that failure to account for the chemokine binding and HIV-adsorption effects of DARC might confound genetic epidemiologic studies that examine the association between chemokine-gene polymorphisms and HIV-AIDS susceptibility in subjects of African descent. Additionally, consistent with our previous contention (Gonzalez et al., 1999
; Moore et al., 2008
), the genotype-phenotype vignettes shown here underscore further the possible impact of differentially distributed transmission and/or disease-modifying genotypes on (1) the heterogeneity of HIV disease burden not just at the level of individuals but also populations and (2) evaluation of vaccine efficacy.
The HIV/AIDS-modifying effects of CCR5-Δ32 and DARC −46C, two population-specific alleles, highlight that the impact of ancestral evolutionary factors—including infectious diseases—on the human genome are influencing outcomes of present-day infections. However, these outcomes can be complex. For example, in addition to the DARC −46C allele, polymorphisms such as sickle-cell trait can confer protection against malaria, which can be costly by causing genetic disease in the homozygous state. Thus, it should not be surprising then that what might be beneficial in one context (e.g., protection from malaria by DARC −46C/C genotype) can be detrimental in other settings (e.g., association between DARC −46C/C genotype and increased risk of HIV acquisition).
Of broad interest, our studies underscore that DARC impacts on chemokines, malaria, and HIV, a ménage à trois. However, given the importance of the chemokine system in host defense, immune responses, and inflammation, it will be important to determine the contribution of DARC to the pathogenesis of other infectious disease and inflammatory disease states.