The expression of DC-SIGN and CD206, molecules implicated in cell-mediated transmission of HIV-1 from monocytes, MDM and mDC to T cells [8,9,26,27], was differentially affected by macrophage polarization. CD206, similar to CD16 and CD163, was expressed on the majority of control and M2a MDM and minimally expressed on M1-MDM. In contrast, DC-SIGN was expressed on 5–17% of control MDMs; it was almost undetectable on the surface of M1-MDM and was expressed by most M2a-MDM (nine-fold increase in the percentage of cells expressing DC-SIGN above background, Table 1, Fig. 1a and b). Thus, among cell surface molecules investigated in this study, DC-SIGN (and not CD206) was the best marker for discriminating among control, M1-MDM and M2a-MDM.
Anti-dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin mAb reduces HIV-1 binding to M2a-MDM, but not to M1-MDM or control monocyte-derived macrophage
DC-SIGN, when expressed on dendritic cell, serves as a high-affinity attachment molecule for various pathogens including HIV-1 [9,27]. In order to investigate whether MDM can also bind HIV-1 via DC-SIGN, we incubated MDM with HIV-1BaL (moi = 1) at 4oC to prevent virus entry; we then measured the amount of HIV-1 p24 Gag antigen attached to the cell surface. Both control and polarized MDM bound HIV-1BaL (Fig. 2a), with control and M2a-MDM exhibiting higher mean levels of binding than M1-MDM (35.7 ± 3.7 vs. 34.9 ± 5.8 vs. 29.6 ± 3.9 pg/ml of HIV-1 p24 Gag, respectively, P < 0.05; n = 8). Incubation of these MDM populations with Leu3a, an anti-CD4 mAb that interferes with HIV-1 binding CD4, led to a mean 47% decrease (interdonor range: 36–67%) of virus binding to control MDM, but had no effect on HIV-1 binding to either M1-MDM and M2a-MDM, presumably due to the low-level expression of CD4 on both polarized MDM (Fig. 2b, as reported) . In contrast, preincubation with anti-DC-SIGN mAb led to a mean 52.5% (interdonor range: 29–74%) reduction in the amount of HIV-1 bound to M2a-MDM, but had no effect on virus binding to both control and M1-MDM (Fig. 2b). Preincubation with a combination of anti-DC-SIGN and anti-CD4 mAbs failed to induce any further decrease in HIV-1 binding to control, M1-MDM or M2a-MDM, when compared with MDM subsets preincubated with only anti-CD4 or anti-DC-SIGN mAbs (Fig. 2b). This finding is consistent with the paucity of DC-SIGN on control and M1-MDM and of CD4 on M2a-MDM (Fig. 1a) .
To determine whether DC-SIGN expressed on M2a-MDM could bind R5 viruses other than HIV-1BaL, we incubated control, M1-MDM and M2a-MDM with a panel of NL4-3 viruses expressing macrophage-tropic R5 primary Envs (ADA, YU2, UK7BR1 and UK1BR15) [19,20]. Control, M1-MDM and M2a-MDM bound equal levels of these viruses. Preincubation with anti-DC-SIGN mAb led to a mean 29%, 35% and 45% reduction in HIV-1 binding to M2a-MDM by ADA, YU2 and UK1BR15 envelopes, respectively, whereas binding of UK7BR1 was not affected by pretreatment with anti-DC-SIGN mAb (Fig. 2c).
Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin facilitates accumulation of HIV-1 DNA in M2a-MDM, but not in M1-MDM or control MDM
As DC-SIGN has been shown to enhance cis- and trans-mediated infection of dendritic cell and cell lines [27,28], we investigated whether DC-SIGN could also facilitate entry of HIV-1 into M2a cells. Polarized and control MDM were infected with HIV-1BaL and levels of HIV-1 DNA synthesized were quantified by real-time PCR after 48 h of infection, an estimated time required to complete a single round of virus replication in these cells .
MDM polarization inhibited HIV-1 replication in polarized vs. control cells (Fig. 3a), as previously reported . Blockade of DC-SIGN in control MDM led to an average 40% increase in HIV-1 DNA (Fig. 3b). As expected, anti-DC-SIGN mAb did not affect HIV-1 DNA synthesis in M1-MDMs, which express only negligible amounts of DC-SIGN and CD4. As reported , HIV-1 DNA levels in M2a-MDM were equivalent to those of control MDM, indicating that there was no impairment of viral entry or DNA synthesis in spite of lower levels of CD4 expression than those of control MDM. Incubation of M2a-MDM with anti-DC-SIGN mAb prior to infection resulted in a mean 24% decrease in HIV-1 DNA (Fig. 3b), whereas pretreatment with anti-CD4 mAb completely blocked infection. These findings are consistent with previous studies demonstrating that DC-SIGN alone cannot mediate HIV-1 infection but rather facilitates entry via a CD4-dependent mechanism .
Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin expression on M2a-MDM correlates with rapid and efficient transfer of X4 and R5 HIV-1 to CD4+ T cells
Several studies have shown that DC-SIGN expressed on the surface of dendritic cell, breast milk macrophages, activated B-lymphocytes and transfected cell lines (DC-SIGN+/CD4+ cells) can capture HIV-1 and trans-infect CD4+ T lymphocytes [8,29]. Therefore, we investigated whether M2a-MDMs also possess an increased capacity to transfer HIV-1 to CD4+ T cells independent of their productive infection. To investigate this possibility, MDM were incubated for 2 h at 37°C with HIV-1LAI/IIIB, an X4 strain incapable of establishing productive infection in MDM [24,25]. Cells were then thoroughly washed and cocultivated with autologous monocyte-depleted, IL-2-activated PBMC (mainly T cells, referred to hereafter as ‘T cells’). After 6 h of coculture, the nonadherent T cells were removed and incubated an additional 12 days in a medium enriched with IL-2. As expected, no X4 virus replication was detected in control, M1-MDM or M2a-MDM (Fig. 4a). However, M2a-MDM exposed to X4 HIV-1 could mediate trans-infection and establishment of productive infection in T cells more efficiently than either control or M1-MDM (Fig. 4a). Preincubation with anti-DC-SIGN mAb before exposure to HIV-1 resulted in a near complete inhibition of the ability of M2a cells to trans-infect T cells, but had no significant effect on virus transmission by control or M1-MDM (Fig. 4b). However, given the limits of sensitivity of the reverse transcriptase assay, we cannot exclude the possibility of low levels of viral replication that were not blocked by anti-DC-SIGN mAb in these M2a cell transfer experiments.
Finally, we tested whether control, M1-MDM and M2a-MDM could transfer R5 HIV-1 to activated T cells in a DC-SIGN-dependent manner using a panel of macrophage-tropic viruses (AD8, YU2 and UK1BR15) shown to bind to DC-SIGN in the preceding experiments (Fig. 2c). Unlike HIV-1LAI/IIIB, control, M1-MDM and M2a-MDM transferred similar levels of these R5 viruses, resulting in the establishment of low-level R5 virus replication in activated T cells. As observed with the X4 virus, anti-DC-SIGN mAb partially blocked HIV-1 transmission from M2a-MDM, but not from control or M1 cells, to T cells (range: 23–32%; Fig. 4c).
In this study, we examined the effects of M1 and M2a polarization on the expression of membrane receptors that play a role in propagation and pathogenesis of HIV-1 in mucosal tissues. Of the molecules examined, only DC-SIGN, a C-type lectin involved in the attachment and transfer of infectious virions from dendritic cell to CD4+ T cells [8,9,21], exhibited a strong differential response to M1 vs. M2a polarization. DC-SIGN was markedly upregulated on the surface of M2a-MDM and downregulated to near-undetectable levels on that of M1-MDM, a pattern that was maintained for at least 7 days post-polarization. The upregulation of DC-SIGN was associated with increased accumulation of HIV-1 DNA in M2a-MDM, potentially compensating for low levels of CD4 on the surface of these cells. In addition, DC-SIGN+ M2a-MDM showed a superior efficiency in transmitting either R5 or X4 viruses compared with control or M1-MDM, thus mimicking mDC in their capacity to bind and transmit HIV-1 to CD4+ T cells . Together, these findings suggest that M2a macrophages may play an important role in local propagation of HIV-1 in tissues favouring TH2 microenvironments.
There has been limited success in identifying phenotype-restricted markers of human macrophage polarization that can be used as research and clinical tools . Here, we identified several markers, including CD16, CD163 and CD206, which showed modest differential expression in M1 vs. M2a cells. However, DC-SIGN (CD209) was the only receptor significantly and differentially affected by M1 and M2a polarization compared with unpolarized control cells. The strong upregulation of DC-SIGN on the surface of M2a-MDM is consistent with an independent study showing that long-term (5 days) stimulation of MDM with IL-4 is associated with increased DC-SIGN mRNA and protein expression . Similarly, exposure of breast milk macrophages to IL-4 leads to increased DC-SIGN expression . Other investigators reported that DC-SIGN expression on monocytes and macrophages is regulated by specific cytokines and dependent on activation of signal transducer activator of transcription 6 (STAT6), typically induced by IL-4 and IL-13 stimulation [32–34]. Conversely, IFN-α and IFN-γ have been shown to suppress DC-SIGN expression via inhibition of tyrosine phosphorylation and nuclear translocation of STAT6 [33,34]. Thus, a better understanding of mechanisms regulating DC-SIGN expression on MDM may lead to new insights for modulation of M2a responses.
We have previously shown that M1 and M2a polarization inhibits productive R5 HIV-1 infection (whereas X4 infection was not observed in either polarized or unpolarized MDM) . M1 restriction occurs at an early, preintegration level and is associated with strong downregulation of CD4, whereas M2a-associated restriction occurs later without affecting HIV-1 DNA synthesis and accumulation . Here, we found that blockade of DC-SIGN was associated with decreased HIV-1 DNA accumulation in M2a-MDM and increased accumulation in control MDM. The mechanisms facilitating DC-SIGN-mediated entry into MDM are poorly understood, but may reflect differences in abundance of DC-SIGN vs. CD4 on these cells (with M2a-MDM expressing low CD4 and high DC-SIGN, whereas control MDM express high CD4 and low DC-SIGN) . Upregulation of DC-SIGN on M2a-MDM may compensate for low CD4 by facilitating binding and receptor/coreceptor-mediated virus entry) . This interpretation is supported by a study  showing that gp120 Env binding to DC-SIGN enhances access to the CD4-binding site. Increased expression of DC-SIGN on M2a-MDM may also promote oligomerization of DC-SIGN, or changes in the configuration of CD4/CCR5/DC-SIGN complexes .
In addition to enhancing HIV-1 DNA accumulation via cis-infection and/or trans-infection of adjacent M2a-MDM, DC-SIGN+ M2a cells rapidly (6 h) transferred X4 HIV-1 to T cells. M2-MDM also efficiently transmitted R5 HIVBaL and recombinant NL4-3 viruses expressing both ADA and YU2 Env, but not R5 brain-derived Env. These differences likely reflect heterogeneous N-linked glycosylation of primary HIV Env glycoproteins, which effects lectin receptor binding . Unlike X4 HIV-1IIIB, anti-DC-SIGN Abs only partially blocked R5 virus transmission. This finding is consistent with a study by Chehimi et al., indicating that anti-DC-SIGN Ab neutralized 39–48% of MDM-T cell transmission. In addition, Chehimi's study showed that mannin blocked transmission by 67–75%, suggesting that other C-type lectins including the macrophage mannose receptor likely contribute to HIV-1 transmission to T cells . In long-term coculture (72 h), these authors did not observe a significant increase in X4 HIV-1 transmission from IL-4-stimulated MDM to Sup-T1 T-cell lines. This discrepancy may reflect either the use of Sup-T1 cells vs. IL-2-stimulated PBMC in our study, differences in duration of coculture or technical variants in the MDM differentiation protocol. In fact, Saidi et al., using long-term (6-day) coculture conditions, demonstrated DC-SIGN-dependent transfer of R5 HIV-1 from IL-4-stimulated MDM to both activated and nonactivated T cells. Thus, M2a macrophages may mimic dendritic cell in their ability to transmit HIV-1 to CD4+ T via DC-SIGN.
Additional investigations are required to determine the in-vivo significance of our findings. Upregulation of DC-SIGN on M2a-MDM raises the possibility that this receptor may play a role in the M2/TH2 axis of immunity [30,38]. In vivo, DC-SIGN has been detected on dendritic cell and on a select subset of specialized macrophages in the lung and placenta, organs with TH2-biased microenvironments [21,39]. Consistent with this idea, a central feature of pathogens that interact with DC-SIGN, such as Schistosoma mansoni, Leishmania parasites and Mycobacterium tuberculosis, is their capacity to modulate the TH1/TH2 cell balance, leading to establishment of persistent infections .
In summary, our study demonstrates in-vitro conditions that induce persistent upregulation of DC-SIGN on MDM and the capacity of DC-SIGN+ M2a-MDM to bind and facilitate HIV-1 infection in M2a cells and primary T-cells, respectively. Macrophages are well represented in TH2-inducing environments such as the lung and gastrointestinal tract [41,42] and may play an important role not only in transmission of HIV-1 but also in the establishment or expansion of viral reservoirs. Thus, targeting of DC-SIGN may provide an important tool for prevention and treatment of HIV-1 and other infectious agents that exploit DC-SIGN for survival and spread in humans.
E.C. performed this study as partial fulfilment of her joint PhD in ‘Molecular and Cellular Biology’ of the Vita-Salute University of Milan (Milan, Italy) and the Open University of London, UK. L.C. performed some aspects of the study as partial fulfilment of his PhD in ‘Molecular Medicine-Section of Basic and Applied Immunology’ of the Vita-Salute University of Milan (Milan, Italy).
E.C. conceived and performed most of the experiments and significantly contributed to writing of the manuscript; L.C. performed some of the experiments with DC-SIGN and contributed to writing of the manuscript; C.R. optimized and performed PCR-based experiments for quantification of HIV-1 DNA; M.A. contributed to the experimental design, supervised all the initial results and contributed to writing of the manuscript; D.G and G.P. contributed to the design, supervised some of the experimental results and contributed to writing of the manuscript. All authors contributed equally to interpretation of data.
We thank Anna Mondino and Marika Falcone from the San Raffaele Scientific Institute for critical reading of the manuscript and helpful suggestions.
This study was supported in part by the Fondation Dormeur, by Europrise (grant no. LSHP CT-2006-037611 to G.P.) and by the Italian Ministry of Health Grant Program of AIDS Research 2009–2010 (to M.A. and G.P.). E.C. was supported in part by a CIHR fellowship. D.G. was supported by NIH R01 MH83588.
Conflicts of interest
The authors have no financial conflicts of interest.
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Keywords:© 2013 Lippincott Williams & Wilkins, Inc.
dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin; HIV transmission; HIV-1; macrophage polarization; T cells