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. 2013 Mar 13;27(5):707-16.
doi: 10.1097/QAD.0b013e32835cfc82.

Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin mediates HIV-1 infection of and transmission by M2a-polarized macrophages in vitro

Edana Cassol  1 , Luca Cassetta, Chiara Rizzi, Dana Gabuzda, Massimo Alfano, Guido Poli
Affiliations

Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin mediates HIV-1 infection of and transmission by M2a-polarized macrophages in vitro

Edana Cassol et al. AIDS. .

Abstract

Objective: To assess in-vitro effects of monocyte-derived macrophage (MDM) polarization into M1 and M2a cells on HIV-1 replication and transmission and obtain new insights into the potential importance of macrophage polarization in vivo.

Design: Human peripheral blood monocytes were differentiated into MDM for 7 days. Control and MDM polarized into M1 or M2a cells were exposed to different strains of HIV-1 and assessed for their ability to bind and transmit virus to CD4 T lymphocytes.

Methods: MDM were incubated with either tumour necrosis factor-alpha (TNF-α) along with interferon-gamma (IFN-γ) or with interleukin-4 (IL-4) for 18 h to obtain M1 or M2a cells, respectively. Expression of cell surface antigens, including CD4 and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN), was evaluated by flow cytometry. C-C chemokine receptor type 5 (CCR5)-dependent (R5) HIV-1 binding, DNA synthesis and viral replication were assessed in the presence or absence of anti-DC-SIGN blocking mAbs. Transmission of C-X-C chemokine receptor type 4 (CXCR4)-dependent (X4) and R5 HIV-1 from MDM to IL-2 activated CD4 T cells was also investigated.

Results: DC-SIGN was strongly upregulated on M2a-MDM and downregulated on M1-MDM compared with control MDM. DC-SIGN facilitated HIV-1 entry and DNA synthesis in M2a-MDM, compensating for their low levels of CD4 cell expression. M2a-MDM efficiently transmitted both R5 and X4 HIV-1 to CD4 T cells in a DC-SIGN-dependent manner.

Conclusion: DC-SIGN facilitates HIV-1 infection of M2a-MDM, and HIV-1 transfer from M2a-MDM to CD4 T cells. M2a-polarized tissue macrophages may play an important role in the capture and spread of HIV-1 in mucosal tissues and placenta.

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Conflict of interest statement

Conflicts of interest

The authors have no financial conflicts of interest.

Figures

Fig. 1
Fig. 1. Regulation of dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin and CD4 expression on the surface of human M1- and M2a-MDM
(a) MDM populations of a single donor were labelled with anti-DC-SIGN or anti-CD4 mAbs 18 h after polarizing or control (unpolarized) conditions and were analysed by flow cytometry. (b) These results were confirmed in six independent MDM donors; downregulation of CD4 from the cell surface of both M1 and M2a-MDM is shown in the right panel. DC-SIGN and CD4 expression levels in polarized cells were normalized to those of autologous control MDM indicated as 1 (dashed line). DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin; MDM, monocyte-derived macrophage.
Fig. 2
Fig. 2. Differential binding of R5 HIV-1 strains to control (unpolarized) and polarized monocyte-derived macrophage
(a) HIV-1 binding to control (CD4highDC-SIGNlow), M1-MDM (CD4lowDC-SIGNneg) and M2a-MDM (CD4lowDC-SIGNhigh) was measured 18 h after polarizing or control conditions as the amount of HIV-1 p24 Gag antigen bound to the cell surface after removal of excess viral inoculums and extensive washing. The results are presented as the average cell surface bound p24 Gag antigen detected in MDM cultures established from eight individual donors. Significantly higher levels of binding of HIV-1 were observed in control and M2a-MDM vs. M1 cells. Differences were assessed using one-way ANOVA and Tukey post-tests. *P ≤ 0.05. (b) Anti-DC-SIGN mAb reduce HIV-1BaL binding to M2a, but not to control or M1-MDM, independently of CD4. Values represent the percentage positivity for p24 Gag antigen vs. control and polarized populations that were not incubated with the indicated mAb (dotted line). The results show the mean + SD of three replicates per each MDM culture established from eight independent donors. The statistical difference in p24 Gag binding between cultures incubated in the presence or absence of the indicated mAb was evaluated by a paired t-test. *P ≤ 0.05. (c) Anti-DC-SIGN mAb reduced binding of NL4-3 viruses expressing macrophage-tropic envelopes from HIV-1ADA, HIV-1YU2 and HIV-1UK1BR15, but not HIV-1UK7BR1, in M2a. As discussed above, values represent the percentage of bound p24 Gag on MDM preincubated with anti-DC-SIGN mAb compared with untreated cells. Shown is the mean + SD of experiments performed in triplicate from two independent donors. The statistical difference in p24 Gag binding between cultures incubated in the presence or absence of the indicated mAb was evaluated by a paired t-test. ANOVA, analysis of variance; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin; MDM, monocyte-derived macrophage. *P ≤ 0.05.
Fig. 3
Fig. 3. Effect of dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin expression on viral DNA synthesis in polarized and control MDM
(a) HIV-1 replication kinetics in control, M1-MDM and M2a-MDM quantified by measuring RT activity of culture supernatants over a 28-day infection period. Results are representative of the average replication (±SD) in MDM cultures established from a representative donor. (b) Dichotomous effect of anti-DC-SIGN mAb on HIV-1 DNA accumulation in control vs. M1-MDM and M2a-MDM. Following MDM activation in 18 h of polarizing or control conditions, cells were washed and preincubated with anti-DC-SIGN mAb for 20 min at room temperature before infection with R5 HIV-1BaL. The results shown (48 h post-infection) were obtained from MDM cultures established from a single donor out of eight independent donors tested in duplicate. The statistical difference between cultures incubated in the presence or absence of the indicated mAb was evaluated by a paired t-test. DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin. *P ≤ 0.05.
Fig. 4
Fig. 4. DC-SIGN+ M2a transfer of X4 and R5 HIV-1 to activated CD4+ T cells
(a) After 18 h in polarizing or control conditions, control, M1-MDM and M2a-MDM were washed and incubated with X4 HIV-1LAI/IIIB for 2 h at 37° C. MDM were then washed and cocultured for 6 h with IL-2 activated, monocyte-depleted PBMC (T cells). HIV-1 transfer was determined by the productive infection of T cells, as measured by RT activity in culture supernatants. Results represent the average ± SD from MDM established from four independent donors. Neither control nor polarized MDMs were productively infected by X4 HIV-1 (left panel), whereas M2a-MDM transferred infectious X4 HIV-1 to T cells (right panel) with much greater efficiency than control or M1-MDM (*P > 0.05 by ANOVA and Tukey post-test). (b) Preincubation of MDM with anti-DC-SIGN mAb prior to exposure to HIV-1LAI/IIIB prevented the ability of these cells, but not that or M1-MDM, to transmit infectious HIV-1 to IL-2 lymphocytes (mean ± SD of MDM cultures established from four individual donors; paired t-test. *P ≤ 0.05, **P ≤ 0.01). (c) Preincubation of MDM with anti-DC-SIGN mAb prior to exposure to viruses expressing Env from HIV-1ADA, HIV-1YU2 and HIV-1UK1BR15 blocked 23–32% of transmission from M2a-MDM to IL-2-stimulated lymphocytes. Results represent average p24 Gag levels ± SD from MDM from two independent donors at day 12 of coculture. We detected an average of 300, 281 and 274 ng/ml of p24 Gag in supernatants from PBMC cocultured with untreated MDM (no anti-DC-SIGN Ab) exposed to NL4-3 viruses with ADA, YU2 and UK1BR15 Envs, respectively. The difference in p24 Gag in culture supernatant between MDM incubated in the presence or absence of the indicated mAb was evaluated by a paired t-test. DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin; MDM, monocyte-derived macrophage; PBMC, peripheral blood mononuclear cell. *P ≤ 0.05.
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