Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity

Abstract

Background: HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14(+) monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1), a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.

Methodology/principal findings: We analyzed sialoadhesin expression on CD14(+) monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14(+) monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-alpha and interferon-gamma but not tumor necrosis factor-alpha. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.

Conclusions/significance: Increased sialoadhesin expression on CD14(+) monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14(+) monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.

Figure 1. Sn expression on CD14⁺ monocytes from HIV seropositive individuals.

(A) Representative frequency histograms of relative Sn expression on CD14⁺ monocytes isolated from subjects with high viral load (HVL, 214,000 RNA copies/ml, thick black line), low viral load (LVL, 6,350 RNA copies/ml, thin black line) and a seronegative control (dotted line). The isotype-matched control mAb is shown in the shaded profile. (B) Correlation analysis of Sn expression and viral load. Sn expression on CD14⁺ monocytes from HIV seropositive subjects (Table 1, n = 24) was determined by flow cytometry and quantified as a geometric mean for each subject. Pearson's correlation analysis showed statistical significance between Sn expression and the log of the subject's viral load (p<0.0017). (C) Correlation analysis of Sn expression and CD4 (counts/ml) revealed no significant relationship (p<0.08).

Figure 2. Interferon-α and -γ induce Sn expression on CD14⁺ monocytes and THP-1 cells.

Cells were cultured in 500 U/ml IFN-α, 100 U/ml IFN-γ or 10 ng/ml TNF-α at 37°C for 48 h and analyzed for Sn expression by flow cytometry. Sn expression on IFN-α-, IFN -γ- or TNF-α-treated cells (thick black lines) and untreated cells (thin black line) were relative to an isotype-matched mAb control (shaded region). Results shown are representative histograms from three independent experiments using monocytes from three seronegative donors.

Figure 3. Constitutive Sn expression in THP-1 by gene transduction (TSn).

(A) Immunoblot analysis of Sn protein expression. THP-1 cell line was transduced with a plasmid encoding Sn cloned downstream of the high-level constitutive promoter CMV. Cell lysates were standardized, reduced with DTT and 10 µg of protein were loaded into each well: monocytes (lane 1), IFN-α-induced monocytes (lane2), THP-1 (lane 3) and TSn (lane 4) (M, molecular size marker). (B) Histogram of relative distribution of Sn on the cell surface of TSn clone. TSn (thick black line) and THP-1 cells (thin black line) were evaluated for Sn expression by flow cytometry using anti-Sn mAb 7D2 relative to the background isotype-matched control mAb (shaded region).

Figure 4. Sn binds HIV-1 in vitro.

(A) TSn binds HIV-1 in an Sn-dependant manner. TSn and THP-1 were incubated with lab-adapted HIV-1_NL4-3, an HIV-1 clade B primary isolate or clade C primary isolate for 1 h at 37°C, washed and then assayed for HIV-1 p24 by ELISA. HIV-1 binding to Sn was abrogated by pretreatment of cells with Sn mAb 7D2 or by pretreatment of HIV-1 with broad-spectrum sialidase. Pretreatment with IgG1 isotype control or CD4 mAbs did not reduce binding. Data presented are the average of 3 separate experiments. (B) IFN-α-treated CD14⁺ monocytes bind HIV-1 in an Sn-dependant manner. HIV-1 binding assays were also performed on CD14⁺ monocytes from seronegative controls treated with 500 U/ml IFN-α to induce Sn expression. Pretreatment with Sn mAb 7D2 and sialidase dramatically reduced HIV-1 binding while pretreatment with IgG1 isotype control or CD4 mAbs had little effect. Data represents monocytes from four separate donors. Error bars represent SD.

Figure 5. Sn-dependent trans infection of reporter cells TZM-bl.

(A) Sn-expressing cells, TSn and IFN-α-induced monocytes, bind HIV-1 and infect TZM-bl cells in trans. Cells were incubated with HIV-1_NL4-3 for 1 h, washed and then cocultured with TZM-bl cells for 48 h. HIV-1 infection of TZM-bl was defined by luciferase expression and quantified as relative light units (RLU). Cells pretreated with Sn mAb 7D2 showed significantly reduced capacity to facilitate trans infection of TZM-bl cells while mAb IgG1 isotype control had no effect. (B) HIV-1 receptor inhibitors block trans infection. Receptor and coreceptor requirements for trans infection of TZM-bl cells were tested by incubating TZM-bl cells with receptor inhibitors including mAbs to CD4, CXCR4 or CCR5, and small molecules AMD3100 or TAK779 prior to adding TSn with bound HIV-1_NL4-3. The CD4, CXCR4, CCR5, AMD3100 and TAK779 receptor inhibitors were tested individually with TZM-bl and did not induce luciferase expression (data not shown). As a control, productive infection of TSn cells was prevented by addition of indinavir (100 µM). Data presented are the average of three separate experiments. Error bars represent SD.

Figure 6. Sn-expressing cells capture HIV-1_NL4-3 and enhance infectivity.

TZM-bl cell cultures were seeded with various concentrations of HIV-1_NL4-3 (800–8000 pg/ml). Monocytic cells, TSn or THP-1 cells, were added and cocultured for 48 h. The capacity of Sn to capture HIV-1_NL4-3 in the cell culture medium and trans infect TZM-bl cells was analyzed for TSn, THP-1 and cell-free virus. Luciferase expression in HIV-1_NL4-3-infected TZM-bl cells was quantified as relative light units (RLU). Results were compiled from 3 separate experiments. Error bars represent SD.