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Placental Hofbauer cells assemble and sequester HIV-1 in tetraspanin-positive compartments that are accessible to broadly neutralizing antibodies.

Johnson EL, Chu H, Byrareddy SN, Spearman P, Chakraborty R - J Int AIDS Soc (2015)

Bottom Line: Following infection, we observed HIV-1 accumulation in potentially acidic compartments, which stained intensely with Lysotracker-Red.Remarkably, these compartments are readily accessible via the cell surface and can be targeted by exogenously applied small molecules and HIV-1-specific broadly neutralizing antibodies.In addition, broadly neutralizing antibodies (4E10 and VRC01) limited viral replication by HIV-1-infected HCs, which may be mediated by FcγRI.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.

ABSTRACT

Introduction: Within monocyte-derived macrophages, HIV-1 accumulates in intracellular virus-containing compartments (VCCs) that are inaccessible to the external environment, which implicate these cells as latently infected HIV-1 reservoirs. During mother-to-child transmission of HIV-1, human placental macrophages (Hofbauer cells (HCs)) are viral targets, and have been shown to be infected in vivo and sustain low levels of viral replication in vitro; however, the risk of in utero transmission is less than 7%. The role of these primary macrophages as viral reservoirs is largely undefined. The objective of this study is to define potential sites of viral assembly, accumulation and neutralization in HCs given the pivotal role of the placenta in preventing HIV-1 infection in the mother-infant dyad.

Methods: Term placentae from 20 HIV-1 seronegative women were obtained following caesarian section. VCCs were evaluated by 3D confocal and electron microscopy. Colocalization R values (Pearson's correlation) were quantified with colocalization module of Volocity 5.2.1. Replication kinetics and neutralization studies were evaluated using p24 ELISA.

Results: We demonstrate that primary HCs assemble and sequester HIV-1(BaL) in intracellular VCCs, which are enriched in endosomal/lysosomal markers, including CD9, CD81, CD63 and LAMP-1. Following infection, we observed HIV-1 accumulation in potentially acidic compartments, which stained intensely with Lysotracker-Red. Remarkably, these compartments are readily accessible via the cell surface and can be targeted by exogenously applied small molecules and HIV-1-specific broadly neutralizing antibodies. In addition, broadly neutralizing antibodies (4E10 and VRC01) limited viral replication by HIV-1-infected HCs, which may be mediated by FcγRI.

Conclusions: These findings suggest that placental HCs possess intrinsic adaptations facilitating unique sequestration of HIV-1, and may serve as a protective viral reservoir to permit viral neutralization and/or antiretroviral drug entry in utero.

No MeSH data available.


Related in: MedlinePlus

HC internal VCCs are accessible to antibodies. Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 4°C (a) or 37°C (b) prior to fixation/permeabilization and labelled with primary antibodies against tetraspanins (red, anti-CD9 and anti-CD81). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green, anti-CA183). Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from 10 donors. Scale bar=7 µm. (c) HIV-1 Gag colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. in non-permeabilized cells at 4°C (white) and 37°C (grey), in comparison to permeabilized cells (black). Data in bar graphs are expressed as the mean+SE (R value) of triplicate sections from 10 donors. Asterisks (*p<0.01) indicate significant differences between the R values of non-permeabilized cells compared to permeabilized cells.
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Figure 0005: HC internal VCCs are accessible to antibodies. Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 4°C (a) or 37°C (b) prior to fixation/permeabilization and labelled with primary antibodies against tetraspanins (red, anti-CD9 and anti-CD81). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green, anti-CA183). Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from 10 donors. Scale bar=7 µm. (c) HIV-1 Gag colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. in non-permeabilized cells at 4°C (white) and 37°C (grey), in comparison to permeabilized cells (black). Data in bar graphs are expressed as the mean+SE (R value) of triplicate sections from 10 donors. Asterisks (*p<0.01) indicate significant differences between the R values of non-permeabilized cells compared to permeabilized cells.

Mentions: To investigate whether the tetraspanin-enriched VCCs in HCs can be accessed from the plasma membrane, we tested the ability of antibodies against CD9 and CD81 to stain the compartments before permeabilization. Staining was performed at 4°C to reduce membrane movement and phagocytosis, while labelling at 37°C mimics in vivo conditions and allows for active uptake. HIV-1BaL infected HCs were immunolabelled at 4°C and 37°C prior to fixation. To control for antibody specificity, we repeated all immunofluorescence experiments with monoclonal anti-TfR, an unrelated protein that does not colocalize with Gag in HCs (Supplementary file 2). At 4°C, tetraspanins were limited to the plasma membrane of infected HCs, while Gag was readily detected in the VCCs (Figure 5a). In contrast, HIV-infected HCs first immunolabelled with antibodies against CD9 and CD81 at 37°C and then permeabilized displayed prominent labelling and colocalization with Gag within the VCCs (Figure 5b). We compared the degree of colocalization between the tetraspanins and Gag in HIV-1-infected HCs immunolabelled at 4°C and 37°C prior to fixation, using cells from individual donors (Figure 5c). Unlike what we have previously shown in MDMs [13], these results confirm that VCCs within primary HCs are accessible to antibodies in the external environment.


Placental Hofbauer cells assemble and sequester HIV-1 in tetraspanin-positive compartments that are accessible to broadly neutralizing antibodies.

Johnson EL, Chu H, Byrareddy SN, Spearman P, Chakraborty R - J Int AIDS Soc (2015)

HC internal VCCs are accessible to antibodies. Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 4°C (a) or 37°C (b) prior to fixation/permeabilization and labelled with primary antibodies against tetraspanins (red, anti-CD9 and anti-CD81). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green, anti-CA183). Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from 10 donors. Scale bar=7 µm. (c) HIV-1 Gag colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. in non-permeabilized cells at 4°C (white) and 37°C (grey), in comparison to permeabilized cells (black). Data in bar graphs are expressed as the mean+SE (R value) of triplicate sections from 10 donors. Asterisks (*p<0.01) indicate significant differences between the R values of non-permeabilized cells compared to permeabilized cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4308659&req=5

Figure 0005: HC internal VCCs are accessible to antibodies. Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 4°C (a) or 37°C (b) prior to fixation/permeabilization and labelled with primary antibodies against tetraspanins (red, anti-CD9 and anti-CD81). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green, anti-CA183). Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from 10 donors. Scale bar=7 µm. (c) HIV-1 Gag colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. in non-permeabilized cells at 4°C (white) and 37°C (grey), in comparison to permeabilized cells (black). Data in bar graphs are expressed as the mean+SE (R value) of triplicate sections from 10 donors. Asterisks (*p<0.01) indicate significant differences between the R values of non-permeabilized cells compared to permeabilized cells.
Mentions: To investigate whether the tetraspanin-enriched VCCs in HCs can be accessed from the plasma membrane, we tested the ability of antibodies against CD9 and CD81 to stain the compartments before permeabilization. Staining was performed at 4°C to reduce membrane movement and phagocytosis, while labelling at 37°C mimics in vivo conditions and allows for active uptake. HIV-1BaL infected HCs were immunolabelled at 4°C and 37°C prior to fixation. To control for antibody specificity, we repeated all immunofluorescence experiments with monoclonal anti-TfR, an unrelated protein that does not colocalize with Gag in HCs (Supplementary file 2). At 4°C, tetraspanins were limited to the plasma membrane of infected HCs, while Gag was readily detected in the VCCs (Figure 5a). In contrast, HIV-infected HCs first immunolabelled with antibodies against CD9 and CD81 at 37°C and then permeabilized displayed prominent labelling and colocalization with Gag within the VCCs (Figure 5b). We compared the degree of colocalization between the tetraspanins and Gag in HIV-1-infected HCs immunolabelled at 4°C and 37°C prior to fixation, using cells from individual donors (Figure 5c). Unlike what we have previously shown in MDMs [13], these results confirm that VCCs within primary HCs are accessible to antibodies in the external environment.

Bottom Line: Following infection, we observed HIV-1 accumulation in potentially acidic compartments, which stained intensely with Lysotracker-Red.Remarkably, these compartments are readily accessible via the cell surface and can be targeted by exogenously applied small molecules and HIV-1-specific broadly neutralizing antibodies.In addition, broadly neutralizing antibodies (4E10 and VRC01) limited viral replication by HIV-1-infected HCs, which may be mediated by FcγRI.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.

ABSTRACT

Introduction: Within monocyte-derived macrophages, HIV-1 accumulates in intracellular virus-containing compartments (VCCs) that are inaccessible to the external environment, which implicate these cells as latently infected HIV-1 reservoirs. During mother-to-child transmission of HIV-1, human placental macrophages (Hofbauer cells (HCs)) are viral targets, and have been shown to be infected in vivo and sustain low levels of viral replication in vitro; however, the risk of in utero transmission is less than 7%. The role of these primary macrophages as viral reservoirs is largely undefined. The objective of this study is to define potential sites of viral assembly, accumulation and neutralization in HCs given the pivotal role of the placenta in preventing HIV-1 infection in the mother-infant dyad.

Methods: Term placentae from 20 HIV-1 seronegative women were obtained following caesarian section. VCCs were evaluated by 3D confocal and electron microscopy. Colocalization R values (Pearson's correlation) were quantified with colocalization module of Volocity 5.2.1. Replication kinetics and neutralization studies were evaluated using p24 ELISA.

Results: We demonstrate that primary HCs assemble and sequester HIV-1(BaL) in intracellular VCCs, which are enriched in endosomal/lysosomal markers, including CD9, CD81, CD63 and LAMP-1. Following infection, we observed HIV-1 accumulation in potentially acidic compartments, which stained intensely with Lysotracker-Red. Remarkably, these compartments are readily accessible via the cell surface and can be targeted by exogenously applied small molecules and HIV-1-specific broadly neutralizing antibodies. In addition, broadly neutralizing antibodies (4E10 and VRC01) limited viral replication by HIV-1-infected HCs, which may be mediated by FcγRI.

Conclusions: These findings suggest that placental HCs possess intrinsic adaptations facilitating unique sequestration of HIV-1, and may serve as a protective viral reservoir to permit viral neutralization and/or antiretroviral drug entry in utero.

No MeSH data available.


Related in: MedlinePlus