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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

HIV-1-neutralizing antibodies access VCCs and inhibit HIV-1 replication in HCs in an FcγRI-dependent manner. (a) Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 37°C prior to fixation/permeabilization and labelled with primary antibodies against (a) FcγRI (red, anti-CD64), (b) 4E10 (red) and VRC01 (red). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green). Scale bar=7 µm. Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from eight donors. Colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. (c) Neutralization activities of anti-HIV-1 neutralizing antibodies, VRC01 and 4E10, were determined in HCs at day 10 post-infection. Infected HCs were exposed to bNAbs alone. In competition experiments, HIV-1-infected HCs were also pre-incubated with the monoclonal antibody against FcγRI (anti-CD64). The percent of inhibition is defined as the reduction of p24 release in the supernatant of Ab-treated HIV-1-infected HCs compared with the control untreated HIV-1-infected HCs. Data in bar graphs are expressed as the mean+SE (% inhibition) of triplicate sections from eight donors. Asterisks (*p<0.01) indicate values that are significantly higher in VRC01- and 4E10-treated HCs compared to hIgG-treated controls. Symbols (*p<0.01) indicate significant differences due to the addition of anti-CD64.
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Figure 0006: HIV-1-neutralizing antibodies access VCCs and inhibit HIV-1 replication in HCs in an FcγRI-dependent manner. (a) Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 37°C prior to fixation/permeabilization and labelled with primary antibodies against (a) FcγRI (red, anti-CD64), (b) 4E10 (red) and VRC01 (red). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green). Scale bar=7 µm. Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from eight donors. Colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. (c) Neutralization activities of anti-HIV-1 neutralizing antibodies, VRC01 and 4E10, were determined in HCs at day 10 post-infection. Infected HCs were exposed to bNAbs alone. In competition experiments, HIV-1-infected HCs were also pre-incubated with the monoclonal antibody against FcγRI (anti-CD64). The percent of inhibition is defined as the reduction of p24 release in the supernatant of Ab-treated HIV-1-infected HCs compared with the control untreated HIV-1-infected HCs. Data in bar graphs are expressed as the mean+SE (% inhibition) of triplicate sections from eight donors. Asterisks (*p<0.01) indicate values that are significantly higher in VRC01- and 4E10-treated HCs compared to hIgG-treated controls. Symbols (*p<0.01) indicate significant differences due to the addition of anti-CD64.

Mentions: It is unknown whether infected HCs are exposed to the maternal HIV-1 immune response in utero. To examine the role of bNAbs at the foeto-maternal interface, we administered anti-HIV-1 gp120 and gp41 antibodies (VRC01 and 4E10, respectively) at 37°C. HIV-1-infected HCs were also incubated with the monoclonal antibody against FcγRI (CD64), a receptor abundantly expressed on HC with great affinity for IgG [27,28]. HIV-1-infected HCs, first immunolabelled with antibodies against FcγRI (CD64) at 37°C and then permeabilized, displayed very specific FcγRI labelling within the VCCs (HIV-1 Gag colocalization R value of 0.63±0.03) (Figure 6a). In addition, 4E10 showed a strong pattern of colocalization with Gag (R value of 0.46±0.04), while VRC01 colocalization with Gag was present but less precise (R value of 0.38±0.02) (Figure 6b). However, the degree of colocalization was substantial for both bNAbs, compared with the anti-TfR controls (HIV-1 Gag colocalization R value of 0.2±0.03) (Supplementary file 2). To test the neutralizing activity of VRC01 and 4E10 in infected HCs, cells were productively infected overnight. Virus was removed, and cells were subsequently treated with the HIV-1-specific bNAbs. On day 4, the antibodies were removed, and viral replication was monitored and analyzed on day 10. Exposure to VRC01 and 4E10 following viral uptake strongly inhibited HIV-1BaL replication in HCs (Figure 6c). These in vitro studies demonstrate that internal accumulations of HIV-1 by HCs may be accessible and permissive to the inhibiting activities of HIV-1-specific bNAbs.


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)

HIV-1-neutralizing antibodies access VCCs and inhibit HIV-1 replication in HCs in an FcγRI-dependent manner. (a) Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 37°C prior to fixation/permeabilization and labelled with primary antibodies against (a) FcγRI (red, anti-CD64), (b) 4E10 (red) and VRC01 (red). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green). Scale bar=7 µm. Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from eight donors. Colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. (c) Neutralization activities of anti-HIV-1 neutralizing antibodies, VRC01 and 4E10, were determined in HCs at day 10 post-infection. Infected HCs were exposed to bNAbs alone. In competition experiments, HIV-1-infected HCs were also pre-incubated with the monoclonal antibody against FcγRI (anti-CD64). The percent of inhibition is defined as the reduction of p24 release in the supernatant of Ab-treated HIV-1-infected HCs compared with the control untreated HIV-1-infected HCs. Data in bar graphs are expressed as the mean+SE (% inhibition) of triplicate sections from eight donors. Asterisks (*p<0.01) indicate values that are significantly higher in VRC01- and 4E10-treated HCs compared to hIgG-treated controls. Symbols (*p<0.01) indicate significant differences due to the addition of anti-CD64.
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Related In: Results  -  Collection

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Figure 0006: HIV-1-neutralizing antibodies access VCCs and inhibit HIV-1 replication in HCs in an FcγRI-dependent manner. (a) Human HCs were infected with HIV-1BaL for three days. Infected HCs were immunolabelled at 37°C prior to fixation/permeabilization and labelled with primary antibodies against (a) FcγRI (red, anti-CD64), (b) 4E10 (red) and VRC01 (red). Labelled HCs were then permeabilized and immunolabelled for HIV-1 Gag (green). Scale bar=7 µm. Image acquisition was performed with an Applied Precision Deltavision deconvolution microscope. Sections shown represent a minimum of 30 cells for each condition from eight donors. Colocalization and partial colocalization R values (using Pearson's correlation) were quantified with the colocalization module of Volocity 5.2.1. (c) Neutralization activities of anti-HIV-1 neutralizing antibodies, VRC01 and 4E10, were determined in HCs at day 10 post-infection. Infected HCs were exposed to bNAbs alone. In competition experiments, HIV-1-infected HCs were also pre-incubated with the monoclonal antibody against FcγRI (anti-CD64). The percent of inhibition is defined as the reduction of p24 release in the supernatant of Ab-treated HIV-1-infected HCs compared with the control untreated HIV-1-infected HCs. Data in bar graphs are expressed as the mean+SE (% inhibition) of triplicate sections from eight donors. Asterisks (*p<0.01) indicate values that are significantly higher in VRC01- and 4E10-treated HCs compared to hIgG-treated controls. Symbols (*p<0.01) indicate significant differences due to the addition of anti-CD64.
Mentions: It is unknown whether infected HCs are exposed to the maternal HIV-1 immune response in utero. To examine the role of bNAbs at the foeto-maternal interface, we administered anti-HIV-1 gp120 and gp41 antibodies (VRC01 and 4E10, respectively) at 37°C. HIV-1-infected HCs were also incubated with the monoclonal antibody against FcγRI (CD64), a receptor abundantly expressed on HC with great affinity for IgG [27,28]. HIV-1-infected HCs, first immunolabelled with antibodies against FcγRI (CD64) at 37°C and then permeabilized, displayed very specific FcγRI labelling within the VCCs (HIV-1 Gag colocalization R value of 0.63±0.03) (Figure 6a). In addition, 4E10 showed a strong pattern of colocalization with Gag (R value of 0.46±0.04), while VRC01 colocalization with Gag was present but less precise (R value of 0.38±0.02) (Figure 6b). However, the degree of colocalization was substantial for both bNAbs, compared with the anti-TfR controls (HIV-1 Gag colocalization R value of 0.2±0.03) (Supplementary file 2). To test the neutralizing activity of VRC01 and 4E10 in infected HCs, cells were productively infected overnight. Virus was removed, and cells were subsequently treated with the HIV-1-specific bNAbs. On day 4, the antibodies were removed, and viral replication was monitored and analyzed on day 10. Exposure to VRC01 and 4E10 following viral uptake strongly inhibited HIV-1BaL replication in HCs (Figure 6c). These in vitro studies demonstrate that internal accumulations of HIV-1 by HCs may be accessible and permissive to the inhibiting activities of HIV-1-specific bNAbs.

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