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The immunomodulatory role of syncytiotrophoblast microvesicles.

Southcombe J, Tannetta D, Redman C, Sargent I - PLoS ONE (2011)

Bottom Line: Immune adaptation is a critical component of successful pregnancy.Other cytokines are down-modulated, such as IP-10 which is associated with 'type 1' immunity.We also observed that PBMC from third trimester normal pregnant women produce more TNFα and IL-6 in response to STBM than PBMC from non-pregnant women, confirming that maternal immune cells are primed by pregnancy, possibly through their interaction with STBM.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom. jen.southcombe@obs-gyn.ox.ac.uk

ABSTRACT
Immune adaptation is a critical component of successful pregnancy. Of primary importance is the modification of cytokine production upon immune activation. With the discovery that normal pregnancy itself is a pro-inflammatory state, it was recognised that the classical Th1/Th2 cytokine paradigm, with a shift towards 'type 2' cytokine production (important for antibody production), and away from 'type 1' immunity (associated with cell mediated immunity and graft rejection), is too simplistic. It is now generally agreed that both arms of cytokine immunity are activated, but with a bias towards 'type 2' immunity. Many factors are released from the placenta that can influence the maternal cytokine balance. Here we focus on syncytiotrophoblast microvesicles (STBM) which are shed from the placenta into the maternal circulation. We show that STBM can bind to monocytes and B cells and induce cytokine release (TNFα, MIP-1α, IL-1α, IL-1β, IL-6, IL-8). Other cytokines are down-modulated, such as IP-10 which is associated with 'type 1' immunity. Therefore STBM may aid the 'type 2' skewed nature of normal pregnancy. We also observed that PBMC from third trimester normal pregnant women produce more TNFα and IL-6 in response to STBM than PBMC from non-pregnant women, confirming that maternal immune cells are primed by pregnancy, possibly through their interaction with STBM.

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pSTBM bind to monocytes and B cells.106 PBMC from normal pregnant women were incubated with 50 µg/ml pSTBM for one hour at 37°C. Cells were then washed in PBS and stained for antibodies towards CD3, CD14, CD19, CD56 and CD19, and the proportion of cells with bound pSTBM identified with the trophoblast specific marker NDOG-2. Gates were set by staining PBMC with all six antibodies without the addition of pSTBM. NDOG-2 staining of cell populations was also compared to an isotype control (representative staining profile shown in A). Binding of pSTBM to cell populations shown in B (n = 3; mean +/− S.E.M.). C) Image stream analysis of B cell (grey bars) and monocyte (black bars) internalisation of pSTBM (labelled with BODIPY FL Maleimide) with IDEAS software (n = 3; mean +/− S.E.M.; * = p<0.05). D) Representative images of cells with internal or external pSTBM - B cells showing brightfield image, CD19 (red), pSTBM (green) and overlay (top panel) and monocytes with brightfield image, pSTBM (green) and overlay (bottom panel).
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pone-0020245-g004: pSTBM bind to monocytes and B cells.106 PBMC from normal pregnant women were incubated with 50 µg/ml pSTBM for one hour at 37°C. Cells were then washed in PBS and stained for antibodies towards CD3, CD14, CD19, CD56 and CD19, and the proportion of cells with bound pSTBM identified with the trophoblast specific marker NDOG-2. Gates were set by staining PBMC with all six antibodies without the addition of pSTBM. NDOG-2 staining of cell populations was also compared to an isotype control (representative staining profile shown in A). Binding of pSTBM to cell populations shown in B (n = 3; mean +/− S.E.M.). C) Image stream analysis of B cell (grey bars) and monocyte (black bars) internalisation of pSTBM (labelled with BODIPY FL Maleimide) with IDEAS software (n = 3; mean +/− S.E.M.; * = p<0.05). D) Representative images of cells with internal or external pSTBM - B cells showing brightfield image, CD19 (red), pSTBM (green) and overlay (top panel) and monocytes with brightfield image, pSTBM (green) and overlay (bottom panel).

Mentions: Next, we determined the binding profile of pSTBM to PBMC by using six colour flow cytometry. We examined the T cell, B cell, NKdim, NKbright and monocyte cell populations of normal pregnant women after incubation with pSTBM for 1 hour. STBM were identified using the trophoblast specific antibody NDOG2. PBMC were stained with antibodies towards CD14, CD3, CD19, CD56 and CD16 to identify T cells, B cells, monocytes, NKdim (CD56+CD16+) and NK bright (CD56brightCD16-) cells, figures 4A. pSTBM binding was detected on small numbers of T and NK cells, and to a greater extent to B cells (40%) and Monocytes (82%), figure 4B. Next we used Image Stream (imaging flow cytometry) and IDEAS analysis software to determine if pSTBM were phagocytosed by the monocytes and B cells. pSTBM were labelled with BODIPY FL maleimide dye and pSTBM incubated with 3×106 PBMC from non-pregnant donors (n = 3). Cells were incubated with pSTBM for 1, 6 or 20 hours then images acquired by Image Stream. Briefly, image stream captures fluorescence microscopy images of cells as the suspension of cells passes through the machine, which enables statistical analysis of pSTBM internalisation on a large number of cells, something which is not possible by conventional confocal microscopy. We found that after one hour of incubation with pSTBM, approximately 60% of B cells and monocytes that had bound pSTBM had phagocytosed the vesicles. Over time, monocytes continued to internalise pSTBM, 90% of monocytes had internalised the pSTBM after 20 hours incubation, whereas no more B cells internalised vesicles, figure 4C. Representative images of B cells and monocytes with internalised or external pSTBM are shown in figure 4D.


The immunomodulatory role of syncytiotrophoblast microvesicles.

Southcombe J, Tannetta D, Redman C, Sargent I - PLoS ONE (2011)

pSTBM bind to monocytes and B cells.106 PBMC from normal pregnant women were incubated with 50 µg/ml pSTBM for one hour at 37°C. Cells were then washed in PBS and stained for antibodies towards CD3, CD14, CD19, CD56 and CD19, and the proportion of cells with bound pSTBM identified with the trophoblast specific marker NDOG-2. Gates were set by staining PBMC with all six antibodies without the addition of pSTBM. NDOG-2 staining of cell populations was also compared to an isotype control (representative staining profile shown in A). Binding of pSTBM to cell populations shown in B (n = 3; mean +/− S.E.M.). C) Image stream analysis of B cell (grey bars) and monocyte (black bars) internalisation of pSTBM (labelled with BODIPY FL Maleimide) with IDEAS software (n = 3; mean +/− S.E.M.; * = p<0.05). D) Representative images of cells with internal or external pSTBM - B cells showing brightfield image, CD19 (red), pSTBM (green) and overlay (top panel) and monocytes with brightfield image, pSTBM (green) and overlay (bottom panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102084&req=5

pone-0020245-g004: pSTBM bind to monocytes and B cells.106 PBMC from normal pregnant women were incubated with 50 µg/ml pSTBM for one hour at 37°C. Cells were then washed in PBS and stained for antibodies towards CD3, CD14, CD19, CD56 and CD19, and the proportion of cells with bound pSTBM identified with the trophoblast specific marker NDOG-2. Gates were set by staining PBMC with all six antibodies without the addition of pSTBM. NDOG-2 staining of cell populations was also compared to an isotype control (representative staining profile shown in A). Binding of pSTBM to cell populations shown in B (n = 3; mean +/− S.E.M.). C) Image stream analysis of B cell (grey bars) and monocyte (black bars) internalisation of pSTBM (labelled with BODIPY FL Maleimide) with IDEAS software (n = 3; mean +/− S.E.M.; * = p<0.05). D) Representative images of cells with internal or external pSTBM - B cells showing brightfield image, CD19 (red), pSTBM (green) and overlay (top panel) and monocytes with brightfield image, pSTBM (green) and overlay (bottom panel).
Mentions: Next, we determined the binding profile of pSTBM to PBMC by using six colour flow cytometry. We examined the T cell, B cell, NKdim, NKbright and monocyte cell populations of normal pregnant women after incubation with pSTBM for 1 hour. STBM were identified using the trophoblast specific antibody NDOG2. PBMC were stained with antibodies towards CD14, CD3, CD19, CD56 and CD16 to identify T cells, B cells, monocytes, NKdim (CD56+CD16+) and NK bright (CD56brightCD16-) cells, figures 4A. pSTBM binding was detected on small numbers of T and NK cells, and to a greater extent to B cells (40%) and Monocytes (82%), figure 4B. Next we used Image Stream (imaging flow cytometry) and IDEAS analysis software to determine if pSTBM were phagocytosed by the monocytes and B cells. pSTBM were labelled with BODIPY FL maleimide dye and pSTBM incubated with 3×106 PBMC from non-pregnant donors (n = 3). Cells were incubated with pSTBM for 1, 6 or 20 hours then images acquired by Image Stream. Briefly, image stream captures fluorescence microscopy images of cells as the suspension of cells passes through the machine, which enables statistical analysis of pSTBM internalisation on a large number of cells, something which is not possible by conventional confocal microscopy. We found that after one hour of incubation with pSTBM, approximately 60% of B cells and monocytes that had bound pSTBM had phagocytosed the vesicles. Over time, monocytes continued to internalise pSTBM, 90% of monocytes had internalised the pSTBM after 20 hours incubation, whereas no more B cells internalised vesicles, figure 4C. Representative images of B cells and monocytes with internalised or external pSTBM are shown in figure 4D.

Bottom Line: Immune adaptation is a critical component of successful pregnancy.Other cytokines are down-modulated, such as IP-10 which is associated with 'type 1' immunity.We also observed that PBMC from third trimester normal pregnant women produce more TNFα and IL-6 in response to STBM than PBMC from non-pregnant women, confirming that maternal immune cells are primed by pregnancy, possibly through their interaction with STBM.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom. jen.southcombe@obs-gyn.ox.ac.uk

ABSTRACT
Immune adaptation is a critical component of successful pregnancy. Of primary importance is the modification of cytokine production upon immune activation. With the discovery that normal pregnancy itself is a pro-inflammatory state, it was recognised that the classical Th1/Th2 cytokine paradigm, with a shift towards 'type 2' cytokine production (important for antibody production), and away from 'type 1' immunity (associated with cell mediated immunity and graft rejection), is too simplistic. It is now generally agreed that both arms of cytokine immunity are activated, but with a bias towards 'type 2' immunity. Many factors are released from the placenta that can influence the maternal cytokine balance. Here we focus on syncytiotrophoblast microvesicles (STBM) which are shed from the placenta into the maternal circulation. We show that STBM can bind to monocytes and B cells and induce cytokine release (TNFα, MIP-1α, IL-1α, IL-1β, IL-6, IL-8). Other cytokines are down-modulated, such as IP-10 which is associated with 'type 1' immunity. Therefore STBM may aid the 'type 2' skewed nature of normal pregnancy. We also observed that PBMC from third trimester normal pregnant women produce more TNFα and IL-6 in response to STBM than PBMC from non-pregnant women, confirming that maternal immune cells are primed by pregnancy, possibly through their interaction with STBM.

Show MeSH