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. 2011 May;164(2):180-92.
doi: 10.1111/j.1365-2249.2011.04330.x. Epub 2011 Feb 24.

Incomplete activation of peripheral blood dendritic cells during healthy human pregnancy

S Della Bella  1 , S Giannelli, V Cozzi, V Signorelli, M Cappelletti, I Cetin, M L Villa
Affiliations

Incomplete activation of peripheral blood dendritic cells during healthy human pregnancy

S Della Bella et al. Clin Exp Immunol. 2011 May.

Abstract

Successful pregnancy relies on the adaptation of immune responses that allow the fetus to grow and develop in the uterus despite being recognized by maternal immune cells. Dendritic cells (DCs) are central to the control of immune tolerance, and their state of activation at the maternal-decidual interface is critical to the feto-maternal immunological equilibrium. So far, the involvement of circulating DCs has been investigated poorly. Therefore, in this study we investigated whether, during healthy human pregnancy, peripheral blood DCs (PBDCs) undergo changes that may be relevant to the adaptation of maternal immune responses that allow fetal tolerance. In a cross-sectional study, we analysed PBDCs by six-colour flow cytometry on whole blood samples from 47 women during healthy pregnancy progression and 24 non-pregnant controls. We demonstrated that both myeloid and plasmacytoid PBDCs undergo a state of incomplete activation, more evident in the third trimester, characterized by increased expression of co-stimulatory molecules and cytokine production but lacking human leucocyte antigen (HLA)-DR up-regulation. To investigate the contribution of soluble circulating factors to this phenomenon, we also performed culture experiments showing that sera from pregnant women added to control DCs conditioned a similar incomplete activation that was associated with reduced DC allostimulatory capacity, supporting the in vivo relevance of our findings. We also obtained evidence that the glycoprotein hormone activin-A may contribute to DC incomplete activation. We suggest that the changes of PBDCs occurring during late pregnancy may aid the comprehension of the immune mechanisms operated by the maternal immune system to maintain fetal tolerance.

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Figures

Fig. 1
Fig. 1
Representative flow cytometric analysis showing the gating strategy used to identify peripheral blood dendritic cell (PBDC) subsets in whole blood samples and the immunophenotype of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) from a non-pregnant woman compared with a woman in the third trimester of pregnancy. (a) Mononuclear cells were gated by their characteristic forward-scatter (FSc) and side-scatter (SSc) excluding death cells, granulocytes and debris. (b) Gated on mononuclear cells, PBDCs were identified as cells positive for HLA-DR and negative for a cocktail of lineage markers. (c) Gated on PBDCs, mDCs were defined as CD11c-positive and CD123-negative cells; pDCs were defined as CD123-positive and CD11c-negative cells. (d) Gated on mDCs or pDCs, the expression of CD80, CD86, CD40 or CD83 (solid lines) by each DC subset was compared with unstained controls (dashed lines). Analysis performed by FlowJo software.
Fig. 2
Fig. 2
Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) are activated along healthy pregnancy progression. (a) The levels of the co-stimulatory molecules CD80 (left axis), CD86 and CD40 and the maturation marker CD83 (right axis) on mDCs and pDCs analysed directly in whole blood samples were expressed as mean fluorescence intensity (MFI) and compared among non-pregnant women and women during healthy pregnancy. (b) The frequency of activated mDCs and pDCs, defined as cells with levels of CD40 and/or CD86 outlining the gate of constitutive expression, was higher in women in the third trimester of pregnancy than in controls and was compared between non-pregnant women and women in the third trimester of pregnancy. (c) The levels of HLA-DR molecules on mDCs and pDCs were expressed as MFI and compared among non-pregnant women and women during healthy pregnancy. (d) The levels of HLA-DR molecules on either activated or non-activated mDCs were compared between non-pregnant women and women in the third trimester of pregnancy. Data shown as mean ± standard error of the mean from 24 non-pregnant and seven, 16 and 24 pregnant women in the first, second and third trimesters, respectively. *P < 0·05, **P < 0·01 and ***P < 0·001 compared with non-pregnant women.
Fig. 3
Fig. 3
Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) from pregnant women express increased amounts of inflammatory and regulatory cytokines in basal conditions, with a prevalent proinflammatory cytokine pattern. The levels of unstimulated and Toll-like receptor (TLR)-stimulated cytokine production by (a) mDCs and (c) pDCs were expressed as mean fluorescence intensity (MFI) and compared among non-pregnant women and women along healthy pregnancy. (b,d) The ratio between the unstimulated MFI values of the indicated cytokines is shown. Data shown as mean ± standard error of the mean from 24 non-pregnant and seven, 11 and 19 pregnant women in the first, second and third trimesters, respectively. *P < 0·05, **P < 0·01 and ***P < 0·001 compared with non-pregnant women. LPS: lipopolysaccharide; IQ: imiquimod.
Fig. 4
Fig. 4
Plasma from pregnant women inhibit the up-regulation of HLA-DR molecules during activation of control myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). (a) Kinetics of the expression of CD80, CD86, CD40, CD83 and HLA-DR molecules on mDCs and pDCs upon incubation of peripheral blood mononuclear cells (PBMCs) with or without plasma from women in the third trimester of pregnancy and non-pregnant women. A culture-induced activation, observed in the absence of plasma, was evident after 5 h incubation. One representative of three independent experiments is shown. (b) Comparison of the effects of 5 h incubation with plasma from either non-pregnant women or pregnant women in the third trimester of pregnancy on the expression of CD80, CD86, CD40, CD83 and HLA-DR on culture-activated mDCs and pDCs from non-pregnant controls. At this time plasma from pregnant, but not from non-pregnant, women inhibited HLA-DR up-regulation. Plasma from pregnant and non-pregnant women did not affect differently the expression of the other molecules. Data shown as mean ± standard error of the mean from 24 experiments. ***P < 0·001 compared with plasma from non-pregnant women.
Fig. 5
Fig. 5
Recombinant human activin-A (ActA) inhibits the up-regulation of HLA-DR molecules during activation of control myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). (a) Representative dose–response curve showing the inhibitory effect of ActA on culture-induced HLA-DR up-regulation on mDCs and pDCs. Control peripheral blood mononuclear cells (PBMCs) were incubated for 5 h in the presence of control plasma plus ActA at the indicated concentrations. One representative of three independent experiments is shown. (b) Comparison of the effects of 5 h incubation with plasma from non-pregnant women with or without 100 ng/ml ActA on the expression of CD80, CD86, CD40, CD83 and HLA-DR on culture-activated mDCs and pDCs from non-pregnant controls. The addition of ActA inhibited HLA-DR up-regulation but did not affect differently the expression of the other molecules. Data shown as mean ± standard error of the mean from seven experiments. *P < 0·05 compared with control plasma without ActA.
Fig. 6
Fig. 6
The inhibition of HLA-DR up-regulation promoted by sera from pregnant women and human activin-A (ActA) is paralleled by lower allostimulatory activity. Myeloid DCs (mDCs) obtained from non-pregnant controls were stimulated with monocyte-conditioned medium (MCM) for 24 h in the presence of either sera from non-pregnant women, or sera from pregnant women in the third trimester or sera from non-pregnant women plus 100 ng/ml ActA; their effects were compared on (a) the expression of CD80, CD86, CD40, CD83 and HLA-DR and (b) the allostimulatory capacity of monocyte-derived DCs (moDCs). Incubation of moDCs with MCM up-regulated significantly the expression of all the activation markers (P < 0·05) and the allostimulatory capacity of moDCs (P < 0·05). Sera from pregnant women and sera from non-pregnant women plus ActA inhibited the MCM-induced HLA-DR up-regulation and allostimulatory activity of moDCs. The proliferation of alloreactive lymphocytes was determined by flow cytometry as percentage of divided carboxyfluorescein succinimidyl ester (CFSE)low/CD4+/CD3+ viable T cells. Data shown as mean ± standard error of the mean from seven experiments. *P < 0·05 compared with MCM stimulation performed in sera from non-pregnant women.
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