Alpha-defensins 1-3 release by dendritic cells is reduced by estrogen

Abstract

Background: During pregnancy the immune system of the mother must protect any activation that may negatively affect the fetus. Changes in susceptibility to infection as well as resolution of some autoimmune disorders represent empirical evidence for pregnancy related alterations in immunity. Sex hormones reach extremely high levels during pregnancy and have been shown to have direct effects on many immune functions including the antiviral response of dendritic cells. Among the immunologically active proteins secreted by monocyte derived DCs (MDDC) are the alpha-defensins 1-3. This family of cationic antimicrobial peptides has a broad spectrum of microbicidal activity and has also been shown to link innate to adaptive immunity by attracting T cells and immature DCs, which are essential for initiating and polarizing the immune response.

Methods: We compare culture-generated monocyte derived DCs (MDDCs) with directly isolated myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) and measure their alpha-defensins 1-3 secretion by ELISA both, in basal situations and after hormone (E2 or PG) treatments. Moreover, using a cohort of pregnant women we isolated mDCs from blood and also measure the levels of these anti-microbial peptides along pregnancy.

Results: We show that mDCs and pDCs constitutively produce alpha-defensins 1-3 and at much higher levels than MDDCs. Alpha-defensins 1-3 production from mDCs and MDDCs but not pDCs is inhibited by E2. PG does not affect alpha-defensins 1-3 in any of the populations. Moreover, alpha-defensins 1-3 production by mDCs was reduced in the later stages of pregnancy in 40% of the patients.

Conclusions: Here, we demonstrate that mDCs and pDCs secrete alpha-defensins 1-3 and present a novel effect of E2 on the secretion of alpha-defensins 1-3 by dendritic cells.

Figure 1

MDDCs secrete α-defensins 1-3: E2 and PG effect. A) ELISA quantification of a-defensins 1-3 secreted by MDDCs, mDCs and pDCs (left panel) or PBMCs and monocytes (right panel) in the supernatant after 6 h of culture. Data are presented as mean plus or minus SD from 3 independent experiments.*P < 0.01 and *P < 0.001 versus MDDCs levels. Data are presented in a log scale. Levels of α-defensins 1-3 measured by ELISA in: (B) MDDCs treated for 24 h with 10, 1 and 0.1 ug/ml of E2 (left panel) or PG (right panel). (C) MDDCs pretreated for 24 h with E2 or PG (10 ug/ml) before NDV-B1 infection for 10 h. Data are presented as mean plus or minus SD from 3 independent experiments. *P < 0.05versus MDDCs levels.

Figure 2

mDCs and pDCs secrete α-defensins 1-3: E2 and PG effect. Levels of α-defensins 1-3 measured by ELISA in: (A) mDCs treated for 24 h with 10, 1 and 0.1 ug/ml of E2 (left panel) or PG (right panel). (B) pDCs treated with 10 ug/ml of E2 for 8 h. Data are presented as mean plus or minus SD from 3 independent experiments.*P < 0.05 versus Mock treated DCs.

Figure 3

ICI 182,780 revert E2-inhibitory effect in the release of a-defensins 1-3 by DCs. CD14⁺ cells isolated from fresh blood were cultured for 5 days in the presence of IL-4 and GM-CSF to differentiate into DCs. The cells were treated with 10, 100 and 1000 nM of ICI 182,780 for the last 3 days of the differentiation process. (A) Levels of ERa measured by RT-PCR in DCs treated or not with ICI 182,780 in the presence of E2 (10 ug/ml) for 24 h. The data presented as mean ± SD are representative of three independent experiments. (B) Percentage of reduction induces by E2-treatment in the levels of a-defensins 1-3 secreted by MDDCs and mDCs pre-treated or not for 3 days (MDDCs) or 24 h (mDCs) with 1000 nM of ICI 182,780 or DMSO, prior to 24 h of Estrogen or Mock treatment.. The data presented as mean ± SD are representative of three independent experiments. (C) Percentage of CD11c+CD14+ cells measured by FACS after 5 days of differentiation in the presence of ICI 182,780 or DMSO during the last 3 days of the process. These data are representative of three independent experiments.

Figure 4

Release of a-defensins 1-3 by mDC during pregnancy. (A) Levels of Estradiol measured by ELISA in serum samples from pregnant women (n = 18) during first and third trimester. *p < 0.001 vs first trimester. (B) Levels of a-defensins 1-3 secreted by mDC from all the individual patients (n = 18) at first and third trimester. The data are presented as mean ± SD.