Influence of reproductive hormones on the induction of CD4(+)CD25 (bright)Foxp (3+) regulatory T cells.
In the context of immunology, pregnancy is a phys
iological state of tolerance of the immune system of
the maternal body to a genetically foreign fetus [1].
This temporary state of the maternal immune system
stems from an increased expansion of regulatory T
cells (Treg cells), which are characterized by the ability
to suppress proliferation of effector cells by direct con
tact or excreting regulatory cytokines [1]. Regulatory
T cells (CD4+CD25brightFoxp3+) embrace adaptive, or
induced Treg cells, which develop at the periphery
from CD4+CD25– T cells, along with natural Treg
cells, which mature in the thymus [1, 2]. Transcription
of the foxp3 gene in CD4+CD25– T cells under the
influence of certain cytokines and hormones results in
them acquiring suppressive activity [1, 2]. During
pregnancy, the expansion of the Treg pool takes place
normally; a decrease in the amount of these cells is
accompanied by gestoses and spontaneous abortions
[1]. It is known that the major reproductive hor
mones—chorionic gonadotropin (CG), estradiol
(E2), estriol (E3), and progesterone (Pr)—play an
important role in the regulation of immunity during
pregnancy [3, 4]. Moreover, recent research has shown
that the peptide hormones leptin and ghrelin, recipro
cal regulators of energy homeostasis, are critical mod
ulators of the reproductive function and immune reac
tions during pregnancy [3, 4]. Therefore, it is impor
tant to clarify the role of these hormones in the
induction of Treg cells during pregnancy.
The goal of this study was to analyze the influence
of CG, E3, E2, Pr, leptin, and ghrelin at the concentra
tions modeling the levels of these hormones during
pregnancy on the induction of Treg cells in peripheral
blood in vitro.
The results show that CG, E3, E2, Pr, leptin, and
ghrelin play a significant role in the induction of adap
tive Treg cells. Changes in the amount of Treg cells in
peripheral blood during pregnancy may stem from the
effect of these hormones, and hormone ensemble is
one of probable mechanisms of modulation of periph
eral tolerance during pregnancy.
We used a fractionated suspension of mononuclear
cells of peripheral blood of healthy women of repro
ductive age (n = 14) taken during the early follicular
phase of the menstrual cycle (days 4 to 7). The suspen
sion of mononuclear cells was obtained by density gra
dient centrifugation using ficoll–verografin as a
medium (1.077 g/cm3). Isolated cells were incubated
in a complete medium (medium 199 supplemented
with 10 mM HEPES, 2 mM Lglutamine, 100 μg/ml
gentamicin, and 10% bovine serum) for one day at
37°C with hormones and 5% CO2. Hormones were
added into cultures at the concentrations modeling
their levels in peripheral blood in the corresponding
trimesters of pregnancy (table). In control samples,
saline used for dissolution of hormones was added
instead of hormones. Tcell viability, assessed by the
eosin test after oneday incubation period with hor
mones, was 95–98%.After incubation with hormones, the lymphocyte
phenotype was assessed using a flow cytometer (Bec
ton Dickinson, United States). Surface and intracel
lular markers were stained as recommended by the
manufacturer of monoclonal antibodies (BioLegend,
United States). When estimating the results, no fewer
than 100 000 cells were examined. We used appropri
ate isotype controls to check for nonspecific binding
and set the lymphocyte gate with negative staining.
Because the expression of the transcription factor
Foxp3 is the main marker of Treg cells [9], the amount
of Treg cells was estimated as the percentage of
CD4+CD25bright Foxp3+ cells (antihuman Foxp3
Alexa Fluor® 488, 206D) in the CD4+CD25bright gate
CD25, BC96) (Fig. 1).
Statistical treatment was performed using paired
Student’s t test.
As can be seen in Fig. 2, a oneday incubation of
peripheral blood mononuclear cells with CG, E2, and
E3 at the concentrations comparable with the hor
mone levels during the corresponding trimesters of
pregnancy (table) significantly increased the percent
age of CD4+CD25bright Foxp3+ cells in the culture.
Ghrelin had a similar effect; however, the Tregacti
vating effect of ghrelin did not manifest itself until the
concentrations comparable with those observed dur
ing the third trimester of pregnancy were reached.
Leptin at the concentration typical of the first trimes
ter of pregnancy, on the other hand, significantly
decreased the percentage of CD4+CD25bright Foxp3+
T cells. Progestorone was not found to have any statis
tically significant effect on the Foxp3 expression in
CD4+CD25bright T cells.
Analyzing the results, we ought to point out that,
normally, there are natural and adaptive Treg cells in
human peripheral blood [1, 2, 9]. During pregnancy,
the proliferation of natural Treg cells and induction of
adaptive Treg cells (Foxp3 expression) [1, 2]. In our
study, only the issue of whether the hormones could
influence the Foxp3 expression was addressed, so the
result was estimated after a oneday incubation period
with hormones. Proliferation, however, is generally
estimated on the fourth to seventh days of incubation
[10]. Therefore, the stimulating effect of the hormones
can be interpreted as the ability to increase the per
centage of adaptive Treg cells. At the same time, that
leptin decreases their percentage, in our opinion,
should be interpreted as a direct influence of leptin on
constitutive Treg cells that are present in the culture
from the outset.
It is noteworthy that all of the mentioned hor
mones that cause an increase in the percentage of
adaptive Treg cells in vitro (including estrogens) use a
cAMPbased mechanism of signal transduction [4,
11, 12], which is one of the mechanisms of increasing
the Foxp3 expression and inducing the adaptive Treg
cells [11, 12]. That Pr has no influence on the Foxp3
expression in CD4+CD25bright T cells of peripheral
blood seems to be explained by that, as opposed to
estrogens, nongenomic effects of Pr are not associ
ated with the cAMP level [4].
Considering that leptin interacting with Treg cells
activates the mammalian target of rapamycin (mTOR)
signaling pathway by reducing their proliferative activ
ity and causing anergy [13], this mechanism may
account for the suppressive action of leptin on consti
tutive Treg cells. It is possible that this effect of leptin
is the reason of a higher amount of spontaneous abor
tions in overweight women [3].
Thus, suppressing constitutive Treg cells, leptin
“neutralizes” the Tregactivating effect of CG and
estrogens during the first half of pregnancy. In the third
trimester of pregnancy, its effect is overrun because
ghrelin adds its adaptive Tregstimulating effect to that
of CG and estrogens. Fine hormonal modulation of
the induction of adaptive and constitutive Treg cells
appears to underlie and/or be one of the mechanisms
of development of the peripheral tolerance to fetus
during pregnancy.
ACKNOWLEDGMENTS
The study was supported by the Program for Basic
Research of the Presidium of the Russian Academy of
Sciences “Molecular and Cell Biology.”
