J Huazhong Univ Sci Technol [Med Sci]

33(5):730-734,2013
730 DOI 10.1007/s11596-013-1188-6
J Huazhong Univ Sci Technol [Med Sci] 33(5):2013

Role of Progesterone in TLR4-MyD88-dependent Signaling Pathway

in Pre-eclampsia
Ying ZHU (朱 颖), Min WU (吴 敏), Chao-ying WU (吴超英), Ge-qing XIA (夏革清)#
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430022, China

© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2013

Summary: The role of progesterone in the Toll-like receptor 4 (TLR4)-MyD88-dependent signaling

pathway in pre-eclampsia was studied. Peripheral blood mononuclear cells (PBMCs) from
pre-eclampsia (PE) patients were subjected to primary culture, and stimulated with different concentra-
tions of progesterone (0, 10-8, 10-6, and 10-4 mol/L). The mRNA expression of TLR4, MyD88 and nu-
clear factor-kappaB (NF-κB) was detected by using real-time PCR. The Ikappa-B protein expression
was detected by using Western blotting. The expression of tumor necrosis factor-α (TNF-α) and inter-
leukin-6 (IL-6) in the supernatant was determined by using ELISA. With the concentrations of proges-
terone increasing, the mRNA expression levels of TLR4, MyD88 and NF-κB in 2-∆∆CT value were sig-
nificantly decreased, and the IkappaB protein expression levels were significantly increased. The TNF-α
and IL-6 expression showed a downward trend when the progesterone concentration increased, and
there were significant differences among all of the groups (P<0.05). It was suggested that progesterone
can inhibit the TLR4-MyD88-dependent signaling pathway in PE significantly and benefit for the preg-
nancy.
Key words: progesterone; pre-eclampsia; Toll-like receptor 4; tumor necrosis factor-α; nuclear fac-
tor-κB; Ikappa-B; interleukin-6


Pre-eclampsia (PE) remains to be the leading cause
of both maternal and fetal morbidity and mortality. So
far, the pathogenesis of PE is not clear, which can not be
explained by monism. It is generally believed that PE is
caused by generous inflammatory factors which lead to
shallow placental implantation, vascular endothelial
injury, multiple organ damages, vasospasm and clinical
symptoms under the impact of polygene and envir-
onment[1]. Many researches have reported that the infla-
mmatory response induced by Toll-like receptor 4 (TLR4)
signaling pathway was one of the most important
pathogenesis of pre-eclampsia[2, 3]. Toll-like receptors
(TLRs) are a kind of pattern recognition receptors. Now,
there are at least 12 members of TLRs family, which can
recognize different legends. TLR4 belongs to TLRs
family and is expressed wildly at the maternal-fetal inter-
face. Following recognizing bacterial lipopolysaccha-
rides (LPS) and heat shock protein-60, TLR4 can
mediate a kind of chronic immune response character-
ized by generous inflammatory factors production[4]
through MyD88-dependent and -independent signaling
pathways[5, 6]. In this study, we focus on the MyD88-
dependent signaling pathway. After recognizing the
ligands, TLR4 recruits MyD88 and activates a series of
kinases downstream then the nuclear factor-κappaB
(NF-κB) is transposed into the nucelus, accordingly
initiating generous cytokines synthesis and releasing,
Ying ZHU, E-mail: caozhuying@163.com
#
Corresponding author, E-mail: xgq67@yahoo.com.cn
such as interleukin (IL)-1, IL-6 and tumor necrosis
factor-α (TNF-α)[7] , which mediates acute inflammatory
responses to kill the invading pathogenic microor-
ganisms directly. TLR4 activation has dual effects: prot-
ective effects through the innate immune response and
adaptive immunity, or injury effects through the persis-
tent inflammatory response[8]. Progesterone is one of the
most important hormones in the pregnancy. At the same
time, progesterone participates in immune response.
Some reports showed that progesterone could inhibit the
TLR4 signaling pathway in central nervous system disea-
ses such as brain damage[9]. Up to now, the role of proge-
sterone in PE was still unknown. In this study, the
peripheral blood mononuclear cells (PBMCs) in patients
with PE were treated with progesterone to explore the
role of progesterone in the TLR-4-MyD88-dependent
signaling pathway in PE.
1 MATERIALS AND METHODS
1.1 Peripheral Venous Blood in PE Patients
PE patients without other complications, aged
(28.0±5.0) years and gestational age of (34.1±4.1) weeks,
were recruited from Department of Obstetrics, Union
Hospital, Tongji Medical College, Huazhong University
of Science and Technology (China) from March, 2010 to
July, 2011. The study obtained the Medical Science
Ethics Committee’s license, and the patients signed info-
rmed consents. The diagnostic criteria of PE were refer-
red to the Williams Obstetrics (23rd edition). A total of
J Huazhong Univ Sci Technol [Med Sci] 33(5):2013
10 mL peripheral venous blood was taken in aseptic
condition, then anticoagulated with heparin (for cells
total protein extraction) or EDTA-Na2 (for cells RNA
extraction).
1.2 Reagents and Antibodies
Ikappa-B antibody was purchased from Santa Co.
(USA). TRIZOL kits were purchased from GIBCO
(USA). The primers were synthetized by Invitrogen Co.
(USA). RNA reverse transcription reagents and real-time
PCR reaction reagents were purchased from Takara
(Japan). Lymphocyte separation medium Ficoll-hypaque
(specific gravity: 1.077±0.002) was purchased from GE
(USA). TNF-α and IL-6 enzyme-linked immunosorbent
assay (ELISA) kits were purchased from Neobioscience
Co. (USA). BCA protein Assay kits were purchased
from Pierce Chemical Co. (USA). BeyoECL plus was
purchased from Beyotime (USA).
1.3 Preparation, Isolation, and Culture of PBMCs
PBMCs were prepared from buffer coats by
Ficoll-hypaque density gradient centrifugation (purity
>95%). PBMCs were resuspended in RP-1640 supple-
mented with 10% fetal calf serum, 1.5 mmol/L L-
glutamine, 100 U/mL penicilin, and 100 µg/mL strept-
omycin. Counts and viability of cells were determined by
0.2% trypan blue. Acquired cells (viability>95% ) were
inoculated into a 6-well culture plate at a density of
4.5×106/mL, cultured at 37ºC in an incubator with 5%
CO2 for 24 h. After 24-h culture the PBMCs developed
into floating growth condition.
1.4 Progesterone Treatment
Progesterone was resupended in ethanol to make a 1
mmol/L stock solution. Prior to experiment,
Progesterone was diluted to different concentrations (10-4,
10-6, 10-8 mol/L) with serum-free RP-1640, and the
control group was serum-free RP-1640. PBMCs were
resuspended and inoculated into a 6-well culture plate at
a density of 4.5×106/mL, cultured with different
concentrations of Progesterone at 37ºC in an incubator
with 5% CO2 for 6 h.
1.5 Detection of TLR4, MyD88 and NF-κB mRNA
Expression by Real-time PCR
The TLR4, MyD88 and NF-κB mRNA expression
levels were detected by using real-time PCR. Their
primers and real-time PCR conditions were as follows:
TLR4 (forward primer: 5'-AGTGTGTGTGTCCGCA-
TGAT-3'; reverse primer: 5'-CCACTTGGGGTCTAAG-
AACG-3'), beforehand degeneration at 95ºC for 30 s,
degeneration at 95ºC for 5 s and renaturation at 58ºC for
30 s, 40 cycles; MyD88 (forward primer: 5′-CGCCGG-
ATGGTGGTGGTTGT-3′; reverse primer: 5′-TGAGG-
TCGCAGACAGTGATGAACC-3′), beforehand degen-
eration at 95ºC for 30 s, degeneration at 95ºC for 5 s and
renaturation at 56ºC for 30 s, 40 cycles; NF-κB (forward
primer: 5'-CCACAAGCAAGAAGCTGAAG-3'; reverse
primer: 5'-AGATACTATCTGTAAGTGAACC-3'), bef-
orehand degeneration at 95ºC for 30 s, degeneration at
95ºC for 5 s and renaturation at 56ºC for 30 s, 40 cycles;
β-actin (forward primer: 5'-TCCTGTGGCATCCACG-
AAACT-3'; reverse primer: 5'-GAAGCATTTGCGGTG-
GACGAT-3').
1.6 Detection of IκB-α by Western Blotting
After the treatment with progesterone, cells were
washed twice with cold PBS, then lysed by adding
731
ice-cold RIPA lysis buffer with PMSF (100:1). After
shaking on ice for 30 min, the lysates were centrifuged at
12 000 g for 10 min at 4ºC. The supernatants were stored
at –80ºC until use. Western blotting was performed.
Briefly, proteins were resolved by SDS-polyacrylamide
gel electrophoresis under reducing conditions in 10%
gels and then electro-transferred to nitrocellulose
membranes. After blocking in TBST containing 5% fat-
free milk, each blot was incubated with IκB-α antibody
for 2 h, and subsequently with HRP-conjugated seco-
ndary antibody for 1 h. Then for exposure (10 s, 30 s, 1
min, 5 min, and up to 1 h) we used BeyoECL plus.
Bound antibodies were detected by a chemiluminescent
detection system. The protein concentrations were meas-
ured by BCA protein Assay kit.
1.7 Determination of TNF-α and IL-6 by ELISA
After the treatment with progesterone, the super-
natants were harvested, and the concentrations of IL-6
and TNF-α were measured by using ELISA.
1.8 Statistical Analysis
Data were expressed as ±s. SPSS 11.0 was used
for χ2 test. The significance was designed as α=0.05.
2 RESULTS
2.1 Effects of Progesterone on TLR4 mRNA in PE
The relative expression levels (2-∆∆CT) of TLR4
mRNA in the control group, 10-8 mol/L progesterone
group, 10-6 mol/L progesterone group, and 10-4 mol/L
progesterone group were 1.455±0.102, 1.055±0.063,
0.730±0.012 and 0.514±0.044 respectively, showing
significant differences among the groups (P<0.05),
which suggested that the expression levels of TLR4
mRNA were significantly reduced with progesterone
concentrations growing (fig. 1).
Fig. 1 The relative expression of TLR4 mRNA in each group
*
P<0.05 among the groups
2.2 Effects of Progesterone on MyD88 mRNA
Expression in PE
The relative expression levels (2-∆∆CT) of MyD88
mRNA in in the control group, 10-8 mol/L progesterone
group, 10-6 mol/L progesterone group, and 10-4 mol/L
progesterone group were 1.052±0.269, 0.705±0.037,
0.294±0.019 and 0.097±0.011 respectively, showing sig-
nificant differences among the groups (P<0.05), which
suggested that the expression levels of MyD88 mRNA
were significantly reduced with progesterone concentra-
 732
tions growing (fig. 2).
Fig. 2 The relative expression levels of MyD88 mRNA in each
group
*
P<0.05 among the groups
2.3 Effects of Progesterone on NF-κB mRNA in PE
The relative expression levels (2-∆∆CT) of NF-κB
mRNA in the control group, 10-8 mol/L progesterone
group, 10-6 mol/L progesterone group, and 10-4 mol/L
progesterone group were 2.341±0.326, 1.045±0.393,
0.283±0.028 and 0.116±0.013 respectively, showing sig-
nificant differences among the groups (P<0.05), which
suggested that the expression of NF-κB mRNA was sig-
nificantly reduced with progesterone concentration
growing (fig. 3).
Fig. 3 The relative expression levels of NF-κB mRNA in each
group
*
P<0.05, **P<0.01 among the groups
2.4 Effects of Progesterone on IκB-α in PE
The expression levels (absorbance, A) of IκB-α pro-
tein in the control group, 10-8 mol/L progesterone group,
10-6 mol/L progesterone group, and 10-4 mol/L progester-
one group were 0.187±0.015, 0.777±0.047, 1.420±0.092
and 2.300±0.243 respectively, showing significant differ-
ences among the groups (P<0.05), which suggested that
the expression of IκB-α protein was significantly increased
with progesterone concentration growing (fig. 4). IκB-α is
the inhibitor of NF-κB. As a transcription factor, NF-κB
can activate transcription of specific target genes follow-
ing IκB-α phosphorylation and degradation[10, 11]. In our
study, the expression of IκB-α protein increased with pro-
gesterone concentration growing, which suggested that the
J Huazhong Univ Sci Technol [Med Sci] 33(5):2013
activation of NF-κB was decreased with the increase of
progesterone concentration.
Fig. 4 The expression of IκB-α protein in each group
*
P<0.05 among the groups
2.5 Effects of Progesterone on TNF-α in PE
The expression levels of TNF-α in the control group,
10-8 mol/L progesterone group, 10-6 mol/L progesterone
group, and 10-4 mol/L progesterone group were
5.733±0.208 pg/mL, 3.100±0.201 pg/mL, 2.101±0.098
pg/mL and 1.100±0.057 pg/mL respectively, showing
significant differences among the groups (P<0.05),
which suggested that the expression levels of TNF-α
were significantly reduced with progesterone concentra-
tions growing (fig. 5).
Fig. 5 The expression levels of TNF-α in each group
*
P<0.05 among the groups
2.6 Effects of Progesterone on IL-6 in PE
The expression levels of IL-6 in the control group,
10-8 mol/L progesterone group, 10-6 mol/L progesterone
group, and 10-4 mol/L progesterone group were 215.80±
34.67 pg/mL, 97.84±2.57 pg/mL, 67.74±0.94 pg/mL and
30.48±0.67 pg/mL respectively, showing significant dif-
ferences among the groups (P<0.05), which suggested
that the expression levels of IL-6 were significantly re-
duced with progesterone concentrations growing (fig. 6).
J Huazhong Univ Sci Technol [Med Sci] 33(5):2013
Fig. 6 The expression levels of IL-6 in each group
*
P<0.05, **P<0.01 among the groups
3 DISCUSSION
TLR4, one of the first discovered mammalian TLRs,
can recognize exogenous ligand such as LPS and en-
dogenous ligand such as heat shock protein-60. TLR4 is
widely expressed in the maternal-fetal interface. TLR4
can activate NF-κB through MyD88, resulting in im-
mune inflammatory response characterized by release of
generous cytokines such as IL-6, IL-2, TNF-α[12, 13]. In
1994, Faas found that the TNF-α produced by the
mononuclear cells was significantly increased in the
animal PE model which was established with the low
doses of LPS, suggesting that the cytokines released by
the monocytes and macrophages were closely related to
the PE[14, 15]. Abrahams reported that the inflammatory
chemotactic factors produced by trophoblasts increased,
generous mononuclear cells and neutrophils gathered to
the maternal-fetal interface after LPS binding to TLR4,
then the balance between trophoblasts and the immune
cells was broken up, accordingly the role of immune
cells in pregnancy changed, which resulted in the disor-
ders of the vascular endothelial cells and maternal-fetal
circulation. These alterations were the most important
pathophysiological changes in PE[3]. Similarly, Kim re-
ported that the expression of TLR4 protein in tro-
phoblasts and placenta of PE patients was higher than
that in the normal pregnant females, which suggested
that the trophoblasts could recognize the “danger sig-
nals” through TLR4, then the maternal cytokine envi-
ronment balance was broken up, which resulted in PE[2].
Our earlier studies also showed that the TLR4 expression
in the umbilical cord blood mononuclear cells of PE
pregnant women was higher than that in the normal
pregnant women[16]. These studies suggest that TLR4
signaling pathway may play an important role in PE.
Progesterone is an important immune response
modulators in pregnancy, which has immunostimulating
or immunosuppressive effects dependent on different
concentrations and treatment durations[17, 18]. Many stud-
ies about progesterone immune mechanism focus on the
TLR4 signaling pathway in the recent years. Some stud-
ies showed that the expression of TLR4, NF-κB, IL-6
and TNF-α was significantly increased, and inflamma-
tory injury mediated by TLR4 played an important role
in nervous system diseases such as subarachnoid hemor-
rhage (SAH), meanwhile progesterone could inhibit the
TLR4 signaling pathway and benefit for the SAH pa-
tients [19]. One study reported that PE symptoms were
733
alleviated after the progesterone treatment, and proges-
terone could inhibit the macrophages infected by
Leishmania donovani to produce IL-6, NO and other
cytokines through the glucocorticoid receptors and TLR4
signaling pathway[20].
Up to now, we have no idea about the role of pro-
gesterone in the TLR4 signaling pathway in PE.
In this study, PBMCs were isolated, and treated
with different concentrations of progesterone. The ex-
pression of TLR4 mRNA, MyD88 mRNA, NF-κB
mRNA, IκB-α protein, TNF-α and IL-6 was detected. It
was found that the mRNA expression levels of TLR4,
MyD88 and NF-κB were significantly decreased with the
concentrations of progesterone increasing, but those of
IκB-α protein significantly increased. Moreover, the ex-
pression levels of TNF-α and IL-6 were also significantly
decreased, which suggested that TLR4 downstream sig-
naling pathway was opened after TLR4 recognized the
“danger signals”, MyD88 gathered, NF-κB was activated
and translocated into the nucleus, leading to generous
cytokines releasing which injured the blood vessels of
placenta, thus induced PE. After progesterone inhibited
the expression of TLR4, the downstream signaling path-
way was closed, MyD88 expression reduced, the activa-
tion of NF-κB also restrained, and the damage caused by
inflammatory factors was alleviated. These studies dem-
onstrated that progesterone is involved in the pathogene-
sis of PE by inhibiting TLR4-MyD88-dependent signal-
ing pathway and it benefits the pregnancy.
Conflict of Interest Statement
The authors declare that there is no conflict of interest
with any financial organization or corporation or individual that
can inappropriately influence this work.
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