The effect of lipopolysaccharide on the expression level of immunomodulatory and immunostimulatory factors of human amniotic epithelial cells
Taheri et al. BMC Res Notes
The effect of lipopolysaccharide on the expression level of immunomodulatory and immunostimulatory factors of human amniotic epithelial cells
Ramezan Ali Taheri 3
Hossein Motedayyen 2
Somayeh Ghotloo 1
Mohsen Masjedi 2
Nariman Mosaffa 4
Abbas Mirshafiey 1
Mahmood Saffari 0 1
0 Department of Microbiology and Laboratory Medicine, Kashan University of Medical Sciences , 5th kilometer of Ravand Road, Kashan , Iran
1 Department of Pathobiology, Faculty of Public Health, Tehran University of Medical Sciences , Tehran , Iran
2 Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences , Isfahan , Iran
3 Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences , Tehran , Iran
4 Department of Immunology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences , Tehran , Iran
Objective: Human amniotic epithelial cells (hAECs) are a novel source of stem cells and have immunomodulatory effects on both the innate and adoptive immune system. hAECs can differentiate into multiple cell lineages that make them a suitable cell source for regenerative medicine. These cells express multiple toll-like receptors (TLRs) and respond to various TLR ligands. This study aimed to evaluate the effect of lipopolysaccharide (LPS), a TLR4 ligand, on the level of immunomodulatory and immunostimulatory factors of hAECs. Results: Our results indicated that LPS had the ability to up-regulate the expression of prostaglandin E2 synthase and transforming growth factor-beta1 in hAECs. However, there was no change in the level of interleukin-1beta, interleukin-6 and interleukin-10 in hAECs when were stimulated with LPS. In addition, we observed tumor necrosis factoralpha was only expressed at very low level in some of hAECs samples which its expression was independent of the effects of LPS.
Human amniotic epithelial cells; Toll-like receptors; Lipopolysaccharide; Immunomodulatory effects; Regenerative medicine
The amnion membrane is the innermost layer of the fetal
membranes and consists of epithelial cells and
avascular stroma [
]. Human amniotic epithelial cells (hAECs)
have unique properties that distinguish them from other
human cell sources. hAECs express HLA-G which is an
inhibitory ligand for B and T lymphocytes, natural killer
(NK), and dendritic cells [
] These cells have the ability
to modulate immune responses through the secretion of
soluble molecules and cell–cell contact . hAECs utilize
various immunosuppressive molecules such as TGF- β1,
prostaglandin E2 (PGE2), and Fas ligand (Fas L) [
These immunosuppressive agents play a crucial role in
regulating immune response and preventing the
development of autoimmune disorders through the inhibition of
T cell proliferation, the shift of immune responses toward
Th2-type responses and the reduction of
pro-inflammatory responses .
In addition to immunomodulatory activities, these cells
are proposed as a novel source of stem cells that
differentiate into different cell types originating from three
germ layers without any of the ethical concerns related to
human stem cells [
]. hAECs express some of the
functional toll-like receptors (TLRs) such as TLR2/TLR6,
TLR4, and TLR5, which are important regulators of the
innate immune system and recognize pathogens by
conserved pathogen associated molecular patterns (PAMPs)
]. TLR2/TLR6 and TLR5 stimulation on hAECs induce
the production of IL-8 and IL-6 [
]. Previous studies
indicated that the human placenta responds to different
PAMPs by TLRs expression [
]. Activation of TLRs by
PAMPs triggers intracellular signaling cascades resulting
in the activation of nuclear factor-kappa B (NF-κB) and
activation protein-1(AP-1), which are known as key
transcription factors responsible for the expression of
proinflammatory cytokines including IL-1, IL-6 and TNF-α.
Pro-inflammatory cytokines stimulate the production of
prostaglandins in the fetal membranes, thereby causing
uterine contractions [
]. Therefore, engagement of TLRs
by PAMP and, subsequently, the production of different
factors of hAECs suggest that the fetal membranes play a
pivotal role in the immune system.
Regarding the fact that the immunosuppressive and
immunostimulatory effects of hAECs are mainly
mediated by soluble molecules such as TGF-β1, PGE2, TNF-α
and IL-6 [
], this study aimed to examine the effect
of lipopolysaccharides (LPS), a ligand for TLR4, on the
expression of soluble factors of hAECs such as TGF-β1,
PGE2, IL-10, IL-1β, TNF-α and IL-6, which mediate the
immunomodulatory and immunostimulatory effects of
Term placentas were obtained from 10 healthy pregnant
women during uncomplicated elective cesarean
deliveries. hAECs were isolated using a method described
]. Briefly, the amnion membrane was manually
peeled off from the chorion and washed several times
with phosphate buffered saline (PBS). The amnion was
then digested at 37 °C for 10 min with 0.05% EDTA/
trypsin (Gibco, USA). The digestion of the amnion layer
was followed twice at 37 °C for 30 min with 0.05% EDTA/
trypsin. The isolated cells from the second and third
digests were pooled and washed with ice-cold RPMI
Assessment of the purity of hAECs by flow cytometry
To determine the purity of hAECs isolated from five
amnion membranes, the cells (8 × 105) were stained with
FITC anti-human CD105, FITC anti-human CD90, and
matched-isotype control IgG antibodies, as negative
controls, at 4 °C for 25 min (Additional file 1: Table S1).
Mesenchymal stem cells (MSCs) were used as positive control
for anti-CD90 and anti-CD105 antibodies. Next, fixation
and permeabilization of the cells were preformed for
intracellular staining with Alexa Fluor® 488 anti-human
cytokeratin or matched-isotype control IgG antibodies
(Additional file 1: Table S1) according to the
manufacturere’s protocol (eBioscience, USA). The cells were then
washed three times with cell staining buffer (Biolegend,
USA) and the purity of the cells was analyzed using a
FACSCalibur flow cytometer (Becton–Dickinson, CA).
hAECs obtained from ten healthy pregnant women
were cultured in 25 cm2 tissue culture flasks at a density
of 2.5 × 105 cells/cm2 in DMEM/F12 medium (Gibco,
USA) supplemented with 10% FBS (Gibco, USA) and
1% penicillin/streptomycin (Sigma-Alderich, USA). LPS
(1 mg, Sigma-Alderich, USA) was dissolved in 1 ml RPMI
medium to yield a stock concentration of 1 mg/ml. After
24 h of incubation at 37 °C, one set of the cultured hAECs
were stimulated with LPS (5 µg/ml) and incubated at
37 °C with 5% CO2. All assays were performed in
duplicate and randomization was used while performing the
experiment. After 6 h, adhered hAECs which were
cultured in the presence or absence of LPS were
dissociated by trypsin and washed twice with PBS for total RNA
RNA extraction, reverse transcription and quantitative
polymerase chain reaction (qPCR)
For gene expression analysis, total RNAs from ten hAEC
samples which were cultured in the presence or absence
of LPS were extracted using the RNeasy Mini RNA
isolation kit according to the manufacturer’s protocol
(Qiagen, USA). The investigators were blinded to sample
information. RNA yield was determined and the purity
was assessed using a spectrophotometer (NanoDrop
8000 spectrophotometer, Thermo scientific, USA).
Complementary deoxyribonucleic acid (cDNA) synthesis was
done using RevertAid First Strand cDNA Synthesis Kit
(Thermo scientific, USA) following the manufacturer’s
instructions. q PCR assay was done using an ABI7700
machine (Applied Biosystems, Foster City, USA) and the
SYBR® Premix Ex Taq™ II Master Mix (Takara, Japan)
according to the manufacturer’s instructions. Each
reaction was initiated at 95 °C for 15 s, followed by 45 cycles
of 95 °C for 5 s and 58 °C for 40 s. All analyses were
performed in triplicate. Threshold cycles (Ct) and melting
curves were generated automatically by the Applied
Biosystems software. The expression levels of each sample
were normalized to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as an endogenous control. The
cycling parameters for GAPDH were similar to those
used for the cytokines. The Relative Expression
Software Tool 2009 (REST 2009) [
] was used to calculate
the relative expression of the target genes, using the ratio
of the Ct values and the PCR amplification efficiencies
of the target genes and the GAPDH gene. REST 2009
uses randomization and bootstrapping methods to test
the statistical significance of the gene expression ratios
and calculate 95% confidence intervals for relative fold
]. Primer sequences are shown in Additional
file 1: Table S2.
The results are expressed as mean± standard error (SE)
and mean ± standard error of mean (SEM). REST 2009
was used for group-wise comparison and statistical
analysis of relative expression results in real-time PCR.
p < 0.05 was considered statistically significant.
The purity of hAECs
To assess the purity of hAEC, the percentage of cells
which were positive for cytokeratin, CD90 and CD105
was measured by flow cytometry. Our results revealed
that more than 97% of the isolated cells expressed
cytokeratin and less than 1% of the cells were positive for
CD90 and CD105 (Fig. 1a–c).
LPS effects on expression of immunosuppressive factors
Since immunomodulatory effects of hAECs make them
as a cell source for cellular therapy, LPS effects on
expression of immunosuppressive mediators in hAECs was
evaluated. Our data showed a significant increase in the
expression level of PGE2 synthase, an enzyme which
produces PGE2, and TGF-β1 in the sample group (hAECs
treated with LPS) compared to the control group (hAECs
cultured in the absence of LPS) (Fig. 2, p < 0.001–0.05).
Although there was a numerical increase in the mean
of IL-10 expression level in the sample group compared
to the control group, LPS could not induce the IL-10
expression in the sample group (Fig. 2).
LPS effects on induction of pro‑inflammatory cytokines
Regarding pro-inflammatory cytokines play an
important role in the induction of immune response against the
cells used with therapeutic purposes, we evaluated LPS
effect on the expression of pro-inflammatory cytokines in
hAECs. In spite of a numerical decrease in IL-6 level and
an increase in IL-1β level in the sample group, the results
of the study indicated that LPS did not influence the
expression of IL-6 and IL-1β in hAECs (Fig. 3). Moreover,
we found that TNF-α was expressed at very low level in
hAECs and was only detectable in some samples such as
sample 6, 7 and 10.
Immune-rejection and tumorigenic upon
transplantation are two major problems which challenge the use of
cell therapy for the treatment of different diseases [
]. To date, multiple cell sources have been employed in
regenerative medicine and the treatment of diseases with
immune pathophysiology, but these treatments have not
been uniformly successful and the evidence is insufficient
to support the efficacy of these therapeutic approaches
. hAECs which are stem cells with the
immunomodulatory effects may be considered as a potential candidate
in the regenerative medicine and immunotherapy for
inflammatory diseases. In addition, some unique
characteristics of these cells make them an interesting source of
cells for use as therapy in regenerative medicine. These
cells express very low level of human leukocyte antigens
(HLA) class I and are negative for HLA-class II and
costimulatory molecules, which may potentially reduce the
risk of immune-rejection after clinical applications [
hAECs do not express telomerase and thus cannot
tumorigenic upon clinical use [
]. Therefore, the use of hAECs
as a therapeutic approach has not challenged with the
two major cell therapy issues, immunological rejection
and tumor formation after transplantation.
TGF-β, IL-10, and PGE2 are known as powerful
immunosuppressive molecules that play a pivotal role in
regulation of the immune system [
]. These factors
impair the proliferation and differentiation of immune
cells and inhibit the production of pro-inflammatory
4, 15, 19
]. Several studies indicated that
hAECs suppressed immune cells proliferation through
TGF-β1 and PGE2 [
]. Regarding the fact that hAECs
express different TLRs and engagement of TLRs by
PAMP influences hAECs function, the critical question
was whether TLR-4 stimulation by LPS, as an important
TLR for hAECs, affects the production of
immunosuppressive and immunostimulatory mediators of hAECs.
In the current study, we observed that LPS had the
ability to induce the expression of TGF-β1 and PGE2
synthase in hAECs but not for IL-10. For the first time,
these results showed that the stimulation of hAECs with
LPS may be considered as an approach for enhancing
the immunoregulatory effects of these cells. However,
the molecular mechanism involved in LPS effects on
upregulation of immunosuppressive molecules in hAECs
is unknown. Therefore, this is a question that must be
addressed in future studies. As mentioned before, no
statistically significant increase of IL-10 level was observed
in the LPS-treated hAECs. This finding may be explained
in the context of very low expression of IL-10 in these
cells, as some reports have shown that hAECs are unable
to produce IL-10 [
This study unexpectedly indicated that LPS did not
affect the level of IL-1β and IL-6 in the hAECs. These
results were in contrast with the known mechanism of
TLR4 function, leading to activation of the NF-κB
pathway and an increase in the production of
pro-inflammatory cytokines [
]. However, there is a report that
indicated the activation of TLR4 on hAECs by LPS
influenced the viability of the cells, while cannot affect the
production of pro-inflammatory cytokines by these cells
]. In addition, it has been demonstrated that hAECs
activation by TLR6/2, TLR 5 and TLR9 agonists produces
IL-6 and IL-8, but not with LPS [
]. In an effort to
discover the effects of LPS on TNF-α expression, we found
that there was an inconsistency in the expression of
TNF-α in hAECs. In contrast with some studies
indicating TNF-α expression by hAECs [
], the results of this
study demonstrated that the expression level of IFN-γ
le was below the detection limit in hAECs, and the level
was unchanged after stimulation with LPS. The IFN-γ
level was only detectable in some of hAECs samples.
Taken together, the results of this study provide
evidence to show that LPS may enhance the
immunomodulatory effects of hAECs through
up-regulating immunosuppressive factors and down-regulating
pro-inflammatory cytokines in hAECs that may
represent an advantageous cell source with potential
applications for regenerative medicine. However, further
studies are required to confirm the effects of LPS on
immunomodulatory effects of hAECs and also explain
the molecular mechanisms of LPS effects on hAECs.
The study was unable to determine whether LPS also
affects the production of immunomodulatory and
immunostimulatory factors of hAECs.
Additional file 1: Table S1. Antibodies used to determine the purity
of hAECs by Flow cytometry. Table S2. Primer sequences used in
SYBRGreen Based Real-Time PCR.
DMEM: Dulbecco’s modified eagle’s medium; RPMI: Roswell park memorial
institute; PBS: phosphate-buffered saline; FBS: fetal bovine serum; hAECs:
human amniotic epithelial cells; HBSS: Hanks’balanced salt solution; LPS:
lipopolysaccharide; IL-1β: interleukin-1beta; TLR: toll-like receptor; PGE2:
prostaglandin E2; TGF-β1: transforming growth factor-beta1; IL-6: interleukin-6;
IL-10: interleukin-10; TNF-α: tumor necrosis factor-alpha; NF-κB: nuclear
factorkappa B; AP-1: activation protein 1; PAMPs: pathogen associated molecular
RT and HM carried out some of the experiments and obtained funding for the
work. SG participated in the design of the experiments and preformed
statistical analysis. NM and AM participated in the study design and performed some
of the experiments. MM provided the placenta tissue and participated in the
design of the experiments. MS drafted the manuscript and participated in the
study design. All authors read and approved the final manuscript.
The authors would like to thank Baqiyatallah University of Medical Sciences,
the deputy of clinical development medical center of Baqiyatallah hospital for
their help and all individuals who participated in this study.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published
Consent for publication
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Isfahan University of
Medical Science. All participants signed informed consent forms prior to
entering in the study.
This study was financially supported by Kashan University of Medical Sciences
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. McDonald CA , Payne NL , Sun G , Moussa L , Siatskas C , Lim R , et al. Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis . J Neuroinflammation . 2015 ; 12 : 112 - 26 .
2. Ilancheran S , Moodley Y , Manuelpillai U . Human fetal membranes: a source of stem cells for tissue regeneration and repair? Placenta . 2009 ; 30 : 2 - 10 .
3. Insausti CL , Blanquer M , García-Hernández AM , Castellanos G , Moraleda JM . Amniotic membrane-derived stem cells: immunomodulatory properties and potential clinical application . Stem Cells Cloning . 2014 ; 7 : 53 - 63 .
4. Liu YH , Vaghjiani V , Tee JY , To K , Cui P , Oh DY , et al. Amniotic epithelial cells from the human placenta potently suppress a mouse model of multiple sclerosis . PLoS ONE . 2012 ; 7 : e35758 .
5. Li H , Niederkorn JY , Neelam S , Mayhew E , Word RA , McCulley JP , et al. Immunosuppressive factors secreted by human amniotic epithelial cells . Invest Ophthalmol Vis Sci . 2005 ; 46 : 900 - 7 .
6. Miki T , Lehmann T , Cai H , Stolz DB , Strom SC . Stem cell characteristics of amniotic epithelial cells . Stem Cells . 2005 ; 23 : 1549 - 59 .
7. Gillaux C , Méhats C , Vaiman D , Cabrol D , Breuiller-Fouché M. Functional screening of TLRs in human amniotic epithelial cells . J Immunol . 2011 ; 187 : 2766 - 74 .
8. Abrahams V , Mor G . Toll-like receptors and their role in the trophoblast . Placenta . 2005 ; 26 : 540 .
9. Motedayyen H , Esmaeil N , Tajik N , Khadem F , Ghotloo S , Khani B , et al. Method and key points for isolation of human amniotic epithelial cells with high yield, viability and purity . BMC Res Notes . 2017 ; 10 : 552 .
10. Pfaffl MW , Horgan GW , Dempfle L . Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR . Nucleic Acids Res . 2002 ; 30 : e36 .
11. Pfaffl M. Rest 2009 software user guide . Hilden: Qiagen; 2009 .
12. Kling C , Steinmann J , Westphal E , Magez J , Kabelitz D. Adverse effects of intradermal allogeneic lymphocyte immunotherapy: acute reactions and role of autoimmunity . Hum Reprod . 2005 ; 21 : 429 - 35 .
13. Ronaghi M , Erceg S , Moreno-Manzano V , Stojkovic M. Challenges of stem cell therapy for spinal cord injury: human embryonic stem cells, endogenous neural stem cells, or induced pluripotent stem cells? Stem Cells . 2010 ; 28 : 93 - 9 .
14. Pratama G , Vaghjiani V , Tee JY , Liu YH , Chan J , Tan C , et al. Changes in culture expanded human amniotic epithelial cells: implications for potential therapeutic applications . PLoS ONE . 2011 ; 6 : e26136 .
15. Lebman DA , Edmiston JS . The role of TGF-β in growth, differentiation, and maturation of B lymphocytes . Microbes Infect . 1999 ; 1 : 1297 - 304 .
16. Chang CJ , Yen ML , Chen YC , Chien CC , Huang HI , Bai CH , et al. Placentaderived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-γ . Stem Cells . 2006 ; 24 : 2466 - 77 .
17. Li C , Zhang W , Jiang X , Mao N. Human-placenta-derived mesenchymal stem cells inhibit proliferation and function of allogeneic immune cells . Cell Tissue Res . 2007 ; 330 : 437 - 46 .
18. Alcaraz A , Mrowiec A , Insausti CL , García-Vizcaíno EM , López-Martínez MC , Moraleda JM , et al. Autocrine TGF-β induces epithelial to mesenchymal transition in human amniotic epithelial cells . Cell Transpl . 2013 ; 22 : 1351 - 67 .
19. Wahl SM , Hunt D , Wong HL , Dougherty S , McCartney-Francis N , Wahl L , et al. Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation . J Immunol . 1988 ; 140 : 3026 - 32 .
20. Triantafilou M , De Glanville B , Aboklaish AF , Spiller OB , Kotecha S , Triantafilou K. Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells . PLoS ONE . 2013 ; 8 : e61199 .