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Citation: Wang, Y.; Wang, Y. Palmitic
Acid Upregulates CD96 Expression to
Mediate Maternal–Foetal Interface
Immune Tolerance by Inhibiting
Cytotoxic Activity and Promoting
Adhesion Function in Human
Decidual Natural Killer Cells.
Bioengineering 2023,10, 1008.
https://doi.org/10.3390/
bioengineering10091008
Academic Editor: Andrea Cataldo
Received: 29 May 2023
Revised: 20 July 2023
Accepted: 8 August 2023
Published: 25 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
bioengineering
Article
Palmitic Acid Upregulates CD96 Expression to Mediate
Maternal–Foetal Interface Immune Tolerance by Inhibiting
Cytotoxic Activity and Promoting Adhesion Function in Human
Decidual Natural Killer Cells
Yingjie Wang and Yun Wang *
Department of Assisted Reproduction, School of Medicine, Shanghai Ninth People’s Hospital Affiliated to
Shanghai Jiao Tong University, No. 500 Zhizaoju Road, Huangpu District, Shanghai 200025, China;
wangyj_20@sjtu.edu.cn
*Correspondence: wangy1860@sh9hospital.org.cn or sammy20080228@icloud.com
Abstract:
Decidual natural killer cells (dNK cells) are an essential component of the immune cells
present at the maternal–foetal interface during early pregnancy, and they play a vital role in various
physiological processes. Abnormalities in the ratio or function of dNK cells have been linked to
recurrent miscarriages. CD96 has been previously shown to regulate NK cell function in the tumour
microenvironment; however, its role and mechanism at the maternal–foetal interface remains unclear.
The present study aimed to investigate the immunomodulatory role of CD96 in dNK cells and its
function at the maternal–foetal interface. Immunofluorescence staining and flow cytometry were
used to detect the expression of cellular markers such as CD96. Furthermore, the secretory function,
adhesion-function-related molecules, and cell proliferation markers of CD96+ and CD96
−
dNK
cells were detected using flow cytometry. In addition, we performed cell culture experiments via
the magnetic bead sorting of NK cells to detect changes in the expression of the aforementioned
functional molecules in dNK cells after the CD96 blockade. Furthermore, we examined the functional
characteristics of dNK cells after palmitic acid treatment at a concentration of 10
µ
M. We also examined
the changes in dNK cell function when subjected to the combined effect of palmitic acid and CD96
antagonists. The results indicated that CD96, TIGIT, CD155, and CD112 were highly expressed at
the maternal–foetal interface, with dNK cells predominantly expressing CD96, whereas TIGIT was
mainly expressed on T cells, and CD155 and CD112 were mainly present in metaphase stromal and
trophoblast cells. CD96+ dNK cells displayed low cytotoxic activity and a high adhesion phenotype,
which mediated the immunosuppressive effect on dNK cells at the maternal–foetal interface. Palmitic
acid upregulated CD96 expression on the surface of dNK cells in the coculture system, inhibiting dNK
cell activity and increasing their adhesion molecule expression. CD96 antagonist treatment blocked
the inhibitory effect of trophoblasts on dNK cells, resulting in enhanced cytokine secretion and
reduced adhesion. The results of this study provide valuable insight into the immunomodulatory role
of CD96 in dNK cells and its mechanism at the maternal–foetal interface, particularly in metaphase
NK cells. This study sheds light on the mechanisms of immune regulation at the maternal–foetal
interface and their implications for the study of recurrent miscarriages of unknown origin.
Keywords:
CD96; dNK cells; immune adhesion; maternal–foetal immunology; recurrent spontaneous
abortion
1. Introduction
The maternal–foetal interface is a key site for the establishment and maintenance of
normal pregnancy, and it is mainly composed of trophoblast cells, decidual immune cells,
and decidual stromal cells [
1
,
2
]. More than 30% of decidual cells in early pregnancy are
immune cells, which are one of the main components of the maternal–foetal interface [
3
].
Bioengineering 2023,10, 1008. https://doi.org/10.3390/bioengineering10091008 https://www.mdpi.com/journal/bioengineering
Bioengineering 2023,10, 1008 2 of 17
A variety of immune cells participate in the maintenance of maternal and foetal immune
homeostasis during pregnancy, such as NK cells, T cells, macrophages, and dendritic
cells [
4
]. The proportion and phenotype of these cells in the process of pregnancy change
dynamically, indicating that the composition of immune cells is different during pregnancy,
in different stages of pregnancy, and in different states of pregnancy [
5
,
6
]. It has been
shown that immune homeostasis at the maternal–foetal interface is usually disrupted in
patients with recurrent pregnancy failure [7].
Interactions with trophoblast cells through surface molecules or secreted substances
affect the process of pregnancy and are important for tolerating and maintaining normal
foetal growth [
8
]. Studies have found that a large number of dNK cells that appear at
the maternal–foetal interface aggregate, but they do not only exert cytotoxic effects like
peripheral NK cells. Moreover, there is growing evidence to suggest that the existence
of dNK cells is also an important factor in maintaining pregnancy [
9
]. CD56, as a charac-
teristic marker molecule of NK cells, exhibits a CD56dim phenotype on highly cytotoxic
NK cells in peripheral blood, though it displays a CD56bright phenotype on decidual
NK cells in the decidua, indicating a close association between CD56 expression and the
functional activity of NK cells. Accordingly, the composition of immune cells and cytokines
at the maternal–foetal interface maintains dynamic changes. As pregnancy progresses,
the microenvironment during embryo implantation and placental development gradually
changes from a proinflammatory phenotype to an anti-inflammatory phenotype; for ex-
ample, dNK cells [
10
]. Williams et al. found that the number of decidual CD56+ cells did
not change in the first and second trimester, and CD56+ cells were reduced in the third
trimester [
11
]. Chen et al. found that the proportion of CD56+ dNK cells with perforin and
granzymes in the decidua gradually decreased as pregnancy progressed, suggesting that
dNK cells function in a more moderate manner during pregnancy [12].
CD96 is a member of the poliovirus receptor (PVR, CD155)-connexin family, which
includes the T-cell Ig and ITIM domains (TIGIT) and CD226 [
13
]. CD96, TIGIT, and CD155
play an important role in regulating NK cell activity, which relies on a large number of
receptor combinations to initiate effector functions [
14
]. PVR, functioning as a high-affinity
ligand for CD96, directly binds to CD96 receptors and mediates the inhibitory effect on the
cytotoxicity of NK and T cells. These effects have been widely validated in tumour tissue,
but it is unclear whether CD96 exhibits inhibitory or stimulatory functions in decidual NK
cells [15].
Palmitic acid (PA) is one of the most common 16-carbon long-chain fatty acids. It is
rich in the human body, and it is one of the most important raw materials for cholesterol
synthesis and the main component of bile acids [
16
]. Palmitic acid is not only a main
component of the constituent cell membrane, but it also exhibits biological activity in the
immune system [
17
]. Previous studies have shown that high concentrations of palmitic
acid can activate the inflammatory response through multiple pathways [
18
]. The excessive
accumulation of palmitic acid induces an inflammatory response at the maternal–foetal in-
terface, leading to adverse pregnancy events such as eclampsia or miscarriage [
19
]. Another
previous study demonstrated that a concentration of 10
µ
M palmitic acid upregulated the
expression of CXCL12 and IL15 at the maternal–foetal interface, potentially contributing to
the prevention of recurrent implantation failure [
20
]. We suspect that a low dose of palmitic
acid has more benefits for pregnancy maintenance at this time, but no studies have yet
confirmed this conjecture.
The aims of this study were to investigate the role and molecular mechanism of
CD96 in resident dNK cells during pregnancy, to investigate the influencing factors on
the pathogenesis of spontaneous abortion, and to explore the function of palmitic acid in
this process.
Bioengineering 2023,10, 1008 3 of 17
2. Materials and Methods
2.1. Tissues
All tissues were collected with the permission of the ethics committee and the consent
of patients in the Obstetrics and Gynecology Hospital of Fudan University. Endometrial
tissue was collected from women of childbearing age (25–40 years) with a normal history
of pregnancy who underwent hysterectomy or diagnostic curettage for benign causes
unrelated to endometrial dysfunction. All samples were evaluated by a histopathologist to
rule out endometrial pathology and identify the cycle phase, specifically the secretory phase.
None of the subjects had received hormonal medication in the preceding 6 months prior
to surgery. Normal pregnancy decidual tissue was obtained from women with selective
termination of pregnancy in the normal first trimester or with nongenetic or nonendocrine
factors (age, 21–35 years; gestational age, 7–9 weeks). The decidua of spontaneous abortion
came from women who underwent evacuation at 6–10 weeks of gestation when the foetal
heart stopped. All tissues were collected under sterile conditions, and 10% foetal bovine
serum (FBS) in DMEM/F-12 (HyClone, Logan, UT, USA, SH30023.01B) was added within
30 min along with 10% foetal bovine serum (FBS; Gibco, Grand Island, NY, USA, 26140-079)
for further isolation of ESCs, DSCs, and dNK cells. ESCs, DSCs, and dNK cells were
isolated and cultured according to our previously established protocol [21].
2.2. Cell Culture
The DSCs or dNK cells used in the experiment were primary cells obtained through
the following process. Firstly, the tissues mentioned in Section 2.1 were cut into pieces
and digested using collagenase. The resulting cell suspension was then filtered through a
strainer to remove any debris. Subsequently, the cell suspension was centrifuged using a
density gradient with Percoll reagent. This centrifugation step allowed for the separation
of cells based on their density. After centrifugation, the cells were collected and cultured
in well plates for further cell culture studies. The cell processing in this paper includes
treatment with the CD96 antibody (Ultra-LEAF
™
Purified anti-human CD96; 338422,
Biolegend, San Diego, CA, USA) at a concentration of 10
µ
g/mL or palmitic acid (PA;
SYSJ-KJ003/KC003, Kunchuang Biotechnology, Xi’an, China) at a concentration of 10
µ
M
for 24 h. Afterwards, the protein levels of specific molecules or the adhesion of stromal
cells to NK cells were detected using flow cytometry staining. Cytokines were detected for
subsequent intracellular flow cytometry staining assays.
2.3. Lysis of Erythrocytes
If necessary, the sample was diluted 10
×
with deionized water, red blood cell (RBC)
lysis buffer (BioLegend, San Diego, CA, USA, 420301) was diluted to a 1
×
working concen-
tration, and then the precipitate was resuspended in 3 mL of 1
×
RBC lysis buffer. The cells
were incubated on ice for 5 min. Cell lysis was stopped by adding 10 mL of cell staining
buffer to the tube. The mixture was centrifuged for 5 min at 350
×
g, and the supernatant
was discarded. The wash was repeated as described above.
2.4. Flow Cytometry Assays
Human antibodies for flow cytometry assays were used for the measurement of cell
markers. Matched immunoglobulin G (IgG) antibodies were used as isotype controls. Flow
cytometry assays were performed according to the manufacturer’s instructions. For the
detection of intracellular molecules such as cytokines, we used Cell Activation Cocktail
(with Brefeldin A) (Biolegend, San Diego, CA, USA, 423303) to stimulate the cells for 6 h
prior to flow cytometry antibody staining of the cell surface. Fixation Buffer (Biolegend, San
Diego, CA, USA, 420801) was used for cell fixation and Intracellular Staining Perm Wash
Buffer (Biolegend, San Diego, CA, USA, 421002) was used for permeabilization followed by
intracellular staining. The cells were subsequently stained with flow cytometry antibodies
and detected on the machine. Cell sorting was performed with the NK cell magnetic bead
Bioengineering 2023,10, 1008 4 of 17
sorting kit (Miltenyi Biotec, Shanghai, China, 130-092-657). The flow cytometry antibodies
used are listed in Table 1. Data were analysed using FlowJo V10 software.
Table 1. Flowcytometry antibodies used in this article.
Antibody Fluorescence Manufactory Clone
Anti-human CD45 antibody BV510 Biolegend 2D1
Anti-human CD3 antibody AF700 Biolegend SK7
Anti-human CD56 antibody PE/Cy7 Biolegend HCD56
Anti-human TIGIT antibody PE Biolegend A15153G
Anti-human CD96 antibody BV421 Biolegend NK92.39
Anti human CD96 antibody AO Abcam NK92.39
Anti human CD155 antibody PE Biolegend TX24
Anti human CD112 antibody APC Biolegend TX31
Anti-human Vimentin antibody AF488 BD RV202
Anti-human Granzyme B antibody APC Biolegend QA18A28
Anti-human CD54 antibody FITC Biolegend HA58
Anti-human CD62E antibody PE Biolegend HAE-1f
Anti-human CD106 antibody APC Biolegend STA
Anti-human IL10 antibody APC Biolegend JES3-9D7
Anti-human Ki67 antibody APC Biolegend Ki-67
Anti-human Ki67 antibody FITC Biolegend 11F6
Anti-human IFN-γantibody AF700 Biolegend 4S.B3
Anti-human IFN-γantibody BV421 Biolegend 4S.B3
2.5. Paraffin Section Preparation
Fresh tissue was fixed with a fixative for more than 24 h. The tissue was removed
from the fixation fluid in the ventilator, smoothed with a scalpel, and placed with the
corresponding label in the dehydration box, which was placed in the dehydrator with
alcohol gradients for dehydration: 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol
for 2 h, 95% alcohol for 1 h, absolute ethanol I for 30 min, absolute ethanol II for 30 min,
alcohol benzene for 5–10 min, xylene I for 5–10 min, xylene II for 5–10 min, 65
◦
C melted
paraffin for 1 h, 65
◦
C melted paraffin II for 1 h, and 65
◦
C melted paraffin III for 1 h.
Once the dehydration process was completed, the wax-soaked tissue was embedded in
the embedding machine. The melted wax was first placed into the embedded box. Before
the wax solidified, the tissue was removed from the dehydration box and placed into the
embedded box according to the embedded face, and the corresponding label was attached.
The frozen table was cooled to
−
20
◦
C, and the wax block was solidified, removed from
the embedded box, and trimmed. The finished wax block was placed in a
−
20
◦
C frozen
table to cool, and then the cooled wax block was placed in a paraffin-slicing microtome and
cut to 4
µ
m thick. Floating slices were placed in 40
◦
C warm water to flatten the tissue, and
the tissue was removed and baked in a 60 ◦C oven. After baking with water and dry wax,
the sample was removed and kept at room temperature.
2.6. Immunofluorescence in Paraffin Sections
The paraffin wax-to-water procedure involved several sequential steps. Firstly, envi-
ronmentally friendly dewaxing was performed twice for 10 min each time, followed by
successive immersions in absolute ethanol for 10 min, 15 min, and 5 min, and finally in
distilled water for 5 min. For antigen repair, tissue sections were placed in a repair box
containing EDTA antigen repair buffer (pH 8.0) and subjected to microwave heating. The
samples were initially heated at medium power until boiling for 8 min, followed by an
8 min
cooling period, and finally heating at low power for 7 min. This heating cycle was
designed to prevent excessive buffer evaporation while ensuring that the samples did not
dry out. After natural cooling, the slides were washed in PBS (pH 7.4) for 5 min each. To
facilitate serum closure, a circle was drawn around the tissue on the slightly dried section
to prevent antibody flow. The sample was dried using PBS, followed by the addition
Bioengineering 2023,10, 1008 5 of 17
of BSA. The sample was then sealed for 30 min, using 10% donkey serum for blocking
antibodies from goat sources and 3% BSA for blocking antibodies from other sources. Next,
the blocking solution was gently removed, and PBS was added to the section in a specific
proportion. The slides were then placed flat in a wet box and incubated overnight at
4
◦
C. To prevent antibody evaporation, a small amount of water was added to the wet
box. Following the primary antibody incubation, the slides were washed three times in
PBS (pH 7.4) using a counter colour shaker for 5 min each time. After slight drying, the
corresponding species-specific secondary antibody was added to the circle, and the sections
were incubated for 50 min at room temperature. For DAPI counter-staining of the nuclei,
the slides were washed three times in PBS (pH 7.4) on a colour shaker for 5 min each
time. The sections were then slightly dried, and DAPI dye solution was added to the circle,
followed by a 10 min incubation at room temperature. To quench spontaneous fluorescence
of the tissue, the slides underwent three washes in PBS (pH 7.4) for 5 min each time. A
spontaneous fluorescent quencher was added to the circle for 5 min, and the slides were
subsequently washed with running water for 10 min. After slight drying, the sections
were sealed with an antifluorescence quenching agent. Finally, the sections were examined
under a fluorescence microscope, and images were collected using specific excitation and
emission wavelengths: DAPI UV excitation at 330–380 nm and emission at 420 nm (blue
light), FITC excitation at 465–495 nm and emission at 515–555 nm (green light), and CY3
excitation at 510–560 nm and emission at 590 nm (red light). The immunofluorescence
results of the paraffin sections showed blue fluorescence of cell nuclei stained with DAPI
under UV excitation, along with the corresponding red or green fluorescence from the
fluorescein-labelled antibodies.
2.7. Cell Adhesion Assays
Stromal cells after different treatments of lentivirus infection were collected, washed,
and cultured in a 24-well plate (1
×
10
5
cells per well) with or without staining with
PKH67 (Sigma-Aldrich, St. Louis, MO, USA, PKH67GL) overnight. PKH26-labelled (Sigma
Aldrich, PKH26GL) dNK cells were cocultured with HTR8s or DSCs for 24 h. Then, for
removal of unattached dNK cells, the medium was removed and the cells were washed
gently with PBS twice. Pictures were obtained under a fluorescence microscope (Olympus,
Tokyo, Japan, IXplore Standard) at five random fields of view and are shown as the ratio of
dNK cells to the stromal cell layer [21].
2.8. Statistical Analysis
All experiments were repeated at least in triplicate. All data were analysed using
GraphPad Prism version 8. All parameters were analysed using an unpaired ttest, Mann-
Whitney U test, or one-way ANOVA. Data that were normally distributed are presented as
the mean ±STD. Statistical significance is indicated by p< 0.05.
3. Results
3.1. CD96 Is Enriched in the Maternal–Foetal Interface during Pregnancy
To clarify CD96 and CD155 expression at the maternal–foetal interface, we performed
immunofluorescence staining of the normal endometrium (NE) tissues and decidua tis-
sues from normal pregnancy (NP) women and spontaneous abortion (SA) women. In
the decidua of normal pregnancies, the expression of CD96 and CD155 at the maternal–
foetal interface was significantly higher compared to spontaneous abortion and normal
endometrium (Figure 1). The expression of CD155 in the decidua and villi of patients with
spontaneous abortion was relatively lower compared to normal pregnancy.
Bioengineering 2023,10, 1008 6 of 17
Bioengineering 2023, 10, x FOR PEER REVIEW 6 of 18
Figure 1. Immunofluorescence staining of the maternal–foetal interface: (A) Immunofluorescence
staining of villi in normal pregnancy and spontaneous abortion and vimentin-labelled chorionic
stromal cells. (B) Immunofluorescence staining of normal endometrium, decidua of normal preg-
nancy, and decidua of spontaneous abortion and vimentin-labelled DSCs or ESCs. (C) Immunoflu-
orescence staining of normal endometrium, decidua of normal pregnancy, and decidua of sponta-
neous abortion and NCAM1-labelled dNK cells. All fields of view were shot under 400× lenses. VIM:
vimentin; SA: spontaneous abortion; NE: normal endometrium; NP: normal pregnancy. *: p < 0.05;
**: p < 0.01.
3.2. CD96 Is Highly Expressed in Normal Gestational Decidual NK Cells, While There Is No
Difference in the Expression of TIGIT in the Endometrium and Normal Pregnancy Decidual NK
Cells
To detect the cell specificity of CD96 expression in decidual and endometrial tissues,
we labelled CD45 for lymphocytes, CD3 for T cells, and CD56 for NK cells with a flow
cytometry antibody, and differences in CD96 expression were compared between cell
groups (Figure 2A–D). We found that the expression of CD96 in various groups of cells in
the decidua of normal pregnancy was significantly higher than that of the endometrial
tissue. We also assessed the CD96-associated TIGIT marker, which was found to be in-
volved in the immunosuppression of CD155 on NK cells in tumour tissue. Within the
same tissue, TIGIT expression was significantly higher on T cells and total lymphocytes
than on NK cells, either in endometrial tissue or in decidual tissue of normal pregnancy.
In contrast, there was no difference in TIGIT expression on NK cells or T cells when com-
paring endometrial and decidual tissues. Since CD155 and CD96 are a pair of interacting
molecules, it is necessary to detect the expression of CD155 at the maternal–foetal inter-
Figure 1.
Immunofluorescence staining of the maternal–foetal interface: (
A
) Immunofluorescence
staining of villi in normal pregnancy and spontaneous abortion and vimentin-labelled chorionic
stromal cells. (
B
) Immunofluorescence staining of normal endometrium, decidua of normal pregnancy,
and decidua of spontaneous abortion and vimentin-labelled DSCs or ESCs. (
C
) Immunofluorescence
staining of normal endometrium, decidua of normal pregnancy, and decidua of spontaneous abortion
and NCAM1-labelled dNK cells. All fields of view were shot under 400
×
lenses. VIM: vimentin; SA:
spontaneous abortion; NE: normal endometrium; NP: normal pregnancy. *: p< 0.05; **: p< 0.01.
3.2. CD96 Is Highly Expressed in Normal Gestational Decidual NK Cells, While There Is No
Difference in the Expression of TIGIT in the Endometrium and Normal Pregnancy Decidual
NK Cells
To detect the cell specificity of CD96 expression in decidual and endometrial tissues,
we labelled CD45 for lymphocytes, CD3 for T cells, and CD56 for NK cells with a flow
cytometry antibody, and differences in CD96 expression were compared between cell
groups (Figure 2A–D). We found that the expression of CD96 in various groups of cells
in the decidua of normal pregnancy was significantly higher than that of the endometrial
tissue. We also assessed the CD96-associated TIGIT marker, which was found to be involved
in the immunosuppression of CD155 on NK cells in tumour tissue. Within the same tissue,
TIGIT expression was significantly higher on T cells and total lymphocytes than on NK cells,
either in endometrial tissue or in decidual tissue of normal pregnancy. In contrast, there
was no difference in TIGIT expression on NK cells or T cells when comparing endometrial
and decidual tissues. Since CD155 and CD96 are a pair of interacting molecules, it is
necessary to detect the expression of CD155 at the maternal–foetal interface, and we also
measured the expression of CD112, the binding site of TIGIT, on decidual stromal cells to
determine the expression of this family of molecules on the decidual surface. We labelled
DSCs (decidual stromal cells) and ESCs (endometrial stromal cells) with Vimentin to detect
CD155 and CD112 expression on the surface of DSCs and ESCs, as shown in Figure 2E–G.
Bioengineering 2023,10, 1008 7 of 17
Flow cytometry showed a higher expression of CD155 (p< 0.05) and CD112 (p< 0.01) in
decidual tissue in normal pregnancy compared to endometrial tissue.
Bioengineering 2023, 10, x FOR PEER REVIEW 7 of 18
face, and we also measured the expression of CD112, the binding site of TIGIT, on decid-
ual stromal cells to determine the expression of this family of molecules on the decidual
surface. We labelled DSCs (decidual stromal cells) and ESCs (endometrial stromal cells)
with Vimentin to detect CD155 and CD112 expression on the surface of DSCs and ESCs,
as shown in Figure 2E–G. Flow cytometry showed a higher expression of CD155 (p < 0.05)
and CD112 (p < 0.01) in decidual tissue in normal pregnancy compared to endometrial
tissue.
Figure 2. Expression of CD96 and CD155 at the decidual interface in normal pregnancy: (A) Gating
strategies for NK cells, T cells, and total immune cells. (B) Histogram of differences in the expression
Figure 2.
Expression of CD96 and CD155 at the decidual interface in normal pregnancy: (
A
) Gating
strategies for NK cells, T cells, and total immune cells. (
B
) Histogram of differences in the expres-
sion of CD96 and TIGIT in different cell subsets in decidual and endometrial tissues of normal
pregnancy. (
C
) Differences in the average fluorescence intensity of CD96 expression in different
subpopulations of cells. (
D
) Differences in the mean fluorescence intensity of TIGIT expression in
different cell subpopulations. (
E
) Entrapment gating strategies for stromal cells in deciduous tissue
and endometrial tissue. (
F
) Histogram of differences in the expression of CD155 and CD112 in DSCs
and ESCs. (
G
) Statistics of the difference in the expression of CD155 and CD112 in DSCs and ESCs.
DSCs: deciduous stromal cells; ESCs: endometrial stromal cells. TL: total lymphocyte; DSC: decidual
stromal cell; ESC: endometrial stromal cell; MFI: mean fluorescence intensity. ns: p> 0.05; *: p< 0.05;
**: p< 0.01; ***: p< 0.001.
Bioengineering 2023,10, 1008 8 of 17
3.3. Phenotype and Characteristics of CD96+ dNK Cells
Primary decidual cells were labelled via flow cytometry, and the dNK cells of CD96+
and CD96
−
were windowed separately to detect the functional receptors of dNK cells. We
investigated whether CD96 expression on the surface of dNK cells affects their function
and phenotype, similar to the role of CD96 in pNK cells, laying down the foundation for
subsequent research. We used flow cytometry staining to label the characteristic molecules
CD56 and CD3 of NK cells and gated NK cells, and then further identified the negative
and positive groups of CD96 on the surface of the NK cells. The results showed that
the proportion of NK cells in primary immune cells was approximately 57% (Figure 3A).
Among NK cells, the proportion of CD96+ NK cells was significantly higher than that
of CD96
−
NK cells (Figure 3B). Various types of adhesion molecules exist according to
their structural characteristics. In addition to some adhesion molecules that have not yet
been classified, they can also be grouped into families such as the integrin family, selectin
family, immunoglobulin superfamily, cadherin, etc. Here, we selected three representative
adhesion molecules for determination: ICAM1 (CD54), VCAM1 (CD106), and selectin
SELE (CD62E) of the immunoglobulin superfamily. Previous studies have found that these
molecules are abundantly expressed on the surface of dNK cells localized to the decidua
during pregnancy, thereby mediating the adhesion of dNK cells at the maternal–foetal
interface. In our examination, CD96+ and CD96
−
NK cells were separately analysed,
revealing that three adhesion molecules were markedly elevated on CD96+ NK cells
(Figure 3C,D). NK cells can express a variety of cellular molecules, such as IFN-
γ
, granzyme
B, TNF-
α
, and perforin, and the expression of cytokines is positively correlated with
the killing activity of NK cells, especially in inflammatory environments. To verify the
relationship between CD96 and dNK cell cytokine function, we detected CD96+ and
CD96
−
NK cells and found that cytokines were significantly reduced on CD96+ NK cells
(Figure 3E,F).
3.4. After CD96 Antagonists Block NK Cells, the Adhesion of NK Cells to Stromal Cells and
Trophoblasts Decreases
To detect the role of CD96 at the maternal–foetal interface, we used CD96 antibodies
as antagonists and added them to the coculture system at a concentration of 10
µ
g/mL.
The adherent and suspended cells were separately labelled using the Cell Adhesion Assay
Kit. Here, we labelled adherent cells with green fluorescence, i.e., DSCs or HTR8 cells, and
suspended cells, i.e., NK cells, with red fluorescence (Figure 4A–D). The results showed
that the adhesion of NK cells to stromal cells decreased after the addition of antagonists. In
addition, we collected NK cells for adhesion molecular assays using flow cytometry and
found that the expression of the three representative adhesion molecules decreased after
the addition of CD96 antagonists (Figure 4E). This finding suggests that CD96 mediates the
adhesion function of NK cells in situ, helping to maintain normal pregnancy. Adhesion
decreased after the addition of antagonists. In the context of the NK cell and HTR8
coculture, changes in NK cell activity after the addition of CD96 antagonists were detected.
We used flow cytometry to detect three cytokines within NK cells. Among them, IFN-
γ
is a
cytokine associated with cytotoxicity and Ki-67 is a proliferation marker that is positively
correlated with NK cell activity. The IL-10 inhibitory receptor molecule was negatively
correlated with NK cell activity. After coculture for 24 h, the cells were stimulated with
the cell activation cocktail. The results of flow cytometry showed that after the addition
of antagonists, the expression intensity of IFN-
γ
increased significantly, the expression
intensity of IL-10 decreased significantly, and the expression intensity of Ki-67 increased
significantly (Figure 4F).
Bioengineering 2023,10, 1008 9 of 17
Bioengineering 2023, 10, x FOR PEER REVIEW 9 of 18
Figure 3. Functional molecule expression on CD96+ dNK cells and CD96− dNK cells: (A) The pop-
ulation and proportion of NK cells in primary cells. (B) The expression of CD96 on NK cells circled
in (A). (C) Histogram of the expression intensity of the three adhesion factors: CD54, CD62E, and
CD106, as tested via flow cytometry. (D) The mean fluorescence intensity of the three adhesion fac-
tors was measured via flow cytometry, and the differences were calculated. (E) Histogram of the
expression intensity of IFN-γ and granzyme B as measured via flow cytometry. (F) The mean fluo-
rescence intensity of the two cytokines was measured via flow cytometry, and the difference was
calculated. MFI: mean fluorescence intensity. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
3.4. After CD96 Antagonists Block NK Cells, the Adhesion of NK Cells to Stromal Cells and
Trophoblasts Decreases
To detect the role of CD96 at the maternal–foetal interface, we used CD96 antibodies
as antagonists and added them to the coculture system at a concentration of 10 μg/mL.
The adherent and suspended cells were separately labelled using the Cell Adhesion Assay
Kit. Here, we labelled adherent cells with green fluorescence, i.e., DSCs or HTR8 cells, and
suspended cells, i.e., NK cells, with red fluorescence (Figure 4A–D). The results showed
that the adhesion of NK cells to stromal cells decreased after the addition of antagonists.
In addition, we collected NK cells for adhesion molecular assays using flow cytometry
and found that the expression of the three representative adhesion molecules decreased
after the addition of CD96 antagonists (Figure 4E). This finding suggests that CD96 medi-
ates the adhesion function of NK cells in situ, helping to maintain normal pregnancy. Ad-
hesion decreased after the addition of antagonists. In the context of the NK cell and HTR8
Figure 3.
Functional molecule expression on CD96+ dNK cells and CD96
−
dNK cells: (
A
) The
population and proportion of NK cells in primary cells. (
B
) The expression of CD96 on NK cells
circled in (
A
). (
C
) Histogram of the expression intensity of the three adhesion factors: CD54, CD62E,
and CD106, as tested via flow cytometry. (
D
) The mean fluorescence intensity of the three adhesion
factors was measured via flow cytometry, and the differences were calculated. (
E
) Histogram of
the expression intensity of IFN-
γ
and granzyme B as measured via flow cytometry. (
F
) The mean
fluorescence intensity of the two cytokines was measured via flow cytometry, and the difference was
calculated. MFI: mean fluorescence intensity. *: p< 0.05; **: p< 0.01; ***: p< 0.001.
Bioengineering 2023,10, 1008 10 of 17
Bioengineering 2023, 10, x FOR PEER REVIEW 10 of 18
coculture, changes in NK cell activity after the addition of CD96 antagonists were de-
tected. We used flow cytometry to detect three cytokines within NK cells. Among them,
IFN-γ is a cytokine associated with cytotoxicity and Ki-67 is a proliferation marker that is
positively correlated with NK cell activity. The IL-10 inhibitory receptor molecule was
negatively correlated with NK cell activity. After coculture for 24 h, the cells were stimu-
lated with the cell activation cocktail. The results of flow cytometry showed that after the
addition of antagonists, the expression intensity of IFN-γ increased significantly, the ex-
pression intensity of IL-10 decreased significantly, and the expression intensity of Ki-67
increased significantly (Figure 4F).
Bioengineering 2023, 10, x FOR PEER REVIEW 11 of 18
Figure 4. After the addition of CD96 antagonists, the function of NK cells in the coculture system
decreased significantly. (A) Cell fluorescence staining assay to detect the adhesion of NK cells to
DSCs before and after the addition of antagonists (400× magnification). (B) Cell fluorescence stain-
ing to detect the adhesion of NK cells to HTR8 cells before and after the addition of antagonists
(400× magnification). (C) Number of dNK cells adhered to the surface of DSCs. (D) Number of dNK
cells adhered to the surface of HTR8. (E) Flow cytometry to detect the effect of adding antagonists
on the expression of NK cell adhesion molecules. (F) Flow cytometry to detect the expression inten-
sity histogram of three cytokines. (G) Flow cytometry was used to detect the average fluorescence
intensity of the three cytokines and count their differences. MFI: mean fluorescence intensity. *: p <
0.05; **: p < 0.01; ***: p < 0.001.
3.5. Low-Dose Palmitic Acid Can Regulate CD96 Expression, Thereby Affecting Cellular Oxida-
tive Function
We detected CD155 molecules on the surface of HTR8 cells and CD96 molecules on
the surface of dNK cells via flow cytometry after the addition of palmitic acid to the cul-
ture system with single culture or coculture of dNK cells and HTR8 cells. To further in-
vestigate the beneficial effects of low-dose palmitic acid at the maternal–foetal interface,
we treated the cells with a low concentration of 10 μM palmitic acid. The expression of
surface CD155 molecules was significantly higher in the PA group when 10 μM palmitic
acid was added to HTR8 cells compared to the control group of HTR8, where no addi-
tional reagents were added or other treatments were performed in addition to the con-
ventional medium (Figure 5A). And when HTR8 cells were cocultured with dNK cells,
the addition of palmitic acid in the PA group was not significantly different from the con-
trol group without palmitic acid in terms of the expression of CD155 on the surface of
HTR8 (Figure 5B). The PA group of dNK cells with palmitic acid addition showed no
difference in CD96 expression compared to the control group of dNK cells cultured alone.
In contrast, when dNK cells were cocultured with HTR8 cells, the expression of CD96 on
dNK cells was significantly higher in the PA group with palmitic acid addition compared
with the control group without palmitic acid addition (Figure 5C,D). To further explore
the effect of palmitic acid on NK cells, we added CD96 antagonists to the culture system
to investigate whether palmitic acid can affect the function of NK cells through its inter-
action with CD96. We tested the oxidative function of NK cells under different conditions
using a Reactive Oxygen Species (ROS) probe kit, and the results showed that the addition
of palmitic acid reduced the ROS content of NK cells both in the PA group and in the PA-
Figure 4.
After the addition of CD96 antagonists, the function of NK cells in the coculture system
decreased significantly. (
A
) Cell fluorescence staining assay to detect the adhesion of NK cells
to DSCs before and after the addition of antagonists (400
×
magnification). (
B
) Cell fluorescence
staining to detect the adhesion of NK cells to HTR8 cells before and after the addition of antagonists
(
400×magnification
). (
C
) Number of dNK cells adhered to the surface of DSCs. (
D
) Number of dNK
Bioengineering 2023,10, 1008 11 of 17
cells adhered to the surface of HTR8. (
E
) Flow cytometry to detect the effect of adding antagonists on
the expression of NK cell adhesion molecules. (
F
) Flow cytometry to detect the expression intensity
histogram of three cytokines. (
G
) Flow cytometry was used to detect the average fluorescence intensity
of the three cytokines and count their differences. MFI: mean fluorescence intensity.
*: p< 0.05
;
**: p< 0.01; ***: p< 0.001.
3.5. Low-Dose Palmitic Acid Can Regulate CD96 Expression, Thereby Affecting Cellular
Oxidative Function
We detected CD155 molecules on the surface of HTR8 cells and CD96 molecules on
the surface of dNK cells via flow cytometry after the addition of palmitic acid to the culture
system with single culture or coculture of dNK cells and HTR8 cells. To further investigate
the beneficial effects of low-dose palmitic acid at the maternal–foetal interface, we treated
the cells with a low concentration of 10
µ
M palmitic acid. The expression of surface
CD155 molecules was significantly higher in the PA group when 10
µ
M palmitic acid was
added to HTR8 cells compared to the control group of HTR8, where no additional reagents
were added or other treatments were performed in addition to the conventional medium
(Figure 5A). And when HTR8 cells were cocultured with dNK cells, the addition of palmitic
acid in the PA group was not significantly different from the control group without palmitic
acid in terms of the expression of CD155 on the surface of HTR8 (Figure 5B). The PA group of
dNK cells with palmitic acid addition showed no difference in CD96 expression compared
to the control group of dNK cells cultured alone. In contrast, when dNK cells were
cocultured with HTR8 cells, the expression of CD96 on dNK cells was significantly higher in
the PA group with palmitic acid addition compared with the control group without palmitic
acid addition (Figure 5C,D). To further explore the effect of palmitic acid on NK cells, we
added CD96 antagonists to the culture system to investigate whether palmitic acid can affect
the function of NK cells through its interaction with CD96. We tested the oxidative function
of NK cells under different conditions using a Reactive Oxygen Species (ROS) probe kit,
and the results showed that the addition of palmitic acid reduced the ROS content of NK
cells both in the PA group and in the PA-HTR8 group cocultured with HTR8, compared
to the control group in which NK cells were cultured without any treatment, indicating
that palmitic acid inhibited the oxidative function of NK cells. After the addition of CD96
antagonists, the ROS content of NK cells was partially restored (Figure 5E). In addition,
we used a mitochondrial membrane potential kit to identify changes in the mitochondrial
membrane potential of cells by detecting changes in the ratio of JC-1 polymers/monomers,
thereby reflecting alterations in cell activity. In general, a higher mitochondrial membrane
potential indicates higher cell activity, and a lower mitochondrial membrane potential
indicates possible apoptosis, while the three experimental groups after the addition of
palmitic acid showed no statistic difference. The results showed that the addition of palmitic
acid could potentially inhibit the mitochondrial membrane potential of NK cells compared
to the control group, in which NK cells were cultured alone. However, when coculturing
dNK cells with HTR8 cells, the addition of palmitic acid and the CD96 antagonist did
not yield significant differences in the detection of mitochondrial membrane potential
(Figure 5F). We examined changes in the expression of functional molecules in NK cells
after the addition of palmitic acid and/or CD96 antagonists, using dNK cells and HTR8
cocultures as a control group. We used flow cytometry to assess three adhesion molecules
within NK cells. These adhesion molecules were negatively correlated with NK cell activity.
After a total of 24 h of incubation, flow cytometry was performed. The results of flow
cytometry showed that after the addition of palmitic acid, the three adhesion molecules
were upregulated as expected, indicating that the adhesion function of NK cells increased
and shifted to a more stable phenotype. When CD96 antagonists were added, all three
adhesion molecules decreased, suggesting that CD96 antagonists relieved the effect of
palmitic acid on the enhancement of NK cell adhesion function (Figure 5G,H). In addition,
we examined the expression levels of molecules in NK cells under the above conditions,
and found that the expression levels of proliferation marker Ki-67 and cytokine IFN-
γ
Bioengineering 2023,10, 1008 12 of 17
decreased significantly after the addition of palmitic acid, while the expression levels of the
two increased significantly after the addition of CD96 antagonists, which was not different
from the control group. The expression level of inhibitory cytokine IL-10 is the opposite of
the former (Figure 5I,J).
Bioengineering 2023, 10, x FOR PEER REVIEW 13 of 18
Figure 5.
Palmitic acid may inhibit NK cell activity through CD96 in dNK cells. (
A
) Effect of palmitic
acid on CD155 expression in HTR8 T cells. (B) Effect of palmitic acid on CD155 expression in HTR8
Bioengineering 2023,10, 1008 13 of 17
cells cocultured with dNK cells. (
C
) Effect of palmitic acid on CD96 expression in dNK cells. (
D
) Effect
of palmitic acid on CD96 expression in dNK cells cocultured with HTR8 cells. (
E
) ROS content of dNK
cells under different conditions. (
F
) Ratio of JC-1 polymer/monomer of dNK cells under different
conditions. (
G
) Histogram of the expression intensity of the three adhesion molecules detected via
flow cytometry. (
H
) The mean fluorescence intensity of the three adhesion molecules was measured
via flow cytometry, and the differences were calculated. (
I
) Histogram of the expression intensity of
the three adhesion cytokines detected via flow cytometry. (
J
) The mean fluorescence intensity of the
three cytokines were measured via flow cytometry, and the differences were calculated. PA: palmitic
acid; MFI: mean fluorescence intensity. ns: p> 0.05; *: p< 0.05; **: p< 0.01; ***: p< 0.001.
4. Discussion
In this study, we found that the expression of CD96 in dNK cells in the uterine
decidua was significantly different from that in uNK cells in the endometrium. The low
expression of CD96 in spontaneous abortion patients indicates the enhanced activity of
NK cells, which may be one of the possible factors contributing to pregnancy failure. On
the other hand, there is a strong correlation between spontaneous abortion and increased
production of proinflammatory factors, such as cytokines TNF-
α
and IFN-
γ
, and conversely,
a reduction in the anti-inflammatory cytokine IL-10 is also associated with spontaneous
abortion. The associated cytokines and immune status were also verified in this experiment,
demonstrating the decrease in granzyme B and IFN
γ
caused by the immunosuppressive
state mediated by CD96. Notably, a recent study by Habets et al. found increased expression
of CD96 on PBMCs from recurrent pregnancy loss samples, in contrast to the current study
that found reduced CD96 expression on dNK cells. Many phenotypic differences between
peripheral blood immune cells and maternal–foetal interface immune cells have been
reported, and the underlying mechanisms of the differential phenotypic changes in CD96
at the peripheral blood and maternal–foetal interfaces need further exploration [22].
Indeed, CD155, CD96, CD112, and TIGIT form a subfamily of associated IgSF receptors
that make up the stimulus/inhibition network. Some scholars classify these receptors as
members of the CD155 family, and multiple intricate interactions exist among them [
23
].
Especially in tumour tissues, the interaction of TIGIT with CD155 has been applied to
clinical tumour treatment [
24
]. Therefore, in this chapter, we conducted further testing of
this family of molecules using flow cytometry, and the results showed that the expression of
CD96 on immune cells was consistent with the results of immunofluorescence, while there
was no significant difference in the expression of TIGIT between the endometrium tissue
and normal pregnancy decidua. This led us to consider that CD96 may exert an immune tol-
erance function at the maternal–foetal interface by binding to CD155. Correspondingly, we
measured the expression of CD155 and CD112 in the endometrium and decidua of normal
pregnancy and found that CD155 expression was higher in the decidua, which was consis-
tent with the aforementioned immunofluorescence results. Additionally, the expression of
CD112 in the decidua was also significantly higher than that in the endometrium. However,
a literature review revealed that CD112 primarily interacts with CD112R and TIGIT to exert
immunosuppressive effects, while single-cell sequencing of the maternal–foetal interface
shows that CD112R is scarcely expressed at this site, leading to the limited focus on CD112
here [
25
]. Based on the above findings, we directed our research towards the functional
expression of CD96 in the presence of CD155 at the maternal–foetal interface.
Trophoblast cells share many characteristics with tumour cells in inducing and main-
taining immune tolerance, especially in inducing immune tolerance in NK cells [
26
]. There
are a considerable number of dNK cells in the decidua, accounting for approximately
70% of the total number of deciduous lymphocytes [
27
]. During pregnancy, the decidua
maintains close contact with trophoblasts without causing damage because trophoblasts ex-
ert an immunosuppressive effect on dNK cells [
28
]. NK cells in the decidua are a dominant
subpopulation, and various factors with inhibitory effects on NK cells are locally present. It
has been confirmed that trophoblast cells are resistant to the killing effect of NK cells, and
in vitro
experiments have shown that purified decidual NK cells fail to produce a cytotoxic
Bioengineering 2023,10, 1008 14 of 17
response against freshly isolated trophoblast cells. After activation by interleukin-2/IL-2,
certain cytotoxic activity against choriocarcinoma cells can be obtained, but trophoblast
cells with activated decidual NK cells still retain some degree of resistance [
29
]. Our experi-
ments also showed that under the conditions of trophoblast cell coculture with NK cells,
the cytokine secretion function of NK cells was significantly inhibited, and the expression
of cytokines and adhesion molecules was significantly altered. In contrast, the expression
of the immune tolerance molecule IL-10 was increased in the coculture.
In this study, we found abnormalities in the expression of CD96 at the pathological
maternal–foetal interface of pregnancy, and we also found that CD96 plays an important
role in immunosuppression and inducing immune tolerance on the surface of NK cells.
In the experiments in this chapter, after we blocked the action of trophoblasts and decid-
ual stromal cells with CD96 using CD96-specific antagonists, the adhesion of dNK cells
decreased significantly, and the expression of the corresponding tolerant cytokine IL-10
was also significantly reduced, while the expression of killer cytokines increased, and the
proliferative activity of NK cells showed a substantial increase. This change marks the
transformation of dNK cells from a robust immune-tolerant phenotype to an active im-
munoaggressive phenotype, thus demonstrating the substantial impact of CD96 expression
on the functional status of dNK cells. If the expression of CD96 in NK cells can be increased
in some way to inhibit the function of dNK cells, it could potentially have a beneficial
impact on maintaining pregnancy.
Although decidual NK cells exhibit low activity, they can still exert cytotoxic effects
on trophoblast cells under certain conditions [
18
]. In the early stage of embryonic develop-
ment, decidual NK cells can regulate the growth of the placenta via direct anti-placental
trophoblast cytotoxic activity, and as mentioned earlier, spontaneous abortion is also as-
sociated with excessive cytotoxic activity [
30
]. Studies have shown that the cytotoxic
activity of IL-2-activated decidual dNK cells against trophoblast cell tumours surpasses
that against normal trophoblast cells. This observation suggests that decidual dNK cells
likely contribute to regulating trophoblast cell invasion [
31
,
32
]. It has also been reported
that trophoblast cells interact with dNK cells, resulting in the synthesis and secretion
of certain growth factors that stimulate trophoblast cell proliferation, indicating that the
cytotoxic activity of dNK cells is widely inhibited during successful pregnancies [33].
An abnormal dNK cell ratio or function is closely related to the occurrence of recurrent
pregnancy loss [
34
]. The cause of the other 50% of cases is unknown, and this condition is
called unexplained RPL [
35
]. These unexplained cases are often associated with immune
disorders. Multiple studies have proposed a potential connection between the abnormal
number and subpopulation of NK cells, which may relate to uRPL. Most studies have
shown that NK cell concentrations are higher in women with uRPL than in healthy fertile
women. In addition, NK cells and trophoblast cells interfere with each other. Trophoblast
cells can transmit signals to dNK cells through direct contact or regulate the function of
NK cells by expressing and secreting a variety of cytokines and chemokines. IL-8, secreted
by dNK cells, has been reported to play an important role in placental formation and
trophoblast cell invasion [
26
,
36
]. Overall, the abnormal interaction between dNK cells and
trophoblast cells exhibited a strong correlation with pregnancy failure and miscarriage.
Previous studies and the above experiments showed that the enrichment of NK cells during
pregnancy will disrupt the homeostasis of pregnancy, and, of course, the intervention of
external factors can significantly affect this process [37,38].
Interventional factors can be useful as targets for clinical treatment. Rapamycin, for
example, has been shown to mediate autophagy in immune cells during pregnancy while
maintaining pregnancy homeostasis [
21
,
27
]. Previous studies have shown that palmitic acid
exerts a proinflammatory effect at the maternal–foetal interface, which partially explains
why adverse pregnancy events are high in obese patients. Studies have also found that
fatty acids accumulate early in the maternal–foetal interface [
19
,
39
]. Therefore, we studied
the role of palmitic acid in the maternal–foetal interface. Palmitic acid, in this study, played
a special role, and we found that palmitic acid can affect the oxidative activity of NK cells
Bioengineering 2023,10, 1008 15 of 17
and inhibit the activity by downregulating the secretion of toxic cytokines. However, the
expression of the immune tolerance molecule IL 10, as well as the adhesion molecules
ICAM-1, VCAM, and SELE, was significantly increased. This may mean that a low dose of
palmitic acid has the potential to keep early pregnancies in a stable state.
Our previous study showed that the adhesion function of NK cells during pregnancy
is highly correlated with pregnancy status. In the normal endometrium, the activity
function of NK cells is significantly increased, thus mediating immune rejection to protect
the endometrium against external harmful factors. At this time, NK cell adhesion is
poor, and motility is increased. However, after pregnancy, NK cells transform to a dNK
phenotype with a low cytokine secretion function, thus ensuring the stable implantation
and development of embryonic tissues containing heterologous genes. Moreover, due to
the increased adhesion molecules of NK cells, such as ICAM-1, ICAM-2, VCAM, SELE,
SELL, and SELP, NK cells remain more active locally and play a role in maintaining
immune homeostasis.
In summary, as shown in Figure 6, maternal–foetal interface trophoblasts can reduce
and regulate decidual NK cells to promote the secretion of cytokines such as IFN-
γ
and
granzyme B; upregulate the expression of the inhibitory cytokine IL-10; upregulate the
expression of adhesion factors such as CD54, CD106, and CD62E; mediate the in situ
adhesion of decidual NK cells; and exert an inhibitory effect on the immunotoxicity of
dNK cells. The value of the results of this study for clinical application needs to be further
explored in future research.
Bioengineering 2023, 10, x FOR PEER REVIEW 16 of 18
acid exerts a proinflammatory effect at the maternal–foetal interface, which partially ex-
plains why adverse pregnancy events are high in obese patients. Studies have also found
that fatty acids accumulate early in the maternal–foetal interface [19,39]. Therefore, we
studied the role of palmitic acid in the maternal–foetal interface. Palmitic acid, in this
study, played a special role, and we found that palmitic acid can affect the oxidative ac-
tivity of NK cells and inhibit the activity by downregulating the secretion of toxic cyto-
kines. However, the expression of the immune tolerance molecule IL 10, as well as the
adhesion molecules ICAM-1, VCAM, and SELE, was significantly increased. This may
mean that a low dose of palmitic acid has the potential to keep early pregnancies in a
stable state.
Our previous study showed that the adhesion function of NK cells during pregnancy
is highly correlated with pregnancy status. In the normal endometrium, the activity func-
tion of NK cells is significantly increased, thus mediating immune rejection to protect the
endometrium against external harmful factors. At this time, NK cell adhesion is poor, and
motility is increased. However, after pregnancy, NK cells transform to a dNK phenotype
with a low cytokine secretion function, thus ensuring the stable implantation and devel-
opment of embryonic tissues containing heterologous genes. Moreover, due to the in-
creased adhesion molecules of NK cells, such as ICAM-1, ICAM-2, VCAM, SELE, SELL,
and SELP, NK cells remain more active locally and play a role in maintaining immune
homeostasis.
In summary, as shown in Figure 6, maternal–foetal interface trophoblasts can reduce
and regulate decidual NK cells to promote the secretion of cytokines such as IFN-γ and
granzyme B; upregulate the expression of the inhibitory cytokine IL-10; upregulate the
expression of adhesion factors such as CD54, CD106, and CD62E; mediate the in situ ad-
hesion of decidual NK cells; and exert an inhibitory effect on the immunotoxicity of dNK
cells. The value of the results of this study for clinical application needs to be further ex-
plored in future research.
Figure 6. Low-dose palmitic acid upregulates the surface expression of CD155 on trophoblast cells
and CD96 on decidual natural killer (dNK) cells, resulting in decreased expression of cytotoxic cy-
tokines in dNK cells. Moreover, it leads to an increase in inhibitory cytokine IL-10 and adhesion
molecules ICAM1, VCAM1, and SELE. These changes signify the transition of dNK cell function
Figure 6.
Low-dose palmitic acid upregulates the surface expression of CD155 on trophoblast cells
and CD96 on decidual natural killer (dNK) cells, resulting in decreased expression of cytotoxic
cytokines in dNK cells. Moreover, it leads to an increase in inhibitory cytokine IL-10 and adhesion
molecules ICAM1, VCAM1, and SELE. These changes signify the transition of dNK cell function
towards an immune-tolerant phenotype. Additionally, there is a significant decrease in the expression
of the proliferation marker Ki-67 in dNK cells. These findings suggest the potential involvement of
these factors in the regulation of early-pregnancy maternal–foetal immune tolerance.
Bioengineering 2023,10, 1008 16 of 17
Author Contributions:
Y.W. (Yun Wang) supervised the entire study, including the procedures,
conception, design, and completion. Y.W. (Yun Wang) participated in the interpretation of the
study data and in revisions to the article. Y.W. (Yingjie Wang) was responsible for conducting the
experiments, data analysis, and article writing. All authors have read and agreed to the published
version of the manuscript.
Funding:
This study was supported by the National Natural Science Foundation of China (NSFC)
(31770989 to Y.W.).
Institutional Review Board Statement:
This study protocol was approved by the ethical committee
of the hospital (reference number 2017-211) and was carried out in accordance with the Helsinki
Declaration.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy or ethical reasons.
Acknowledgments:
We gratefully acknowledge all the staff of the Department of Assisted Reproduc-
tion in Shanghai Ninth People’s Hospital for their support and cooperation.
Conflicts of Interest: The authors declare no conflict of interest.
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