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In-Vitro Evaluation of Immunomodulation Effects of Mesenchymal Stem Cell-Derived Exosomes in Refractory Chronic Spontaneous Urticaria

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doi: 10.21911/aai.120
Asthma Allergy Immunol 2023;21:45-54
RESEARCH ARTICLE
ASTHMA
ALLERGY
IMMUNOLOGY
ASTIM
ALLERJİ
İMMÜNOLOJİ
45
Copyright © 2023 The Author(s). This is an open-access article published by Turkish National Society of Allergy and Clinical Immunology under the terms of the Creative Commons Attribution
License (CC BY NC) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No use, distribution or reproduction
is permitted which does not comply with these terms.
In-Vitro Evaluation of Immunomodulation Effects of
Mesenchymal Stem Cell-Derived Exosomes in Refractory
Chronic Spontaneous Urticaria
Alper Tunga OZDEMIR1 , Cengiz KIRMAZ2 , Rabia Bilge OZGUL OZDEMIR3 , Mustafa OZTATLICI4 ,
Pinar KILICARSLAN SONMEZ4 , Mehmet Ibrahim TUGLU4
ABSTRACT
Objective: Approximately half of chronic spontaneous urticaria (CSU) patients are thought to have an autoimmune pathology, and they
are resistant to current treatment approaches. Mesenchymal stem cells (MSCs) are adult cells that have been shown to be useful in many
autoimmune pathologies due to their immunomodulation properties. This study aimed to investigate the immunomodulatory effects of
MSCs, and exosomes isolated from refractory CSU patients.
Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 5 refractory CSU patients and 5 healthy
volunteers. The effects of MSCs isolated from CSU patients and healthy MSCs were compared. Co-culture experiments were performed
to evaluate the efficacy of Mesenchymal Stem Cells and exosomes on PBMCs of CSU patients and healthy volunteers. To compare the
resulting effects, changes in IFN-γ, IL-4, IL-10, IL-17a, and TGF-β cytokines were detected by the ELISA method. Cell proliferations
were detected with the CCK-8 kit.
Results: The effects of autologous and allogeneic MSCs on IFN- γ expressions were similar, both providing significant suppression at all
cell ratios. However, IL-4 and IL-10 expression of PBMCs co-cultured with allogeneic MSCs significantly decreased while IL-17a and
TGF-β expression increased significantly. In addition, our findings indicated that exosomes were capable of significant suppression at
low PBMC ratios, regardless of autologous or allogeneic origin, but MSCs were more effective as the number of PBMCs increased.
Conclusion: These preliminary findings from in-vitro experiments suggested that allogeneic MSC, or high-dose exosome administration
may be a potential approach for treatment in CSU patients, most of whom are regarded as suffering from an autoimmune disease and
resistant to current treatments. However, our findings need to be supported by clinical studies.
Keywords: Chronic spontaneous urticaria, mesenchymal stem cells, exosome, immunomodulation
1Medical Biochemistry Laboratory, Merkezefendi State Hospital, Manisa, Turkey
2Department of Allergy and Clinical Immunology, Manisa Celal Bayar University, School of Medicine, Manisa, Turkey
3Clinic of Allergy and Clinical Immunology, Manisa City Hospital, Manisa, Turkey
4Department of Histology and Embryology, Manisa Celal Bayar University, School of Medicine, Manisa, Turkey
INTRODUCTION
Urticaria is a skin disease characterized by itchy, edem-
atous, round, or oval shaped, red, and raised lesions on the
skin ranging in size from a few millimeters to centimeters.
The disease occurs in the form of episodes and if this con-
dition lasts longer than 6 weeks it is called chronic urti-
caria (CU). Skin findings may be caused by certain stimuli
such as cold, pressure, sun exposure and this condition is
called inducible urticaria. However, in most cases, there is
no identifiable trigger, and this group of patients is defined
as chronic spontaneous urticaria (CSU) (1). The interac-
tion of allergen-IgE complexes with the FcεRI receptor
on the surface of mast cells causes degranulation, and the
resulting mediators such as histamine, tryptase and leu-
ORCID Alper Tunga Ozdemir / 0000-0002-7708-077X, Cengiz Kirmaz / 0000-0001-8873-1681, Rabia Bilge Ozgul Ozdemir / 0000-0002-8171-3402,
Mustafa Oztatlici / 0000-0001-9914-7122, Pinar Kilicarslan Sonmez / 0000-0002-9413-1735, Mehmet Ibrahim Tuglu / 0000-0002-0569-8415
Corresponding Author: Alper Tunga Ozdemir * alpertungaozdemir@outlook.com
Received: 10.10.2022 • Accepted: 17.01.2023
Online Published: 13.03.2023
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The Effects of MSC-Derived Exosomes on CSU
Asthma Allergy Immunol 2023;21:45-54
kotrienes cause skin manifestations. Although there is no
history of allergy, mast cell activation and skin symptoms
are the main findings in CSU cases. It has been reported
that mast cells are activated either by an autoantibodies
of type IgG against FcεRI (type IIb autoimmune CSU) or
by auto-IgEs that complex with the auto-allergen (type I
autoimmune CSU) (2,3). Specifically, autoantibodies to
IgE or IgE receptors are thought to be present in 30-40% of
CSU cases (3,4). Omalizumab is a biological therapy agent
that blocks the interaction of IgE and FcεRI and has been
shown to be effective in many CSU patients (5). However,
there is a significant group of CSU patients who do not
even respond to omalizumab therapy, and autoantibod-
ies to FceRI may be the cause of this resistance. Although
there are studies showing that tumor necrosis factor alpha
inhibitors and intravenous immunoglobulin treatments
can be beneficial, apart from the treatment algorithm
recommended in the current guideline, these costly treat-
ments may not provide a complete cure and may cause
serious side effects (6,7). The skin symptoms of CSU have
a direct impact on patients’ quality of life, and the chronic-
ity of the disease results in the perception that the patient
will never recover, resulting in anxiety and serious damage
to social relationships and the emotional state (8). For this
reason, the search for an effective treatment alternative
for resistant CSU patients continues intensively. One of
these is Mesenchymal stem cells (MSCs), which have been
shown to be beneficial in autoimmune diseases.
MSCs are multipotent adult cells, and they can be iso-
lated from various tissues such as adipose tissue, bone
marrow, and dental pulp. Since they are a heterogene-
ous group of cells, some criteria have been determined to
define them. MSCs are cells that adhere to plastic surfac-
es; they can differentiate into cell types of mesenchymal
origin such as chondrocytes, adipocyte, and osteocytes;
and in terms of surface markers they must express CD73,
CD90, and CD105 molecules, but not express CD11b,
CD14, CD19, CD34, CD45, and HLA-DR (9). MSCs are
immunomodulatory cells that suppress immune cells like
T cells (10), B cells, natural killer (NK) cells (11), and anti-
gen-presenting cells such as macrophages and dendritic
cells (12), but activate regulatory T and regulatory B cells
(10,13). The roles of indolamine 2.3 dioxygenase (IDO),
prostaglandin E2 (PGE2), interleukin (IL)-10, hepato-
cyte growth factor, human leukocyte antigen (HLA)-G,
and transforming growth factor (TGF) -β on the immu-
nomodulation effects of MSCs are well elucidated (14).
Therefore, MSCs have become an attractive research
area for immune-related pathological conditions such as
Crohn’s disease, systemic lupus erythematosus, multiple
sclerosis, and even graft-versus-host disease (15). Recent-
ly, it has been reported that not only MSCs but also extra-
cellular vesicles obtained from MSCs can provide similar
immunomodulation. Exosomes originating from MSCs
provide a paracrine effect in the form of the delivery of
their cytoplasmic contents consisting of DNA, RNA, and
proteins to target cells (16). Considering the potential
effects of exosomes that can be as effective as MSCs, it is
understood that they are of great importance in the devel-
opment of cell-free and effective treatment approaches.
MSCs may be a potential option in the treatment of refrac-
tory CSU cases considered to have an autoimmune back-
ground. In a clinical trial with a small study group, we
showed the positive effects of MSCs on refractory CSU
cases (17). However, there is no study showing the effects
of MSC-derived exosomes on refractory CSU. Therefore,
we investigated the immunomodulatory effects of MSCs
and MSC-derived exosomes on immune cells belonging
to refractory CSU cases in this study. We aimed to obtain
preliminary and comparative data on the cell-free treat-
ment approaches for CSU in this way.
MATERIALS and METHODS
Patients and control subjects:
The protocol and content of this project was reviewed
and approved by the Manisa Celal Bayar University Fac-
ulty of Medicine Ethics Committee (Approval number:
27-09-2017 / 20.478.486). In this study, adipose tissue-
derived MSCs isolated from patients with refractory CSU
in our previous study, and the ATCC adipose-derived
MSC cell line were used (17). Venous blood samples were
taken from three female and two male CSU patients whose
MSCs were used for co-culture experiments and PBMC
isolation was performed. All patients were diagnosed
with CSU for at least one year (according to the EAACI/
GA2LEN/EDF/WAO guidelines) (18), and they were those
who could not experience disease control despite using
omalizumab and/or cyclosporine for six months or long-
er. Since the patients were unresponsive to omalizumab/
cyclosporine, their treatment was continued with high-
dose antihistamine drugs, and yet the weekly urticaria
activity score (UAS7) was greater than 20. Patients with
a history of chronic inducible urticaria, atopic dermatitis,
another underlying itchy skin disease, parasitic infection,
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Ozdemir AT, Kirmaz C, Ozgul Ozdemir RB, Oztatlici M, Kilicarslan Sonmez P, Tuglu MI
Asthma Allergy Immunol 2023;21:45-54
The protein content of the exosomes was extracted using
RIPA lysis buffer (ThermoFisher Scientific, USA), fol-
lowed by determining the amount of protein using the
BCA protein assay (ThermoFisher Scientific, USA). To
verify the exosomes, the Exo-FACS kit (Hansa Biomed,
Estonya) was used, and the exosomes loaded into the
latex-beads were labeled with CD63 Alexa 488 (Hansa
Biomed, Estonya), CD9 FITC (Clone: MEM-61, Exbio,
Czech Republic), and CD105 (Clone: MEM-226, Exbio,
Czech Republic) antibodies, and then analyzed with the
Accuri C5 Flow cytometry device (BD Biosciences, USA).
Co-Culture
To evaluate immunomodulatory interactions, 1x104
cells/well MSCs were seeded in appropriate positions of a
96-well plate and incubated for 24 hours to attach. CSU
patients (uP) and healthy control PBMCs (hP) were acti-
vated with v/v 2% phytohemagglutinin (PHA) M (Gibco,
USA) for 48 hours before the co-culture, and then 1x104
(1:1), 5x104 (5:1) and 1x105 (10:1) cells/well were added to
appropriate positions. To evaluate the effects of exosomes,
1x104, 5x104, and 1x105 activated PBMCs were added to
the appropriate wells of another 96-well culture dish and
10 μg/mL of exosomes isolated from each patient’s own
MSCs (uExo) or ATCC-MSCs (hExo) were added; the
cells were then incubated for 24 hours. The same number
of PHA-activated and unstimulated PBMCs was deter-
mined as positive and negative controls, and then all cells
were incubated for 24 hours. The co-culture experiment
plan is summarized in Figure 1.
and malignancy were excluded from the study. Five age-
and sex-matched healthy subjects without any allergies,
urticaria, itchy skin disease, autoimmune disease, parasitic
infection, or a history of malignancy were included in the
study as a control group.
Cell culture
To evaluate autologous interactions, adipose tissue-
derived MSCs isolated from refractory CSU patients in our
previous study were used (urticaria patient-derived MSC;
uMSC). Human adipose-derived MSCs (ATCC® PCS-500-
011 ™) were purchased from the American Type Culture
Collection to evaluate allogeneic interactions (healthy
donor MSC; hMSC). All MSCs were cultured by using
Dulbecco’s Modified Eagle’s medium-F12 (Biosera, USA)
medium that including 10% fetal bovine serum (FBS)
(Biosera, USA), 100 U/ml penicillin, 100 μg/ml streptomy-
cin (Biosera, USA) and 1% 2 mM L-glutamate (Biosera,
USA) at 37 oC and 5% CO2 incubator. Cells were con-
trolled daily, and sub-cultured when they reached 70-80 %
confluence. The 3rd passage of MSCs was used to perform
exosome isolation and co-culture experiments. Venous
blood samples of all patients were taken into BD Vacu-
tainer® Plus Plastic K2 EDTA tubes, and peripheral blood
mononuclear cells (PBMCs) were isolated using Lym-
phosep lymphocyte separation medium (density: 1.077,
Biosera, USA). PBMCs were cultivated in a culture medi-
um composed of Roswell Park Memorial Institute -1640
medium (Biosera, USA), 10% FBS (Biosera, USA), 100 U/
ml penicillin, 100 μg/ml streptomycin (Biosera, USA) and
1% 2 mM L-glutamate (Biosera, USA) at 37 oC and 5% CO2
incubator.
Exosome isolation
MSCs were grown to 90% confluency, the old medium
was removed, fresh medium containing exosome-depleted
FBS (Exo-FBS, System Biosciences, USA) was added, and
then cultured for another 3 days. Medium supernatants
were collected at the end of the incubation, and centrifu-
gation was performed for 30 minutes at speeds of 300g,
1.200g and 10.000g, respectively, in order to remove cells
and apoptotic residues in the medium supernatants. After
each centrifugation, the medium on the top was carefully
collected and transferred to another tube. Subsequently,
exosome isolation of each case was performed using the
Exo-Prep One-step Exosome Isolation kit and according
to the manufacturer’s protocol (Hansa Biomed, Estonya).
Figure 1. Schematic summary of co-culture experiments.
(u: CSU patient, h: Healthy control, MSC: Mesenchymal stem cell,
PBMC: Peripheral blood mononuclear cell)
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The Effects of MSC-Derived Exosomes on CSU
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groups with Shapiro-Wilk normality test results p<0.05.
The one-way ANOVA (Kruskal-Wallis for nonparamet-
ric groups) method was used to compare groups with each
other. Comparison data found as p <0.05 were considered
statistically significant.
RESULTS
Cell culture
Cultured cells were plastic adherent and had spindle
morphology. In flow cytometry analysis of the isolated
exosomes, CD9, CD63, and CD105 markers were found
to be positive (Figure 2). In PBMCs cultured with MSCs at
1:1 and 1:5 ratios, clustering, which is the indicator of acti-
vation, was not evident, but was present in the culture with
a ratio of 1:10. On the contrary, clusters were observed at
all cell ratios in PBMCs cultured with exosomes (Figure 3).
Cytokine alterations
First, we compared the cytokine levels of CSU patients
and healthy subjects (Figure 4). We observed that the
IFN-γ and IL-4 levels of hP cells at 1x104 cell ratios were
significantly higher than uPs (p = 0.01 and p= 0.02 respec-
tively), but there was no significant difference for 5x104 (p
= 0.46 and p = 0.91 respectively) and 1x105 cell ratios (p
= 0.98 and p= 0.86 respectively). For all cell ratios, IL-10
and TGF-β levels of uPs were significantly lower than hPs
(for 1x104: p<0.001 and p<0.001; for 5x104: p= 0.005 and
p<0.001; for 1x105: p<0.001 and p<0.001 respectively),
but IL-17a levels were significantly higher (for cell ratios
p<0.001).
Cell proliferation assays
The effects of MSCs and exosomes on the prolifera-
tion of PBMCs were determined by the Cell Counting
Kit-8 (CCK-8) (Abbkine Scientific Co. China). For this,
the number of cells used in the co-culture experimental
setup was used and after 24 hours of incubation, 10 μL of
CCK-8 reagent was added to each well and the cells were
re-incubated for 4 hours. Absorbances were measured at
450 nm using the ELX800 microplate reader.
ELISA
ELISA analyzes were performed to evaluate the effects
of MSCs and exosomes on inflammatory cytokine expres-
sions. The collected cell culture supernatants were cen-
trifuged at 4000 rpm for 10 minutes and cell debris was
removed. ELISA kits capable of measuring human IFN-γ,
IL-4, IL-6, IL-10, IL-17a, and TGF-β levels were purchased
from Diaclone, France. Cytokine changes were measured
in accordance with the manufacturer’s protocol. Absorb-
ances were determined using the Multiskan™ FC Micro-
plate Photometer (Thermo Scientific™, USA) instrument.
The PRISM v8 (GraphPad San Diego, USA) software was
used for the creation of standard curves and calculations.
Statistical Analysis
The data obtained were analyzed using the PRISM v8
(GraphPad San Diego, USA) statistical software. Normal-
ity and lognormality tests were performed to determine
whether the groups were in normal distribution and
nonparametric calculation methods were applied for the
Figure 2. Microscopic images of mesenchymal stem cells of chronic spontaneous urticaria patients (A-E) and ATCC cell line (F). Flow
cytometry histogram graphics of exosomes isolated from mesenchymal stem cells (G).
(PM: CSU patient mesenchymal stem cells, HM: Healthy mesenchymal stem cells)
D E F G
A B C
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Ozdemir AT, Kirmaz C, Ozgul Ozdemir RB, Oztatlici M, Kilicarslan Sonmez P, Tuglu MI
Asthma Allergy Immunol 2023;21:45-54
by u/hMSCs only. At all cell ratios, hPs’ IL-4 expressions
were significantly reduced by u/hMSCs and u/hExos. In
contrast, IL-4 expressions of uP were significantly reduced
at 1:1 and 1:5 cell ratios and only by hMSCs. IL-10 expres-
sions of uP were significantly reduced by u/hMSCs and
u/hExos at 1:1 cell ratios. However, while a significant
reduction was achieved with hMSCs and uMSCs at 1:5
We then compared the effects of MSCs and exosomes
on cytokine expressions. At 1:1 cell ratios, IFN-γ expres-
sion of uP and hP was significantly reduced by both u/
hMSCs and u/hExos. At 1:5 cell ratios, IFN-γ expression
of hP was significantly reduced by u/hMSCs and u/hExos,
but only u/hMSCs for uP led to significant reduction. As
for the 1:10 cell ratios, significant reduction was achieved
Figure 3. MRepresentative microscopy images of co-cultured mesenchymal stem cells and PBMCs at 1:1 (A), 1:5 (B) and 1:10 (C) cell
ratios, and PBMCs at 1x104 (D), 5x104 (E) and 1x105 (F) numbers cultured by adding 10 µg/mL exosome.
Figure 4. Comparison graphs of cytokine levels of activated peripheral blood mononuclear cell (PBMC) at dierent cell ratios of chronic
spontaneous urticaria patients (uPBMC) and healthy subjects (hPBMC). Data are presented as mean and standard deviation.
(*: p<0.05 statistically signicant)
A B C
D E F
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The Effects of MSC-Derived Exosomes on CSU
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IL-17a and TGF-β expression of uP or hP. The “p” val-
ues of the statistical comparison of the groups are given in
Table I, and comparison charts of the groups are presented
in Figure 5.
Cell proliferations
The optical density changes determined by the CCK-
8 assay are presented in Figure 5. Proliferations of PHA-
activated uP cells at 1:1 cell ratios were significantly
cell ratios, only hMSCs led to a significant reduction at
1:10 ratios. Interestingly, we found that the resulting sig-
nificant increase in IL-17a and TGF-β expressions of uP
were only caused by hMSCs across all cell ratios. How-
ever, both hMSCs and uMSCs led to a significant increase
in IL-17 expression of hP. At all cell proportions, TGF-β
expressions of hPs increased significantly with hMSCs, but
uMSCs only provided a significant increase at 1:1 and 1:5
cell ratios. Exosomes did not cause a significant change in
Table I: Table of “p” values obtained from statistical analysis.
Cytokine Groups hMSCs uMSCs hEXOs uEXOs
IFN-γ
uPBMCs (1:1) p<0.001 p= 0.001 p= 0.01 p= 0.04
hPBMCs (1:1) p<0.001 p<0.001 p= 0.001 p= 0.001
uPBMCs (1:5) p= 0.03 p= 0.02 p>0.99 p>0.99
hPBMCs (1:5) p<0.001 p<0.001 p<0.001 p<0.001
uPBMCs (1:10) p= 0.007 p= 0.003 p= 0.93 p= 0.89
hPBMCs (1:10) p<0.001 p<0.001 p= 0.14 p= 0.72
IL-4
uPBMCs (1:1) p=0.004 p= 0.94 p>0.99 p= 0.98
hPBMCs (1:1) p= 0.001 p= 0.001 p= 0.01 p= 0.007
uPBMCs (1:5) p=0.03 p= 0.96 p= 0.95 p>0.99
hPBMCs (1:5) p= 0.002 p= 0.007 p= 0.02 p= 0.01
uPBMCs (1:10) p>0.99 p= 0.90 p= 0.37 p= 0.51
hPBMCs (1:10) p<0.001 p<0.001 p= 0.005 p= 0.01
IL-10
uPBMCs (1:1) p<0.001 p<0.001 p= 0.002 p<0.001
hPBMCs (1:1) p<0.001 p<0.001 p<0.001 p<0.001
uPBMCs (1:5) p= 0.003 p= 0.02 p= 0.80 p= 0.81
hPBMCs (1:5) p<0.001 p= 0.005 p= 0.19 p= 0.22
uPBMCs (1:10) p= 0.02 p= 0.27 p>0.99 p>0.99
hPBMCs (1:10) p<0.001 P= 0.006 p= 0.14 p= 0.34
IL-17
uPBMCs (1:1) p= 0.02 p= 0.54 p= 0.48 p= 0.39
hPBMCs (1:1) p<0.001 p<0.001 p>0.99 p>0.99
uPBMCs (1:5) p= 0.02 p= 0.90 p= 0.98 p>0.99
hPBMCs (1:5) p<0.001 p<0.001 p= 0.19 p= 0.22
uPBMCs (1:10) p<0.001 p= 0.15 p= 0.14 p= 0.28
hPBMCs (1:10) p<0.001 p<0.001 p>0.99 p>0.99
TGF-β
uPBMCs (1:1) p= 0.003 p>0.99 p>0.99 p>0.99
hPBMCs (1:1) p= 0.006 p= 0.01 p>0.99 p>0.99
uPBMCs (1:5) p<0.001 p= 0.36 p= 0.82 p= 0.93
hPBMCs (1:5) p<0.001 p=0.04 p>0.99 p>0.99
uPBMCs (1:10) p<0.001 p= 0.94 p= 0.83 p= 0.52
hPBMCs (1:10) p= 0.04 p= 0.80 p>0.99 p>0.99
u: Chronic spontaneous urticaria patients, h: Healthy control, PBMC: Peripheral blood mononuclear cells, MSC: Mesenchymal stem cell,
EXO: Exosome
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Asthma Allergy Immunol 2023;21:45-54
significantly suppressed with both hMSCs (p<0.001) and
uMSCs (p<0.001), and the suppression induced by hMSCs
was significantly higher than for uMSCs (p= 0.02).
DISCUSSION
In this study, we investigated the in vitro immu-
nomodulation effects of MSCs isolated from adipose tis-
sues of refractory CSU patients and exosomes obtained
from these cells. As a control, we used allogeneic adipose
tissue MSCs, and exosomes obtained from a healthy indi-
vidual. We evaluated how these interactions changed at
different MSC:PBMC or exosome:PBMC ratios. IFN-γ
is a pro-inflammatory cytokine that enhances the activa-
tion of immune cells, major histocompatibility complex
(MHC) expressions, immunoglobulin productions, and
production of reactive oxygen derivatives (19). In stud-
ies performed with serum samples of CSU cases, it was
reported that IFN-γ levels were significantly higher than in
healthy controls, and this elevation decreased significantly
suppressed by hMSCs (p<0.001), uMSCs (p<0.001), hExo
(p<0.001), and uExo (p<0.001). The suppression created
by hMSCs and uMSCs was significantly higher compared
to hExo (p<0.001 and p<0.001 respectively) and uExo
(p<0.001 and p<0.001 respectively). At the 1:5 cell ratios,
proliferations of uP cells were significantly suppressed
with both hMSCs (p= 0.004) and uMSCs (p= 0.02), but
at a 1:10 cell ratio, only hMSCs produced significant sup-
pression (p= 0.005). Proliferations of PHA-activated hP
cells at 1:1 and 1:5 cell ratios were significantly suppressed
by hMSCs (p<0.001 and p<0.001 respectively), uMSCs
(p<0.001 and p<0.001 respectively), hExo (p<0.001 and
p<0.001 respectively), and uExo (p<0.001 and p<0.001
respectively). Like uP cells, suppression formed by hMSCs
and uMSCs was significantly higher compared to hExo
(for 1:1 ratio p<0.001 and p<0.001; for 1:5 ratio p<0.001
and p<0.001 respectively) and uExo (for 1:1 ratio p<0.001
and p<0.001; for 1:5 ratio p<0.001 and p<0.001 respec-
tively). At the 1:10 cell ratio, the proliferation of hPs was
Figure 5. Comparison graphics of cytokine measurements obtained from co-culture experiments. Data are presented as mean and
standard deviation. Which cell or exosome is used in co-culture groups is indicated with a (+) sign. ere is a statistically signicant
dierence (p<0.05) between the “o” symbols and points having the same color.
(p: patient, h: healthy)
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Asthma Allergy Immunol 2023;21:45-54
did not cause a significant change in both hPs and uPs.
It has been reported that the interaction of MSCs with
monocytes/macrophages increases IL-17 expression of
T cells, and suppression of IL-17a levels occurs with the
removal of monocytes from the environment (10,12,32).
In addition, the increase in IL-17a that occurs in the pres-
ence of MSCs has been shown to potentiate the effect of
the resulting immune suppression (33). In our study, the
significant increase in IL-17a in hPs interacting with both
hMSCs and uMSCs was consistent with the data in the lit-
erature. However, this increase in uPs was only provided
by hMSCs. These data lead to two different interpreta-
tions. One of them is that allogeneic interaction may be
an important factor in increases in IL-17a expression of
PBMCs. The other is that MSCs of CSU patients may not
have an effect on the IL-17a expression of own PBMCs.
IL-6 stands out as an important cytokine in differentiation
of naive T cells to Th17. However, simultaneous TGF-β
stimulation with IL-6 causes these cells to differentiate
from the proinflammatory Th17 type to anti-inflammato-
ry Treg cells. Interestingly, it has been shown that there
is an increase in IL-17 and IL-10 expression simultane-
ously in Th17 cells (34,35). As with IL-17a expressions, we
detected a similar interaction in TGF-β expressions. Con-
sidering the effect of TGF-β on Th17/Treg plasticity, our
findings revealed that allogeneic MSCs may have especial-
ly contributed to the immunosuppressive differentiation
of Th17 cells. However, there was no significant change
induced by exosomes for both IL-17a and TGF-β.
IL-10 is a strong immunosuppressive cytokine. Degir-
menci et al. have reported in their study that the IL-10
expressions of PBMCs of CSU patients were significantly
lower than healthy individuals (26). Another study has
shown that the use of omalizumab causes a decrease in IL-
10-producing T cell frequencies in CSU patients (36). We
also observed that PBMCs of CSU patients had significant-
ly lower IL-10 expressions than healthy subjects. However,
the responses of PBMCs of patients and healthy subjects to
MSCs and exosomes for IL-10 were similar. We observed
that at 1:1 cell ratios, IL-10 expression of uPs and hPs were
significantly suppressed by both MSCs and exosomes,
but the effect of exosomes disappeared as the cell ratios
increased. Another important finding we observed was
that IL-10 expressions of hPs were suppressed by hMSCs
and uMSCs at all cell ratios, whereas in uPs this was only
provided by hMSCs. These findings suggested that the
allogeneic interaction could play an important role in the
manifestation of the effects of MSCs. We detected a sup-
after the use of omalizumab, a human anti-IgE antibody
(20,21). However, in our previous study with refractory
CSU cases, we reported that IFN-γ levels were not affected
by the use of omalizumab (17). In other words, it is under-
stood that there may be aberrant differences in cytokine
levels between omalizumab-responsive and resistant CSU
patients. In this study, we observed that IFN-γ secretions
of PHA-activated uPs were significantly suppressed by
both autologous and allogeneic MSCs, but the same effect
could not be produced by exosomes, except for a 1:1 ratio.
As for the IFN-γ expression of hPs, we found that both
u/h MSCs and u/h exosomes (except 1:10 ratio) were
capable of significant suppression at all ratios. However,
the effect formed by u/hMSCs was significantly superior
to exosomes. It has been shown in many studies that both
MSCs and MSC-derived exosomes can effectively suppress
IFN-γ expression of PBMCs (10,12,22-24). However, our
findings suggested that the suppression caused by the
direct interaction of MSCs with immune cells could be
superior.
In studies performed for IL-4, a cytokine closely asso-
ciated with allergic diseases, it has been reported that the
IL-4 expressions of the PBMCs of CSU patients are sig-
nificantly lower and this situation is more pronounced in
cases with positive ASST (25-27). There are many stud-
ies showing that IL-4 expressions of PBMCs from both
healthy and allergic patients are effectively suppressed by
MSCs and also exosomes (10,12,23,28,29). We observed
that IL-4 expression of hPs was significantly suppressed by
both u/hMSCs and u/h exosomes at all cell ratios. Howev-
er, we found that this suppression in uPs occurs at 1:1 and
1:5 cell ratios and only by hMSCs. This finding suggested
that interaction with autologous MSCs may not influence
IL-4 expression of uPs, and this may also apply to autolo-
gous and allogeneic exosomes.
Approximately half of the refractory CSU cases are
thought to be an autoimmune pathology, and Th17
cells and its cytokine IL-17 are prominent in autoim-
mune diseases (3). There are studies revealing the pres-
ence of high Th17/IL-17 in both serum and skin biopsies
of CSU patients and its correlation with disease severity
(17,30,31). Consistent with the literature, we also observed
that IL-17 levels of uPs were significantly higher than hPs.
In our experiments with MSCs and exosomes, we found
that hPs’ IL-17a expression was significantly increased
by both hMSCs and uMSCs, but in uPs this increase was
caused only by hMSCs. We observed that u/h exosomes
53
Ozdemir AT, Kirmaz C, Ozgul Ozdemir RB, Oztatlici M, Kilicarslan Sonmez P, Tuglu MI
Asthma Allergy Immunol 2023;21:45-54
Authorship Contributions
Concept: Alper Tunga Ozdemir, Cengiz Kirmaz, Rabia Bilge
Ozgul Ozdemir, Design: Alper Tunga Ozdemir, Cengiz Kirmaz,
Rabia Bilge Ozgul Ozdemir, Mustafa Oztatlici, Mehmet Ibrahim
Tuglu, Data collection or processing: Alper Tunga Ozdemir,
Rabia Bilge Ozgul Ozdemir, Mustafa Oztatlici, Pinar Kilicarslan
Sonmez, Mehmet Ibrahim Tuglu, Analysis or Interpretation: Alper
Tunga Ozdemir, Mustafa Oztatlici, Mehmet Ibrahim Tuglu,
Literature search: Alper Tunga Ozdemir, Cengiz Kirmaz, Rabia
Bilge Ozgul Ozdemir, Mustafa Oztatlici, Writing: Alper Tunga
Ozdemir, Cengiz Kirmaz, Rabia Bilge Ozgul Ozdemir, Mustafa
Oztatlici, Pinar Kılıcarslan Sonmez, Mehmet Ibrahim Tuglu,
Approval: Alper Tunga Ozdemir, Cengiz Kirmaz, Rabia Bilge
Ozgul Ozdemir, Mustafa Oztatlici, Pinar Kılıcarslan Sonmez,
Mehmet Ibrahim Tuglu.
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Conflict of Interest
The authors declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this article.
Funding
The Scientific Research Projects Commission of Manisa Celal
Bayar University financially supported the project whose reference
number is 2017-171.
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