ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 7, pp. 1230-1240 © The Author(s), 2026. This article is an open access publication.
1230
Secreted Cyclophilin A Induces M1 and M2 Macrophage
Polarization in the THP-1 Model in vitro
Anastasiia A. Kalinina
1
, Marina V. Kubekina
2
, Maria A. Zamkova
1
,
Nadezhda A. Persiyantseva
1
, Alexandra V. Bruter
1,2
, Dmitry B. Kazansky
1
,
and Ludmila M. Khromykh
1,a
*
1
N. N. Blokhin National Medical Research Center of Oncology,
Ministry of Health of the Russian Federation, 115478 Moscow, Russia
2
Center for Precision Genome Editing and Genetic Technologies for Biomedicine,
Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia
a
e-mail: lkhromykh@list.ru
Received February 18, 2026
Revised April 17, 2026
Accepted April 23, 2026
Abstract— Fate of monocytes that migrate into tissues is determined by various mediators at the site of
inflammation. The microenvironment governs monocyte differentiation and polarization into macrophages,
which acquire different surface markers and opposite functions. Secreted cyclophilin A (CypA) is a pro-in-
flammatory factor and a component of the monocyte humoral microenvironment. In these studies, effects
of recombinant human CypA (rhCypA) on differentiation of human monocytic cells were evaluated in the
THP-1 in vitro model. RhCypA induced polarization of M1 macrophages in the early stages of THP-1 onto-
genesis without additional stimuli, whereas it promoted M2 polarization only in the cells differentiated to
M0 monocytes and in the presence of IL-4 and IL-13. Under conditions of M2 induction, rhCypA significantly
upregulated expression of the genes of characteristic M2 markers CD163 and IL-10 in the THP-1 cells, and
decreased the level of p38 MAP kinase activation, suggesting diminished pro-inflammatory activity of the
generated cells. The RhCypA-induced M1 and M2 macrophages inhibited proliferation of activated CD4+
T cells via direct cell-cell contact. These findings suggested that secreted CypA could induce generation of
suppressor M2 macrophages and/or myeloid-derived suppressor cells at the site of inflammation during in-
fection or cancer progression. This new immunoregulatory effect of rhCypA could be of particular clinical
relevance for the development of strategies for controlling chronic inflammation and reprogramming the
tumor microenvironment.
DOI: 10.1134/S0006297926600432
Keywords: cyclophilin A, M1/M2 polarization, THP-1, suppressor macrophages
* To whom correspondence should be addressed.
INTRODUCTION
Macrophages are known to possess high plas-
ticity and can acquire different functional states by
receiving diverse activating stimuli in their milieu
facilitating both pro-inflammatory (destructive) and
anti-inflammatory (regenerative) processes. The di-
versity of macrophages is complex, and in addition
to the classical (M1) macrophages and alternatively
activated (M2) macrophages, cells with the mixed
M1/M2 phenotypes were described [1]. Lipopolysac-
charide (LPS) and interferon-γ (IFNγ) induce differen-
tiation of immature monocytes into pro-inflammato-
ry M1 macrophages, while IL-4, IL-13, tumor growth
factor β (TGFβ), and IL-10 induce alternatively acti-
vated M2 cells, which are involved in regeneration
processes and tumor development [2]. Still, analysis
of macrophage inductors and macrophage cellular
markers shows that some mediators that induce M1
polarization are also involved to a certain extent in
M2 polarization (LPS, tumor necrosis factor α (TNFα),
IFNγ, IL-1β, IL-6, IL-8, IL-12, IL-18, etc.) [3].
CypA INDUCES M1/M2 POLARIZATION 1231
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026
Secreted cyclophilin A (CypA) is a pro-inflam-
matory factor, which acts as a chemoattractant for
different immune cells (dendritic cells, macrophages,
neutrophils, and activated T cells) and takes part in
the development of the site of inflammation. This
factor is known to induce acute inflammation, and
a number of studies found its involvement in the
M1 macrophage polarization [4]. Still, acute inflam-
mation can progress into a chronic form, defined by
the presence of M2 macrophages. Chronic inflamma-
tion is frequently linked to cancer, and CypA can also
contribute to its development [5]. Yet, the effects of
secreted CypA on M2 induction and conditions re-
quired for this remain unknown as of today. In this
study, involvement of CypA in generation of different
macrophage subpopulations and conditions required
for this were elucidated using the THP-1 cell model.
MATERIALS AND METHODS
Recombinant human CypA (rhCypA). The full-
length PPIA gene encoding human CypA was cloned
into a pET22b(+) vector (Novagen, USA), which was
next used for transformation of the E. coli strain
BL21(DE3)Gold. RhCypA was extracted from the bac-
terial biomass and purified. Isolated rhCypA was
dissolved in Na-K-phosphate buffered saline (PBS;
pH 7.3). The protein purity was >95%; the endotoxin
content in rhCypA samples was below 0.038 ng per
1 mg of the protein [6].
Cell line, culture conditions. Human monocyt-
ic THP-1 cells were cultured in RPMI-1640 medium
(PanEco, Russia), supplemented with 10% fetal bovine
serum (HyClone, GE Healthcare, USA), 2 mM L-glu-
tamine (PanEco), 0.01 mg/mL ciprofloxacin (KRKA,
Slovenia), 0.05 mM 2-mercaptoethanol (Merck, Ger-
many), and 0.01 M HEPES (PanEco) (complete medi-
um, CM) at 37°C, 5% CO
2
. Cells were seeded in 6-well
plates (Corning Costar, USA) at a density of 0.5×10
6
cells/well in 2 mL of CM.
Induction of M1 and M2 macrophages from
THP-1 cells. To generate M1 macrophages, THP-1 cells
were cultured with rhCypA and phorbol 12-myristate
13-acetate (PMA, Sigma, USA). To generate M2 mac-
rophages, THP-1 cells were cultured with PMA and
recombinant human IL-4 (Goldbio, China) and IL-13
(Goldbio).
Real-time PCR (qPCR). RNA was extracted using
the TRIzol reagent (Thermo Scientific, USA) according
to the manufacturer’s recommendations. To obtain
complementary DNA (cDNA), reverse transcription
was performed using a MMLV RT kit according to the
manufacturer’s protocol (Evrogen, Russia). Levels of
gene expression were analyzed by qPCR with a CFX96
amplifier (Bio-Rad, USA) using a qPCRmix-HS SYBR
mix (Evrogen). All cDNA samples of THP-1 cells were
amplified in three technical repeats. Primers used in
this study are listed in the Online Resource 1. The
data were analyzed using the CFX Manager software
(Bio-Rad). Relative gene expression levels were cal-
culated using the ΔΔCt method [7]. The level of gene
expression in the control group was considered as 1.
Monoclonal antibodies (mAbs). The following
mAbs were used in this study: anti-CD68-PerCp-Cy5.5
(clone Y1.82/A, BioLegend, USA), anti-CD11b-PE (clone
D12 (ASR), BD Bioscience, USA), anti-CD147–APC (clone
HIM6, BioLegend), anti-CD206–APC (clone 19.2 (RUO),
BD Bioscience), anti-CD86–BV421 (clone 2331 FUN1,
BD Bioscience), anti-CD163-PE-Cy7 (clone GHI/61, Bio-
Legend). Purified anti-CD3 mAb (clone HIT3a, BD Bio-
science) and anti-CD28 mAb (clone 2.2, BD Bioscience)
were also used in this work.
Flow cytometry analysis. Cell samples (0.5-1×10
6
cells) were incubated with 10% human serum
(10 min, 4°C) for Fc blocking and stained with fluo-
rescent mAbs for 40 min at 4°C. Cells were analyzed
with a FACS Canto II cytometer (BD Bioscience) using
the FACSDiva 6.0 software (BD Bioscience). Cells were
gated based on the parameters of forward (FSC) and
side (SSC) scatter, followed by the singlet gating based
on the parameters FSC-H vs. FSC-A. Dead cells were
excluded by the staining with the LIVE/DEAD Fixable
Yellow Dead Cell Stain kit (Invitrogen, USA). The sur-
face marker expression was analyzed in the popu-
lation of live singlet cells. The data were processed
with the FlowJo 7.6 software (TreeStar Inc., USA).
Western blot analysis. THP-1 cells were seeded
in 6-well plates as described above and cultured for
96 h. Cells were next lysed in a RIPA buffer with a
protease inhibitor cocktail (Sigma-Aldrich, USA). The
total protein concentration was quantified by the
Bradford method. Absorbance at 560 nm was mea-
sured with a plate reader Tecan (Austria). Proteins
were separated using SDS-PAGE and transferred
onto a 0.2 μm nitrocellulose membrane (Bio-Rad).
After blocking with 5% skimmed milk, membranes
were treated with primary anti-p38 MAPK antibodies
(#9212, Cell Signaling Technology, USA) or anti-phos-
pho-p38 (pp38) MAPK antibodies (#4511, Cell Sig-
naling Technology) and incubated at 4°C overnight.
Membranes were next washed with Tris-borate saline
with Tween 20 (TBS-T) and incubated for 1 h at room
temperature with secondary antibodies (#7074, Cell
Signaling Technology). Membranes were visualized
using a Clarity Western ECL Substrate or ClarityMax
Western ECL Substrate (Bio-Rad) with an iBright-
FL1500 Imaging System (Invitrogen). Densitometric
analysis of protein expression levels was performed
using the open-source software ImageJ 1.54k (USA).
Analysis of IL-10 levels in THP-1 growth
medium. The level of IL-10 in the growth medium
KALININA et al.1232
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026
of THP-1 cells cultured under the conditions of M1
and M2 generation was measured using an ELISA kit
(Cloud-Clone Corp., USA) according to the manufac-
turer’s recommendations.
Mixed lymphocyte reaction (MLR). Human
CD4+ T cells sorted from a peripheral blood (ATCC,
USA) were used as responders. For this, T cells were
cultured in triplicates in 96-well plates (Corning
Costar) at a density of 1×10
5
cells/well in 150 µL
of CM and activated by anti-CD3 mAbs (10 µg/mL),
anti-CD28 mAbs (2 µg/mL), and IL-2 (10 U/mL, Ron-
coleukin, Russia). THP-1 cells were seeded in 6-well
plates as described above, cultured for 96 h, and used
as stimulators. For this, THP-1 cells were treated with
mitomycin C (McC, 100 µg/mL, Kyowa Hakko Kogyo
Co., Ltd., Japan) for 60 min at 37°C, washed three
times with PBS by centrifugation (200g, 5 min, 4°C),
and added to CD4+ T cells at a responder-to-stimula-
tor ratio 1 : 3 or 1 : 5. Cells were cultured for 5 days
at 37°C, 5% CO
2
. To evaluate the background level of
proliferation, T cells were similarly cultured without
stimulators. The level of CD4+ T cell proliferation was
measured using an Alamar blue staining (Thermo
Fisher Scientific) added for the last 4 h according to
the manufacturer’s recommendations. Optical den-
sity (OD) was measured using a Tecan reader at
570/600 nm.
Statistical analysis. The data were presented as
a mean ± standard deviation (mean ± SD). Statistical
analysis was performed with the unpaired Student’s
t-test using the Prism software (v. 8.1.2, GraphPad,
USA). Datasets were tested for normal distribution by
the Shapiro–Wilk test to confirm relevance of using
the Student’s t-test. Differences were considered sig-
nificant at p ≤ 0.05.
RESULTS
Effects of rhCypA on THP-1 differentiation de-
pend on the maturation stage of these cells. First,
possible polarizing effects of rhCypA were analyzed
with intact THP-1 cells used as a model of early im-
mature monocytes. For this, intact THP-1 cells were
cultured with different doses of rhCypA for 18 h.
Table 1 shows that exposure to rhCypA at the dose
of 5 µg/mL induced marked expression of the M1
marker genes in the THP-1 cells: the level of expres-
sion of the CD80 gene increased 14-fold, IL1b– 9-fold,
CXCL10– 20-fold, CCL2– 15-fold, and IL8– 13-fold vs.
the intact control. At higher concentrations, rhCypA
induced strong upregulation of these genes along
with the induction of TNFa, IL6, and CXCL2 expres-
sion, whose levels were elevated 14-, 5-, and 17-fold,
respectively, in the presence of 50 µg/mL of rhCypA
relative to the intact control. The induction of ex-
pression of the M2 genes was nearly absent in this
experimental system. At the dose of rhCypA above
5 µg/mL, we observed the most stable tendency in up-
regulation of expression of the CD163 and IL10 genes
(2.5-fold at 5 µg/mL, Table 2).
At the maximal dose of 50 µg/mL, rhCypA also in-
duced upregulation of expression of the CD206, IL-18,
IL-4, and CXCL-1 genes 2-3-fold compared to the con-
trol level in the intact cells. Weak rhCypA-mediated
induction of expression of some of these genes was
found earlier in the similar experimental setup [8].
Thus, these findings indicated that rhCypA could
significantly promote upregulation of the complex
of genes characteristic of M1 macrophages in very
early, poorly differentiated, and immature monocytes,
which are intact THP-1 cells. These data are in agree-
ment with other studies [9].
Next, we analyzed the effects of rhCypA on M1
and M2 polarization in more mature THP-1 cells.
PMA is known to induce maturation of THP-1 cells to
the stage corresponding to M0 macrophages. Analysis
of expression of the M1 marker genes in PMA-treat-
ed THP-1 cells revealed increased sensitivity of more
mature cells to rhCypA. Even at the dose of 1 µg/mL,
rhCypA-induced upregulation of the CD80, CXCL10,
and IL1b genes was approximately 6-, 9-, and 5.6-fold
vs. the corresponding gene expression levels in the
control cells (the PMA group) (Fig. 1a).
An increase of the rhCypA doses up to 10 and
50 µg/mL further enhanced expression of the ana-
lyzed genes: CD80 12- and 17-fold and IL1b 17- and
19-fold, respectively, vs. the PMA group. The CXCL-10
gene was an exception: in the presence of 10 µg/mL
of rhCypA, expression of this gene increased 10-fold
compared to the PMA control, but in the presence
of 50 µg/mL of rhCypA, expression of the CXCL10
gene was upregulated only 6-fold vs. the PMA group
(Fig. 1a). Suppression of the CXCL10 gene expression
could be due to the high sensitivity of the CXCL-10
to rhCypA, which resulted in the drop of this gene
expression when rhCypA was used at a maximal dose.
Activation with PMA also increased sensitivity
of the M2 markers CD163 and IL-10 to the effects of
rhCypA. At the dose of 1 µg/mL, rhCypA upregulat-
ed expression of the CD163 and IL-10 genes 7- and
9-fold, respectively, compared to the PMA-treated
control. This effect was absent without PMA induc-
tion (Table 2). When the rhCypA concentration was
increased to 50 µg/mL, the level of CD163 expres-
sion was not markedly changed, while expression of
the IL10 gene increased 12-fold vs. the PMA group
(Fig. 1b).
These data showed that THP-1 cells at the M0
stage had higher sensitivity to rhCypA compared to
more immature intact THP-1 cells and strongly up-
regulated expression of the genes of the markers
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Table 1. Effects of rhCypA on expression of the M1 marker genes in the intact THP-1 cells
Groups M1 marker
CD80 IL1b CXCL10 CCL2 IL8 TNFa IL6 CXCL2
Control THP-1 intact 1 ± 0.11 1 ± 0.03 1 ± 0.12 1 ± 0.05 1 ± 0.03 1 ± 0.07 1 ± 0.08 1 ± 0.10
rhCypA 0.01 µg/mL 1.80 ± 0.28 0.94 ± 0.28 0.93 ± 0.10 0.97 ± 0.07 0.75 ± 0.03 0.85 ± 0.13 1.48 ± 0.17 1.67 ± 0.09
rhCypA 0.1 µg/mL 4.12 ± 0.15* 1.01 ± 0.13 1.35 ± 0.16 1.15 ± 0.10 0.84 ± 0.01 1.027 ± 0.12 1.47 ± 0.15 2.03 ± 0.09
rhCypA 1 µg/mL 1.67 ± 0.14 1.66 ± 0.13 3.17 ± 0.25* 2.34 ± 0.07 1.53 ± 0.01 1.16 ± 0.15 2.63 ± 0.19 2.32 ± 0.30
rhCypA 5 µg/mL 14.48 ± 2.27* 8.90 ± 0.77* 19.95 ± 2.69* 14.96 ± 0.26* 13.26 ± 1.22* 3.86 ± 0.48* 1.33 ± 0.20 2.43 ± 0.42
rhCypA 10 µg/mL 39.86 ± 3.14* 24.21 ± 0.22* 34.96 ± 1.41* 52.51 ± 4.25* 30.06 ± 0.23* 7.28 ± 0.46* 2.40 ± 0.14 4.55 ± 0.33*
rhCypA 50 µg/mL 101.29 ± 6.99* 29.72 ± 1.97* 21.62 ± 1.90* 68.43 ± 1.95* 68.33 ± 2.80* 14.72 ± 1.18* 5.22 ± 0.25* 17.43 ± 1.47*
Note. THP-1 cells were cultured with indicated doses of rhCypA for 18 h. The data are presented as a mean ± SD for three technical repeats. * p ≤ 0.05 vs. the control
(THP-1 intact) (unpaired Student’s t-test).
Table 2. Effects of rhCypA on expression of the M2 marker genes in the intact THP-1 cells
Groups M2 marker
CD163 CD206 IL4 IL10 IL18 CXCL1
Control THP-1 intact 1 ± 0.03 1 ± 0.09 1 ± 0.31 1 ± 0.10 1 ± 0.01 1 ± 0.11
rhCypA 0.01 µg/mL 1.12 ± 0.05 1.26 ± 0.09 1.30 ± 0.24 1.61 ± 0.04 1.15 ± 0.08 1.72 ± 0.16
rhCypA 0.1 µg/mL 0.95 ± 0.06 1.5 ± 0.08 0.97 ± 0.08 1.08 ± 0.04 0.94 ± 0.08 1.47 ± 0.11
rhCypA 1 µg/mL 1.83 ± 0.17 1.86 ± 0.08 1.38 ± 0.21 1.12 ± 0.09 1.052 ± 0.09 1.92 ± 0.09
rhCypA 5 µg/mL 2.51 ± 0.01* 1.44 ± 0.09 0.83 ± 0.27 2.59 ± 0.75* 1.10 ± 0.09 1.12 ± 0.15
rhCypA 10 µg/mL 3.53 ± 0.60* 1.52 ± 0.14 1.22 ± 0.11 2.03 ± 0.43 1.35 ± 0.27 1.81 ± 0.14
rhCypA 50 µg/mL 2.6 ± 0.39* 2.95 ± 0.15* 3.50 ± 0.23* 2.11 ± 0.35* 1.99 ± 0.08 3.36 ± 0.14*
Note. THP-1 cells were cultured with indicated doses of rhCypA for 18 h. The data are presented as a mean ± SD for three technical repeats. * p ≤ 0.05 vs. the control
(THP-1 intact) (unpaired Student’s t-test).
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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026
Fig. 1. Effects of rhCypA on expression of the M1 and M2 marker genes in PMA-activated THP-1 cells. Analysis of effects of
rhCypA at different doses on induction of expression of the M1 marker genes (a) and of the M2 marker genes (b). THP-1
cells were cultured with 50ng/mL PMA for 72 h, washed, and cultured with rhCypA at indicated concentrations for anoth-
er 18 h. PMA-induced cells were used as the control. The data are presented as a mean ± SD for three technical repeats.
* p ≤ 0.05 (unpaired Student’s t-test).
characteristic of M1 and M2 macrophages when
exposed to lower concentrations of rhCypA (Fig. 1,
a and b).
Effects of rhCypA on expression of M2 mac-
rophage markers in THP-1 cells. Next, we sought
to determine whether CypA participates in genera-
tion of M2 macrophages and under what conditions.
Cytokines IL-4 and IL-13 are known to induce the
M2 macrophage generation [1, 2], and we aimed to
ascertain if CypA could also be involved in this pro-
cess. For this, THP-1 cells were cultured with PMA,
IL-4, IL-13, and different doses of rhCypA for 96 h
(Fig. 2). Cells exposed to PMA, IL-4, and IL-13 were
used as the positive control. Compared to the con-
trol cells, rhCypA induced strong upregulation of the
IL-10, CD163, and MMP9 genes in THP-1 cells (Fig. 2).
In this experimental setup, significant upregula-
tion of these genes was detected in the cells exposed
even to 1 µg/mL of rhCypA: 9-, 19-, and 12-fold, re-
spectively, vs. the positive control. When rhCypA was
used at the dose of 50 µg/mL, expression of the IL10,
CD163, and MMP9 genes was enhanced 136-, 43-, and
29-fold, respectively, vs. the positive control (Fig. 2).
Upregulation of other genes characteristic of M2 mac-
rophages, MRC1 and ARG1, was observed in the pres-
ence of 25 µg/mL rhCypA (5.5- and 3.7-fold, respec-
tively, compared to the positive control). Moderate
sensitivity of the TGFβ and CD200R genes to rhCypA
was also found: expression of these genes was upreg-
ulated 2-fold relative to the positive control (Fig. 2).
Of note, lowered doses of IL-4 and IL-13 were
used in this experimental system. No marked M2
gene expression was detected in the THP-1 cells
treated with rhCypA and IL-4 without IL-13 (data
not shown). Hence, our studies indicated that
rhCypA could induce expression of the M2 genes only
in a certain microenvironment, which provides a suf-
ficient initial level of monocyte maturity (by PMA)
and contains IL-4 and IL-13 at low concentrations.
Further analysis showed that along with the M2
markers, these cells expressed the genes character-
istic of M1 macrophages (Online Resource 2). In the
cells exposed to rhCypA at doses of 10-50 µg/mL, we
found significant upregulation of expression of the
IL8 (13-10-fold), IL1b (10-75-fold), and, to a lesser
extent, of the CXCL10 (5-7-fold) gene vs. the positive
control.
Thus, under the conditions of M2 generation,
rhCypA promoted maturation of the cells with high
levels of expression of the IL-10, CD163, MMP9, and,
to a lesser extent, of the MRC1, ARG1, and TGFb
genes. Furthermore, significant expression of the IL8,
IL1b, and, to a lesser degree, of the CXCL10 genes was
induced in these cells.
Effects of rhCypA on signaling in THP-1 cells
cultured under the conditions of M2 generation.
It was shown that the pro-inflammatory activity of
CypA is mediated by activation of NF-kB and its trans-
location into the nucleus, as well as by phosphoryla-
tion and activation of the p38 MAP kinase [10]. Thus,
we next analyzed the effects of rhCypA on the levels
of total p38 (p38) and phosphorylated p38 (pp38) in
THP-1 cells cultured under conditions of M2 macro-
phage generation. Online Resource 3 shows that in
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Fig. 2. Combined effects of rhCypA, IL-4, and IL-13 on induction of expression of the genes of M2 macrophage markers.
THP-1 cells were cultured with PMA, IL-4, IL-13, and different doses of rhCypA for 96 h. Gene expression levels were ana-
lyzed using qPCR. The PMA group was used as the negative control, and the PMA+IL-4+IL-13 group was used as the positive
control. The data of one representative experiment are presented as a mean ± SD for three technical repeats. * p ≤ 0.05
(unpaired Student’s t-test).
the cells treated with PMA, IL-4, IL-13, and rhCypA,
the amounts of pp38 and p38 were decreased 2.7-
and 1.4-fold, respectively, vs. the positive control (the
PMA+IL-4+IL-13 group) (Online Resource 3b). Further-
more, in the cells exposed to PMA, IL-4, IL-13, and
rhCypA, the pp38/p38 ratio was 1 : 1 compared to
3 : 1 in the positive control. These data implied that
in THP-1 cells cultured under the conditions of M2
generation with rhCypA, the p38 MAPK pathway was
not activated, and this could indicate lowered pro-
inflammatory potential of these cells.
Effects of rhCypA on the functional activity of
THP-1 cells generated under conditions of M1 and
M2 macrophage induction. To analyze functionality
of the THP-1 cells cultured under the conditions of
M1 and M2 macrophage generation, their contact-de-
pendent effects on proliferation of activated human
CD4+ T cells were evaluated in MLR. Intact THP-1
cells, as allogeneic stimulators, significantly (by 70%)
enhanced proliferation of CD4+ T cells (Fig. 3).
The PMA-treated THP-1 cells slightly (by 22%)
promoted proliferation of CD4+ T cells in MLR vs. the
intact THP-1 cells (Fig. 3). Yet, THP-1 cells activated
by PMA with 10 and 25 µg/mL of rhCypA exhibited
regulatory functions manifested as suppression of the
proliferative activity of the responders 4- and 3-fold,
respectively, compared to the level of CD4+ T cell pro-
liferation in MLR with the PMA-treated cells (Fig. 3).
THP-1 cells cultured under the conditions of M2 gen-
eration (PMA+IL-4+IL-13) exhibited different effects
depending on the dose of rhCypA used for their in-
duction. When generated with the M2 inductors and
10 µg/mL of rhCypA, the THP-1 cells showed no reg-
ulatory activity (Fig. 3). However, the THP-1 cells gen-
erated under the conditions of M2 macrophage gen-
eration with the higher dose of rhCypA (25 µg/mL)
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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026
Fig. 3. Effects of induction regimens of THP-1 cells on the proliferative activity of activated CD4+ T cells. THP-1 cells were
cultured under the conditions of M1 and M2 macrophage generation for 96 h and used as stimulators in the mixed lym-
phocyte culture with activated human CD4+ T cells. The level of CD4+ T cell proliferation was evaluated in 5 days. Intact
THP-1 cells treated with McC were used as the negative control. The data of one representative experiment are shown as
a mean ± SD. * p ≤ 0.05 (unpaired Student’s t-test).
exhibited strong suppressor activity and inhibited
proliferation of CD4+ T cells 4.6-fold compared to the
proliferation levels in MLR with the THP-1 cells treat-
ed with PMA, IL-4, and IL-13 (Fig. 3).
As shown above (Fig. 2), rhCypA enhanced ex-
pression of the IL-10 gene in the THP-1 cells cultured
under the conditions of M2 macrophage generation
(PMA+IL-4+IL-13). Secretion of this cytokine could
probably also mediate suppression in MLR with the
THP-1 cells generated under these conditions (Fig. 3,
PMA+IL-4+IL-13+rhCypA 25 µg/mL). Yet, this hypoth-
esis was not confirmed, since the level of IL-10 in
the growth medium of these cells was not changed
compared to the control levels in the cultures of in-
tact THP-1 cells (data not shown). Furthermore, the
conditioned medium of the THP-1 cells cultured un-
der different regimens of M1 and M2 induction had
no effects on proliferation of activated CD4+ T cells
(data not shown). These findings indicated that the
suppressor effects of THP-1 cells induced under var-
ious modes with rhCypA were mediated by the con-
tact-dependent mechanisms.
Phenotypic features of THP-1 cells cultured un-
der the conditions of M2 generation. To ascertain
the role of rhCypA in generation of M2 macrophages
in more detail, we analyzed the phenotype of the
THP-1 cells cultured under the conditions of M2 in-
duction (treatment with PMA, IL-4, and IL-13) (Fig. 4).
For this, percentages of THP-1 cells that expressed
CD163 and CD206 markers characteristic of M2 mac-
rophages were evaluated. RhCypA at doses of 10 and
25 µg/mL increased the relative count of CD163+ cells
4.8-fold and of CD206+ cells – 3.9- and 6-fold, respec-
tively, vs. the positive control (PMA). Furthermore,
the percentages of the double positive CD163+CD206+
cells increased 6- and 7-fold in the presence of 10
and 25 µg/mL of rhCypA, respectively, compared to
the positive control (Fig. 4a). The combined action of
CypA INDUCES M1/M2 POLARIZATION 1237
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026
Fig. 4. Phenotypic analysis of THP-1 cells cultured under the conditions of M2 macrophage induction. The percentages (%)
of the cells expressing CD163 and CD206 (a), CD86 and CD68 (b). PMA – the negative control; PMA+IL-4+IL-13– the positive
control. Different doses of rhCypA were used in experimental groups. The data of one representative staining are shown.
rhCypA and M2 inductors had no effect on the rela-
tive count of THP-1 cells expressing CD86, the mole-
cule required for regulation of T cell activity (Fig. 4b).
Yet, the proportions of CD68+ cells increased 3- and
5-fold under the exposure to 10 and 25 µg/mL of rh-
CypA, respectively, compared to the positive control
(PMA+IL-4+IL-13) (Fig. 4b). Previous studies showed
that upregulation of the adhesion molecule CD68 was
linked to macrophage differentiation.
The CD147 molecule is a receptor of CypA, and
signaling through it mediates pro-inflammatory re-
sponses [11]. Thus, we evaluated changes in the
levels of CD147 expression in THP-1 cells cultured
under the conditions of M2 generation. RhCypA in-
creased the relative count of CD147
int
cells (~2-fold
vs. the positive control) (Fig. 5a). This finding could
indicate a somewhat decline in the pro-inflammatory
potential of the THP-1 cells, although 90% of the cells
in these culture conditions were still CD147
hi
under
both doses of rhCypA (Fig. 5a). CD147
hi
cells were fur-
ther characterized based on expression of CD11b as
a marker of negative regulation of pro-inflammatory
processes [12]. RhCypA induced accumulation of the
CD147
hi
CD11b+ cells, whose proportion was increased
5- and 8-fold in the presence of 10 and 25 µg/mL of
rhCypA, respectively, vs. the positive control (PMA+
IL-4+IL-13) (Fig. 5b).
DISCUSSION
Macrophages differentiate from monocytes in tis-
sues and simultaneously acquire the functional phe-
notype, which depends on the stimuli from their mi-
croenvironment. Macrophages have a wide range of
functions that are ultimately shaped by multiple fin-
est humoral and cellular signals in the milieu, which
macrophages encounter under normal conditions,
in stress conditions, or during different pathological
processes [3].
CypA is a pro-inflammatory factor, which is in-
volved in both acute and chronic inflammation and
is one of the components at the site of inflammation.
The results of this study suggest that without addi-
tional mediators, rhCypA itself can induce differen-
tiation of monocytes into M1 macrophages at a very
KALININA et al.1238
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026
Fig. 5. Analysis of CD11b and CD147 expression in THP-1 cells cultured under the conditions of M2 macrophage gener-
ation. PMA – the negative control; PMA+IL-4+IL-13 – the positive control. a) The percentage (%) of CD147
int
and CD147
hi
cells. b) The percentage (% of the CD147
hi
parental population) of CD11b+CD147+ and CD11b-CD147+ cells. The data of one
representative staining are shown.
early stage of their ontogenesis (intact THP-1 cells).
By attracting monocytes to the site of inflammation
[13] and inducing their polarization into M1 macro-
phages, CypA promotes their rapid conversion into
pro-inflammatory macrophages and effectors, which,
in turn, facilitate effective development of the im-
mune response.
This study showed that CypA can induce M2
polarization under specific conditions that include
a certain initial level of THP-1 differentiation (PMA-
induced maturation) and a complex of additional hu-
moral factors (IL-4 and IL-13). The most noticeable
upregulation of the IL-10 and CD163 genes, one of
the characteristic markers of M2 macrophages, was
detected under the effect of rhCypA. Furthermore,
under the conditions of M2 macrophage generation,
exposure to rhCypA decreased the level of p38 MAP
kinase (both total and phosphorylated) in the THP-1
cells, which is involved in pro-inflammatory signal-
ing pathways. However, these cells simultaneously
expressed the genes of IL-8, IL-1β, etc. that were
identified as markers of M1 macrophages. Previous
research described similar macrophages that were at-
tributed to the M2b subpopulation and produced both
pro- and anti-inflammatory cytokines [3].
The phenotypic analysis of THP-1 cells cultured
under the conditions of M2 generations with rhCypA
showed that these cells did not have an upregulated
expression of CD86, which would support T cell acti-
vation and the pro-inflammatory immune response.
Still, these induction conditions promoted generation
of the cells that expressed the M2 markers CD163 and
CD206, as well as CD68. Previous studies found that
macrophages expressing CD206 and CD68 were of-
ten associated with intratumoral localization (tumor-
associated macrophages, TAMs) and considered as the
key immunosuppressive components of the tumor
microenvironment [14].
Furthermore, rhCypA facilitated formation of
CD147
hi
CD11b
+
THP-1 cells. CD147 is a receptor of
CypA, while CD11b regulates immune processes. Sig-
naling through CD11b inhibits production of pro-in-
flammatory cytokines [15] and suppresses leukocyte
activation [16]. CD11b is also involved in the gener-
ation of immunosuppressive TAMs, which promote
tumor growth [17].
CypA is known to upregulate expression of its
receptor by autocrine-paracrine mechanisms [5, 18].
The findings of this study suggest that high concen-
trations of secreted CypA increase expression of its
CypA INDUCES M1/M2 POLARIZATION 1239
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026
receptor on a macrophage (CD147
hi
cells). This, in
turn, could significantly enhance intracellular signal-
ing and, hence, induce expression of the suppressor
molecule CD11b and formation of a macrophage with
regulatory activity.
The functional analysis of THP-1 cells cultured
under the conditions of M1 and M2 generation
showed that rhCypA promoted induction of the cells
with suppressor activity, which inhibited proliferation
of activated CD4+ T cells by the contact-dependent
mechanisms. Thus, our study implied that rhCypA
could be involved in formation of mononuclear my-
eloid-derived suppressors.
Today much effort is put into research of my-
eloid-derived suppressor cells (MDSC) and factors
of their generation. Under infection, inflammation,
or tumor progression, immature myeloid cells are
attracted to the sites of inflammation to substitute
damaged or exhausted cells. Yet, under the patholog-
ical microenvironment and in the presence of vari-
ous pro-inflammatory stimuli, these cells change the
differentiation pathway, acquire immunosuppressive
functions, and form MDSC [19]. The data of this study
showed that rhCypA could generate suppressor cells
from immature monocytes (intact THP-1 cells). Thus,
it seems plausible that CypA, as one of the factors at
the site of inflammation, could be involved in gen-
eration of MDSC along with other pro-inflammatory
mediators.
As noted above, macrophages acquired various
functional states depending on the signals in their
milieu. This largely depends on a phase of inflam-
mation, since in its different stages, monocytes en-
ter a distinct microenvironment. In this context, we
assume that monocytes migrating into the site of
inflammation in the late stages are exposed to the
microenvironment rich in humoral and cellular acti-
vation stimuli. This creates conditions favorable for
the M2 macrophage polarization and acquisition of
the regulatory immunosuppressive functions that me-
diate the contraction of the inflammatory responses.
Our research indicated that secretory CypA could
play a significant role in this process along with
other factors.
Abbreviations
CM complete medium
CypA cyclophilin A
IFNγ interferon-γ
IL interleukin
LPS lipopolysaccharide
mAb monoclonal antibody
MDSC myeloid-derived suppressor cells
MLR mixed lymphocyte reaction
PMA phorbol 12-myristate 13-acetate
qPCR real-time polymerase chain reaction
rhCypA recombinant human CypA
TAM tumor-associated macrophage
TGFβ tumor growth factor β
Supplementary information
The online version contains supplementary material
available at https://doi.org/10.1134/S0006297926600432.
Contributions
L.M.K. developed concept and design of the study, su-
pervised the study, and wrote the manuscript; A.A.K.,
M.V.K., M.A.Z., and A.V.B. performed experiments;
N.A.P. analyzed the data; D.B.K. edited the manuscript.
Funding
This work was financially supported by the Russian
Science Foundation (grant no.25-25-00043, 2025-2026).
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest
The authors of this work declare that they have no
conflicts of interest.
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