S-1-Propenylcysteine promotes IL-10-induced M2c macrophage polarization through prolonged activation of IL-10R/STAT3 signaling

概要

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S‑1‑Propenylcysteine promotes
IL‑10‑induced M2c macrophage
polarization through prolonged
activation of IL‑10R/STAT3
signaling
Satomi Miki1,2*, Jun‑ichiro Suzuki2, Miyuki Takashima2, Mari Ishida1, Hiroki Kokubo1 &
Masao Yoshizumi1*
Atherosclerosis is a chronic inflammatory disease that may lead to the development of serious
cardiovascular diseases. Aged garlic extract (AGE) has been reported to ameliorate atherosclerosis,
although its mode of action remains unclear. We found that AGE increased the mRNA or protein
levels of arginase1 (Arg1), interleukin-10 (IL-10), CD206 and hypoxia-inducible factor 2α (HIF2α)
and decreased that of CD68, HIF1α and inducible nitric oxide synthase in the aorta and spleen of
apolipoprotein E knockout mice. We also found that S-1-propenylcysteine (S1PC), a characteristic
sulfur compound in AGE, increased the level of IL-10-induced Arg1 mRNA and the extent of M2clike macrophage polarization in vitro. In addition, S1PC increased the population of M2c-like
macrophages, resulting in suppressed the population of M1-like macrophages and decreased
lipopolysaccharide-induced production of pro-inflammatory cytokines. These effects were
accompanied by prolonged phosphorylation of the IL-10 receptor α (IL-10Rα) and signal transducer
and activator of transcription 3 (STAT3) that inhibited the interaction between IL-10Rα and Src
homology-2-containing inositol 5’-phosphatase 1 (SHIP1). In addition, administration of S1PC
elevated the M2c/M1 macrophage ratio in senescence-accelerated mice. These findings suggest that
S1PC may help improve atherosclerosis due to its anti-inflammatory effect to promote IL-10-induced
M2c macrophage polarization.
Atherosclerosis is a chronic inflammatory disease that may lead to the development of life-threatening illnesses
such as coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular d
­ isease1,2. In the
early stages of atherosclerosis, oxidized low density lipoproteins (oxLDL) induce inflammation in arterial walls,
causing augmented infiltration of monocytes into the sub-endothelial space and differentiation of monocytes
into macrophages. Subsequently, the arterial macrophages are transformed into foam cells, a process that is
accompanied by the upregulation of several genes, such as CD36 and C
­ D682–6. The foam cells then secrete proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), into the arterial intima, promoting the formation of atherosclerotic p
­ laques1,7. The resulting chronic inflammation promotes
the release of damage-associated molecular patterns (DAMPs), such as nucleic acids and proteins from dead cells,
as well as senescence-associated secretory phenotype (SASP) factors, such as inflammatory cytokines, growth
factors, and proteases from senescent cells. These accelerate vascular aging by creating a positive feedback loop
and eventually exacerbating plaque f­ ormation8,9.
Macrophages can be polarized to two major phenotypes depending on the microenvironmental conditions:
pro-inflammatory M1 and anti-inflammatory M2 m
­ acrophages10–12. It has been reported that the development
of atherosclerosis alters the ratio of polarized ­macrophages13,14. M1 macrophages promote the formation of atherosclerotic plaques by sustaining inflammation, whereas M2 macrophages aid the regression of atherosclerotic

1

Department of Cardiovascular Physiology and Medicine, Graduate School of Biomedical and Health Sciences,
Hiroshima University, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima‑shi, Hiroshima  734‑8551, Japan. 2Central Research
Institute, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi, Koda‑cho, Akitakata‑shi, Hiroshima 739‑1195,
Japan. *email: miki_s@wakunaga.co.jp; yoshizum-tky@umin.ac.jp
Scientific Reports |

(2021) 11:22469

| https://doi.org/10.1038/s41598-021-01866-3

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www.nature.com/scientificreports/
plaques by promoting tissue repair, anti-inflammatory cytokine release, and efferocytosis through phagocytosis
of apoptotic c­ ells4,15–17. Specifically, M2c macrophages inhibit the accumulation of foam cells by inducing mer
tyrosine kinase (MerTK)-dependent e­ fferocytosis18,19. The polarization of macrophages to pro-inflammatory
M1 macrophages expressing TNF-α, CD68, IL-18, hypoxia-inducible factor 1α (HIF1α), C–C motif chemokine
ligand 5 (CCL5), CD86, and nitric oxide (NO) is elicited by exposure to pro-inflammatory cytokines such as
interferon-γ (IFN-γ) and TNF-α11,12,20–24. On the other hand, anti-inflammatory cytokine IL-10 induces polarization to anti-inflammatory M2c macrophages that produce IL-10, transforming growth factor-β (TGF-β),
arginase-1 (Arg1), CD206, HIF2α, scavenger receptor AI (SR-AI), Suppressor of cytokine signaling 3 (SOCS3),
and ­CD15011,12,22,24–27.
The IL-10 receptor (IL-10R) consists of a tetrameric complex with two ligand-binding subunits (IL-10Rα)
and two accessory signaling subunits (IL-10Rβ)28–31. The binding of IL-10 to the extracellular domain of IL-10Rα
results in the phosphorylation of janus kinase-1 (JAK1) and tyrosine kinase-2 (TYK2), which interact with
IL-10Rα and IL-10Rβ, ­respectively29–31. Activated JAK1 then phosphorylates tyrosine residues such as ­Tyr446 and
­Tyr496 in the intracellular domain of the IL-10Rα subunits, leading to interactions between transcription factor
signal transducer and activator of transcription 3 (STAT3) via the Src homology-2 (SH2) ­domain31–33. Additionally, phosphorylation of STAT3 at ­Tyr705 by JAK1 causes STAT3 dimerization in the cytoplasm. The dimerized
STAT3 translocates to the nucleus and binds to the promoter region of IL-10-responsive genes, resulting in
augmented transcription of M2c macrophage-associated g­ enes31,33,34.
Aged garlic extract (AGE) has been shown to demonstrate various pharmacological actions including
­antihypertensive35–37, cardiovascular ­protective38,39, and immunomodulatory ­effects40,41. It is prepared by aging
raw garlic (Allium sativum L.) in a water–ethanol mixture for more than 10 months at room ­temperature42,43.
AGE contains several water-soluble sulfur compounds such as S-1-propenylcysteine (S1PC)42,43. Recently, S1PC
has been shown to inhibit IL-6 production through autophagy activation, improve peripheral blood circulation
by increasing the NOx production, and maintain endothelial barrier ­function44–46. We have previously reported
that AGE inhibits lipid deposition in apolipoprotein E knockout (ApoE-KO) mice, an atherosclerosis ­model47,48.
However, the underlying molecular mechanism and active constituents by which AGE improves atherosclerosis
in vivo have not yet been identified.
Here, we aimed to explore the mechanism underlying the anti-atherosclerotic effect of AGE.

Results

AGE inhibited the formation of atherosclerotic plaques and the M1/M2‑like macrophage ratio
in ApoE‑KO mice.  To confirm the anti-atherosclerotic effect of AGE, we evaluated lipid accumulation in the

vascular tissue of ApoE-KO mice fed a standard diet containing 3% AGE for 17 weeks. As shown in Fig. 1a, Supplemental Fig. S1a and b, and Table S1, AGE decreased the Oil Red O-positive areas in the aorta of the ApoE-KO
mice compared with those of the mice fed normal diets, without altering the systemic lipid profiles.
We then investigated whether AGE affected macrophage polarization. Immunohistological staining showed
that AGE reduced the number of macrophages expressing iNOS, an M1 macrophage marker, in the atherosclerotic lesions of the mice (Supplemental Fig. S1d). In addition, AGE significantly suppressed the levels of Cd68
and Hif1α mRNA, out of five M1 macrophage marker genes (Cd68, Tnfα, Il-18, Hif1α, and Ccl5), in the whole
aorta (Fig. 1b). Furthermore, AGE also reduced the protein level of inducible nitric oxide synthase (iNOS) in the
spleen of the mice (Fig. 1d). On the other hand, AGE increased the number of macrophages expressing Arg1,
an M2 macrophage marker, in the atherosclerotic lesions of the mice (Supplemental Fig. S1e). Moreover, out of
five M2 macrophage marker genes (Arg1, Mrc1 coding CD206, Epas1 coding HIF2α, Msr1 coding SR-AI, and
Slamf1 coding CD150), AGE significantly increased the levels of Arg1, Mrc1 and Epas1 mRNA in the whole aorta
(Fig. 1c). It also elevated the levels of Arg1, IL-10, and CD206 proteins in the spleen of the mice (Fig. 1d). These
results suggested the possibility that AGE retards the progression of atherosclerosis partly through altering the
M1/M2 macrophage ratio in several tissues.

S1PC expanded the population of IL‑10‑induced M2c‑like macrophages.  IL-10 induces the

expression of Arg1, resulting in the polarization of macrophages to M2c ­macrophages49,50. Since AGE increased
the expression of Arg1 and IL-10 in ApoE-KO mice, we evaluated the effect of AGE on IL-10-induced increase
in Arg1 mRNA level in macrophage colony-stimulating factor (M-CSF)-induced bone marrow-derived macrophages (BMDMs). We found that AGE significantly enhanced the level of Arg1 mRNA in recombinant mouse
IL-10 (mIL-10)-treated BMDMs but not without mIL-10 (Supplemental Fig. S2a).
We then examined the effect of S1PC (Supplemental Fig. S2b) on the expression of four M2 macrophage
maker genes in mIL-10-treated BMDMs. As shown in Fig. 2a, Supplemental Fig. S2c and d, S1PC upregulated
the levels of Il-10, Arg1, Socs3, and Epas1 mRNA in the mIL-10-treated BMDMs, whereas S1PC had no effect on
the expression of these mRNAs without mIL-10. Next, we assessed whether S1PC promoted the polarization of
macrophages to M2c-like macrophages and found that S1PC increased the population of M2c-like macrophages
­(CD11b+, F4/80+, ­CD86-, ­CD206+, and ­CD150+ cells) when treated with mIL-10 for 48 h, but not 24 h and without
mIL-10 (Fig. 2b and Supplemental Fig. S2e). These results suggest that S1PC is a major active constituent in AGE
that promotes M2c-like macrophage polarization. ...

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