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Interferon-γ enhances the therapeutic effect of mesenchymal stem cells on experimental renal fibrosis

金井亮広島大学

2021.02.22

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Interferon‑γ enhances
the therapeutic effect
of mesenchymal stem cells
on experimental renal fibrosis

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Ryo Kanai1, Ayumu Nakashima1,2*, Shigehiro Doi1, Tomoe Kimura1, Ken Yoshida1,
Satoshi Maeda2,3, Naoki Ishiuchi1, Yumi Yamada1, Takeshi Ike1, Toshiki Doi1, Yukio Kato2,3 &
Takao Masaki1*

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Mesenchymal stem cells (MSCs) administered for therapeutic purposes can be activated by
interferon-γ (IFN-γ) secreted from natural killer cells in injured tissues and exert anti-inflammatory
effects. These processes require a substantial period of time, leading to a delayed onset of MSCs’
therapeutic effects. In this study, we investigated whether pretreatment with IFN-γ could potentiate
the anti-fibrotic ability of MSCs in rats with ischemia–reperfusion injury (IRI) and unilateral ureter
obstruction. Administration of MSCs treated with IFN-γ strongly reduced infiltration of inflammatory
cells and ameliorated interstitial fibrosis compared with control MSCs without IFN-γ treatment.
In addition, conditioned medium obtained from IFN-γ-treated MSCs decreased fibrotic changes in
cultured cells induced by transforming growth factor-β1 more efficiently than that from control MSCs.
Most notably, secretion of prostaglandin E2 from MSCs was significantly increased by treatment with
IFN-γ. Increased prostaglandin E2 in conditioned medium obtained from IFN-γ-treated MSCs induced
polarization of immunosuppressive CD163 and CD206-positive macrophages. In addition, knockdown
of prostaglandin E synthase weakened the anti-fibrotic effects of MSCs treated with IFN-γ in IRI rats,
suggesting the involvement of prostaglandin E2 in the beneficial effects of IFN-γ. Administration of
MSCs treated with IFN-γ might represent a promising therapy to prevent the progression of renal
fibrosis.

The morbidity rate of chronic kidney disease (CKD) is estimated to be 8%–16% w
orldwide1. Etiological studies of CKD have reported multiple causes of disease initiation including hypertension, diabetes mellitus, and
glomerulonephritis2. Despite differences in disease initiation, renal fibrosis exacerbated by persistent inflammation is a histological change common to all these etiologies3,4. Currently there are few effective treatments
that prevent the progression of CKD and many patients eventually develop renal failure, which requires renal
replacement therapy, resulting in a heavy social and economic burden. Therefore, there is an urgent need for the
development of novel therapeutic strategies to treat renal fibrosis associated with CKD.
The pathogenesis of CKD is mediated by inflammatory cells, which cause renal fibrosis via fibroblast activation and increased extracellular matrix deposition5. Damage-associated molecular patterns (DAMPs) released
from damaged tissues activate the local immune system and several studies reported DAMPs were involved in
promoting renal fibrosis6,7. In CKD patients, the inflammatory microenvironment is maintained by infection,
uremic toxins, or tissue i schemia9, and this chronic inflammation contributes to the sustained release of DAMPs
from injured kidney tissues, which induces further inflammation and fibrosis. Therefore, the inhibition of inflammation is expected to ameliorate renal fibrosis.
Mesenchymal stem cells (MSCs) isolated from various tissues including bone marrow, blood, and adipose tissue10, have multipotency and self-renewal ability11,12. They exert their beneficial effects by suppressing

1

Department of Nephrology, Hiroshima University Hospital, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima,
Hiroshima 734‑8551, Japan. 2Department of Stem Cell Biology and Medicine, Graduate School of Biomedical
and Health Sciences, Hiroshima University, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima, Hiroshima 734‑8553,
Japan. 3TWOCELLS Company, Limited, 16‑35 Hijiyama‑honmachi, Minami‑ku, Hiroshima 732‑0816,
Japan. *email: ayumu@hiroshima‑u.ac.jp; masakit@hiroshima‑u.ac.jp
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inflammation and fibrosis via a paracrine mechanism13,14. Several studies have reported that MSCs or extracellular vesicles derived from MSCs have beneficial effects for renal fi
brosis15–18.
High mobility group box-1 protein (HMGB1) and interleukin-18 (IL-18) are members of DAMPs— HMGB1
was reported to promote the migration of M
SCs19,20, whereas IL-18 contributed to the secretion of interferon-γ
21
(IFN-γ) from natural killer c ells . Furthermore, IFN-γ released from immune cells at sites of damaged tissues
stimulated MSCs to secrete anti-inflammatory mediators including prostaglandin E2 (PGE2)22–26. PGE2 has been
reported to induce the polarization of immunosuppressive M2 macrophages that produce anti-inflammatory
cytokines and inhibit the persistence of inflammation27,28. In addition, we previously reported that MSCs promoted macrophage differentiation from an M1 pro-inflammatory phenotype to an immunosuppressive M2
phenotype, and that the administration of ex vivo-expanded MSCs suppressed the progression of fi
brosis29,30.
Taken together, this suggests that IFN-γ-preconditioned MSCs have a strong immunosuppressive effect and
therefore might ameliorate renal fibrosis. However, the utility of MSCs cultured with IFN-γ for the treatment of
kidney disease has not been investigated.
This study investigated the therapeutic effect of MSCs cultured in IFN-γ-containing medium on inflammation and fibrosis using rat ischemia–reperfusion injury (IRI) and unilateral ureter obstruction (UUO) models.

Results

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Expression of HMGB1, IL‑18, and IFN‑γ in the kidney after the IRI procedure. HMGB1 and
IL-18, which are known as DAMPs, are released from damaged tissues. Released IL-18, which is also induced
by HMGB18, contributes to secretion of IFN-γ19. To confirm expression of HMGB1, IL-18, and IFN-γ induced
by IRI, rats were sacrificed to evaluate their expression in the kidney at 1 day (Post IRI Day 1) and 7 days (Post
IRI Day 7) after the IRI procedure. As shown in a previous s tudy31, the protein level of HMGB1 was increased
by the IRI procedure (Fig. 1a). Immunostaining revealed that IL-18 and IFN-γ-positive areas were increased
strongly in the kidney at 1 day after IRI. However, their increase was attenuated at 7 days after IRI (Fig. 1b,c).
These changes are similar to those seen in a previous study32. Furthermore, because IL-18 promotes IFN-γ secretion from natural killer cells, IFN-γ expression might be associated with IL-18 secretion. MSCs are activated by
released IFN-γ and exert immunosuppressive effects22–26. Therefore, administration of MSCs cultured in IFN-γcontaining medium might have beneficial effects on preventing the progression of renal fibrosis.
IFN‑γ enhances the ability of MSCs to attenuate fibrosis induced by IRI. To evaluate anti-fibrotic

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effects of MSCs, we injected PBS, rat MSCs (rMSCs) treated with IFN-γ (IFN-γ rMSCs) or untreated rMSCs
(control rMSCs) into the abdominal aorta of rats after ischemic reperfusion. Twenty-one days later, the rats were
sacrificed, and injured kidneys were collected to evaluate the degree of fibrosis by western blotting. The protein
levels of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1), markers for drivers of
fibrosis, were increased in the kidney of PBS-injected rats and their levels were suppressed by injection of control
rMSCs (Fig. 2a). Furthermore, injection of IFN-γ rMSCs decreased IRI-induced fibrotic changes more significantly than that of control rMSCs (Fig. 2a). Immunostaining of α-SMA, collagen type I (Col-I), and collagen
type III (Col-III) (extracellular matrix proteins) was also performed to assess renal fibrosis. α-SMA, Col-I, and
Col-III-positive areas were increased in the PBS group. Similar to the results from western blotting, administration of IFN-γ rMSCs reduced α-SMA, Col-I, and Col-III-positive areas more strongly compared with that of
control rMSCs (Fig. 2b,c). These results suggest that IFN-γ-preconditioned rMSCs have a strong anti-fibrotic
effect. ...

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参考文献

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Acknowledgements

We would like to express our gratitude to Dr Mikihito Kajiya of the Department of Periodontal Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University for his insightful advice on the experimental

design. We would like to thank Prof. Takeshi Kawamoto of the Writing Center, Hiroshima University and Mitchell Arico from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. We also thank

Ms. Miki Kagiya for technical assistance. This study was supported in part by JSPS KAKENHI Grant Number

JP17K09699. A part of this work was carried out at the Analysis Center of Life Science, Natural Science Center

for Basic Research and Development, Hiroshima University.

Author contributions

A.N. and T.M. designed the study; R.K., A.N., T.K., K.Y., S.M., N.I, Y.Y., T.I., and T.D. carried out experiments;

R.K., S.D., N.I. and Y.K. analyzed the data; R.K. and A.N. drafted and revised the paper. All authors read and

approved the final manuscript.

Competing interests The Department of Stem Cell Biology and Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University is a collaborative research laboratory funded by TWOCELLS Company, Limited. Dr. Maeda

is the Deputy Division Manager of R&D Division II, Head Office of Research and Development of TWOCELLS

Company, Limited. Emeritus Prof. Kato is the Vice President of TWOCELLS Company, Limited. Except for the

abovementioned disclosures, all authors have declared that no conflict of interest exists.

Additional information

Supplementary Information The online version contains supplementary material available at https://doi.

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