Prostaglandins and Other Lipid Mediators 144 (2019) 106363

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Prostaglandins and Other Lipid Mediators

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Role of acyl-CoA synthetase ACSL4 in arachidonic acid metabolism T

⁎
Hiroshi Kuwata, Shuntaro Hara
Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University, Tokyo, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: The activation of long-chain free fatty acids is the first step reaction of their usage in the cells and tissues, which
acyl-CoA synthetase are catalyzed by a family of enzymes called acyl-coenzyme A synthetases long-chain isoform (ACSL). The five
ACSL4 ACSL enzymes identified in mammals are thought to have specific and differing functions. Among them, ACSL4
Arachidonic acid is a unique isozyme that preferentially catalyzes several polyunsaturated fatty acids (PUFAs) such as arachidonic
Eicosanoid
acid (AA), and ACSL4 is thought to be an important isozyme for PUFA metabolism. Recent studies revealed that
Polyunsaturated fatty acid
ACSL4 is involved in biological responses including inflammation, steroidogenesis, cell death, female fertility,
and cancer. ACSL4 and its substrate PUFAs are thus likely to contribute to these responses. However, the roles of
ACSL4 in PUFA metabolism are not fully understood. In this review, we describe the recent progress in ACSL4
research including the involvement of this enzyme in AA metabolism.

1. Introduction
Long-chain fatty acids are important nutrients that are used as fuel
for energy, the post-translational modification of proteins, components
of membrane lipids, precursors of bioactive lipid mediators, and more.
For most biological events (anabolic and catabolic cellular responses),
free long-chain fatty acids must be converted to their respective acyl-
coenzyme A (acyl-CoA) forms. The enzymes catalyzing the first step of
the activation of fatty acids are acyl-CoA synthetases (ACS) which can
activate short-, medium-, long-, and very-long-chain fatty acids [1], and
are referred to as ACSS, ACSM, ACSL, and ACSVL, respectively. The
ACS family also includes ACS bubblegum (ACSBG) and ACS family
(ACSF) enzymes [2,3]. Because the use of the fatty acids is essential for
cell homeostasis, the members of the ACS family are evolutionarily
conserved enzymes and are expressed in bacteria, yeasts, protozoans,
insects, fish, and mammals [3].
Among the ACS family enzymes, ACSL — which catalyzes a broad
range of fatty acid substrates from 10 to 22 carbons — consists of five
ACSL isozymes (ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6) in mam-
mals. The substrate specificities of the ACSL enzymes differ among the
five isozymes [4–6], and in particular, ACSL4 prefers polyunsaturated
fatty acid (PUFAs) such as arachidonic acid (AA) and eicosapentaenoic
acid (EPA) as its substrate [7]. Indeed, several studies have shown that
ACSL4 plays a role in AA metabolism in a manner that is dependent on
the cell types (Table 1). The PUFAs are precursors of bioactive lipid
mediators such as eicosanoids, and the unique feature of ACSL4
suggests a link between it and various pathological events (e.g., in-
flammation and cancer).
Because the characteristics and functions of ACS family proteins are
discussed in other reviews [1,8], in this review we summarize the re-
cent progress regarding the functions of ACSL4, with a focus on roles of
ACSL4 in PUFA metabolism.
2. Roles of ACSL4 in eicosanoid biosynthesis following
proinflammatory stimulation
The regulation of eicosanoid biosynthesis is quite complex, and
several AA-metabolizing enzymes such as phospholipase A2 (PLA2),
cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450
(CYP) enzymes are known to be involved in this regulation. In general,
AA released after cell activation is metabolized by COX or LOX, and
then the oxidized AA is converted into bioactive eicosanoids by each
terminal eicosanoid synthase (Fig. 1). Cytochrome P450 (CYP) mono-
oxygenase catalyzes the production of epoxyeicosatrienoic acids (EETs)
and hydroxyeicosatetraenoic acids (HETEs), and both EETs and HETEs
are activated by ACSL enzymes [9,10], resulting in the production of
EET- and HETE-CoAs. These oxidized acyl-CoAs are esterified into
phospholipids [11], which cause changes in properties of the mem-
brane lipid bilayer [12]. Studies of eicosanoid biosynthesis have fo-
cused on the regulation of eicosanoid-producing enzymes, but it re-
mains unknown whether all of the released free AA uses these
metabolic pathways.

⁎
Corresponding author at: Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University, 1-5-8
Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
E-mail address: haras@pharm.showa-u.ac.jp (S. Hara).

https://doi.org/10.1016/j.prostaglandins.2019.106363
Received 31 March 2019; Received in revised form 15 June 2019; Accepted 11 July 2019
Available online 12 July 2019
1098-8823/ © 2019 Elsevier Inc. All rights reserved.
H. Kuwata and S. Hara Prostaglandins and Other Lipid Mediators 144 (2019) 106363

Table 1
Relationships between ACSL and eicosanoid biosynthesis.
Cell-Types Stimulation Eicosanoids Experimental methods Eicosanoid release Ref.

2+
Rat neutrophiles Ca -ionophore (A23187) 5-HETE 6-keto-PGF1α PGE2 Triacsin C upregulated [14]
Human endothelial cells vasopressin 6-keto-PGF1α Triacsin C no effect [16]
angiotensin-II
bradykinin
Ca2+-ionophore (ionomycin)
Human breast cancer MDA-MB-231 no 5-HETE 12-HETE 15-HETE Knockdown of ACSL4 downregulated [31]
cells
Human breast cancer MCF-7 cells no 5-HETE 12-HETE 15-HETE Overexpression of ACSL4 upregulated
Human arterial smooth muscle cells IL-1β PGE2 Overexpression of ACSL4v1 downregulated [19]
no Triacsin C or Rosiglitazone upregulated
IL-1β Knockdown of ACSL4 downregulated
Rat fibroblastic 3Y1 cells IL-1β PGs 15-HETE 11-HETE (5,11-diHETE) Triacsin C or Knockdown of upregulated [15,21]
ACSL4
Mouse embryonic fibroblasts IL-1β PGE2 PGD2 PGF2α Triacsin C
Human fibroblastic WI-38 cells IL-1β
Human monocytic THP-1 cells lipopolysaccaride
Mouse gonadal white adipose tissue high fat diet COX-, LOX-, and CYP450-derived adipocyte-specific ACSL4 no effect [36]
oxylipins knockout

HETE, hydroxyeicosatetraenoic acid; 6-keto-PGF1α, stable metabolite of PGI2; ACSL4v1, ACSL4 transcript variant 1.

Fig. 1. ACSL4 in eicosanoid biosynthesis.

ACSL4: acyl-CoA synthetase 4, COX: cycloox-
ygenase, CYP: cytochrome P450, EETs: epox-
yeicosatrienoic acids, HETEs: hydro-
xyeicosatetraenoic acids, LOX: lipoxygenase,
LTs: leukotrienes, MAGL: monoacylglycerol li-
pase, PGs: prostaglandins, PLA2: phospholipase
A2, TX: thromboxane.

Oh-ishi et al. demonstrated that following calcium-ionophore
A23187 stimulation, the treatment of peritoneal neutrophils with
triacsin C (which inhibits ACSL activity [13]) markedly enhanced the
release of eicosanoids as well as AA [14]. The enhancement of the re-
lease of eicosanoids by triacsin C treatment was also observed in several
cell types, including human fibroblastic WI-38 cells (Table 1 [15],).
These results suggest that an inhibition of the activation of AA fol-
lowing stimulation may cause the increased levels of free AA and then
the conversion of AA into eicosanoids. However, this hypothesis may
not apply to all cell types, such as human endothelial cells [16]. The
reason for this is not yet known, but there may be intricate mechanisms
underlying the biosynthesis of eicosanoids.
Because triacsin C is now known to inhibit the activities of ACSL1,
ACSL3, and ACSL4 [17,18], it is apparent that the above-mentioned
effects of triacsin C are mediated by the inhibition of these enzymes.
Further, ACSL4 is likely to be involved in the incorporation of free AA
following stimulation, since this isoform prefers PUFAs as its substrate.
Consistent with this notion, Golej et al. [19] have shown that the
overexpression of ACSL4 in human arterial smooth muscle cells reduced
the cytokine-dependent release of PGE2 compared to the release by
mock-transfected cells. In addition, the treatment of these cells with
rosiglitazone (which inhibits ACSL4 activity [20]) as well as treatment
with triacsin C enhanced the release of PGE2.
We also observed that the knockdown of ACSL4 markedly enhances
the eicosanoid biosynthesis from interleukin (IL)-1β-treated rat fibro-
blastic 3Y1 cells [15,21]. This effect is specific to the ACSL4 isoform,
because the knockdown of ACSL1 or ACSL3 failed to enhance the ei-
cosanoid release. The knockdown of ACSL4 not only enhanced the le-
vels of eicosanoids; it also accelerated the release of eicosanoids after
stimulation. In light of these results, we speculated that most of the AA
released from phospholipids after IL-1β-stimulation is eliminated
promptly via the ACSL4 pathway.
3. Involvement of ACSL4 in the release of AA into the
intramitochondrial compartment
In contrast to the biosynthesis of eicosanoids via the PLA2 pathway
mentioned above, the regulation of the release of AA from steroidogenic
cells may use an alternative AA-releasing pathway mediated by the
ACSL4-mitochondrial acyl-CoA thioesterase (ACOT2) pathway rather
than the PLA2 pathway [22,23]. In this alternative pathway, ACSL4
catalyzes the conversion of intracellular free AA into AA-CoA and
provides it to ACOT2, which releases AA in mitochondria. The released
AA in turn is metabolized by the lipoxygenase pathway to induce the

2
H. Kuwata and S. Hara
steroidogenic acute regulatory (StAR) protein [24,25]. StAR protein is a
rate-limiting enzyme of steroid hormone biosynthesis, which controls
the transport of cholesterol into the inner mitochondrial membrane,
and the expression of StAR is regulated by AA or its metabolites in these
cells [24,26,27].
Several studies demonstrated that ACSL4 localizes on the mi-
tochondria-associated membrane, endoplasmic reticulum, or peroxi-
somes of the liver and certain cancer cells [28–30]. Although further
research is needed to clarify how ACSL4 can cross-talk with ACOT2, it
has been speculated that AA-CoA produced by ACSL4 at specific cellular
compartments may translocate to mitochondria via the acyl-CoA
binding protein DBI and the mitochondrial outer membrane protein
TSPO [23].
The perturbation of the regulation of AA concentration by the
knockdown of ACSL4 and ACOT2 markedly attenuated the adreno-
corticotropin (ACTH)- or cAMP-induced release of steroid hormone
such as progesterone, and supplementation with AA recovered the
steroid hormone release from ACTH-treated steroidogenic cells [26,31].
Thus, the AA released by the ACSL4-ACOT2 pathway in mitochondria is
essential for steroidogenesis. Cho et al. demonstrated that female mice
heterozygous for ACSL4 deficiency had reduced fertility accompanied
by morphological changes of and prostaglandin accumulation in uterine
tissue [32]. Although the mechanism underlying the reduced fertility
observed in these mice is uncertain, it is possible that both perturbed
prostaglandin production and reduced AA supplementation via the
ACSL4-ACOT2 pathway (both of which attenuate steroid hormone
production) may affect female fertility.
Little is known about the cell types that use the ACSL4-ACOT2 axis
for AA release, but it has been established that the ACSL4-ACOT2 axis is
used in breast cancer cells and in steroidogenic cells. Maloberti et al.
showed that the expression levels of ACSL4 are correlated with the
malignancy of breast cancer [33]. In that study, the knockdown of
ACSL4 in the highly aggressive human breast cancer cell line MDA-MB-
231 decreased the cells' proliferation, invasion, and migration, accom-
panied by a reduction of several lipoxygenase products. The addition of
these LOX products restored the reduced cell proliferation induced by
knockdown of ACSL4. Thus, lipid mediators produced via the ACSL4-
ACOT2 pathway may regulate the progression of certain cancers.
4. The effects of ACSL4 on AA uptake and phospholipid
composition
Because ACSL4 prefers PUFAs such as AA as a substrate, this pre-
ference seems to affect the fatty acid composition of several lipids.
Indeed, there are several reports about the roles of ACSL4 in the cellular
uptake of extracellular AA. The overexpression of ACSL4 in human
arterial smooth muscle cells markedly increased the incorporation of
exogenous AA into phosphatidylethanolamines and phosphatidylinosi-
tols [19]. Similarly, the overexpression of ACSL4 in fibroblast-like COS-
7 cells facilitated the incorporation of exogenous AA as well as oleic
acid (OA) in phosphatidylinositols [34]. In COS-7 cells, exogeneous AA,
but not OA, is also incorporated into phosphatidylcholines. Thus, the
effects of ACSL4 on exogenous fatty acid incorporation may depend on
the cell type.
One of our investigations revealed that the knockdown of en-
dogenous ACSL4 expression in 3Y1 cells reduced the levels of several
phosphatidylcholine and phosphatidylinositol species bearing AA fol-
lowing IL-1β stimulation [15]. In this case, the levels of AA-containing
phospholipids are minimally affected by the knockdown of ACSL4.
More recently, it was observed that not only the levels of AA-containing
phospholipids but also those of other PUFA-bearing phospholipids are
regulated by ACSL4 [35,36]. The ablation of ACSL4 in mouse em-
bryonic fibroblasts markedly decreased the levels of PUFA-containing
phosphatidylethanolamines [35], suppressing ferroptosis (see below).
Further, adipocytes obtained from adipocyte-specific ACSL4-deficient
mice fed a high-fat diet had reduced ability to incorporate PUFA into
3
Prostaglandins and Other Lipid Mediators 144 (2019) 106363
phospholipids compared to the adipocytes from control mice [36]. The
lipidomic studies not only defined the role of ACSL4 in arachidonic acid
incorporation within phospholipids; they also showed that the resulting
linoleic acid incorporation increased with ACSL4 deficiency. Deficiency
of ACSL4 in adipocytes reduces the incorporation of AA into phos-
pholipids and AA pools, and also reduces the levels of the AA lipid
peroxidation product 4-hydroxynonenal [36]. These alternations of
lipid composition by ACSL4 deficiency resulted in the protection of the
high-fat diet-induced accumulation of adipose and liver fat, adipocyte
death, gonadal white adipocyte tissue inflammation, and insulin re-
sistance, suggesting that ACSL4 is involved in the obesity-associated
adipocyte dysfunction. Thus, the control of the levels of PUFA-con-
taining phospholipids by ACSL4 enzyme may be involved in various
physiological and pathological processes.
5. ACSL4 in the brain
ACSL4 transcripts are detected in a wide range of tissues, with
marked differences in their expression levels. Two splicing variants
have been identified in ACSL4; ACSL4 variant 1 is a ubiquitous variant
of ACSL4, and ACSL4 variant 2 appears to be restricted to the brain
[6,7]. The ACSL4 variant 2 contains an N-terminal 41-amino acids ex-
tension sequence, which is predicted to form an integral membrane-
associated domain. When these ACSL4 variants were transfected in
COS-7 cells, the ACSL4 variant 1 localized to the inside of the plasma
membrane, whereas ACSL4 variant 2 located at the endoplasmic re-
ticulum and on lipid droplets [34]. Those results also showed that the
overexpression of ACSL4 variants significantly increased the cellular
uptake of free AA to the same degree, suggesting that the ability of AA
trapping is independent of the subcellular localization of ACSL4 var-
iants.
Although the functional differences between ACSL4 splicing var-
iants are currently unknown, the differences in the subcellular locali-
zation of ACSL4 variants may contribute to their specific functions.
Meloni et al. showed that ACSL4 variant 2 is expressed in the human
brain, and the cerebellum and corpus callosum in particular express
only ACSL4 variant 2 [37]. ACSL4 variant 2 may exert its specific
functions in those brain regions, Other regions (e.g., the hippocampus)
express both ACSL4 variants. It is thus important to determine why
ACSL4 variants are distributed in specific regions of the brain and to
elucidate their functions.
Mutations of ACSL4 gene in human were found in two families with
nonspecific mental retardation [38,39]. One of the mutations was
missense and the other was a splice-site change in ACSL4 gene. The
level of the ACSL activity of lymphoblastoid cell lines from the affected
subjects of both families was low levels compared to normal cells. Thus,
both mutations are null mutations. In the brain, the expression of
ACSL4 is localized in specific regions [39,40]. In the cerebellum, ACSL4
is expressed in the soma of Purkinje and granular cells and in the
proximal dendritic region of Purkinje cells. In the hippocampus, ACSL4
is expressed in the soma of pyramidal cells. These results suggest that
ACSL4 is expressed in neurons and not in glial cells, and the decreased
expression of ACSL4 appears to be related to development of the two
families’ mental retardation. It is unclear how the reduced ACSL4 ac-
tivity could lead to mental retardation, and it is not known whether the
overproduction of eicosanoids or an alternation of the phospholipid
composition in ACSL4 deficiency contributes to mental retardation.
However, because the inhibition of ACSL4 enhances AA-induced
apoptosis [41], it has been speculated that a decreased expression of
ACSL4 in brain may lead to precocious apoptosis in neurons and to a
disruption of brain development [39]. Meloni et al. showed that ACSL4
plays an important role in dendritic spine generation [37]. The me-
chanisms underlying the roles of ACSL4 in neural developments are
thus of great interest.
H. Kuwata and S. Hara
6. ACSL4 in cancer
In addition to breast cancer, ACSL4 has been found to be upregu-
lated in several cancers including, colon, liver, and prostate cancer
[33,42–45]. Cao et al. [42] reported that they detected ACSL4 expres-
sion predominantly in colon epithelium, and the signals were much
higher in adenocarcinoma. In contrast, the expression level of ACSL4 in
colon adenoma was comparable to that in the adjacent normal tissues,
suggesting that the upregulation of ACSL4 expression is correlated with
colon cancer development. A high expression level of ACSL4 is also
observed in human hepatocellular carcinoma and several hepatoma cell
lines [46]. The forced expression of ACSL4 in ACSL4-negative hepato-
cellular carcinoma SNU398 cells promoted cancer cell growth and in-
hibited ACSL inhibitor triacsin C-induced cell death; conversely,
triacsin C inhibited the growth of Hep 3B cells (which express high
levels of ACSL4), attenuating cell proliferation [43].
Similar to hepatocellular carcinoma, the expression level of ACSL4
protein is upregulated in malignant prostatic tissues compared to be-
nign tissues. It was revealed that the expression of ACSL4 in both breast
and prostate cancer cells is inversely correlated with sex hormone re-
ceptor expression [47]. In prostate cancer LNCaP cells, which express a
trace amount of ACSL4, the transfection of ACSL4 cDNA increased cell
proliferation, migration, invasion, and anchorage-independent cell
growth, whereas the knockdown of ACSL4 in prostate cancer cells ex-
pressing endogenous ACSL4 attenuated cell proliferation, migration,
and invasion [45].
In human gastric cancer, however, the expression of ACSL4 is fre-
quently down-regulated. Ye et al. [48] showed that approx. 50% of
gastric cancer specimens decreases in the expression levels of ACSL4
mRNA by 67% of noncancerous gastric mucosa. The overexpression of
ACSL4 in gastric cancer cells inhibits cell growth and cell migration,
and, conversely, the knockdown of ACSL4 increases them. These results
are inconsistent with the ACSL4 functions observed in the above-men-
tioned cancers, and further studies are needed to explain these dis-
crepancies.
More recently, Chen et al. analyzed the roles of ACSL4 as well as
other ACSL isozymes in various cancers by performing a systematic
analysis of gene alterations and clinical outcomes [49]. The analysis
revealed that ACSL4 is upregulated in colorectal, head and neck,
kidney, myeloma, and liver cancer, but downregulated in bladder,
brain, breast, leukemia, and lung cancer. The data also showed that a
high expression of ACSL4 is associated with poor survival of patients
with colorectal cancer: in contrast, a low expression of ACSL4 is asso-
ciated with poor survival of patients with brain, breast, or lung cancer.
These data clearly indicate a relationship between ACSL4 expression
pattern and cancer progression. However, the results obtained with
breast cancer tissue are controversial because some findings are in-
consistent with these results [50], and more detailed investigations are
needed to better understand these relationships. In addition, the in-
ducible expression of ACSL4 in these cancerous tissues seems to facil-
itate or inhibit cancer cell progression, and ACSL4 may thus become the
molecular target for particular types of cancer drug development.
7. ACSL4 in cell death
PUFAs have the potential to cause cytotoxicity that is either de-
pendent on or independent of oxidative stress against various types of
cells, and the signaling pathways causing cytotoxic effects differ be-
tween cell types [41,51–53]. In some cases, ceramide- or unfolded
protein response (UPR)-mediated apoptosis is involved in the PUFA-
induced cytotoxicity [52,54]. Because these cell death signals are in-
itiated by an excess amount of PUFA, the removal of unesterified PUFAs
by their metabolic enzymes is a plausible mechanism of the suppression
of PUFA-induced cytotoxic effects. Indeed, the overexpression of a
PUFA metabolic enzyme (such as COX-2 or ACSL4) reduces AA-induced
apoptosis [41].
4
Prostaglandins and Other Lipid Mediators 144 (2019) 106363
Ferroptosis, one of the regulated cell death processes that is distinct
from other types of cell-regulated cell death (e.g., apoptosis, necrosis,
and autophagic cell death) is executed via reactive oxygen (ROS)- and
iron-dependent mechanisms [55–57]. Several ferroptosis-inducing
molecules have been discovered, and it has been revealed that the
disturbances of the appropriate controls of intracellular redox-related
product levels, including glutathione as well as lipid hydroperoxide, are
important for the execution of this process of regulated cell death. For
example, the inhibition of glutamate/cysteine antiporter system Xc−, a
key regulator of intracellular glutathione levels that causes a reduction
of cellular antioxidant glutathione, leads to ferroptosis. In line with this,
the depletion of glutathione inactivates glutathione peroxidases (GPXs)
such as GPX4, which leads to the accumulation of oxidized phospho-
lipids and the induction of ferroptosis [58]. Treatments with lipophilic
antioxidants suppressed the ferroptosis induced by the depletion of
glutathione or by the inhibition of GPX4 [55]; thus, the accumulation of
peroxidized phospholipid species is one of the critical events inducing
ferroptosis.
Because GPX4 is a unique enzyme that can remove lipid peroxides
from membrane phospholipids [59] and since PUFA-containing phos-
pholipids are potential targets for lipid peroxidation, the biosynthetic
pathway for PUFA-containing phospholipids is important for the ex-
ecution of ferroptosis. Indeed, by performing a massive insertional
mutagenesis analysis, Dixon et al. identified ACSL4 and lysopho-
sphatidylcholine acyl transferase 3, both of which are important for the
biosynthesis of PUFA-containing phospholipids as key enzymes for
ferroptosis [60]. In addition, Yuan et al. showed that erastin dose-de-
pendently induced ferroptotic cell death in ACSL4-expressing cells
(such as HepG2 and HL60 cells) but had little effect on ACSL4-negative
cells (such as LNCaP and K562 cells); further, the knockdown of ACSL4
in the former cell lines protected erastin-induced ferroptosis, and the
overexpression of ACSL4 in the latter cell lines facilitated ferroptosis
[61]. Thus, phospholipid peroxides produced via the ACSL4 pathway
are considered the ‘executioners’ of ferroptosis, and researchers have
tried to find the enigmatic lipid peroxidation products inducing fer-
roptosis. AA- and adrenic acid (AdA)-containing phosphatidylethano-
lamines, both of which are produced via the ACSL4 pathway, are oxi-
dized during ferroptosis, and their peroxidized products act as the
executioners of ferroptosis [35,62].
8. Conclusions
Long-chain fatty acids are important molecules that are active in
various metabolic and catabolic processes including energy storage and
membrane formation, and as a source of bioactive lipid mediators. The
long-chain fatty acids in these processes must be activated by ACSL
enzymes in order to be used. The dietary intake of n-3 and n-6 PUFAs
affects human health. Under these circumstances, ACSL4 activates
PUFAs and participates in several biological responses such as in-
flammation, pregnancy, and cancer development and progression as
well as cell death, via the synthesis of PUFA-derived CoAs. ACSL4 is
thus one of the principal enzymes that regulate the availability of
PUFAs, and ACSL4 might therefore become a potential target in the
treatment of PUFA-mediated exacerbations of disease.
Acknowledgments
We thank the members of our lab for their helpful discussions and
comments. This work was supported in part by Grants-in-Aid for
Scientific Research (B) (nos. 25293033 and 16H05108) from the Japan
Society for the Promotion of Science, and by a Grant-in-Aid for
Scientific Research on Innovative Areas (no. 25116720) and a grant for
a Private University High Technology Research Center Project from the
Ministry of Education, Sports, Science, Culture and Technology of
Japan.
H. Kuwata and S. Hara
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