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ARTICLE
Lysosomal cholesterol overload in macrophages
promotes liver fibrosis in a mouse model of NASH
Michiko Itoh
1,2,3,4
*, Atsushi Tamura
5
*, Sayaka Kanai
2,3
, Miyako Tanaka
1,6,7
,YoheiKanamori
1
, Ibuki Shirakawa
1
,AyakaIto
1,6
,
Yasuyoshi Oka
8
, Isao Hidaka
9
, Taro Takami
9
, Yasushi Honda
10
, Mitsuyo Maeda
11,12
, Yasuyuki Saito
13
, Yoji Murata
13
,
Takashi Matozaki
14
,AtsushiNakajima
10
, Yosky Kataoka
11,12
, Tomoo Ogi
8
, Yoshihiro Ogawa
1,15
, and Takayoshi Suganami
1,6,7,16
Accumulation oflipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis
in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have
previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse
and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal
cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of
NASH. β-cyclodextrin polyrotaxane (βCD-PRX), a unique supramolecule, isdesigned to elicit free cholesterol from lysosomes.
Treatment with βCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead
hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic
cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in
macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol
metabolism in macrophages would be a novel mechanism of NASH.
Introduction
Nonalcoholic fatty liver disease (NAFLD) is a clinical spectrum
that encompasses simple steatosis to nonalcoholic steatohepatitis
(NASH), the latter of which is considered among the top etiologies
for hepatocellular carcinoma and indications for liver transplan-
tation (Younossi et al., 2018,2019). Although simple steatosis is
generally benign and reversible, 10–30% of NAFLD patients de-
velop NASH and cirrhosis. According to the two-hit or multiple-
hithypothesis,thepathogenesisofNASHinvolvesdysregulated
lipid metabolism and inflammatory and profibrotic cues (Tilg and
Moschen, 2010). Numerous clinical trials targeting each process
have been conducted worldwide; however, there are no approved
therapeutic strategies for NASH (Vuppalanchi et al., 2021).
The concept of “lipotoxicity”has been pointed out that cyto-
toxic lipids, such as cholesterol, fatty acids, and their metabolites,
may directly cause cell death or act in a proinflammatory and
profibrotic manner in the pathogenesis of NASH (Neuschwander-
Tetri, 2010). Lipidomic analysis of human livers revealed that
levels of free cholesterol are increased in NASH, while those of
esterified cholesterol do not change compared to simple steatosis
(Caballero et al., 2009;Van Rooyen et al., 2011). Recent evidence
has shown that excessive dietary cholesterol exaggerates stea-
tohepatitis in rodents and humans (Farrell et al., 2019;Musso
et al., 2003;Savard et al., 2013). Moreover, free cholesterol is an
important lipotoxic lipid inducing hepatocyte death (Gan et al.,
2014;Mar´
ıet al., 2006), upregulation of profibrotic factors in
hepatocytes (Wang et al., 2020), and activation of hepatic stellate
cells, all of which lead to overproduction of extracellular matrix
(Teratani et al., 2012;Tomita et al., 2014). Interestingly, there
.............................................................................................................................................................................
1
Department of Molecular Medicine and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan;
2
Department of Bioelectronics,
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan;
3
Kanagawa Institute of Industrial Science and Technology, Kawasaki,
Japan;
4
Department of Metabolic Syndrome and Nutritional Science, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan;
5
Department of
Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan;
6
Department of Immunometabolism, Nagoya
University Graduate School of Medicine, Nagoya, Japan;
7
Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Nagoya, Japan;
8
Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan;
9
Department of Gastroenterology, Yamaguchi University
Graduate School of Medicine, Yamaguchi, Japan;
10
Department of Gastroenterology and Hepatology, Yokohama City University Graduate School of Medicine, Yokohama,
Japan;
11
Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Kobe, Japan;
12
Laboratory for Cellular Function Imaging, RIKEN Center for
Biosystems Dynamics Research, Kobe, Japan;
13
Division of Molecular and Cellular Signaling,Department of Biochemistry and Molecular Biology, Kobe University Graduate
School of Medicine, Kobe, Japan;
14
Division of Biosignal Regulation, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine,
Kobe, Japan;
15
Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;
16
Center for One Medicine
Innovative Translational Research, Gifu University Institute for Advanced Study, Gifu, Japan.
*M. Itoh and A. Tamura contributed equally to this paper. Correspondence to Michiko Itoh: mito@riem.nagoya-u.ac.jp; Takayoshi Suganami: suganami@riem.nagoya-
u.ac.jp.
© 2023 Itoh et al. This article is available under a CreativeCommons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
Rockefeller University Press https://doi.org/10.1084/jem.20220681 1of20
J. Exp. Med. 2023 Vol. 220 No. 11 e20220681
exist cholesterol crystals within the lipid droplets of steatotic
hepatocytes in both NASH patients and NASH models (Ioannou
et al., 2013,2017). Of note, cholesterol crystals are observed in
almost all the patients with fibrosing NASH but only in a few
patients with simple steatosis (Ioannou et al., 2019), suggesting
the pathological significance of cholesterol crystallization in the
development of NASH.
We and others have reported unique histological structures
termed crown-like structures (CLS), where macrophages sur-
round and engulf dying or dead hepatocytes with large lipid
droplets in murine and human NASH (Ioannou et al., 2013;Itoh
et al., 2013,2017). Activated fibroblasts and collagen deposition
are observed around CLS, and the number of CLS has been
positively correlated with the fibrosis area, suggesting that CLS
are the site of interaction of dead hepatocytes and stromal cells,
which exerts as a driving force of liver fibrosis (Itoh et al., 2013).
Indeed, hepatocyte death triggers phenotypic changes in mac-
rophages constituting CLS thereby acquiring profibrotic
properties (Itoh et al., 2017). We also characterized these mac-
rophages as a disease-specific macrophage subset with a gene
expression profile distinct from other scattered macrophages
(Itoh et al., 2017;Kanamori et al., 2021). However, it still re-
mains unclear how these macrophages undergo phenotypic
changes interacting with dead hepatocytes.
In this study, we focused on the lysosomal accumulation of
free cholesterol and subsequent lysosomal dysfunction in CLS-
constituting macrophages in our NASH model using genetically
obese melanocortin 4 receptor–deficient (MC4R-KO) mice
(Farrell et al., 2019;Itoh et al., 2011). We employed
β-cyclodextrin polyrotaxane (βCD-PRX), a unique supramolec-
ular compound, to excrete free cholesterol specifically from ly-
sosomes (Tamura and Yui, 2015,2018). When administered to
MC4R-KO mice, βCD-PRX effectively ameliorated liver fibrosis
at least partly by decreasing free cholesterol content in macro-
phages and suppressing the activation of profibrotic pathways in
a NASH model, without affecting the hepatic and serum levels of
cholesterol. In vitro experiments revealed that loading of cho-
lesterol crystals induces lysosomal dysfunction and profibrotic
changes in macrophages, which was reversed by βCD-PRX ad-
ministration. This study demonstrates that lysosomal cholesterol
overload triggers phenotypic changes and profibrotic activation
of macrophages interacting with dead hepatocytes, which would
be a novel mechanism of NASH development.
Results
Cholesterol crystallization in hepatocytes and cholesterol
loading to macrophages in NASH
Because CLS formation precedes the onset of liver fibrosis in a
murine model of NASH, we compared the number of CLS with
histological scores in liver biopsy specimens and measurements
of FibroScan, a noninvasive imaging modality, in patients at an
earlier stage of NAFLD/NASH. A total of 98 patients with mild
liver dysfunction and hyperglycemia were analyzed (Table S1).
The score of the controlled attenuation parameter (CAP), which
represents hepatic lipid content, was increased in parallel with
the NAFLD activity score (NAS) and decreased in patients with
advanced fibrosis (Fig. S1 A). The score of liver stiffness mea-
surement (LSM) was increased only in advanced fibrosis (fi-
brosis stage > 3; Fig. S1 B)asreported(Eddowes et al., 2019). The
number of CLS was elevated at the early stages of NASH (cor-
responding to a NAS score of 5 and a fibrosis stage of 1 or 2),
whereas that was rather decreased in the advanced stages of
NASH (Fig. S1 C), consistent with our previous data (Itoh et al.,
2013,2017). These findings indicate that CLS is an earlier marker
for liver fibrosis than LSM and raise a strong need to investigate
the underlying mechanisms of CLS formation for better under-
standing the initial stage of NASH.
Thus, we performed electron microscopic analysis of the
livers of MC4R-KO mice fed a Western diet (WD) for 20 wk,
which exhibited obesity, insulin resistance, and NASH-like liver
phenotypes (Farrell et al., 2019;Itoh et al., 2011). Fine cholesterol
crystals were observed within the lipid droplets of hepatocytes
(Fig. 1 A). Cholesterol crystals were also present in the remnant
lipids of dead hepatocytes surrounded by macrophages, and lipid
accumulation was conversely observed in macrophages when
the remnant lipids were reduced (Fig. 1 B). Unlike macrophages
in the normal liver, numerous lipid droplets, lysosomes, and
autolysosomes were observed in the cytoplasm of macrophages
in NASH livers (Fig. 1 C). Polarized light microscopy also re-
vealed the presence of large cholesterol crystals in CLS (Fig. 1 D).
Cellular cholesterol content was increased only in the CD11c-
positive F4/80
hi
CD11b
lo
macrophages from NASH livers,
which form CLS (Itoh et al., 2017), compared with the CD11c-
negative macrophages from normal and NASH livers (Fig. 1 E).
On the other hand, cholesterol content remained unchanged
in F4/80
lo
CD11b
hi
macrophages (Fig. 1 E). In atherosclerotic
plaques, cholesterol taken up by macrophages undergoes
degradation in lysosomes and induces lysosomal stress (Sergin
et al., 2015). Similarly, the enhanced immunostaining of lyso-
somal enzyme cathepsin D (CTSD) was observed in the macro-
phages forming CLS in MC4R-KO mice and human NASH (Fig. 1,
F and G). These data suggest that remnant lipids, including
cholesterol crystals, in dead hepatocytes induce lysosomal stress
in CLS-constituting macrophages both in mice and humans.
Optimization of chemically modified βCD-PRX targeting
the liver
To elucidate the role of lysosomal cholesterol overload in CLS-
constituting macrophages, we came up with a unique supra-
molecule, βCD-PRX (Tamura et al., 2016;Tamura and Yui, 2018).
2-Hydroxypropyl-βCD (HP-βCD) is known to form the inclusion
complex with free cholesterol and remove it from the cells
(Kilsdonk et al., 1995). βCD-threaded acid-degradable PRXs are
preferentially distributed to endosomes and lysosomes through
endocytosis, and the stopper molecules in βCD-PRXs are
designed to release βCD under lysosomal acidic conditions
(Fig. 2 A). Thus, βCD-PRXs serve as carriers to deliver βCD to
lysosomes efficiently. Our group has developed various
chemically modified βCD-PRXs because the chemical modifi-
cation plays an essential role in imparting solubility in aqueous
media and modulating body disposition (Nishida et al., 2016;
Ohashi et al., 2021;Tamura et al., 2022;Tamura and Yui, 2014,
2015;Tonegawa et al., 2020;Zhang et al., 2021).
Itoh et al. Journal of Experimental Medicine 2of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
To identify the optimal chemical modification for liver tar-
geting, seven series of βCD-PRXs with different chemical mod-
ifications, including nonionic, anionic, cationic, and zwitterionic
groups were synthesized and tested (Fig. 2 B and Table S2).
When the inclusion ability of cholesterol was tested at pH 7.4, all
chemically modified βCD-PRXs showed negligible ability to
solubilize free cholesterol, unlike HP-βCD (Fig. 2 C), because the
inclusion of cholesterol was inhibited by the occupation of the
hydrophobic cavity of threaded βCDs with an axial polymer
chain. In contrast, all chemically modified PRXs solubilized
cholesterol at pH 5.0 because the released βCDs formed an in-
clusion complex with free cholesterol (Fig. 2 C). Cytotoxicity
tests revealed cytotoxicity of several chemically modified βCD-
PRXs at high concentrations (methyl, carboxymethyl carbamate,
and 2-(N,N-dimethylamino)ethyl carbamate [DMAE]), and HP-
βCD (Fig. 2 D). We also examined the toxicity of various
chemically modified βCD-PRX in vivo 24 h after subcutaneous
administration into WT mice. Serum levels of aspartate
Figure 1. Cholesterol crystallization and lysosomal stress in CLS-constituting macrophages in NASH livers. (A–C) Electron micrographs of NASH livers
from MC4R-KO mice fed a WD for 20 wk (MC/WD) and WT mice kept on an SD (WT/SD). (A) Fine cholesterol crystals were observed in lipid droplets of
hepatocytes (insets). Asterisks, CLS. Scale bar, 10 μm. (B) Cholesterol crystallization in the remnant lipids of dead hepatocyte surrounded by macrophages
(left), and lipid accumulation in CLS-constituting macrophages (right). Scale bars, 10 μm. (C) Macrophages in sinusoids of normal liver (left) and CLS-
constituting macrophage (right). N, nucleus; LD, lipid droplets; RL, remnant lipid of dead hepatocyte; arrowheads, lysosomes; white arrows, autolyso-
somes. Scale bars, 5 μm. (D) Representative image of polarized light microscope of the liver from MC4R-KO mice transplanted with bone marrow cells from
GFP-transgenic mice and fed a WD for 20 wk. Scale bar, 10 μm. (E) Total cholesterol content of macrophages isolated from normal (WT/SD) and NASH livers
(MC/WD). Gatingstrategies for F4/80
hi
CD11b
lo
macrophages (Mφ): CD45
+
Ly6G
−
SiglecF
−
F4/80
hi
CD11b
lo
;F4/80
lo
CD11b
hi
Mφ:CD45
+
Ly6G
−
SiglecF
−
F4/80
lo
CD11b
hi
.F4/80
hi
CD11b
lo
macrophages were separated based on the expression levels of CD11c. n= 4. **P < 0.01; n.s., not significant. (F) Serial sections of the
livers from WT mice fed an SD and MC4R-KO mice fed a WD stained with F4/80 and CTSD antibodies. Arrows, CLS; C, central veins; P, portal veins. Scale bars,
50 μm. (G) Serial sections of the livers from NASH patients stained with CD68, CD11c, and CTSD. Arrows, CLS. Scale bars, 50 μm. Data and images are
representative of two independent experiments (A–F). Error bars represent means ± SEM.
Itoh et al. Journal of Experimental Medicine 3of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Figure 2. Optimization of chemical modification for βCD-PRXs. (A) Schematic illustration showing the mechanism of action of βCD-PRX. (B) Structure of
chemically modified βCD-PRXs. Me, methyl; Ac, acetyl; MEEE, 2-(2-(2-methoxyethoxy)ethoxy)ethyl carbamate; CM, carboxymethyl carbamate; and SPAE, 2-(N-
3-sulfopropyl-N,N-dimethylammonium)ethyl carbamate. (C) Cholesterol binding capacity evaluated by the solubility of cholesterol in the presence of each
βCD-PRX and HP-βCD (βCD) under neutral and acidic pH conditions. Open bar, pH 7.4; solid bar, pH 5.0. n= 3. **P < 0.01, n.s.; not significant. (D) Cytotoxicity
of chemically modified βCD-PRXs and βCD in RAW264 macrophages. n=5.(E) Representative images ofhematoxylin and eosin staining of the livers from WT
mice treated with chemically modified βCD-PRXs at a dose of 200 mg/kg for 24 h. (F and G) Hepatic mRNA expression of inflammatory genes (F) and tissue
Itoh et al. Journal of Experimental Medicine 4of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
aminotransferase, alanine aminotransferase (ALT), and alka-
line phosphatase were elevated in mice treated with DMAE-
modified βCD-PRX (Table S3). Histological examinations revealed
immune cell infiltration into the livers in DMAE-modified βCD-
PRX, and hepatic expression of inflammatory genes was up-
regulated in methyl, acetyl, and DMAE-modification groups
consistent with the cytotoxicity test (Fig. 2, E and F). Among
others, 2-(2-hydroxyethoxy)ethyl carbamate (HEE)–modified
βCD-PRX showed only minimal effects on these parameters. A
biodistribution study of chemically modified βCD-PRXs reveled
that HEE modification demonstrated the highest accumulation
in the liver in WT mice (Fig. 2 G). Thus, the HEE group-
modified βCD-PRX (hereafter indicated as βCD-PRX) was
used in the subsequent studies.
We examined cellular distribution of BODIPY-labeled βCD-
PRX in the livers of WT mice treated with a single injection of
βCD-PRX at different doses. βCD-PRX uptake was observed
mainly in hepatocytes, sinusoidal endothelial cells, and macro-
phages in a dose-dependent manner, whereas little uptake was
detected in the (CD45 and CD146-negative) “others”fraction,
including fibroblasts and biliary epithelial cells (Fig. 3 A).
Moreover, we confirmed a hepatotrophic biodistribution in a
NASH model (MC4R-KO mice fed a WD for 20 wk; Fig. 3 B). The
uptake rate was significantly increased in several cell types after
the onset of NASH, but not in the other fractions (Fig. 3 C).
Taken together, we considered that the HEE group is the optimal
chemical modification for further study.
βCD-PRX attenuates liver fibrosis by reducing cholesterol
accumulation in macrophages
To elucidate the pathophysiological role of lysosomal cholesterol
accumulation in CLS-constituting macrophages in the develop-
ment of NASH, MC4R-KO mice received subcutaneous admin-
istration of βCD-PRX for 6 wk, after they developed NASH (Fig. 4
A). The liver weight and serum ALT levels were decreased with
βCD-PRX treatment, whereas the serum and hepatic levels of
triglyceride and (total, free, and esterified) cholesterol and the
area of cholesterol crystals in the liver did not change (Fig. 4,
B–D;Fig. S2 A;andTable 1). Using isolated hepatocytes from
MC4R-KO mice treated with βCD-PRX, we found that βCD-PRX
did not affect mRNA expression of lipid metabolism–related
genes in hepatocytes (Fig. S2 B). βCD-PRX also showed only
marginal effects on cholesterol contents in primary cultured
hepatocytes prepared from normal and steatotic livers (Fig. S2
C). In contrast, βCD-PRX treatment markedly inhibited the in-
crease in the free cholesterol content of the CD11c-positive
macrophages (localizing to CLS) from NASH livers (P < 0.01,
Fig. 4 E). Moreover, βCD-PRX treatment markedly suppressed
hepatic mRNA expression of Ccl2 and profibrotic factors, along
with their serum cytokine levels, in the NASH model (Fig. 4 F
and Table S4) without remarkable impact on hepatic expression
of genes related to cholesterol metabolism, de novo lipogenesis,
β-oxidation, and glucose metabolism, except Mttp, which me-
diates very-low-density lipoprotein secretion from the livers
(Fig. 4 F). Moreover, Sirius red staining and desmin im-
munostaining (representing activated fibroblasts) revealed that
βCD-PRX effectively suppressed liver fibrosis (Fig. 4, G and H).
These observations, taken together, indicate that βCD-PRX
ameliorates the development of liver fibrosis by mainly acting
on macrophages rather than hepatocytes and fibroblasts.
Effect of βCD-PRX on lysosomal stress in CLS-constituting
macrophages
Next, we evaluated the effect of βCD-PRX on the macrophages
constituting CLS because these macrophages are supposed to
interact with dead hepatocytes containing cholesterol crystals.
F4/80 immunostaining revealed the comparable formation of
CLS, regardless of βCD-PRX treatment (Fig. 5 A). We have pre-
viously identified increased lysosomal stress and subsequent
activation of transcription factor E3 (TFE3) in the CLS-
constituting macrophages by which CD11c expression was in-
duced in these cells (Kanamori et al., 2021). In this study, nuclear
TFE3 immunostaining in CLS was markedly suppressed by βCD-
PRX treatment, along with CD11c expression (Fig. 5 B). These
CLS-constituting macrophages were positive for C-type lectin
domain family 4 member F (Clec4f) immunostaining in our
NASH model as previously reported (Itoh et al., 2017). Inter-
estingly, TIM4 immunostaining was almost absent in the mac-
rophages forming the CLS in the control group. In contrast, there
was a modest positive signal observed in the βCD-PRX–treated
group, suggesting that βCD-PRX treatment affects the gene ex-
pression profiles related to Kupffer cell (KC) identity (Fig. 5 C).
βCD-PRX treatment also suppressed gene expression of
Atp6v0d2 (a lysosomal stress marker), Itgax (CD11c), profibrotic
Spp1 (osteopontin), and Pdgfb in F4/80
hi
CD11b
lo
macrophages
isolated from the livers at the end of the experiment (Fig. 5 D).
Histological analysis revealed osteopontin immunostaining
in CLS and the osteopontin-positive area was reduced with
βCD-PRX treatment in parallel with mRNA expression levels
(Fig. 5 E).
Moreover, we confirmed the effect of βCD-PRX in another
mouse model of NASH, in which WT mice were fed a high-
cholesterol (HC) diet for 20 wk (Boland et al., 2019), and re-
ceived βCD-PRX treatment (30 mg/kg/d) during the last 6 wk
(Fig. S3 A). There were no significant changes in body weight,
liver weight, and liver triglyceride and cholesterol contents
(Fig. S3 B and Table S5). βCD-PRX suppressed TFE3 nuclear
translocation in CLS and fibrotic changes in the liver (Fig. S3,
C–E). These observations support the therapeutic effect of
βCD-PRX on NASH development independently of MC4R
signaling. Collectively, these observations indicate that βCD-
PRX treatment ameliorates lysosomal stress response and
suppresses profibrotic phenotypes of the CLS-constituting
macrophages.
distribution evaluated by fluorescence intensities (G) in WT mice 24 h after subcutaneous injection of Cy5.5-labeled βCD-PRXs at a dose of 200 mg/kg. Emr1,
EGF-like module-containing mucin-like hormone receptor-like1 (F4/80); TNF, tumor necrosis factor-α.n= 5. *P < 0.05, **P < 0.01versus PBS. Data and images
are representative of two independent experiments. Error bars represent means ± SEM.
Itoh et al. Journal of Experimental Medicine 5of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Cholesterol crystals induce inflammatory and profibrotic
changes in macrophages from steatotic livers
We next performed cholesterol loading experiments in vitro
using hepatic macrophages prepared from WT mice on a stan-
dard diet (SD; normal livers) and MC4R-KO mice fed a WD for
6–8 wk (steatotic livers; Fig. 6 A). Cholesterol crystals were
added to hepatic macrophages, and then lysosomes and choles-
terol were stained with lysosome-associated membrane protein
(LAMP) 1 and Filipin, respectively (Fig. 6 B). Treatment with
cholesterol crystals resulted in increased expression of galectin-
3, a marker of lysosomal membrane damage, and nuclear
translocation of TFE3. These effects were reversed by βCD-PRX
treatment (Fig. 6, C and D). RNA sequencing (RNA-seq) was
conducted to compare gene expression profiles between hepatic
macrophages from normal livers and steatotic livers treated
with cholesterol crystals (n= 2). Gene ontology analysis of 1,049
genes upregulated (>twofold) in macrophages from steatotic
livers relative to those from normal livers revealed activation of
inflammatory pathways in steatotic livers (Fig. 6 E). The number
of genes, whose expression was increased by the treatment with
cholesterol crystals, was much higher in macrophages from
steatotic livers (Fig. 6 F). Unsupervised hierarchical clustering
revealed differential responses to cholesterol crystals between
the macrophages from normal and steatotic livers (Fig. 6 G).
Several inflammatory cytokines and lysosome-related genes
(Atp6v0d2) were similarly upregulated by the treatment with
cholesterol crystals in the macrophages from normal and stea-
totic livers (cluster E), whereas genes characteristic for scar-
associated macrophage (SAM) or NASH-associated macrophage
(Ramachandran et al., 2019;Xiong et al., 2019), such as Itgax,
Vegfa,Fabp4,Spp1,andKcnn4, were upregulated only in the
macrophages from steatotic livers (clusters B and C). We con-
firmed the data by quantitative real-time PCR (Fig. 7 A). In-
triguingly, ingenuity pathway analysis using RNA-seq data
revealed early growth response 1 (Egr1) activation as an up-
stream transcription regulator specific for steatotic liver–derived
hepatic macrophages treated with cholesterol crystals (Fig. 7 B).
We also found increased mRNA expression and nuclear trans-
location of Egr1 in hepatic macrophages isolated from steatotic
livers (Fig. 7, C and D). It has been reported that Egr1 induces
production of adhesion molecules, cytokines, and growth factors
including osteopontin and platelet-derived growth factor (PDGF)
Figure 3. Biodistribution of βCD-PRX in the liver. (A) Cellular uptake of BODIPY-modified HEE-βCD-PRX at various dosages in WT mice. (B) Tissue dis-
tribution of HEE-βCD-PRX evaluated by fluorescence intensities in MC4R-KO mice fed a WD for 20 wk. n=3.(C) Comparison of HEE-βCD-PRX distribution at a
dose of 200 mg/kg in WT mice fed an SD (WT/SD) and MC4R-KO mice fed a WD for 20 wk (MC/WD). n= 3. *P < 0.05, **P < 0.01 versus WT/SD. Data are
representative of two independent experiments. Error bars represent means ± SEM. MFI, mean fluorescence intensity.
Itoh et al. Journal of Experimental Medicine 6of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Figure 4. Effect of βCD-PRX onliver fibrosis in a mouse model of NASH. (A) Experimental protocol of βCD-PRX treatment in a NASH model using MC4R-
KO mice. After the development of NASH with 18-wk WD feeding, MC4R-KO mice were received implantation of osmotic minipumps at a dose of 30 mg/kg/d
Itoh et al. Journal of Experimental Medicine 7of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
in certain cell types (Khachigian and Collins, 1997;Wang et al.,
2021;Wu et al., 2009). These data indicate that cholesterol
crystals induce profibrotic changes only in macrophages from
steatotic livers, which may acquire proinflammatory traits after
exposure to the microenvironmental milieu of steatosis.
βCD-PRX alleviates cholesterol crystal–induced lysosomal
dysfunction by promoting cholesterol excretion
Next, we examined the molecular mechanisms of action of
βCD-PRX using RAW264 macrophages because RAW264
macrophages showed similar gene expression profiles (RNA-
seq data, n= 4) and immunostaining patterns to hepatic
macrophages from steatotic livers when treated with choles-
terol crystals (Fig. 8, A and B;andFig. S4, A–D). Acridine
orange emits fluorescence in acidic environments, indicating
the intact function of lysosome membranes (Kanamori et al.,
2021). Flowcytometric analysis revealed increased side scatter
intensity, reflecting the uptake of cholesterol crystals (Ioannou
et al., 2019), and decreased fluorescence intensity of acridine
orange, indicating lysosomal dysfunction (Fig. 8 C). βCD-PRX
treatment prevented cholesterol overload–induced lysosomal
dysfunction (Fig. 8 C) and restored lysosomal function even
after cholesterol crystals were once taken up by macrophages
(Fig. 8 D). βCD-PRX treatment also suppressed the otherwise
increased protein levels of microtubule-associated protein
1 light chain 3-II form (LC3-II), which localizes autophagosomal
membrane, and p62, a selective substrate for autophagic pro-
tein degradation (Fig. S4 E). Similar results were obtained using
a tandem fluorescence-tagged vector (mRFP-GFP-LC3), which
emits both mRFP and GFP signals in autophagosomes and only
an mRFP signal in autolysosomes (Kimura et al., 2007;Fig.
S4 F). All these data indicate that βCD-PRX treatment
protects against cholesterol crystal–induced lysosomal
stress/injury in macrophages. Although cholesterol loading
affected endoplasmic reticulum stress and mitochondrial func-
tion, βCD-PRX treatment showed only limited effects (Fig. S4, G
and H).
We next compared the effect of βCD-PRX with that of HP-
βCD unthreaded in linear polymer and simvastatin, a 3-hy-
droxy-3-methylglutaryl-CoA reductase inhibitor. Treatment
with HP-βCD and simvastatin showed only limited or no ef-
fects on intracellular cholesterol accumulation and cholesterol
excretion in the media (Fig. 8, E and F). In line with this result,
the expression of TFE3-target genes was not suppressed by
HP-βCD or simvastatin (Fig. 8 G). FACS analysis using cholera
toxin subunit B, a lipid raft marker, revealed that HP-βCD
disrupted the integrity of lipid rafts, but βCD-PRX did not (Fig.
S4 I). Moreover, we examined the effect of βCD-PRX on
cholesterol-independent inflammatory stimuli, such as TNFα
and L-leucyl-leucine methyl ester, a lysosome-destabilizing
agent, and found that βCD-PRX treatment did not suppress
mRNA expression of proinflammatory and profibrotic genes
under these stimuli (Fig. S5, A and B). These results suggest
that βCD-PRX exerts its anti-profibrotic effects via enhanced
lysosomal cholesterol excretion and subsequent amelioration
of lysosomal stress/injury.
Consistent with the data using hepatic macrophages isolated
from steatotic macrophages (Figs. 6 and 7), increased mRNA
expression and nuclear translocation of Egr1 were observed in
RAW264 macrophages treated with cholesterol crystals (Fig. S5,
C and D). Knockdown of Egr1 almost completely inhibited the
expression of osteopontin and PDGF without affecting the ex-
pression of lysosome-related genes (Fig. 8 H). We also found that
knockdown of MiT/TFE transcription factors (TFE3/TFEB
[transcription factor EB]) markedly suppressed the expression
of Egr1, lysosome-related genes, and Egr1-target genes including
Spp1 and Pdgfb (Fig. 8 I and Fig. S5 E).Theseobservationssuggest
that TFE3/TFEB drives fibrogenic pathways at least partly through
activation of Egr1.
of βCD-PRX or normal saline as a control (Cont) for an additional 6 wk. PRX, βCD-PRX. WT/SD-cont, n= 6; MC/WD-cont, n=9;MC/WD-PRX,n=8.(B) Body
weight and liver weight after βCD-PRX treatment. (C) Hepatic TG and total cholesterol (TC) content. (D) Area of cholesterol crystals in the liver evaluated by
polarized light microscope. (E) Free and esterified cholesterol content of F4/80
hi
CD11b
lo
macrophages isolated from the livers at the end of the experiment.
Open bars, CD11c-negative macrophages; closed bars, CD11c-positive macrophages. (F) Hepatic mRNA expression of genes related to lipid metabolism, in-
flammation, and fibrogenesis. Nr1h3, nuclear receptor subfamily 1 group H member 3 (LXRα); Nr1h2, nuclear receptor subfamily 1 group H member 2 (LXRβ);
Srebf, sterol regulatory element binding transcription factor; Ppara, peroxisomal proliferator-activated receptor α;Abca1, ATP binding cassette subfamily A
member 1; Agcg1, ATP biding cassette subfamily G member 1; Ldlr, low density lipoprotein receptor; Msr1, macrophage scavenger receptor 1; Hmgcr,
hydroxymethylglutaryl-CoA reductase; Hmgcs, hydroxymethylglutaryl-CoA synthase; G6pase, glucose 6-phosphatase; Pfk, 6-phosphofructokinase; Acox,per-
oxisomal acyl-coenzyme A oxidase 1; Cpt1a; carnitine palmitoyltransferase 1A; Mttp, microsomal triglyceride transfer protein; Fasn, fatty acid synthase; Acc1,
acetyl-CoA carboxylase 1; Itgax, integrin subunit αX(CD11c);IL1β, interleukin-1β;Ccl2, C-C motif chemokine ligand 2; Tgfβ1, transforming growth factor β1;
Pdgfb, platelet-derived growth factor subunit B; Spp1, secreted phosphoprotein 1; Timp1, tissue inhibitor of metalloproteinase 1; Col1a1, collagen type I αchain;
and Col4a1, collagen type IV αchain. (G and H) Fibrosis area evaluated by Sirius red staining (G) and quantification of desmin-positive area (H). C,central veins.
Scale bars, 50 μm. *P < 0.05, **P < 0.01 versus WT/SD-Cont;
#
P < 0.05,
##
P < 0.01. Data and images are representative of two independent experiments. Error
bars represent means ± SEM.
Table 1. Serological parameters of MC4R-KO and WT micetreated with
βCD-PRX for 6 wk
WT/SD MC/WD
Cont Cont PRX
TG (mg/dl) 112.5 ± 1.8 120.1 ± 8.8 134.4 ± 16.5
TC (mg/dl) 93.7 ±4.4 351.3 ± 20.0** 317.3 ± 18.2**
ALT (U/liter) 35.8 ± 1.8 287.9 ± 38.3** 173.9 ± 34.1**
#
Insulin (ng/ml) 0.4 ± 0.1 33.6 ± 11.9* 23.0 ± 7.9
MC, MC4R-KO mice; Cont, control; PRX, βCD-PRX; TC, total cholesterol.
WT/SD-Cont, n= 6; MC/WD-Cont, n=9;MC/WD-PRX,n=8.**P<0.01
versus WT/SD-Cont;
#
P < 0.05 versus MC/WD-Cont. Data are
representative of two independent experiments. Data are expressed as the
mean ± SEM.
Itoh et al. Journal of Experimental Medicine 8of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Figure 5. Effect of βCD-PRX on inflammatory and profibrotic phenotypes of CLS-constituting macrophages in NASH. Immunohistochemistry of the
livers of MC4R-KO mice treated with βCD-PRX for 6 wk. (A) F4/80 immunostaining and quantification of CLS number. C,central veins. Scale bars, 50 μm. WT/
SD-Cont, n= 6; MC/WD-Cont, n= 9; MC/WD-PRX, n=8.(B) Immunofluorescence staining of TFE3, F4/80, and CD11c, and quantification of the rates of TFE3
nuclear translocation and CD11c-positive CLS. Nuclei were counterstained with DAPI. Scale bars, 50 μm. MC/WD-Cont, n=9;MC/WD-PRX,n=8.**P<0.01
versus MC/WD-Cont. (C) Representative images of Clec4f and Tim4 immunostaining of the livers from WT and MC4R-KO mice. Arrows, CLS. Scale bars, 50 μm.
(D) mRNA expression levels of genes related to NASH-specific macrophage phenotypes, including Itgax,Atp6v0d2,Spp1,andPdgfb in F4/80
hi
CD11b
lo
mac-
rophages sorted from livers of MC4R-KO mice after 6-wk βCD-PRX treatment. n=3.(E) Representative images of serial sections stained with anti-F4/80 and
Osteopontin antibodies. Scale bars,50 μm. WT/SD-Cont, n=6;MC/WD-Cont,n= 9; MC/WD-PRX, n= 8. **P < 0.01 versus WT/SD-Cont.
#
P < 0.05,
##
P < 0.05.
Data and images are representative of two independent experiments. Error bars represent means ± SEM.
Itoh et al. Journal of Experimental Medicine 9of20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Figure 6. Effect of cholesterol crystals and βCD-PRX on gene expression profiles in macrophages isolated from normal and steatotic livers.
(A) Experimental protocol using primary hepatic macrophages. Hepatic macrophages were isolated using magnetic columns from normal livers (WT
mice fed an SD) and steatotic livers (MC4R-KO mice fed a WD for 6–10 wk), and were stimulated with cholesterol crystals (CC, 500 μg/ml) and βCD-PRX
(1 mM) for 24 h. (B) Representative images of immunostaining of macrophages from normal livers and steatotic livers treated with CC and βCD-PRX for
24 h. Fixed cells were stained with LAMP1 (a lysosome marker, green), filipin (free cholesterol, red), and PI (propidium iodide, nuclei, blue). Scale bars, 20
μm. (C and D) Immunostaining of Galectin 3 (a marker of lysosomal membrane damage, green), LAMP1 (red), and DAPI (blue; C) and TFE3 (green), Filipin
(blue), and PI (red; D) in hepatic macrophages. Scale bars, 20 μm. Images are representative of two independent experiments. (E) RNA-seq was
conducted using hepatic macrophages isolated from normal livers and steatotic livers (n= 2). Gene ontology analysis of the genes twofold upregulated in
hepatic macrophages isolated from steatotic livers compared with those from normal livers using Metascape. (F) Venn diagram showing the twofold
upregulated genes in CC-treated macrophages compared to each Veh. (G) Unsupervised hierarchical clustering analysis using RNA-seq data (k=5).
Genes belonging to each cluster are indicated on the right.
Itoh et al. Journal of Experimental Medicine 10 of 20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Phagocytosis of dead cells induces lysosomal dysfunction
in macrophages
Next, we examined the involvement of phagocytosis of dead
cells in lysosomal dysfunction in macrophages. Unstimulated
(indicated as normal) or lipid-loaded Hepa1-6 cells were
stained with pHrodo, rendered necrotic by freeze and thaw,
and then added to RAW264 macrophages (Fig. 9 A). There was
no difference in the clearance of dead cells, regardless of the
type of dead cells or the existence of βCD-PRX (Fig. 9 B). The
free cholesterol content was markedly increased in macro-
phages by adding lipid-loaded Hepa1-6 compared to normal
Hepa1-6, and the increase was considerably suppressed by
βCD-PRX (Fig. 9 C).Afterengulfmentoflipid-loadedHepa1-6
cells, nuclear translocation of TFE3 was observed in a time-
dependent manner, whereas it was observed only transiently
and mildly after engulfment of normal Hepa1-6 cells (Fig. 9, D
and E). In parallel with these changes, nuclear translocation
Egr1, along with increased expression of osteopontin, was
observed in macrophages phagocytosing lipid-loaded Hepa1-6
cells (Fig. 9, F and G). Taken together, these findings suggest
that phagocytosis of dead cells, as well as cholesterol crystals,
induces lysosomal stress in macrophages to acquire profi-
brotic properties.
Discussion
Evidence has accumulated regarding the macrophage subsets
responsible for the development of liver fibrosis termed SAM,
NASH-associated macrophage, and lipid-associated macrophage
(Ramachandran et al., 2019;Remmerie et al., 2020;Xiong et al.,
2019). Recently, Fabre et al. reported that CD9 and triggering
receptor expressed on myeloid cells 2–positive macrophages
expressing Spp1, glycoprotein nonmetastatic melanoma protein
B, fatty acid binding protein 5, and CD63 are highly profibrotic
and enriched in the fibrotic niche across species and organs
using multiple single-cell RNA-seq datasets (Fabre et al., 2023).
We have also found a macrophage subset localizing to CLS in
murine NASH models, harboring gene expression patterns
similar to the above profibrotic macrophages, based on a
completely different approach (Itoh et al., 2017). In this
study, using a unique supramolecule, βCD-PRX, we dem-
onstrated that lysosomal-free cholesterol accumulation
Figure 7. Differential response to cholesterol crystal (CC)–induced lysosomal stress in macrophages isolated from normal and steatotic livers.
(A) mRNA expression levels analyzed by quantitative real-time PCR in hepatic macrophages. Gpnmb, glycoprotein nonmetastatic melanoma protein B;
Vegfa, vascular endothelial growth factor A); Kcnn4, potassium calcium–activated channel, subfamily N, member 4. (B) Top five upstream transcription
regulators specific for CC-treated macrophages from steatotic livers analyzed by ingenuity pathway analysis. (C) Egr1 expression in hepatic macrophages.
n= 4. *P < 0.05, **P < 0.01 versus each Veh;
##
P < 0.01. (D) Immunostaining of Egr1 (red) in hepatic macrophages (Mφ). Scale bars, 20 μm. Data and images
are representative of two independent experiments.
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Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Figure 8. Effect of βCD-PRX on lysosomal dysfunction and profibrotic changes in RAW264 macrophages. (A) mRNA expression of genes related to
NASH-specific macrophage phenotypes after incubation with cholesterol crystal (CC) and βCD-PRX for 24 h. **P < 0.01 versus Veh;
##
P<0.01.(B) Oste-
opontin and PDGF-BB secretion from RAW264 macrophages in response to CC and βCD-PRX. (C and D) Evaluation of lysosomal function using acridine orange
in RAW264 macrophages. Side scatter (SSC) intensity represents uptake of CCs, and fluorescence of acridine orange was detected by PerCP channel. βCD-PRX
was added at the same time with CC (C) or 2 h after CC treatment (D). Blue line, CC; orange line, CC + βCD-PRX. n= 4. *P < 0.05, **P < 0.01 versus CC at each
timing. (E) Immunocytochemistry of RAW264 macrophages treated with CC in the presence of βCD-PRX (1 mM), HP-βCD (unthreaded in linear polymer; βCD,
1 mM), and Simvastatin (Sim, 1 μM) for 24 h. TFE3 (green), Filipin (blue), and PI (red). (F and G) Free cholesterol concentrations in the culture media (F) and
mRNA expression of genes related to lysosomal stressin RAW264 treated with CC and βCD-PRX, βCD, and Sim (G). n= 4. *P < 0.05, **P < 0.01 versus Veh;
#
P<
0.05,
##
P < 0.01. (H) The effect of Egr1 knockdown on expression of Spp1 and Pdgfb. Macrophages were transfected with siRNA targeting Egr1 and negative
Itoh et al. Journal of Experimental Medicine 12 of 20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
triggers lysosomal stress/injury and profibrotic activation of
macrophages. Of note, such lysosomal stress was also observed
in the macrophages constituting CLS in human NASH (Fig. 1;
Kanamori et al., 2021).Several studies have pointed to the foamy
appearance of hepatic macrophages in hyperlipidemic mice fed a
WD, in which cholesterol overload in macrophages has been
considered as a result of high levels of circulating low-density
lipoprotein similar to atherosclerotic plaques (Sano et al.,
2004;Yoshimatsu et al., 2004). MC4R-KO mice exhibited
hypercholesterolemia, whereas cholesterol accumulation and
lysosomal stress were observed only in the CD11c-positive macro-
phages in CLS, suggesting that free cholesterol is derived from dead
hepatocytes. Accordingly, this study sheds light on the novel mech-
anism for lipotoxicity as the pathogenesis of NASH and provides
evidence that the microenvironmental milieu in the liver modulates
stress response of macrophages, thereby activating profibrotic pro-
grams during the development of NASH.
As HP-βCD promotes cholesterol removal from the cells, the
efficacy of HP-βCD has been reported in mouse models of
Niemann-Pick type C (NPC) disease and atherosclerosis (Liu
et al., 2009;Ory et al., 2017;Zimmer et al., 2016). However, a
substantial amount of HP-βCD is required ranging from 4,000 to
8,000 mg/kg body weight to achieve therapeutic effects. In
addition, a high prevalence of adverse effects such as acute
toxicity, pulmonary injury, and ototoxicity may hinder the
clinical application of HP-βCD when it is used for lifestyle-
related diseases that require high safety in the long term. In
contrast, βCD-PRX is designed to avoid such toxic effects be-
cause βCD-PRX does not directly act on free cholesterol of the
plasma membranes (Tamura and Yui, 2014). In this study, we
confirmed that HEE-modified βCD-PRX is hepatotropic, low
toxic, and capable of expelling free cholesterol from macro-
phages at a low dose (30 mg/kg) in vivo. Although βCD-PRX was
distributed to hepatocytes as well as macrophages, it did not af-
fect the cholesterol content in hepatocytes. This is probably be-
cause cholesterol is mainly stored in lipid droplets in hepatocytes,
whereas βCD-PRX is delivered to lysosomes (Tamura et al., 2016).
Cholesterol-lowering medications, such as statins and ezetimibe,
have been tested in NAFLD/NASH patients because of the high
prevalence of hypercholesterolemia and cardiovascular diseases
(Horn et al., 2022). Statins inhibit 3-hydroxy-3-methylglutaryl-
CoA reductase, the rate-limiting enzyme of cholesterol synthesis
in hepatocytes, and ezetimibe inhibits intestinal cholesterol ab-
sorption by binding to the NPC-like 1 sterol transporter, both of
which are expected to reduce the cholesterol burden on the liver.
However, large randomized placebo-controlled trials are still
lacking to prove their efficacy in liver fibrosis. On the other hand,
βCD-PRX targets accumulated free cholesterol in lysosomes, so
that lysosomal function is restored in macrophages constituting
CLS, thereby leading to suppression of profibrotic activation.
Thus, βCD-PRX could be a novel therapeutic agent for NASH with
auniquemechanismofaction.
During the development of NASH, KCs derived from yolk sac
are activated and partially undergo cell death, while monocyte-
derived macrophages, in turn, occupy the niche and differenti-
ate into those with KC-like signatures (F4/80
hi
CD11b
lo
;Seidman
et al., 2020;Xiong et al., 2019). In this study, since the F4/80
hi
CD11b
lo
macrophage fraction was separated regardless of their
origin, they may include both yolk sac–derived KCs and
monocyte-derived macrophages. Given that βCD-PRX affected
the KC-like signature, improvement of lysosomal function
would regulate cell death or macrophage differentiation.
Of note, profibrotic genes were upregulated only in cultured
macrophages isolated from steatotic livers in response to ly-
sosomal stress, but not from normal livers, suggesting that the
predisposition of macrophages in the microenvironment of
steatotic livers is required for sensing metabolic stress and
subsequent activation of profibrotic pathways. In line with this,
Seidman et al. reported that hepatic macrophages possess
disease-specific enhancer landscapes that suppress KC identity
and promote SAM-like phenotype (Seidman et al., 2020). In-
triguingly, we demonstrated a novel mechanism that choles-
terol overload induces activation of TFE3/TFEB, leading to
upregulation of Egr1 and subsequent activation of profibrotic
pathways. Since TFE3/TFEB plays important roles in various
cell types, future research would clarify the significance of
TFE3/TFEB and/or Egr1 in macrophages in the pathogenesis of
NASH. On the other hand, as shown in Fig. 5 B, treatment with
βCD-PRX significantly but partially suppressed the number of
TFE3-positive macrophages in CLS, suggesting the involvement
of other factors in the induction of lysosomal stress in macro-
phages during the development of NASH. For instance, changes
in lipid composition, such as cholesterol or sphingosine levels
and reactive oxygen species, can lead to increased lysosomal
membrane permeabilization (Gómez-Sintes et al., 2016;Kirkegaard
et al., 2010). Our previous data revealed that iron accumulation
induces lysosomal stress and profibrotic activation in CLS-
forming macrophages (Kanamori et al., 2021). It is likely that
multiple metabolic changes, including cholesterol and iron, are
involved in lysosomal stress/injury observed in this process.
A substantial amount of cholesterol is accumulated in mac-
rophages after ingestion of apoptotic cells, and lysosomal acid
lipase is required to hydrolyze esterified cholesterol from in-
gested apoptotic cells to activate liver X receptor (Mota et al.,
2021;Viaud et al., 2018). In this study, we observed increased
content of free cholesterol in RAW264 macrophages after en-
gulfment of Hepa1-6 cells possessing lipid droplets filled with
esterified cholesterol, lysosomal degradation of esterified cho-
lesterol results in free cholesterol overload to macrophages,
which induced Egr1 activation. Our in vitro and in vivo data
support the notion that accumulated cholesterol in macrophages
is derived from dead hepatocytes and activates profibrotic
pathways upon phagocytosis, which could be reversed by βCD-
PRX treatment. However, the limitation of this study is that
control, and treated with CC for 6 h. (I) Role of TFE3 and TFEB transcription factors in CC-induced Egr1 expression in RAW264 macrophages. Macrophages
were transfected with siRNA targeting Tfe3/Tfeb and negative control, and treated with CC for 6 h n= 4. **P < 0.01 versus Cont-Veh;
##
P < 0.01. Data and
images are representative of three (A–G) or two (H and I) independent experiments. Error bars represent means ± SEM.
Itoh et al. Journal of Experimental Medicine 13 of 20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
phagocytosis assay was conducted with immortalized cell lines,
and the experimental settings are not sufficient to recapitulate
the cellular interaction in CLS. For the next step, it is important to
investigate the molecular mechanisms of how dead cell clearance
induces profibrotic changes in macrophages, and how free cho-
lesterol crystalizes in lipid droplets of dying hepatocytes.
In summary, we demonstrated that cholesterol derived
from dead hepatocytes induces lysosomal dysfunction in
Figure 9. Phagocytosis of dead cells induces lysosomal dysfunction and profibrotic changes in macrophages. (A) Experimental protocol for macro-
phage phagocytosis of dead cells. Hepa1-6 cells were incubated with the inclusion complex of cholesterol with randomly methylated βCD (50 μM) and oleic
acid (500 μM) for 48 h. Unstimulated (indicated as normal) and lipid-loaded Hepa1-6 cells were stained with pHrodo, which emits fluorescence in an acidic
environment in lysosomes, and induced necrotic cell death by freeze and thaw. RAW264 macrophages were incubated with dead Hepa1-6 cells for the in-
dicated time. (B) Clearance rate was evaluated by the number of dead cells in the media at the indicated time. Orange, normal Veh; pale orange, normal PRX;
blue, lipid-loaded Veh; pale blue, lipid-loaded PRX. n=4.(C) Macrophages were incubated with dead cells with or without βCD-PRX for 6 h and pHrodo-
positive and -negative macrophages were subjected to measurement of free cholesterol content. n=3–4. *P < 0.05, **P < 0.01. (D) Immunostaining of TFE3
(green) in RAW264 macrophages after supplementation of dead cells and βCD-PRX. hr, hour. (E) Time course of TFE3 nuclear translocation. (F and G) Im-
munostainingof TFE3 (green), Osteopontin (red; F), and Egr1 (red; G) inRAW264 macrophages. Scale bars, 20 μm. Data and images are representative of two
independent experiments. Error bars represent means ± SEM.
Itoh et al. Journal of Experimental Medicine 14 of 20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
the macrophages constituting CLS, leading to upregulation of
profibrotic genes, thereby promoting liver fibrosis (Fig. S5 F).
Treatment with βCD-PRX ameliorated liver fibrosis at least
partly through decreasing free cholesterol content in macro-
phages and suppressing the activation of profibrotic pathways
in a NASH model. This study provides evidence that lysosomal
cholesterol overload triggers macrophage phenotypic changes
and promotes the development of NASH, which could be a
novel therapeutic target for NASH.
Materials and methods
Reagents
All reagents were purchased from Sigma-Aldrich or Nacalai
Tesque unless otherwise noted. Chemically modified βCD-PRXs
were synthesized and characterized as described previously
(Nishida et al., 2016;Ohashi et al., 2021;Tamura and Yui, 2014,
2015;Tonegawa et al., 2020;Zhang et al., 2021). The number of
threading βCD, the number of modified functional groups, and
the number-average molecular weight of each chemically mod-
ified βCD-PRX are shown in Table S2. The HEE-group modified
βCD-PRX used in the mouse experiment of NASH was as follows:
the number of threading βCDs, 11.8; the number of modified
HEE groups, 5.29 per threaded βCD (62.4 per βCD-PRX); and the
average molecular weight per βCD-PRX, 27,900.
Animals
MC4R-KO mice on a C57BL/6J background were kindly provided
by Joel K. Elmquist (University of Texas Southwestern Medical
Center, Dallas, TX, USA). Age-matched C57BL/6J WT mice were
purchased from CLEA Japan. 8-wk-old male MC4R-KO and WT
mice were fed a WD (D12079B; 468 kcal/100 g, 41% energy as fat,
34.0% sucrose, and 0.21% cholesterol; Research Diets) for 24 wk
to induce NASH. WT mice were maintained on a standard diet
(CE-2; CLEA) for the same period. HC diet–induced NASH
model, 8-wk-old male WT mice were fed an HC diet (D09100310;
40 kcal% fat, 20 kcal% fructose, 2% cholesterol; Research Diets)
for 20 wk. At the end of the experiment, the animals were
sacrificed under anesthesia when fed ad libitum.
βCD-PRX administration
βCD-PRX was subcutaneously administered using osmotic
minipumps at a dose of 30 mg/kg/d (Alzet model 2002; Palo
Alto). After 18-wk WD or HC feeding, osmotic minipumps filled
with βCD-PRX or normal saline were implanted under the back
skin of mice. They were kept on the WD or HC for an additional
6 wk, with the replacement of minipumps every 2 wk. To an-
alyze the cellular distribution of βCD-PRX in the liver, βCD-PRX
was administered intraperitoneally at the indicated doses, and
the livers were analyzed after 24 h. The Cy5.5-labeled chemi-
cally modified βCD-PRXs were administered subcutaneously at
200 mg/kg, and the fluorescence intensities of each tissue were
analyzed after 24 h.
Blood analysis
Blood glucose concentrations were measured using a blood
glucose test meter (Glutest PRO R; Sanwa Kagaku). Serum
concentrations of ALT, tryglyceride (TG), and TC were measured
by the biochemical analyzer (DRI-CHEM NX500V; Fujifilm).
Serum concentrations of insulin and cytokines were determined
using the ELISA kit (Morinaga Co. Ltd.). Serum concentrations
of TNFα, IL-1β, MCP-1, Osteopontin, and PDGF-BB were mea-
sured by each ELISA kit (R&D).
FACS analysis and sorting experiments
The mice were perfused with PBS or HBSS without calcium and
magnesium to remove blood from the liver. To isolate hep-
atocytes, the livers were dispersed in HBSS with calcium and
magnesium supplemented with 1 mg/ml type IV collagenase
(Sigma-Aldrich), and the cell suspensions were centrifuged at
50–100 × gfor 2 min. The livers were digested using a gen-
tleMACS dissociator (Miltenyi Biotech) to isolate the non-
parenchymalcellfraction,andcelldebriswasremovedby
Percoll density gradient centrifugations. Non-parenchymal
cells were stained with antibodies against CD45 (30-F11), F4/
80 (BM8), CD11b (M1/70), CD3 (17A2), CD11c (N418), CD146
(ME-9F1), Ly6G (1A8), and SiglecF (S17007A; BioLegend). Dead
cells were separated using 7-AAD or DAPI. Cells were analyzed
using FACSCanto II (BD Biosciences) or sorted using FACSAria
II (BD Biosciences). Cell plots were analyzed using FlowJo v10
software.
Measurement of lipid content of livers and isolated cells
Total lipids in the liver were extracted using chloroform/
methanol. The total cholesterol and triglyceride concentrations
in the livers were measured by enzymatic assay kits (Fujifilm
Wako Pure Chemicals). The cellular cholesterol content of iso-
lated primary macrophages, hepatocytes, and RAW264 was
measured by gas chromatography–mass spectrometry (GC-MS).
Briefly, the cells were suspended in PBS (500 μl), and 5α-
cholestane (50 μg/ml in pyridine, 12 μl) was added as an inter-
nal standard. Cholesterol was extracted with chloroform and
methanol, and the organic phases were collected and evaporated
to dryness via nitrogen flow. The dried samples were dissolved
in dehydrated pyridine (150 μl; Fujifilm Wako Pure Chemical)
and derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide
(50 μl) for 30 min at 60°C for the quantification of cholesterol. GC-
MS measurements were performedonaGCMS-QP2020(Shi-
madzu) equipped with an AOC-20i autoinjector and a DB-5MS
capillary column (30 m × 0.25 mm internal diameter, 0.25 μm
phase thickness; Agilent Technologies). Ultrahigh-purity he-
lium (>99.999%) was used as the carrier gas. Sample solutions
(1 μl) were injected in splitless mode, and the measurements
were performed in a selected-ion monitoring mode. The oven
temperature was held initially at 50°C for 2 min (0–2 min),
increased to 260°C at a rate of 40°C/min (2–7.25 min), increased
further to 310°C at a rate of 2.5°C/min (7.25–27.25 min), and
finally held at 310°C for 2 min (27.25–29.25 min). The ions used
for the quantification were as follows: cholesterol (22.010 min)
m/z = 458 and 5α-cholestane (17.405 min) m/z = 217. For the
quantification of cholesteryl esters, the samples were hydro-
lyzed with KOH (150 mg/ml in ethanol) for 1 h at 70°C to
quantify total cholesterol. The amount of cholesteryl esters was
determined by subtracting the free cholesterol values from the
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Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
total cholesterol values. The amounts of cellular free cholesterol
and esterified cholesterol were expressed by normalizing with
the cell number.
Histological analysis
The livers were fixed with 10% neutral-buffered formalin and
embedded in paraffin. 4-μm-thick sections of the liver were
stained with Sirius red. Immunohistochemical staining was
performed for F4/80 (MCA497GA; Serotec), desmin (ab15200;
Abcam), CTSD (ab75852; Abcam), osteopontin (AF808; R&D),
and type III collagen (1330-01; SouthernBiotech). Positive areas
for Sirius red, osteopontin, desmin, and type III collagen were
measured on the whole area of each slice using ImageJ software
(National Institutes of Health). For immunofluorescent staining,
the livers were embedded in an OCT compound and frozen in
liquid nitrogen. 10-μm-thick frozen sections were stained with
antibodies against F4/80, TFE3 (HPA023881; Sigma-Aldrich),
CD11c (14-0114; eBioscience), Clec4f (MAB2784; R&D), Tim4
(130002; BioLegend), and secondary antibodies. Sections were
mounted in Vectashield mounting medium with DAPI (Vector
Labs) and photographed under identical settings for each
staining using a C2 confocal microscope (Nikon). TFE3-positive
ratio was quantified by counting the number of TFE3-positive
nuclei out of DAPI spots in all the CLS observed on each slice.
CD11c-positive CLS was determined when some or all of the
macrophages forming CLS expressed CD11c. To observe cho-
lesterol crystals in NASH livers from bone marrow chimeric
MC4R-KO mice with GFP-Tg mice fed a WD for 20 wk, 1-mm-
thick sliced liver samples were fixed with 4% paraformalde-
hyde, embedded in OCT compound, and frozen in liquid ni-
trogen. 10-μm-thick frozen sections were dried and observed
using BX53 microscope with a polarizing filter (Olympus).
Electron microscopy analysis
MC4R-KO mice fed a WD for 20 wk were perfused with 2.5%
glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4. Liver tissue was cut into 1-mm cubes and post-
fixed in 2.5% glutaraldehyde and 4% paraformaldehyde over-
night. Fixed samples were cut with a microslicer at 500 μm
thick. The slices were then fixed with 1% OsO4 for 1 h, dehy-
drated in graded ethanol, and flat-embedded on siliconized glass
slides in epoxy resin. Serial ultrathin sections of the livers at 70-
nm thickness were mounted on pieces of silicon wafers and
contrasted with uranyl acetate and lead citrate. Scanning elec-
tron microscopy images were obtained using a backscattered
electron detector (BED-C; voltage: 7 kV; PC current, 1.8 nA; work
distance: 6) in a JSM-7900F scanning electron microscope
(JEOL).
Cholesterol preparation
Cholesterol crystals were generated as previously described
(Emanuel et al., 2014). Briefly, cholesterol was dissolved in
ethanol at a concentration of 50 mg/ml by heating to 60°C.
Cholesterol crystals were formed at −30°C and centrifuged at
10,000 × gfor 20 min. The crystals were resuspended in PBS at
50 mg/ml and sonicated extensively. The inclusion complex
of cholesterol with randomly methylated βCD (βCD/Chol)
was prepared by stirring cholesterol powder (38.6 mg) in
100 mM randomly methylated βCD solution for 24 h at room
temperature. The solution was passed through a 0.22-μm filter
before use.
Solubility of cholesterol by inclusion complexation with βCDs
The cholesterol solubility of differentially modified βCD-PRXs
was measured using high-performance liquid chromatography,
according to the previous reports (Tamura et al., 2016).
Cell culture
RAW264 murine macrophage cells and Hepa1-6 murine hepa-
toma cells were maintained in Dulbecco’s minimal essential
media supplemented with 10% FBS and antibiotics. For primary
hepatic macrophage culture, non-parenchymal cell fractions
isolated from the livers of WT mice fed an SD and MC4R-KO
mice fed a WD for 6–10 wk were stained with PE-conjugated F4/
80 antibody (REA126; Miltenyi) and MojoSort Mouse anti-PE
Nanobeads (BioLegend), and separated using an autoMACS
separator (Miltenyi; Kanamori et al., 2021). Primary hepatocytes
were isolated by centrifugation at 50–100 × gfrom the liver
suspensions ofWT mice fed an SD and MC4R-KO mice fed a WD
for 10 d. Primary hepatic macrophages and hepatocytes were
seeded on collagen-coated plates. The cytotoxicity of chemically
modified βCD-PRXs on RAW264 macrophages was assessed us-
ing Cell TiterGlo 2.0 (Promega). RAW264 macrophages and he-
patic macrophages were treated with cholesterol crystals (500
μg/ml) for the indicated times with or withoutβCD-PRX (1 mM),
HP-βCD (1 mM), or simvastatin (1 μM). Hepa1-6 cells were in-
cubated with βCD/Chol (50 μM) and oleic acid (500 μM) for 48 h
to produce lipid droplets in the cytoplasm.
Cell transfection experiments
RAW264 macrophages were reverse-transfected with small
interfering RNA (siRNA) targeting TFE3 (Invitrogen), TFEB
(Sigma-Aldrich) for 48 h, and Egr1 (Invitrogen) for 24 h using
lipofectamine RNAiMax (Invitrogen). After siRNA transfection,
cells were treated with cholesterol crystals with or without
βCD-PRX for 6 h. RAW264 macrophages plated on glass bottom
dishes were transfected with plasmid DNA encoding mRFP-GFP
tandem fluorescence-tagged LC3 (21074; Addgene) using lip-
ofectamine 3000 (Invitrogen). After 48 h incubation, the cells
were treated with cholesterol crystals for 24 h and analyzed by
C2 confocal microscope.
Immunocytochemistry of macrophages
Primary hepatic macrophages and RAW264 macrophages
were seeded on chambered cover glasses (Nalge Nunc) and
stimulated as indicated. Cells were fixed with 4% parafor-
maldehyde and stained with anti-LAMP1 (121601; BioLegend),
LAMP2 (ab13524; Abcam), Galectin-3 (14979-1-AP; Proteintech),
TFE3 (HPA023881; Sigma-Aldrich), Egr1 (4153; Cell Sig-
naling), osteopontin (AF808; R&D), and Filipin (50 μg/ml;
Polysciences). Photographs were captured under identical
settings for each staining. TFE3-positive ratio was quanti-
fied by counting the number of TFE3-positive nuclei out of
DAPI spots.
Itoh et al. Journal of Experimental Medicine 16 of 20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
Evaluation of lysosomal function using acridine orange
RAW264 macrophages were treated with cholesterol crystals
(500 μg/ml) and βCD-PRX for indicated times. Acridine orange
was added at a concentration of 5 μg/ml for 30 min before FACS
analysis.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from the liver or cultured cells using
Sepasol reagent or PureLink RNA Micro Kit (Invitrogen).
Quantitative real-time PCR was performed with the StepOne-
Plus Real-time PCR System using the Fast SYBR Green Master
Mix Reagent (Applied Biosystems) as previously described. The
primers used in this study are listed in Table S6. Data were
normalized to 36B4levels and analyzed using the comparative Ct
method.
RNA-seq analysis
Hepatic macrophages isolated from normal and steatotic livers
were treated with cholesterol crystals for 24 h (n= 2), and
RAW264 macrophages were treated with cholesterol crystals
with or without βCD-PRX (n= 4). Total RNA was purified using
RNeasy MiniElute Cleanup Kit (Qiagen). RNA was quantified
with Qubit RNA BR Assay Kit (Invitrogen) and the integrity was
evaluated with Agilent Bioanalyzer 2100 (Agilent Technologies).
RNA-seq libraries were constructed by NEBNext Poly (A) mRNA
Magnetic Isolation Module (NEB) and MGIEasy RNA Directional
Library Prep Set (MGI), according to the manufacturer’sin-
structions. Libraries were sequenced with 150 bp paired-end
reads on a DNBSEQ-G400 sequencer (MGI Tech). Low-quality
reads and adapter sequences were trimmed using fastp software
(Chen et al., 2018). The reads were mapped to the mm10 ref-
erence genome using HISAT2 (Kim et al., 2019)followedby
transcript assembly and quantification using StringTie (Pertea
et al., 2015). Genes expressed below 0.5 counts per million were
filtered out from analysis. Fold changes of gene expression levels
were analyzed between hepatic macrophages from normal and
steatotic livers or RAW264 macrophages treated with choles-
terol crystals with or without βCD-PRX using iDEP v.0.94 (Ge
et al., 2018), Metascape, and IPA (QIAGEN).
Western blotting analysis
RAW264 macrophages were lysed in radioimmunoprecipitation
assay buffer (FujiFilm Wako) supplemented with Protease In-
hibitor Cocktail (Sigma-Aldrich). Proteins were separated by
SDS-PAGE and immunoblotted with anti-LC3 antibody (PM0369;
MBL), anti-p62 antibody (PM045; MBL), and anti-α-tubulin an-
tibody (T5168; Sigma-Aldrich). Immunoblots were detected and
analyzed with ECL Prime (Cytiva) and ChemiDoc XRS Plus (Bio-
Rad).
Human study
Japanese NAFLD/NASH patients who had sustained liver dys-
function, dyslipidemia, and insulin resistance were recruited at
Yamaguchi University Hospital and Yokohama City University
Hospital. We measured body mass index and serum parameters
according to the standard procedures. LSM and CAP values
were determined by FibroScan (Echosens). Liver samples
were obtained by ultrasound-guided liver biopsy to evaluate
liver histology. NAS score and fibrosis stage were assessed
according to the NASH clinical research network scoring system.
Formalin-fixed and paraffin-embedded liver specimens were
stained with antibodies against CD68 (M0876; Dako), CD11c
(EP1347Y, Abcam), and CTSD.
Statistics
Data are presented as mean ± standard error of mean (SEM), and
statistical significance was set at P < 0.05. Statistical analysis
was performed using analysis of variance, followed by Tukey-
Kramer test. Two-tailed unpaired Student’sttest was used to
compare the two groups. Statistical analyses were performed
with JMP Pro 15 (SASInstitute, Cary, NC, USA). In human study,
box plots were created for CAP values by NAS and fibrosis stage,
and LSM values by fibrosis stage to evaluate associations. Cor-
relations were evaluated by calculating Spearman’srankcor-
relation coefficient. To evaluate the association of NAS, fibrosis
stage, CAP, and LSM with respect to CLS number, we used a
generalized linear model with CLS number as the dependent
variable and the logarithm of the link function. In this analysis, a
first-order linear model was constructed for NAS, fibrosis stage,
CAP, and LSM, and a model including a quadratic term to
evaluate the curvilinear relationship. R2 values were calculated
as the goodness of fit of the models.
Study approval
All animal experiments were approved by the Guidelines for the
Care and Use of Laboratory Animals of Nagoya University and
the Animal Care and Use Committee, Research Institute of En-
vironmental Medicine, Nagoya University (No. 20253), and the
Institutional Animal Care and Use Committee of Tokyo Medical
and Dental University (A2018-158A, A2020-114C and A2022-
072A). All animal experiments were carried out according to the
Animal Research: Reporting of In Vivo Experiments guidelines.
The clinical study complied with the principles of the Declara-
tion of Helsinki. The protocol was approved by the Ethical
Committee for Human and Genome Research of Research In-
stitute of Environmental Medicine, Nagoya University (No.
388), the Medical Research Ethics Committee of Tokyo Medical
and Dental University (M2015-544), the Institutional Review
Board of Yamaguchi University Hospital (2020-063), and the
Ethics Committee of Yokohama City University Hospital
(B151101010). Although written informed consent was not ob-
tained for the current study, we obtained approval from the
Ethics Committee/Institutional Review Board of each institu-
tion based on the Japanese Ethical Guidelines for Clinical
Studies, disclosed detailed information on the study protocol,
and provided all participants with an opportunity to refuse
their inclusion in the study.
Online supplemental material
Fig. S1 shows the relationship between crown-like structure
number and clinical parameters in NAFLD/NASH subjects (re-
lated to Table S1). Fig. S2 shows the effect of βCD-PRX on cho-
lesterol accumulation in hepatocytes. Fig. S3 shows the effect of
βCD-PRX on liver fibrosis in a mouse model of NASH with high-
Itoh et al. Journal of Experimental Medicine 17 of 20
Cholesterol overload to macrophages promotes NASH https://doi.org/10.1084/jem.20220681
fat and HC diet. Fig. S4 shows the effect of cholesterol crystal–
indued lysosomal stress and the impact on cellular function
(related to Fig. 8). Fig. S5 shows the molecular mechanism of
action of βCD-PRX. Table S1 shows clinical data of NAFLD/NASH
patients. Table S2 shows molecular characteristics of chemically
modified βCD-PRXs (related to Fig. 2). Table S3 shows serolog-
ical parameters of WT mice treated with chemically modified
βCD-PRXs (related to Fig. 2). Table S4 shows serum cytokine
levels in MC4R-KO mice treated with βCD-PRX for 6 wk (related
to Fig. 4). Table S5 shows the effect of 6-wk βCD-PRX treatment
on serological parameters of WT mice fed an HC diet for 20 wk
(related to Fig. S3). Table S6 lists primers used in this study.
Data availability
The RNA-seq data are available on the website of the Gene Ex-
pression Omnibus at the National Center for Biotechnology In-
formation (GEO accession no. GSE235024 and GSE235222).
Acknowledgments
We thank Dr. Joel K. Elmquist (University of Texas Southwest-
ern Medical Center, Dallas, TX, USA) for his generous gift of
MC4R-KO mice, Dr. Isao Sakaida (Yamaguchi University) for his
support on the clinical study, Dr. Hajime Yamakage (Satista Co.,
Ltd.) for his help on statistical analysis, Dr. Yasuhiro Murakawa
(Kyoto University) for his support on electron microscopy
analysis, and Dr. Mashito Sakai (Nippon Medical School) for his
help on interpretation of transcriptome data. We also thank
the members of the Suganami laboratory for their helpful
discussions.
This work was supported in part by Grants-in-Aid for Sci-
entific Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (20H04944, 20H03447,
22K08667, 22K19524, 22H04806), and Japan Agency for Medi-
cal Research and Development (CREST [JP21gm1210009s0103],
Research Program on Hepatitis [JP22fk0210094s0202], Project
Promoting Clinical Trials for Development of New Drugs
[A172], and Research Program on Rare and Intractable Diseases
[JP20ek0109488]). This study was also supported by research
grants from MSD Life Science Foundation, Public Interest In-
corporated Foundation, the Kurata Grants by the Hitachi Global
Foundation, SEI Group CSR Foundation, Smoking Research
Foundation, Ono Medical Research Foundation, Kobayashi
Foundation, Uehara Memorial Foundation for Life Sciences,
and Takeda Science Foundation. Open Access funding provided
by Nagoya University.
Author contributions: M. Itoh, A. Tamura, and T. Suganami
designed the experiments. M. Itoh, A. Tamura, S. Kanai, M.
Tanaka, Y. Kanamori, and I. Shirakawa performed experiments.
A. Tamura and A. Ito prepared reagents. Y. Oka and T. Ogi
performed RNA-seq analysis. M. Maeda and Y. Kataoka per-
formed electron microscopic analysis. I. Hidaka, T. Takami, Y.
Honda, and A. Nakajima provided human samples. Y. Saito, Y.
Murata, T. Matozaki, and Y. Ogawa supervised experimental
data. M. Itoh, A. Tamura, and T. Suganami reviewed data and
drafted the manuscript.
Disclosures: M. Itoh reported grants from the Ministry of Edu-
cation, Culture, Sports, Science and Technology of Japan, MSD
Life Science Foundation, the Kurata Grants by the Hitachi Global
Foundation, Uehara Memorial Foundation for Life Sciences, and
Takeda Science Foundation outside the submitted work. M.
Tanaka reported grants from Grants-in-Aid for Scientific Re-
search from the 996 ministry of Education, Culture, Sports,
Science and Technology of Japan (21K08526) and the Japan
Agency for Medical 998 Research and Development
(22fk0210094h0002) outside the submitted work. Y. Murata
reported grants from the Japan Society for the Promotion of
Science outside the submitted work. T. Matozaki reported
grants from a Grant-in-Aid for Scientific Research (A) from
Japan Society for the Promotion of Science and P-PROMOTE of
the Japan Agency for Medical Research and Development out-
side the submitted work. Y. Kataoka reported non-financial
support from JEOL Ltd. outside the submitted work. T. Suga-
nami reported grants from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, Japan Agency for
Medical Research and Development, SEI Group CSR Foundation,
Smoking Research Foundation, Ono Medical Research Founda-
tion, Kobayashi Foundation, Uehara Memorial Foundation for Life
Sciences, Suzuken Memorial Foundation, and KOS´
ECosmetology
Research Foundation outside the submitted work; in addition, T.
Suganami received a donation department from Seaknit Bio-
technology Co. Ltd., which does nothaveanyconflictofinterests
related to this work. No other disclosures were reported.
Submitted: 18 April 2022
Revised: 27 April 2023
Accepted: 20 July 2023
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Supplemental material
Figure S1. Relationship between CLS number and clinical parameters in NAFLD/NASH subjects. (A) Correlation of the CAP value measured by FibroScan,
which represents hepatic lipid content, and histological scores of the livers (NAS and fibrosis stage). (B) Correlation of the LSM value measured by FibroScan
and histological fibrosis stage. (C) Correlation of CLS number and NAS, fibrosis stage, CAP, and LSM values. Error bars represent means ± SEM.
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Figure S2. Effect of βCD-PRX on cholesterol accumulation in hepatocytes. (A) Free and esterified cholesterol content of the livers from WT mice and
MC4R-KO mice fed a WD for 24 wk. Cont, control. WT/SD-Cont, n= 6; MC/WD-Cont, n=9;MC/WD-PRX,n=8.**P<0.01versusWT/SD-Cont.(B) mRNA
expression ofgenes related to cholesterol metabolism inhepatocytes isolated from WTmice fed an SD and MC4R-KO mice fed a WD for 4 wk, and treated with
βCD-PRX for the last 1 wk. n=5.*P<0.05,**P<0.01versusWT/SD-Cont.(C) Free and esterified cholesterol content of primary hepatocytes treated with
βCD-PRX for 24 h. Primary hepatocytes were isolated from WT mice fed an SD and MC4R-KO mice fed a WD for 10 d, and treated with βCD-PRX for 24 h n=3.
**P < 0.01 versus WT/SD-Veh;
##
P < 0.01. Data are representative of two (A and B) or three (C) independent experiments. Error bars represent means ± SEM.
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Figure S3. Effect of βCD-PRX on liver fibrosis in a mouse model of NASH with high-fat and HC diet. (A) Experimental protocol of βCD-PRX treatment in
a NASH model with an HC diet for 20 wk. βCD-PRX was subcutaneously administered by osmotic minipumps at a dose of 30 mg/kg/d of βCD-PRX or normal
saline as a control (Cont) for the last 6 wk. PRX, βCD-PRX. SD-Cont, n=6;HC-Cont,n=7;HC-PRX,n=7.(B) Body weight and liver weight after βCD-PRX
treatment. (C) Immunofluorescence staining of TFE3 and F4/80. Scale bars, 50 μm. (D) Fibrosis area evaluated by type III collagen immunostaining. C, central
veins. Scale bars, 50 μm. (E) Hepatic mRNA expression of genes related to inflammation, fibrogenesis, and lipid metabolism. *P < 0.05, **P < 0.01 versus SD-
cont;
#
P < 0.05,
##
P < 0.01. Error bars represent means ± SEM.
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Figure S4. Effect of cholesterol crystals (CCs) on lysosomal stress functions in RAW264 macrophages. (A) RAW264 macrophages treated with CC and
βCD-PRX for 24 h were subjected to RNA-seq analysis (n= 4). Heatmap showing fold changes of the genes extracted by an unsupervised hierarchical clustering
of hepatic macrophages shown in Fig. 6 G compared with vehicle-treated RAW264 macrophages. (B and C) Representative images of immunostaining of
RAW264 macrophages treated with CC and βCD-PRX. (B) LAMP2 (a lysosome marker,green), filipin (free cholesterol, red), and PI (nuclei, blue). (C) Galectin 3
(green), LAMP1 (red), and DAPI (blue). Scale bars, 20 μm. (D) mRNA expression levels of genes related to inflammatory cytokines and chemokines. n=4.*P<
0.05, **P < 0.01 versus Veh;
#
P<0.05,
##
P < 0.01. (E) Western blot analysis of LC-3 and p62 protein levels. n= 4. **P < 0.01 versus Veh;
##
P < 0.01. (F) Images
of RAW264 macrophages transiently expressing mRFP-GFP-LC3 treated with CC and βCD-PRX for 24 h and GFP-positive ratio of mRFP-positive puncta. **P <
0.01 versus CC. (G) mRNA expression levels of genes related to endoplasmic reticulum stress. n= 4. **P <0.01 versus Veh;
#
P<0.05,
##
P<0.01.(H) Oxygen
consumption rate (OCR) of RAW264 macrophages treated with CC and βCD-PRX for 18 h. n= 2. FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone;
R/A, rotenone/antimycin A. (I) Effect of HP-βCD (βCD) and βCD-PRX on the levels of lipid raft integrity evaluated by FACS analysis using cholera toxin subunit
B, a marker for lipid rafts. n=3–4. **P < 0.01 versus Veh. Data and images are representative of two (B, C, F, H, and I) or three (D, E, and G) independent
experiments. Error bars represent means ± SEM. Source data are available for this figure: SourceData FS4.
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Figure S5. Molecular mechanism of action of βCD-PRX. (A and B) Effect of βCD-PRX on TNFα(20 μg/ml, 24 h; A) and L-leucyl-L-leucine methyl ester
(LLOMe, 1 mM, 3 h), an artificial lysosomal membrane damage reagent (B). n=4.**P<0.01versusVeh;
#
P<0.05,
##
P<0.01.(C and D) Representative images
of Egr1 immunostaining (C) and mRNA expression of Egr1 (D) in RAW264 macrophages treated with cholesterol crystals (CCs) and βCD-PRX for 24 h n=4.
**P<0.01versusVeh;
##
P<0.01.(E) mRNA expression in RAW264 macrophages treated with CC for 6 h under knockdown of Tfe3 and Tfeb. n=4.**P<0.01
versus Cont-Veh. Data and images are representative of three (A) or two (B–E) independent experiments. Error bars represent means ± SEM. (F) Potential role
of free cholesterol overload in profibrotic transformation of hepatic macrophages interacting with dead hepatocytes. Duringthe course of NASH development,
macrophages aggregate around dead hepatocytes with CCs in the lipid droplets, and macrophages engulf the corpses of hepatocytes and remnant lipids. In the
microenvironment of steatotic livers, macrophages undergo profibrotic activation at least partly through TFE3/TFEB-Egr1 axis. βCD-PRX excretes free cho-
lesterol from lysosomes and reverses the phenotypic changes of macrophages. Created with BioRender.
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Provided online are six tables. Table S1 shows clinical data of NAFLD/NASH patients. Table S2 shows molecular characteristics of
chemically modified βCD-PRXs. Table S3 shows serological parameters of WT mice treated with chemically modified βCD-PRXs.
Table S4 shows serum cytokine levels in MC4R-KO mice treated with βCD-PRX for 6 wk. Table S5 shows effect of 6-wk βCD-PRX
treatment on serological parameters of WT mice fed an HC diet for 20 wk. Table S6 shows primers used in this study.
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