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\begin{document}

\title{Bardoxolone methyl improves non-alcoholic steatohepatitis through
inhibition of macrophage infiltration.}



\author[1]{Kazuhiro  Onuma}%
\author[1]{Kenji Watanabe}%
\author[1]{Keishiro Isayama}%
\author[2]{Sayaka  Ogi}%
\author[2]{Yasunori  Tokunaga}%
\author[1]{Yoichi Mizukami}%
\affil[1]{Yamaguchi University}%
\affil[2]{UBE Corporation}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and purpose: Bardoxolone methyl
(2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester,
CDDO-Me) is a potent activator of nuclear factor erythroid 2-related
factor (Nrf)2, which induces anti-oxidative-associated genes. CDDO-Me is
known to exert protective effects against chronic inflammatory diseases
in the kidney and lungs. However, its pharmacological effects on
non-alcoholic steatohepatitis (NASH) caused by fat accumulation remain
unknown. In this study, we examined the hepatoprotective effects of
CDDO-Me in a diet-induced NASH mouse model, and elucidated its
pharmacological mechanisms using RNA-seq analysis. Experimental
approach: CDDO-Me was orally administered to mice fed a
choline-deficient, L-amino acid-defined, high-fat diet, and
histological, biochemical, and transcriptome analyses were performed on
the livers of mice that developed NASH. Key results: CDDO-Me
administration induced the expression of antioxidant genes and
cholesterol transporters downstream of Nrf2 and significantly improved
the symptoms of NASH. Whole-transcriptome analysis revealed that CDDO-Me
inhibited the inflammatory pathway that leads to phagocyte recruitment,
in addition to activating the Nrf2-dependent pathway. Among inflammatory
pathways, CC chemokine ligand (CCL)3 and CCL4 in the downstream of
NF-?B, which are associated with the recruitment of macrophages
expressing CC chemokine receptor (CCR)1 and CCR5, were released into
blood in NASH mice. In contrast, CDDO-Me directly inhibited the
expression of CCL3-CCR1 and CCL4-CCR5 in macrophages. Conclusions and
Implications: Overall, this study revealed the potent hepatoprotective
effect of CDDO-Me in a NASH mouse model, and demonstrated that its
pharmacological effects were closely associated with the reduction of
macrophage infiltration through CCL3-CCR1 and CCL4-CCR5 inhibition, in
addition to Nrf2-mediated hepatoprotective effects.%
\end{abstract}%



\sloppy


\textbf{Bardoxolone methyl improves non-alcoholic steatohepatitis
through inhibition of macrophage infiltration}

Kazuhiro Onuma\textsuperscript{1,2}, Kenji Watanabe\textsuperscript{1},
Keishiro Isayama\textsuperscript{1}, Sayaka Ogi\textsuperscript{2},
Yasunori Tokunaga\textsuperscript{2}, Yoichi
Mizukami\textsuperscript{1}*

\textsuperscript{1} Institute of Gene Research, Yamaguchi University
Science Research Center, Yamaguchi, Japan,

\textsuperscript{2} Pharmaceutical Research Laboratory, Pharmaceutical
Division, UBE Corporation, Yamaguchi, Japan

*\textbf{Address Correspondence to} : Yoichi Mizukami

Institute of Gene Research, Yamaguchi University Science Research
Center, 1-1-1, Minami-kogushi, Ube, Yamaguchi, Japan 755-8505

Tel: +81-836-22-2183 /Fax: +81-836-22-2185

E-mail: mizukami@yamaguchi-u.ac.jp

\textbf{Data availability statement} : All data are presented in the
manuscript or supplementary information.

The raw RNA-sequencing data are submitted to GEO under accession number
GSE239861. Any further data will be made available to the corresponding
author upon reasonable request.

\textbf{Funding statement} : This work was the result of using research
equipment shared in MEXT Project for promoting public utilization of
advanced research infrastructure (Program for supporting construction of
core facilities) Grant Number JPMXS0440400021-23 and grants-in-aid for
scientific research (Grant Nos. 21H03358 and 22K19711) from of the
Ministry of Education, Culture, Sports, Science and Technology of Japan.

\textbf{Author contribution statement} : KO designed the study,
conducted the experiments, and wrote the manuscript. SO conducted the
experiments. KW and KI supported the gene analysis and YT designed the
study. YM designed the study and wrote the manuscript. All authors
discussed the results and provided input to the manuscript.

\textbf{Conflict of interest disclosure} : The authors declare no
conflicts of interest associated with this manuscript.

\textbf{Ethics approval statement} : All human and animal studies were
approved by the appropriate ethics committee and have therefore been
performed in accordance with the ethical standards of the 1964
Declaration of Helsinki and its later amendments.

\textbf{Abstract}

\textbf{Background and purpose:} Bardoxolone methyl
(2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester,
CDDO-Me) is a potent activator of nuclear factor erythroid 2-related
factor (Nrf)2, which induces anti-oxidative-associated genes. CDDO-Me is
known to exert protective effects against chronic inflammatory diseases
in the kidney and lungs. However, its pharmacological effects on
non-alcoholic steatohepatitis (NASH) caused by fat accumulation remain
unknown. In this study, we examined the hepatoprotective effects of
CDDO-Me in a diet-induced NASH mouse model, and elucidated its
pharmacological mechanisms using RNA-seq analysis.

\textbf{Experimental approach:} CDDO-Me was orally administered to mice
fed a choline-deficient, L-amino acid-defined, high-fat diet, and
histological, biochemical, and transcriptome analyses were performed on
the livers of mice that developed NASH.

\textbf{Key results:} CDDO-Me administration induced the expression of
antioxidant genes and cholesterol transporters downstream of Nrf2 and
significantly improved the symptoms of NASH. Whole-transcriptome
analysis revealed that CDDO-Me inhibited the inflammatory pathway that
leads to phagocyte recruitment, in addition to activating the
Nrf2-dependent pathway. Among inflammatory pathways, CC chemokine ligand
(CCL)3 and CCL4 in the downstream of NF-\selectlanguage{greek}κ\selectlanguage{english}B, which are associated with
the recruitment of macrophages expressing CC chemokine receptor (CCR)1
and CCR5, were released into blood in NASH mice. In contrast, CDDO-Me
directly inhibited the expression of CCL3-CCR1 and CCL4-CCR5 in
macrophages.

\textbf{Conclusions and Implications:} Overall, this study revealed the
potent hepatoprotective effect of CDDO-Me in a NASH mouse model, and
demonstrated that its pharmacological effects were closely associated
with the reduction of macrophage infiltration through CCL3-CCR1 and
CCL4-CCR5 inhibition, in addition to Nrf2-mediated hepatoprotective
effects.



\textbf{Introduction}

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic
liver disease caused by excess fat accumulation and is closely
associated with obesity and metabolic syndrome (Tessari, Coracina,
Cosma, \& Tiengo, 2009). The NAFLD often progresses to non-alcoholic
steatohepatitis (NASH) accompanied by chronic liver injury, fibrosis,
and inflammation, a part of which occurs liver cirrhosis and
hepatocellular carcinoma (Bellentani, 2017). During progression to NASH,
excess lipid accumulation in the liver results in the activation of
hepatic macrophages and hepatic stellate cells and induces the
infiltration of immune cells. These activated cells release active
oxygen along with various cytokines and chemokines, thereby enhancing
immune cell migration, hepatic inflammation, and fibrosis. Recently, a
single-cell RNA-seq (scRNA-seq) analysis in NASH mice showed the
presence of several types of macrophages in different active states,
such as resident Kupffer cells and monocyte-derived Kupffer cells in
NASH (Daemen et al., 2021; Remmerie et al., 2020; Seidman et al., 2020).
Thus, activated macrophages appear to play an essential role in NASH
progression.

Nuclear factor erythroid 2-related factor (Nrf)2, a transcription factor
that induces antioxidant-associated gene expression under oxidative
stress, might function as a therapeutic target for NASH (Bataille \&
Manautou, 2012). Kelch-like ECH-associated protein (Keap)1 binds the
Neh2 domain of Nrf2 in the cytoplasm and prevents its translocation to
the nucleus (Itoh et al., 2013). Active oxygen produced during
inflammation induces the dissociation of Nrf2 from Keap1, leading to the
nuclear translocation of Nrf2 and its transcriptional activity for
antioxidant gene expression. Notably, NASH caused by a methionine- and
choline-deficient diet is considerably aggravated in Nrf2-deficient mice
(Chowdhry et al., 2010). In contrast, Nrf2 activation by Keap1-knockdown
attenuates steatosis in NASH (Zhang, Yeager, Tanaka, \& Klaassen, 2010).
Further, several pharmacological approaches for activating Nrf2 are also
known to attenuate NASH progression (Sharma et al., 2018; Shimozono et
al., 2013). Bardoxolone (CDDO), a potent pharmacological activator of
Nrf2, is reported to inhibit the induction of various cytokines such as
interferon-1 and interleukin-1 in mouse peritoneal macrophages and rat
brain microglia (Tran, McCoy, Sporn, \& Tansey, 2008) and to induce
monocyte differentiation from myeloid leukaemia cells (Suh et al., 1999)
These findings suggest that CDDO may inhibit macrophage-related
inflammation. Notably, CDDO-Me markedly improves obesity-induced
inflammatory diseases in the kidney and heart and has also been used in
clinical trials for treating chronic kidney disease and pulmonary
arterial hypertension (Chin et al., 2018). However, the hepatoprotective
effects of CDDO-Me on NASH remain unknown.

Therefore, in this study, we investigated the hepatoprotective effects
of CDDO-Me in a diet-induced NASH mouse model and its pharmacological
mechanisms using whole-transcriptome analysis with RNA-seq.

\textbf{Methods}

\textbf{Animals}

Five-week-old male C57BL/6J mice were obtained from Japan SLC, Inc.
(Shizuoka, Japan) to remove the effects of the estrous cycle in female
mice. The species, age, and sex were selected based on a previous study
(Matsumoto et al., 2013). All animals were acclimated to the laboratory
environment for one week and were confirmed to be healthy prior to the
experiments. The mice were housed in barrier facilities and bred at a
room temperature of 23 \selectlanguage{ngerman}± 2 °C with 50 ± 10\% humidity, on a 12-h
light/dark cycle with ventilation. All experimental protocols were
reviewed and approved by the Animal Care and Use Committee of the
Pharmaceutical Research Laboratory of the Ube Corporation (Yamaguchi,
Japan). Approval No. is P-18175. Animal studies were conducted in
compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and
the recommendations of the \emph{British Journal of Pharmacology}(Lilley
et al., 2020).

\textbf{Materials}

Bardoxolone methyl (CDDO-Me; Figure 1a) was purchased from Shanghai
Heat-Biochem, Inc. (Shanghai, China), and methyl cellulose was purchased
from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). A
choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)
consisting of 60 kcal\% from fat was obtained from Research Diets, Inc.
(New Brunswick, NJ).

\textbf{Choline-deficient, L-amino acid defined, high-fat mouse model of
NASH}

C57BL/6J mice were randomized into groups of equal size and stratified
into four groups according to body weight. The mice were fed a regular
\selectlanguage{greek}γ-\selectlanguage{english}ray-sterilized chow diet or CDAHFD and 3 or 10
mg*kg\textsuperscript{-1} CDDO-Me suspended in 0.5
w*v\textsuperscript{-1}\% methylcellulose (Fujifilm Wako Pure Chem.
Corp.) or 0.5 w*v\textsuperscript{-1}\% methyl cellulose as the vehicle
were orally administered every day to 8 mice in each group during the
experiment. After 8 weeks of treatment with the regular diet or CDAHFD,
all mice were weighed and euthanized under isoflurane anaesthesia. The
livers were weighed and divided into sections, which were fixed in 10\%
phosphate-buffered formalin, frozen in liquid nitrogen, or placed in
RNAlater solution (Thermo Fisher Scientific Inc., Waltham, MA) for
further evaluation. Serum samples were collected and stored at -80 \selectlanguage{ngerman}°C
until further analyses.

\textbf{Biochemical analyses}

Serum levels of aspartate aminotransferase, alanine aminotransferase,
total bilirubin, triglycerides, LDL cholesterol, and HDL cholesterol
were measured using a 7180 automatic biochemistry analyser (Hitachi
Ltd., Tokyo, Japan).

\textbf{Cell culture and \emph{in vitro} treatment}

Hepa1c1c7 cells (RRID:CVCL\_0328) were obtained from DS Pharma
Biomedical Co. Ltd. (Osaka, Japan), and maintained in MEM\selectlanguage{greek}α \selectlanguage{english}containing
10\% FBS and 1\% penicillin/streptomycin/amphotericin B (Thermo Fisher
Scientific Inc.). Hepa1c1c7 cells were seeded onto plates for 24 h and
treated with the indicated concentrations of CDDO-Me for 6, 24, and 48
h. RAW264.7 cells (RRID:CVCL\_0493) were obtained from the ECACC General
Collection and maintained in DMEM containing 10\% FBS and 1\%
penicillin/streptomycin/amphotericin B (Thermo Fisher Scientific Inc.).
RAW264.7 cells were seeded onto plates for 24 h and treated with the
indicated concentrations of CDDO-Me for 6 h. After incubation with
CDDO-Me, cells were collected for quantitative PCR.

\emph{In vitro} \textbf{NAD(P)H quinone dehydrogenase 1 enzyme activity
induction assay}

NAD(P)H Quinone Dehydrogenase 1 (NQO1) activity was measured as
previously described (Park et al., 2011) with slight modifications.
Briefly, Hepa1c1c7 cells were seeded in 96-well plates for 24 h and
treated with the indicated concentrations of CDDO-Me for 48 h. The cells
were then lysed with shaking for 20 min in 50 \selectlanguage{greek}μ\selectlanguage{english}L of cell lysis buffer
(Cell Signaling Tech, Inc., Danvers, MA) and reacted for 5 min with 200
\selectlanguage{greek}μ\selectlanguage{english}L of reaction buffer (25 mM Tris-HCl; 0.07\% albumin; 0.01\% Tween-20;
2 U*mL\textsuperscript{-1} Glucose-6-phosphate dehydrogenase; 5 \selectlanguage{greek}μ\selectlanguage{english}M
flavin adenine dinucleotide; 1 \selectlanguage{greek}μ\selectlanguage{english}M Glucose-6-phosphate; 30 \selectlanguage{greek}μ\selectlanguage{english}M
nicotinamide adenine dinucleotide phosphate; 0.03\% MTT; and 50 \selectlanguage{greek}μ\selectlanguage{english}M
Menadione). After adding 50 \selectlanguage{greek}μ\selectlanguage{english}L of stop solution (0.3 mM dicumarol/5 mM
potassium dihydrogenphosphate), absorbance was measured at 540 nm. The
number of cells was measured using the CellTiter-Glo assay (Promega
Corp, Madison, WI) and the absorbance value indicating NQO1 activity was
corrected using another plate seeded under the same conditions as
described above. The EC\textsubscript{150} value, the concentration that
increased the NQO1 activity, was also calculated.

\emph{In vivo} \textbf{pharmacokinetics and pharmacodynamics study}

Non-fasting C57BL/6J mice were orally administered CDDO-Me at doses of 3
and 10 mg*kg\textsuperscript{-1}. Blood samples were collected via the
abdominal vein into blood collection tubes containing EDTA-2K (Terumo
Corp, Tokyo, Japan) under anaesthesia at 1, 4, 6, and 24 h after oral
administration. Liver samples were also collected to analyse the
expression of \emph{Nqo1} using quantitative PCR. The plasma
concentration of CDDO-Me was measured using the API 3200 triple
quadrupole mass spectrometer (AB Sciex LLC., Redwood City, CA) under the
following conditions: column, Kinetex C18 (Phenomenex Inc., Torrance,
CA, 50 \selectlanguage{ngerman}× 2.1 mm, 2.6 \selectlanguage{greek}μ\selectlanguage{english}m); column temperature, 40 \selectlanguage{ngerman}°C; flow rate, 0.4
mL\selectlanguage{english}*min\textsuperscript{-1}; mobile phase A, 0.1\% formic acid/MilliQ
aqueous solution; mobile phase B, 0.1\% formic acid in 50
v\selectlanguage{english}*v\textsuperscript{-1}\% acetonitrile/methanol solution; and gradient
-- 0 min: A / B=90 / 10, 1 min: A / B=10 / 90, 1-3 min: A / B=10 / 90,
3.01 min, A / B: 90 / 10. CDDO-Me was quantified in the negative
ionization mode using multiple reaction monitoring with niflumic acid as
the internal standard, as previously described (Resham et al., 2015).
Data were acquired and integrated using Analyst software version 1.7 (AB
Sciex LLC., RRID: not yet assigned).

\textbf{Quantitative PCR}

Total RNA was isolated using a NucleoSpin RNA kit (Takara Bio Inc.,
Shiga, Japan). RNA was reverse transcribed using a PrimeScript RT
reagent kit with a gDNA eraser (Takara Bio Inc.). cDNA was amplified
with a TB Green Premix Ex Taq II kit (Takara Bio Inc.) using a Thermal
Cycler Dice Real Time System (Takara Bio Inc.). All primers were
purchased from Takara Bio Inc. The primer sequences are listed in Table
S1. Relative mRNA levels were normalized to \emph{Gapdh} mRNA levels.

\textbf{Histology}

Paraffin-embedded liver samples were cut into approximately 4 \selectlanguage{greek}μ\selectlanguage{english}m
sections and stained with haematoxylin and eosin (HE) and Sirius red.
For Sirius red staining, deparaffinized sections were incubated for 15
min with Picrosirius red solution prepared by mixing 5 mL of 1\% Sirius
red solution (Muto Pure Chem. Co., Ltd., Tokyo, Japan) with 100 mL of
saturated picric acid solution (Fujifilm Wako Pure Chem. Corp). The
NAFLD activity score (NAS) of each slide was evaluated as previously
described (Kleiner et al., 2005).

\textbf{Western blotting}

T-PER (Thermo Fisher Scientific Inc.) and Halt protease and phosphatase
inhibitor cocktail (Thermo Fisher Scientific Inc.) were mixed at a ratio
of 99:1 to prepare the lysis buffer. Liver samples were homogenized in
the lysis buffer and centrifuged at 10,000 \selectlanguage{ngerman}× \emph{g} for 10 min. The
supernatants were collected, each sample was mixed with Lane marker
reducing sample buffer (Thermo Fisher Scientific Inc.) at a ratio of
4:1, and heated at 95 ºC for 5 min to prepare the blotting samples. The
prepared samples were separated using sodium dodecyl
sulphate-polyacrylamide gel electrophoresis. Proteins were transferred
from the gel onto a PVDF membrane using an iBlot Dry Blotting System
(Thermo Fisher Scientific Inc.). The primary antibodies reactions
{[}anti-Vinculin antibody (Cat\# 13901S, RRID:AB\_2728768) was purchased
from Cell Signaling Tech, Inc.; anti- NQO-1 antibody (Cat\# ab34173,
RRID:AB\_2251526), anti-Glutamate-cysteine ligase catalytic subunit
(GCLC) antibody (Cat\# ab207777, RRID: none yet assigned), and
anti-alpha smooth muscle actin (\selectlanguage{greek}α\selectlanguage{english}SMA) antibody (Cat\# ab32575,
RRID:AB\_722538) were purchased from Abcam Inc. (Cambridge, UK) {]} were
performed using the iBind\textsuperscript{TM} Western Device (Thermo
Fisher Scientific Inc.). The bound primary antibodies were detected
using an HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat \#
HAF008, RRID: AB\_357235; R\&D Systems Inc., Minneapolis, MN). Then,
Amershan\textsuperscript{TM} ECL Prime western blotting detection
reagent (GE Healthcare Inc., Chicago, IL) and
ChemiDoc\textsuperscript{TM} XRS Plus (Bio-Rad Laboratories Inc.,
Hercules, CA) were used for protein detection. All immunological
procedures were performed according to the recommendations of
the\emph{British Journal of Pharmacology} (Alexander et al., 2018).

\textbf{Quantification of malondialdehyde}

Hepatic malondialdehyde (MDA) levels were measured using a lipid
peroxidation assay kit (Abcam Inc., Cat\# ab118970) according to the
manufacturer's protocol. Briefly, liver homogenates were mixed with
thiobarbituric acid (TBA) to generate MDA-TBA adducts. The MDA-TBA
adducts were then quantified colourimetrically.

\textbf{Quantification of hydroxyproline}

Hepatic hydroxyproline content was measured as previously described
(Matsumoto et al., 2013), with slight modifications. Briefly,
homogenized liver samples were hydrolysed overnight in 6N HCl at 105 \selectlanguage{ngerman}°C,
neutralized with 6N NaOH, and filtered through a 0.2 \selectlanguage{greek}µ\selectlanguage{english}m PFTE filter
(Advantec Toyo, Ltd., Tokyo, Japan). Aliquots of the hydroxylate were
diluted in citrate buffer, oxidized with chloramine-T dissolved in
citrate buffer, and incubated for 20 min at room temperature. The
reaction was terminated with 20\% perchloric acid. After incubation, 20
w*v\textsuperscript{-1}\% p-dimethylaminobenzaldehyde dissolved in
ethylene glycol mono-methyl ether was added, the samples were incubated
for 1 h at 60 \selectlanguage{ngerman}°C, dispensed into a 96-well plate, and measured for
absorbance at 570 nm. The hydroxyproline levels were determined using a
standard curve. All reagents were purchased from Fujifilm Wako Pure
Chem. Corp.

\textbf{Enzyme-Linked Immunosorbent Assay (ELISA)}

Serum concentrations of CC chemokine ligand (CCL)3 and CCL4 were
measured using ELISA kits (R\&D Systems Inc.) according to the
manufacturer's protocol.

\textbf{Immunohistochemistry}

Paraffin-embedded liver samples were cut into approximately 4 \selectlanguage{greek}μ\selectlanguage{english}m
sections and incubated for 6 min with proteinase K (Agilent tech. Inc.,
Santa Clara, CA) or autoclave-heated for 10 min in 0.01 M citrate buffer
for antigen retrieval. Sections were washed thrice in PBS and blocked by
incubation with 3\% BSA in PBS for 30 min or with a blocking reagent
(Cat \#414321; Nichirei Bioscience Inc., Tokyo, Japan) for 60 min at
room temperature. The expression levels of \selectlanguage{greek}α\selectlanguage{english}SMA, F4/80, and Lymphocyte
antigen 6 complex, locus G (Ly-6G) were determined using anti-\selectlanguage{greek}α\selectlanguage{english}SMA
(Agilent tech. Inc., Cat\# MO851, RRID: not yet assigned), anti-F4/80
(Bio-Rad Laboratories Inc., Cat\# MCA497R, RRID: AB\_323279), and
anti-Ly-6G (BioLegend Inc., San Diego, CA, Cat\# 127602, RRID:
AB\_1089180) antibodies, respectively. After washing, the sections used
for F4/80 and Ly-6G detection were incubated with biotinylated anti-rat
IgG (Vector laboratories Inc., Newark, CA, Cat\# BA-9400, RRID:
AB\_2336202) and analysed based in the formation of a
streptavidin-biotin complex (Vector lab. Inc., Cat\# PK-4000) with
3,3'-diaminobenzidine tetrahydrochloride (DAB-4HCl) solution (Agilent
tech. Inc., Cat\# K3468). Sections used for \selectlanguage{greek}α\selectlanguage{english}SMA detection were
incubated with Simple stain MAX PO (Nichirei Bioscience Inc., Cat\#
414321) and with detection using DAB-4HCL solution. The sections were
counterstained with haematoxylin. The numbers of F4/80- or Ly-6G-
positive cells per \selectlanguage{ngerman}×40 high-power field were counted manually. Ten
high-power fields were examined per mouse.

The expression of C-type lectin domain family 4 member F (CLEC4F),
Glycoprotein nonmetastatic melanoma protein B (GPNMB), CC chemokine
receptor (CCR)2, Sialophorin (SPN), CCR1, and CCR5 was determined using
Ventana discovery ultra (Roche Diagnostics K.K., Basel, Switzerland)
according to Ruo discovery universal (ver. 0.00.0394) staining. Briefly,
for immunohistochemistry, antigen retrieval was performed using CC1
buffer (Roche Diag. K.K., Cat\# 518-108939) for 64 min at 95 °C. The
sections were then incubated for 4 min at 37 °C with Inhibitor CM, which
was included in the Chromomap kit (Roche Diag. K.K., Cat\# 518-100803).
The sections were stained for 32 min at 37 °C with the respective
primary antibody, against CCR1 (Abcam Inc., Cat\# ab140756, RRID: none
yet assigned) and CCR5 (Bioss Inc., Woburn, MA, Cat\# bs-2514R, RRID:
AB\_10857802). They were then incubated for 16 min at 37 °C with the
secondary antibody, Omnimap anti-rabbit HRP (Roche Diag. K.K., Cat\#
518-102135). Next, the sections were incubated for 12 min at 37 °C with
Discovery DAB CM and Discovery H\textsubscript{2}O\textsubscript{2} CM
and for 4 min at 37 °C with Discovery Copper CM, all of which were
included in the Chromomap kit (Roche Diag. K.K., Cat\# 518-100803) to
detect the signal. The sections were counterstained with haematoxylin.

For immunofluorescence, antigen retrieval and inhibition of endogenous
peroxidase activity were performed in the same manner as for
immunohistochemistry. Sections for CLEC4F detection were blocked with
Goat F(ab) anti-mouse IgG H\&L (Abcam Inc., Cat\# ab6668) for 16 min at
37 °C. Sections were stained for 32 min at 37 ºC with the respective
primary antibody against GPNMB (Abcam Inc., Cat\# ab188222, RRID: none
yet assigned), CCR2 (Abcam Inc., Cat\# ab273050, RRID: AB\_2893307),
CLEC4F (R\&D systems Inc., Cat\# MAB2784, RRID: AB\_2081338), and SPN
(Thermo Fisher Scientific Inc., Cat\# PA5-96540, RRID:AB\_2808342). They
were then incubated for 16 min at 37 ºC with the secondary antibody,
Omnimap anti-rabbit HRP (Roche Diag. K.K., Cat\# 518-102135) for
anti-GPNMB, anti-CCR2 and anti-SPN primary antibodies or with Omnimap
anti-rat HRP (Roche Diag. K.K., Cat\# 518-110932) for anti-CLEC4F
primary antibody. The sections were then incubated for 32 min at 37 ºC
with Discovery Cy5 (Roche Diag. K.K., Cat\# 518-113667) for signal
detection.

For multiple staining, the processes of antigen retrieval, inhibition of
endogenous peroxidase activity, and reaction with primary and secondary
antibodies were conducted in same as for the respective
immunohistochemistry or immunofluorescence process, except an incubation
step for 4--8 min at 95 ºC was added between each staining step to
denature the residual antibodies. For signal detection, the sections
were incubated for 32 min at 37 ºC with Discovery Cy5 (Roche Diag. K.K.,
Cat\# 518-113667), Discovery rhodamine (Roche Diag. K.K., Cat\#
518-111861), Discovery FITC (Roche Diag. K.K., Cat\# 518-111878) and
Discovery DCC (Roche Diag. K.K., Cat\# 518-113988) at the end of each
staining step. Positive areas in the sections were quantified using a
BZ-X810 microscope and Hybrid cell count software (Keyence Corp, Osaka,
Japan) as previously described (Yoshimoto \& Shinya, 2022).

\textbf{Whole-transcriptome analysis with RNA-seq}

Total RNA was extracted from the livers of mice using the RNeasy Mini
Kit (Qiagen Inc., Hilden, Germany), libraries were prepared as
previously described (Kohno et al., 2020; Muto et al., 2023), and the
quality and concentration of the libraries were evaluated using Agilent
2200 TapeStation (Agilent tech. Inc., D1000). The libraries mixed to
equal molecular concentrations were sequenced on an Illumina NextSeq500
DNA sequencer with a 75-bp paired-end cycle sequencing kit (Illumina
Inc., San Diego, CA). The data were then trimmed and mapped to the mouse
reference genome GRCm38 release-95 using the CLC Genomics Workbench
software (ver.12.0.3; Qiagen Inc., RRID:SCR\_011853). The mapped read
counts were normalized to transcripts per million (TPM) and converted to
log2 after adding 1. Differentially expressed genes with p-value
\textless{} 0.05 and fold change \textgreater{} \textbar{}1.2\textbar{}
were used for Ingenuity Pathway Analysis (Qiagen Inc.,
RRID:SCR\_008653), which was performed to analyse the detected genes. A
Venn diagram was created using the Calculate and draw custom venn
diagrams software
(\emph{http://bioinformatics.psb.ugent.be/webtools/Venn/}). All heat
maps were plotted as z-scores calculated from the TPM of each gene in
the RNA-seq data using GraphPad Prism software (version 9.0; GraphPad
Software, RRID:SCR\_002798). The marker genes in scRNA-seq were referred
against the mouse NAFLD myeloid cell atlas dataset (Guilliams et al.,
2022). The genes listed in the referenced data were used to confirm
specificity in monocyte and macrophage clusters among mouse NAFLD liver
cells using public databases (\emph{https://www.livercellatlas.org/}),
and genes with higher specificity were selected. UMAP visualization of
mouse NAFLD liver cells or NAFLD myeloid cells was performed using
public datasets (https://www.livercellatlas.org/).

\textbf{Data and Statistical Analysis}

Results were showed as mean ± S.D. Statistical differences were analysed
by one-way ANOVA with subsequent Dunnett's multiple comparison test or
non-parametric Kruskal-Wallis test, followed by Dunn-Bonferroni post-hoc
test using GraphPad Prism software (version 9.0; GraphPad Software,
RRID:SCR\_002798). Statistical significance was set at P \textless{}
0.05. The data and statistical analyses complied with the
recommendations of the \emph{British Journal of Pharmacology} for
experimental design and analysis in pharmacology (Curtis et al., 2018).

\textbf{Results}

\textbf{Determination of the oral administration dose in CDDO-Me
treatment}

Cytoplasmic Nrf2 binds to a Keap1 homodimer that interacts with Cullin
3, constituting a ubiquitin E3 ligase complex, the formation of which
results in Nrf2 degradation through the ubiquitin-proteasome system.
CDDO-Me, an activator of Nrf2, covalently binds to Cys-151 within the
Keap1 sequence, induces Nrf2 translocation to the nucleus, and increases
its transcriptional activity (Figure 1a) (Cleasby et al., 2014; Suzuki
\& Yamamoto, 2017). To determine the optimal concentration of CDDO-Me
required to activate Nrf2, the \emph{Nqo1} mRNA expression level was
examined in Hepa1c1c7 cells in the presence of CDDO-Me. CDDO-Me
upregulated \emph{Nqo1} gene expression in a dose-dependent manner to
the extent of 0.1--3 nM, and the increased expression in 3 nM was
maintained until 10 nM (Figure 1b). Consistent with mRNA expression,
NQO1 enzyme activity in Hepa1c1c7 cells was increased by CDDO-Me, with
an EC\textsubscript{150} value of 0.62 nM (Figure 1c). Single oral
administration of 3 or 10 mg\selectlanguage{english}*kg\textsuperscript{-1} of CDDO-Me to mice
increased \emph{Nqo1} expression in the liver to approximately 3- or
6-fold higher than that in the control mice at 24 h, respectively
(Figure 1d). The maximum plasma concentrations of CDDO-Me attained in
mice after administering 3 and 10 mg\selectlanguage{english}*kg\textsuperscript{-1} were 10.2 +-
1.6 and 32.9 +- 0.6 nM, respectively (Figure 1e), and CDDO-Me disappeared
from the plasma after 24 h of administration (Figure 1e). Based on these
observations, doses of 3 and 10 mg\selectlanguage{english}*kg\textsuperscript{-1} were selected
to investigate the hepatoprotective effects of CDDO-Me.

\textbf{Improvement of liver steatosis in CDAHFD-fed mice upon CDDO-Me
treatment}

To examine the hepatoprotective effects of CDDO-Me, it was orally
administered to CDAHFD-fed mice, once daily for 8 weeks from 6 weeks of
age, and serum and liver samples were collected at the end of the
experiment (Figure 2a). No loss of body weight was observed upon daily
administration of CDDO-Me below 10 mg\selectlanguage{english}*kg\textsuperscript{-1} for 8 weeks
(Figure S1). HE staining of the liver from CDAHFD-fed mice showed marked
features of NASH, accompanied by large lipid droplets and inflammation,
in addition to leukocyte infiltration (Figure 2b). The NAFLD activity
score (NAS) calculated with steatosis, lobular inflammation, and
ballooning in HE staining was greater than 5 (Figure S2 and Table S2),
indicating that NASH occurred in the liver after 8 weeks of CDAHFD
feeding. Administration of CDDO-Me to NASH model mice improved the NAS
in a dose-dependent manner (Figure 2c). Consistent with NAS, the
increased biochemical parameters including aspartate aminotransferase
and alanine aminotransferase were significantly decreased in the mice
injected 10 mg\selectlanguage{english}*kg\textsuperscript{-1} of CDDO-Me (Table 1). The hepatic
expression levels of the inflammatory cytokines \emph{Tnfa, Ifng, IL-6}
, and \emph{IL-1b} , which were upregulated in CDAHFD-fed mice, were
significantly reduced according to the administered dose of CDDO-Me
(Figure 2d,e and Figure S3a,b). The expression of \emph{Abcg5} , a
transporter involved in the excretion of cholesterol into bile (Berge et
al., 2000; Tian, Gao, Zhang, Hackfort, \& Zucker, 2019), and\emph{Ces2c}
and \emph{Ces1g} , which are inhibitory enzymes of lipogenesis (Lian,
Nelson, \& Lehner, 2018), were significantly upregulated by
administering CDDO-Me (Figure 2f,g, and Figure S3c). These observations
revealed that CDDO-ME improved the symptoms of NASH in the livers of
CDAHFD-fed mice.

\textbf{Reduction of oxidative stress by CDDO-Me in the liver of NASH
mice}

Hepatic MDA levels were examined in the livers of NASH model mice fed
CDAHFD and treated with CDDO-Me because oxidative stress participates in
the progression to NASH (Friedman, Neuschwander-Tetri, Rinella, \&
Sanyal, 2018). The MDA level, which was significantly elevated in
CDAHFD-fed mice, was nearly restored to that in the control upon CDDO-Me
treatment at 3 mg\selectlanguage{english}*kg\textsuperscript{-1} of (Figure 3a). The expression
of Nrf2-target genes \emph{Nqo1} and \emph{Gclc} , which have
antioxidative effects, was upregulated in the livers of CDAHFD-fed mice
in a dose-dependent manner after treatment with CDDO-Me (Figure 3b,c),
consistent with the protein expression levels (Figure 3d-f and Figure
S4). These observations indicated that the antioxidative genes elicited
by CDDO-Me at least partially inhibited the progression to NASH.

\textbf{Amelioration of hepatic fibrosis by CDDO-Me in NASH mice}

Nrf2 activation has been reported to inhibit the hepatic fibrosis
induced by HSC activation through inhibition of the TGF\selectlanguage{greek}β/\selectlanguage{english}Smad pathway
(Oh et al., 2012; Prestigiacomo \& Suter-Dick, 2018). Collagen fibres,
which are a marker of fibrosis formation detected by Sirius red
staining, were significantly increased in the livers of CDAHFD-fed mice
compared with those in control livers, consistent with the \selectlanguage{greek}α\selectlanguage{english}SMA staining
observed in HSC activation (Figure 4a). CDDO-Me treatment markedly
reduced the positive staining results (Figure 4a). Accumulation of
hydroxyproline, a major component of collagen in NASH, was significantly
inhibited by treatment with CDDO-Me compared to that in the
vehicle-treated CDAHFD-fed mice (Figure 4b). The expression levels
of\emph{Tgfb} and other profibrotic markers, \emph{Acta2, Col1a1, Mmp2,
Mmp9, Mmp14,} and \emph{Timp1} were significantly upregulated in
CDAHFD-fed mice in a dose-dependent manner when treated with CDDO-Me
(Figure 4c-e and Figure S5). The protein levels of \selectlanguage{greek}α\selectlanguage{english}SMA were also
consistent with the immunohistochemical data (Figure 4f, g and Figure
S4). These results indicate that CDDO-Me inhibits the hepatic fibrosis
induced by HSC activation. All of full-length blot images were shown in
Figure S4.

\textbf{Whole-transcriptome analysis using the livers of NASH mice
treated with CDDO-Me}

We comprehensively analysed the RNA expression in the liver to elucidate
the mechanism underlying the hepato-protective effects of CDDO-Me in
CDAHFD-induced NASH mice, because CDDO-Me is involved in the various
signalling pathways such as NF-\selectlanguage{greek}κ\selectlanguage{english}B or Akt inhibition in addition to Nrf2
activation. Among the 41,326 transcripts detected using RNA-seq
analysis, 2,625 were significantly upregulated in NASH mice by more than
1.2-fold compared to those in the control mice (Figure 5a). Pathway
analysis using the 130 genes upregulated by CDDO-Me in CDAHFD-fed mice
showed activation of Nrf2 (gene symbol \emph{NFE2L2} )-mediated
oxidative stress and xenobiotic metabolism-related pathways (Figure S6).
Of the 2,625 upregulated genes, the expression of 195 genes was
decreased below 80\% by treatment with CDDO-Me (Figure 5a-c), and the
CDDO-Me-regulated genes abundantly included leukocyte-related genes such
as \emph{C5ar1} ,\emph{Ccl3} , \emph{Ccl4,} \emph{Fcgr1} , and
\emph{Lilra5} as shown in Table S3. Pathway analysis using these 195
downregulated genes showed that inhibition of cytokines caused a
reduction in inflammatory pathways, such as recruitment and cell
movement of leukocytes through NF-\selectlanguage{greek}κ\selectlanguage{english}B inhibition (Figure 5d) and immune
cell trafficking (Figure 5e). CDAHFD-fed mice showed 167 downregulated
genes (fold change \textless{} 0.8, p \textless{} 0.05) compared to
those in control mice, and only 17 of the 167 genes were significantly
increased by CDDO-Me treatment (Figure 5b). These data suggest that the
inhibition of inflammatory cell recruitment may play an important role
in the hepatoprotective effect of CDDO-Me.

\textbf{Suppression of macrophage infiltration in the livers of NASH
mice treated with CDDO-Me}

To identify the inflammatory cells regulated by CDDO-Me, the expression
of marker genes in leukocyte subtypes was visualized using a heatmap.
The representative macrophage markers, \emph{Adgre1} and \emph{Cd86} ,
were remarkably elevated in NASH mice and their increased expression was
significantly inhibited by CDDO-Me treatment (Figure 6a). The neutrophil
markers \emph{Ptprc} , \emph{Cxcr4} , and \emph{Itgb2} were upregulated,
similar to those of macrophages, but their inhibition by CDDO-Me
treatment was weak. The markers for T and B cells remained almost
unchanged (Figure 6a and Figure S7). To confirm the leukocyte subtypes
in the liver, immunohistochemical staining with the macrophage marker
F4/80 and the neutrophil marker LY6G, was performed in liver sections
from NASH mice treated with CDDO-Me. CDDO-Me treatment decreased the
number of macrophages in the livers of NASH mice (Figure 6b,c). The
number of neutrophils infiltrating the livers of NASH mice was
comparatively low, and their infiltration was decreased by half by
CDDO-Me treatment (Figure 6b,d). The hepatic macrophage subtypes
targeted by CDDO-Me have been analysed based on marker genes using
scRNA-seq of NASH in previous reports (Guilliams M et al., 2022). Most
marker genes in lipid-associated macrophages were highly expressed in
NASH, and CDDO-Me significantly inhibited their increased expression
along with a decrease in lipid accumulation (Figure 7a,b). The
expression of marker genes for monocytes and monocyte-derived
macrophages, such as monocyte-derived Kupffer cells and patrolling
monocytes, was significantly increased in the liver during NASH, and
this increase was suppressed by CDDO-Me treatment (Figure 7a,b). Marker
genes of tissue-localized macrophages, such as resident Kupffer cells
and peritoneal macrophages were almost constant in the NASH group,
independent of CDDO-Me administration (Figure 7a,b). The localization of
representative macrophage subtypes was examined by immunohistochemistry
using specific antibodies against marker genes. GPNNB, a marker of
lipid-associated macrophages, was not detected in the control liver
sections, whereas GPNNB-positive cells were present in the area
surrounding the lipid droplets in NASH (Figure 7c,d). CDDO-Me strongly
suppressed lipid-associated macrophages staining in NASH. The number of
cells stained for markers of monocytes and patrolling monocytes, CCR2
and SPN, respectively, was significantly increased in NASH and was
decreased by CDDO-Me administration (Figure 7c,e,f). These cells were
frequently observed in the surrounding blood vessels. CLEC4F-positive
cells indicated that resident Kupffer cells widely existed throughout
the normal liver, and that staining was slightly decreased by NASH,
independent of CDDO-Me treatment (Figure 7c,g). These observations
indicated that monocyte-derived macrophages infiltrated the liver
through blood vessels, while CDDO-Me effectively inhibited recruitment.

\textbf{Genes regulated by CDDO-Me treatment in macrophages}

To elucidate the molecules participating in macrophage recruitment
regulated by CDDO-Me during NASH, network analysis was performed using
CDDO-Me-downregulated genes. The network indicated that CDDO-Me
inhibited the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway (Figure S8), leading to inhibition of cell
infiltration through chemokine ligands CCL4, and human CCL3L3
corresponding to mouse CCL3 (Figure 8a). The expression levels of
chemokines and receptors associated with macrophage recruitment were
visualized on a heatmap (Figure 8b). CDDO-Me markedly inhibited the
expression of chemokine ligands, \emph{Ccl3} and \emph{Ccl4} , and
chemokine receptors, \emph{Ccr1} and \emph{Ccr5,} which were increased
in NASH mice; this pattern was consistent with that of NAS, an indicator
of NASH. The expression levels of representative chemokines and their
receptors were confirmed using quantitative PCR (Figure S9). Serum
protein levels of CCL3 and CCL4, which were upregulated in NASH mice
were inhibited in a dose-dependent manner by CDDO-Me treatment (Figure
8c,d), indicating that the chemokine ligands released into blood may
function as migration factors for monocyte-derived macrophages. The
number of cells expressing CCR1 and CCR5 increased in the surrounding
blood vessels during NASH and almost disappeared in the presence of
CDDO-Me (Figure 8e-g). Analysis of mouse NAFLD using scRNA-seq data from
public datasets (https://www.livercellatlas.org/) suggested
that\emph{Ccl3} and the receptor \emph{Ccr1} were expressed in
monocytes/macrophages and neutrophils, and that \emph{Ccl4} and the
receptor \emph{Ccr5} were detected in macrophages/monocytes and Kupffer
cells (Figure S10). CCR1- and CCR5-expressing cells corresponded to
cells expressing CCR2, a monocyte marker (Figure 8h), indicating that
CCR1 and CCR5 expressed in monocyte-derived macrophages were regulated
by CDDO-Me. To examine whether CDDO-Me directly participates in the
expression of chemokine receptors and ligands, we measured their
expression levels in RAW264.7 murine macrophages after CDDO-Me
treatment. CDDO-Me inhibited the expression levels of \emph{Ccr1}
and\emph{Ccr5} , and simultaneously blocked \emph{Ccl3} and \emph{Ccl4}
, the ligands of these receptors, respectively in RAW264.7 cells (Figure
8i-8l). Taken together with these observations, CDDO-Me directly
inhibits the CCL3-CCR1 and CCL4-CCR5 axes in the macrophages of NASH
mice, contributing to the improvement of non-alcoholic steatohepatitis
and fibrosis through the prevention of monocyte-derived macrophage
migration.

\textbf{Discussion}

We showed that CDDO-Me, a potent activator of Nrf2, dramatically
improved NASH with lipid droplets and fibrosis in CDAHFD-fed mice and
markedly suppressed the recruitment of monocyte-derived macrophages from
blood vessels, in addition to activating the Nrf2-dependent pathway.
During this study, the CCL3-CCR1 and CCL4-CCR5 axes involved in the
chemoattraction of macrophages were identified from the regulated genes
in NASH model mice as novel targets of CDDO-Me.

NAFLD occurred in mice fed a choline-deficient, L-amino-acid-defined,
high-fat diet and progressed to NASH with inflammation and hepatic
injury for 8 weeks. Administration of CDDO-Me to NASH mice improved the
NAS calculated from steatosis, inflammation, and hepatic injury. To
identify the molecular targets of CDDO-Me, which regulate NASH
progression, whole-transcriptome analysis was performed using the livers
of NASH mice. The upregulated genes in NASH mice treated with CDDO-Me
showed activation of the Nrf2-mediated oxidative stress response
originating from \emph{NFE2L2} (gene name of Nrf2), indicating that
CDDO-Me-regulated genes were mediated by Nrf2 activation. This
observation was consistent with our quantitative PCR data and previous
studies analysing the downstream factors of Nrf2 activation (Tonelli,
Chio, \& Tuveson, 2018). Pathway analysis using the expressed genes
corresponding to the disease condition in the presence of CDDO-Me during
NASH indicated that CDDO-Me regulated the recruitment of immune cells,
which was confirmed by immunohistochemical observation. Among the immune
cells, numerous macrophages/monocytes were detected in NASH, which were
efficiently reduced by CDDO-Me treatment; however, it did not affect the
infiltration of lymphoid cells. Hepatic macrophages, which release
chemokines and cytokines, considerably affect the progression of NASH
from NAFLD, leading to inflammation and fibrosis (Tacke, Puengel,
Loomba, \& Friedman, 2023). Subtypes with distinct functions are present
in monocytes/macrophages of NASH, and the classification of these
subtypes is based on the diversity of gene expression in macrophages
(Daemen et al., 2021). Using scRNA-seq, we divided hepatic macrophages
into seven subtypes in the NASH model: lipid-associated macrophages,
monocytes, patrolling monocytes, central vein and capsule macrophages,
monocyte-derived Kupffer cells, resident Kupffer cells, and peritoneal
macrophages (Guilliams et al., 2022). Subtype-specific genes not
expressed in other cells within the liver were selected as marker genes
to examine subtype localization. Monocytes and monocyte-derived
macrophages, such as lipid-associated macrophages, monocytes, patrolling
monocytes, central vein and capsule macrophages, and monocyte-derived
Kupffer cells were increased during NASH and were suppressed by CDDO-Me,
in contrast to the constant or slight increase in resident Kupffer cells
and peritoneal macrophages localized in the tissues. Staining with
macrophage subtype-specific antibodies showed that lipid-associated
macrophages, monocytes, and patrolling monocytes infiltrated the vessel
lumen into the space of Disse, and that the macrophages surrounding the
blood vessels disappeared after CDDO-Me treatment. These findings
indicate that the major target of CDDO-Me is monocyte/macrophage
recruitment from the bone marrow. The marker for resident Kupffer cells
showed scattered detection in whole tissues and was not affected by
CDDO-Me treatment. Krenkel et al. showed that monocyte-derived
macrophages specifically activate various growth factors and cytokines
involved in fibrosis progression in NASH (Krenkel et al., 2018).
Inhibition of macrophage recruitment by CDDO-Me was mainly determined to
be mediated by the CCL3-CCR1 and CCL4-CCR5 axes for the following
reasons. Pathway analysis using genes downregulated by CDDO-Me showed
decreased phagocyte recruitment, which included CCL3 and CCL4 in the
network. Second, CCL3 and CCL4 were released into blood during NASH, and
serum CCL3 and CCL4 levels returned to control levels in the presence of
CDDO-Me. Third, CCR1, the receptor for CCL3, and CCR5, the receptor for
CCL4, were localized in monocytes infiltrated during NASH. Finally,
CDDO-Me directly inhibited the expression of \emph{Ccl3-Ccr1}
and\emph{Ccl4-Ccr5} in the mouse macrophage cell line RAW264.7, in a
dose-dependent manner. In NAFLD model mice, CCL3-deficiency has been
reported to attenuate the steatosis and fibrosis associated with
M2-macrophages (Xu et al., 2021). CCR5 critically reduces macrophage
infiltration into the adipose tissue and subsequent insulin resistance
in CCR5-deficient mice (Kitade et al., 2012). Further, CCL2, CCL3, CCL4,
and CCL5, have been previously identified as the relevant chemokines in
NAFLD/NASH (Marra \& Tacke, 2014); we found that CDDO-Me inhibited the
expression of these chemokines, except for CCL2. The absence of CCL2
extensively inhibits hepatic steatosis and macrophage accumulation in
adipose tissue produced by a high-fat diet (Kanda et al., 2006), whereas
CCL2-deficient mice show no effect on macrophage recruitment in obesity
(Inouye et al., 2007). Obese mice with deleted CCR2, the receptor of
CCL2, show ameliorated steatosis and improved glucose homeostasis
(Weisberg et al., 2006), consistent with data obtained using a
pharmacological antagonist of CCR2 (Tamura et al., 2010), indicating
that the inhibiting CCR2 rather than CCL2 is more effective for treating
NASH progression. CCR2 expression was decreased in the presence of
CDDO-Me, which may partially inhibit macrophage infiltration during
NASH. The upstream pathway indicated that CCL3 and CCL4 was regulated by
NF-\selectlanguage{greek}κ\selectlanguage{english}B, which is also a target of CDDO-Me. Ahmad et al. reported that
CDDO-Me was directly associated with Cys-179 on IKK\selectlanguage{greek}β, \selectlanguage{english}causing inhibition
of the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway in U937 myeloid leukaemia cells (Ahmad, Raina,
Meyer, Kharbanda, \& Kufe, 2006). The inhibitory effect of CDDO-Me on
the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway may predominantly work in myeloid cells because
macrophage-specific NF-\selectlanguage{greek}κ\selectlanguage{english}B activation is involved in the numerous
inflammatory diseases (Mussbacher, Derler, Bas\selectlanguage{ngerman}ílio, \& Schmid, 2023).

scRNA-seq data from the NAFLD model indicate that CCR5 is mainly
expressed in monocytes/macrophages, while CCR1 is expressed in
neutrophils and monocytes (Figure S10). Neutrophil infiltration is often
observed in the early phase of NASH from NAFLD (Herrero-Cervera,
Soehnlein, \& Kenne, 2022), and the CCL3/CCR1 axis may be involved in
the progression of the early phase of the disease through neutrophils
and monocytes. CDDO-Me may reduce neutrophil infiltration through CCR1
in the early phase and regulate the recruitment of monocytes/macrophages
expressing CCR5 in the late phase of NASH, which may lead to efficient
inhibition of disease progression. The chemokines CCL3 and CCL4 increase
in macrophages during NASH, stimulating the chemokine receptors CCR1 and
CCR5 in phagocytes, and enhancing the release of CCL3 and CCL4 from
macrophages through autocrine/paracrine mechanisms (Fahey et al., 1992).
This activation could sustain inflammation in the tissues for a long
time, and inhibition of chemokines and receptors by CDDO-Me may be
helpful in arresting chronic inflammatory diseases in various tissue
macrophages, for instance, CDDO-Me significantly improved chronic kidney
disease with type 2 diabetes in clinical trials. CDDO-Me also
ameliorates high-fat diet-induced colon inflammation and
radiation-induced lung inflammation in mice. The novel pharmacological
activities of CDDO-Me may inhibit macrophage recruitment through
autocrine/paracrine mechanisms involving chemokines and their receptors.
However, additional whole-transcriptome analysis will be useful for
elucidating the pharmacological targets of various medicines.

Overall, this study demonstrates the potent hepatoprotective effect of
CDDO-Me in a mouse model of NASH along with the underlying mechanism of
reduced macrophage infiltration via CCL3-CCR1 and CCL4-CCR5 inhibition,
in addition to Nrf2-mediated hepatoprotective effects.

\textbf{Acknowledgments:} We thank Kenji Yoneda (Yoshindo Inc.) for
helpful discussions, Sayuri Kurihara (UBE Corporation) for providing
technical assistance. We also thank Makiko Nakagawa and Yuko Nakatani
(Yamaguchi University) for support with the NGS analysis, and Noriko
Kondo (Yamaguchi University) for assisting with the immunohistochemical
analysis. Part of the research was carried out using research equipment
shared in the MEXT Project for Promoting Public Utilization of Advanced
Research Infrastructure (Program for Supporting the Construction of Core
Facilities), grant number JPMXS0440400021-23. The RNA-seq and
immunohistochemical analyses were supported by the Institute of Gene
Research of the Yamaguchi University Science Research Center.

\textbf{Data and materials availability:} The data and materials
described in this report are available upon request.

\textbf{Figure Legends}

\textbf{Figure 1. CDDO-Me induces \emph{Nqo1} expression and activation}

(a) Pharmacological mechanism of CDDO-Me in Nrf2 activation. (b)
Expression of \emph{Nqo1} mRNA in Hepa1c1c7 murine hepatoma cells after
treatment with the indicated concentrations of CDDO-Me for 6, 24, or 48
h determined using quantitative PCR (mean + S.D., duplicate). (c) NQO1
enzyme activity in Hepa1c1c7 cells after treatment with the indicated
concentrations of CDDO-Me (mean ± S.D., triplicate);
EC\textsubscript{150} was calculated from the activities. (d) Expression
of \emph{Nqo1} mRNA in the livers of C57BL/6J mice after a single oral
administration of CDDO-Me at 3 mg\selectlanguage{english}*kg\textsuperscript{-1} or 10
mg\selectlanguage{english}*kg\textsuperscript{-1} (mean +- S.D., triplicate). (e) The
concentrations of plasma CDDO-Me in C57BL/6J mice after a single oral
administration of CDDO-Me at 3 mg\selectlanguage{english}*kg\textsuperscript{-1} or 10
mg\selectlanguage{english}*kg\textsuperscript{-1} (mean +- S.D., triplicate).

\textbf{Figure 2. CDDO-Me improves NASH in CDAHFD-fed mice}

(a) Experimental design for CDDO-Me treatment of CDAHFD-fed mice (n =
8). (b) Representative images of HE-stained liver sections. Scale bars
show 100 \selectlanguage{greek}μ\selectlanguage{english}m (upper panels) and 20 \selectlanguage{greek}μ\selectlanguage{english}m (lower panels). (c) The scores for
steatosis, lobular inflammation, hepatocyte ballooning, and NAS.
Statistical differences were analysed using a non-parametric
Kruskal-Wallis test followed by a Dunn-Bonferroni post-hoc test (*P
\textless{} 0.05; **P \textless{} 0.01; ***P \textless{} 0.001, ****P
\textless{} 0.0001, versus vehicle-treated CDAHFD-fed mice, mean + S.D.,
n = 8). (d-g) mRNA expression of \emph{Tnfa} (d), \emph{Ifng}
(e),\emph{Abcg5} (f), and \emph{Ces2c} (g) determined using quantitative
PCR (mean + S.D., n = 8). Statistical differences were analysed using
Dunnett's multiple comparison test (*P \textless{} 0.05; **P \textless{}
0.01; ***P \textless{} 0.001, ****P \textless{} 0.0001, versus
vehicle-treated CDAHFD-fed mice). Cont, Control; Veh, Vehicle. ns; not
significant.

\textbf{Figure 3.} \textbf{CDDO-Me reduces oxidative stress in the liver
of CDAHFD-fed mice}

(a) Hepatic MDA levels measured in the liver homogenates of CDAHFD-fed
mice treated without or with the indicated concentrations of CDDO-Me
(mean + S.D., n = 8). (b and c) mRNA expression of \emph{Nqo1} (b)
and\emph{Gclc} (c) determined using quantitative PCR (mean + S.D., n =
8). Statistical differences were analysed using Dunnett's multiple
comparison test (*P \textless{} 0.05; **P \textless{} 0.01; ***P
\textless{} 0.001, ****P \textless{} 0.0001 versus vehicle-treated
CDAHFD-fed mice). (d) NQO1, GCLC, and Vinculin protein levels determined
by immunoblotting. (e and f) Densitometric analyses of Figure. 3d were
normalized to Vinculin levels (mean + S.D., triplicate). Cont, Control;
Veh, Vehicle. ns; not significant.

\textbf{Figure 4. CDDO-Me ameliorates hepatic fibrosis in CDAHFD-fed
mice}

(a) Hepatic sections stained with Sirius red or anti-\selectlanguage{greek}α\selectlanguage{english}SMA antibody.
Scale bars show 100 \selectlanguage{greek}μ\selectlanguage{english}m (upper panels) and 20 \selectlanguage{greek}μ\selectlanguage{english}m (lower panels). (b)
Hydroxyproline levels measured in the livers (mean + S.D., n = 8). (c-e)
mRNA expression of \emph{Acta2} (c), \emph{Col1a1} (d), and
\emph{Tgfb}(e) determined using quantitative PCR (mean + S.D., n = 8).
Statistical differences were analysed using Dunnett's multiple
comparison test (*P \textless{} 0.05; **P \textless{} 0.01; ***P
\textless{} 0.001, ****P \textless{} 0.0001, versus vehicle-treated
CDAHFD-fed mice). (f) \selectlanguage{greek}α\selectlanguage{english}SMA and Vinculin protein levels determined by
immunoblotting. (g) The densitometric analysis of Figure. 4f was
normalized to Vinculin levels (mean + S.D., triplicate). Cont, Control;
Veh, Vehicle.

\textbf{Figure 5.} \textbf{Whole-transcriptome analysis reveals that
CDDO-Me inhibits recruitment of immune cells in NASH mice}

(a) (left panel) Volcano plot showing the expressions of all transcripts
in the livers, where 2625 genes are upregulated (red) and 167 genes are
downregulated (blue) in CDAHFD-fed mice. (right panel) Volcano plot
shows the expressions of 2625 genes upregulated in the left panel, and
the 22 genes upregulated (red) and 195 genes downregulated (blue) by
CDDO-Me treatment in CDAHFD-fed mice (b) Venn diagram shows the numbers
of upregulated and downregulated genes by CDDO-Me treatment in
CDAHFD-fed mice. (c) The expression of 195 genes downregulated by
CDDO-Me among the upregulated genes in CDAHFD-fed mice are shown on the
heatmap. (d) The graphical summary obtained by Ingenuity Pathway
Analysis using 195 genes in Figure 5c. (e) The top five pathways
detected by Ingenuity Pathway Analysis using 195 genes in Figure. 5c are
shown on the bar graph.

\textbf{Figure 6. CDDO-Me suppresses macrophage infiltration in the
liver of NASH mice}

(a) Heatmap showing the expression of marker genes in macrophages,
neutrophils, T cells, and B cells in the liver. (b) Hepatic sections
stained with anti-F4/80 or anti-LY6G antibodies. Scale bars represent
100 \selectlanguage{greek}μ\selectlanguage{english}m in the upper panels and 20 \selectlanguage{greek}μ\selectlanguage{english}m in the lower panels. (c and d)
F4/80 (c) or LY6G (d) positive cells counted in Figure. 6b (mean + S.D.,
n = 8). Statistical differences were analysed using Dunnett's multiple
comparison test (*P \textless{} 0.05, **P \textless{} 0.01, ***P
\textless{} 0.001, ****P \textless{} 0.0001 versus vehicle-treated
CDAHFD-fed mice). Cont, Control; Veh, Vehicle. ns; not significant.

\textbf{Figure 7. CDDO-Me inhibits recruitment of monocyte-derived
macrophages in the liver of NASH mice}

(a) Heatmap showing the expression of marker genes in each macrophage
subtype previously identified using single-cell RNA-seq analysis. (b)
Violin plot showing the expression of marker genes in the subtypes.
Statistical differences were analysed using Dunnett's multiple
comparison test. (*P \textless{} 0.05; **P \textless{} 0.01; ***P
\textless{} 0.001, ****P \textless{} 0.0001 versus vehicle-treated
CDAHFD-fed mice). (c) Hepatic sections stained with anti-GPNMB,
anti-CCR2, anti-SPN, or anti-CLEC4F antibodies. Scale bars represent 50
\selectlanguage{greek}μ\selectlanguage{english}m. (d-g) GPNMB (d), CCR2 (e), SPN (f), or CLEC4F (g)-positive cells
quantified from the hepatic sections in Figure. 7c (mean + S.D.,
quadruplicate). Cont, Control; Veh, Vehicle. ns; not significant.

\textbf{Figure 8. CDDO-Me reduces CCL3-CCR1 and CCL4-CCR5 expression in
macrophages}

(a) The top pathway in Regulatory Effects detected by Ingenuity Pathway
Analysis using 195 genes downregulated in Figure. 5a. (b) Heatmap shows
the gene expressions involved in chemotaxis in the liver. (c and d) The
serum protein levels of CCL3 (c) and CCL4 (d) measured using ELISA (mean
+ S.D., n = 8). Statistical differences were analysed using Dunnett's
multiple comparison test (*P \textless{} 0.05; **P \textless{} 0.01;
***P \textless{} 0.001, ****P \textless{} 0.0001 versus vehicle-treated
CDAHFD-fed mice). (e) Hepatic sections stained with anti-CCR1 or
anti-CCR5 antibody. Scale bars represent 50 \selectlanguage{greek}μ\selectlanguage{english}m. (f and g) CCR1 (f) or
CCR5 (g)-positive cells quantified in the hepatic sections from Figure.
8e (mean + S.D., quadruplicate). (h) Sections from CDAHFD-fed mice
multiple stained with anti-CCR1(green), anti-CCR2 (red), anti-Ly6G
(blue), and anti-CLEC4F antibodies (pink) (left panel) or anti-CCR5
(green), anti-CCR2 (red), anti-GPNMB (blue), and anti-CLEC4F antibodies
(pink) (right panel). Scale bars represent 50 \selectlanguage{greek}μ\selectlanguage{english}m. (i-l) mRNA expression
of \emph{Ccl3} (i), \emph{Ccl4} (k) and the receptors, \emph{Ccr1} (j)
and \emph{Ccr5} (l) determined using quantitative PCR in RAW264.7 cells
treated with CDDO-Me for 6 h (mean + S.D., triplicate). Cont, Control;
Veh, Vehicle.



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