Exposure to Fine Airborne Particulate Matters Induces Hepatic Fibrosis in Murine Models

Abstract

Hepatic fibrosis, featured by accumulation of excessive extracellular matrix in liver tissues, is associated with metabolic disease and cancer. Inhalation exposure to airborne particulate matter in fine ranges (PM2.5) correlates with pulmonary dysfunction, cardiovascular disease, and metabolic syndrome. In this study, we investigated the effect and mechanism of PM2.5 exposure on hepatic fibrogenesis.
Both inhalation exposure of mice and in vitro exposure of specialized cells to PM2.5 were performed to elucidate the effect of PM2.5 exposure on hepatic fibrosis. Histological examinations, gene expression analyses, and genetic animal models were utilized to determine the effect and mechanism by which PM2.5 exposure promotes hepatic fibrosis.
Inhalation exposure to concentrated ambient PM2.5 induces hepatic fibrosis in mice under the normal chow or high-fat diet. Mice after PM2.5 exposure displayed increased expression of collagens in liver tissues. Exposure to PM2.5 led to activation of the transforming growth factor β (TGFβ)-SMAD3 signaling, suppression of peroxisome proliferator-activated receptor γ (PPARγ), and expression of collagens in hepatic stellate cells. NADPH oxidase plays a critical role in PM2.5-induced liver fibrogenesis.
Exposure to PM2.5 exerts discernible effects on promoting hepatic fibrogenesis. NADPH oxidase mediates the effects of PM2.5 exposure on promoting hepatic fibrosis.
Copyright © 2015. Published by Elsevier B.V.

Full text

Exposure to fine airborne particulate matters induces hepatic
fibrosis in murine models
Ze Zheng
1
, Xuebao Zhang
1
, Jiemei Wang
1
, Aditya Dandekar
2
, Hyunbae Kim
1
, Yining Qiu
1
,
Xiaohua Xu
4
, Yuqi Cui
3
, Aixia Wang
3,4
, Lung Chi Chen
5
, Sanjay Rajagopalan
3,
, Qinghua Sun
3,4
,
Kezhong Zhang
1,2,
⇑
1
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA;
2
Department of
Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201, USA;
3
Division of Cardiovascular Medicine,
Davis Heart & Lung Research Institute, College of Medicine, Ohio State University, Columbus, OH 43210, USA;
4
Division of Environmental Health Sciences, College of Public Health, Ohio State University, Columbus, OH 43210, USA;
5
Department of Environmental Medicine, New York University, Tuxedo, NY 10987, USA
Background & Aims: Hepatic fibrosis, featured by the accumula-
tion of excessive extracellular matrix in liver tissue, is associated
with metabolic disease and cancer. Inhalation exposure to air-
borne particulate matter in fine ranges (PM
2.5
) correlates with
pulmonary dysfunction, cardiovascular disease, and metabolic
syndrome. In this study, we investigated the effect and mecha-
nism of PM
2.5
exposure on hepatic fibrogenesis.
Methods: Both inhalation exposure of mice and in vitro exposure
of specialized cells to PM
2.5
were performed to elucidate the
effect of PM
2.5
exposure on hepatic fibrosis. Histological examina-
tions, gene expression analyses, and genetic animal models were
utilized to determine the effect and mechanism by which PM
2.5
exposure promotes hepatic fibrosis.
Results: Inhalation exposure to concentrated ambient PM
2.5
induces hepatic fibrosis in mice under the normal chow or
high-fat diet. Mice after PM
2.5
exposure displayed increased
expression of collagens in liver tissues. Exposure to PM
2.5
led to
activation of the transforming growth factor b-SMAD3 signaling,
suppression of peroxisome proliferator-activated receptor
c
, and
expression of collagens in hepatic stellate cells. NADPH oxidase
plays a critical role in PM
2.5
-induced liver fibrogenesis.
Conclusions: Exposure to PM
2.5
exerts discernible effects on pro-
moting hepatic fibrogenesis. NADPH oxidase mediates the effects
of PM
2.5
exposure on promoting hepatic fibrosis.
Ó2015 Published by Elsevier B.V. on behalf of the European
Association for the Study of the Liver.
Introduction
Recent studies indicated that exposure to fine ambient particu-
late matter (aerodynamic diameter <2.5
l
m, PM
2.5
) is a risk fac-
tor for pulmonary and cardiovascular diseases as well as
metabolic syndrome [1–3]. Traffic-related airborne PM
2.5
is a
complex mixture of particles and gases from gasoline and diesel
engines, together with dust from wear of road surfaces, tires, and
brakes [4,5]. Airborne PM
2.5
demonstrates an incremental capac-
ity to penetrate into the distal airway units and potentially enter
the systemic circulation with diminishing sizes. It has been sug-
gested that the cytotoxic effects of PM
2.5
are more associated
with PM
2.5
as a complex other than single or a few components
of PM
2.5
particles [6]. The particle sizes, charges, and combined
effects of individual components of PM
2.5
are all crucial to the
adverse health impact of PM
2.5
exposure. Studies from our group
and others suggested that PM
2.5
exposure triggers a variety of
maladaptive signaling pathways in the lung, blood vessels, liver,
and adipose tissues that are associated with endoplasmic reticu-
lum (ER) stress, oxidative stress, and inflammatory responses
[1,7–12]. Moreover, we recently demonstrated an important find-
ing that inhalation exposure to PM
2.5
causes a non-alcoholic
steatohepatitis (NASH)-like phenotype and depletion of hepatic
glycogen storage in animals [1]. Through both in vivo and
in vitro analyses, we revealed the signaling pathways through
which PM
2.5
exposure promotes NASH-associated activities and
impairment of hepatic glucose metabolism. We identified the dis-
ruption of hepatic lipid/glucose homeostasis, lobular and portal
inflammation, as well as mild hepatic steatosis in the liver of
the mice exposed to PM
2.5
for 10 weeks [1]. However, the pro-
nounced effect of PM
2.5
exposure on modulating hepatic path-
ways associated with liver fibrogenesis has not been
characterized.
Liver fibrosis and cirrhosis are the advanced stages of chronic
liver injuries caused by chronic hepatitis viral infection, obesity,
alcoholism, or autoimmune diseases. Recent studies showed that
PM
2.5
exposure activates Kupffer cells in murine liver tissues,
indicating that PM
2.5
represents a risk factor for NAFLD progres-
sion [1,10,13]. In this study, we used a ‘‘real-world’’ PM
2.5
Journal of Hepatology 2015 vol. 63 j1397–1404
Keywords: Air pollution; Hepatic fibrosis; PM
2.5
.
Received 9 September 2014; received in revised form 7 July 2015; accepted 16 July
2015; available online 26 July 2015
⇑
Corresponding author. Address: 540 E. Canfield Avenue, Detroit, MI 48201, USA.
Tel.: +1 313 577 2669; fax: +1 313 577 5218.
E-mail address: kzhang@med.wayne.edu (K. Zhang).

Current address: Division of Cardiology, University of Maryland School of
Medicine, Baltimore, MD 21201, USA.
Abbreviations: PM, ambient particulate matter; PM
2.5
, PM with aerodynamic
diameter less than 2.5
l
m; FA, filtered air; OASIS, Ohio’s Air Pollution Exposure
System for the Interrogation of Systemic Effects; PPAR, peroxisome
proliferator-activated receptor; TGFb, transforming growth factor b;HSC,
hepatic stellate cells; p47phox, Neutrophil cytosolic factor 1; NOX, NADPH
peroxidase; ROS, reactive oxygen species.
Research Article

exposure system, ‘‘Ohio’s Air Pollution Exposure System for the
Interrogation of Systemic Effects (OASIS)’’, to perform
whole-body exposure to mice of environmentally relevant
PM
2.5
. We demonstrate that exposure to PM
2.5
causes a dis-
cernible phenotype of hepatic fibrosis in animals. Through both
in vivo and in vitro analyses, we reveal the signaling pathways
through which PM
2.5
exposure promotes hepatic
fibrogenesis-associated activities. The information from this work
has important implications in the understanding and treatment
of air pollution-induced liver diseases.
Materials and methods
Animal experiments
C57BL/6 male mice of six-weeks-old were purchased from the Jackson Laborato-
ries (Bar Harbor, ME), and were equilibrated for two weeks prior to experimental
enrollment. The mice were housed in cages with regular chow or a high-fat diet
(Teklad TD 88137, 42% calories from fat) in an Association for Assessment and
Accreditation of Laboratory Animal Care-accredited animal housing facility. All
the animal experiments were approved by the Ohio State University and the
Wayne State University IACUC committee and carried out under the institutional
guidelines for ethical animal use.
Exposure of animals to ambient PM
2.5
Mice were exposed to concentrated ambient PM
2.5
or filtered air (FA) in the OASIS
in Columbus, OH, where most of the PM
2.5
component is attributed to long-range
transport [10]. The concentrated PM
2.5
was generated using a versatile aerosol
concentration enrichment system (VACES) as we described previously [14]. Mice
under the normal chow or high-fat diet were exposed to concentrated PM
2.5
for
six hours per day, five days per week for 10 weeks or 9 months [1,10]. The control
(FA) mice were exposed to an identical protocol with the exception of a
high-efficiency particulate-air filter positioned in the inlet valve to remove all
of the PM
2.5
in the filtered air stream. Mice deficient in the cytosolic subunit of
the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47phox
/
)
and wild-type control mice of C57BL/6 strain background (both from Jackson
Laboratories) were exposure to FA or PM
2.5
beginning at the age of three weeks
for a duration of 10 weeks.
Histological scoring for hepatic fibrosis
Paraffin-embedded mouse liver tissue sections (5
l
m) were subjected to
Sirius-red or Masson’s trichrome staining for hepatic fibrosis. The histological
analysis of liver fibrosis were as described previously [15,16]. Each section was
examined by a specialist who was blinded to the sample information. Hepatic
fibrosis were scored according to the modified Scheuer scoring system for fibrosis
and cirrhosis [16,17]. The fibrosis stage scores were based on the 0–4 stage sys-
tem: 0, none; 1, zone 3 perisinusoidal fibrosis; 2, zone 3 perisinusoidal fibrosis
plus portal fibrosis; 3, perisinusoidal fibrosis, portal fibrosis, plus bridging fibro-
sis; and 4, cirrhosis.
Statistics
Experimental results are shown as mean ± SEM (for variation between animals or
experiments). All in vitro experiments were repeated with biological triplicates at
least three times independently. The data were analyzed and compared by paired,
two-tailed Student’s ttests. Multiple comparisons were compared with ANOVA
and proceeded by ad hoc statistical test when necessary. Statistical tests with
p<0.05 were considered significant.
For a full description of materials and methods used in this work, see Supple-
mentary data.
β-actin
0
1
2
3
4
5
6
0
20
40
60
80
100
120
140
160
180
200
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
FA
MPAF 2.5
** *
Fibrosis scores
(arbitrary units)
ABC
FA
α-SMA IHC
FA FA
Col1
1 2 3 1 2 3
FA
0
20
40
60
80
100
120
140
160 **
Activated TGFβ1 in mouse
serum (ng/ml)
FA
*
TGFβ1 mRNA in mouse liver
(fold changes)
FA
FA
D
NC
HFD
NC
HFD
NC
HFD
FA
Masson’s trichromeSirius-red
FA
Normal chow
Normal chow HFD
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Normal chow Normal chow
EFG
Col1/actin
0.09
0.06
0.07
0.14
0.19
0.41
0.01
0.02
0.02
1.01
0.71
0.95
HFD
PM 2.5
PM 2.5 PM 2.5
1 2 3 1 2 3
PM 2.5 PM 2.5 PM 2.5 PM 2.5
HFD HFD
PM 2.5 PM 2.5 PM 2.5
Fig. 1. PM
2.5
exposure induces hepatic fibrosis in mouse liver. (A and B) Sirius-red staining of hepatic collagen deposition (A) and Masson’s trichrome staining of collagen
fiber (B) in formalin-fixed liver tissue sections from C57BL/6 mice under the normal chow (NC) or high-fat diet (HFD) exposed to FA or PM
2.5
for 10 weeks. Magnifications:
200. The arrows point out areas of hepatic fibrosis. (C) Immunohistochemical (IHC) staining of the hepatic stellate cell surface marker
a
-SMA in the liver tissue sections
from the NC or HFD fed mice exposed to FA or PM
2.5
for 10 weeks. Magnifications: 200. (D) Hepatic fibrosis grades of the mice exposed to PM
2.5
or FA for 10 weeks.
Hepatic fibrosis grades were determined based on the histological analyses of Sirius-red staining of collagens, according to the Scheuer scoring system for fibrosis and
cirrhosis [16,17]. Data are shown as mean ± SEM (n = 8 FA- or 9 PM
2.5
-exposed animals). (E) Immunoblotting analysis of collagen I (Col1) in the liver tissue from the mice
exposed to PM
2.5
or FA. The values below gel images represent quantification of Col1 protein signal intensities after normalization to those of b-actin. (F) Serum levels of
activated TGFb1 (determined by ELISA) in the mice exposed to PM
2.5
or FA. For A-B, each bar denotes the mean ± SEM (n = 3). (G) Quantitative real-time PCR (qRT-PCR)
analysis of expression levels of the Tgfb1mRNA in the livers of the mice exposed to PM
2.5
or FA for 10 weeks. Fold changes of mRNA levels were shown by comparing to the
FA-exposed control mice. From B-G,
*
p<0.05;
**
p<0.01. (This figure appears in colour on the web.)
Research Article
1398 Journal of Hepatology 2015 vol. 63 j1397–1404

Results
Inhalation exposure to PM
2.5
induces discernible hepatic fibrosis in
mice
To elucidate in vivo effects of PM
2.5
exposure, male C57BL/6 mice
on the normal chow or high-fat diet were exposed to concen-
trated ambient PM
2.5
or FA in exposure chambers of OASIS
located at Columbus, USA, where most of the PM
2.5
is attributed
to long-range transport [1,10] (Supplementary Fig. 1). OASIS is a
VACES through which fine and ultrafine particles are concen-
trated and exposed to the animals in the chambers [10,18].It
has been demonstrated that the distribution and size of concen-
trated PM
2.5
collected from the exposure chamber air truly reflect
that of non-concentrated PM
2.5
present in the ambient air
[14,19]. At the same exposure site, animals were exposed to
PM
2.5
or FA for 10 weeks or 9 months in two different time peri-
ods. During the first exposure period, the ambient mean daily
PM
2.5
concentration at the study site was 6.5
l
g/m
3
, while the
mean concentration of PM
2.5
in the exposure chamber was
74.6
l
g/m
3
[1]. During the second exposure period, the ambient
mean daily PM
2.5
concentration at the site was 15.8
l
g/m
3
, and
the mean concentration of PM
2.5
in the exposure chamber was
111.0
l
g/m
3
[20]. Previously, we reported that the elemental
composition, as measured by energy-dispersive X-ray fluores-
cence (ED-XRF) analysis, include alkali metals, alkaline earth
metals, transition metals, poor metals, non-metals, metalloid,
and halogens [10].
To evaluate effects of PM
2.5
exposure on liver fibrosis, we first
performed histological analyses with liver tissue sections of the
mice exposed to PM
2.5
or FA for 10 weeks. Sirius-red and Mas-
son’s trichrome staining of collagen deposition revealed signifi-
cant perisinusoidal fibrosis in the liver of the mice under
normal chow diet exposed to PM
2.5
(Fig. 1A, B). The fibrosis
grades of the PM
2.5
-exposed mice were significantly higher than
that of the FA-exposed mice (Fig. 1D). We evaluated production
of collagen proteins in the livers of the mice under PM
2.5
expo-
sure. Levels of collagen I, the major collagen protein in liver extra-
cellular matrix, were significantly increased in the livers of the
PM
2.5
-exposed mice (Fig. 1E). These results indicate a discernible
effect of PM
2.5
exposure on promoting hepatic fibrosis. Further-
more, we evaluated hepatic fibrosis in the PM
2.5
-exposed animals
under the high-fat diet. The grades of hepatic fibrosis in the ani-
mals that were simultaneously fed the high-fat diet and exposed
to PM
2.5
for 10 weeks were significantly higher than that of
FA-exposed control animals (Fig. 1A, B, D, E). Notably, the
high-fat fed, PM
2.5
-exposed animals displayed advanced stages
of hepatic fibrosis, including portal fibrosis and bridging fibrosis,
as shown by histological analysis of collagen disposition (Fig. 1A),
suggesting that PM
2.5
exposure may interact with the high-fat
diet to exacerbate its effect in promoting fibrogenesis. Addition-
ally, we examined hepatic fibrosis in the animals under the nor-
mal chow diet exposed to PM
2.5
for 9 months. Interestingly, the
hepatic fibrosis grades in the animals after 9 months PM
2.5
expo-
sure were only insignificantly higher than those of the
FA-exposed animals (Supplementary Fig. 2A, B). This observation
suggests that the animals may possess adaptation mechanisms to
attenuate the effect of a single environmental risk factor during
the chronic PM
2.5
exposure.
Next, we examined expression of
a
-smooth muscle actin
(
a
-SMA), a prominent marker for activation of hepatic stellate
cells (HSC) and fibrosis progression [21], in the livers of PM
2.5
-
or FA-exposed mice under the normal chow or high-fat diet.
Immunohistochemical analysis showed markedly increased
a
-SMA staining in the mouse liver tissues upon PM
2.5
exposure,
suggesting a strong effect of PM
2.5
exposure on HSC activation
(Fig. 1C). In hepatic fibrosis, the release of latent transforming
growth factor b(TGFb) stimulates HSC to produce collagens, a
key event of hepatic fibrogenesis [22,23]. Enzyme-linked
immunosorbent assay (ELISA) indicated that serum levels of acti-
vated TGFb1 in the mice exposed to PM
2.5
were significantly
higher than those of the FA-exposed mice (Fig. 1F). Gene expres-
sion analysis confirmed higher expression levels of Tgfb1mRNA
in the livers of mice exposed to PM
2.5
(Fig. 1G). Together, these
results implicate that TGFb, the inflammatory trigger of hepatic
fibrosis, is upregulated upon PM
2.5
challenge.
Exposure to PM
2.5
stimulates TGFbrelease by macrophages and
collagen production by HSC
In the liver, fibrogenesis is a coordinated process that involves
multiple specialized cell types, including Kupffer cells (resident
macrophages), Ito cells (quiescent adipocytes), HSC, and extracel-
lular matrix [22]. Upon liver injuries or chronic stress, Ito cells
can be activated and differentiate into myofibroblastic HSC with
decreased peroxisome proliferator-activated receptor
c
(PPAR
c
)
expression and fat-storing function [24,25]. In this process, TGFb
produced by inflammatory cells facilitates the differentiation of
myofibroblastic HSC, which is specialized in producing extracel-
lular matrix proteins, especially collagens, to promote hepatic
fibrogenesis [22,23]. It has been reported that three major liver
cell types, Kupffer cells, HSC, and hepatocytes, can produce TGFb
in liver pathogenesis [26,27]. Previously we demonstrated that
PM
2.5
exposure activates Kupffer cells in animal models [1].
Numbers of activated macrophages and induction of
macrophage-associated inflammatory cytokine genes, including
TGFb1,IL1b,IL6,TNF
a
,IL8,CCL2,CCL3, and Gro1, were increased
in the liver of PM
2.5
-exposed mice (Supplementary Fig. 3A–D).
To define the target liver cell types that produce TGFbunder
PM
2.5
exposure, we exposed mouse primary hepatocytes, macro-
phage cell line RAW264.7, and LX-2, a human HSC cell line that
retains the key features of HSC and has been used to study hep-
atic fibrosis and liver disease [28],toPM
2.5
particles. In response
to PM
2.5
challenge, levels of activated TGFbin the culture med-
ium from RAW264.7 cells, but not hepatocytes or LX-2 cells, were
increased in a time-dependent manner (Fig. 2A). Gene expression
analysis confirmed that expression of the mRNAs encoding Tgfb1,
Tgfb2, and Tgfb3in RAW264.7 cells was significantly increased
upon PM
2.5
exposure (Fig. 2B). These results suggest that macro-
phage is the major cell type that produces TGFbunder PM
2.5
exposure.
We further assessed the effects of PM
2.5
on
fibrogenesis-associated signaling in HSC. LX-2 cells were cultured
in the conditioned medium from the macrophage cell line
RAW264.7 exposed to PM
2.5
or vehicle for 12 and 24 h, respec-
tively. Upon PM
2.5
exposure, phosphorylation of SMAD3, a key
mediator of TGFb-triggered fibrotic response [29], was signifi-
cantly increased in LX-2 cells in a time-dependent manner
(Fig. 2C). Correlated to the phosphorylation of SMAD3, expression
levels of both mRNAs and proteins of collagen I and
a
-SMA were
increased in LX-2 cells incubated with the conditioned media
from PM
2.5
-exposed macrophages (Fig. 2D, E; Supplementary
JOURNAL OF HEPATOLOGY
Journal of Hepatology 2015 vol. 63 j1397–1404 1399

Fig. 5). Additionally, we visualized production of collagen I and
collagen IV in LX-2 cells upon PM
2.5
challenge through
immunofluorescent analysis. LX-2 cells cultured in the condi-
tioned medium from PM
2.5
-exposed RAW264.7 cells produced
much more collagen I and collagen IV proteins than those cul-
tured in the control medium (Fig. 2F; Supplementary Fig. 4), thus
confirming the effect of PM
2.5
exposure on collagen production
by HSC. Furthermore, to verify whether the PM
2.5
-induced colla-
gen production from HSC depends on TGFbsignaling, we incu-
bated LX2 cells with the specific TGF receptor (TGFR) inhibitor
SB431542 prior to the culture with the conditioned medium from
PM
2.5
- or vehicle-exposed RAW264.7 cells. Under PM
2.5
-exposed
conditioned medium, pre-treatment of SB431542 significantly
reduced both mRNA and protein levels of collagen I in LX2 cells
(Supplementary Fig. 6), suggesting a crucial role of TGFbsignaling
in PM
2.5
-induced hepatic fibrogenesis as well as the effectiveness
of TGFbreceptor antagonist SB431542 in suppressing
PM
2.5
-induced hepatic fibrosis.
Exposure to PM
2.5
represses PPAR
c
in HSC
It is known that expression of PPAR
c
in HSC is closely associated
with fibrosis in the liver under injuries or chronic stress [24,25].
The decrease in expression of PPAR
c
enables the quiescent adipo-
cytes (Ito cells) to be activated and further differentiated into
myofibroblastic HSC [24,25]. Previously, we showed that inhala-
tion exposure to PM
2.5
reduces expression of PPAR
c
and PPAR
a
in
liver tissues [1]. However, we have not determined whether
PM
2.5
exposure modulates PPAR
c
expression in stellate cells. To
determine whether PM
2.5
exposure can modulate PPAR
c
expres-
sion in HSC, we examined levels of PPAR
c
in the HSC cell line
LX-2 upon PM
2.5
challenge. LX-2 cells were cultured in the condi-
tioned medium from macrophage cell line RAW264.7 exposed to
PM
2.5
for 12 or 24 h. Upon exposure to the conditioned medium,
both protein and mRNA levels of PPAR
c
were gradually decreased
in LX-2 cells in a manner that depends on the PM
2.5
exposure
time of RAW264.7 cells (Fig. 3A, B).
Pioglitazone is a commonly-used PPAR
c
agonist to prevent
inflammation, fibrosis, and insulin resistance in type II diabetes
patient by increasing PPAR
c
levels [30]. To test whether pioglita-
zone can prevent PM
2.5
-triggered downregulation of PPAR
c
in
HSC, LX-2 cells were incubated with pioglitazone for 48 h
followed by incubation with the conditioned medium from
PM
2.5
-exposed RAW264.7 cells. The pre-treatment of pioglitazone
at the concentration of 1
l
M can partially rescue the downregu-
lation of PPAR
c
1and PPAR
c
2mRNA expression in the LX-2 cells
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0
100
200
300
400
500
600
700
800
900
Primary hepatocytes
Raw264.7
LX2
0
1
2
3
4
5
6
7
8
9
0
50
100
150
200
250
300
ABC
Veh 12 h 24 h
p-SMAD3
SMAD3
β-actin
Tgfβ1 Tgfβ2 Tgfβ3
*
***
**
**
**
n.s.
**
*
Ctl
12 h
24 h
24 h 2 h 8 h 24 h
Activated mouse TGFβ
1
(pg/ml)
Vehicle
DAPI
Col1
Merged
D
*
*
*
Col1α1 Col4α4
Veh
12 h
24 h
Col1
Tubulin
Veh 12 h 24 h
E
mRNA levels in RAW264.7
cells (fold changes)
Veh 12 h 24 h
Ctl
Collagen I in LX2 cells
(% of control)
*
0
20
40
60
80
100
120
140
160
FA 12 h 24 h
p-Smad3 / Smad3 in LX2 cells
(% of control)
Collagen mRNA levels in LX2
cells (fold change)
PM 2.5
n.s.
PM 2.5
PM 2.5
PM 2.5
PM 2.5
PM 2.5
PM 2.5
PM 2.5
n.s.
PM 2.5
PM 2.5
n.s.
F
Fig. 2. Exposure to PM
2.5
stimulates TGFbsignaling in macrophages and collagen production in HSC. (A) Levels of secreted TGFb1 in primary hepatocytes, RAW264.7
cells, and LX-2 cells upon the challenge of PM
2.5
(5
l
g/ml) for different time intervals as indicated. The levels of TGFb1 in the culture medium were determined by ELISA and
presented after normalization to cell numbers. Each time point represents the mean ± SEM (n = 3 biological replicates). (B) qRT-PCR analysis of expression levels of the
Tgfb1,Tgfb2, and Tgfb3mRNAs in RAW264.7 cells challenged with PM
2.5
for different time intervals as indicated. Fold changes of mRNA levels were shown by comparing to
the vehicle (PBS)-treated controls. Each bar represents the mean ± SEM (n = 3 biological replicates). (C) Immunoblotting analysis of total and phosphorylated SMAD3 in LX-2
cells cultured in the conditioned medium from RAW264.7 cells exposed to PM
2.5
for 12 or 24 h. The graph beside the images showed fold changes of phosphorylated SMAD3
levels after normalization to total SMAD3 levels. Each bar denotes the mean ± SEM (n = 3 biological replicates). (D) qRT-PCR analysis of expression levels of the Collagen 1
a
1
(Col1
a
1) and Collagen 4
a
4(Col4
a
4) mRNA in LX-2 cells cultured in the conditioned medium from RAW264.7 cells exposed to PM
2.5
for 12 or 24 h. LX-2 cells were incubated
with the conditioned medium for 36 h before subjected to the real-time RT-PCR analysis. Fold changes of mRNA are shown by comparing to the vehicle control. Each bar
denotes the mean ± SEM (n = 3 biological replicates). (E) Immunoblotting analysis of Col1 protein levels in LX-2 cells cultured in the conditioned medium from RAW264.7
cells exposed to PM
2.5
for 12 or 24 h. The graph beside the images showed fold changes of collagen protein levels after normalization to those of Tubulin. Each bar denotes
the mean ± SEM (n = 3 biological replicates). From A–E,
*
p<0.05;
**
p<0.01. (F) Immunofluorescent analysis of Col1 production in the LX-2 cells cultured in the conditioned
medium from RAW264.7 cells exposed to PM
2.5
for 10 h. LX-2 cells were incubated with the conditioned medium for 36 h before subjected to the immunofluorecent
analysis. LX-2 cells were cultured in the conditioned medium from RAW264.7 cells exposed to PBS (vehicle) as the control. The nucleus was stained with DAPI. The
experiments were repeated three times, and representative images were shown. (This figure appears in colour on the web.)
Research Article
1400 Journal of Hepatology 2015 vol. 63 j1397–1404

cultured in the conditioned media from PM
2.5
-exposed RAW264.7
cells (Fig. 3C, D). Interestingly, when the concentration of pioglita-
zone was increased to 2.5
l
M, the pioglitazone pre-treatment was
effective in increasing expression of PPAR
c
2, but not PPAR
c
1,in
LX-2 cells. Immunoblotting analysis confirmed that pioglitazone
treatment at the concentration of 1
l
M can rescue the downreg-
ulation of PPAR
c
protein levels by PM
2.5
in LX-2 cells (Fig. 3E).
These results imply the potential role of the PPAR
c
antagonist
pioglitazone in preventing PM
2.5
-induced hepatic fibrogenesis
by attenuating PM
2.5
-induced PPAR
c
downregulation.
PM
2.5
-induced hepatic fibrosis relies on NADPH oxidase
We explored potential involvements of cell stress sensors in
PM
2.5
-induced hepatic fibrosis. It is known that NADPH oxidase
(NOX) generates large amounts of superoxide anions in phago-
cytes and plays a key role in immune defense [31]. Kupffer cells
highly express NOX and generate high amounts of reactive oxy-
gen species (ROS) in response to early liver injuries [32]. Upon
cellular stress, neutrophil cytosolic factor 1 (NCF1 or p47phox),
a key regulatory subunit of NOX, is phosphorylated and translo-
cate to the cell membrane to form the active NOX. To assess
the involvement of NADPH oxidase in experimental liver fibrosis,
we exposed p47phox
/
and wild-type control mice to PM
2.5
or
FA for 10 weeks. Upon PM
2.5
exposure, p47phox
/
mice exhib-
ited significantly attenuated liver fibrosis, compared to the
wild-type counterparts (Fig. 4A, B), suggesting that NOX plays a
major role in mediating the action of PM
2.5
exposure in hepatic
fibrogenesis. Consistent with the hepatic fibrosis staining result,
expression levels of collagen 1 mRNA in the liver tissues of the
p47phox
/
mice were dramatically decreased, compared to that
of the control mice, under PM
2.5
exposure (Fig. 4C).
Next, we examined production of ROS, an important trigger of
hepatic fibrosis [33], in the liver tissues of p47phox
/
and control
mice after PM
2.5
exposure. While inhalation exposure to PM
2.5
significantly induced ROS in the liver tissues of the wild-type
control mice, PM
2.5
-triggered ROS production was significantly
reduced in the p47phox
/
mice (Fig. 4D, E). Additionally, we
examined hepatic steatosis in the p47phox
/
and wild-type con-
trol mice after PM
2.5
exposure (Fig. 4F). Consistent with our early
finding, PM
2.5
exposure lead to hepatic lipid accumulation in the
wild-type mice. However, hepatic steatosis was relieved in the
PM
2.5
-exposed p47phox
/
mice, compared to that of wild-type
mice under PM
2.5
exposure (Fig. 4F), suggesting a critical role of
NOX in mediating PM
2.5
-induced hepatic steatosis. Furthermore,
we examined expression of PPAR
c
and TGFb1 in the livers of
p47phox
/
and control mice under PM
2.5
exposure. While
PM
2.5
exposure repressed expression of PPAR
c
mRNA in the
wild-type mice, expression of PPAR
c
in the p47phox
/
mouse liv-
ers was not suppressed by PM
2.5
exposure (Fig. 4G). Indeed, the
levels of PPAR
c
mRNA were even increased in the livers of
PM
2.5
-exposed p47phox
/
mice, compared to the PM
2.5
-exposed
wild-type mice or FA-exposed p47phox
/
mice. Moreover, PM
2.5
exposure was not able to increase expression of Tgfb1mRNA in
the p47phox
/
liver (Supplementary Fig. 7). Together, these
results suggested that NOX plays an important role in mediating
the action of PM
2.5
exposure in suppressing PPAR
c
and stimulat-
ing TGFb1 expression in the liver.
Discussion
In this study, we demonstrate that inhalation exposure to PM
2.5
represents a significant risk factor to hepatic fibrosis. Previously,
β-actin
PPARγ
Ctl 12 h 24 h
0
20
40
60
80
100
120
A
**
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Veh
E
**
PPARγ
Tubulin
Vehicle
B
*
**
0
20
40
60
80
100
120
140
160
PPARγ levels in LX2 cells
(fold change %)
Veh
PPARγ protein in LX2
cells (fold change %)
PPARγ1mRNA in LX2 cells
(fold change)
0
1
2
3
4
5
6
7
8DMSO
1 μM Pio
2.5 μM Pio
**
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0 DMSO
1 μM Pio
2.5 μM Pio
*
*
PPARγ1 mRNA levels in
LX2 cells (fold change)
PPAR
γ2 mRNA levels in
LX2 cells (fold change)
PBS PBS
12 h
24 h
PM 2.5
PM 2.5 PM 2.5
PM 2.5
PM 2.5
PM 2.5
PM 2.5
n.s.
n.s.
Veh
12 h
24 h
n.s.
n.s.
n.s. n.s.
DMSO
1 μM Pio
2.5 μM Pio
DMSO
1 μM Pio
2.5 μM Pio
CD
DMSO
1 μM Pio
2.5 μM Pio
Fig. 3. PM
2.5
exposure downregulates expression of PPAR
c
in HSC. (A)
Quantitative real-time PCR analysis of expression levels of PPAR
c
1mRNA in LX-
2 cells cultured in the conditioned medium from RAW264.7 cells exposed to PM
2.5
for 12 or 24 h. Fold changes of mRNA are shown by comparing to the vehicle
control. Each bar denotes the mean ± SEM (n = 3 biological replicates). (B)
Immunoblotting analysis of PPAR
c
levels in LX-2 cells cultured in the conditioned
media from RAW264.7 cells exposed to PM
2.5
for 12 or 24 h. LX-2 cells were
incubated with the conditioned medium for 36 h before subjected to immunoblot-
ting analysis. LX-2 cells were cultured in the conditioned medium from RAW264.7
cells exposed to PBS for 24 h as the control. The graph beside the images shows
fold changes of PPAR
c
protein levels in LX-2 cells. The fold changes of PPAR
c
in
PM
2.5
-exposed LX-2 cells were determined by normalizing to the PPAR
c
signal
intensities in PM
2.5
-exposed LX-2 cells to that of vehicle-exposed LX-2 cells. (C-E)
Pre-treatment of pioglitazone (Pio) prevents PM
2.5
-induced downregulation of
PPAR
c
in HSC. LX-2 cells were cultured in the presence of Pio at the concentration
of 1 or 2.5
l
M for 48 h before they were incubated with the conditioned medium
from RAW264.7 cell exposed to PM
2.5
(50
l
g/ml) for 14 h. LX-2 were incubated
with the conditioned medium for 36 h in the presence of Pio, and then subjected to
extraction of RNAs and proteins for qRT-PCR analyses of PPAR
c
1and PPAR
c
2mRNA
levels (C and D) and Western blot analysis of PPAR
c
protein levels (E). LX-2 cells
treated with vehicles (DMSO, the vehicle for Pio treatment; and PBS, the vehicle for
PM
2.5
). The fold changes of PPAR
c
mRNA levels in PM
2.5
- and/or Pio-treated cells
were determined by normalizing to PPAR
c
levels in vehicle-treated cells. Each bar
denotes the mean ± SEM (n = 3 biological replicates). The graph beside the images
(E) showed fold changes of PPAR
c
levels in LX-2 cells. The fold changes of PPAR
c
in
the Pio-treated LX2 cells were determined by comparing the normalized PPAR
c
signal intensities in the Pio-treated LX2 cells to that in the vehicle-treated LX2
cells (100%).
*
p60.05,
**
p60.01.
JOURNAL OF HEPATOLOGY
Journal of Hepatology 2015 vol. 63 j1397–1404 1401

we revealed important roles of ER stress, oxidative stress, and
inflammation in liver pathogenesis under PM
2.5
exposure [1,10].
Our works demonstrated that animals under PM
2.5
exposure dis-
played a NASH-like phenotype, reduction of hepatic glycogen
storage, and hepatic insulin resistance under normal chow diet
[1]. The current work extends our prior observations on the link
between air pollution exposure and liver pathogenesis. While
PM
2.5
exposure alone can cause mild but discernable hepatic
fibrosis, PM
2.5
can interact with another health risk factor, the
high-fat diet, to facilitate advanced stages of hepatic fibrosis.
Hepatic inflammation is a major contributing factor to the
development of liver fibrosis. Our study showed that inhalation
exposure to PM
2.5
induces TGFbproduction in mouse liver, pre-
sumably from Kupffer cells, which subsequently stimulates
SMAD3 signaling and collagen expression in HSCs (Figs. 1 and
2). As suggested by our previous studies, systemic inflammation
through circulating inflammatory cytokines, macrophage or neu-
trophil infiltration, and direct stimulation of Kupffer cells by
PM
2.5
particles delivered to the liver can all potentially promote
hepatic inflammation [1,10]. Therefore, systemic inflammation
and infiltrated leukocytes, in addition to Kupffer cells, may also
be involved in facilitating hepatic fibrosis through the
TGFb-SMAD3-collagen regulatory axis. Another important event
linking to hepatic fibrosis is the downregulation of PPAR
c
by
PM
2.5
exposure in HSC. In the liver, PPAR
c
plays important roles
in anti-inflammatory response and energy homeostasis
associated with HSC, Kupffer cells, and hepatocytes [34].In
particular, the differentiation of HSCs from Ito cells is inversely
correlated with PPAR
c
levels [24]. Therefore, the downregulation
of PPAR
c
in HSC could be important to the progression of hepatic
fibrogenesis in PM
2.5
-exposed mice (Fig. 3). Additionally, hepatic
steatosis developed under PM
2.5
exposure, partially due to
repression of PPAR
a
and PPAR
c
[1], may also contribute to the
progression of liver fibrosis. Based on these scenarios, delineating
which inflammatory pathways, either systemic or local, are
relatively important in promoting hepatic fibrogenesis, and
defining the extent to which hepatic inflammation or steatosis
contributes to liver fibrosis under PM
2.5
exposure are interesting
subjects to be investigated in the future.
Our study demonstrated that PM
2.5
-induced hepatic steatosis
relies on NOX activity. We previously reported a critical role of
NOX in mediating oxidative stress, as p47phox
/
animals
appeared to be protective from PM
2.5
-mediated insulin resistance
and inflammation [20,35]. Studies from other groups showed that
NOX is critically involved in hepatic fibrosis induced by angioten-
sin II-, bile duct ligation-, or methionine-choline-deficient diet
[36,37]. Our work with PM
2.5
exposure provides new evidence
that NOX is essential for hepatic fibrosis under real-world envi-
ronmental stress. NOX generates large amounts of ROS in phago-
cytic cells, and ROS is known to play an important role in hepatic
fibrosis [31,32]. Under PM
2.5
exposure, p47phox
/
mice dis-
played significantly reduced ROS production and hepatic fibrosis
FA
A
B
D
CG
WT
FA
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0
0.4
0.8
1.2
1.6
2.0
Col1 mRNA levels
(fold changes)
PPARγ1 mRNA levels
(fold changes)
*
FA FA FA FA
FA
E
F
PM 2.5 PM 2.5 PM 2.5 PM 2.5
PM 2.5
PM 2.5 PM 2.5
*
*
p47phox -/- WT
p47phox -/- WT
p47phox -/-
WT p47phox-/- WT
p47phox-/- WT
p47phox-/- WT
p47phox-/-
FA FA PM 2.5 PM 2.5 FA FA PM 2.5 PM 2.5
0.0
0.5
1.0
1.5
2.0
0
50
100
150
DHE fluorescence
(arbitrary unit)
Fibrosis score
arbitrary units
Fig. 4. Hepatic fibrosis under PM
2.5
exposure relies on NOX activity. (A) Histological analysis of hepatic collagen deposition (Sirius-red staining) in formalin-fixed liver
tissue sections from p47phox
/
and wild-type control mice exposed to FA or PM
2.5
for 10 weeks. Magnifications: 200. The arrows point out areas of hepatic fibrosis. (B)
Hepatic fibrosis grades of the p47phox
/
and control mice exposed to PM
2.5
or FA. Hepatic fibrosis grades were determined based on the histological analyses of Sirius-red
staining of collagens, according to the Scheuer scoring system for fibrosis and cirrhosis [16,17]. Data are shown as mean ± SEM (n = 4 animals per group). (C) qRT-PCR
analysis of expression levels of Col1 mRNA in p47phox
/
and control mice exposed to FA or PM
2.5
for 10 weeks. Fold changes of mRNA are shown by normalizing mRNA
levels to that of the FA-exposed control animals. Each bar denotes the mean ± SEM (n = 3 animals per group). (D) DHE staining of ROS signals in the liver tissue sections from
the mice exposed to FA or PM
2.5
. The oxidative red fluorescence was detected by a Zeiss fluorescence microscope. Magnification : 400. (E) Quantification of DHE-stained
ROS signals in the PM
2.5
- and FA-exposed p47phox
/
and control mice. DHE signals were quantified by counting the number of positive stained nuclei in 8 random fields.
Microscopic interference contrast was used to exclude positive signals from non-cell origin. The percentages of DHE-positive nuclei (compared to total nuclei) were shown.
Data are shown as mean ± SEM (n = 4 animals per group).
*
p<0.05. (F) Oil-red O staining of hepatic lipid droplets in the livers of p47phox
/
and wild-type control mice
exposed to FA or PM
2.5
for 10 weeks (magnifications: 200). (G) qRT-PCR analysis of expression levels of PPAR
c
mRNA in the liver tissues of p47phox
/
and control mice
exposed to FA or PM
2.5
for 10 weeks. Fold changes of mRNA are shown by normalizing mRNA levels to that of the FA-exposed control animals. Each bar denotes the
mean ± SEM (n = 4 animals per group). For B, C, E, and G,
*
p<0.05. (This figure appears in colour on the web.)
Research Article
1402 Journal of Hepatology 2015 vol. 63 j1397–1404

(Fig. 4). Importantly, PM
2.5
-induced downregulation of PPAR
c
and upregulation of TGFb1 were attenuated in the livers of
p47phox
/
mice (Fig. 4G; Supplementary Fig. 7), suggesting that
NOX plays a major role in mediating PM
2.5
-triggered fibrogenesis
through modulating PPAR
c
and TGFbsignaling.
The significance of our study needs to be placed in the context
of real-world PM
2.5
exposure. The PM
2.5
concentration in the ani-
mal exposure chambers was approximately 10 times that of daily
ambient PM
2.5
concentration in most US and European cities. In
the developing countries, such as China, India, and Latin Ameri-
can, where the daily PM
2.5
levels range from 100 to 200
l
g/m
3
(approximately 10–20-fold higher than those in major U.S. cities)
[38], the detrimental effects of PM
2.5
exposure on public health
have been grossly underestimated. Even in the U.S. or European
countries, the prevalence of metabolic syndrome was increased
with increasing PM
2.5
concentrations, as evidenced by a 1%
increase in diabetes prevalence seen with a 10
l
g/m
3
increase
in PM
2.5
exposure [39]. Therefore, the PM
2.5
levels at high ranges,
as obtained through our experimental expose system, not only
recapitulate true air pollution environment in the developing
countries but also can be translated into cumulative exposures
in the U.S. or European countries. Our studies suggest that the
liver is an important target organ and a key player in pathophys-
iology under PM
2.5
exposure. This finding has important implica-
tions in the medical care from the prevention to treatment of
systemic diseases associated with air pollution.
Conflict of interest
The authors who have taken part in this study declared that they
do not have anything to disclose regarding funding or conflict of
interest with respect to this manuscript.
Authors’ contributions
Study concept and design: K.Z., Z.Z., Q.S.; acquisition of data,
analysis and interpretation of data: Z.Z., K.Z., Q.S., X.Z., J.W.,
A.D., H.K., Y.Q., A.W.; drafting of the manuscript: K.Z., Z.Z.; critical
revision of the manuscript for important intellectual content:
K.Z., Z.Z., Q.S.; statistical analysis: Z.Z., X.Z., J.W., A.D., X.X., Q.S.;
obtained funding: Z.Z., Q.S.; administrative, technical, or material
support: X.X., Y.C., A.W., L.C.C., S.R.; study supervision: K.Z., Q.S.
Acknowledgement
Portions of this work were supported by National Institutes of
Health (NIH) grants DK090313 and ES017829 to KZ, American
Heart Association Grants 0635423Z and 09GRNT2280479 to KZ,
NIH grant ES018900 to QS, and NIH grants R01ES019616,
R01ES017290, and R01ES015146 to SR. We thank Dr. Scott Fried-
man for kindly providing LX-2 cells and Dr. Todd Leff for provid-
ing pioglitazone.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jhep.2015.07.
020.
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