Atherogenic diet-induced hepatitis is partially dependent on
Moreshwar S. Desai,*,†M. Michele Mariscalco,*,†Ahmad Tawil,†J. G. Vallejo,‡
and C. W. Smith†,1
*Pediatric Critical Care Medicine,†Section of Leukocyte Biology, Children’s Nutrition Research Center, and
‡Infectious Diseases and Winters Center For Heart Failure Research, Department of Pediatrics, Baylor College of
Medicine, Houston, Texas, USA
such as the Paigen diet have been used to study
atherogenesis, lithogenesis, and proinflammatory
microvascular changes induced by nutritional hy-
percholesterolemia. Although these diets lead to
chronic hepatic inflammation and fibrosis, the
early inflammatory changes have been poorly char-
acterized. TLR4, a known receptor for LPS, is also
a receptor for a variety of endogenous ligands and
has been implicated in atheroma formation. Here,
we specifically examined the early inflammatory
response of the liver to the atherogenic (ATH) diet
and the possible contribution of TLR4. Animals fed
the high-cholesterol/cholate diet for 3 weeks devel-
oped a significant, predominantly mononuclear
leukocyte infiltration in the liver, hepatic steatosis,
elevated hepatic expression of MCP-1, RANTES,
and MIP-2, and increased serum levels of liver
enzymes. In TLR4-deleted animals, there was a
30% attenuation in the serum alanine transami-
nase levels and a 50% reduction in the leukocyte
infiltration with a fourfold reduction in chemokine
expression. In contrast, hepatic steatosis did not
differ from wild-type controls. TLR2 deletion had
no effect on diet-induced hepatitis but increased
the amount of steatosis. We conclude that the early
inflammatory liver injury but not hepatic lipid load-
ing induced by the ATH diet in mice is mediated in
part by TLR4. J. Leukoc. Biol. 83: 1336–1344;
Diets high in cholesterol and cholate
Key Words: cholesterol/cholate diet ? inflammation ? TLR2 ? che-
A diet high in cholesterol (1.25%), fat (15%), and cholate
(0.5%) described by Paigen and co-workers  has been used
to induce proinflammatory changes in the microvasculature ,
increase production of reactive oxygen species , elevate
expression of adhesion molecules in the endothelial cells ,
enhance adherence and emigration of granulocytes , en-
hance T cell-mediated release of the proinflammatory cytokine
IFN-? , and increase the platelet–leukocyte interaction .
In addition, this diet has been shown to induce nutritional
hypercholesterolemia, atherogenesis , and cholesterol gall-
stone disease in inbred mouse strains [9, 10]. Three weeks
diet-fed mice demonstrate increased plasma cholesterol, low-
density lipoprotein (LDL)/very LDL cholesterol, nonesterified
cholesterol, and reduced high-density lipoprotein cholesterol
compared with chow-fed animals .
In rats, this diet results in lipid-laden hepatic parenchymal
cells and Kupffer cells , mild hepatic fibrosis , and
oxidative hepatocellular injury [14, 15]. At the molecular level,
this diet at 5–15 weeks has been shown to activate hepatic
NF-?B and induce mRNA for the mouse homologue of MCP-1
(CCL2), colony-stimulating factors, heme oxygenase, and mem-
bers of the serum amyloid A family in the liver. A correlation
was also reported between hepatic inflammatory gene induction
and susceptibility to fatty-streak development . However,
there has been little characterization of the early stages of
hepatic inflammation. The liver plays a critical role in the
metabolism of cholesterol [17, 18] and bile acids , which
influence atherogenesis [20, 21] and lithogenesis .
TLR4, a transmembrane protein, is known to activate proin-
flammatory pathways in response to LPS, leading to production
of inflammatory cytokines in various tissues including the liver
. It is also clear that endogenous ligands exist for TLR4. It
is thought that these endogenous substances are responsible for
instituting the host innate immune response during noninfec-
tious stress events [24, 25]. It has also been shown that dietary
hypercholesterolemia sensitizes the liver to endotoxemia, and
it has been postulated that overexpression of hepatic TLR4
may contribute to the observed phenomenon . As such,
TLR4 has been implicated in the process of atheroma gener-
ation [27–30]. Recently, there has also been literature support-
ing a role for TLR2 in atherogenesis [31, 32]. As liver-medi-
ated processes along with hypercholesterolemia are critical for
atheroma generation, we assessed the possible contributions of
TLR4 and TLR2 to the early liver injury associated with
1Correspondence: Section of Leukocyte Biology, Department of Pediatrics,
Baylor College of Medicine, 1100 Bates Ave., Suite 6014, Houston, TX
77030-2600, USA. E-mail: firstname.lastname@example.org
Received June 11, 2007; revised January 17, 2008; accepted February 5,
1336 Journal of Leukocyte Biology
Volume 83, June 2008
0741-5400/08/0083-1336 © Society for Leukocyte Biology
MATERIALS AND METHODS
Male mice, 8–10 weeks old, 25–28 g, were used for all of the experiments.
C57BL/6J (strain #664) mice were used to establish the model. For evaluating
the role of TLR4, C57BL/10SNJ-TLR4-deleted (TLR4del) mice were raised in
our animal facility from homozygous mating (strain #3752). C57BL/10SnJ
(strain #666) were the controls for the TLR4delstrain (Jackson Laboratories,
Bar Harbor, ME, USA). For the TLR2 experiments, heterozygous, TLR2-
deficient mice were backcrossed six generations on a C57BL/6NHsd (Harlan
Sprague Dawley Inc., Indianapolis, IN, USA) background. After genotyping
using the appropriate primers, 8- to 10-week-old male, TLR2delmice were
used with age- and gender-matched C57BL/6NHsd controls (Harlan Sprague
Dawley Inc.). Mice were housed in a room with a 12-h light cycle and had free
access to food and water. The Institutional Animal Care and Use Committee at
Baylor College of Medicine (Houston, TX, USA) approved all protocols.
Mice were fed an atherogenic (ATH) “Paigen” diet [1.25% (w/w) cholesterol,
0.5% (w/w) cholic acid (CA), and 16% (w/w) fats in the form of soybean oil,
cocoa butter, and coconut oil] or isocaloric (ISO) control chow (0.3% w/w
cholesterol, no CA, and 5% w/w fats) for 3 weeks for each experiment. Both
diets were obtained from Research Diets Inc. (New Brunswick, NJ, USA). They
were irradiated and stored per recommendations from the manufacturer. Feeds
were weighed and changed twice a week, and animals were weighed each week
to monitor weight gain. Rodent chow supplemented with 0.5% CA was spe-
cially formulated and ordered separately from Harlan (Harlan Teklad, Madi-
son, WI, USA) for separate sets of experiments to evaluate effects of dietary
Four sets of experiments were performed. 1) To study the effects of diet on liver
inflammation, C57BL/6J mice were assigned to an ATH diet or ISO chow
group. After 3 weeks of diet, animals were anesthetized, blood was collected
from vena cava, and the animals were killed. Liver was obtained for further
analysis. Animals were analyzed in two groups: ISO (n?4) and ATH (n?5). 2)
To study the effects of cholate supplementation on liver inflammation,
C57BL/6J mice were assigned to cholate supplemented or standard rodent
chow. After 3 weeks of diet, animals were anesthetized, blood was collected
from vena cava, and the animals were killed. Liver was obtained for further
analysis. Animals were analyzed in two groups: chow (n?4) and CA-supple-
mented chow (n?5). 3) To assess the role of TLR4, TLR4del(C57BL/10SNJ)
mice and their wild-type (WT) controls (C57BL/10SnJ) were randomly as-
signed to an ATH diet or ISO chow for a duration of 3 weeks. Mice were fasted
for 6 h prior to sacrifice. Results were analyzed according to their respective
groups: WT ? ISO (n?5); WT ? ATH (n?6); TLR4del? ISO (n?5); and
TLR4del? ATH (n?6). 4) For TLR2 experiments, TLR2delmice and their WT
controls (C57BL/6NHsd) were randomly assigned to ATH or ISO chow for a
duration of 3 weeks. Mice were fasted for 6 h prior to sacrifice. Results were
analyzed according to their respective groups: WT ? ISO (n?4); WT ? ATH
(n?6); TLR2del? ISO (n?4); and TLR2del? ATH (n?6).
Serum alanine transaminase (ALT) activity and
Nonhemolyzed serum from blood samples collected from the vena cava was
used for determination of ALT activity and serum cholesterol levels using a
kinetic spectrophotometric assay (Thermo Electron, Louisville, CO, USA).
Portal venous endotoxin measurement
C57BL/6 mice (n?12) were separately fed an ATH diet (n?8) or ISO chow
(n?4) for 3 weeks. Blood was collected from the portal vein prior to sacrifice.
Platelet-rich plasma fraction was isolated from pooled, heparinized blood
samples, which were prepared as described previously , and endotoxin was
detected using a kinetic chromogenic assay using the kinetic QCL kit (Cam-
brex, Walkersville, MD, USA).
Sections of liver preserved in formalin-free zinc fixative (BD PharMingen, San
Diego, CA, USA) were embedded in paraffin, sectioned, and stained with H&E.
To demonstrate hepatic lipid accumulation, additional sections of liver were
embedded in OCT and frozen at –80°C and subsequently stained with Oil
Red-O (Sigma Chemical Co., St. Louis MO, USA).
Immunohistochemistry was performed on 4 ?m-thick sections of paraffin-
embedded liver tissue. The following were used: Pan-leukocyte marker, anti-
mouse CD45 (leukocyte common antigen, Ly-5, clone 30-F11, BD PharMin-
gen) at 1:20 dilution; rat anti-mouse F4/80 for monocyte/macrophages includ-
ing Kupffer cells (Serotec, Raleigh, NC, USA) at a dilution of 1:50; and rat
anti-mouse neutrophil antibody (Serotec) at a dilution of 1:50. Biotinylated,
HRP-tagged rabbit anti-rat antibody (Vectastain ABC kit, Vector Laboratories,
Burlingame, CA, USA) was used as a secondary antibody at a dilution of 1:200.
Brown staining of target cells was obtained by using a working solution of
3,3?-diaminobenzidene substrate (Vector Laboratories). Liver was counter-
stained with Gill’s hematoxylin (Fisher Diagnostics, Middletown, VA, USA)
and visualized directly under 10? and 60? magnification under light micro-
scope (Leitz, Germany) and evaluated using a SPOT camera (Diagnostics
Instruments Inc., Sterling Heights, MI, USA). The contribution of platelets was
evaluated by staining paraffin-embedded liver sections (4 ?-thick) with anti-
mouse, CD41-PE labeled antibody (BD PharMingen; 1:100; clone MWreg30)
and mounting in Airvol (Celenase, Ltd., Dallas, TX, USA) containing 1 ?mol/L
4?,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.). Slides were
counterstained with DAPI to assess nuclear morphology. Sections were exam-
ined by immunofluorescence (Delta Vision, Applied Precision, Issaquah, WA,
Quantification of inflammatory cells
Ten high-power fields (60?) per 4 ?m-thick section of stained liver per animal
were randomly selected (http://www.randomizer.org). The level of inflammation
was assessed morphometrically as the volume percent of hepatic parenchyma
comprised of the inflammatory cells (percent volume) per high-power field
using an unbiased, stereological technique described by Howard and Reed
. To derive an unbiased estimate of the volume fraction (percent surface
area per 4 ?m-thick section) of hepatic tissue composed of the cell of interest,
a cycloid grid (ref. , C-1, Page 210, Appendix B) was randomly positioned
on the field, and the number of points of the grid hitting the nucleus of the
stained inflammatory cell divided by the number of points hitting the hepatic
parenchyma (including the sinusoids) gave an unbiased estimate of volume
fraction (volume percent). Volume fraction (percent volume) ? P(Y)/P (ref).
Where P(Y) is the number of points on the grid hitting the nuclei of interest,
and P (ref) is the total number of points hitting the hepatic parenchyma
(including the sinusoids) per high-power field. Care was taken not to count the
cells in the areas of the central vein, the portal vein, and the hepatic artery.
Hepatic lipid extraction and analysis
The Bligh-Dyer lipid extraction method was used to quantify the amount of
lipids in the liver tissue . Briefly, 0.4 g liver in 1 ml PBS was mechanically
disrupted in a sonicator, and then 0.5 ml chloroform and 1 ml methanol (1:20)
were added and vortexed vigorously for 1 min. Chloroform (0.5 ml) was added
again and vortexed for 1 min. The mixture was centrifuged at 3000 rpm 5 min
to separate the phases. The bottom (organic lipid) phase was transferred to
preweighed glass tubes and dried in a hood for 48 h. The dried lipid layer was
weighed and was reported in mg of lipids per gram of liver tissue. Methyl esters
of the total lipid fraction were prepared with boron trifluoride-methanol as
described earlier  and quantified by gas liquid chromatography (Hewlett-
Packard 5890 gas chromatograph) on a DB-225 capillary column (J & W
Scientific, Folsom, CA, USA). Fatty acids were identified by comparison with
the retention times of fatty acid methyl ester standards.
IFN-? and IL-6 levels in liver tissue lysate
Quantitative assessment of liver tissue IFN-? and IL-6 content was done using
ELISA kits specific for tissue lysate (Ray-Biotech, Inc., Norcross, GA, USA).
Liver tissue lysate was prepared per kit instructions. Protein levels in the
Desai et al.
TLR4 in liver injury with atherogenic diet1337
tissue extracts were measured using the Micro-bicinchoninic acid protein
assay kit (Pierce Chemical Co., Rockford, IL, USA). Results were reported in
picograms per gram of protein loaded.
Quantitative real-time PCR
Snap-frozen liver samples were homogenized in QIAzol lysis reagent, and total
RNA was isolated with the RNeasy Mini kit (Qiagen, Valencia, CA, USA).
Quantity and quality of the extracted RNA were verified using a Nanodrop-
1000 spectrophotometer (Nanodrop Technology, Wilmington, DE, USA). Syn-
thesis of cDNA was performed with the TaqMan reverse transcription kit
(Applied Biosystems, Foster City, CA, USA) using 2 ?g total RNA and random
hexamer primers per the manufacturer’s recommendations. For the amplifica-
tion of ICAM-1 and MCP-1, 5 ?l cDNA was added to a corresponding 20?
TaqMan MGB probe primer set for each message, multiplexed with primers for
GAPDH and 2? TaqMan Universal PCR master mix (Applied Biosystems).
PCR was performed in a 7500 real-time PCR system (Applied Biosystems)
using the manufacturer’s suggested thermal settings: one cycle of 2 min at
50°C, followed by 10 min at 95°C and 40 cycles of 15 s at 95°C and 60 s at
60°C. Relative mRNA expression was calculated as the ? comparative thresh-
old (Ct) method. GAPDH was used as internal control.
RNase protection assay
RANTES (CCL5), eotaxin (CCL11), lymphotactin (XCL1), MIP-2 (CXCL2),
IFN-?-induced protein (IP-10; CXCL10), and T cell activation gene 3 (TCA-3;
CCL1) gene expression was determined by the use of a custom-designed
multiprobe RNase protection assay system (mck-5, RiboQuant, PharMingen),
which was used according to the manufacturer’s protocol. Total RNA was
extracted from snap-frozen livers as described above, and probe synthesis,
RNA hybridization, RNase treatment, and specific probe purification, electro-
phoresis, and imaging were done as described previously . Signals were
quantified by use of software (Image QuaNT, Molecular Dynamics, Sunnyvale,
CA, USA) and were normalized to L32.
Data are presented as means ? SEM. Data were analyzed using ANOVA, and
differences between groups were identified using Neuman-Keuls post hoc,
unless specified. All statistical calculations were done using the PRISM 3.0
software program (Graphpad Prism, San Diego, CA, USA). P ? 0.05 was
selected as the level of significance.
ATH diet induces hypercholesterolemia, a
predominant mononuclear cell infiltrate, liver
injury, and hepatic steatosis
Serum cholesterol levels were increased significantly in the
C57BL6/J mice fed the ATH diet for 3 weeks compared with
animals fed ISO chow (180.5?14.85 mg/dl vs. 128?5.5 mg/dl
in C57BL6/J; P?0.05). Leukocyte infiltrates were evident in
the periportal area in the animals on the ATH diet, compared
with the animals on an ISO diet (Fig. 1, A–B). The majority of
these cells in the liver of animals on the ATH diet was F4/80?,
consistent with increases in monocyte/macrophage and Kupffer
cells. Neutrophil influx was also seen (Fig. 1E). Oil Red-O
staining revealed microvesicular steatosis with lipid deposition
in the hepatocytes (Fig. 1, C–D). Serum ALT (Fig. 1E) levels
were increased in C57BL6/J after 3 weeks on the ATH diet,
consistent with diet-mediated liver injury. These results were
reproduced in the C57BL10/SnJ mice, which were separately
used as WT controls for the TLR4delanimals (Figs. 2a and 3).
Cholate added to the chow diet alone for 3 weeks also
induced mild liver elevation of the liver enzymes (serum ALT
levels: 147?33 ?L vs. 36?3 ?L; P?0.028; unpaired t-test)
and a modest increase in leukocytes (percent volume of
CD45? cells: 1.4?0.25 vs. 0.4?0.05; P?0.024) when com-
pared with standard rodent chow-fed C57BL/6J animals. It
should be noted that this amount of injury and inflammation
alone was ?50% of that seen in animals eating the ATH diet
for 3 weeks (Fig. 1E). However, unlike those animals, animals
whose diet was supplemented with cholate alone had no evi-
dence of hepatic lipid loading [lipid levels: 17.50?2.44 mg/g
liver in cholate-fed vs. 21.65?1.144 mg/g liver in chow-fed;
P?not significant (ns)].
Fig. 1. Effect of an ATH diet on liver injury in C57BL6/J mice. Repre-
sentative photomicrographs of livers of mice fed ISO chow (A) and an ATH
diet (B) stained with H&E; (arrow) peri-portal mononuclear cell infiltrates.
(C and D) Oil Red-O staining. Red staining (D) indicates intracellular lipid
deposition after 3 weeks of an ATH diet (10? original magnification;
original scale bar?100 ?). (E; Left) Morphometric quantification of hepatic
parenchyma stained by immunohistochemistry for monocytes/macro-
phages/Kupffer cells (F4/80?) and polymorphonuclear cells (PMN) cells.
Values denote volume fraction (% Volume) of cells of interest per hepatic
parenchyma per high-power field (60? original). A total of 10 high-power
fields counted per liver per mouse. (Right) ALT levels in the serum after feeding an ATH diet or ISO chow. Results are means ? SEM (n?4?5 per group); *, P
? 0.002, versus ISO-fed groups using unpaired two-tailed t-test.
1338 Journal of Leukocyte Biology
Volume 83, June 2008
There was no evidence of portal venous endotoxin in
C57BL/6J mice, irrespective of their diet, as measured by
kinetic chromogenic assay (data not shown).
TLR4 deletion attenuates the inflammatory
response and hepatic injury in mice on an ATH
TLR4delanimals fed the ATH diet had attenuation of the serum
ALT levels by ?30% compared with their WT C57BL10/SnJ
controls (186?22 ?L vs. 247?18 ?L; P?0.05; Fig. 2a). There
were also 50% fewer CD45? cells (Fig. 2b), F4/80? cells, and
neutrophils in the ATH diet-fed TLR4delanimals compared
with their ATH diet-fed WT controls (Fig. 3). TLR4delanimals
fed an ATH diet showed a small but significant (fourfold)
increase in the F4/80? cells compared with the ISO-fed
groups. The number of CD45? cells and polymorphonuclear
cells in the TLR4delmice fed an ATH diet was also increased
(threefold) compared with the ISO chow-fed groups but did not
reach statistical significance (Figs. 2b and 3).
ATH-fed mice showed significantly more platelets in their
liver (mostly in the sinusoidal spaces) than the ISO-fed mice
(percent volume CD41? cells: 6.1%?0.9 vs. 12.9 ?0.4;
P?0.02 by t-test). There was, however, a relative paucity of
platelets in the inflammatory foci in the livers of the WT
ATH-fed animals (Fig. 4). The deletion of TLR4 did not
appear to change the amount of platelets compared with WT
animals (percent volume CD41? cells: 12.70%?0.3 vs. 12.9
There was no increase in hepatic IL-6 and IFN-? protein
expression in control or TLR4delmice, irrespective of ATH or
chow diet (data not shown).
TLR4 deletion-attenuated expression of MCP-1,
RANTES, and MIP-2
Hepatic mRNA levels for MCP-1 (CCL2), ICAM-1 (CD54),
RANTES (CCL5), MIP-2 (CXCL2), lymphotactin (XCL1),
eotaxin (CCL11), TCA-3 (CCL1), and IP-10 (CXCL10) were
increased in the livers of WT (C57BL10/SnJ) mice after feed-
Fig. 2. Effect of an ATH diet on control
and TLR4delanimals. (a) Serum ALT levels
measured in the C57BL/10SnJ (WT) and
TLR4delmice at the end of 3 weeks of
feeding an ATH diet or ISO chow. Results
are means ? SEM (n?5–6 per group); *,
P ? 0.05, versus TLR4del? ATH; #, P ?
0.01, versus ISO-fed TLR4deland WT an-
imals. (b) Representative photomicrographs
of livers of WT and TLR4delmice stained
for CD45-positive cells. (b, B) Arrow shows
CD45 ? brown-stained cells. (b, A) WT ?
ISO chow; (b, B) WT ? ATH chow; (b, C)
TLR4del? ISO chow; (b, D) TLR4del?
ATH chow. Note higher number of brown-
stained cells in b, B compared with b, D
(60? original magnification; original scale
bar?50 ?). (Right) Morphometric quantifi-
cation of hepatic parenchyma stained by
immunohistochemistry for all leukocytes
(CD45? cells). Values denote volume frac-
tion (% Vol) of cells of interest per hepatic
parenchyma per high-power field (60? original). A total of 10 high-power fields counted per liver per mouse; *, P ? 0.05, versus all groups. (c) Representative
photomicrographs of frozen sections of livers of WT (c, A and B) and TLR4delmice (c, C and D) stained with Oil Red-O. Red staining (c, B and D) indicates
intracellular lipid deposition after 3 weeks of an ATH diet (original magnification, 10?; original scale bar?100 ?). (Right) Quantification of the lipid content
of the liver in mg of lipid per gram of liver. Results are mean ? SEM (n?4–6); *, P ? 0.01, versus respective ISO-fed animals.
Desai et al.
TLR4 in liver injury with atherogenic diet1339
ing the ATH diet (Figs. 3 and 5); ribonuclease protection
assay (RPA) analysis of WT and TLR4delISO chow-fed ani-
mals revealed no detectable bands (data not shown). Livers of
TLR4delmice had significantly less mRNA for MCP-1 (Fig. 3),
RANTES, and MIP-2 (Fig. 5).
Expression of ICAM-1 (Fig. 3), lymphotactin, eotaxin, IP-10,
and TCA-3 was similar between the two diet-fed groups, irre-
spective of TLR4 deletion (data not shown).
TLR4 deletion did not affect the hepatic lipid
loading, serum cholesterol levels, or the fatty
acid profile of diet-fed mice
Serum cholesterol levels (200?10 mg/dl vs. 196?13 mg/dl;
P?ns), hepatic lipid content as assessed by Oil Red-O
staining, and lipid extraction by the Bligh-Dyer method
were similar in the WT and TLR4delgroups fed the ATH diet
As nutritional saturated fatty acids, especially lauric acid
(C12:0), has been implicated as a ligand for TLR4 signaling
[38, 39], and the Paigen diet has a high content of lauric acid
and saturated fats (information from Research Diets Inc.), we
analyzed the fatty acid methyl esters from lipid extracts in each
group (n?5–6) by gas chromatography. We found statistically
higher content of lauric acid in the livers of mice fed the ATH
diet, irrespective of the presence or absence of TLR4 (Fig. 6)
The hepatic concentrations of palmitoleic acid (C16:1), oleic
acid (C18:1), linoleic acid (C18:2,n-6), ?-linolenic acid (C18:
3,n-3), and ?-linolenic acid (C18:3,n-6) were significantly
higher in ATH diet-fed livers than their chow-fed counterparts
(Fig. 6). The hepatic contents of myristic acid (C14:0), palmitic
(C16:0), stearic (C18:0), arachidic (C20:0), behenic (C22:0),
arachidonic (C20:4), eicosapentanoic, and docasahexaenoic
acids were similar among all groups, irrespective of diet or
TLR4 presence (data not shown). Except for linoleic (C18:2,n-
6), which was found to be higher in TLR4delATH-fed mice, the
fatty acid profile of the WT and the TLR4delmice was similar
within their diet groups (Fig. 6).
TLR2 deletion did not attenuate the inflammatory
response or liver injury in ATH diet-fed animals
The TLR2delmice and their WT controls, C57BL/6NHsd (Har-
lan strain), showed evidence of liver injury (ALT), inflamma-
tion (CD45? cells), hepatic steatosis, and hypercholesterol-
emia when compared with their chow-fed counterparts (Fig.
7). There was, however, no statistical difference among the
serum cholesterol levels (243.6?14.8 mg/dl vs. 225.3?17.8
mg/dl; P?ns), ALT levels, and the volume percent of hepatic
CD45? cells between the WT and the TLR2del, diet-fed groups
(Fig. 7). On Bligh-Dyer analysis, livers of diet-fed, TLR2del
mice showed a significantly higher lipid content as compared
with the diet-fed WT mice (Fig. 7).
In this paper, we demonstrate that microvesicular steatohepa-
titis occurs within weeks of feeding Paigen’s ATH diet. There
is a five- to sevenfold increase in the percent of the hepatic
parenchyma of infiltrating neutrophils and over a tenfold in-
crease in the F4/80? cells in those animals fed a high-
cholesterol/cholate diet. In addition, we show that the leuko-
Fig. 3. Quantification of monocyte/macrophages, neutrophils, and quantita-
tive PCR for expression of MCP-1 and ICAM in livers of TLR4deland their WT
controls. Using immunohistochemistry and morphometric analysis, the volume
of the hepatic parenchyma, which stained for monocytes/macrophages/Kupffer
cells (F4/80?) and neutrophils, was quantified. Values denote volume fraction
(% vol) of cells of interest per hepatic parenchyma per high-power field (60?
original). A total of 10 high-power fields counted per liver per mouse. Results
are mean ? SEM (n?4–6). (Right) Bar graphs show quantitative real-time PCR
results from RNA extracted from snap-frozen livers, using probes for ICAM
and MCP-1. Relative mRNA expression was calculated by the ? Ct method
with GAPDH as endogenous control. Results are means ? SEM (n?5–6); *,
P ? 0.05, versus all groups; #, P ? 0.05, versus ISO-fed groups; ?, P ? 0.05,
versus WT ? ISO group.
Fig. 4. Platelet staining by immunofluo-
rescence. Representative photomicrographs
of livers of ATH diet-fed WT (C57BL/
10SnJ; A) and TLR4del(B) mice stained for
CD41-positive cells (original magnification,
20?; original scale bars?40 ?). Note the
relative paucity of CD41? red staining seen
in the inflammatory cells (C) of the WT mice
(original magnification, 40?; original scale
1340 Journal of Leukocyte Biology
Volume 83, June 2008
cyte infiltrate is primarily a result of monocyte/macrophages,
although we could not rule out that there was also an increase
in the number of Kupffer cells. There was also an increase in
the number of platelets found, primarily in the hepatic sinu-
osoids, in those animals fed the ATH diet. It is important to
highlight that in this study, we excluded cells that were in the
lumen of central or portal veins and arteries; thus, our counts
more accurately reflect cells in the hepatic architecture itself.
Mechanistically, the inflammatory changes in the liver are
primarily a result of the downstream effects of TLR4 activation,
as TLR4delanimals had an abrogation of leukocyte influx and
inhibition of inflammatory mediators including MCP-1, RAN-
Fig. 5. RPA analysis of proinflammatory chemokines. (Left) Gene expression of
lymphotactin (XCL1), RANTES (CCL5), eotaxin (CCL11), MIP-2 (CXCL2), IP-10
(CXCL10), and TCA-3 (CCL1) in the livers of WT and TLR4delATH-fed mice.
Signals were compared with large ribosomal (L32) mRNA content. (Right) Bar graphs
show relative intensities of RANTES and MIP-2 compared with L32; *, P ? 0.02,
versus TLR4delmice using Mann-Whitney rank sum analysis.
Fig. 6. Quantification of hepatic fatty ac-
ids. After 3 weeks of feeding ISO or ATH
diets in WT and TLR4del, fatty acid content
was determined as described in Materials
and Methods. Results reported as nano-
moles per gram of liver; means ? SEM
(n?5–6 per group); *, P ? 0.05, versus
#, P ? 0.05, versus
Desai et al.
TLR4 in liver injury with atherogenic diet1341
TES, and MIP-2. There is little if any effect of TLR2 deletion
on the inflammatory response. It appears that TLR4 and likely
TLR2 have little or no role in the development of hepatic
steatosis, which occurs with the ATH diet. Additionally, TLR4
deletion resulted in greater reduction in hepatic inflammation
than hepatic injury (as measured by ALT), suggesting that
other mechanisms are responsible for injury in addition to
inflammation. Finally, the accumulation of platelets in the liver
as a result of feeding the ATH diet does not appear to be a
result of TLR4-dependent mechanisms.
Several studies demonstrate the critical role of TLR4 in the
development of atheroma in animals with hypercholesterolemia
[27–29]. Apolipoprotein-E (APO-E)-deficient mice, which also
lack TLR4 or have a targeted deletion of MyD88, a downstream
effector of TLR4, have reduced atherosclerosis and altered
plaque size compared with APO-E-deficient mice with intact
TLR4 [28, 29]. In these studies, hypercholesterolemia is a
result of the APO-E defect. The current study is the first
demonstration of the role of TLR4 in the hepatic response to an
TLR4 is expressed on hepatocytes, stellate cells, Kupffer
cells, sinusoidal endothelial cells, dendritic cells, as well as
bile-duct epithelium . Thus, it is not possible to delineate
which cell/cells may have been affected by TLR4 deletion. We
verified previous observations [41, 42] that MCP-1, RANTES,
and ICAM-1 were significantly up-regulated upon feeding the
Paigen diet for 3 weeks. We have further demonstrated the
up-regulation of other proinflammatory chemokines, such as
lymphotactin, eotaxin, MIP-2, IP-10, and TCA-3 with 3 weeks
of diet, which contribute to the inflammatory cell infiltrates and
subsequent liver injury. MCP-1 and RANTES are expressed by
all the cells in the liver, especially the Kupffer cells, and are
shown to be up-regulated by inflammatory stimuli . MCP-1
and RANTES are critical to the recruitment of monocytes, T
lymphocytes, and NK cells . We did not specifically stain
for T lymphocytes in the liver. However, work by Stokes et al.
 has shown that T lymphocytes may be responsible for the
early changes that occur in the microvasculature with the
Paigen diet. We hypothesize that signaling through TLR4 on
hepatocytes/Kupffer/stellate cells results in increased MCP-1,
RANTES, and MIP-2. This in turn leads to monocyte and to a
lesser extent, neutrophilic infiltrate, resulting in inflammation
and injury, which eventually lead to chronic inflammation and
The Paigen diet is complex. Cholesterol, cholate, and the fat
components each have a unique role to play in the inflamma-
tory process . It is not possible to delineate in this exper-
iment the ligand for TLR4, which initiates the inflammatory
events. We did not detect portal venous endotoxin in the ATH
diet-fed mice. However, this does not rule out the possibility of
other bacterial translocation products, which could be ligands
for TLR4, or amount of endotoxin, which could not be detected.
It is clear that oxidized LDL is a ligand for TLR4 [27, 30], and
it has been reported that this ATH diet, when fed to mice,
induced the hepatic formation of oxidized phospholipids re-
sponsible for biological activity of mildly oxidized LDL [45,
46]. Lauric acid (C12:0), a saturated fatty acid also found in
LPS, is a known ligand for TLR4 . Lauric acid was present
in the ATH diet used here, although the relative concentration
was minimal compared with the rest of the lipid content. We
found increased hepatic content of lauric acid in the ATH
diet-fed animals, and this was not different between the WT
compared with the TLR4delmice. However, to signal through
TLR4, we believe that increased serum/hepatic levels of lauric
acid would need to be present. Cholic acid has also been shown
to increase hepatic mRNA levels of ICAM-1, VCAM-1, and
TNF-?  and contribute to liver injury, although this effect
has not been shown to be mediated through TLR4 . We
have demonstrated hepatitis but not steatosis by cholate sup-
plementation alone. The inflammatory response and injury
seen with cholate supplementation were approximately half of
that seen with the ATH diet. This suggests that the fat and
cholesterol components also account for the observed response
separately, as suggested by the study done by Vergnes et al.
, or together with the presence of cholate. This effect may
be ameliorated by the absence of TLR4.
As TLR2 has also been implicated in cholesterol, fat me-
tabolism, and atherogenesis [31, 32], we evaluated the role of
TLR2 in this model. Our results demonstrate that TLR2 dele-
tion did not afford hepatic protection, and there is increased
hepatic lipid content after 3 weeks of diet. The increase in
steatosis seen in the TLR2delmice is novel. Few studies have
been done examining the effects of TLR2 on steatosis. Re-
cently, it has been demonstrated that CD36 (fatty acid trans-
Fig. 7. Effect of an ATH diet on control
and TLR2delanimals. Analysis of serum
ALT levels, morphometric cell count of he-
patic CD45? cells, and hepatic lipid ex-
traction in diet-fed, TLR2delmice and their
WT controls at the end of 3 weeks of feeding
an ATH diet or ISO chow. Results are
means ? SEM (n?4–6 per group); *, P ?
0.05, versus ATH-fed groups;#, P ? 0.05,
versus WT ? ATH.
1342 Journal of Leukocyte Biology
Volume 83, June 2008
porter protein) acts as a facilitator or a coreceptor for diacyl-
glyceride recognition through the TLR2/6 complex . It is
this CD36–TLR2 interaction that has been proposed as a
mechanism, whereby an endogenous lipid ligand can facilitate
TLR2 signaling in atherosclerosis . CD36 also plays an
important role in lipid metabolism and regulates fatty acid flux
among the muscle, adipose tissue, and liver. Lack of CD36
causes increased fatty acid delivery to the liver and results in
steatosis [50–52]. We speculate that global deletion of TLR2
probably alters the CD36–TLR interaction, leading to in-
creased flux of free fatty acids to the liver, thereby leading to
We emphasize that there were strain differences in C57BL/
6NHsd (Harlan) used in TLR2 experiments and C57BL6 and
C57BL/10SnJ (Jackson strain) used in establishing the model
and TLR4 experiments. Although the diet was the same, the
amount of liver injury and inflammatory response was less in
the C57BL/6NHsd (Harlan) and highlights the critical role of
selection of control animals.
In conclusion, our studies show for the first time that TLR4
but not TLR2 is responsible in part for the hepatitis seen with
short-term feeding of a high-cholesterol/high-fat/CA-contain-
ing ATH diet. TLR4 deletion did not affect hepatic lipid
loading or the fatty acid profile. Although CA feeding leads to
mild liver injury, it does not affect lipid accumulation and is
insufficient to produce steatohepatitis.
Funding was from the training grant NIH-HL007939 and
USDA 6250-51000-046. We thank Dr. William C. Heird and
Vijayalaksmi Nannegari for their help in the analysis of hepatic
fatty acid content. We are thankful for Dr. Akira for theTLR2del
mice. We also thank Elizabeth Priest for the breeding and
housekeeping of our mice strains.
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