The anti-inflammatory effects of adiponectin are mediated via a heme oxygenase-1-dependent pathway
in rat Kupffer cells
Palash Mandal1, Pil-Hoon Park1, Megan R. McMullen1, Brian T. Pratt1 and Laura E. Nagy1, 2.
1Departments of Pathobiology and 2Gastroenterology, Cleveland Clinic,
Cleveland, OH 44195
Short title: Heme oxygenase-1 mediates the anti-inflammatory effect of adiponectin
Acknowledgements: This work was supported in part by NIH grant RO1AA0011975.
Address corresponds to:
Laura E Nagy
Cleveland Clinic Foundation
Lerner Research Institute/NE-40
9500 Euclid Avenue
Cleveland, OH 44195
Page 1 of 37
Abbreviations: ALD: Alcoholic liver disease; AdipoR: adiponectin receptor; BSA: bovine serum albumin;
BV: biliverdin IX; CO: carbon monoxide; CoPP: cobalt protoporphyrin; DMSO: dimethyl sulfoxide; gAcrp:
globular adiponectin; EtOH: ethanol; Fcγ: Fc-gamma receptor; fl-Acrp: full-length adiponectin; HO-1:
heme oxygenase-1; IgG: immunoglobulin G; IL-1β: interleukin 1β; IL-6: interleukin 6; IL-10: interleukin 10;
IL-10R: interleukin 10 receptor; JAK1: janus kinase 1; LPS: lipopolysaccharide; MAPK: mitogen activated
protein kinase; PBS: phosphate buffered saline; PMSF: phenylmethylsulphonyl fluoride; ROS: reactive
oxygen species; Scrb siRNA: scrambled small interfering RNA; SDS: sodium dodecyl sulfate: SEM:
standard error of the mean; SOCS-3: suppressor of cytokine signaling-3; STAT3: signal transducers and
activators of transcription protein 3; TNF-α: tumor necrosis factor α; TNFR-1: tumor necrosis factor
receptor superfamily, member 1A; ZnPP: zinc protoporphyrin.
Page 2 of 37
Altered expression and activity of immunomodulatory cytokines plays a major role in the pathogenesis of
alcoholic liver disease. Chronic ethanol feeding increases the sensitivity of Kupffer cells, the resident
hepatic macrophage, to lipopolysaccharide (LPS), leading to increased tumor necrosis factor-α (TNF-α)
expression. This sensitization is normalized by treatment of primary cultures of Kupffer cells with
adiponectin, an anti-inflammatory adipokine. Here we tested the hypothesis that adiponectin-mediated
suppression of LPS signaling in Kupffer cells is mediated via an interleukin-10 (IL-10)/heme oxygenase-1
(HO-1) pathway after chronic ethanol feeding. Knock-down of IL-10 expression in primary cultures of
Kupffer cells with siRNA prevented the inhibitory effect of globular adiponectin (gAcrp) on LPS-stimulated
TNF-α expression. gAcrp increased IL-10 mRNA and protein expression, as well as expression of the IL-
10 inducible gene, HO-1; expression was higher in Kupffer cells from ethanol-fed rats compared to pair-
fed controls. While IL-10 receptor surface expression on Kupffer cells was not affected by ethanol
feeding, IL-10-mediated phosphorylation of STAT3 and expression of HO-1 was higher in Kupffer cells
after ethanol feeding. Inhibition of HO-1 activity, either by treatment with the HO-1 inhibitor, zinc
protoporphyrin, or by siRNA knock-down of HO-1, prevented the inhibitory effect of gAcrp on LPS-
stimulated TNF-α expression in Kupffer cells. LPS-stimulated TNF-α expression in liver was increased in
mice after chronic ethanol exposure. When mice were treated with cobalt protoporphyrin to induce HO-1
expression, ethanol-induced sensitivity to LPS was ameliorated. Conclusion: gAcrp prevents LPS-
stimulated TNF-α expression in Kupffer cells via the activation of the IL-10/STAT3/HO-1 pathway. Kupffer
cells from ethanol-fed rats are highly sensitive to the anti-inflammatory effects of gAcrp; this sensitivity is
associated with both increased expression and sensitivity to IL-10.
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The innate and adaptive immune systems have been implicated in the progression of alcoholic liver
disease (ALD). Disruption in the regulation of the innate immune response is thought to be particularly
important in the early stages of ethanol-induced liver injury (1). Accumulating evidence suggests that an
imbalance between the activities of pro- and anti-inflammatory mediators contributes to ethanol-induced
liver injury. For example, ethanol consumption leads to elevated lipopolysaccharide (LPS)/endotoxin in
the portal blood, as well as a sensitization of Kupffer cells to activation, resulting in production of a
number of inflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin (IL)-6 and
reactive oxygen species (ROS). Among the pro-inflammatory mediators, TNF-α plays a critical role in the
pathogenesis of ALD (1); treatment with TNF-α neutralizing antibody reduces ethanol-induced liver injury
in animals and TNF-α receptor 1 (TNFR-1) knock-out mice are resistant to the toxic effects of ethanol
Loss of anti-inflammatory mediators may also contribute to a pro-inflammatory state in the liver and
facilitate injury. For example, IL-10 is an immunomodulatory cytokine with potent anti-inflammatory and
immunosuppressive properties. IL-10 decreases production of pro-inflammatory cytokines, including TNF-
α and IL-1β (2). While little is known about the regulation of IL-10 expression and activity in the liver in
response to chronic ethanol, impaired expression of IL-10 contributes to inflammation in alcoholic
cirrhotics (3) and IL-10 deficient mice are more sensitive to ethanol-induced liver injury (4). Disruption in
the expression and activity of adiponectin, an abundant 30-kDa adipokine with potent anti-inflammatory
properties (5), may also contribute to a pro-inflammatory imbalance during chronic ethanol exposure.
Adiponectin suppresses macrophage activity via a number of mechanisms. For example, adiponectin
inhibits the proliferation of myelomonocytic progenitor cells, dampens the upregulation of endothelial
adhesion molecules in response to inflammatory signals, suppresses phagocytic activity, as well as
reduces LPS-stimulated cytokine production in macrophages (6-8). Chronic ethanol exposure decreases
adiponectin concentrations in rats and mice (9, 10); treatment of mice with adiponectin during chronic
ethanol exposure prevents the development of liver injury, decreasing both steatosis and TNF-α
expression in the liver (10). While the mechanisms for these therapeutic effects of adiponectin are not
Page 4 of 37
well understood, the decrease in steatosis is most likely related to the critical role of adiponectin in
regulation of glucose and lipid homeostasis. Further, we have previously reported that adiponectin
treatment normalizes LPS-induced TNF-α production in primary cultures of Kupffer cells after chronic
ethanol exposure (9) suggesting that adiponectin therapy may directly suppress the pro-inflammatory
activity of Kupffer cells after chronic ethanol feeding.
Recent data suggest an important link between adiponectin and IL-10, two critical anti-inflammatory
mediators which may contribute to ethanol-induced liver injury. For example, adiponectin induces the
expression of IL-10 mRNA and protein in cultured macrophages (11, 12). Expression of IL-10 is required
for the anti-inflammatory effects of adiponectin in RAW 264.7 macrophages since immunoneutralization
of IL-10 prevents gAcrp-mediated desensitization to LPS (11). IL-10 mediates its anti-inflammatory
functions via induction of IL-10-inducible genes, including heme oxygenase-1 (HO-1) and suppressor of
cytokine signaling 3 (SOCS3)(2). Induction of these genes involves the activation of STAT3 signaling
pathways (2). Adiponectin and HO-1 pathways also interact. For example, increased adiponectin
expression is associated with increased expression of HO-1 and enhanced cardiac protection in diabetic
rats (13). Further, induction of HO-1 increases adiponectin expression in Zucker rats, leading to
decreased TNF-α expression and reduced adipogenesis (14).
HO-1 has anti-apoptotic, anti-inflammatory and anti-proliferative properties (15). There is a growing
appreciation that HO-1, in particular, is an important down-stream mediator of the anti-inflammatory
effects of IL-10 in macrophages (15). HO-1, and its down-stream mediator carbon monoxide (CO), both
inhibit LPS-induced expression of pro-inflammatory cytokines and increase LPS-induced expression of IL-
10 in macrophages (15). Induction of HO-1 prevents ethanol-induced oxidative damage in cultured
hepatocytes (16) and also decreases complement-mediated injury in the endothelium (17, 18).
Since a failure in the ability to induce adequate anti-inflammatory responses likely contributes to chronic
inflammation during long-term ethanol exposure, here we tested the hypothesis that there is a beneficial
interplay between gAcrp, IL-10 and HO-1 in the regulation of LPS-induced TNF-α expression by Kupffer
Page 5 of 37
cells after chronic ethanol exposure. We found that induction of both IL-10 and HO-1 expression are
required for the anti-inflammatory effects of gAcrp in Kupffer cells. Importantly, the increased sensitivity
of Kupffer cells from ethanol-fed rats to gAcrp was associated with increased expression of IL-10, as well
as enhanced IL-10 receptor signaling, leading to the greater expression of HO-1. When HO-1 expression
was increased in mice by treatment with cobalt protoporphyrin, chronic ethanol-induced sensitization of
LPS-stimulated TNF-α expression in liver was normalized. These data suggest that therapeutic strategies
to enhance IL-10 and/or HO-1 expression or signaling may be effective strategies for dampening the
sensitivity of Kupffer cells to stimulation after chronic ethanol.
Page 6 of 37
Materials and Methods
Adult male Wistar rats weighing 140–150 g were purchased from Harlan Sprague Dawley (Indianapolis,
IN). Lieber-DeCarli ethanol diet (regular, no.710260) was purchased from Dyets (Bethlehem, PA). Cell
culture reagents were from Invitrogen (Grand Island, NY). Recombinant human gAcrp expressed in E. coli
and full-length adiponectin expressed in HEK293 cells were purchased from Peprotech, Inc. (Rocky Hill,
NJ) and BioVendor Lab Medicine (Candler, NC), respectively. Additional materials are described in
Chronic ethanol feeding and Kupffer cell isolation
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at
the Cleveland Clinic. Chronic ethanol-feeding to rats and mice, as well as the isolation and culture of
Kupffer cells, were performed as previously described (9, 19, 20) (see Supplemental Material for further
details). Isolated Kupffer cells were then either plated immediately or used for nucleofection prior to
plating. One to four hours after plating, nonadherent cells were removed by aspiration and fresh media
with or without 1 µg/ml gAcrp added. After 18h in culture, cells were treated with or without 100 ng/ml
LPS or 10 ng/ml IL-10, as indicated in the figure legends. In some experiments, inhibitors were added to
the Kupffer cell culture media 30 min prior to the IL-10 treatment. The dose and time of exposure of
Kupffer cells to gAcrp and LPS were based on previous studies (9, 19).
Nucleofection in rat Kupffer cells
Freshly isolated Kupffer cells were transfected using the Amaxa mouse macrophage Nucleofector kit
according to the instructions of the manufacturer using the Y-001 program (Lonza, Cologne, Germany),
except for the following modifications. Samples were processed individually and the entire nucleofection
procedure for each sample was completed in less than 5 min. For each nucleofection sample, 2 x 106
Kupffer cells were centrifuged for 10 min at 300 x g. The pellet was washed with 1 ml PBS, collected at
300 x g for 5 min and then resuspended in 105 µl nucleofector solution and transferred to a 1.5 mL
eppendorf tubes for a final concentration of ~2 x 106 cells/ 100 µl. Cells were then treated or not with 2.0
Page 7 of 37
µg specific or scrambled siRNA (siRNA sequences are provided in Supplemental Materials), transferred
into the electroporation cuvette and placed in the Nucleofector device. After nucleofection, cells were
immediately removed from the cuvette and plated in a 96-well plate (150 µl/well) at 0.5 x 106 cells/well.
After 4 h, the cell culture medium was replaced with fresh medium with or without 1 µg/ml gAcrp or 10
ng/ml IL-10 for 18 h and then treated with or without LPS or IL-10, as described in the figure legends.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated, reverse transcribed and qRT-PCR amplification was performed as previously
described (9). The relative amount of target mRNA was determined using the comparative threshold (Ct)
method by normalizing target mRNA Ct values to those of 18S. Details of the procedure and primer
sequences are provided in Supplemental Material.
The quantity of secreted IL-10 protein was measured in the media from Kupffer cells after treatment with
gAcrp for 18 h using a rat IL-10 ELISA kit (Biosource, Camarillo, CA).
Western blot analysis
Western blot analysis was performed using enhanced chemiluminescence for signal detection. Signal
intensities were quantified by densitometry using Image J software (NIH).
Flow cytometry Analysis
After 18h culture with or without gAcrp, Kupffer cells were gently scraped and adjusted to 1 million cells
per ml with culture media. Cells were greater than 90% viable as determined by Trypan blue exclusion.
Expression of IL-10 receptor A subunit was then measured by flow cytometry, as described in the figure
legend. Data were acquired and processed using FlowJo software (Becton Dickinson).
Page 8 of 37
Because of the limited number of Kupffer cells available from each animal, data from several feeding
trials are presented in this study. Values are means ± standard error of the mean (SEM). Data were
analyzed by general linear models procedure (SAS; Carey, IN). Data were log transformed, if needed, to
obtain a normal distribution. Follow-up comparisons were made by least square means testing.
Page 9 of 37
Chronic ethanol feeding increases the sensitivity of Kupffer cells to LPS-stimulated TNF-α expression;
LPS increased TNF-α mRNA accumulation was 2.7-fold higher in Kupffer cells from ethanol-fed rats
compared to pair-fed rats (Figure 1)(9). Treatment of primary cultures of rat Kupffer cells with gAcrp for
18 h suppressed LPS-stimulated responses in Kupffer cells isolated from both pair-fed and ethanol-fed
rats (Figure 1)(9). In other cellular model systems, adiponectin exerts its anti-inflammatory actions
through induction of IL-10 (11). Therefore, we tested if knock-down of IL-10 expression with siRNA
ameliorated the ability of gAcrp to suppress LPS-stimulated TNF-α expression in Kupffer cells.
Transfection of Kupffer cells with siRNA against IL-10 effectively suppressed IL-10 mRNA accumulation
(Supplementary Figure 1A) and prevented the suppression of LPS-stimulated TNF-α mRNA accumulation
by gAcrp (Figure 1). Scrambled siRNA had no effect on IL-10 mRNA (Supplementary Figure 1A) or the
response to gAcrp (Figure 1).
Primary cultures of Kupffer cells from ethanol-fed rats are more sensitive than cells from pair-fed rats to
the anti-inflammatory actions of both gAcrp and full-length adiponectin to suppress LPS-dependent
responses (9). Since IL-10 is required for gAcrp to suppress LPS-stimulated TNF-α mRNA accumulation
in Kupffer cells, the more potent effects of adiponectin after ethanol feeding may be due to increased
gAcrp-stimulated expression of IL-10 and/or increased sensitivity of the Kupffer cells to stimulation by IL-
10. To test these hypotheses, isolated Kupffer cells were treated with increasing concentrations of gAcrp
for 18h and IL-10 protein secreted in the media was measured by ELISA. Accumulation of IL-10 protein
was higher in Kupffer cells from ethanol-fed rats compared to cells from pair-fed controls (Figure 2A).
Similarly, IL-10 mRNA expression was also higher in Kupffer cells from ethanol-fed rats compared to cells
from pair-fed rats when incubated with gAcrp (Figure 2B) or full-length adiponectin (Figure 2C). These
data suggested that increased gAcrp-stimulated IL-10 expression may contribute, at least in part, to the
higher sensitivity of Kupffer cells from ethanol-fed rats to gAcrp. siRNA knock-down of adiponectin
receptors (AdipoR) revealed that the effects of gAcrp on IL-10 mRNA were dependent on the expression
of AdipoR1, but not AdipoR2 (Figure 2D).
Page 10 of 37
IL-10 mediates its anti-inflammatory effects via interactions with IL-10 receptors and activation of specific
signaling pathways; STAT3 activation is required for IL-10-mediated signaling (2). Surface expression of
the IL-10 receptor subunit A, the ligand binding subunit of the IL-10 receptor, on Kupffer cells was not
affected by either ethanol feeding or gAcrp treatment (Figure 3). Stimulation with IL-10 increased the
phosphorylation of JAK1 within 30 min in Kupffer cells from ethanol-fed, but not pair-fed, rats (Figure 4).
Phosphorylation of STAT3 in response to IL-10 was both more rapid (within 10 min) and more robust in
Kupffer cells from ethanol-fed rats compared to pair-fed rats (Figure 4). IL-10 had no effect on
phosphorylation of JAK2, p38 or ERK1/2 mitogen-activated protein kinases in Kupffer cells under these
conditions (data not shown).
IL-10R activation of the STAT3 pathway increases expression of STAT3 responsive genes, such SOCS3
and HO-1 (2). Culture of Kupffer cells with gAcrp increased the expression of SOCS3 and HO-1 mRNA
(Figure 5A/B). Consistent with the increased gAcrp-stimulated IL-10 expression and phosphorylation of
STAT3 after chronic ethanol feeding, gAcrp treatment increased HO-1 and SOCS3 mRNA expression to
a greater extent in Kupffer cells from ethanol-fed compared to pair-fed rats (Figure 5A/B). gAcrp
increased HO-1 protein expression in Kupffer cells from ethanol-fed rats (Figure 5C), but not in Kupffer
cells from pair-fed rats. Despite the increase in SOCS3 mRNA, SOCS3 protein was not significantly
increased in response to gAcrp in Kupffer cells from either ethanol- or pair-fed rats (Figure 5C).
Because HO-1 is a critical mediator of the anti-inflammatory effects of IL-10 (15), we further investigated
the mechanisms by which gAcrp increased HO-1 expression in Kupffer cells. To test whether gAcrp
induces HO-1 expression through an IL-10 dependent pathway, Kupffer cells were transfected with siRNA
against IL-10 in order to knock-down IL-10 expression. When IL-10 expression was inhibited, gAcrp-
stimulated HO-1 mRNA expression was suppressed in Kupffer cells from both pair- and ethanol fed rats
(Figure 6A). Scrambled siRNA administration had no effect on gAcrp-stimulated HO-1 mRNA expression
(Figure 6A). The signaling pathways down-stream of gAcrp-stimulated IL-10 expression were
investigated with the use of selective inhibitors. gAcrp stimulated HO-1 mRNA expression was
attenuated when Kupffer cells were pre-treated with a selective inhibitor of STAT3 (JSI-124) (Figure 6B).
Page 11 of 37
Finally, IL-10-stimulated HO-1 mRNA expression was suppressed in Kupffer cells transfected with siRNA
against STAT3; scrambled siRNA had no effect on IL-10-dependent HO-1 expression (Figure 6C). siRNA
knock-down of STAT3 decreased STAT3 protein expression (Supplementary Figure 1C). Taken together,
these data demonstrate that gAcrp induces HO-1 expression via an IL-10/STAT3-dependent pathway.
Since HO-1 has potent anti-oxidant and anti-inflammatory activity, we investigated the role of HO-1 in
mediating the effect of gAcrp using both biochemical and siRNA knock-down strategies. First, when
Kupffer cells were treated with zinc protoporphyrin (ZnPP), a competitive inhibitor of HO-1 activity, prior to
culture with gAcrp, the inhibitory effect of gAcrp on LPS-stimulated TNF-α expression was ameliorated
(Figure 7A). Similar results were obtained using a siRNA strategy. When Kupffer cells were transfected
with siRNA against HO-1, expression of HO-1 protein was decreased compared to Kupffer cells
transfected with scrambled siRNA (Supplementary Figure 1B). Knock-down of HO-1 with siRNA
prevented the inhibitory effect of gAcrp on LPS-stimulated TNF-α mRNA, whereas scrambled siRNA had
no effect (Figure 7B).
Previous studies have shown that treatment of mice with supra-physiological concentrations of
adiponectin during chronic ethanol exposure protects from ethanol-induced steatosis and inflammation
(10). Based on our novel findings in Kupffer cells that HO-1 is a down-stream mediator of the anti-
inflammatory effects of adiponectin, we designed an in vivo experiment to ascertain whether induction of
HO-1 would normalize LPS-stimulated TNF-α expression in liver after chronic ethanol exposure. HO-1
mRNA and protein expression in mouse liver were not affected by chronic ethanol feeding (Figure 8A);
however, treatment with cobalt protoporphyrin (CoPP) increased HO-1 expression in liver of both ethanol-
and pair-fed mice (Figure 8A). After chronic ethanol feeding, LPS-stimulated TNF-α mRNA expression
was increased 2-fold compared to pair-fed controls (Figure 8B). However, when mice were pre-treated
with CoPP to induce HO-1 expression, LPS-stimulated TNF-α expression was reduced and did not differ
between ethanol- and pair-fed mice (Figure 8B).
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Increased expression of TNF-α contributes to ethanol-induced liver injury (1). Treatment of mice with
adiponectin, a potent adipokine with anti-inflammatory properties, prevents ethanol-induced steatosis and
TNF-α expression (10). Kupffer cells isolated from rats exposed to chronic ethanol exhibit increased
sensitivity to LPS-stimulated TNF-α expression and are used as a model system to understand the
interaction between ethanol and LPS-mediated responses in macrophages (21). Interestingly, the anti-
inflammatory actions of adiponectin are enhanced in Kupffer cells isolated from rats chronically exposed
to ethanol, compared to pair-fed controls (9). Despite the efficacy of adiponectin in decreasing LPS-
mediated responses, both in mouse models (10) and primary cultures of Kupffer cells (9), the
development of adiponectin for therapeutic interventions in patients with alcoholic liver disease is likely of
limited utility, due to the high concentration of adiponectin in the circulation, as well as the complex
oligomeric structure of adiponectin. Therefore, here we made use of primary cultures of Kupffer cells to
investigate the molecular mechanisms for the anti-inflammatory effects of adiponectin after chronic
ethanol exposure. Understanding the mechanisms of adiponectin action, particularly in ethanol-treated
macrophages, could illuminate molecular targets of adiponectin action that are more amenable to
pharmacological intervention. Here we have identified an IL-10/STAT3/HO-1 dependent pathway that
mediates the anti-inflammatory effects of adiponectin in Kupffer cells. The activity of this pathway is
enhanced in Kupffer cells from ethanol-fed rats due to both an increased gAcrp-mediated expression of
IL-10, as well as a greater IL-10 stimulated phosphorylation of STAT3 and expression of HO-1.
Importantly, induction of HO-1 was also effective at normalizing LPS-stimulated TNF-α expression in an in
vivo model of chronic ethanol exposure.
Adiponectin has potent anti-inflammatory properties, both in vivo and in cultured macrophages.
Initially, treatment of macrophages with adiponectin increases the expression of inflammatory cytokines,
such as TNF-α and IL-6 (11, 22). However, upon continued exposure to gAcrp, the expression of anti-
inflammatory mediators, such as IL-10 and IL-1 receptor antagonist, is increased (11, 12). Increased
expression of IL-10 is critical for the anti-inflammatory effects of adiponectin in macrophages;
Page 13 of 37
immunoneutralization of IL-10 prevents the suppression of LPS-stimulated TNF-α production by 1 µg/ml
gAcrp in RAW 264.7 macrophages (11). However, in one recent report from the Libby group, IL-10 was
not critical in mediating the anti-inflammatory effects of 10 µg/ml full-length adiponectin in human
macrophages (23). Here we report that knock-down of IL-10 in primary cultures of Kupffer cells
prevented gAcrp-mediated suppression of LPS-stimulated TNF-α mRNA accumulation, demonstrating
that IL-10 is necessary and sufficient to mediate the anti-inflammatory effects of gAcrp in primary cultures
of Kupffer cells. We also demonstrated that the induction of IL-10 by gAcrp in Kupffer cells was
dependent on AdipoR1, but not AdipoR2, expression. The contribution of AdipoR1, which has a higher
affinity for globular adiponectin compared to full-length adiponectin (24), may explain the differences
between our results indicating an essential role of IL-10 and that of the Libby group (23), using higher
concentrations of full-length adiponectin, that reported the induction of multiple anti-inflammatory
Kupffer cells isolated from ethanol-fed rats are more sensitive to the long-term anti-inflammatory effects of
either gAcrp or full-length adiponectin, exhibiting decreased LPS-stimulated NFκB and MAPK activation,
as well as decreased TNF-α expression relative to Kupffer cells from pair-fed controls (9). Because IL-10
is essential to the anti-inflammatory role of gAcrp in Kupffer cells, we hypothesized that ethanol feeding
increased the sensitivity to gAcrp via increased IL-10 expression and/or increased sensitivity to IL-10
mediated responses. Our data demonstrate that chronic ethanol feeding increased the sensitivity of
Kupffer cells to gAcrp-stimulated IL-10 expression; expression of both IL-10 mRNA as well as the quantity
of secreted IL-10 protein is increased in Kupffer cells from ethanol-fed rats compared to cells from control
rats. Kupffer cells from ethanol-fed rats also exhibited enhanced IL-10-dependent signaling (Figure 4),
independent of any effect of chronic ethanol on the cell surface expression of IL-10RA, the ligand binding
subunit of the IL-10 receptor complex (Figure 3). Chronic ethanol accelerated and enhanced IL-10-
stimulated phosphorylation of STAT3 (Figure 4) and increased expression of IL-10 dependent genes,
including HO-1 and SOCS-3 mRNA (Figure 5).
Page 14 of 37
Very little is known about the impact of acute or chronic ethanol on IL-10 expression and signaling. After
chronic ethanol exposure, plasma IL-10 concentrations are reduced in mice and IL-10 deficient mice
exhibit an even greater sensitivity to LPS after ethanol feeding compared to wild type mice (4). Short
term/acute ethanol exposure increases IL-10 expression by monocytes in human subjects, as well as in
mice in response to LPS. When human subjects consume a single dose of alcohol, the production of IL-
10 by isolated monocytes in response to LPS is increased compared to controls (25). This increase can
be prevented by inhibiting HO-1 by pre-treatment with zinc protoporphyrin (26). Taken together with the
current data, it appears that while chronic ethanol exposure decreases circulating concentrations of IL-10
(4), both short term/acute and chronic ethanol exposure contribute to an enhanced IL-10 expression in
monocytes/macrophages in response to immunoregulatory signals, such as LPS or gAcrp.
IL-10 binds to a heterodimeric IL-10R, which undergoes transphosphorylation and then activates the
Jak1/STAT3 pathway (27). Activation of STAT3 is essential for IL-10-dependent signaling (2). Chronic
ethanol feeding increased IL-10 stimulated phosphorylation of JAK1 and STAT3 in Kupffer cells.
Furthermore, inhibition of STAT3 signaling via chemical inhibitors or via siRNA knock-down ameliorated
IL-10-dependent expression of HO-1 (Figure 6). Reports in the literature suggest that the impact of
chronic ethanol on the regulation of STAT3 is complex, and is likely to have ligand- and cell-type specific
effects. Exposure of primary cultures of hepatocytes to ethanol suppresses IL-6-stimulated STAT3
activation (28). Gao and colleagues have identified cell specific roles for STAT3 in hepatocytes
compared to monocytes/ macrophages in the liver (29). Expression of STAT3 in hepatocytes had a
negative impact on liver injury and promoted inflammation, while expression of STAT3 in
monocytes/macrophages suppressed inflammation during ethanol exposure (29). The anti-inflammatory
role of STAT3 in monocytes/ macrophages during chronic ethanol exposure is consistent with our
identification of a critical contribution of STAT3 in Kupffer cells in mediating the anti-inflammatory effects
Accumulating evidence suggests that HO-1 plays an important anti-inflammatory role in chronic
inflammatory diseases and protects cells from oxidative insult (15). Heme oxygenase catalyzes the initial
Page 15 of 37
and rate limiting step in oxidative degradation of heme, yielding equimolar amounts of biliverdin IXα,
carbon monoxide, and free iron (30). There are three isoforms of HO: HO-2 and HO-3 are constitutive
forms, while HO-1 (also known as heat shock protein 32) is an inducible isozyme, with high expression
levels in spleen and Kupffer cells (31). HO-1 is a stress-responsive protein whose expression is
upregulated by a broad spectrum of inducers, including heme, heavy metals, nephrotoxins, cytokines,
endotoxins and oxidative stress. Interestingly, HO-1 expression was not increased by chronic ethanol
exposure alone in either isolated Kupffer cells (Figure 5) or mouse liver (Figure 8). However, literature
suggests that HO-1 expression in response to ethanol may be dependent on the age of the animals
studied (32, 33). In Kupffer cells, pharmacological inhibition of HO-1 or siRNA knock-down of HO-1
expression completely ameliorated the ability of gAcrp to inhibit LPS-stimulated TNF-α expression.
Pharmacological induction of HO-1 in mice reduced LPS-stimulated TNF-α expression in the livers of
ethanol-fed mice to that of pair-fed controls. Taken together, these data demonstrated a critical role for
HO-1 in dampening the pro-inflammatory response to LPS both in Kupffer cells and in vivo.
In summary, these data provide strong evidence for an essential role of IL-10/STAT3/HO-1 in mediating
the anti-inflammatory function of gAcrp, demonstrating that gAcrp-dependent responses utilize two critical
anti-inflammatory pathways. Importantly, after chronic ethanol exposure, Kupffer cells exhibit an
increased sensitivity to the anti-inflammatory effects of both gAcrp and IL-10 and induction of HO-1 in vivo
protects mice from the sensitizing effects of ethanol on LPS-stimulated TNF-α expression. The
identification of HO-1 as a down-stream effector of gAcrp provides an exciting path for the design and
development of novel therapeutic approaches for the resolution of chronic inflammation associated with
alcoholic liver disease.
Page 16 of 37
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13. L'Abbate A, Neglia D, Vecoli C, Novelli M, Ottaviano V, Baldi S, Barsacchi R, et al. Beneficial
effect of heme oxygenase-1 expression on myocardial ischemia-reperfusion involves an increase in
adiponectin in mildly diabetic rats. Am J Physiol Heart Circ Physiol 2007;293:H3532-3541.
14. Kim DH, Burgess AP, Li M, Tsenovoy PL, Addabbo F, McClung JA, Puri N, et al. Heme
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stem cells. J Pharmacol Exp Ther 2008;325:833-840.
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Figure 1: IL-10 siRNA prevents globular adiponectin (gAcrp)-induced suppression of TNF-α mRNA
expression in LPS-stimulated Kupffer cells. Kupffer cells isolated from pair- and ethanol-fed rats were
transfected or not with 2.0 µg of IL-10 siRNA or scrambled siRNA and then cultured with or without 1
µg/ml gAcrp for 18 h. Kupffer cells were then stimulated with 100 ng/ml of LPS for 60 min and TNF-α and
18S mRNA measured by qRT-PCR. In control experiments, siRNA knock-down of IL-10 decreased
gAcrp-induced IL-10 mRNA expression after treatment with gAcrp for 5 h (see Supplementary Figure 1A).
Knock-down efficiency did not differ between Kupffer cells from pair- and ethanol-fed rats. Values
represent means ± SEM, n=4, *p<0.05 ethanol-fed compared to pair-fed, +p<0.05 compared to cells not
treated with gAcrp.
Figure 2: Chronic ethanol feeding increases the sensitivity of Kupffer cells to adiponectin-
stimulated IL-10 protein and mRNA expression. A) Kupffer cells isolated from pair- and ethanol-fed
rats were cultured with 0-1000 ng/ml gAcrp for 18 h and IL-10 peptide secreted into the media measured
by ELISA. Values represent means ± SEM, n=7, *p<0.05 ethanol-fed compared to pair-fed, +p<0.05
compared to cells not treated with gAcrp. B/C) Kupffer cells isolated from pair- and ethanol-fed rats were
cultured overnight and then treated with 1 µg/ml gAcrp (B) or 1 µg/ml full-length adiponectin (C) for 0-5h
and the quantity of IL-10, β-actin and 18S mRNA measured by qRT-PCR. Expression of IL-10 mRNA
was normalized to β-actin or 18S and then expressed relative to expression in Kupffer cells from pair-fed
rats not treated with gAcrp. Values represent means ± SEM, n=6-8 in B and 4 in C, *p<0.05 ethanol-fed
compared to pair-fed, +p<0.05 compared to cells not treated with adiponectin. D) Kupffer cells were
transfected or not with 2.0 µg of adiponectin R1 (AdipoR1), adiponectin R2 (AdipoR2) siRNA or
scrambled siRNA and then cultured for 18 h. Kupffer cells were then stimulated or not with 1 µg/ml gAcrp
for 5 h and IL-10 and 18S mRNA measured by qRT-PCR. siRNA knock-down decreased expression of
AdipoR1 and AdipoR2 mRNA equally in Kupffer cells from pair- and ethanol-fed rats (see Supplementary
Figure 1D/E). Values represent means ± SEM, n=4, *p<0.05 ethanol-fed compared to pair-fed, +p<0.05
compared to cells not treated with gAcrp.
Page 21 of 37
Figure 3: IL-10 receptor A (IL-10RA) expression in Kupffer cells after chronic ethanol feeding.
Kupffer cells isolated from pair- and ethanol-fed rats were treated with or without 1 µg/ml gAcrp for 18 h.
and cell surface expression of IL-10RA (solid line), relative to isotype controls (dotted line), was measured
by flow cytometry. Kupffer cells were harvested by gentle scraping and the cells collected by
centrifugation at 300 x g for 10 minutes. The pellet was washed with PBS and resuspended in 100 µL
PBS+ 0.1% sodium azide and then blocked with 1.0 µg anti-mouse CD32/CD16 Fcγ Block antibodies for
15 min at 4ºC. Then the cells were stained with ~0.5 µg fluorochrome conjugated IL-10 receptor A (PE-
conjugated IL-10 RA) or isotype control (PE-conjugated IgG1) diluted in PBS containing 0.1% sodium
azide for 30 min. Cells were then washed twice with PBS and resuspended in 0.5 ml wash buffer (final
concentration ~106 cells in 0.5 ml) and held on ice until flow cytometric measurements were performed on
a FACScan flow cytometer (Becton Dickinson Immunocytometry systems, Mountain View, CA). The
percentage of IL-10RA positive cells (50 ± 3 for pair-fed and 53 ± 3 for ethanol-fed) and the median
fluorescence intensity (20 ± 8 for pair-fed and 26 ± 13) did not differ between Kupffer cells from pair- and
ethanol-fed rats. Values represent means ± SEM, n=4. Traces shown are representative of four
Figure 4: Chronic ethanol feeding increases IL-10-stimulated phosphorylation of JAK1 and STAT3
in rat Kupffer cells. Kupffer cells isolated from pair-and ethanol-fed rats were cultured overnight and
then stimulated with 10 ng/ml IL-10 for 0-30 min. Kupffer cells were then washed twice in ice-cold PBS
and lysed at 4° C in 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS containing 1 mM
sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1 mM
PMSF, 10 µg/ml aprotinin and protease inhibitor (Complete-EDTA freeTM). After 30 min, lysates were
centrifuged at 16,000 X g for 15 min at 4° C. 20 µg of cellular extract was separated on 10% SDS-PAGE
gels and then transferred to PVDF membranes for detection. Membranes were blocked in BSA and
probed with antibodies against phospho-JAK1 and phospho-STAT-3. Membranes were then probed with
antibodies to total JAK1 (data not shown), STAT3 and heat shock cognate protein 70 (hsc70), as a
Page 22 of 37
loading control. A) Representative images. B-C) Values represent means ± SEM, n=8-9, *p<0.05
ethanol-fed compared to pair-fed, +p<0.05 compared to cells (time 0) not treated with IL-10.
Figure 5: Chronic ethanol feeding increases gAcrp-mediated induction of HO-1 and SOCS-3 mRNA
and HO-1 protein expression in rat Kupffer cells. Kupffer cells isolated from pair- and ethanol-fed rats
were cultured with 1µg/ml gAcrp for 18h and quantity of A) heme-oxygenase-1 (HO-1) or B) suppressor
of cytokine signaling-3 (SOCS-3) and 18S mRNA measured by qRT-PCR. HO-1 and SOCS-3 mRNA
were normalized to 18S mRNA and values are expressed relative to Kupffer cells from pair-fed rats not
treated with gAcrp. Values represent means ± SEM, n=4, values with different superscripts are
significantly different from each other, p<0.05. C) Kupffer cells isolated from pair-and ethanol-fed rats
were cultured with 1 µg/ml gAcrp for 18 h and HO-1 and SOCS-3 protein measured by Western blot.
Images are representative of three independent experiments. gAcrp increased HO-1 protein by 1.66 ±
0.13 in pair-fed and 5.28 ± 1.73 (p<0.05 for ethanol-fed only, n=4).
Figure 6: Adiponectin induces HO-1 mRNA expression through an IL-10- and STAT3-dependent
pathway. A) Kupffer cells isolated from pair- and ethanol-fed rats were transfected or not with 2.0 µg of
IL-10 siRNA or scrambled siRNA and then cultured with or without 1 µg/ml gAcrp for 18 h. HO-1 and 18S
mRNA were measured by qRT-PCR. HO-1 mRNA was normalized to 18S mRNA and values are
expressed relative to Kupffer cells from pair-fed rats not treated with gAcrp. Values represent means ±
SEM, n=3, +p<0.05 compared to gAcrp-treated cells not transfected with siRNA or transfected with
scrambled siRNA. B) Kupffer cells isolated from pair- and ethanol-fed rats were pretreated with 10 µM
JSI-124, an inhibitor of STAT3 signaling, or vehicle (DMSO) for 30 min and then cultured with or without
10 ng/ml IL-10 for 18h. HO-1 and 18S mRNA were measured by qRT-PCR. HO-1 mRNA was normalized
to 18S mRNA and values are expressed relative to Kupffer cells from pair-fed rats not treated with IL-10.
Values represent means ± SEM, n=3, +p<0.05 compared to IL-10-treated cells not treated with inhibitor.
C) Kupffer cells isolated from pair- and ethanol-fed rats were transfected or not with 2.0 µg of STAT3
siRNA or scrambled siRNA and then cultured with or without 10 ng/ml IL-10 for 18 h. HO-1 and 18S
mRNA were measured by qRT-PCR. HO-1 mRNA was normalized to 18S mRNA and values are
Page 23 of 37
expressed relative to Kupffer cells from pair-fed rats not treated with gAcrp. Values represent means ±
SEM, n=3, +p<0.05 compared to gAcrp-treated cells not transfected with siRNA or transfected with
Figure 7: HO-1 mediates the inhibitory effects of gAcrp on LPS-stimulated TNF-α expression.
A) Kupffer cells isolated from pair- and ethanol-fed rats were cultured with or without 0.5 µM zinc
protoporphyrin (ZnPP) in the presence or absence of 1µg/ml gAcrp for 18h. Kupffer cells were then
stimulated with 100 ng/ml LPS for 1h and TNF-α and 18S mRNA measured by qRT-PCR. Values
represent means ± SEM, n=4, +p<0.05 compared to control cells not treated with gAcrp. B) Kupffer cells
isolated from pair- and ethanol-fed rats were transfected or not with 2.0 µg of HO-1 siRNA or scrambled
siRNA and then cultured with or without 1 µg/ml gAcrp for 18 h. Kupffer cells were then stimulated with
100 ng/ml of LPS. TNF-α and 18S mRNA were measured by qRT-PCR. Values represent means ± SEM,
n=5, +p<0.05 compared to cells within a treatment group not cultured with gAcrp.
Figure 8. Induction of HO-1 decreases LPS-stimulated TNF-α expression in vivo after chronic
ethanol feeding to mice. A/B Mice were allowed free access to increasing concentrations of ethanol as
part of a complete liquid diet to a maximum concentration of 32% of kcal over 25 days or pair-fed control
diets (see Supplemental materials for detailed ethanol exposure protocol). Mice were then injected or not
with 5 mg/kg cobalt protoporphyrin (CoPP) or vehicle (saline). After 24 h, mice were injected with 0.7
µg/kg LPS or saline. Expression of HO-1 (A) and TNF-α mRNA (B) was measured by qRT-PCR after 60
min and normalized to 18S mRNA. HO-1 protein was measured by Western blot (representative images
are shown in the inset to panel A). Values represent means ± SEM, n=4-7 (A) values with difference
superscripts are signficantly different from each other (B) *p<0.05 compared to pair-fed, +p<0.05
compared to LPS treated without treatment.
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Antibody and reagent sources
Antibodies were from the following sources: STAT3 (Santa Cruz Biotechnology; Santa Cruz, CA),
phospho-STAT3, phospho-JAK1, total JAK1 and SOCS-3 (Cell Signaling Technology, Inc., Danvers, MA),
HO-1 (Assay Designs, Ann Arbor, MI), Hsc70 (Alpha Diagnostic; San Antonio, TX). Anti-rabbit and anti-
mouse IgG-peroxidase was purchased from Boehringer Mannheim (Indianapolis, IN). LPS from
Escherichia coli serotype 026:B6 (tissue culture-tested, L-2654) was purchased from Sigma; all
experiments were carried out with a single lot of LPS (Lot number 064K4077). gAcrp preparations
contained less than 0.2 ng of LPS/µg of protein. Endotoxin contamination was routinely monitored in the
laboratory using a kinetic chromogenic test based on the Limulus amebocyte lysate assay (Kinetic-QCL,
BioWhittaker, and Walkersville, MD). Recombinant rat IL-10 was purchased from R & D Systems, Inc.
(Minneapolis, MN). Mouse bone marrow nucleofection kit was purchased from Lonza (Cologne,
Germany). PE-conjugated IL-10 RA antibody and mouse IgG1 monoclonal antibodies were purchased
from Abcam (Cambridge, MA). CD16/32 antibody was purchased from eBioscience (San Diego, CA). Zinc
protoporphyrin and Co (III) protoporphyrin IX chloride were purchased from Frontier Scientific, Inc (Logan,
Utah). Rat IL-10 ELISA kit was purchased from Biosource (Camarillo, CA).
Ethanol feeding and Kupffer cell isolation to rats
Rats were allowed free access to the Lieber-DeCarli high-fat complete liquid diet for 2 days. Rats were
then randomly assigned to pair-fed or ethanol-fed groups. Ethanol-fed rats were allowed free access to a
liquid diet containing 17% of the calories from ethanol for 2 days and then a liquid diet containing 35% of
the calories from ethanol for 4 weeks(1). Control rats were pair-fed a liquid diet in which maltose dextrins
were substituted isocalorically for ethanol over the entire feeding period. Kupffer cells were isolated and
cultured as previously described (1, 2). Briefly, isolated Kupffer cells were suspended in CMRL media
with 10% FBS, L-glutamine and antibiotic-antimycotic (CMRL-FBS) at a concentration of 2 x 106 cells/ml.
Cell suspensions were either nucleofected prior to plating or immediately plated onto 96-well (0.2 x 106
cells/well for analysis of mRNA or IL-10 by ELISA) or 24-well (0.75 x 106 cells/well for flow cytometry and
1.0 x 106 Western blot analysis) culture plates.
Page 33 of 37
Ethanol feeding to mice
Female C57BL/6 mice were randomly assigned to ethanol- or pair-fed groups. Ethanol-fed mice were
allowed free access to a complete Lieber-DeCarli high fat diet with increasing concentrations of ethanol
(5.5% of calories for 2 days, 11% of calories for 2 days, 22% of calories for 7 days, 27% of calories for 7
days and finally, 32% of calories for 7 days)(3). Control mice were pair-fed diets that isocalorically
substituted maltose dextrins for ethanol. At the end of the feeding protocol, mice were treated with 5
mg/kg CoPP or vehicle (saline) via intraperitoneal injection. Twenty-four hours later, mice were injected
intraperitoneally with 0.7 µg LPS/g body weight or an equivalent volume of sterile, endotoxin-free saline
(0.09%). Mice were then anesthetized, livers blanched with saline via the portal vein and excised.
Portions of each liver were then stored in RNAlater (Qiagen, Valencia, CA) and stored at -20oC until
further analysis. Characteristics of the mice are shown in Supplemental Table 1.
siRNA nucleofection in rat Kupffer cells
Following nucleofection, gene expression was analyzed at different times, as indicated in the figure
legends. siRNA were silencer select pre-designed sequences that were validated by Ambion/Applied
Biosystems. Rat HO-1 siRNA sequence: sense 5’-GGA AAA UCC CAG AUC AGC Att-3’ and antisense
5’-UGC UGA UCU GGG AUU UUC Ctc-3’ Rat IL-10 siRNA sequence: sense 5’-GCC UUG UCA GAA
AUG AUC Att-3’ and antisense 5-UGA UCA UUU CUG ACA AGG Ctt-3’. Rat STAT-3 siRNA sequence:
sense 5’ GGC UAA GUU UUG CAA AGA Att-3’ and antisense: 5’ UUC UUU GCA AAA CUU AGC Cca-3’.
Rat AdipoR1 sequences: sense: 5’-UAAUCAGUAGAGCAAUCCCtg-3’ and antisense: 5’-UUUUCUGAG
CCUU AUAUCUgg-3’. Rat AdipoR2 sequences: sense: 5’-AGAUAUAAGGCUCAGAAAAtt-3’ and
antisense: 5’- UUUUCUGAGCCUUAUAUCUgg-3’. Nonspecific siRNA scrambled duplex (sense: 5’-
GCG CGC UUU GUA GGA UUC G-3’, antisense: 5’-CGA AUC CUA CAA AGC GCG C-3’) were
purchased from Ambion. Efficiency of knock-down was determined by Western blot analysis and qRT-
PCR, as indicated in the figure legends.
RNA isolation and quantitative real-time PCR
Page 34 of 37
RNA was isolated from Kupffer cells using the RNeasy Micro Kit (Qiagen), with on-column DNA digestion
using the RNase-free DNase set (Qiagen) according to the manufacturer’s instructions. RNA from mouse
liver was isolated using RNeasy Mini-kit (Qiagen) . Total RNA (200–300 ng) was reverse-transcribed using
the RETROscript kit (Ambion, Austin, TX) with random decamers as primers. Real-time PCR amplification
was performed in a Mx3000p (Stratagene, La Jolla, CA) using SYBR Green PCR Core Reagents (Applied
Biosystems; Warrington, UK). The primer sequences for rat Kupffer cells were as follows:18S, forward 5’-
ACG GAA GGG CAC CAC CAG GA-3’ and reverse 5’-CAC CAC CAC CC A CGG AAT CG-3’; TNF-α,
forward 5’-CAA GGA GGA GAA GTT CCC AA-3’ and reverse 5’- CTC TGC TTG GTG GTT TGC TA -3’;
β-actin, forward 5’ CGG TCA GGT CAT CAC TAT CG-3’ and reverse 5’ TTC CAT ACC CAG GAA GGA
AG-3’; HO-1, forward 5’-AGA GTT TCC GCC TCC AAC CA-3’ and reverse 5‘-CGG GAC TGG GCT AGT
TCA GG -3’; and IL-10, forward 5’-ACG CTG TCA TCG ATT TCT CC-3’ and reverse 5’-CGG GTG GTT
CAA TTT TTC AT-3’. Primer sequences for mouse liver were as follows: 18S, forward 5’-ACG GAA GGG
CAC CAC CAG GA-3’ and reverse 5’-CAC CAC CAC CC A CGG AAT CG-3’; TNF-α ,forward 5’ –
CCCTCACACTCAGATCATCTTCT and reverse 5’- GCTACGACG TGGGCTACAG ; HO-1, forward 5’-
AAGCCGAGAATGCTGAGTTCA and reverse 5’- CGGGTGTAGATATGGTACAAGGA. All primers used
for real-time PCR analysis were synthesized by Integrated DNA Technologies (Coralville, IA) Statistical
analysis of real-time PCR data was performed using ∆Ct values.
Supplemental Table 1: Characteristics of ethanol- and pair-fed mice (n=4-7 per group).
Pair-fed EtOH-fed Pair-fed EtOH-fed Pair-fed EtOH-fed
CoPP treatment none none saline saline CoPP CoPP
LPS treatment saline saline LPS LPS LPS LPS
Initial body weight (g)
17.5 ± 0.5 17.5 ± 0.5 16.93 ± 0.5 17.0 ± 0.3 17.4 ± 0.1 17.4 ± 0.1
Final body weight (g)
21.5 ± 0.8 20.3 ± 0.3 20.3 ± 0.7 18.2 ± 0.5* 20.8 ± 0.5 18.6 ± 0.2*
0.050 ± 0.003 0.053 ± 0.002* 0.046 ± 0.002 0.052 ± 0.001* 0.050 ± 0.002 0.054 ± 0.001*
*p<0.05 compared to pair-fed mice in the same treatment group.
Page 35 of 37
Supplemental Figure 1 : siRNA knock-down of gene expression in primary Kupffer cells. Kupffer
cells isolated from pair- and ethanol-fed rats were transfected or not with 2.0 µg target siRNA or
scrambled siRNA. Efficiency of siRNA knock-down was determined by measuring A) IL-10 mRNA
relative to 18S mRNA (values represent means ± SEM, n=3, +p<0.05 compared to non-transfected cells
and cells transfected with scrambled siRNA) or B) HO-1 protein expression or C) STAT3 protein
expression by Western blot. Hsc70 was used as a loading control. Representative Western blots are
shown. D) Adiponectin receptor 1 (AdipoR1) and E) adiponectin receptor 2 (AdipoR2) mRNA relative to
18S mRNA (values represent means ± SEM, n=3, +p<0.05 compared to non-transfected cells and cells
transfected with scrambled siRNA).
1. Aldred A, Nagy LE. Ethanol dissociates hormone-stimulated cAMP production from inhibition of
TNF-alpha production in rat Kupffer cells. Am J Physiol 1999;276:G98-G106.
2. Kishore R, Hill JR, McMullen MR, Frenkel J, Nagy LE. ERK1/2 and Egr-1 contribute to increased
TNF-alpha production in rat Kupffer cells after chronic ethanol feeding. Am J Physiol Gastrointest Liver
3. Roychowdhury S, McMullen MR, Pritchard MT, Hise AG, van Rooijen N, Medof ME, Stavitsky AB,
et al. An early complement-dependent and TLR-4-independent phase in the pathogenesis of ethanol-
induced liver injury in mice. Hepatology 2009;49:1326-1334.
Page 36 of 37
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