Omega-3 fatty acids alleviate chemically induced acute hepatitis by suppression

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Omega-3 fatty acids alleviate chemically induced acute hepatitis by

suppression of cytokines

Hepatology March 2007

Christoph Schmöcker 1§, Karsten H. Weylandt 2, Lena Kahlke 1,

Jingdong Wang 1, Hartmut Lobeck 3, Gisa Tiegs 4, Berg 2, Jing

X. Kang 1 *¶

1Department of Medicine, Massachusetts General Hospital and Harvard

Medical School, Boston, MA

2Department of Hepatology and Gastroenterology, Charité University

Medicine Berlin, Campus Virchow, Berlin, Germany

3Department of Pathology, Klinikum Ernst von Bergmann, Potsdam,

Germany

4Institute of Experimental and Clinical Pharmacology and Toxicology,

University of Erlangen-Nuremberg, Erlangen, Germany

Funded by:

Boehringer Ingelheim Fonds Travel Allowance Program

American Cancer Society; Grant Number: RSG-03-140-01-CNE

NIH; Grant Number: R01CA-113605

“….The results presented here indicate that increasing the

hepatic content of n-3 PUFA could decrease inflammatory activity in

acute hepatitis…. Future studies will be necessary to analyze in

more detail the optimal fatty acid ratios and lipid mediators

involved in n-3 PUFA-associated inflammation dampening in the liver

and also to expand the data presented here into models of clinically

important chronic hepatitis.â€

Abstract

Cytokines such as tumor necrosis factor alpha (TNF-a) are key factors

in liver inflammation. Supplementation with essential omega-3

polyunsaturated fatty acids (n-3 PUFA) has been demonstrated to lower

TNF-a and IL-1 production in mononuclear cells. An inflammation-

dampening effect has been observed with increased omega-3 fatty acid

supplementation in several inflammatory diseases. In this study, we

used the transgenic fat-1 mouse, expressing a Caenorhabditis elegans

desaturase endogenously forming n-3 PUFA from n-6 PUFA, to analyze

the effect of an increased n-3 PUFA tissue status in the macrophage-

dependent acute D-galactosamine/lipopolysaccaride (D-GalN/LPS)

hepatitis model. We show less severe inflammatory liver injury in fat-

1 mice with a balanced n-6/n-3 PUFA ratio as evidenced by reduced

serum alanine aminotransferase levels and less severe histological

liver damage. This decreased inflammatory response was associated

with decreased plasma TNF-a levels and with reduced hepatic gene

expression of TNF-, IL-1, IFN- and IL-6 in fat-1 mice, leading to a

decreased rate of apoptosis in livers from fat-1 animals, as measured

by DAPI-staining.

Conclusion: The results of this study offer evidence for an

inflammation dampening effect of omega-3 polyunsaturated fatty acids

in the context of liver inflammation.

Article Text

Acute hepatitis due to viral, toxic, or autoimmune pathogenesis is

characterized by an activation of macrophages and T cells with an

increased production of cytokines that leads to parenchymal liver

damage and liver dysfunction. An increase in tumor necrosis factor

alpha (TNF-a) is one of the early events in liver inflammation. TNF-a

has been implicated in liver damage in alcoholic hepatitis and

steatohepatitis,[1] and a study examining TNF-a blockade has shown

improvement in alcoholic hepatitis.[2]

Growing evidence indicates that omega-3 polyunsaturated fatty acids

(n-3 PUFA) and their specific lipid mediators can reduce the activity

of inflammatory processes.[3] High concentrations of n-3 PUFA reduce

the lipopolysaccharide (LPS)-dependent activation of nuclear factor

kappaB (NF- in monocytes,[4] which leads to a decreased production

of TNF-a.[5] Similar effects also have been observed in human

mononuclear cells.[6] Recent studies implicated newly identified n-3

PUFA-derived lipid mediators such as resolvin E1 in these processes.

[7][8] Our recent results in the macrophage-dependent dextrane sodium

sulfate colitis model showed suppression of experimental colitis by

increased omega-3 fatty acid tissue content and formation of n-3 PUFA-

derived lipid mediators.[9] These observations suggest that omega-3

fatty acids might lower inflammation susceptibility in general.

Hence, they could also dampen the inflammatory response in liver

tissue, probably by regulating Kupffer cell activation and

suppressing cytokine production.

We therefore evaluated the role of high n-3 PUFA content in the

pathogenesis of D-galactosamine/lipopolysaccaride (D-GalN/LPS)-

induced hepatitis in transgenic fat-1 mice. These mice carry the fat-

1 gene from the roundworm Caenorhabditis elegans and are able to

convert omega-6 into omega-3 fatty acids.[10] Therefore, these mice

have an endogenously elevated n-3 PUFA tissue content as compared

with their wild-type littermates, even when maintained on a low n-3

PUFA diet. This is in contrast to feeding procedures that may bring

in confounding factors as different diets need to be fed. Use of the

transgenic fat-1 mouse model eliminates confounding factors of diet

because only one diet is needed as the genetic approach using the fat-

1 gene modifies the n-6/n-3 fatty acid ratio (converts n-6 to n-3)

endogenously.

D-GaIN/LPS hepatitis is a well-established model for macrophage-

dependent liver injury in mice.[11] D-GalN is known as a specific

hepatotoxic transcriptional inhibitor that sensitizes the liver

toward LPS treatment in subtoxic amounts[12] and leads to an acute

cytokine-dependent liver inflammation.[13] D-GaIN/LPS hepatic injury

is T cell-independent: LPS binds to Kupffer cells and activates them,

resulting in a liberation of large amounts of cytokines, such as TNF-

a, IL-1, and IL-6.[14-16] TNF-a is a critical mediator of apoptotic

liver damage in this model.[17] Interferon gamma (IFN-y), produced by

activated natural killer cells, is also a pro-inflammatory cytokine

involved in this model.[18]

The results presented here show that transgenic fat-1 mice with a

balanced n-6/n-3 PUFA ratio developed less severe D-GalN/LPS-induced

inflammatory liver damage than wild-type mice, as evidenced by

decreased production of pro-inflammatory cytokines and significantly

lower serum alanine aminotransferase as well as less severe liver

pathology.

Results and Discussion

Although liver tissue of wild-type mice showed high levels of n-6

PUFA with a very low n-3 PUFA content (ratio n-6/n-3 PUFA 64.61 ±

16.78, n= 5), the transgenic fat-1 mice with a balanced AA/EPA ratio

had significant endogenous amounts of n-3 PUFA in their liver tissue

(ratio n-6/n-3 PUFA 5.90 ± 0.21, n = 5) (Table 1). The most notable

differences between the two groups were found in a-linolenic acid

(18:3 n-3), eicosapentaenoic acid (20:5 n-3), docosapentaenoic acid

(22:5 n-3), and docosahexaenoic acid (22:6 n-3). Slightly lower

levels of arachidonic acid were seen in the liver tissue of fat-1

animals as compared with the wild-type mice; however, the difference

was not statistically significant.

Administration of LPS to D-GaIN-sensitized mice induced severe

hepatic damage as detected by increased serum ALT at 6 hours after

injection. Serum ALT activities were found to be significantly lower

in the fat -1 group than in wild-type mice (Fig. 1). All mice treated

with D-GaIN/LPS showed histopathological signs of acute hepatitis 6

hours after challenge, reflected by necrosis, apoptosis, inflammatory

cell infiltrate, and hemorrhage. Histological examinations of liver

sections showed severe confluent and focal necrosis, apoptosis, and

focal inflammation in wild-type mice. Liver damage and histological

changes were found to be significantly less severe in fat-1 mice

(Fig. 2). The scores of the HAI were significantly different between

the wild-type and fat-1 group (P < 0.05 for the comparison between

fat-1 mice versus wild-type mice; Table 2).

Of particular interest was analyzing the pathways of cell death in

this context, because the examination of particularly apoptotic

activity in the inflamed hepatic tissue can contribute to the

understanding of the damage processes in the liver. Previous results

have indicated that TNF-a is a strong inductor of apoptosis in D-Gal-

sensitized liver tissue.[22] DAPI staining was used to detect

apoptotic hepatocytes. Administration of D-GaIN/LPS to wild-type mice

induced high numbers of apoptotic cells (109.3 ± 8.3 per 300 nuclei;

n = 3), whereas fat-1 mice showed remarkably less hepatocellular

apoptosis (41.5 ± 5.1 per 300 nuclei; n = 3), indicating less severe

cellular damage in the fat-1 animals (Fig. 3).

TNF-a has been shown to be a crucial pro-inflammatory mediator in

acute liver inflammation.[1][13][17] Plasma levels of TNF-a were

therefore determined at 90 minutes after D-GaIN/LPS challenge and

found to be significantly higher in wild-type mice (2,216 ± 684.6

pg/ml; n = 4) than in fat-1 mice (455.5 ± 145.1 pg/ml; n = 4).

Furthermore, intrahepatic TNF-a expression, as measured by real-time

RT-PCR 6 hours after hepatitis induction, was significantly different

between wild-type and fat-1 animals (wild-type mice, 30.65 ± 6.09, -

fold induction as compared with control animals, n = 5, fat-1 mice

13.31 ± 2.68, -fold induction, n = 5) (Fig. 4). Hence, plasma and

intrahepatic TNF-a levels correlated with the severity of liver

disease.

These findings are consistent with previous studies in animal models

and in humans, which showed that n-3 PUFA decreased TNF-a production.

[4-6] This could be due to n-3 PUFA-derived lipid mediators, the

resolvins and protectins, which have been shown to be potent

antiinflammatory mediators.[7] A recent study analyzing the formation

and the molecular effect of n-3 PUFA has identified a G protein-

coupled receptor-specific pathway involved in NF-kB down-regulation

by the n-3 PUFA-derived resolvin E1,[8] which in turn could also down-

regulate NF-kB-induced TNF-aformation.

We next examined the hepatic expression of the inflammatory cytokines

IL-6, IFN-y, and IL-1beta by using quantitative real-time RT-PCR. The

fat-1 group showed a significant reduction in IL-6 mRNA compared with

the wild-type animals (Fig. 5A). The proinflammatory IL-6 is elevated

in the D-GalN/LPS hepatitis model[13]; therefore, the significantly

decreased expression of IL-6 mRNA in fat-1 mice could contribute to

the reduced inflammatory response observed in the fat-1 mice. Higher

expression of IFN-y and IL-1beta was also seen in the wild-type

animals, demonstrating dampening of macrophage-associated pro-

inflammatory cytokines in fat-1 mice (Fig. 5B,C). Particularly with

regard to IFN-y expression, examining underlying mechanisms further

in the future will be interesting, because cytokines such as IL-18

and IL-12 have been implicated in the upstream, leading to expression

of IFN-y in macrophages.[23]

The results presented here indicate that increasing the hepatic

content of n-3 PUFA could decrease inflammatory activity in acute

hepatitis. A limitation of our study might be the narrow range of the

AA/EPA ratio chosen for the mice in the experimental group. Whether

higher or lower n-6/n-3 PUFA ratios modify the inflammation dampening

effect observed here is not clear.

The n-3 fatty acids might exert an anti-inflammatory effect via

competitive inhibition of the n-6 PUFA-derived pro-inflammatory

eicosanoids. However, in this study, we found only small and not

significant differences in the content of arachidonic acid (AA, 20:4

n-6) in the liver tissue between fat-1 transgenic and wild-type mice.

Indeed, we found higher levels of arachidonic acid as compared with

the direct n-3 PUFA competitor eicosapentaenoic acid (EPA, 20:5 n-3)

also in the fat-1 mice. A significant difference was seen in the

amounts of n-3 fatty acids such as EPA and DHA, between fat-1 mice

and their wild-type littermates. Therefore, based on the results of

our previous study in a colitis model,[9] lipid mediators formed from

the n-3 PUFA may be responsible for the inflammation-dampening effect

seen in this population.

Although the D-GalN/LPS model of acute hepatitis in mice is not

directly comparable to liver inflammation in humans, it is a well

established model of hepatitis, mirroring activation of macrophages

and cytokine release, factors crucial also in human hepatitis of

various causes.[1][13] Our results indicate a role for n-3 PUFA in

alleviation of hepatic injury and inflammation. Indeed, a recent case

report suggests that n-3 PUFA might be beneficial in infants with

intestinal failure and parenteral nutrition-related liver disease.

[24] Future studies will be necessary to analyze in more detail the

optimal fatty acid ratios and lipid mediators involved in n-3 PUFA-

associated inflammation dampening in the liver and also to expand the

data presented here into models of clinically important chronic

hepatitis.