differences in innate immune signaling between alcoholic and non-alcoholic steatohepatitis

REVIEW

Differences in innate immune signaling between alcoholicand non-alcoholic steatohepatitisJan Petrasek, Timea Csak, Michal Ganz and Gyongyi Szabo

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Key words

alcoholic liver disease, alcoholicsteatohepatitis, inflammasome, innateimmunity, interleukin-1 beta, interleukin-1receptor antagonist, Kupffer cells,non-alcoholic steatohepatitis, Toll-likereceptors.

Accepted for publication 12 October 2012.

Correspondence

Dr Gyongyi Szabo, University ofMassachusetts Medical School, Departmentof Medicine, LRB 208, 364 Plantation Street,Worcester, MA 01605, USA. Email:[email protected]

AbstractThe similar histopathological characteristics of alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH), and the crucial role of the innate immune response inboth conditions may lead to the assumption that ASH and NASH represent the samepathophysiological entities caused by different risk factors. In this review paper, weelaborate on the pathophysiological differences between these two entities and highlightthe disease-specific involvement of signaling molecules downstream of the Toll-like recep-tor 4, and the differential mechanism by which the inflammasome contributes to ASHversus NASH. Our findings emphasize that ASH and NASH have disease-specific mecha-nisms and therefore represent distinct biological entities. Further studies are needed todissect the emerging differences in pathogenesis of these two conditions.

IntroductionLiver diseases represent a significant cause of morbidity and mor-tality worldwide, ranking as the ninth leading cause of death.1–3

Only second to viral hepatitis, alcoholic liver disease (ALD), andnon-alcoholic fatty liver disease (NAFLD) represent the mostprevalent liver diseases in the United States and developedcountries.4–6 Both entities have a broad clinical spectrum, rangingfrom simple steatosis to steatohepatitis with or without fibrosis,cirrhosis, and hepatocellular carcinoma. Steatosis, observed insimple ALD and in NAFLD is a benign and self-limited condition,but in 10–20% cases, the condition progresses to alcoholic steato-hepatitis (ASH) or non-alcoholic steatohepatitis (NASH), which

share a component of liver inflammation and injury mediated bythe innate immune response.7 This is of a clinical importancebecause inflammation determines the long-term prognosis ofpatients with these diseases, whereas steatosis per se does notappear to have an adverse impact on long-term outcome.8–11

The concept of dysregulated innate immunity as an indispens-able component of ASH and NASH is supported by the findingsthat patients with ASH have increased antibodies against Escheri-chia coli in plasma,12 patients with NASH have increased serumantibodies against endotoxin,13 and that consumption of alcohol orintake of a high-fat or high-carbohydrate diet leads to an increasein gut-derived endotoxin in the portal circulation, activating resi-dent liver macrophages to produce several pro-inflammatory

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93Journal of Gastroenterology and Hepatology 2013; 28 (Suppl. 1): 93–98

© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

cytokines.14–18 Recognition of Toll-like receptors (TLR) as the keycomponents involved in activation of the innate immune systemenabled substantial progress in understanding the mechanismsmediating ASH and NASH.

Gut-derived bacterial components arecritical in the pathogenesis of ASH andNASHDue to its unique blood supply via the portal system, the liverreceives blood from the intestine, exposing hepatocytes and liverimmune cells not only to nutrients but also to gut-derived micro-bial products, including the lipopolysaccharide (LPS, endotoxin),a component of Gram-negative bacterial wall.19 Multiple lines ofevidence support the hypothesis that gut-derived endotoxin isinvolved in ASH and NASH. First, it has been shown that exces-sive intake of alcohol increases gut permeability of normallynon-absorbable substances.11 Second, intestinal Gram-negativebacteria, as well as blood endotoxin, are increased in acute12,13 andchronic12,14,15 alcohol feeding models, and in human and animalstudies of NAFLD/NASH.14,20–22 The mechanisms involve bacte-rial overgrowth, increased intestinal permeability and transloca-tion of endotoxin,23–26 which is increased 5 to 20-fold in the serumof patients with ASH,8,16 3-fold in healthy individuals on a high-fatdiet,14 and 6 to 20-fold in individuals with NAFLD,21,22 comparedto normal subjects. Third, intestinal sterilization with antibiotics oradministration of probiotics resulted in decreased LPS levels andreduced liver inflammation, injury and fibrosis in ASH and NASHin experimental settings.25–31

Activation of Kupffer cells has been identified as one of the keyelements in the pathogenesis of ASH and NASH. Kupffer cells arethe largest population of tissue macrophages, predominantly dis-tributed in the lumen of hepatic sinusoids and exhibit endocyticactivity against blood-borne materials entering the liver.10,24 Trig-gering of toll-like receptor signaling drives Kupffer cells toproduce inflammatory cytokines and chemokines and to initiatethe inflammatory cascade.25 Indeed, the essential role of Kupffercells as a central component of the pathomechanism of ASH orNASH has been demonstrated in studies in mice and rats that showthat inactivation of Kupffer cells with gadolinium chloride orliposomal clodronate can almost fully ameliorate inflammation,steatosis, and damage in ASH and NASH.24,32–34

The role of Toll-like receptor 4 in thepathogenesis of ASH and NASHThe innate immune system recognizes conserved pathogen-associated molecular patterns, which are released during bacterialmultiplication or when bacteria die or lyse,35 through pattern rec-ognition receptors, including TLRs.36 For example, TLR4 recog-nizes LPS from Gram-negative bacteria, and is a potent activatorof innate immune responses through its binding to the TLR4complex via the co-receptors CD14 and MD-2.37 Activation ofKupffer cells via a TLR4-dependent mechanism plays a crucialrole in the pathogenesis of ASH and NASH.15,24,28,38–40 Alcoholicliver injury was prevented in C3H/HeJ mice,41 which have func-tional mutation in the TLR4 gene and have a defective response tobacterial endotoxin, or in mice with a genetic deficiency ofTLR4.42,43 Similarly, deficiency in TLR4 prevented development

of NASH.24,40,44 Prevention of ASH or NASH-associated liverinflammation and injury in TLR4-deficient mice was associatedwith decreased expression of inflammatory cytokines, compared towild-type mice.

Different pathways mediate thepathogenic effects of TLR4 signaling inASH and NASHTLR4 is unique among TLRs in its ability to activate two distinctpathways. One pathway is activated by the adaptors TIR domain-containing adaptor protein (TIRAP) and MyD88, which leads toactivation of NF-kB and to the induction of inflammatory cytok-ines. The second pathway (MyD88-independent) is activated byTIR-domain-containing adapter-inducing interferon-b (TRIF) andTRIF-related adaptor molecule (TRAM), which activates theTANK-binding kinase 1/IkappaB kinase epsilon (TBK/IKKe) andthe interferon regulatory factor 3 (IRF3) to induce Type I interfer-ons (IFNs), as well as NF-kB activation.45,46 The two TLR4-dependent signaling pathways are induced sequentially, and theTRAM-TRIF pathway is only operational from early endosomesfollowing endocytosis of TLR4.47

Both MyD88-dependent and MyD88-independent pathways ofTLR4 signaling were activated in mouse models of ASH orNASH, as documented by increased serum and liver inflamma-tory cytokines, increased nuclear binding of NF-kB to itsDNA response element, and upregulation of Type I IFNs andinterferon-stimulated genes in the liver.42,48,49 In addition,intraperitoneal administration of LPS to alcohol-fed mice orsteatogenic diet further activated both branches of the TLR4pathway.42,44,50 Based on these data, it would be tempting tohypothesize that a similar biological scheme determines respon-siveness to LPS in ASH and NASH. However, our studies do notsupport this notion.

Using the Lieber-DeCarli model of ASH, we observed thatalcohol-fed mice deficient in MyD88 exhibited the same extent ofinflammation, steatosis, and injury as their wild-type controls,which contrasted with a full protection from ASH in TLR4-deficient mice.42 Further analyses showed that deficiency ofMyD88 did not abrogate activation of NF-kB in the liver, and thatWT or MyD88-deficient but not TLR4-deficient mice on anethanol diet demonstrated upregulation of Type I IFNs and IFN-dependent genes in whole livers and in isolated Kupffer cells.These data suggested that TLR4, but not MyD88, leads to activa-tion of signaling mechanisms, including the NF-kB pathway,during the development of ASH. Furthermore, these data, alongwith the findings of others46 suggested a functional activation ofthe MyD88-independendent, IRF3-dependent pathway. We con-firmed this hypothesis and observed abrogation of Type I IFNsignaling along with a complete protection from alcohol-inducedinflammation, steatosis, and damage in alcohol-fed, IRF3-deficientmice, compared to alcohol-fed wild-type controls.48 Thus, our datademonstrated that the pathogenic effect of TLR4 signaling in ASHis mediated via the TRIF/IRF3-dependent, MyD88-independentpathway.

Similar to ASH, there is ample evidence supporting the impor-tant role of TLR4 signaling, including NF-kB activation andupregulation of inflammatory cytokines in the pathogenesis ofNASH.24,40,44,51–53 In contrast to the mechanisms involved in ASH,

Differences in innate immune signaling J Petrasek et al.

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there seems to be a crucial role of MyD88-dependent signaling inNASH. This observation is based on data demonstrating thatinflammation, steatosis, liver damage, and fibrosis were remark-ably inhibited in MyD88-deficient mice fed with choline-deficientsteatogenic diet (54 and G. Szabo, unpublished data). The role ofthe MyD88-dependent pathway was further supported by a signifi-cant protection from NASH that was observed in mice deficient inTLR9, which requires MyD88 for its downstream signaling.54,55 Incontrast, although deficiency of IRF3 in mice abrogated inductionof Type I IFNs, it did not provide any protection from NASH-associated liver inflammation, steatosis, or damage (G. Szabo,manuscript in preparation).

The differential contribution of MyD88-dependent and MyD88-independent pathways of TLR4 signaling in the pathogenesis ofASH and NASH may be attributable to multiple factors. Forexample, the development of NASH, in contrast to ASH, involvesinsulin resistance and an endocrine crosstalk between adiposetissue and the liver. It has been shown that adiponectin, an anti-inflammatory adipokine secreted by adipose tissue, inhibits theTLR4/MyD88-dependent pathway in macrophages.56 A recentmeta-analysis demonstrated approximately 35% decrease ofserum adiponectin in patients with NAFLD, and more than 50%decrease in patients in NASH.57 In contrast, reports on the rela-tionship of adiponectin and ASH show either increase,58–61 nochange,62 or minimal decrease that poorly correlated with theextent of liver injury.63 Based on these reports, demonstratingassociation of adiponectin levels with NAFLD/NASH versus nocorrelation in ASH, we cannot exclude that downregulation ofadiponectin in NAFLD/NASH may contribute to inflammatorysignaling in liver macrophages with preferential induction ofMyD88-dependent pathways. Therefore, signaling from theadipose tissue could potentially modulate the preference for asignaling pathway downstream of TLR4.

Another factor contributing to the differential induction ofTLR4 downstream pathways in ASH and NASH may relate tothe differences in dynamics between these two entities. Althoughboth of them take years to develop in humans, animal modelssuggest that excessive consumption of alcohol may induce liverinflammation at an earlier time point than consumption of ste-atogenic diet. For example, it takes only one intragastric gavageof ethanol to elicit significant liver steatosis in mice (64 and G.Szabo, unpublished observations), or less than seven days of theethanol-containing Lieber-DeCarli diet to initiate liver inflamma-tion,65 but it takes at least 18–24 weeks of feeding with highfat/Western-style diet or the choline-deficient amino acid-defineddiet (CDAA,54) to induce liver inflammation in mice.51,66

Although artificial diets such as the methionine-choline deficient(MCD) diet induce inflammation within a week, these diets rep-resent experimental models for mechanisms involved in NASHbut do not necessarily reflect liver disease development inhumans (49,66 and G. Szabo, unpublished observations). There-fore, it cannot be excluded that different pathways may beresponsible for early versus late stages of pathogenesis of ASHand NASH. This notion is supported by our findings that phar-macological blocking of IL-1 receptor, which signals throughMyD88, could achieve a pronounced protective effect in micewith advanced ASH,67 and that deficiency of MyD88 or IL-1signaling showed protective effect only in later stages of fattyliver disease in mice.51,54,66,68

Differential activation of theinflammasome and IL-1 signaling inASH and NASH

In the pathogenesis of ASH and NASH, activated Kupffer cellsexert their pathogenic effects predominantly via inflammatorycytokines, such as TNF-a, IL-1b, IL-8, or MCP-1.51,53,69,70

Although TLR4-dependent mechanisms are involved in upregula-tion of inflammatory mediators, IL-1b is specific because it isproduced as inactive pro-IL-1b and requires inflammasomes forprocessing. Caspase-1, the effector component of the inflamma-some, cleaves pro-IL-1b into the bioactive IL-1b,71 which acts inan autocrine/paracrine manner via the Type-I IL-1 receptor (IL-1R1). The activation of IL-1R1 is inhibited by its binding to theIL-1 receptor antagonist (IL-1Ra), a naturally occurring cytokinewhose function is to prevent the biologic response to IL-1.72

Studies have demonstrated a pathogenic role of IL-1 signaling inthe murine model of NASH,51,54 upregulation of inflammasomecomponents in the liver and increased serum levels of IL-1Ra inpatients with NASH66,73 and increased levels of IL-1b in patientswith ASH.74 However, there were no data supporting the causalrole of IL-1 signaling in ASH, and there were only limited data onthe cellular source and mechanism of IL-1b activation in NASH.

In our studies, we observed that inflammasome and IL-1b wereactivated in ASH, as documented by increased expression ofinflammasome components NALP3 (NLR family, pyrin domain-containing 3), ASC (apoptosis-associated speck-like protein con-taining a CARD), and caspase-1 in the livers of alcohol-fed mice,and by increased activity of liver caspase-1 and elevated levels ofcleaved IL-1b in the liver and in the serum.67 Deficiency of inflam-masome components ASC or caspase-1, significantly amelioratedalcohol-induced liver inflammation, steatosis, and damage.Similar protection was observed in mice deficient in IL-1R1 whichlack IL-1 signaling, and in mice treated with recombinant IL-1Rawhich inhibits IL-1 signaling.67

Similar to ASH, the methionine-choline deficient diet (MCD)-based mouse model of NASH demonstrated activation ofcaspase-1 in the liver and increased levels of cleaved IL-1b in theliver and in the serum after six weeks of treatment.66,68 Using thehigh-fat diet model of NASH, we observed that caspase-1 andIL-1b became activated at a later time point of nine months alongwith increased inflammation, but not at four weeks when liverpathology was dominated by steatosis only.66 This finding con-trasted with our ASH data which demonstrated that inflammasomeactivation occurs very early in the course of alcohol treatment.67

Furthermore, deficiency of caspase-1 significantly amelioratedonly liver inflammation induced by the MCD diet but did notalleviate liver damage.68 Some protection from MCD diet-inducedliver damage was observed in mice lacking IL-1a or IL-1b;however, this protection was observed only after 18 weeks of theexperiment and not at earlier time points.51 In the context of earlyactivation of the inflammasome in ASH and a significant protec-tion from all components of alcoholic liver disease observed inmice deficient in inflammasome components or IL-1 signaling, theavailable data suggested differential role of inflammasomes in thepathogenesis of ASH and NASH.

In search for mechanisms that would explain this discrepancy,we investigated the cellular source of activated inflammasome andIL-1b. Both in ASH and NASH, the baseline levels of caspase-1

J Petrasek et al. Differences in innate immune signaling



protein or pro-Casp-1, ASC, Nlrp3, and pro-IL-1b mRNA weresubstantially higher in liver immune cells, compared to hepato-cytes.66,67 Specific for ASH, analysis of liver immune cells orprimary hepatocytes isolated from alcohol-fed mice showed thatalcohol increased the active fragment of caspase-1 and IL-1b onlyin liver immune cells but not in primary hepatocytes. As these datasuggested that liver immune cells were the predominant cell typethat activates caspase-1 and IL-1b in ASH, we generated caspase-1-chimeric mice using a combination of clodronate-mediatedKupffer cell depletion, irradiation, and bone marrow transplanta-tion. Using this model, we confirmed our hypothesis thatcaspase-1 expressed in Kupffer cells was involved in alcohol-induced liver inflammation, steatosis, and injury, and we did notfind any evidence for a pathogenic role for caspase-1 in liverparenchymal cells in the development of ASH.67

In addition to Kupffer cell-specific inflammasome activation inASH,67 we observed that activation of the inflammasome occurredalso in isolated hepatocytes in NASH.66 Specifically, primaryhepatocytes isolated from the MCD-fed mice had increasedexpression of NALP3, ASC, caspase-1, and pro-IL-1b mRNA.49,66

Taking into account that fatty livers had elevated the expression ofinflammasome components and that this process occurred in hepa-tocytes which accumulate lipids, we tested whether fatty acidsexert any effects on inflammasome in hepatocytes. We observedthat in vitro treatment of primary mouse hepatocytes with palmi-toic acid, a saturated fatty acid, resulted in upregulation of theinflammasome component NALP3, priming of caspase-1 for sub-sequent activation by LPS and induction of IL-1b secretion. Usingthe pan-caspase inhibitor Z-VAD, we demonstrated that theseevents were caspase dependent, and we also showed that they werecaused by saturated fatty acids, whereas non-saturated fatty acidshad no effect. We further showed that hepatocytes exposed topalmitoic acid produced inflammasome-mediated danger signals,which in turn activated liver macrophages in a caspase-dependentmanner.66

Taken together, our findings have outlined several differences ininflammasome/IL-1 signaling between ASH and NASH. First,activation of inflammasome seems to be an early event in ASHand late event in NASH. Second, deficiency in inflammasomecomponents or absence of IL-1 signaling significantly amelioratesinflammation, steatosis, and liver damage in ASH, whereas onlyprotection from liver steatosis is consistently observed in NASH(51,54,68 and G. S., unpublished data). Third, whereas inflammasomeactivation in ASH is specific to bone marrow-derived Kupffercells, hepatocytes are involved in inflammasome activation inNASH. We hypothesize that this difference may be due to thepredominance of cytotoxic free fatty acids and increased hepatocytelipoapoptosis in NASH, compared to ASH in which the majorityof fatty acids in hepatocytes is in esterified, less toxic form.75

ConclusionIn spite of the comparable histopathological characteristics ofASH and NASH, their similar pattern of progression to advancedliver disease, and the crucial role of innate immune signaling inboth conditions, it is unlikely that the same immunopathogenicmechanisms contribute to ASH and NASH. Further studies areneeded to dissect the emerging differences in pathogenesis of thesetwo conditions.

AcknowledgmentsThis work was supported by NIH grants AA017729 andDK075635 (to G. Szabo). Core resources supported by the Dia-betes Endocrinology Research Center grant DK32520 from theNational Institute of Diabetes and Digestive and Kidney Diseaseswere used. Dr Gyongyi Szabo is a member of the UMass DERC(DK32520).

Conflict of interestThe authors have no conflicts of interest to declare.

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differences in innate immune signaling between alcoholic and non-alcoholic steatohepatitis

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