Reduced Early Alcohol-Induced Liver Injury inCD14-Deficient Mice1
Ming Yin,* Blair U. Bradford, 2* Michael D. Wheeler,* Takehiko Uesugi,* Matthias Froh,*Sanna M. Goyert,† and Ronald G. Thurman*
Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown thatCD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed toinvestigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice ina model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 6 0.3%) was increased significantly by ethanol(7.36 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normalliver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score5 3.86 0.4). Incontrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score5 1.6 6 0.3,p < 0.05). Additionally, NF-kB,TGF-b, and TNF-a were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanolfeeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting viaCD14 plays a major role in the development of early alcohol-induced liver injury. The Journal of Immunology,2001, 166:4737–4742.
Endotoxins (LPS) represent the major component of theouter membrane of Gram-negative bacteria and have beenimplicated in sepsis, organ failure, and shock (1). Accu-
mulating evidence suggests that endotoxins and proinflammatorycytokines also participate in early alcohol-induced liver injury (2).Elevated circulating endotoxin most likely activates Kupffer cellsto release many potent effectors and cytokines, thus leading totissue injury. The following evidence supports this hypothesis:1) Ethanol increases permeability of the isolated small bowel toendotoxin in a dose-dependent manner (3) and elevates circulatingendotoxin (4). 2) Reduction of Gram-negative bacteria in the in-testines (i.e., sources of endotoxin) with antibiotics (5) or lactoba-cillus administration (6) minimized early alcoholic liver injury.3) Early ethanol-induced liver injury can be prevented by gado-linium chloride, a selective Kupffer cell toxicant (7). 4) Early al-cohol-induced liver injury was attenuated with TNF-a Abs andwas prevented in TNF receptor-1 knockout mice (8, 9).
CD14, a GPI-anchored glycoprotein that is highly expressed onthe surface of human monocytes and most macrophages (10), hasbeen demonstrated to be part of a specific cellular LPS binding site(11) and is a key mediator of septic shock induced by endotoxin
(10, 12, 13). Circulating LPS binds to LPS-binding protein (LBP)3
and activates monocytes/macrophages via binding to the CD14receptor (14, 15). Kupffer cells, the resident liver macrophages, arethe major population of the monocyte-macrophage lineage (16)and contain CD14 receptors (4, 17). Recent studies show that al-cohol exposure increases the expression of CD14 in Kupffer cells(18) and estrogen sensitizes Kupffer cells to LPS via increases inCD14. The interaction of LPS with CD14 triggers a signaling cas-cade and results in induction of cytokines (IL-6, IL-1, and TNF-a)that are known to participate in liver injury (2). It has been shownpreviously that CD14-deficient mice do not produce significantlevels of these cytokines even when exposed to high levels of LPS(12). To test the hypothesis that CD14 is involved in early alcohol-induced liver injury, the response of CD14-deficient mice to eth-anol was compared with that wild-type mice by evaluating param-eters of hepatic injury using a murine enteral ethanol feedingmodel (9). After 4 wk of continuous enteral ethanol feeding, notonly was steatosis observed, but inflammation and necrosis alsooccurred in livers of wild-type mice. However, in mice lackingCD14 receptors, hepatic pathology due to alcohol was largelyblocked. These studies show that CD14, presumably on Kupffercells, plays a critical role in early alcohol-induced liver injury andfurther support a role for endotoxin in this disease. Preliminaryaccounts of this study have been reported elsewhere (19).
Materials and MethodsAnimals
Female CD14-deficient mice generated as described elsewhere (12, 20)were backcrossed 10 times with BALB/c mice (The Jackson Laboratory,Bar Harbor, ME). Age- and sex-matched wild-type BALB/c mice, weigh-ing 21–24 g, served as controls. Mice were screened for the presence of theCD14 receptor mRNA (1/1) in wild-type mice and the absence of theCD14 receptor (2/2) mRNA in knockout mice by RNase protection assay.
*Department of Pharmacology, Laboratory of Hepatobiology and Toxicology, Uni-versity of North Carolina, Chapel Hill, NC 27599; and†Division of Molecular Med-icine, North Shore University Hospital, Cornell University Medical College, Manhas-set, NY 11030
Received for publication November 6, 2000. Accepted for publication January24, 2001.
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported, in part, by grants from National Institute of AlcoholAbuse and Alcoholism (to R.G.T.) and National Institutes of Health (to S.M.G.).2 Address correspondence and reprint requests to Blair U. Bradford, Department ofPharmacology, Laboratory of Hepatobiology and Toxicology, CB 7365, Mary EllenJones Building, University of North Carolina, Chapel Hill, NC 27599-7365. E-mailaddress: [email protected]
3 Abbreviations used in this paper: LBP, LPS-binding protein; ALT, alanine amino-transferase; LT-b, lymphotoxin-b; MIF, migration inhibitory factor.
All animals received humane care in compliance with institutional guide-lines. Body weight was measured before surgery and at necropsy after 4 wkof continuous delivery of control or ethanol-containing diet.
Surgery
The principle surgical procedures were similar to methods previously de-scribed by Tsukamoto et al. (21) in rats, with modifications based on thesize of mice (9). Briefly, mice were anesthetized by injection of pentobar-bital sodium (50 mg/kg; Abbott Laboratories, North Chicago, IL), andlaparotomy was performed under sterile surgical conditions. A PE90 poly-ethylene tube (Becton Dickinson, Sparks, MD) was placed in the squamouspart of the stomach. The tube was anchored to the stomach wall withDacron and fixed to the abdominal wall. It was then tunneled s.c. to thedorsal aspect of the neck followed by closing of the abdominal wall with7-0 prolene sutures. The tube was then pulled through a 250P polysulfoneattachment mouse button (Instech Laboratories, Plymouth Meeting, PA)and spring coil. The button was fixed under the skin with its metal springcoil outside of the body to protect the tube. The feeding tube was attachedto an infusion pump by means of a swivel, allowing complete mobility ofthe mouse within a metabolic cage. Animals were allowed to recover for 1wk with free access to chow diet and water before starting alcohol-con-taining or control high-fat liquid diets.
Diets
A basic liquid diet was prepared according to Thompson and Reitz (22) asdescribed previously and supplemented with lipotropes as described byMorimoto et al. (23). The control diet (1.3 kcal/ml) contained corn oil asfat (34% of total calories), protein (23%), carbohydrate (43%), plus min-erals and vitamins. For the isocaloric ethanol diet, dextrin-maltose wasreplaced by ethanol (9). The liquid diet was fed continuously at the rate of11 ml/day to achieve weight gain. Throughout the experimental periodof liquid diet delivery, mice had free access to cellulose pellets as a sourceof fiber (Harlan Teklad, Madison, WI).
Experimental protocol
CD14-deficient and wild-type mice were randomly allocated into two ex-perimental groups of five to seven mice each and were fed either ethanol-containing or an isocaloric high-fat control diet. Animals received diets byinfusion through an intragastric cannula for up to 4 wk as described pre-viously (9). Usually, the ethanol dose was 28–29 g/kg/day during thefourth week of feeding. CD14-deficient and wild-type mice were sacrificedafter 4 wk and blood samples were collected via the inferior vena cava atnecropsy. Serum was stored at220°C until alanine aminotransferase(ALT) was analyzed by standard enzymatic procedures (24). Livers wereremoved and weighed and tissue samples were divided; some were fixed inFormalin, others were frozen in liquid nitrogen and stored at280°C.
Urine collection and assay for ethanol
Concentrations of ethanol in urine are representative of blood alcohol lev-els (25). Mice were housed in metabolic cages that separated urine fromfeces and urine samples were collected over 24 h for each mouse. Ethanollevels in urine were determined daily by measuring absorbance at 366 nmresulting from the reduction of NAD1 by alcohol dehydrogenase (24).
Pathological evaluation
Formalin-fixed liver samples were embedded in paraffin and stained withhematoxylin and eosin to assess steatosis, inflammation, and necrosis.Liver pathology was scored as described by Nanji et al. (26) as follows:steatosis (the percentage of liver cells containing fat):,25%5 11; ,50%5 21; ,75%5 31; .75%5 41; inflammation and necrosis: 1 focus perlow-power field5 11; 2 or more5 21. One point was given for eachgrade of severity of histological abnormality and a total score was calcu-lated for each liver.
RT-PCR amplification of CD14 cDNA
Mouse Kupffer cell RNA was extracted by standard phenol:chloroformextraction in guanadinium isothiocyanate (RNA-STAT60; Tel-Test,Friendswood, TX) followed by ethanol precipitation. mRNA (5mg) wasadded to reverse transcription buffer containing 0.5 U of RNase (Promega,Madison, WI), 5 mM DTT, 2 mM dNTPs (Life Technologies, Rockville,MD), 1 mM random hexamer primer (Boehringer Mannheim, Indianapolis,IN), and 1 U of avian myleoblastosis virus reverse transcriptase (Promega).The reaction was incubated at 42°C for 45 min and terminated at 95°C for2 min. An aliquot (5ml) of the RT reaction was added to PCR amplificationbuffer containing 2 mM dNTPs (Life Technologies), 0.4mM forward and
reverse primers, and 0.5 U ofTaq DNA polymerase (Boehringer Mann-heim). PCR was initiated by a 7-min incubation at 95°C, followed by 20cycles of 1 min at 95°C, 1 min at 48°C, and 2 min at 72°C. The reactionwas followed by a 10-min incubation at 72°C. The amplification productwas resolved on 1.5% agarose gel. PCR primers for CD14 which generateda 180-bp fragment were as follows: forward, 59-GCAACTTCTCAGATCCGAAGCC-39; and reverse, 59-CAACAGTAAGCCGCTTTAAGAC-39.
RNase protection assay
Total RNA was isolated from hepatic tissue using RNA STAT60 (Tel-Test). Samples of total RNA (30mg) were hybridized with 105 cpm of[a-32P]UTP (3000 Ci/mmol; Amersham, Arlington Heights, IL)-labeledprobe (see below) at 42°C overnight. RNase digestions were conductedwith a mixture of RNase A and RNase T1 (PharMingen, San Diego, CA)at 37°C for 45 min, followed by proteinase K treatment for 15 min, andphenol: chloroform extraction and ethanol precipitation. The digested prod-ucts were separated on a 6% acrylamide gel. The RNA probe used to detectCD14-specific RNA was generated by in vitro transcription of linearizedpCR-rCD14 after cleavage withHindIII. The pCR-rCD14 plasmid wasgenerated by insertion of the RT-PCR amplification fragment (180 bp) ofmouse Kupffer cell cDNA into the pCR-TOPO cloning plasmid (Invitro-gen, San Diego, CA). The RNA probes (PharMingen) used to detectGAPDH-specific RNA (as control), L32 as a ribosomal housekeepinggene, TNF-a, lymphotoxin-b (LT-b), macrophage migration inhibitoryfactor (MIF), and TGF-b were generated by in vitro transcription of mouseGAPDH template (PharMingen). The protected RNA was visualized byautoradiography.
EMSA
A gel mobility shift assay was used in this study to assess the amount ofactive protein involved in protein-DNA interactions. The limitations of thismethod are the amount of nuclear protein needed for assay as well as thenumber of cells isolated from the rat liver. Binding conditions for NF-kBwere characterized and EMSAs were performed as described in detail else-where (27). Briefly, extracts (40mg) from liver tissues were preincubatedfor 10 min on ice with 1mg of poly(dI-dC) and 20mg of BSA (both fromPharmacia Biotech, Piscataway, NJ) in a buffer that contained 1 mMHEPES (pH 7.6), 40 mM MgCl2, 0.1 M NaCl, 8% glycerol, 0.1 mM DTT,0.05 mM EDTA, and 2ml of a 32P-labeled DNA probe (10,000 cpm/ml)that contained 0.4 ng of double-stranded oligonucleotide. Mixtures wereincubated for 20 min on ice and resolved on 5% polyacrylamide (29:1cross-linking) and 0.43Tris-borate-EDTA gels. After electrophoresis, gelswere dried and exposed to Kodak film (Kodak, Rochester, NY). Data werequantitated by scanning autoradiograms with GelScan XL (Pharmacia).
Statistics
ANOVA was used for the determination of statistical significance as ap-propriate. For comparison of pathological scores, the Mann-WhitneyUrank sum test was used. Data are presented as mean6 SEM. A p , 0.05was selected before the study as the level of significance.
ResultsBody and liver weights
Liver and body weight were determined to assess the generalhealth of the animals. All animals survived surgery and gainedweight during 4 wk of continuous delivery of high-fat liquid dietswith or without ethanol. There were no significant differences inbody weight among the groups studied. At necropsy, liver to bodyweight ratios in wild-type mice receiving ethanol were signifi-cantly higher than in wild-type mice fed control diet (Fig. 1). Incontrast, the ratio in CD14-deficient mice given ethanol was sig-nificantly lower than in wild-type mice fed ethanol. Thus, ethanolcaused significantly greater enlargement of livers in wild-type thanin CD14 knockout mice.
Urine ethanol
Urine ethanol concentrations were monitored to index the degreeof intoxication. As reported previously in studies with rats (28) orC57BL mice (7, 9), urine ethanol levels also fluctuated in theCD14-deficient or wild-type BALB/c mice in a cyclic pattern from18 to.500 mg/dl likely due to thyroid hormones and stress as wasrecently reported in rats (29). Average urine ethanol concentrations
during 4 wk of ethanol exposure were 1586 25 mg/dl in wild-typemice and 1626 26 mg/dl in the knockouts, values which were notstatistically different.
Serum transaminases
As a marker of liver injury, serum ALT was monitored throughoutthe study. Before continuous administration of liquid diets, theserum level of ALT in both wild-type and CD14 knockout micewas 446 5 U/L. In wild-type mice, 4 wk of ethanol exposuresignificantly increased the serum ALT;3-fold (126 6 27 U/L;Fig. 2); however, ethanol exposure did not increase ALT inCD14-deficient mice.
Pathological evaluation
Fig. 3 shows representative photomicrographs of livers from wild-type and CD14 knockout mice after 4 wk of exposure to control orethanol diets. There were no pathological changes seen in wild-type or CD14 knockout mice receiving high-fat control diet (Fig.3, A andB). Accordingly, the pathology score of livers from wild-type and knockout mice fed control high-fat diet was minimal (Fig.4). However, marked fatty accumulation and mild to moderateinflammation and necrosis were observed in wild-type mice givenethanol (Fig. 3C), with an average pathology score of 3.86 0.6(Fig. 4D). This value was significantly greater than values ob-served in wild-type mice given control diet (Fig. 4D). Fatty accu-mulation in wild-type mice receiving ethanol was panlobular, withmassive large droplets of fat in pericentral areas and midzonalregions near central veins. In livers from CD14-deficient mice,however, only mild fatty accumulation was detected (Fig. 3D),with mild inflammation but no necrosis. As a result, the pathologyscores of livers from CD14 knockout mice receiving ethanol dietwere only 1.76 0.4 (Fig. 4D), values which were significantlylower than ethanol-treated, wild-type mice (p , 0.05).
Inflammatory markers in liver
An RNase protection assay was used to determine the tissue levelsof message for TNF-a, LT-b, MIF, and TGF-b. Ethanol caused
FIGURE 2. Effect of continuous diet delivery with or without ethanolon serum ALT levels. Blood samples were collected at necropsy, i.e., 4 wkafter high-fat liquid diet feeding in the presence or absence of ethanol. ALTlevels were determined as described inMaterials and Methods. Data pre-sented are mean6 SEM, n 5 5–7 mice/group. Two-way ANOVA wasused for determination of statistical differences.p, p , 0.05 compared withthe other three groups of mice by one-way ANOVA with Tukey’s post hoctest. KO, Knockout.
FIGURE 3. Representative photomicrographs of livers from wild-typeand CD14 knockout mice after 4 wk of continuous diet delivery with orwithout ethanol. Animals were treated as described inMaterials and Meth-ods. Livers from wild-type mice receiving high-fat control diet (A), CD14knockouts fed high-fat control diet (B), wild-type mice given ethanol diet(C), and CD14 knockouts given ethanol diet (D) are shown. Original mag-nification,3100. With higher magnification,E andF show inflammationand necrosis in wild-type animals fed ethanol;G and H depict histologywithout inflammation and necrosis in CD14-deficient mice receiving eth-anol. H-F represents high-fat; EtOH represents animals given the ethanoldiet. Representative photomicrographs.
FIGURE 1. Changes in liver to body weight ratio in mice after 4 wk ofenteral high-fat control or ethanol-containing diet. Body and liver weightswere determined at necropsy. H-F is high-fat control diet; EtOH representsethanol-containing diet. There were five to seven mice in each group. Dataare presented as mean6 SEM. a, p , 0.05 compared with the mice thatreceived high-fat control diet.b, p , 0.05 compared with the wild-typemice that received ethanol by one-way ANOVA with Tukey’s post hoctest. KO, Knockout.
about a 2.5-fold increase in tissue levels of TNF-a mRNA in liversfrom wild-type mice (Fig. 5) as well as the inflammatory cytokineLT-b. This phenomenon was not observed in CD14 knockout
mice. TGF-b and LT-b were doubled after ethanol in the wild-typebut not in CD14 knockout mice (Fig. 5). MIF was detected in allgroups. To assess the involvement of NF-kB after alcohol treat-ment in both strains of mice, activation was measured by theEMSA in liver (Fig. 6). A doubling of NF-kB activation occurredafter ethanol treatment in wild-type mice. In CD14 knockout mice,however, activation of NF-kB did not occur after ethanoltreatment.
DiscussionThe role of LBP and CD14 in early alcohol-induced liver injury
Activation of monocytes and macrophages by endotoxin (LPS)from Gram-negative bacteria has been extensively studied to de-fine the mechanisms that underlie innate immune responses againstbacterial pathogens (30). Numerous studies have revealed thatLPS, instead of being directly toxic to cells or organs, exerts itsbiological effects indirectly through stimulation of host cells toproduce endogenous mediators which elicit a response that canlead to severe tissue injury and/or death. The mechanism throughwhich endotoxin stimulates cells is a multistep process that in-cludes the initial binding of endotoxin to a serum protein, LBP(14). The binding of endotoxin to LBP forms monomers and al-lows LPS to efficiently bind to CD14 on the surface of monocytes/macrophages (11, 14). The binding of LPS to CD14 triggers therelease of endogenous inflammatory mediators including cytokinesand free radicals (15, 30–33), leading to inflammation and requiresadditional molecules for signal transduction, including Toll-likereceptors (34, 35). It was very recently reported that alcohol-in-duced liver injury, using the same model used in this study, isblunted in the Toll-like receptor 4 mutant C3H/HeJ mouse (36).Taken together, this study and the work presented here support thehypothesis that a signaling pathway comprised of CD14 and Toll-like receptor 4 is required for development of pathology due toethanol.
Ethanol and endotoxin
Studies demonstrating the strong resistance of CD14-deficientmice to endotoxin suggest that CD14 plays a predominant rolein endotoxin shock (12, 37). The development of the CD14knockout mice provides a powerful tool for studies of the roleof endotoxin in various diseases. It is well known that ethanolexposure increases circulating endotoxin (4). However, ethanol-induced hepatic injury was minimized significantly here inCD14-deficient mice (Fig. 4). This indicates that cells of theimmune system respond to LPS through a pathway involvingthe CD14 receptor (13, 38) and supports the hypothesis that
FIGURE 4. Effect of 4 wk of continuous diet delivery with or withoutethanol on hepatic pathology in wild-type and CD14 knockout (KO) mice.Pathology was scored as described inMaterials and Methods. EtOH rep-resents mice receiving ethanol-containing diet. Data are presented asmean6 SEM, n 5 5–7 mice/group. The Mann-WhitneyU rank sum testwas used for determination of statistical differences.a, p , 0.05 comparedwith wild-type mice given control diet;b, p , 0.05 compared with wild-type mice given the ethanol diet
FIGURE 5. Effects of 4 wk of continuous diet delivery with ethanol ontissue levels of TNF-a, LT-b, TGF-b, and MIF mRNA in livers fromwild-type and CD14 knockout mice. TNF-a, LT-b, TGF-b, and MIFmRNA levels were measured by standard RNase protection assay as de-tailed inMaterials and Methods(A). TNF mRNA was quantitated on gelswith densitometry (B).
FIGURE 6. EMSA for NF-kB in liver from wild-type (WT) andCD14 knockout (KO) mice treated for 4 wk with continuous ethanol.The optimal binding conditions for NF-kB were characterized and EM-SAs were performed as described inMaterials and Methods. Gels weredried and exposed to Kodak film and data were quantitated by densi-tometric analysis.
elevated circulating endotoxin by excessive alcohol intake ac-tivates residential hepatic macrophages (Kupffer cells) to re-lease endogenous inflammatory mediators via activation ofNF-kB (Figs. 6 and 7).
The activation of NF-kB by enteral ethanol treatment in therat is known (39). It has been shown that NF-kB activation isnot increased after 1 wk of ethanol treatment (40). This suggeststhat longer exposure to ethanol is critical as was observed here(Fig. 6). The increase in NF-kB activation is consistent with thehypothesis that endotoxin binds to CD14, activates the Kupffercells, and elicits the production of reactive oxygen specieswhich increases NF-kB (Fig. 7). The absence of activation ofNF-kB in the CD14 knockout mouse after enteral ethanolstrengthens this hypothesis.
Recently, it was reported that chronic ethanol increases mRNAfor TGF-b expression in rats;2 fold after 2 wk of enteral ethanol(41). In these experiments, mRNA for LBP and CD14 were notincreased by chronic endotoxin but were increased after acute en-dotoxin. The authors conclude that down-regulation of mRNA forCD14 and LBP may occur as an adaptation in chronic alcoholabuse. The results in mice presented here using a chronic model ofethanol delivery provide an alternative explanation. TGF-b proteinis doubled after ethanol treatment (see Fig. 5 andResults) in wild-type mice but is not elevated in the CD14 knockout mouse fedcontrol or ethanol-containing diet, suggesting that CD14 is neces-sary for TGF-b activation and not for down-regulation.
Cytokines
TNF-a is a central proinflammatory cytokine (42). TNF-a act-ing through its receptor-1 pathway plays a predominant role inLPS-induced inflammatory diseases (43) and mediates the le-thal effects of endotoxin (44). TNF-a is increased in alcoholicswith hepatitis and levels correlate with survival (35) and ap-pears to be the principle mediator of early alcohol-induced liverinjury since injury was blocked in mice lacking the TNF recep-
tor-1 (9). In the present study, increased levels of TNF-a (Fig.5) along with the most severe hepatic injury among the groupsstudied were observed in the livers from wild-type mice fedethanol, whereas these effects were blocked in CD14-deficientmice. Therefore, these results are consistent with the hypothesisthat excessive intake of alcohol increases circulating endotoxinwhich activates Kupffer cells via the CD14 receptor to releaseTNF-a, leading to liver injury (Fig. 7).
In this study, macrophage MIF was detected in all treatmentgroups studied (Fig. 5) and is commonly expressed in liver andother tissues (45) as an inflammatory cytokine that can alsoinduce TNF-a (46). Studies with anti-MIF Ab demonstrate aprotective effect in models of endotoxin-induced injury (47).After 4 wk of dietary treatment with or without ethanol, a mildinflammatory infiltration was observed in mice receiving ahigh-fat control diet. This is different from the results of a pre-vious study using mice on a C57BL/6J background (9), whereno inflammation was detected in animals receiving a high-fatcontrol diet. This phenomenon is possibly due to the fact thatBALB/c mice are more susceptible to inflammation (48) involv-ing, in part, the inflammatory cytokine MIF.
In summary, the results of the present study are consistent withthe hypothesis that CD14 receptors play a major role in vivo in thepathogenesis of alcohol-induced liver injury; thus, drugs or genesthat target CD14 signaling pathways may prove beneficial in treat-ing alcoholic hepatitis.
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