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Hepatology international 17(3), 698-708 (2023). doi:10.1007/s12072-022-10473-x, Published by Springer, Heidelberg

Up to 30% of patients with severe alcoholic hepatitis (sAH) die within 3 months of presentation. The degree of ductular reaction, characterized by accumulation of biliary and liver progenitor cells, confers a poor prognosis. Keratin fragments are established serological surrogates of liver injury and keratin 19 (K19) is a histological marker of the ductular reaction. We assessed the relationship between serum K19 levels (viz. CYFRA21-1), histology and outcome in patients with sAH.Serum CYFRA21-1 was quantified in pre-treatment serum samples from 824 patients enrolled in the STOPAH trial. The cohort was randomly divided into two groups to test mortality associations; histological analyses were performed using the 87 cases with suitable samples.CYFRA21-1 levels were elevated in sAH and strongly predicted alcoholic steatohepatitis (ASH) on biopsy (area under the receiver operated characteristic [AUROC] 0.785 [95% Confidence Interval 0.602-0.967]) and 90-day survival (AUROC 0.684/0.693). The predictive ability of CYFRA21-1 was comparable with the model of end-stage liver disease (MELD) score and was independently associated with survival in multivariable analysis. CYFRA21-1 moderately correlated with hepatocellular injury markers M30/M65 but displayed a more robust predictive ability. A combination of MELD and CYFRA21-1 conferred a modest improvement in the AUROC value (0.731/0.743).In sAH serum, CYFRA21-1 levels associate with the presence of ASH on biopsy and independently predict 90-day survival. As a single marker performance is comparable to established scoring systems. Therefore, CYFRA21-1, which is available in many clinical laboratories, may become a useful component of prognostic models.

BMC medicine 19, 39 (2021). doi:10.1186/s12916-021-01917-6 special issue: "Editor���s Choice: World's Digestive Health Day 2021", Published by BioMed Central, London

Journal of Clinical Medicine 9(4), 1140 (2020). doi:10.3390/jcm9041140, Published by MDPI, Basel

Objectives Severe alcoholic hepatitis (sAH) confers substantial mortality, but the disease course is difficult to predict. As iron parameters are attractive outcome predictors in other liver diseases, we tested their prognostic ability in sAH. Methods Serum ferritin, transferrin, iron, transferrin saturation, nontransferrin-bound iron, soluble transferrin receptor, and hepcidin were measured in 828 patients with sAH recruited prospectively through the STOPAH trial. The cohort was randomly divided into exploratory (n = 200) and validation sets (n = 628). Results Patients with sAH had diminished serum transferrin but increased transferrin saturation. Among iron parameters, baseline transferrin was the best predictor of 28-day (area under the receiver operated characteristic 0.72 [95% confidence interval 0.67-0.78]) and 90-day survival (area under the receiver operated characteristic 0.65 [0.61-0.70]). Transferrin's predictive ability was comparable with the composite scores, namely model of end-stage liver disease, Glasgow alcoholic hepatitis score, and discriminant function, and was independently associated with survival in multivariable analysis. These results were confirmed in a validation cohort. Transferrin did not correlate with markers of liver synthesis nor with non-transferrin-bound iron or soluble transferrin receptor (as markers of excess unbound iron and functional iron deficiency, respectively). Discussion In patients with sAH, serum transferrin predicts mortality with a performance comparable with commonly used composite scoring systems. Hence, this routinely available parameter might be a useful marker alone or as a component of prognostic models.

Background Keratins (Ks) represent tissue-specific proteins. K18 is produced in hepatocytes while K19, the most widely used ductular reaction (DR) marker, is found in cholangiocytes and hepatic progenitor cells. K18-based serum fragments are commonly used liver disease predictors, while K19-based serum fragments detected through CYFRA21-1 are established tumor but not liver disease markers yet. Since DR reflects the severity of the underlying liver disease, we systematically evaluated the usefulness of CYFRA21-1 in different liver disease severities and etiologies. Methods Hepatic expression of ductular keratins (K7/K19/K23) was analyzed in 57 patients with chronic liver disease (cohort i). Serum CYFRA21-1 levels were measured in 333 Austrians with advanced chronic liver disease (ACLD) of various etiologies undergoing hepatic venous pressure gradient (HVPG) measurement (cohort ii), 231 French patients with alcoholic cirrhosis (cohort iii), and 280 hospitalized Germans with decompensated cirrhosis of various etiologies (cohort iv). Results (i) Hepatic K19 levels were comparable among F0–F3 fibrosis stages, but increased in cirrhosis. Hepatic K19 mRNA strongly correlated with the levels of other DR-specific keratins. (ii) In ACLD, increased serum CYFRA21-1 associated with the presence of clinically significant portal hypertension (CSPH; HVPG ≥ 10 mmHg) (OR = 5.87 [2.95–11.68]) and mortality (HR = 3.02 [1.78–5.13]; median follow-up 22 months). (iii) In alcoholic cirrhosis, elevated serum CYFRA21-1 indicated increased risk of death/liver transplantation (HR = 2.59 [1.64–4.09]) and of HCC (HR = 1.74 [1.02–2.96]) over the long term (median follow-up 73 months). (iv) In decompensated cirrhosis, higher serum CYFRA21-1 predicted 90-day mortality (HR = 2.97 [1.92–4.60]) with a moderate accuracy (AUROC 0.64), independently from established prognostic scores. Conclusions Hepatic K19 mRNA and serum CYFRA21-1 levels rise in cirrhosis. Increased CYFRA21-1 levels associate with the presence of CSPH and reliably indicate mortality in the short and long term independently of conventional liver biochemistry markers or scoring systems. Hence, the widely available serum CYFRA21-1 constitutes a novel, DR-related marker with prognostic implications in patients with different settings of advanced liver disease.

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.

Background & Aims Heat shock protein (Hsp) 72 is a molecular chaperone that has broad cytoprotective functions and is upregulated in response to stress. To determine its hepatic functions, we studied its expression in human liver disorders and its biological significance in newly generated transgenic animals. Methods Double transgenic mice overexpressing Hsp72 (gene Hspa1a ) under the control of a tissue-specific tetracycline-inducible system ( Hsp72-LAP mice) were produced. Acute liver injury was induced by a single injection of acetaminophen (APAP). Feeding with either a methionine choline-deficient (MCD; 8 weeks) or a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet (DDC; 12 weeks) was used to induce lipotoxic injury and Mallory–Denk body (MDB) formation, respectively. Primary hepatocytes were treated with palmitic acid. Results Patients with non-alcoholic steatohepatitis and chronic hepatitis C infection displayed elevated HSP72 levels. These levels increased with the extent of hepatic inflammation and HSP72 expression was induced after treatment with either interleukin (IL)-1β or IL-6. Hsp72-LAP mice exhibited robust, hepatocyte-specific Hsp72 overexpression. Primary hepatocytes from these animals were more resistant to isolation-induced stress and Hsp72-LAP mice displayed lower levels of hepatic injury in vivo . Mice overexpressing Hsp72 had fewer APAP protein adducts and were protected from oxidative stress and APAP-/MCD-induced cell death. Hsp72-LAP mice and/or hepatocytes displayed significantly attenuated Jnk activation. Overexpression of Hsp72 did not affect steatosis or the extent of MDB formation. Conclusions Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions. Lay summary HSP72 constitutes a stress-inducible, protective protein. Our data demonstrate that it is upregulated in patients with chronic hepatitis C and non-alcoholic steatohepatitis. Moreover, Hsp72-overexpressing mice are protected from various forms of liver stress.

The presence of the homozygous &lsquo, Pi*Z&rsquo, variant of alpha-1 antitrypsin (AAT) (&lsquo, Pi*ZZ&rsquo, genotype) predisposes to liver fibrosis development, but the role of iron metabolism in this process remains unknown. Therefore, we assessed iron metabolism and variants in the Homeostatic Iron Regulator gene (HFE) as the major cause of hereditary iron overload in a large cohort of Pi*ZZ subjects without liver comorbidities. The human cohort comprised of 409 Pi*ZZ individuals and 254 subjects without evidence of an AAT mutation who were recruited from ten European countries. All underwent a comprehensive work-up and transient elastography to determine liver stiffness measurements (LSM). The corresponding mouse models (Pi*Z overexpressors, HFE knockouts, and double transgenic [DTg] mice) were used to evaluate the impact of mild iron overload on Pi*Z-induced liver injury. Compared to Pi*Z non-carriers, Pi*ZZ individuals had elevated serum iron, transferrin saturation, and ferritin levels, but relevant iron overload was rare. All these parameters were higher in individuals with signs of significant liver fibrosis (LSM &ge, 7.1 kPa) compared to those without signs of significant liver fibrosis. HFE knockout and DTg mice displayed similar extent of iron overload and of fibrosis. Loss of HFE did not alter the extent of AAT accumulation. In Pi*ZZ individuals, presence of HFE mutations was not associated with more severe liver fibrosis. Taken together, Pi*ZZ individuals display minor alterations in serum iron parameters. Neither mild iron overload seen in these individuals nor the presence of HFE mutations (C282Y and H63D) constitute a major contributor to liver fibrosis development.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated keratins 8/18 (K8/K18). MDBs are characteristic of alcoholic and nonalcoholic steatohepatitis (NASH) and discriminate between the relatively benign simple steatosis and the more aggressive NASH. Given the emerging evidence for a genetic predisposition to MDB formation and NASH development in general, we studied whether high-fat (HF) diet triggers MDB formation and liver injury in susceptible animals. Mice were fed a high-fat (HF) or low-fat (LF) diet plus a cofactor for MDB development, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Additionally, we fed nontransgenic and K8 overexpressing mice (K8tg) with the HF diet. The presence of MDB and extent of liver injury was evaluated using biochemical markers, histological staining, and immunofluorescence microscopy. In DDC-fed animals, an HF diet resulted in greater liver injury and up-regulation of inflammation-related genes. As a potential mechanism, K8/K18 accumulation and increased ecto-5′-nucleotidase (CD73) levels were noted. In the genetically susceptible K8tg mice, HF diet triggered hepatocellular injury, ballooning, apoptosis, inflammation, and MDB development by way of 1) decreased expression of the major stress-inducible chaperone Hsp72 with appearance of misfolded keratins; 2) elevated levels of the transglutaminase 2 (TG2); 3) increased K8 phosphorylation at S74 with subsequent TG2-mediated crosslinking of phosphorylated K8; and 4) higher production of the MDB-modifier gene CD73. Conclusion: Our data demonstrate that HF diet triggers aggregate formation and development of liver injury in susceptible individuals through misfolding and crosslinking of excess K8. (Hepatology 2014;60:169–178)

Background & Aims Alpha-1 antitrypsin deficiency (AATD) is among the most common genetic disorders. Severe AATD is caused by a homozygous mutation in the SERPINA1 gene that encodes the Glu342Lys substitution (called the Pi*Z mutation, Pi*ZZ genotype). Pi*ZZ carriers may develop lung and liver diseases. Mutation-associated lung disorders have been well studied, but less is known about the effects in liver. We assessed the liver disease burden and associated features in adults with this form of AATD. Methods We collected data from 554 Pi*ZZ adults (403 in an exploratory cohort, 151 in a confirmatory cohort), in 9 European countries, with AATD who were homozygous for the Pi*Z mutation, and 234 adults without the Pi*Z mutation (controls), all without pre-existing liver disease. We collected data on demographic parameters, comorbidities, lung- and liver-related health, and blood samples for laboratory analysis. Liver fibrosis was assessed non-invasively via the serum tests Aspartate Aminotransferase to Platelet Ratio Index and HepaScore and via transient elastography. Liver steatosis was determined via transient elastography-based controlled attenuation parameter. We performed histologic analyses of livers from transgenic mice that overexpress the AATD-associated Pi*Z variant. Results Serum levels of liver enzymes were significantly higher in Pi*ZZ carriers vs controls. Based on non-invasive tests for liver fibrosis, significant fibrosis was suspected in 20%–36% of Pi*ZZ carriers, whereas signs of advanced fibrosis were 9- to 20-fold more common in Pi*ZZ carriers compared to non-carriers. Male sex; age older than 50 years; increased levels of alanine aminotransferase, aspartate aminotransferase, or γ-glutamyl transferase; and low numbers of platelets were associated with higher liver fibrosis burden. We did not find evidence for a relationship between lung function and liver fibrosis. Controlled attenuation parameter ≥280 dB/m, suggesting severe steatosis, was detected in 39% of Pi*ZZ carriers vs 31% of controls. Carriers of Pi*ZZ had lower serum concentrations of triglyceride and low- and very-low-density lipoprotein cholesterol than controls, suggesting impaired hepatic secretion of lipid. Livers from Pi*Z-overexpressing mice had steatosis and down-regulation of genes involved in lipid secretion. Conclusions In studies of AATD adults with the Pi*ZZ mutation, and of Pi*Z-overexpressing mice, we found evidence of liver steatosis and impaired lipid secretion. We identified factors associated with significant liver fibrosis in patients, which could facilitate hepatologic assessment and counseling of individuals who carry the Pi*ZZ mutation. ClinicalTrials.gov Number NCT02929940 .

We focus on hepatic inclusions, which are defined as intracellular aggregates of stainable substances. They represent established hallmarks of their respective human disorders, but unlike aggregates found in neurodegenerative disorders are often not well studied. Hepatic inclusions can be subdivided into primary liver aggregates and aggregates found in multiple tissues. The former ones consist of inclusions found in endoplasmic reticulum storage diseases such as α 1-antitrypsin aggregates or ground-glass hepatocytes, p62-containing (Mallory-Denk bodies and intracellular hyaline bodies) and porphyrin-containing inclusions. p62-containing aggregates are not restricted to the liver but are found in multiple other disorders such as Parkinson or Alzheimer disease. Inclusions such as pale bodies or intracellular hyaline bodies are typical for malignant disorders while others (ground-glass hepatocytes and α1-antitrypsin aggregates) are predominantly seen in non-neoplastic tissues. The inclusions, which are not restricted to the liver, are often due to a systemic viral infection, but also due to disruption of glycogen metabolism or systemic inclusion-forming diseases such as polyglutamine disorders or sarcoidosis. Despite their heterogeneity, inclusions share several pathogenic principles such as an imbalance between protein damage/misfolding on one side and repair/degradation on the other side. This is why hepatic aggregates represent a valuable tool to study the aggregation process in general and to improve our understanding of inclusions found in multiple human disorders. © 2013 American Physiological Society. Compr Physiol 3:1393-1436, 2013.

The liver is the central regulator of iron metabolism and accordingly, chronic liver diseases often lead to systemic iron overload due to diminished expression of the iron-regulatory hormone hepcidin. To study the largely unknown regulation of iron metabolism in the context of hepatic disease, we used two established models of chronic liver injury, ie repeated carbon tetrachloride (CCl(4)) or thioacetamide (TAA) injections. To determine the impact of CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) on hepcidin production, the effect of a single TAA injection was determined in wild-type and CHOP knockout mice. Furthermore, CHOP and hepcidin expression was assessed in control subjects and patients with alcoholic liver disease. Both chronic injury models developed a distinct iron overload in macrophages. TAA-, but not CCl(4) - injected mice displayed additional iron accumulation in hepatocytes, resulting in a significant hepatic and systemic iron overload which was due to suppressed hepcidin levels. C/EBPα signalling, a known hepcidin inducer, was markedly inhibited in TAA mice, due to lower C/EBPα levels and overexpression of CHOP, a C/EBPα inhibitor. A single TAA injection resulted in a long-lasting (> 6 days) suppression of hepcidin levels and CHOP knockouts (compared to wild-types) displayed significantly attenuated hepcidin down-regulation in response to acute TAA administration. CHOP mRNA levels increased 5-fold in alcoholic liver disease patients versus controls (p < 0.005) and negatively correlated with hepcidin expression. Our results establish CHOP as an important regulator of hepatic hepcidin expression in chronic liver disease. The differences in iron metabolism between the two widely used fibrosis models likely reflect the differential regulation of hepcidin expression in human liver disease.

Simple epithelial keratins (SEKs) are the cytoplasmic intermediate filament proteins of single-layered and glandular epithelial cells as found in the liver, pancreas, intestine, and lung. SEKs have broad cytoprotective functions, which are facilitated by dynamic posttranslational modifications and interaction with associated proteins. SEK filaments are composed of obligate heteropolymers of type II (K7, K8) and type I (K18-K20, K23) keratins. The multifaceted roles of SEKs are increasingly appreciated due to findings obtained from transgenic mouse models and human studies that identified SEK variants in several digestive diseases. Reorganization of the SEK network into aggregates called Mallory-Denk bodies (MDBs) is characteristic for specific liver disorders such as alcoholic and nonalcoholic steatohepatitis. To spur further research on SEKs, we here review the methods and potential caveats of their isolation as well as possibilities to study them in cell culture. The existing transgenic SEK mouse models, their advantages and potential drawbacks are discussed. The tools to induce MDBs, ways of their visualization and quantification, as well as the possibilities to detect SEK variants in humans are summarized.

Simple epithelial keratins (SEKs) are the cytoplasmic intermediate filament proteins of single-layered and glandular epithelial cells as found in the liver, pancreas, intestine, and lung. SEKs have broad cytoprotective functions, which are facilitated by dynamic posttranslational modifications and interaction with associated proteins. SEK filaments are composed of obligate heteropolymers of type II (K7, K8) and type I (K18-K20, K23) keratins. The multifaceted roles of SEKs are increasingly appreciated due to findings obtained from transgenic mouse models and human studies that identified SEK variants in several digestive diseases. Reorganization of the SEK network into aggregates called Mallory-Denk bodies (MDBs) is characteristic for specific liver disorders such as alcoholic and nonalcoholic steatohepatitis. To spur further research on SEKs, we here review the methods and potential caveats of their isolation as well as possibilities to study them in cell culture. The existing transgenic SEK mouse models, their advantages and potential drawbacks are discussed. The tools to induce MDBs, ways of their visualization and quantification, as well as the possibilities to detect SEK variants in humans are summarized.

Background & Aims Keratins (K) constitute the epithelial intermediate filaments. Among them, K7/K19 are widely used markers of the regenerative liver response termed ductular reaction (DR) that consists of activated biliary epithelial cells (BECs) and hepatic progenitor cells (HPCs) and correlates with liver disease severity. In the present study we aimed to characterize K23 in the liver. Methods We analyzed the expression and localization of K23 in the digestive system under basal conditions as well as in various human and mouse liver diseases/stress models. Cell culture studies were used to study factors regulating K23 expression. Results In untreated mice, K23 was restricted to biliary epithelia. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e., multidrug resistance protein 2 ( Mdr2 ) knockouts, animals treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine or subjected to bile duct ligation. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct co-localization with K7/K19. In chronic human liver disease, K23 expression increased in patients with a more prominent inflammation/fibrosis. A dramatic upregulation (>200times) was observed in patients with acute liver failure (ALF) and end-stage primary biliary cholangitis (PBC). Patients with alcoholic liver cirrhosis displayed increased K23 serum levels. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TNF-related weak inducer of apoptosis (TWEAK), and the type I acute phase inducer interleukin (IL)-1β but not the type II inducer IL-6 elevated K23 expression. Conclusions K23 represents a specific, stress-inducible DR marker, whose levels correlate with liver disease severity. K23 may represent a useful non-invasive DR marker. Lay summary Ductular reaction represents a basic response to liver injury and correlates with liver disease severity. Our study identifies K23 as a novel ductular reaction marker in mice and humans.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Keratins 8 and 18 (K8/K18) are the intermediate filaments proteins of simple-type digestive epithelia and provide important cytoprotective function. K8/K18 variants predispose humans to chronic liver disease progression and poor outcomes in acute acetaminophen (APAP)-related liver failure. Given that K8 G62C and R341H/R341C are common K8 variants in European and North American populations, we studied their biological significance using transgenic mice. Mice that overexpress the human K8 variants, R341H or R341C, were generated and used together with previously described mice that overexpress wild-type K8 or K8 G62C. Mice were injected with 600 mg/kg of APAP or underwent bile duct ligation (BDL). Livers were evaluated by microarray analysis, quantitative real-time polymerase chain reaction, immunoblotting, histological and immunological staining, and biochemical assays. Under basal conditions, the K8 G62C/R341H/R341C variant-expressing mice did not show an obvious liver phenotype or altered keratin filament distribution, whereas K8 G62C/R341C animals had aberrant disulphide cross-linked keratins. Animals carrying the K8 variants displayed limited gene expression changes, but had lower nicotinamide N-methyl transferase (NNMT) levels and were predisposed to APAP-induced hepatotoxicity. NNMT represents a novel K8/K18-associated protein that becomes up-regulated after K8/K18 transfection. The more pronounced liver damage was accompanied by increased and prolonged JNK activation; elevated APAP protein adducts; K8 hyperphosphorylation at S74/S432 with enhanced keratin solubility; and prominent pericentral keratin network disruption. No differences in APAP serum levels, glutathione, or adenosine triphosphate levels were noted. BDL resulted in similar liver injury and biliary fibrosis in all mouse genotypes. Conclusion: Expression of human K8 variants G62C, R341H, or R341C in mice predisposes to acute APAP hepatotoxicity, thereby providing direct evidence for the importance of these variants in human acute liver failure. (Hepatology 2015) Hepatology 2015;62:876–886)

Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated keratins 8/18 (K8/K18). MDBs are characteristic of alcoholic and nonalcoholic steatohepatitis (NASH) and discriminate between the relatively benign simple steatosis and the more aggressive NASH. Given the emerging evidence for a genetic predisposition to MDB formation and NASH development in general, we studied whether high-fat (HF) diet triggers MDB formation and liver injury in susceptible animals. Mice were fed a high-fat (HF) or low-fat (LF) diet plus a cofactor for MDB development, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Additionally, we fed nontransgenic and K8 overexpressing mice (K8tg) with the HF diet. The presence of MDB and extent of liver injury was evaluated using biochemical markers, histological staining, and immunofluorescence microscopy. In DDC-fed animals, an HF diet resulted in greater liver injury and up-regulation of inflammation-related genes. As a potential mechanism, K8/K18 accumulation and increased ecto-5'-nucleotidase (CD73) levels were noted. In the genetically susceptible K8tg mice, HF diet triggered hepatocellular injury, ballooning, apoptosis, inflammation, and MDB development by way of 1) decreased expression of the major stress-inducible chaperone Hsp72 with appearance of misfolded keratins; 2) elevated levels of the transglutaminase 2 (TG2); 3) increased K8 phosphorylation at S74 with subsequent TG2-mediated crosslinking of phosphorylated K8; and 4) higher production of the MDB-modifier gene CD73.Our data demonstrate that HF diet triggers aggregate formation and development of liver injury in susceptible individuals through misfolding and crosslinking of excess K8.

Background & Aims: Keratins (Ks) 7, 8, 18 and 19 constitute important markers and modifiers of liver disease. In mice, K8 and K18 are stress inducible and a dysregulated K8 > K18 stoichiometry predisposes to formation of Mallory–Denk bodies (MDBs), i.e. aggregates characteristic of chronic liver disorders such as alcoholic liver disease (ALD). In our study, we analyse the expression and the regulation of keratins in context of human liver disease. Methods: K7, K8, K18 and K19 mRNA levels were determined in liver biopsies from patients with ALD, non-alcoholic steatohepatitis (NASH), chronic hepatitis B (HBV), hepatitis C (HCV) and from control subjects. HepG2 and Hep3B cells were treated with IL-1b, IL-6 and TNF-a. Mice were injected with turpentine, an established IL-6 inducer. Results: K7, K8 and K18 were 1.5- to 3-fold upregulated in livers of ALD and HCV patients with a more active disease, but not in HBV/NASH subjects, while K19 was significantly elevated in all analysed disorders. K8 and K18 expression displayed a strong correlation (r = 0.89), but dysregulated levels with the K8 > K18 state were seen in ALD. All keratins were overexpressed in subjects with moderate vs. minimal inflammation, while K7, K8 and K18 were upregulated in patients with advanced liver fibrosis. In HepG2/Hep3B cells, IL-6 treatment but not IL-1b or TNF-a significantly increased K8 and K18 expression and elevated K18 levels were seen after turpentine injection. Conclusions: Keratins represent type II acute-phase responsive genes overexpressed in specific human liver disorders. A K8 > K18 state occurs in ALD and predisposes to MDB formation.

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117–1128)