Mallory-Denk Bodies Formation Research Articles

Male LEW.1WR1 Rats Develop Metabolic Dysfunction, Steatohepatitis, and Liver Damage

Most patients with non-alcoholic steatohepatitis (NASH) have insulin resistance, and there is a near-universal association between NASH and insulin resistance. Insulin resistance induces lipid accumulation in the liver, leading to the development of metabolic syndrome. However, most NASH rodent models fail to develop metabolic syndrome. LEW.1WR1 rats that are 23 weeks old showed increased body mass, epididymal fat, and liver mass, suggesting obesity-driven metabolic dysfunction. We have characterized steatosis, inflammation, Mallory–Denk body formation with hematoxylin and eosin (H&E), and fibrosis with Trichome blue staining. The presence of hepatic fibrosis with other features of NASH described above is one of the major strengths of this model since most of the currently available NASH models do not develop microvesicular steatosis or fibrosis. Together with the other important features of NASH described above, we confirm that male LEW.1WR1 rats develop NASH and insulin resistance with a standard diet.

Mallory-Denk bodies and hepatocellular senescence: a causal relationship?

Mallory-Denk bodies (MDBs) are hepatocellular cytoplasmic inclusions, which occur in certain chronic liver diseases, such as alcohol-related (ASH) and metabolic dysfunction-associated (MASH) steatohepatitis, copper toxicosis, some drug-induced liver disorders, chronic cholangiopathies, and liver tumors. Our study focused on the expression of the senescence markers p21WAF1/cip1 and p16INK4a in hepatocytes containing MDBs in steatohepatitis, chronic cholangiopathies with fibrosis or cirrhosis, Wilson’s disease, and hepatocellular carcinomas. Cytoplasm and nuclei of MDB-containing hepatocytes as well as MDB inclusions, except those associated with carcinoma cells, were strongly p16-positive, p21-positive, as well as p21-negative nuclei in MDB-containing hepatocytes which were observed whereas MDBs were p21-negative. Expression of the senescence marker p16 suggests that MDB formation reflects an adaptive response to chronic stress resembling senescence with its consequences, i.e., expression of inflammation- and fibrosis-prone secretome. Thus, senescence can be regarded as “double-edged sword” since, on the one hand, it may be an attempt of cellular defense, but, on the other, also causes further and sustained damage by inducing inflammation and fibrosis related to the senescence-associated secretory phenotype and thus progression of chronic liver disease.

Single-cell transcriptomes identifies characteristic features of mouse macrophages in liver Mallory-Denk bodies formation

Mallory-Denk bodies (MDBs) consist of intracellular aggregates of misfolded proteins in ballooned hepatocytes and serve as important markers of progression in certain liver diseases. Resident hepatic macrophage-mediated inflammation influences the development of chronic liver diseases and cancer. Here, the first systematic study of macrophages heterogeneity in mice was conducted to illustrate the pathogenesis of MDB formation using single-nucleus RNA sequencing (snRNA-seq). Furthermore, we provided transcriptional profiles of macrophages obtained from the fractionation of mouse liver tissues following chronic injury. We equally identified seven discrete macrophage subpopulations, each involved in specific cellular activated pathways such as basal metabolism, immune regulation, angiogenesis, and cell cycle regulation. Among these, a specific macrophage cluster (Cluster4), a subpopulation specifically expressing genes that regulate cell division and the cell cycle, was identified. Interestingly, we found that CCR2 was significantly induced in Cluster2, thereby inducing monocytes to migrate to macrophages to promote MDB pathogenesis. Thus, our study is the first to demonstrate the heterogeneity of macrophages associated with liver MDB formation in mice through single-cell resolution. This serves as the basis for further insights into the pathogenesis of liver MDB formation and molecular mechanisms of chronic liver disease progression.

Altered regulation of LncRNA analysis of human alcoholic hepatitis with Mallory-Denk Bodies (MDBs) is revealed by RNA sequencing

Mallory-Denk Bodies (MDBs) are prevalent in a variety of liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Long noncoding RNAs (lncRNAs) are considered as emerging new gene regulators, which participates in many functional activities through diverse mechanisms. We previously reported the mechanisms involved in the formation of liver MDBs in mouse model and in AH livers where MDBs had formed. To investigate the regulation of mRNAs expression and the probable role of lncRNAs in AH livers with MDBs, RNA-Seq analyses was further conducted to determine the mRNA and lncRNA expression profiles of the AH livers compared with the normal livers. It showed that different lncRNAs have different information contribution degrees by principal component analysis, and the integrated analysis of lncRNA-mRNA co-expression networks were linked to endocytosis, cell cycle, p53 signaling pathways in the human. Based on the co-expression networks, we identify 36 mRNAs that could be as potential biomarkers of alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). To our knowledge, this is the first report on the regulatory network of lncRNAs associated with liver MDB formation in human, and these results might offer new insights into the molecular mechanisms of liver MDB formation and the progression of AH to HCC.

Trehalose alleviates oxidative stress-mediated liver injury and Mallor-Denk body formation via activating autophagy in mice.

Autophagy is a degradation pathway for long-lived cytoplasmic proteins or damaged organelles and also for many aggregate-prone and disease-causing proteins. Endoplasmic reticulum (ER) stress and oxidative stress are associated with the pathophysiology of various liver diseases. These stresses induce the accumulation of abnormal proteins, Mallory-Denk body (MDB) formation and apoptosis in hepatocytes. A disaccharide trehalose had been reported to induce autophagy and decrease aggregate-prone proteins and cytotoxicity in neurodegenerative disease models. But the effects of trehalose in hepatocytes have not been fully understood. We examined the effect of trehalose on autophagy, ER stress and oxidative stress-mediated cytotoxicity and MDB formation in hepatocytes using mice model with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment for 3months. We administered trehalose by intraperitoneal injection of water containing 10% trehalose (0.02mg/g body weight) every other day for 3months. Our results demonstrated that trehalose induced autophagy and reduced ER stress, oxidative stress, MDB formation and apoptosis in hepatocytes of DDC-fed mice by Western blotting and immunostaining analyses. Electron microscopy revealed that trehalose induced autolysosome formation, which located is close to the MDBs. Thus, our findings suggest that trehalose can become a therapeutic agent for oxidative stress-related liver diseases via activating autophagy.

High Keratin 8/18 Ratio Predicts Aggressive Hepatocellular Cancer Phenotype

BACKGROUND & AIMS: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termed Mallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation. METHODS: We analyzed livers of aged Krt18−/− mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18−/− mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis. RESULTS: Our analysis of the genetic profile of Krt18−/− mice with 26 human hepatoma cell lines and with data sets of >300 patients with HCC, where Krt18−/− gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype. CONCLUSIONS: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC.

The PPAR\u03b1 Agonist Fenofibrate Prevents Formation of Protein Aggregates (Mallory-Denk bodies) in a Murine Model of Steatohepatitis-like Hepatotoxicity

Chronic intoxication of mice with the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to morphological and metabolic changes closely resembling steatohepatitis, a severe form of metabolic liver disease in humans. Since human steatohepatitis (both the alcoholic and non-alcoholic type) is characterized by reduced expression of PPARα and disturbed lipid metabolism we investigated the role of this ligand-activated receptor in the development of DDC-induced liver injury. Acute DDC-intoxication was accompanied by early significant downregulation of Pparα mRNA expression along with PPARα-controlled stress-response and lipid metabolism genes that persisted in the chronic stage. Administration of the specific PPARα agonist fenofibrate together with DDC prevented the downregulation of PPARα-associated genes and also improved the stress response of Nrf2-dependent redox-regulating genes. Moreover, oxidative stress and inflammation were strongly reduced by DDC/fenofibrate co-treatment. In addition, fenofibrate prevented the disruption of hepatocyte intermediate filament cytoskeleton and the formation of Mallory-Denk bodies at late stages of DDC intoxication. Our findings show that, like in human steatohepatitis, PPARα is downregulated in the DDC model of steatohepatitis-like hepatocellular damage. Its downregulation and the pathomorphologic features of steatohepatitis are prevented by co-administration of fenofibrate.

Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling

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. 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. 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. Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions. 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 role of Tec kinase signaling pathways in the development of Mallory Denk Bodies in balloon cells in alcoholic hepatitis

Several research strategies have been used to study the pathogenesis of alcoholic hepatitis (AH). These strategies have shown that various signaling pathways are the target of alcohol in liver cells. However, few have provided specific mechanisms associated with Mallory-Denk Bodies (MDBs) formed in Balloon cells in AH. The formation of MDBs in these hepatocytes is an indication that the mechanisms of protein quality control have failed. The MDB is the result of aggregation and accumulation of proteins in the cytoplasm of balloon degenerated liver cells. To understand the mechanisms that failed to degrade and remove proteins in the hepatocyte from patients suffering from alcoholic hepatitis, we investigated the pathways that showed significant up regulation in the AH liver biopsies compared to normal control livers (Liu et al., 2015). Analysis of genomic profiles of AH liver biopsies and control livers by RNA-seq revealed different pathways that were up regulated significantly. In this study, the focus was on Tec kinase signaling pathways and the genes that significantly interrupt this pathway. Quantitative PCR and immunofluorescence staining results, indicated that several genes and proteins are significantly over expressed in the livers of AH patients that affect the Tec kinase signaling to PI3K which leads to activation of Akt and its downstream effectors.

Up regulation of Spleen Tyrosine Kinase in ballooned hepatocytes with Mallory‐Denk Bodies in Alcoholic Liver Disease

Liver injury from alcohol abuse causes balloon hepatocytes and MDB formation. Mallory‐Denk bodies (MDBs) are found in 70% to 75% of patients with alcoholic hepatitis (AH). MDBs are composed of intracellular aggregations of misfolded proteins in ballooned hepatocytes. To determine the mechanism of balloon degeneration and MDB formation in hepatocytes, we evaluated the role of spleen tyrosine kinase (Syk) which is associated with transmembrane receptors to mediate numerous signal transductions downstream of these receptors. Spleen tyrosine kinase is widely expressed in hematopoietic cells and modulates several signaling pathways including PIK3/AKT and p7056 pathways. These pathways are important in regulating the cell cycle and are actively involved in mediating cell adhesion. Dysregulation of the PI3K/Akt pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. In this study, liver biopsy sections fixed in formalin and embedded in paraffin (FFPE) were used from patients with alcoholic hepatitis (AH). The results show that the expression of Syk protein is up regulated by RNAseq and real time PCR analyses in the alcoholic hepatitis patients compared to controls. The results were also supported by using IHC fluorescent intensity staining morphometric system. Morphometric quantification of fluorescent intensity showed a two fold increase in Syk in the cells forming MDBs compared to surrounding normal hepatocytes. Interestingly, the upregulation of Syk protein is co‐localized with ubiquitinated proteins in the cytoplasm of balloon cell hepatocytes which have formed MDBs. This observation demonstrates the role of Syk as a potential multifunctional target in protein quality control mechanism of hepatocytes when ER stress is activated.

Role of Protein Quality Control Failure in Alcoholic Hepatitis Pathogenesis.

The mechanisms of protein quality control in hepatocytes in cases of alcoholic hepatitis (AH) including ufmylation, FAT10ylation, metacaspase 1 (Mca1), ERAD (endoplasmic reticulum-associated degradation), JUNQ (juxta nuclear quality control), IPOD (insoluble protein deposit) autophagocytosis, and ER stress are reviewed. The Mallory–Denk body (MDB) formation develops in the hepatocytes in alcoholic hepatitis as a consequence of the failure of these protein quality control mechanisms to remove misfolded and damaged proteins and to prevent MDB aggresome formation within the cytoplasm of hepatocytes. The proteins involved in the quality control pathways are identified, quantitated, and visualized by immunofluorescent antibody staining of liver biopsies from patients with AH. Quantification of the proteins are achieved by measuring the fluorescent intensity using a morphometric system. Ufmylation and FAT10ylation pathways were downregulated, Mca1 pathways were upregulated, autophagocytosis was upregulated, and ER stress PERK (protein kinase RNA-like endoplasmic reticulum kinase) and CHOP (CCAAT/enhancer-binding protein homologous protein) mechanisms were upregulated. In conclusion: Despite the upregulation of several pathways of protein quality control, aggresomes (MDBs) still formed in the hepatocytes in AH. The pathogenesis of AH is due to the failure of protein quality control, which causes balloon-cell change with MDB formation and ER stress.

P62/Sequestosome-1 Is Indispensable for Maturation and Stabilization of Mallory-Denk Bodies.

Mallory-Denk bodies (MDBs) are hepatocytic protein aggregates found in steatohepatitis and several other chronic liver diseases as well as hepatocellular carcinoma. MDBs are mainly composed of phosphorylated keratins and stress protein p62/Sequestosome-1 (p62), which is a common component of cytoplasmic aggregates in a variety of protein aggregation diseases. In contrast to the well-established role of keratins, the role of p62 in MDB pathogenesis is still elusive. We have generated total and hepatocyte-specific p62 knockout mice, fed them with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce MDBs and allowed the mice to recover from DDC intoxication on a standard diet to investigate the role of p62 in MDB formation and elimination. In the absence of p62, smaller, granular and less distinct MDBs appeared, which failed to mature to larger and compact inclusions. Moreover, p62 deficiency impaired the binding of other proteins such as NBR1 and Hsp25 to MDBs and altered the cellular defense mechanism by downregulation of Nrf2 target genes. Upon recovery from DDC intoxication on a standard diet, there was an enhanced reduction of p62-deficient MDBs, which was accompanied by a pronounced decrease in ubiquitinated proteins. Our data provide strong evidence that keratin aggregation is the initial step in MDB formation in steatohepatitis-related mouse models. Interaction of p62 with keratin aggregates then leads to maturation i.e., enlargement and stabilization of the MDBs as well as recruitment of other MDB-associated proteins.

Aberrant Modulation of the BRCA1 and G1/S Cell Cycle Pathways in Alcoholic Hepatitis Patients with Mallory Denk Bodies Present Revealed by RNA Sequencing

Mallory‐Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Liver injury from alcohol administration causes balloon hepatocytes and MDB formation impeding liver regeneration. By comparing AH livers where MDBs had formed with normal liver transcriptomes obtained by RNA sequencing (RNA‐Seq), there was significant up regulation of BRCA1‐mediated signaling and G1/S cell cycle checkpoint pathways. The transcriptional architecture of differentially expressed genes from AH patients reflected step‐wise transcriptional changes progressing to AH. Key molecules such as BRCA1, p15 and p21 involved in BRCA1 signaling and G1/S cell cycle checkpoints were significantly up regulated both in patients’ livers and in the livers of the DDC re‐fed mice model where MDBs had formed. MiR‐483‐3p was down regulated in AH livers. The down regulation of miR‐483‐3p may be the mechanism for the up regulation of BRCA1 since miR‐483‐3p is an inhibitor of BRCA1 expression. The hyperactivation of G1/S cell cycle checkpoint inhibitors p15 and p21 results in cell cycle arrest and liver regeneration inhibition.ConclusionsProvided here for the first time is the RNA‐Seq data that represents the fully annotated catalogue of the expression of mRNAs. The most prominent alterations observed were the changes in BRCA1‐mediated signaling and G1/S cell cycle checkpoint pathways. These new findings expand previous and related knowledge in the search for gene changes that might be critical in the understanding of the underlying progression to the development of AH.Support or Funding InformationNIH (AAU01021898‐04).

Mallory Denk Body Formation in Alcoholic Hepatitis: The Pivotal Role of Interleukin-8 Signaling.

Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding diethyl 1,4-dehydro-2,4,6-trimethyl-3,5-pyridine-dicarboxylate (DDC). The chemokine CXCL8, also known as interleukin-8 (IL-8) and its receptors are involved in oncogenesis and in tumor progression, invasion, and metastasis. We reported previously the marked upregulation of IL-8 signaling in AH and DDC fed mice with MDBs present by RNA sequencing (RNA-Seq) analyses. Central molecules including IL-8 and the chemokine (C-X-C motif) receptor 2 (CXCR2) of this pathway were significantly upregulated in the livers of DDC refed mice and human liver biopsies from AH livers. MDB containing balloon hepatocytes in AH livers have increased intensity of staining of the cytoplasm for both IL-8 and CXCR2. Taken conjointly, these data indicates a crucial role of IL-8 signaling during MDB formation, and IL-8 and CXCR2 may be targeted as biomarkers for personalized treatment of AH.

Aberrant modulation of the BRCA1 and G1/S cell cycle pathways in alcoholic hepatitis patients with Mallory Denk Bodies revealed by RNA sequencing.

Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Liver injury from alcohol administration causes balloon hepatocytes and MDB formation impeding liver regeneration. By comparing AH livers where MDBs had formed with normal liver transcriptomes obtained by RNA sequencing (RNA-Seq), there was significant upregulation of BRCA1-mediated signaling and G1/S cell cycle checkpoint pathways. The transcriptional architecture of differentially expressed genes from AH livers reflected step-wise transcriptional changes progressing to AH. Key molecules such as BRCA1, p15 and p21 were significantly upregulated both in AH livers and in the livers of the DDC re-fed mice model where MDBs had formed. The increase of G1/S cell cycle checkpoint inhibitors p15 and p21 results in cell cycle arrest and inhibition of liver regeneration, implying that p15 and p21 could be exploited for the identification of specific targets for the treatment of liver disease. Provided here for the first time is the RNA-Seq data that represents the fully annotated catalogue of the expression of mRNAs. The most prominent alterations observed were the changes in BRCA1-mediated signaling and G1/S cell cycle checkpoint pathways. These new findings expand previous and related knowledge in the search for gene changes that might be critical in the understanding of the underlying progression to the development of AH.

Altered regulation of miR-34a and miR-483-3p in alcoholic hepatitis and DDC fed mice

MicroRNAs are small noncoding RNAs that negatively regulate gene expression by binding to the untranslated regions of their target mRNAs. Deregulation of miRNAs is shown to play pivotal roles in tumorigenesis and progression. Mallory–Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. By comparing AH livers where MDBs had formed with normal livers, there were significant changes of miR-34a and miR-483-3p by RNA sequencing (RNA-Seq) analyses. Real-time PCR further shows a 3- and 6-fold upregulation (respectively) of miR-34a in the AH livers and in the livers of DDC re-fed mice, while miR-483-3p was significantly downregulated in AH and DDC re-fed mice livers. This indicates that miR-34a and miR-483-3p may be crucial for liver MDB formation. P53 mRNA was found to be significantly downregulated both in the AH livers and in the livers of DDC re-fed mice, indicating that the upregulation of miR-34a is permitted by the decrease of p53 in AH since miR-34a is a main target of p53. Overexpression of miR-34a leads to an increase of p53 targets such as p27, which inhibits the cell cycle leading to cell cycle arrest. Importantly, BRCA1 is a target gene of miR-483-3p by RNA-Seq analyses and the downregulation of miR-483-3p may be the mechanism for liver MDB formation since the BRCA1 signal was markedly upregulated in AH livers. These results constitute a demonstration of the altered regulation of miR-34a and miR-483-3p in the livers of AH and mice fed DDC where MDBs formed, providing further insight into the mechanism of MDB formation mediated by miR-34a and miR-483-3p in AH.

IL-8 signaling is up-regulated in alcoholic hepatitis and DDC fed mice with Mallory Denk Bodies (MDBs) present

Chemokines and their receptors are involved in oncogenesis and in tumor progression, invasion, and metastasis. Various chemokines also promote cell proliferation and resistance to apoptosis of stressed cells. The chemokine CXCL8, also known as interleukin-8 (IL-8), is a proinflammatory molecule that has functions within the tumor microenvironment. Deregulation of IL-8 signaling is shown to play pivotal roles in tumorigenesis and progression. Mallory–Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. By comparing AH livers where MDBs had formed with normal livers, there were significant changes of IL-8 signaling by RNA sequencing (RNA-Seq) analyses. Real-time PCR analysis of CXCR2 further shows a 6-fold up-regulation in AH livers and a 26-fold up-regulation in the livers of DDC re-fed mice. IL-8 mRNA was also significantly up-regulated in AH livers and DDC re-fed mice livers. This indicates that CXCR2 and IL-8 may be crucial for liver MDB formation. MDB containing balloon hepatocytes in AH livers had increased intensity of staining of the cytoplasm for both CXCR2 and IL-8. Overexpression of IL-8 leads to an increase of the mitogen activated protein kinase (MAPK) cascade and exacerbates the inflammatory cycle. These observations constitute a demonstration of the altered regulation of IL-8 signaling in the livers of AH and mice fed DDC where MDBs formed, providing further insight into the mechanism of MDB formation mediated by IL-8 signaling in AH.

Alleviation mechanisms against hepatocyte oxidative stress in patients with chronic hepatic disorders.

Autophagy induction and Mallory-Denk body (MDB) formation have been considered to have cytoprotective effects from cellular stress in liver diseases. We investigated the relations among oxidative stress, autophagy and MDB formation in patients with chronic hepatitis B (CHB), chronic hepatitis C (CHC) and non-alcoholic fatty liver disease (NAFLD) to clarify the alleviation mechanisms against oxidative stress of hepatocytes. First, we treated cultured cells with proteasome inhibitor (PI) or free fatty acid (FFA) and evaluated endoplasmic reticulum (ER) stress, oxidative stress, ubiquitinated proteins and p62 by western blotting. Then, we used human liver biopsy samples to evaluate oxidative stress, autophagy and MDB formation by immunohistochemical analysis. Treatment with PI or FFA increased ER stress, oxidative stress, ubiquitinated proteins and p62 in cultured cells. Human liver biopsy samples of CHC and NAFLD showed that MDB formed in areas with strong oxidative stress and that the MDB-containing cells circumvented oxidative stress. Keratin 8 (K8) expression was strong in MDB-containing cells in CHC and NAFLD. However, in CHB samples, the expression of K8 was not increased in response to oxidative stress and MDB aggregates did not appear. Aminotransferase values were significantly lower in patients with CHC and NAFLD in whom light chain 3 antibody expression was increased in response to oxidative stress. Strong expression of K8 was considered to be important for MDB formation. MDB protect liver cells from oxidative stress at a cellular level and autophagy reduced hepatic damage when it was induced in the hepatocytes exposed to strong oxidative stress.

Mallory–Denk Body (MDB) formation modulates ufmylation expression epigenetically in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH)

Promoter CpG island hypermethylation is an important mechanism for inactivating key cellular enzymes that mediate epigenetic processes in hepatitis-related hepatocellular carcinoma (HCC). The ubiquitin-fold modifier 1 (Ufm1) conjugation pathway (Ufmylation) plays an essential role in protein degradation, protein quality control and signal transduction. Previous studies showed that the Ufmylation pathway was downregulated in alcoholic hepatitis (AH), non-alcoholic steatohepatitis (NASH) and in mice fed DDC, resulting in the formation of Mallory–Denk Bodies (MDBs). In this study, we further discovered that betaine, a methyl donor, fed together with DDC significantly prevents the increased expression of Ufmylation in drug-primed mice fed DDC. Betaine significantly prevented transcript silencing of Ufm1, Uba5 and UfSP1 where MDBs developed and also prevented the increased expression of FAT10 and LMP7 caused by DDC re-fed mice. Similar downregulation of Ufmylation was observed in multiple AH and NASH biopsies which had formed MDBs. The DNA methylation levels of Ufm1, Ufc1 and UfSP1 in the promoter CpG region were significantly increased both in AH and NASH patients compared to normal subjects. DNA (cytosine-5-)-methyltransferase 1 (DNMT1) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) mRNA levels were markedly upregulated in AH and NASH patients, implying that the maintenance of Ufmylation methylation might be mediated by DNMT1 and DNMT3B together. These data show that MDB formation results from Ufmylation expression epigenetically in AH and NASH patients. Promoter CpG methylation may be a major mechanism silencing Ufmylation expression.

The inflammasome in alcoholic hepatitis: Its relationship with Mallory–Denk body formation

Recent studies indicate that the inflammasome activation plays important roles in the pathogenesis of alcoholic hepatitis (AH). Nod-like receptor protein 3 (NLRP3) is a key component of the macromolecular complex that is so called the inflammasome that triggers caspase 1-dependent maturation of the precursors of IL-1β and IL-18 cytokines. It is also known that the adaptor proteins including apoptosis-associated speck-like protein containing CARD (ASC) and the mitochondrial antiviral signaling protein (MAVS) are necessary for NLRP3-dependent inflammasome function. Steatohepatitis frequently includes Mallory–Denk body (MDB) formation. In the case of alcoholic steatohepatitis, MDB formation occurs in 80% of biopsies (French 1981; French 1981). While previous studies have focused on in vitro cell lines and mouse models, we are the first group to investigate inflammasome activation in AH liver biopsy specimen and correlate it with MDB formation. Expression of NOD1, NLRP3, ASC, NAIP, MAVS, caspase 1, IL-1β, IL-18, and other inflammatory components including IL-6, IL-10, TNF-α, IFN-γ, STAT3, and p65 was measured in three to eight formalin-fixed paraffin-embedded AH specimens and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. The specimens were double stained with ubiquitin to demonstrate the relationship between inflammasome activation and MDB formation. MAVS, caspase1, IL-18, and TNF-α showed increases in expression in AH compared to the controls (p<0.05), and NAIP expression markedly increased in AH compared to the controls (p<0.01). There was a trend that levels of NLRP3, ASC, caspase1, IL-18, IL-10, and p65 expression correlated with the number of MDBs found in the same field of measurement (correlation coefficients were between 0.62 and 0.93, p<0.05). Our results demonstrate the activation of the inflammasome in AH and suggest that MDB could be an indicator of the extent of inflammasome activation.