Kupffer cell diversity maintains liver function in alcohol-associated liver disease.

BACKGROUND AND AIMS: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western diet alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by single cell RNAseq and targeted KC ablation to understand the diversity and function of liver macrophages in ALD. APPROACH AND RESULTS: In the WDA liver, KCs and infiltrating monocytes/macrophages each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor-mediated endocytosis and lipid metabolism, but most were predicted to be noninflammatory and antifibrotic with 1 minor KC cluster having a proinflammatory and extracellular matrix degradation gene signature. Infiltrating monocyte/macrophage clusters, in contrast, were predicted to be proinflammatory and profibrotic. In vivo, diphtheria toxin-based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK7, and Igf2bp3. Gene set enrichment analysis of whole-liver RNAseq from the KC-ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. CONCLUSIONS: In this ALD model, KCs are anti-inflammatory and are critical for the maintenance of hepatocyte differentiation. Infiltrating monocytes/macrophages are largely proinflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to the treatment of liver failure and fibrosis in ALD.

Alcohol-induced epigenetic changes prevent fibrosis resolution after alcohol cessation in miceresolution.

BACKGROUND AND AIMS: Alcohol-associated liver disease is a major cause of alcohol-associated mortality. Recently, we identified hepatic demethylases lysine demethylase (KDM)5B and KDM5C as important epigenetic regulators of alcohol response in the liver. In this study, we aimed to investigate the role of KDM5 demethylases in alcohol-associated liver disease resolution. APPROACH AND RESULTS: We showed that alcohol-induced liver steatosis rapidly resolved after alcohol cessation. In contrast, fibrosis persisted in the liver for up to 8 weeks after the end of alcohol exposure. Defects in fibrosis resolution were in part due to alcohol-induced KDM5B and KDM5C-dependent epigenetic changes in hepatocytes. Using cell-type-specific knockout mice, we found that adeno-associated virus-mediated knockout of KDM5B and KDM5C demethylases in hepatocytes at the time of alcohol withdrawal promoted fibrosis resolution. Single-cell ATAC sequencing analysis showed that during alcohol-associated liver disease resolution epigenetic cell states largely reverted to control conditions. In addition, we found unique epigenetic cell states distinct from both control and alcohol states and identified associated transcriptional regulators, including liver X receptor (LXR) alpha (α). In vitro and in vivo analysis confirmed that knockout of KDM5B and KDM5C demethylases promoted LXRα activity, likely through regulation of oxysterol biosynthesis, and this activity was critical for the fibrosis resolution process. Reduced LXR activity by small molecule inhibitors prevented fibrosis resolution in KDM5-deficient mice. CONCLUSIONS: In summary, KDM5B and KDM5C demethylases prevent liver fibrosis resolution after alcohol cessation in part through suppression of LXR activity.

Sepsis-induced endothelial dysfunction drives acute-on-chronic liver failure through Angiopoietin-2-HGF-C/EBPß pathway.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is an acute liver and multisystem failure in patients with previously stable cirrhosis. A common cause of ACLF is sepsis secondary to bacterial infection. Sepsis-associated ACLF involves a loss of differentiated liver function in the absence of direct liver injury, and its mechanism is unknown. We aimed to study the mechanism of sepsis-associated ACLF using a novel mouse model. APPROACH AND RESULTS: Sepsis-associated ACLF was induced by cecal ligation and puncture procedure (CLP) in mice treated with thioacetamide (TAA). The combination of TAA and CLP resulted in a significant decrease in liver synthetic function and high mortality. These changes were associated with reduced metabolic gene expression and increased CCAAT enhancer binding protein beta (C/EBPß) transcriptional activity. We found that C/EBPß binding to its target gene promoters was increased. In humans, C/EBPß chromatin binding was similarly increased in the ACLF group compared with control cirrhosis. Hepatocyte-specific Cebpb knockout mice had reduced mortality and increased gene expression of hepatocyte differentiation markers in TAA/CLP mice, suggesting that C/EBPß promotes liver failure in these mice. C/EBPß activation was associated with endothelial dysfunction, characterized by reduced Angiopoietin-1/Angiopoietin-2 ratio and increased endothelial production of HGF. Angiopoietin-1 supplementation or Hgf knockdown reduced hepatocyte C/EBPß accumulation, restored liver function, and reduced mortality, suggesting that endothelial dysfunction induced by sepsis drives ACLF through HGF-C/EBPß pathway. CONCLUSIONS: The transcription factor C/EBPß is activated in both mouse and human ACLF and is a potential therapeutic target to prevent liver failure in patients with sepsis and cirrhosis.

The polymorphism rs975484 in the protein arginine methyltransferase 1 gene modulates expression of immune checkpoint genes in hepatocellular carcinoma.

Protein arginine methyltransferase 1 (PRMT1) is a key regulator of hepatic immune responses. Recently, we reported that PRMT1 regulates the tumor immune response in hepatocellular carcinoma (HCC). Here we found that PRMT1 expression in human HCC correlates with that of programmed cell death 1 ligand 1 (PD-L1), PD-L2, and other checkpoint genes. PRMT1 deletion in mice reduced PD-L1 and PD-L2 expression in tumors and reduced the efficiency of PD-1 antibody treatment in a diethylnitrosamine-induced HCC mouse model, suggesting that PRMT1 regulates the hepatic immune checkpoint. Mice had reduced PD-L1 and PD-L2 expression when PRMT1 was specifically deleted in tumor cells or macrophages, but PRMT1 deletion in dendritic cells did not alter PD-L1 and PD-L2 expression. rs975484 is a common polymorphism in the human PRMT1 gene promoter, and we found that it alters PRMT1 expression in blood monocytes and tumor-associated macrophages in human HCC. PRMT1 expression was higher in individuals with a GG genotype than in individuals with a CC genotype, and heterozygous carriers had intermediate expression. Luciferase reporter assays indicated that this differential expression is due to an extra C/EBPß-binding site in the PRMT1 promoter of individuals carrying the minor G allele. The rs975484 genotype also correlated with PRMT1 target expression in HCC. Individuals with the GG genotype had significantly higher levels of the PRMT1 targets PD-L1, PD-L2, and VISTA than those with the CC genotype. We conclude that PRMT1 critically controls immune checkpoints in mice and humans and that the PRMT1 polymorphism rs975484 affects checkpoint gene expression in HCC.

A Western diet with alcohol in drinking water recapitulates features of alcohol-associated liver disease in mice.

BACKGROUND: Mouse models of alcohol-associated liver disease vary greatly in their ease of implementation and the pathology they produce. Effects range from steatosis and mild inflammation with the Lieber-DeCarli liquid diet to severe inflammation, fibrosis, and pyroptosis seen with the Tsukamoto-French intragastric feeding model. Implementation of all of these models is limited by the labor-intensive nature of the protocols and the specialized skills necessary for successful intragastric feeding. We thus sought to develop a new model to reproduce features of alcohol-induced inflammation and fibrosis with minimal operational requirements. METHODS: Over a 16-week period, mice were fed ad libitum with a pelleted high-fat Western diet (WD; 40% calories from fat) and alcohol added to the drinking water. We found the optimal alcohol consumption to be that at which the alcohol concentration was 20% for 4 days and 10% for 3 days per week. Control mice received WD pellets with water alone. RESULTS: Alcohol consumption was 18 to 20 g/kg/day in males and 20 to 22 g/kg/day in females. Mice in the alcohol groups developed elevated serum transaminase levels after 12 weeks in males and 10 weeks in females. At 16 weeks, both males and females developed liver inflammation, steatosis, and pericellular fibrosis. Control mice on WD without alcohol had mild steatosis only. Alcohol-fed mice showed reduced HNF4α mRNA and protein expression. HNF4α is a master regulator of hepatocyte differentiation, down-regulation of which is a known driver of hepatocellular failure in alcoholic hepatitis. CONCLUSION: A simple-to-administer, 16-week WD alcohol model recapitulates the inflammatory, fibrotic, and gene expression aspects of human alcohol-associated steatohepatitis.

Protein arginine methyl transferase 1- and Jumonji C domain-containing protein 6-dependent arginine methylation regulate hepatocyte nuclear factor 4 alpha expression and hepatocyte proliferation in mice.

Alcohol is a well-established risk factor for hepatocellular carcinoma (HCC), but the mechanisms by which it promotes liver cancer are not well understood. Several studies have shown that cellular protein arginine methylation is inhibited by alcohol. Arginine methylation is controlled by the reciprocal activity of protein arginine methyltransferases, primarily protein arginine methyl transferase 1 (PRMT1), and a demethylase Jumonji C domain-containing protein 6 (JMJD6). The aim of this study was to explore the role of arginine methylation changes in alcohol pathogenesis. We found that PRMT1 activity is inhibited in livers of mice fed with alcohol compared to pair-fed mice. Using hepatocyte-specific PRMT1 knockout mice, we identified that loss of PRMT1 results in enhanced hepatocyte proliferation and a 33% increase in liver size. This increased hepatocyte proliferation was associated with reduced expression of hepatocyte nuclear factor 4 alpha (Hnf4α), an important regulator of liver tumorigenesis. We found that PRMT1 regulates Hnf4α expression directly through arginine methylation at the (Hnf4α) promoter. In the absence of PRMT1, JMJD6 can demethylate the Hnf4α promoter and suppress its expression. We were able to restore Hnf4α expression and abolish the increase in hepatocyte proliferation by knockdown of JMJD6 in PRMT1 knockout mice. Knockdown of JMJD6 in alcohol-fed mice similarly increased Hnf4α expression. We then examined whether loss of arginine methylation might play a role in alcohol-associated liver cancers. We examined 25 human HCC specimens and found a strong correlation (R = 0.8; P < 0.01) between arginine methylation levels and Hnf4α expression in these specimens, suggesting that the above mechanism is relevant in patients. CONCLUSION: Taken together, these data suggest that PRMT1 inhibition, such as induced by alcohol, may result in epigenetic changes leading to loss of Hnf4α. This effect may contribute to alcohol's ability to promote liver tumors. (Hepatology 2018;67:1109-1126).

Arginine methylation regulates c-Myc-dependent transcription by altering promoter recruitment of the acetyltransferase p300.

Protein arginine methyltransferase 1 (PRMT1) is an essential enzyme controlling about 85% of the total cellular arginine methylation in proteins. We have shown previously that PRMT1 is an important regulator of innate immune responses and that it is required for M2 macrophage differentiation. c-Myc is a transcription factor that is critical in regulating cell proliferation and also regulates the M2 transcriptional program in macrophages. Here, we sought to determine whether c-Myc in myeloid cells is regulated by PRMT1-dependent arginine methylation. We found that PRMT1 activity was necessary for c-Myc binding to the acetyltransferase p300. PRMT1 inhibition decreased p300 recruitment to c-Myc target promoters and increased histone deacetylase 1 (HDAC1) recruitment, thereby decreasing transcription at these sites. Moreover, PRMT1 inhibition blocked c-Myc-mediated induction of several of its target genes, including peroxisome proliferator-activated receptor γ (PPARG) and mannose receptor C-type 1 (MRC1), suggesting that PRMT1 is necessary for c-Myc function in M2 macrophage differentiation. Of note, in primary human blood monocytes, p300-c-Myc binding was strongly correlated with PRMT1 expression, and in liver sections, PRMT1, c-Myc, and M2 macrophage levels were strongly correlated with each other. Both PRMT1 levels and M2 macrophage numbers were significantly lower in livers from individuals with a history of spontaneous bacterial peritonitis, known to have defective cellular immunity. In conclusion, our findings demonstrate that PRMT1 is an important regulator of c-Myc function in myeloid cells. PRMT1 loss in individuals with cirrhosis may contribute to their immune defects.

Protein arginine methyltransferase 1 modulates innate immune responses through regulation of peroxisome proliferator-activated receptor γ-dependent macrophage differentiation.

Arginine methylation is a common posttranslational modification that has been shown to regulate both gene expression and extranuclear signaling events. We recently reported defects in protein arginine methyltransferase 1 (PRMT1) activity and arginine methylation in the livers of cirrhosis patients with a history of recurrent infections. To examine the role of PRMT1 in innate immune responses in vivo, we created a cell type-specific knock-out mouse model. We showed that myeloid-specific PRMT1 knock-out mice demonstrate higher proinflammatory cytokine production and a lower survival rate after cecal ligation and puncture. We found that this defect is because of defective peroxisome proliferator-activated receptor γ (PPARγ)-dependent M2 macrophage differentiation. PPARγ is one of the key transcription factors regulating macrophage polarization toward a more anti-inflammatory and pro-resolving phenotype. We found that PRMT1 knock-out macrophages failed to up-regulate PPARγ expression in response to IL4 treatment resulting in 4-fold lower PPARγ expression in knock-out cells than in wild-type cells. Detailed study of the mechanism revealed that PRMT1 regulates PPARγ gene expression through histone H4R3me2a methylation at the PPARγ promoter. Supplementing with PPARγ agonists rosiglitazone and GW1929 was sufficient to restore M2 differentiation in vivo and in vitro and abrogated the difference in survival between wild-type and PRMT1 knock-out mice. Taken together these data suggest that PRMT1-dependent regulation of macrophage PPARγ expression contributes to the infection susceptibility in PRMT1 knock-out mice.

Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling.

Arginine methylation is a common post-translational modification, but its role in regulating protein function is poorly understood. This study demonstrates that, TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase involved in innate immune signaling, is regulated by reversible arginine methylation in a range of primary and cultured cells. Under basal conditions, TRAF6 is methylated by the methyltransferase PRMT1, and this inhibits its ubiquitin ligase activity, reducing activation of toll-like receptor signaling. In response to toll-like receptor ligands, TRAF6 is demethylated by the Jumonji domain protein JMJD6. Demethylation is required for maximal activation of NF-κB. Loss of JMJD6 leads to reduced response, and loss of PRMT1 leads to basal pathway activation with subsequent desensitization to ligands. In human primary cells, variations in the PRMT1/JMJD6 ratio significantly correlate with TRAF6 methylation, basal activation of NF-κB, and magnitude of response to LPS. Reversible arginine methylation of TRAF6 by the opposing effects of PRMT1 and JMJD6 is, therefore, a novel mechanism for regulation of innate immune pathways.

Regulation of FOXO3 by phosphorylation and methylation in hepatitis C virus infection and alcohol exposure.

UNLABELLED: Hepatitis C virus (HCV) infection produces chronic liver injury that is significantly exacerbated by alcohol consumption. While multiple mechanisms contribute to this synergy, a viral-induced loss of antioxidant responses has been shown to play an important role. This study examined the effects of HCV infection and alcohol on the regulation of the transcription factor FOXO3, an important regulator of Mn-superoxide dismutase (SOD2) expression, a tumor suppressor, and a component of the hepatic antioxidant response system. FOXO3 was activated by either HCV or alcohol alone but suppressed by the combination. To understand this paradoxical result, we applied a capillary isoelectric focusing (IEF) method to determine the pattern of FOXO3 posttranslational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used antiphosphoserine, acetyl-lysine, methylarginine, and ubiquitin antibodies to identify the PTM patterns present in each species. HCV caused multiple changes including phosphorylation of FOXO3 at S-574, a novel c-Jun N-terminal kinase (JNK) site, which promoted nuclear translocation and transcription. Ethanol suppressed arginine-methylation of FOXO3 promoting nuclear export and degradation of the JNK phosphorylated form. Human liver biopsy samples showed the presence of the HCV-specific form of FOXO3 in HCV-infected livers but not in normal liver or nonalcoholic steatohepatitis. CONCLUSION: The development of this novel IEF method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 PTMs that contribute to pathogenesis. This approach will allow further dissection of the role of protein PTMs in viral liver disease.

Hepatitis C and alcohol exacerbate liver injury by suppression of FOXO3.

Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3(-/-) mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2(+/-)) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol-induced liver injury.

Protective role of 17ß-estradiol in alcohol-associated liver fibrosis is mediated by suppression of integrin signaling.

BACKGROUND: Alcohol-associated liver disease is a complex disease regulated by genetic and environmental factors such as diet and sex. The combination of high-fat diet and alcohol consumption has synergistic effects on liver disease progression. Female sex hormones are known to protect females from liver disease induced by high-fat diet. In contrast, they promote alcohol-mediated liver injury. We aimed to define the role of female sex hormones on liver disease induced by a combination of high-fat diet and alcohol. METHODS: Wild-type and protein arginine methyltransferase (Prmt)6 knockout female mice were subjected to gonadectomy (ovariectomy, OVX) or sham surgeries and then fed western diet and alcohol in the drinking water. RESULTS: We found that female sex hormones protected mice from western diet/alcohol-induced weight gain, liver steatosis, injury, and fibrosis. Our data suggest that these changes are, in part, mediated by estrogen-mediated induction of arginine methyltransferase PRMT6. Liver proteome changes induced by OVX strongly correlated with changes induced by Prmt6 knockout. Using Prmt6 knockout mice, we confirmed that OVX-mediated weight gain, steatosis, and injury are PRMT6 dependent, while OVX-induced liver fibrosis is PRMT6 independent. Proteomic and gene expression analyses revealed that estrogen signaling suppressed the expression of several components of the integrin pathway, thus reducing integrin-mediated proinflammatory (Tnf, Il6) and profibrotic (Tgfb1, Col1a1) gene expression independent of PRMT6 levels. Integrin signaling inhibition using Arg-Gly-Asp peptides reduced proinflammatory and profibrotic gene expression in mice, suggesting that integrin suppression by estrogen is protective against fibrosis development. CONCLUSIONS: Taken together, estrogen signaling protects mice from liver disease induced by a combination of alcohol and high-fat diet through upregulation of Prmt6 and suppression of integrin signaling.

Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Stimulation of BK virus DNA replication by NFI family transcription factors.

BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.

Forkhead box class O transcription factors in liver function and disease.

The forkhead box transcription factor class O (FOXO) family represents a group of transcription factors that is required for a number of stress-related transcriptional programs including antioxidant response, gluconeogenesis, cell cycle control, apoptosis, and autophagy. The liver utilizes several FOXO-dependent pathways to adapt to its routine cycles of feeding and fasting and to respond to the stresses induced by disease. FOXO1 is a direct transcriptional regulator of gluconeogenesis, reciprocally regulated by insulin, and has profound effects on hepatic lipid metabolism. FOXO3 is required for antioxidant responses and autophagy and is altered in hepatitis C infection and fatty liver. Emerging evidence suggests dysregulation of FOXO3 in some hepatocellular carcinomas. FOXOs are notable for the extensive number of functionally significant posttranslational modifications that they undergo. Recent advances in our understanding how FOXOs are regulated are providing a more detailed picture of how specific combinations of posttranslational modifications alter both nuclear translocation as well as transcriptional specificity under different conditions. This review summarizes emerging knowledge of FOXO function in the liver, FOXO changes in liver disease, and the posttranslational modifications responsible for these effects.

Arginine Methylation of Integrin Alpha-4 Prevents Fibrosis Development in Alcohol-Associated Liver Disease.

BACKGROUND & AIMS: Alcohol-associated liver disease (ALD) comprises a spectrum of disorders including steatosis, steatohepatitis, fibrosis, and cirrhosis. We aimed to study the role of protein arginine methyltransferase 6 (PRMT6), a new regulator of liver function, in ALD progression. METHODS: Prmt6-deficient mice and wild-type littermates were fed Western diet with alcohol in the drinking water for 16 weeks. Mice fed standard chow diet or Western diet alone were used as a control. RESULTS: We found that PRMT6 expression in the liver is down-regulated in 2 models of ALD and negatively correlates with disease severity in mice and human liver specimens. Prmt6-deficient mice spontaneously developed liver fibrosis after 1 year and more advanced fibrosis after high-fat diet feeding or thioacetamide treatment. In the presence of alcohol Prmt6 deficiency resulted in a dramatic increase in fibrosis development but did not affect lipid accumulation or liver injury. In the liver PRMT6 is primarily expressed in macrophages and endothelial cells. Transient replacement of knockout macrophages with wild-type macrophages in Prmt6 knockout mice reduced profibrotic signaling and prevented fibrosis progression. We found that PRMT6 decreases profibrotic signaling in liver macrophages via methylation of integrin α-4 at R464 residue. Integrin α-4 is predominantly expressed in infiltrating monocyte derived macrophages. Blocking monocyte infiltration into the liver with CCR2 inhibitor reduced fibrosis development in knockout mice and abolished differences between genotypes. CONCLUSIONS: Taken together, our data suggest that alcohol-mediated loss of Prmt6 contributes to alcohol-associated fibrosis development through reduced integrin methylation and increased profibrotic signaling in macrophages.

A Pathogenic Role of Non-Parenchymal Liver Cells in Alcohol-Associated Liver Disease of Infectious and Non-Infectious Origin.

Now, much is known regarding the impact of chronic and heavy alcohol consumption on the disruption of physiological liver functions and the induction of structural distortions in the hepatic tissues in alcohol-associated liver disease (ALD). This review deliberates the effects of alcohol on the activity and properties of liver non-parenchymal cells (NPCs), which are either residential or infiltrated into the liver from the general circulation. NPCs play a pivotal role in the regulation of organ inflammation and fibrosis, both in the context of hepatotropic infections and in non-infectious settings. Here, we overview how NPC functions in ALD are regulated by second hits, such as gender and the exposure to bacterial or viral infections. As an example of the virus-mediated trigger of liver injury, we focused on HIV infections potentiated by alcohol exposure, since this combination was only limitedly studied in relation to the role of hepatic stellate cells (HSCs) in the development of liver fibrosis. The review specifically focusses on liver macrophages, HSC, and T-lymphocytes and their regulation of ALD pathogenesis and outcomes. It also illustrates the activation of NPCs by the engulfment of apoptotic bodies, a frequent event observed when hepatocytes are exposed to ethanol metabolites and infections. As an example of such a double-hit-induced apoptotic hepatocyte death, we deliberate on the hepatotoxic accumulation of HIV proteins, which in combination with ethanol metabolites, causes intensive hepatic cell death and pro-fibrotic activation of HSCs engulfing these HIV- and malondialdehyde-expressing apoptotic hepatocytes.

Inhibition of human BK polyomavirus replication by small noncoding RNAs.

Small noncoding RNAs regulate a variety of cellular processes, including genomic imprinting, chromatin remodeling, replication, transcription, and translation. Here, we report small replication-regulating RNAs (srRNAs) that specifically inhibit DNA replication of the human BK polyomavirus (BKV) in vitro and in vivo. srRNAs from FM3A murine mammary tumor cells were enriched by DNA replication assay-guided fractionation and hybridization to the BKV noncoding control region (NCCR) and synthesized as cDNAs. Selective mutagenesis of the cDNA sequences and their putative targets suggests that the inhibition of BKV DNA replication is mediated by srRNAs binding to the viral NCCR, hindering early steps in the initiation of DNA replication. Ectopic expression of srRNAs in human cells inhibited BKV DNA replication in vivo. Additional srRNAs were designed and synthesized that specifically inhibit simian virus 40 (SV40) DNA replication in vitro. These observations point to novel mechanisms for regulating DNA replication and suggest the design of synthetic agents for inhibiting replication of polyomaviruses and possibly other viruses.

Alcohol-associated fibrosis in females is mediated by female-specific activation of lysine demethylases KDM5B and KDM5C.

Alcohol-associated liver disease is a major cause of alcohol-related mortality. However, the mechanisms underlying disease progression are not fully understood. Recently we found that liver molecular pathways are altered by alcohol consumption differently in males and females. We were able to associate these sex-specific pathways with two upstream regulators: H3K4-specific demethylase enzymes KDM5B and KDM5C. Mice were fed the Lieber-DeCarli alcohol liquid diet for 3 weeks or a combination of a high-fat diet with alcohol in water for 16 weeks (western diet alcohol model [WDA] model). To assess the role of histone demethylases, mice were treated with AAV-shControl, AAV-shKdm5b, and/or AAV-shKdm5c and/or AAV-shAhR vectors. Gene expression and epigenetic changes after Kdm5b/5c knockdown were assessed by RNA-sequencing and H3K4me3 chromatin immunoprecipitation analysis. We found that less than 5% of genes affected by Kdm5b/Kdm5c knockdown were common between males and females. In females, Kdm5b/Kdm5c knockdown prevented fibrosis development in mice fed the WDA alcohol diet for 16 weeks and decreased fibrosis-associated gene expression in mice fed the Lieber-DeCarli alcohol liquid diet. In contrast, fibrosis was not affected by Kdm5b/Kdm5c knockdown in males. We found that KDM5B and KDM5C promote fibrosis in females through down-regulation of the aryl hydrocarbon receptor (AhR) pathway components in hepatic stellate cells. Kdm5b/Kdm5c knockdown resulted in an up-regulation of Ahr, Arnt, and Aip in female but not in male mice, thus preventing fibrosis development. Ahr knockdown in combination with Kdm5b/Kdm5c knockdown restored profibrotic gene expression. Conclusion: KDM5 demethylases contribute to differences between males and females in the alcohol response in the liver. The KDM5/AhR axis is a female-specific mechanism of fibrosis development in alcohol-fed mice.