Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage‐associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in ...hepatocytes and macrophage‐driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C‐X‐C motif) ligand 10 (CXCL10)‐laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC‐treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)‐tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)‐tagged EV marker, CD63, after LPC treatment of cotransfected Huh‐7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone‐marrow–derived macrophages with lipotoxic hepatocyte‐derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10‐neutralizing antisera. MLK3‐deficient mice fed a NASH‐inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild‐type mice. Conclusions: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10‐bearing vesicles from hepatocytes, which are chemotactic for macrophages. (Hepatology 2016;63:731–744)
In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in ...mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells.
Pre-existing anti-adenovirus (Ad) neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using Ad vectors and oncolytic Ads; however, it has not been fully elucidated which Ad ...capsid protein-specific antibodies are involved in AdNAb-mediated inhibition of Ad infection in vivo. In this study, mice possessing antibodies specific for each Ad capsid protein were prepared by intramuscular electroporation of each Ad capsid protein-expressing plasmid. Ad vector-mediated hepatic transduction was efficiently inhibited by more than 100-fold in mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid. An Ad vector pre-coated with FX before administration mediated more than 100-fold lower transduction efficiencies in the liver of warfarinized mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid, compared with those in the liver of warfarinized non-immunized mice. These data suggest that anti-fiber protein and anti-penton base antibodies bind to an Ad vector even though FX has already bound to the hexon, and inhibit Ad vector-mediated transduction. This study provides important clues for the development of a novel Ad vector that can circumvent inhibition with AdNAbs.
We have previously reported that epiregulin is a growth factor that seems to act on liver progenitor cells (LPCs) during liver regeneration. However, the relationship between epiregulin and LPCs has ...remained unclear. The aim of the present study was to clarify the role of epiregulin during liver regeneration. The serum levels of epiregulin in patients with acute liver failure were examined. A liver injury model was developed using mice fed a diet containing 0.1% 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) to induce LPCs. We then evaluated the expression of epiregulin and LPCs in these mice. The proliferation of epithelial cell adhesion molecule + LPCs cultured with epiregulin was examined in vitro, and finally epiregulin was overexpressed in mouse liver. In patients with acute liver failure, serum epiregulin levels were elevated significantly. In DDC mice, LPCs emerged around the portal area. Epiregulin was also detected around the portal area during the course of DDC-induced liver injury and was partially coexpressed with Thy1. Serum epiregulin levels in DDC mice were also significantly elevated. Recombinant epiregulin augmented the proliferative capacity of the LPCs in a dose-dependent manner. In mice showing overexpression of epiregulin, the expression of PCNA on hepatocytes was increased significantly. Finally, LPCs emerged around the portal area after epiregulin gene delivery. We concluded that epiregulin promotes the proliferation of LPCs and DNA synthesis by hepatocytes and is upregulated in the serum of patients with liver injury. Furthermore, induction of epiregulin leads to the appearance of LPCs. Epiregulin would be a useful biomarker of liver regeneration.
Aim
The number of Japanese patients with anorexia nervosa (AN) is increasing as society changes. Mild liver injury is a complication of AN in around 30% of cases. In some rare instances, patients ...present with severe liver injury similar to acute liver failure. However, there are numerous uncertainties over the clinical characteristics of this condition. The objective of the present study was to clarify the clinical characteristics of AN complicated by liver injury and to investigate the factors related to hepatic complications.
Methods
Thirty‐seven patients hospitalized at our institution with a diagnosis of AN were enrolled as the study subjects. The study used clinical data obtained at the time of hospitalization. The enrolled patients underwent subgroup analysis and were categorized into three groups: (i) normal alanine aminotransferase (ALT), (ii) moderately elevated ALT, and (iii) highly elevated ALT.
Results
All of the study subjects were female with a median age of 24 years and presenting with marked weight loss (mean body mass index, 13 kg/m2). Thirteen of the subjects had liver injury. We found that patients in the highly elevated ALT group had a significantly high blood urea nitrogen (BUN)/creatinine ratio, and a low blood sugar level.
Conclusions
Our present findings indicate that AN patients with highly elevated ALT have a severe dehydration. This suggests that dysfunction of hepatic circulation accompanying severe dehydration due to malnutrition may be an important factor in the development of liver injury in AN patients.
With the epidemic of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The influence of a perinatal obesity‐inducing diet (OID) on the development ...and progression of NAFLD in offspring is important but incompletely studied. Hence, we fed breeding pairs of C57BL/6J mice during gestation and lactation (perinatally) either chow or an OID rich in fat, fructose, and cholesterol (FFC). The offspring were weaned to either chow or an FFC diet, generating four groups: perinatal (p)Chow‐Chow, pChow‐FFC, pFFC‐Chow, and pFFC‐FFC. Mice were sacrificed at 10 weeks of age. We examined the whole‐liver transcriptome by RNA sequencing (RNA‐seq) and whole‐liver genome methylation by reduced representation bisulfite sequencing (RRBS). Our results indicated that the pFFC‐FFC mice had a significant increase in hepatic steatosis, injury, inflammation, and fibrosis, as assessed histologically and biochemically. We identified 189 genes that were differentially expressed and methylated in the pFFC‐FFC mice versus the pChow‐FFC mice. Gene set enrichment analysis identified hepatic fibrosis/hepatic stellate cell activation as the top canonical pathway, suggesting that the differential DNA methylation events in the mice exposed to the FFC diet perinatally were associated with a profibrogenic transcriptome. To verify that this finding was consistent with perinatal nutritional reprogramming of the methylome, we exposed pFFC‐Chow mice to an FFC diet in adulthood. These mice developed significant hepatic steatosis, injury, inflammation, and more importantly fibrosis when compared to the appropriate controls. Conclusion: Perinatal exposure to an OID primes the immature liver for an accentuated fibrosing nonalcoholic steatohepatitis (NASH) phenotype, likely through nutritional reprogramming of the offspring methylome. These data have potential clinical implications for monitoring children of obese mothers and risk stratification of children with NAFLD.
Perinatal exposure to high fat, fructose, and cholesterol (FFC) in mice primes the fetal liver to fibrosing NASH, likely secondary to nutritional reprograming of the liver methylome. This association is further confirmed, upon re‐exposure to the FFC diet in adulthood, where mice develop an accentuated, rapidly progressive NASH phenotype.
Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel ...CTC detection system using a green fluorescence protein (GFP)-expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus-adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell-specific microRNA, miR-142-3p, were incorporated into the 3'-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.
In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into ...the 3′-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3′-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.
PDF
