Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis.

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

Unique Biology of Pancreatic Ductal Adenocarcinoma Accompanied by Rapidly Impaired Diabetes: A Favorable Long-Term Survival Following Curative Resection.

BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are sometimes diagnosed accompanied by rapidly impaired diabetes (PDAC-RID). Although this type of PDAC may have unusual biological features, these features have not been explained. METHODS: Patients with PDAC who underwent upfront pancreatectomy between 2010 and 2018 were retrospectively reviewed. PDAC-RID was defined as a glycated hemoglobin (HbA1c) value of ≥ 8.0% of newly diagnosed diabetes, and acute exacerbation of previously diagnosed diabetes. Other patients were classified as PDAC with stable glycometabolism (PDAC-SG). Clinicopathological factors, long-term survival rates, and recurrence patterns were evaluated. RESULTS: Of the 520 enrolled patients, 104 were classified as PDAC-RID and 416 as PDAC-SG. There was no significant difference regarding TNM staging, resectability, or adjuvant chemotherapy rate between the groups. However, 5-years cancer-specific survival (CSS) was significantly higher in the PDAC-RID group than in the PDAC-SG group (45.3% vs. 31.1%; p = 0.02). This survival difference was highlighted in relatively early-stage PDAC (≤ pT2N1) (CSS: 60.8% vs. 43.6%; p = 0.01), but the difference was not significant for advanced-stage PDAC. A multivariate analysis of early-stage PDAC showed that PDAC-SG was an independent risk factor of shorter CSS (hazard ratio 1.76; p = 0.02). The hematogenous metastatic rate in early-stage PDAC was lower in the PDAC-RID group than in the PDAC-SG group (18.3% vs. 35.8%; p = 0.01). CONCLUSIONS: PDAC-RID showed a favorable long-term survival rate after curative resection with low hematogenous metastases, which may be due to its unique biology.

Impact of electrical muscle stimulation on serum myostatin level and maintenance of skeletal muscle mass in patients undergoing living-donor liver transplantation: Single-center controlled trial.

AIM: Sarcopenia is reportedly associated with a poor prognosis in patients who undergo living-donor liver transplantation (LDLT), most of whom are not able to tolerate muscle strengthening exercise training. Myostatin is one of the myokines and a negative regulator of skeletal muscle growth. The clinical feasibility of an electrical muscle stimulation (EMS) system, which exercises muscle automatically by direct electrical stimulation, has been reported. In this study, we aimed to determine the effect of perioperative application of SIXPAD, which is a type of EMS system, with reference to the serum myostatin and sarcopenia in LDLT patients. METHOD: Thirty patients scheduled for LDLT were divided into a SIXPAD group (n = 16) and a control group (n = 14). In the SIXPAD group, EMS was applied to the thighs twice daily. The serum myostatin was measured in samples obtained before use of SIXPAD and immediately before LDLT. The psoas muscle index (PMI) at the level of the third lumbar vertebra and the quadriceps muscle area were compared on computed tomography images before use of SIXPAD and 1 month after LDLT. RESULTS: The preoperative serum myostatin was found to be higher in LDLT patients than in healthy volunteers and EMS significantly reduced the serum myostatin. Electrical muscle stimulation prevented a postoperative reduction not only in the area of the quadriceps muscles but also in the PMI despite direct stimulation of the thigh muscles. CONCLUSION: Stimulation of muscles by EMS decreases the serum myostatin and helps to maintain skeletal muscle in patients who have undergone LDLT.

Hepatitis C virus modulates signal peptide peptidase to alter host protein processing.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

Sphingosine-1-phosphate promotes tumor development and liver fibrosis in mouse model of congestive hepatopathy.

BACKGROUND AND AIMS: Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and HCC. However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood. APPROACH AND RESULTS: Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce LSECs capillarization in mice and in vitro. LSEC capillarization was also confirmed in patients with FALD. Mitogenic factor, sphingosine-1-phosphate (S1P), was increased in congestive liver and expression of sphingosine kinase 1, a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR) 1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobial treatment lowered portal blood LPS concentration, LSEC capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver. CONCLUSIONS: In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for patients with RHF with primary or metastatic liver cancer.

Toll-like receptor 7 agonist, GS-986, is an immune-stimulant inducing follicular helper T cells and expanding HBs antigen-specific B cells in vitro.

BACKGROUNDS AND AIMS: Toll-like receptor (TLR) agonists have been developed as adjuvants to efficiently induce antiviral immune responses. Specificity and potency of these compounds are essential requirements for clinical trial applications. In patients with hepatitis B virus (HBV) infections, sustained loss of hepatitis B surface antigen (HBsAg) is a therapeutic goal, which may be achievable by the sequential activation of follicular helper T cells (Tfh) and antibody-secreting B cells. We aimed to elucidate whether novel TLR7 agonist, GS-986, could activate immune responses involved in HBV elimination. METHODS: To clarify the impact of GS-986 on pDCs, we quantified the expression levels of surface markers and evaluated for Tfh induction in a culture model consisting of human pDCs with allogeneic naïve CD4+ T cells. In addition, we examined whether GS-986 could enhance HBs antibody production capacity using PBMC from CHB patients. RESULTS: pDCs from CHB patients had lower OX40L expression and as well as impaired capacity for Tfh induction compared with those from healthy donors. However, GS-986-stimulated pDCs from CHB patients expressed OX40L and produced IL-6 and IL-12, resulting in the induction of IL-21-producing Tfh cells (CXCR5+ PD-1+ CD4+ ) from naïve CD4+ T cells. The Tfh-inducing capacity of GS-986 was reduced in the presence of an anti-OX40L blocking antibody. Furthermore, GS-986 promoted HBsAg-specific antibody production in PBMCs from CHB patients. CONCLUSIONS: GS-986 is an adjuvant that stimulates pDCs to induce Tfh differentiation and antigen-specific B-cell production. This immune profile may be beneficial for therapeutic application as an immune modulator in CHB patients.

Preoperative serum brain-derived neurotrophic factor as a predictive biomarker for sepsis after living-donor liver transplantation.

AIM: Although the survival rate after living-donor liver transplantation (LDLT) is improving, sepsis still limits the prognosis. Immune dysfunction and sarcopenia are often observed in LDLT patients, and increase susceptibility to infection. Brain-derived neurotrophic factor (BDNF) is a myokine produced by immune cells and skeletal muscle. We aimed to determine whether serum BDNF could be a feasible biomarker for sepsis of LDLT patients. METHODS: We measured serum samples from 124 patients who underwent LDLT and 9 healthy volunteers for BDNF. We examined its correlation with incidence rate of sepsis. To clarify the source of BDNF, we examined its expression in lymphocytes, skeletal muscle cells, and hepatocytes. RESULTS: Patients who experienced sepsis showed worse short-term survival. Preoperative serum BDNF was lower in LDLT patients compared with healthy volunteers, and was also lower in Child-Pugh C compared with Child-Pugh A or B. Serum BDNF was inversely correlated with Model for End-Stage Liver Disease and controlling nutritional status (CONUT) scores, but had a weak positive correlation with skeletal muscle mass index (SMI). Multivariate analysis revealed that serum BDNF was independently associated with sepsis. Preoperative serum BDNF was a better predictor of sepsis in LDLT patients than CONUT score or SMI. Serum BDNF was positively correlated with lymphocyte counts, especially T cells. In vitro, T cells and skeletal muscle cells produced BDNF. CONCLUSIONS: Preoperative serum BDNF could be a predictive biomarker for sepsis after LDLT, by reflecting the systemic condition including hepatic function, nutritional status, and immune status.

Effects of sodium glucose cotransporter 2 inhibitors and pioglitazone on FIB-4 index in metabolic-associated fatty liver disease.

BACKGROUND: The antidiabetic drugs sodium glucose cotransporter 2 inhibitors (SGLT2is) and thiazolidinediones have beneficial effects on the liver dysfunction of patients with nonalcoholic fatty liver disease and type 2 diabetes mellitus (T2DM). We aimed to determine the efficacy of these drugs for the treatment of liver disease in patients with metabolic dysfunction-associated fatty liver disease (MAFLD) and T2DM. METHODS: We undertook a retrospective study of 568 patients with MAFLD and T2DM. Of these, 210 were treating their T2DM with SGLT2is (n = 95), 86 with pioglitazone (PIO), and 29 with both. The primary outcome was the change in Fibrosis-4 (FIB-4) index between baseline and 96 weeks. RESULTS: At 96 weeks, the mean FIB-4 index had significantly decreased (from 1.79 ± 1.10-1.56 ± 0.75) in the SGLT2i group, but not in the PIO group. The aspartate aminotransferase to platelet ratio index, serum aspartate and alanine aminotransferase (ALT), hemoglobin A1c, and fasting blood sugar significantly decreased in both groups (ALT: SGLT2i group, -17 ± 3 IU/L; PIO group, -14 ± 3 IU/L). The bodyweight of the SGLT2i group decreased, but that of the PIO group increased (-3.2 kg and +1.7 kg, respectively). When the participants were allocated to two groups according to their baseline ALT (>30 IU/L), FIB-4 index significantly decreased in both groups. In patients taking pioglitazone, the addition of SGLT2i improved liver enzymes but not FIB-4 index for 96 weeks. CONCLUSIONS: Treatment with SGLT2i causes a larger improvement in FIB-4 index than PIO in patients with MAFLD over 96 weeks.

Peripheral-dominant liver fibrosis and tumor distribution in a mouse model of congestive hepatopathy.

AIM: Congestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in the periphery of the liver in patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in the murine model of congestive hepatopathy. METHODS: Chronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava. Distribution of liver congestion, fibrosis, and tumors in partial ligation of the suprahepatic inferior vena cava mice were assessed by histological findings, laser microdissection (LMD)-based qPCR and enhanced computed tomography. LMD-based RNA-sequencing was performed to identify causal factors that promote tumor development in congestive hepatopathy. RESULTS: Liver fibrosis was mainly induced in the periphery of the liver and co-localized with distribution of liver congestion. Liver tumors were also induced in the periphery of the liver where liver congestion and fibrosis occurred. LMD-based RNA-sequencing revealed the upregulation of extracellular matrix/collagen fibril-, wound healing-, angiogenesis-, morphogenesis-, and cell motility-related signaling pathways in periphery of liver compared with liver center. CONCLUSIONS: Our findings showed the experimental relevance of liver congestion, fibrosis, and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Metabolic dysfunction-associated fatty liver disease directly related to liver fibrosis independent of insulin resistance, hyperlipidemia, and alcohol intake in morbidly obese patients.

AIM: Hepatic fibrosis is associated with various factors, including metabolic dysfunction-associated fatty liver disease (MAFLD), insulin resistance, and alcohol intake in patients with morbid obesity. We investigated factors directly associated with hepatic fibrosis in patients with morbid obesity using a graphical model. METHODS: We enrolled 134 consecutive patients with morbid obesity who underwent liver biopsy during sleeve gastrectomy (median age 43.5 years; MAFLD 78.4%; homeostasis model assessment of insulin resistance [HOMA-IR] 5.97; >20 g/day alcohol intake 14.2%). Patients were classified into none/mild (F0/1; n = 77) or significant/advanced fibrosis (F2/3; n = 57) groups, based on histology. Factors associated with F2/3 were analyzed using logistic regression analysis and a graphical model. RESULTS: F2/3 was observed in 42.5% of the enrolled patients. The prevalence of MAFLD and HOMA-IR values were significantly higher in the F2/3 group than in the F0/1 group; however, no significant difference in alcohol intake was observed between the two groups. On logistic regression analysis, MAFLD, but not HOMA-IR or alcohol intake, was the only independent factor associated with F2/3 (odds ratio 7.555; 95% confidence interval 2.235-25.544; p = 0.0011). The graphical model revealed that F2/3 directly interacted with MAFLD, diabetes mellitus, HOMA-IR, and low-density lipoprotein cholesterol. Among these factors, MAFLD showed the strongest interaction with F2/3. CONCLUSIONS: We determined that MAFLD was more directly associated with significant/advanced fibrosis than insulin resistance or hyperlipidemia, and alcohol intake was not directly associated with hepatic fibrosis. Metabolic dysfunction-associated fatty liver disease could be the most important factor for hepatic fibrosis in patients with morbid obesity.

Impact of Immune Reconstitution-Induced Hepatic Flare on Hepatitis B Surface Antigen Loss in Hepatitis B Virus/Human Immunodeficiency Virus-1 Coinfected Patients.

BACKGROUND: Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) in hepatitis B virus/human immunodeficiency virus-1 (HBV/HIV-1)-coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss. METHODS: This was a retrospective study of 58 HBV/HIV-1-coinfected subjects HBsAg-positive for ≥6 months before ART initiation and followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV-monoinfected patients, and HBV/HIV-1-coinfected patients with IRIS-HF or acute hepatitis B infection. RESULTS: During ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1-coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF. CONCLUSIONS: IRIS-HF was associated with HBsAg loss in HBV/HIV-1-coinfected patients.

Blood angiopoietin-2 predicts liver angiogenesis and fibrosis in hepatitis C patients.

BACKGROUND: Pathological angiogenesis is involved in the development of hepatocellular carcinoma. In patients with chronic hepatitis C (CHC), the level of angiogenic factor angiopoietin (ANGP)-2 is reported to be increased in the blood, correlating with fibrosis. In this study, we aimed to clarify whether blood ANGP-2 is useful as a biomarker for liver angiogenesis and fibrosis in CHC patients and to further reveal the relationship between such pathology in a carbon tetrachloride (CCl4)-treated liver fibrosis mouse model. METHODS: Plasma levels of ANGP-2, expression of a liver sinusoidal endothelial cell (LSEC) marker (CD31), collagen deposition (Sirius Red staining) in the liver, clinical fibrosis markers (Mac-2 binding protein glycosylation isomer, virtual touch quantification, and liver stiffness measurement), and liver function (albumin bilirubin score) were examined in CHC patients. To determine the effects of an anti-angiogenic agent on liver fibrosis in vivo, sorafenib was administered to the CCl4-treated mice (BALB/c male). RESULTS: The plasma levels of ANGP-2 were increased in CHC patients compared to healthy volunteers and decreased by the eradication of hepatitis C with direct-acting antivirals. In addition, plasma ANGP-2 levels were correlated with CD31 expression, collagen deposition, clinical fibrosis markers, and liver function. Sorafenib inhibited liver angiogenesis and fibrosis in the CCl4-treated mice and was accompanied by decreased ANGP-2 expression in LSECs. CONCLUSIONS: ANGP-2 may serve as a useful biomarker for liver angiogenesis and fibrosis in CHC patients. In addition, angiogenesis and fibrosis may be closely related.

Effects of a low-intensity resistance exercise program on serum miR-630, miR-5703, and Fractalkine/CX3CL1 expressions in subjects with No exercise habits: A preliminary study.

AIMS: Exercise is effective for the prevention of liver cancer. Exercise exerts biological effects through the regulation of microRNAs (miRNAs) and cytokines/myokines. We aimed to investigate the effects of low-intensity resistance exercise on serum miRNA and cytokine/myokine expressions in subjects with no exercise habits. METHODS: We enrolled seven male subjects with no exercise habits in this prospective before-after study. All subjects performed a low-intensity resistance exercise program (three metabolic equivalents, approximately 20 min/session). Serum miRNA expressions were evaluated using microarrays. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed miRNAs before and after exercise. Serum cytokine/myokine expressions were evaluated using a multiplex panel. RESULTS: All subjects completed the exercise program with no adverse events. In the microarray analysis, seven miRNAs showed a significant change between before and after exercise. Of these, microRNA (miR)-630 and miR-5703 showed a >1.5-fold increase (miR-630: 40.7 vs. 69.3 signal intensity, p = 0.0133; miR-5703: 30.7 vs. 55.9 signal intensity, p = 0.0051). KEGG pathway enrichment analysis showed that miR-630- and miR-5703-related genes were enriched in 37 and 5 pathways, including transforming growth factor-beta and Wnt signaling pathways, respectively. In the multiplex analysis, 12 cytokines/myokines showed significant alteration after exercise compared to before exercise. Of these, fractalkine/CX3CL1 showed the most significant up-regulation by exercise (94.5 vs. 109.1 pg/ml, p = 0.0017). CONCLUSIONS: A low-intensity resistance exercise program was associated with upregulation of serum miR-630, miR-5703, and fractalkine/CX3CL1 expressions in subjects with no exercise habits. Thus, even low-intensity exercise may alter miRNA and cytokine/myokine expressions in humans.

Myostatin as a fibroblast-activating factor impacts on postoperative outcome in patients with hepatocellular carcinoma.

AIM: In patients with liver cirrhosis, high levels of serum myostatin are associated with poor prognosis. We aimed to clarify the influence of myostatin on the prognosis of patients with non-alcoholic fatty liver disease-hepatocellular carcinoma (NAFLD-HCC) without cirrhosis and on the progression of liver fibrosis. METHODS: Serum myostatin levels were evaluated in 234 patients who underwent primary surgical resection for single HCC. To clarify the impact of myostatin on liver fibrosis, we established human primary liver fibroblasts from resected livers, and cultured them in the presence of myostatin. RESULTS: The median age was 67.4 years, the median L3 skeletal muscle mass index was 44.4 cm2 /m2 , and the median body mass index was 23.4 kg/m2 . Eighty-two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men <42, women <38 cm2 /m2 ). The etiologies of liver disease were hepatitis B virus (n = 61), hepatitis C virus (n = 86), and non-B non-C hepatitis (n = 87) including NAFLD (n = 74). High preoperative serum myostatin and vascular invasion were independent predictors of poor overall survival (OS). High serum myostatin was associated with poor OS in patients with no sarcopenia (n = 152). In patients without advanced liver fibrosis (Fibrosis stage, 0-2; n = 58), high levels of serum myostatin were also associated with poor OS, regardless of sarcopenia. Serum myostatin levels were increased with the progression of liver fibrosis. Liver fibroblasts were activated and produced collagen following stimulation with myostatin. CONCLUSIONS: In patients with NAFLD-HCC without advanced liver fibrosis, high levels of serum myostatin were associated with poor OS. Myostatin activated primary fibroblasts and stimulated collagen production.

USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation.

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.

Infection with flaviviruses requires BCLXL for cell survival.

BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses.

Serum soluble sialic acid-binding immunoglobulin-like lectin-7 concentration as an indicator of liver macrophage activation and advanced fibrosis in patients with non-alcoholic fatty liver disease.

AIM: Non-alcoholic fatty liver disease (NAFLD) is a leading cause of liver disease worldwide. Because liver fibrosis is associated with the long-term prognosis of patients with NAFLD, there is an urgent need for non-invasive markers of liver fibrosis. Sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is an immunomodulatory molecule expressed on various immune cells, including macrophages, which plays a key role in liver inflammation and fibrosis in NAFLD. We aimed to determine whether serum levels of soluble Siglec-7 (sSiglec-7) could have utility at a marker of fibrosis in this patient population. METHODS: We examined serum samples from 93 NAFLD patients and 19 healthy donors for macrophage-associated protein, including sSiglec-7, soluble CD163, and YKL-40, and examined their correlation with liver fibrosis scores, tissue elastography, and histological findings. Independent factors associated with advanced fibrosis were analyzed using a logistic regression model and a decision tree. To clarify the source of sSiglec-7, we examined its expression in liver tissue-derived macrophages and cultured monocyte-derived macrophages. RESULTS: Serum sSiglec-7 levels were significantly higher in NAFLD patients compared with healthy donors, and correlated positively with sCD163 and YKL-40 levels. Serum sSiglec-7 was an independent diagnostic marker with high specificity (96.3%) for advanced fibrosis (F3 and F4) in NAFLD patients. Siglec-7 was mainly expressed on CCR2+ macrophages in the liver, and sSiglec-7 production by monocyte-derived macrophages in vitro was increased after stimulation by pro-inflammatory factors. CONCLUSIONS: Elevated serum sSiglec-7 could serve as an independent marker with high specificity for advanced liver fibrosis in patients with NAFLD.

High serum interleukin-34 level is a predictor of poor prognosis in patients with non-viral hepatocellular carcinoma.

AIMS: We aimed to investigate the impact of interleukin (IL)-34 and YKL-40, regulators of hepatic fibrosis and tumor growth, on the prognosis of patients with non-viral hepatocellular carcinoma (HCC). METHODS: We enrolled 159 non-viral HCC patients (age, 70.8 ± 8.5 years; female/male, 43/116). Of these, 86 patients were alive and 73 patients had died at the censor time point. Serum IL-34 and YKL-40 levels were quantified by enzyme-linked immunosorbent assay. Patients were stratified by the median level of serum IL-34 to examine its effect on survival. Multivariate analysis and random forest analysis were used to evaluate the impact of IL-34 and YKL-40 on the prognosis of non-viral HCC patients. RESULTS: Interleukin-34 (hazard ratio [HR] 1.30; 95% confidence interval [CI], 1.13-1.49; P ≤ 0.01), tumor size (HR 1.63; 95% CI, 1.37-1.94; P ≤ 0.01), and tumor number (HR 1.53; 95% CI, 1.25-1.87; P ≤ 0.01) were independent predictive factors for survival. Furthermore, the survival rates were significantly lower in the high IL-34 group than in the low IL-34 group (5-year survival rates, 34.7% vs. 59.8%, respectively; P < 0.05). In the random forest analysis for survival, IL-34 was the third-highest ranking factor, following tumor size and number. In a stratification analysis, serum α-fetoprotein level and Fibrosis-4 index were independent positive risk factors for high serum IL-34 level. YKL-40 was not associated with prognosis in either the multivariate or random forest analysis. CONCLUSION: Interleukin-34 was an independent factor for survival of non-viral HCC patients. Interleukin-34 might be associated with prognosis through tumor and hepatic fibrosis factors.