Hyuk Soo Eun

CD11b(+) Gr1(+) bone marrow cells ameliorate liver fibrosis by producing interleukin-10 in mice.

Clinical trials and animal models suggest that infusion of bone marrow cells (BMCs) is effective therapy for liver fibrosis, but the underlying mechanisms are obscure, especially those associated with early effects of BMCs. Here, we analyzed the early impact of BMC infusion and identified the subsets of BMCs showing antifibrotic effects in mice with carbon tetrachloride–induced liver fibrosis. An interaction between BMCs and activated hepatic stellate cells (HSCs) was investigated using an in vitro coculturing system. Within 24 hours, infused BMCs were in close contact with activated HSCs, which was associated with reduced liver fibrosis, enhanced hepatic expression of interleukin (IL)‐10, and expanded regulatory T cells but decreased macrophage infiltration in the liver at 24 hours after BMC infusion. In contrast, IL‐10–deficient (IL‐10−/−) BMCs failed to reproduce these effects in fibrotic livers. Intriguingly, in isolated cells, CD11b+Gr1highF4/80− and CD11b+Gr1+F4/80+ BMCs expressed more IL‐10 after coculturing with activated HSCs, leading to suppressed expression of collagen and α‐smooth muscle actin in HSCs. Moreover, these effects were either enhanced or abrogated, respectively, when BMCs were cocultured with IL‐6−/− and retinaldehyde dehydrogenase 1−/− HSCs. Similar to murine data, human BMCs expressed more IL‐10 after coculturing with human HSC lines (LX‐2 or hTERT), and serum IL‐10 levels were significantly elevated in patients with liver cirrhosis after autologous BMC infusion. Conclusion: Activated HSCs increase IL‐10 expression in BMCs (CD11b+Gr1highF4/80− and CD11b+Gr1+F4/80+ cells), which in turn ameliorates liver fibrosis. Our findings could enhance the design of BMC therapy for liver fibrosis. (HEPATOLOGY 2012;56:1902–1912)

Exosome‐mediated activation of toll‐like receptor 3 in stellate cells stimulates interleukin‐17 production by γδ T cells in liver fibrosis

During liver injury, hepatocytes secrete exosomes that include diverse types of self‐RNAs. Recently, self‐noncoding RNA has been recognized as an activator of Toll‐like receptor 3 (TLR3). However, the roles of hepatic exosomes and TLR3 in liver fibrosis are not yet fully understood. Following acute liver injury and early‐stage liver fibrosis induced by a single or 2‐week injection of carbon tetrachloride (CCl4), increased interleukin (IL)‐17A production was detected primarily in hepatic γδ T cells in wild‐type (WT) mice. However, liver fibrosis and IL‐17A production by γδ T cells were both significantly attenuated in TLR3 knockout (KO) mice compared with WT mice. More interestingly, IL‐17A‐producing γδ T cells were in close contact with activated hepatic stellate cells (HSCs), suggesting a role for HSCs in IL‐17A production by γδ T cells. In vitro treatments with exosomes derived from CCl4‐treated hepatocytes significantly increased the expression of IL‐17A, IL‐1β, and IL‐23 in WT HSCs but not in TLR3 KO HSCs. Furthermore, IL‐17A production by γδ T cells was substantially increased upon coculturing with exosome‐treated WT HSCs or conditioned medium from TLR3‐activated WT HSCs. However, similar increases were not detected when γδ T cells were cocultured with exosome‐treated HSCs from IL‐17A KO or TLR3 KO mice. Using reciprocal bone marrow transplantation between WT and TLR3 KO mice, we found that TLR3 deficiency in HSCs contributed to decreased IL‐17A production by γδ T cells, as well as liver fibrosis. Conclusion: In liver injury, the exosome‐mediated activation of TLR3 in HSCs exacerbates liver fibrosis by enhancing IL‐17A production by γδ T cells, which might be associated with HSC stimulation by unknown self‐TLR3 ligands from damaged hepatocytes. Therefore, TLR3 might be a novel therapeutic target for liver fibrosis. (Hepatology 2016;64:616‐631)

Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4–MD2 complex

Reactive oxygen species (ROS) contribute to the development of non-alcoholic fatty liver disease. ROS generation by infiltrating macrophages involves multiple mechanisms, including Toll-like receptor 4 (TLR4)-mediated NADPH oxidase (NOX) activation. Here, we show that palmitate-stimulated CD11b+F4/80low hepatic infiltrating macrophages, but not CD11b+F4/80high Kupffer cells, generate ROS via dynamin-mediated endocytosis of TLR4 and NOX2, independently from MyD88 and TRIF. We demonstrate that differently from LPS-mediated dimerization of the TLR4–MD2 complex, palmitate binds a monomeric TLR4–MD2 complex that triggers endocytosis, ROS generation and increases pro-interleukin-1β expression in macrophages. Palmitate-induced ROS generation in human CD68lowCD14high macrophages is strongly suppressed by inhibition of dynamin. Furthermore, Nox2-deficient mice are protected against high-fat diet-induced hepatic steatosis and insulin resistance. Therefore, endocytosis of TLR4 and NOX2 into macrophages might be a novel therapeutic target for non-alcoholic fatty liver disease.Reactive species of oxygen promote the development of hepatic steatosis. Here, Kim et al. demonstrate that palmitate stimulates macrophage infiltration and increases oxidative stress during steatosis by binding to the TLR4–MD2 complex, which results in the activation of NOX2.

Clinical Outcomes of Endoscopic Submucosal Dissection for Undifferentiated or Submucosal Invasive Early Gastric Cancer

Background/Aims Early gastric cancer (EGC) that is undifferentiated or shows submucosal invasion has not been generally accepted as an indication for endoscopic treatment. But recently, experiences with endoscopic submucosal dissection (ESD) for undifferentiated EGC or submucosal invasive (SM) EGC have increased. The aim of this study was to evaluate clinical outcomes of ESD for EGC with undifferentiation or submucosal invasion. Methods Between August 2005 and August 2009, among 210 EGCs treated using ESD at our hospital, 18 lesions were diagnosed as undifferentiated gastric cancer and 41 as SM gastric cancer. A retrospective analysis was done on the medical records of these patients. Results Mean follow-up periods were 19.39±11.2 months. During the follow-up period, local recurrence was noted in 4 lesions. Local recurrence rates of the EGC groups (group 1, mucosal cancer with undifferentiation; group 2, SM cancer with differentiation; group 3, SM cancer with undifferentiation) were 10%, 4.5%, and 50%, respectively. Groups 1 and 2 were not significantly different in local recurrence rates compared to the mucosal cancer with differentiation group (p=0.061, p=0.125, respectively). The undifferentiated EGC group was significantly lower in curability using ESD than the differentiated EGC group (55.6% vs. 89.6%, p=0.000). The curability of the SM EGC group was lower than the mucosal EGC group (36.6% vs. 98.9%). Conclusions Complete resection using ESD is difficult in undifferentiated and SM gastric cancers. SM cancer with undifferentiation should be treated immediately by salvage operation. For mucosal cancer with undifferentiation or SM cancer with differentiation, one should consider careful short-term follow-up.

Gene expression of NOX family members and their clinical significance in hepatocellular carcinoma

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex-derived reactive oxygen species (ROS) promote chronic liver inflammation and remodeling that can drive hepatocellular carcinoma development. The role of NOX expression in hepatocellular carcinoma (HCC) has been partially investigated; however, the clinical relevance of collective or individual NOX family member expression for HCC survival remains unclear. Here, we obtained NOX mRNA expression data for 377 HCC samples and 21 normal liver controls from the TCGA data portal and performed Kaplan-Meier survival, gene ontology functional enrichment, and gene set enrichment analyses. Although most NOX genes exhibited little change, some were significantly induced in HCC compared to that in normal controls. In addition, HCC survival analyses indicated better overall survival in patients with high NOX4 and DUOX1 expression, whereas patients with high NOX1/2/5 expression showed poor prognoses. Gene-neighbour and gene set enrichment analyses revealed that NOX1/2/5 were strongly correlated with genes associated with cancer cell survival and metastasis, whereas increased NOX4 and DUOX1 expression was associated with genes that inhibit tumour progression. On the basis of these data, NOX family gene expression analysis could be a predictor of survival and identify putative therapeutic targets in HCC.

Hepatic immunophenotyping for streptozotocin-induced hyperglycemia in mice

Emerging evidence revealed that diabetes induces abnormal immune responses that result in serious complications in organs. However, the effect of hyperglycemia on hepatic immunity remains obscure. We evaluated the population and function of hepatic immune cells in streptozotocin (STZ)-induced hyperglycemic mice. CC chemokine receptor 2 (CCR2)-knockout mice and mice with a depletion of regulatory T cells (DEREG) were used to investigate the migration and role of regulatory T cells (Tregs) in hyperglycemic mice. The inflammatory cytokines and hepatic transaminase levels were significantly increased in the hyperglycemic mice. The population and number of infiltrating monocytes, granulocytes, and Tregs were enhanced in the livers of the hyperglycemic mice. Hepatic monocytes other than macrophages showed the increased expression of inflammatory cytokines and chemokines in the hyperglycemic mice. The CCR2 knockout and DEREG chimeric mice exhibited increased populations of activated T cells and neutrophils compared to the WT chimeric mice, which promoted hepatic inflammation in the hyperglycemic mice. The migration of CCR2 knockout Tregs into the liver was significantly reduced compared to the WT Tregs. We demonstrated that hyperglycemia contributes to increase in infiltrating monocytes and Tregs, which are associated with hepatic immune dysfunction in mice. CCR2-mediated migration of Tregs regulates hyperglycemia-induced hepatic inflammation.

Treatment with 4-Methylpyrazole Modulated Stellate Cells and Natural Killer Cells and Ameliorated Liver Fibrosis in Mice

Background & Aims Accumulating evidence suggests that retinol and its metabolites are closely associated with liver fibrogenesis. Recently, we demonstrated that genetic ablation of alcohol dehydrogenase 3 (ADH3), a retinol metabolizing gene that is expressed in hepatic stellate cells (HSCs) and natural killer (NK) cells, attenuated liver fibrosis in mice. In the current study, we investigated whether pharmacological ablation of ADH3 has therapeutic effects on experimentally induced liver fibrosis in mice. Methods Liver fibrosis was induced by intraperitoneal injections of carbon tetrachloride (CCl4) or bile duct ligation (BDL) for two weeks. To inhibit ADH3-mediated retinol metabolism, 10 μg 4-methylpyrazole (4-MP)/g of body weight was administered to mice treated with CCl4 or subjected to BDL. The mice were sacrificed at week 2 to evaluate the regression of liver fibrosis. Liver sections were stained for collagen and α-smooth muscle actin (α-SMA). In addition, HSCs and NK cells were isolated from control and treated mice livers for molecular and immunological studies. Results Treatment with 4-MP attenuated CCl4- and BDL-induced liver fibrosis in mice, without any adverse effects. HSCs from 4-MP treated mice depicted decreased levels of retinoic acids and increased retinol content than HSCs from control mice. In addition, the expression of α-SMA, transforming growth factor-β1 (TGF-β1), and type I collagen α1 was significantly reduced in the HSCs of 4-MP treated mice compared to the HSCs from control mice. Furthermore, inhibition of retinol metabolism by 4-MP increased interferon-γ production in NK cells, resulting in increased apoptosis of activated HSCs. Conclusions Based on our data, we conclude that inhibition of retinol metabolism by 4-MP ameliorates liver fibrosis in mice through activation of NK cells and suppression of HSCs. Therefore, retinol and its metabolizing enzyme, ADH3, might be potential targets for therapeutic intervention of liver fibrosis.

Blockade of Retinol Metabolism Protects T Cell-Induced Hepatitis by Increasing Migration of Regulatory T Cells

Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis. Con A was injected into wild type (WT), Raldh1 knock-out (Raldh1−/−), CCL2−/− and CCR2−/− mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-γ in T cells. Moreover, interferon-γ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis. These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis.

Risk factors for metachronous gastric carcinoma development after endoscopic resection of gastric dysplasia: Retrospective, single-center study

AIM To determine the gastric adenocarcinoma (GAC) occurrence rate and related factors, we evaluated the follow-up results of patients confirmed to have gastric dysplasia after endoscopic resection (ER). METHODS We retrospectively analyzed the medical records, endoscopic examination records, endoscopic procedure records, and histological records of 667 cases from 641 patients who were followed-up for at least 12 mo, from among 1273 patients who were conformed to have gastric dysplasia after Endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) of gastric mucosal lesions between January 2007 and August 2013 at the Chungnam National University Hospital. RESULTS The mean follow-up period was 33.8 mo, and the median follow-up period was 29 mo (range: 12-87). During the follow-up period, the occurrence of metachronous GAC was 4.0% (27/667). The mean and median interval periods between the occurrence of metachronous GAC and endoscopic treatment of gastric dysplasia were 36.3 and 34 mo, respectively (range: 16-71). The factors related to metachronous GAC occurrence after ER for gastric dysplasia were male sex (5.3% vs 1.0%), open-type atrophic gastritis (9.5% vs 3.4%), intestinal metaplasia (6.8% vs 2.4%), and high-grade dysplasia (HGD; 8.4% vs 3.2%). Among them, male sex [OR: 5.05 (1.18-21.68), P = 0.029], intestinal metaplasia [OR: 2.78 (1.24-6.23), P = 0.013], and HGD [OR: 2.70 (1.16-6.26), P = 0.021] were independent related factors in multivariate analysis. Furthermore, 24 of 27 GAC cases (88.9%) occurred at sites other than the previous resection sites, and 3 (11.1%) occurred at the same site as the previous resection site. CONCLUSION Male sex, intestinal metaplasia, and HGD were significantly related to the occurrence of metachronous GAC after ER of gastric dysplasia, and most GACs occurred at sites other than the previous resection sites.

Pulmonary Sarcoidosis That Developed During the Treatment of a Patient With Crohn Disease by Using Infliximab

For inflammatory bowel disease (IBD), antitumor necrosis factor treatment offers a new direction for both patients and medical doctors. This treatment has dramatically improved the quality of life for patients with ulcerative colitis and Crohn disease (CD). However, with increasing usage and longer follow-up periods, a wider range of possible adverse effects may be encountered. We report an unusual case of pulmonary sarcoidosis developed during the treatment of a patient with CD by using infliximab. A 30-year-old male who had been treated for CD with infliximab for 18 months was admitted due to abnormal opacities on chest radiography. Chest computed tomography displayed clustered small nodules in both lobes and enlarged multiple lymph nodes. The patient was diagnosed with sarcoidosis from the results of a biopsy of the subcarinal lymph node. Lung lesions were improved five months after infliximab was stopped.