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Chaowen Zheng,1,2 Siyuan Li,1 Huanran Lyu,1 Cheng Chen,1 Johannes Mueller,1 Anne Dropmann,3 Seddik Hammad,3,4 Steven Dooley,3 Songqing He,2 Sebastian Mueller1,5 1Center for Alcohol Research, University of Heidelberg, Heidelberg, Germany; 2Division of Hepatobiliary Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 3Molecular Hepatology Section, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany; 4Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Quena, Egypt; 5Viscera AG Bauchmedizin, Bern, SwitzerlandCorrespondence: Sebastian Mueller, Center for Alcohol Research, University Hospital Heidelberg Im Neuenheimer Feld 350 69120 Heidelberg, Germany, Tel +49 6221 567121, Email sebastian.mueller@urz.uni-heidelberg.dePurpose: Both hepatic iron accumulation and hemolysis have been identified as independent prognostic factor in alcohol-related liver disease (ALD); however, the mechanisms still remain poorly understood. We here demonstrate that hepatocytes are able to directly ingest aged and ethanol-primed red blood cells (RBCs), a process termed efferocytosis.Methods: Efferocytosis of RBCs was directly studied in vitro and observed by live microscopy for real-time visualization. RBCs pretreated with either CuSO4 or ethanol following co-incubation with Huh7 cells and murine primary hepatocytes. Heme oxygenase-1 (HO-1) and other targets were measured by q-PCR.Results: As shown by live microscopy, oxidized RBCs, but not intact RBCs, are rapidly ingested by both Huh7 cells and murine primary hepatocytes within 10 minutes. In some cases, more than 10 RBCs were seen within hepatocytes, surrounding the nucleus. RBC efferocytosis also rapidly induces HO1, its upstream regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and ferritin, indicating efficient heme degradation. Preliminary data further suggest that hepatocyte efferocytosis of oxidized RBCs is, at least in part, mediated by scavenging receptors such as ASGPR1. Of note, pretreatment of RBCs with ethanol but also heme and bilirubin also initiated efferocytosis. In a cohort of heavy human drinkers, a significant correlation of hepatic ASGPR1 with the heme degradation pathway was observed.Conclusion: We here demonstrate that hepatocytes can directly ingest and degrade oxidized RBCs through efferocytosis, a process that can be also triggered by ethanol, heme and bilirubin. Our findings are highly suggestive for a novel mechanism of hepatic iron overload in ALD patients.Keywords: alcohol-related liver disease, ASGPR1, efferocytosis, heme oxygenase-1, red blood cells, scavenging receptors, iron metabolism

Background & Aims Large extracellular vesicles, specifically AnnexinV + EpCAM + CD147 + tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, the aim of this study was to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. Methods Fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients’ sera that were associated with the presence of a tumour (AnnexinV + EpCAM + CD147 + taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV + EpCAM + ASGPR1 + taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, and 202 control subjects were enrolled. Results The results indicate that AnnexinV + EpCAM + CD147 + taMPs were elevated in HCC and CCA. Furthermore, AnnexinV + EpCAM + ASGPR1 + CD133 + taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV + EpCAM + ASGPR1 + taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest sizes of successfully detected cancers were ranging between 11–15 mm. AnnexinV + EpCAM + ASGPR1 + taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV + EpCAM + ASGPR1 + taMPs. Conclusion These data provide strong evidence that AnnexinV + EpCAM + ASGPR1 + taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possible extent of these cancers in patients with advanced liver diseases. Lay summary Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. [ABSTRACT FROM AUTHOR]