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882A AASLD ABSTRACTS HEPATOLOGY, October, 2015 sues and accelerates regression of CCl 4 -induced fibrosis by stimulating expression of matrix metalloproteinase (MMP)-13 and MMP-9. A subsequent study has identified opioid growth factor receptor-like 1 (OGFRL1) as a G-CSF-induced novel bone marrow cells-derived factor. To establish the molecular bases for future therapeutic application, we examined, in the present study, tissue distribution of endogenous OGFRL1 and the therapeutic effects of exogenous OGFRL1 administration. We also characterized the functions of OGFRL1 by generating its null mice. Methods: Total RNA was extracted from various organs/tissues of mice, and expression levels of Ogfrl1 gene were quantified using real time RT-PCR. Human bone marrow mesenchymal stem cells were infected with recombinant OGFRL1-expressing lentiviruses and injected into the spleen of CCl 4 -treated nude mice. Hepatic expression of Mmp-13 and Mmp-9 mRNAs was examined by real time RT-PCR, and liver regeneration was estimated by BrdU uptake into hepatocytes. Ogfrl1 gene targeting was conducted using the CRISPR/Cas9 system with a guide RNA set in the first exon of Ogfrl1 gene. Results: Endogenous OGFRL1 was expressed predominantly in neural tissues and, to the lesser extent, in hematopoietic organs such as bone marrow and spleen. Among the peripheral blood cells, monocytes and granulocytes were the major sources of OGFRL1. Liver tissues contained a small but significant amount of Ogfrl1 mRNA, which was decreased following repeated CCl 4 injections. When fibrotic mice underwent partial hepatectomy, expression levels of endogenous Ogfrl1 gene were further declined. An intrasplenic injection of OGFRL1-expressing cells stimulated hepatic expression of MMP-13 and MMP-9, and increased the BrdU uptake into hepatocytes in fibrotic liver after partial hepatectomy. It also caused a remarkable increase in gene expression of cyclins and other cell cycle-related factors as well as that of AFP. OGFRL1 null mice exhibited no abnormalities in their developmental process or liver tissue architecture. However, mitosis of hepatocytes was delayed following partial hepatectomy in OGFRL1-null mice compared with wild type littermates. Conclusions: OGFRL1 plays a critical role in hepatocyte proliferation, and administration of OGFRL1-expressing cells could be a potential regeneration therapy for advanced liver fibrosis. Disclosures: The following authors have nothing to disclose: Takayo Yanagawa, Hideaki Sumiyoshi, Sachie Nakao, Masato Ohtsuka, Minoru Kimura, Yousuke Chiba, Shintaro Satake, Tadashi Moro, Hiromi Chikada, Akihide Kamiya, Yutaka Inagaki 1370 Interleukin 15 regulates transforming growth factor β1-induced collagen production in hepatic stellate cells by controlling a network of suppressors of collagen transcription Kohtaro Ooka 3 , Huanzi Han 1 , Holger Fey 1 , Costica Aloman 1 , Jingjing Jiao 2 ; 1 Division of Gastroenterology/Liver Diseases (MC716), University of Illinois at Chicago, Chicago, IL; 2 University of Texas MD Anderson Cancer Center, Houston, TX; 3 Medicine, University of Illinois at Chicago, Chicago, IL Background & Aim: Interleukin 15 (IL15) signaling is unique in that IL15 binds to the high affinity IL15 receptor a subunit (IL15Rα) which it shuttles to the cell surface and stimulates opposing cells through the β/γ receptor complex. Our group has identified a protective role for IL15 signaling in murine models of liver fibrosis using IL15Rα knockout mice (IL15RαKO). In addition to the established effects of IL15 on maintaining natural killer (NK) cell homeostasis, a cell type well known to limit fibrogenesis, a direct anti-fibrotic effect of IL15 signaling on radio-resistant liver cells has also been observed. We aim to understand in detail the mechanism of the anti-fibrotic effect of IL15 signaling on hepatic stellate cells (HSC), the major type of fibrogenic cells in the liver,. Methods: HSCs were isolated from wild type (WT) and IL15RαKO mice by gradient centrifugation followed by depletion of contaminating leukocytes and endothelial cells. Purity of HSC was validated by the absence of mRNA for CD31 and CD45. HSC from WT and IL15RαKO were assessed by quantitative real time PCR (qPCR) and western blot to measure fibrogenic markers and well-known negative regulators of collagen transcription. Results: IL15RαKO HSCs display enhanced fibrogenesis both in the steady-state and under transforming growth factor β1 (TGF-β1) stimulation as shown by qPCR of collagen 1A1 (COL1A1), collagen 3A1 (COL3A1) and platelet-derived growth factor receptor (PDGFR). Treatment with the IL15/IL15Rα complex reduced expression of COL1A1 and alpha smooth muscle actin (αSMA) in IL15RαKO HSCs and reduced the magnitude of αSMA expression induced by TGF-β1. Expression of several negative regulators involved in the collagen expression pathway including TP53, serine-threonine kinase receptor-associated protein (STRAP) and ski-like (SKIL) were lower in IL15RαKO HSCs and were further down-regulated by TGF-β1 treatment. Conclusion: IL15 signaling regulates suppressors of collagen transcription to have a direct anti-fibrotic effect on HSCs. Our study highlights a possible therapeutic effect of IL15 and IL15/IL15Rα in liver fibrosis. Disclosures: The following authors have nothing to disclose: Kohtaro Ooka, Huanzi Han, Holger Fey, Costica Aloman, Jingjing Jiao 1371 NLRP3 inflammasome modulation of hepatic stellate cell activation: role in liver fibrosis Alexander Wree 1,2 , Matthew D. McGeough 1 , Davide Povero 1 , Akiko Eguchi 1 , Hal M. Hoffman 1 , Ariel E. Feldstein 1 ; 1 Pediatrics, UCSD, La Jolla, CA; 2 Internal Medicine III, University Hospital, RWTH-Aachen, Aachen, Germany Background: NLRP3 inflammasome activation has been increasingly recognized as an important mechanism of liver damage. Our group has recently demonstrated that isolated NLRP3 inflammasome activation is sufficient to induce liver inflammation and fibrosis (Hepatology 2014 Mar). Here we tested the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly modulate their activation and contribution to liver fibrosis. Methods: An immortalized human HSC line (LX-2) was incubated with the NLRP3 inflammasome inducers, LPS (as initial priming signal) and ATP (as activating signal). Additionally, primary HSCs were isolated from Nlrp3 D301NneoR/+ knock-in mice on a C57BL/6 background which were crossed to C57BL/6.Cg-Tg(Cre/Esr1)–Estrogen Receptor Cre (CreT) mice allowing for temporal control of NLRP3 activation. In a third set of experiments, mutant Nlrp3 was activated in the aforementioned mice in vivo for over 16 weeks. Activation of the NLRP3 inflammasome and HSCs was assessed via fluorescent imaging techniques and mRNA analysis. Results: LX-2 cells incubated with LPS (1ug/ml, over 3h) showed increased mRNA levels of Nlrp3 (1.5 fold compared to control), Incubation with ATP (5mM) resulted in an additional increase (2.0 fold). Collagen-1 mRNA levels were only raised in LX-2 cells when incubated with LPS and ATP (1.5 fold compared to control or LPS only). Fluorescently labeled active Caspase 1 was present in LX-2 cells when incubated with LPS + ATP. The NLRP3 inflammasome was induced in vitro in cultured primary HSCs via 4OH-tamoxifen treatment. Immunofluorescence for F-actin showed cytoskeleton reorga-
HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 883A nization as early as 6h after 4OH-tamoxifen incubation with mRNA levels of Nlrp3, alpha smooth muscle actin and tissue inhibitor of metalloproteinase 1 being increased. Activation of the NLRP3 inflammasome in Nlrp3 D301N/+ /CreT mice over 16 weeks resulted in hepatomegaly while tamoxifen-injected control mice showed regular liver weights (liver to body weight % - 6.1% ± 0.32% vs. 4.6% ± 0.03%, p < 0.05). Analysis of liver histology revealed increased inflammation with neutrophilic infiltration and increased mRNA levels of myeloperoxidase (fold-change to WT 52.8 ± 13.75, p
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1312A AUTHOR INDEX HEPATOLOGY, Octo
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