Macrophages and immunity in alcoholic liver disease and regeneration


CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis

Matthias Bartneck1, Josef Ehling2,3, Xiao Wei1, Viktor Fech1, Chrisfer Boeck1, Kanishka Hittotiya4, Tom Luedde1, Fabian Kiessling2, Twan Lammers2,5, Christian Trautwein1, Frank Tacke1

1Department of Medicine III, Medical Faculty, RWTH University, Aachen, Germany; 2Department of Experimental Molecular Imaging, Medical Faculty, RWTH University, Aachen, Germany; 3Institute of Pathology, Medical Faculty, RWTH University, Aachen, Germany; 4Institute of Pathology, Medical Faculty, RWTH University, Bonn, Germany; 5Department of Targeted Therapeutics, MIRA Instifute for Biomedical Technology and Technical Medicine, Enschede, Netherlands

Purpose of the study: In conditions of chronic liver injury, angiogenesis is associated with progressive fibrosis and may contribute to the development of hepatocellular carcinoma. Although hypoxia-induced expression of vascular endothelial growth factor (VEGF) occurs in advanced fibrosis, we hypothesized that inflammation may endorse hepatic angiogenesis already at the early stages of liver fibrosis. Methods: We developed a novel micro-CT-based in vivo imaging approach for the visualization and quantification of hepatic blood volume and studied the development of angiogenesis and fibrosis progression. Angiogenesis was studied after 6 weeks of repetitive carbon tetrachloride (CCl4) liver injury in C57BL/6 mice, with and without inhibition of the chemokine CCL2 by an antagonist (mNOX-E36). Results summary: Angiogenesis and fibrosis progression were closely correlated in chronic liver injury and were determined non-invasively by functional contrastenhanced μCT (Figure). Inflammatory monocyte-derived macrophages (MDM) massively accumulated in injured livers and co-localized with newly formed vessels in portal tracts. MDM expressed high levels of proangiogenic factors like VEGF. Pharmacological inhibition of monocyte infiltration by targeting CCL2 almost completely prevented fibrosis-associated angiogenesis, but not fibrosis progression, as assessed by in vivo μCT scans. Specifically, MDM primarily promoted sprouting angiogenesis within the portal vein tract based on liver endothelial cell staining. Conclusion: We demonstrate that inflammation-associated angiogenesis is promoted by CCL2-dependent monocytes during fibrosis progression and introduce an innovative in vivo μCT methodology to monitor angiogenesis and anti-angiogenic therapy effects in experimental liver fibrosis. Our data suggest that fibrosis progression, at least at its early stages, is largely independent from hepatic angiogenesis.



Christian Trautwein - Grant/Research Support: BMS, Novartis, BMS, Novartis; Speaking and Teaching: Roche, BMS, Roche, BMS

Frank Tacke - Grant/Research Support: Novartis, Noxxon; Speaking and Teaching: Roche, BMS, Gilead, Falk, MSD, Janssen

The following people have nothing to disclose: Matthias Bartneck, Josef Ehling, Xiao Wei, Viktor Fech, Christer Baeck, Kanishka Hittatiya, Tom Luedde, Fabian Kiessling, Twan Lammers


Alcoholic hepatitis not responding to medical therapy is related to a complex defect in liver regeneration

Laurent Dubuquoy1, Alexandre Louvet1,2, Guillaume Lassailly1,2, Stephanie Truonf3, Emmanuel Boleslawski3, David Buob4, Florent Artru1,2, Amélie Cannesson1,2, François Maggiotto1, Emilie Gantier1, Christophe Moreno5, Sebastien Dharancy1,2, Philippe Mathurin1,2

1U995, Inserm/Université Lille2,Lille, France; 2Service des Maladies de I'Appareil Digestif et de la nutrition, CHRU de Lille, Lille, France; 3Service de chirurgie digestive et transplantations, CHRU de Lille, Lille, France; 4lnstitut de Pathologie-Centre de Biologie Pathologie, CHRU de Lille, Lille, France; 5Department of Gastroenterology, Hepatopancreatology and Digestive Oncology, Erasme Hospital, Brussels, Belgium

In alcoholic hepatitis (AH) resistant to medical therapy, mechanisms leading to end-stage liver failure are unknown. Of note, the contribution of the regeneration process is still to be investigated. The availability of whole liver due to a program of early liver transplantation in AH is a unique opportunity to perform a translational study aiming at identifying these mechanisms. Methods: Liver explants from 16 patients transplanted for AH not responding to medical therapy were compared to 16 controls (normal liver after metastasis resection) and 12 patients transplanted for decompensated alcoholic cirrhosis. Liver cytokines were quantified using ELISA. Hepatocyte function and proliferation and liver progenitor biology were evaluated by immunohistochemistry, western-blot or RT-PCR. Mitochondrial morphology was evaluated by electron microscopy. Results: As expected, IL-8, a neutrophil chemoattractant, was strongly induced in AH (p<0.001). In AH, an important decrease in expression of albumin (p<0.001) and HepPar1 staining confirmed a significant defect in functional hepatocytes. Electron microscopy revealed a marked impairment of mitochondria in AH hepatocytes with no significant abnormality in the 2 other groups. TNF and IL-6, cytokines essential for liver regeneration, were reduced in AH compared to normal liver and cirrhosis (p<0.05). Ki-67 staining showed a virtual absence of hepatocyte proliferation in AH and control liver compared to a significant hepatocyte proliferation in cirrhosis (p=0.005). Proliferation of hepatic progenitors assessed by the expression of TWEAK and its receptor Fn14 was high in AH, elevated but to a significant lesser degree in cirrhosis and as expected very weak in the control liver (p<0.05). In hepatic lobule, presence of progenitors stained for cytokeratin 7 was observed only in AH with a biliary phenotype of progenitors as shown by an important cyfokeratin 19 staining. AH was characterized by a specific profile of ductular reaction (CD10-, CD56+, EMA+), that was unexpectedly different from that classically observed in diseases associated with marked regeneration. Conclusions: AH not responding to medical therapy is associated with an inflammatory process and severe mitochondrial dysfunction without compensatory regeneration of mature hepatocytes. Despite a marked proliferation, hepatic progenitors seem to engage only biliary differentiation. Future treatment for severe AH not responding to medical therapy should drive the orientation of regeneration to a mature hepatocyte profile.


Christophe Moreno - Board Membership: JANSSEN, Janssen Therapeutics; Consulting: Gilead, MSD; Grant/Research Support: ROCHE, Janssen, Novartis, Astellas, MSD; Speaking and Teaching: MSD, BMS, JANSSEN

Sebastien Dharancy - Board Membership: NOVARTIS, GENZYME; Speaking and Teaching: ASTELLAS, ROCHE

Philippe Mathurin - Board Membership: Schering-Plough, Janssen-Cilag, BMS, Gilead; Consulting: Roche, Bayer, Boehringer

The following people have nothing to disclose: Laurent Dubuquoy, Alexandre Louvet, Guillaume Lassailly, Stephanie Truant, Emmanuel Boleslawski, David Buob, Florent Artru, Amélie Cannesson, François Maggiotto, Emilie Gantier


γδ T cells promote liver regeneration via Dectin-1 dependent IL-17/IL-22 mediated inflammatory interplay

Raghavendra S. Rao, Christopher S. Graffeo, Rishabh Gulati, Suchithra Narayan, Tasnima Mohaimin, Stephanie Greco, Lena Tomkoetter, Eliza van Heerden, Rocky M. Barilla, Oscar Carazas, Reuven Blobstein, Yisroel Gelbstein, Atsuo Ochi, Constantinos P. Zambirinis, Michael Deutsch, George Miller

Surgery, New York University School of Medicine, New York, NY

Introduction: Inflammation plays a critical role in liver regeneration. γδT cells are recognized to have an emerging role in the innate immune system. We hypothesized that γδT cells could be important in the priming of liver regeneration. Methods: 70% partial hepatectomy was performed in 8 week old TCRδ-/-, Dectin-1-/- and C57BL/6 mice. Analyses were done using flow cytometry, immunohistochemistry, Western Blotting, PCR, ELISA and cytometric bead assays. Results: Partial hepatectomy leads to upregulation of Dectin-1 ligands in the liver with co-incident recruitment and functional activation of γδ T cells, which overexpress the Dectin-1 receptor. γδ T cells upregulate their expression of IL-22 and IL-17 in the regenerating liver in a Dectin-1 dependent manner. Further, we found that γδ T cells and Dectin1 expression are necessary for robust hepatic regeneration as TCRδ-/- and Dectin-1-/- mice had markedly reduced rates of hepatic regeneration. In particular, TCRδ-/- transgenic mice expressed reduced Cyclin D1, immediate early genes (c-fos and c-jun), HGF, and exhibited diminished activation of STAT3, MAP Kinase, and Notch signaling pathways during hepatic regeneration. Dectin-1-/- mice also exhibited depressed hepatocyte proliferation and Cyclin D1 upregulation. We showed that γδ T cells influence liver regeneration both by directly stimulating hepatocyte proliferation via IL-22 production and by inducing the generation of an activated pro-regenerative phenotype in Kupffer cells and DC while suppressing NKT cell activation and anti-regenerative IFN- γ production. We found that the capacity for y5 T cells to effect a pro-regenerative phenotype in DC, Kupffer cells, and NKT cells was dependent on their expression of lL-17 and accentuated by Dectin-1 ligation. More broadly, our observations are the first to implicate Dectin-1 ligation in any model of sterile inflammation and suggest that γδ T cell expression of IL-17 dictate the pro-inflammatory effects of Dectin-1 activation. Conclusion: Dectin-1 dependent IL-17/IL22 production from γδ T cells polarizes hepatic leukocyte subsets towards a pro-regenerative phenotype and directly induces hepatocyte proliferation. This is the first study to ascribe a central role for Dectin-1 signaling in sterile inflammation and highlights the central role of the Dectin-1- γδ T cell - IL-17 axis.


The following people have nothing to disclose: Raghavendra S. Rao, Christopher S. Graffeo, Rishabh Gulati, Suchithra Narayan, Tasnima Mohaimin, Stephanie Greco, Lena Tomkoetter, Eliza van Heerden, Rocky M. Barilla, Oscar Carazas, Reuven Blobstein, Yisroel Gelbstein, Atsuo Ochi, Constantinos P. Zambirinis, Michael Deutsch, George Miller


Myeloid specific deficiency of gp96, a master chaperone of toll-like receptor 4 (TLR4), reduces inflammatory cytokines and protects against alcoholic liver injury

Aditya Ambade, Donna Catalano, Pranoti Mandrekar

Department of Medicine, UMass Medical School, Worcester, MA

Background/Aims: Gut-derived endotoxin plays a significant role in the pathogenesis of alcoholic liver disease [ALD] via increased inflammatory cytokine production such as TNFα. The endoplasmic reticulum resident form of stress-inducible hsp90 (gp96/grp94) serves as a molecular chaperone for receptor TLR4 in the lipopolysaccharide (LPS) pathway. Chronic alcohol exposure increases gp96 mRNA and protein in the liver. Recently, we showed that hsp90/gp96 inhibition using a water soluble geldanamycin analog, 17-DMAG [17-Dimethylaminoethylamino-17-demethoxygeldanamycin], inhibits LPS induced inflammatory cytokines in the liver. Here we sought to determine whether gp96 plays a critical role in augmentation of chronic alcohol induced innate immune responses and liver injury. We hypothesize that myeloid specific deletion of gp96, a master chaperone of TLR4, alleviates alcohol induced liver injury by decreasing TLR4 expression and pro-inflammatory cytokines. Methods: To test our hypothesis, we subjected LysMCre+/-Hsp90b1flox/- (gp96 KO) mice and respective wild type (WT) littermates to chronic-binge alcohol feeding. Serum samples were evaluated for ALT and liver cytokine expression was assessed. TBARS, a marker of oxidative stress was assessed after alcohol feeding. For mechanistic in vitro experiments, we treated chronic alcohol exposed RAW 264.7 macrophages with hsp90/gp96 inhibitor, 17-DMAG and analyzed the effect on TLR4 surface expression. Results: Alcohol fed gp96 KO mice had significantly lower liver to body weight ratio as compared to alcohol fed WT mice (p=0.05). The alcohol fed gp96 KO mice had lower induction of serum ALT (p=0.05) compared to alcohol fed WT mice. Chronic binge alcohol induced triglyceride levels were significantly reduced in alcohol fed gp96 KO mice (p=0.04). Liver histopathology shows lower liver injury in gp96 KO mice. The cytokine gene expression analysis exhibits lower levels of TNFα (p=0.005), MCP-1 (p=0.003) and IL-6 (p=0.004) in alcohol fed gp96 KO mice. TBARS, a marker of oxidative stress was significantly lower (p=0.003) in alcohol fed gp96 mice compared to alcohoi fed WT mice. Further, upon inhibition by 17-DMAG in vitro, TLR4 surface expression was reduced significantly without any effect on viability in chronic alcohol exposed RAW macrophages. Conclusion: Our results support our hypothesis that myeloid specific deletion of gp96 protects mice from alcoholic liver injury. Further, gp96 inhibition in vitro results in down regulation of chronic alcohol induced TLR4 surface expression in macrophages.


The following people have nothing to disclose: Aditya Ambade, Donna Catalano, Pranoti Mandrekar


Alcohol-induced defects in transcytosis may be explained by decreased dynein processivity along hyperacetylated microtubules

Jennifer L. Groebner1,David J. Fernandez1,Dean J. Tumo2, Pomelo L. Tumo1

1 Biology Department, The Catholic University of America, Washington, DC; 2Department of Internal Medicine, University of Nebraska, Omaha, NE

Alcoholic liver disease has been clinically well described, but the molecular mechanisms leading to hepatoxicity remain to be fully elucidated. Previously, we determined that microtubules are hyperacetylated and more stable in ethanol-treated WIF-B cells, liver slices and in livers from ethanol-fed rats. From our recent studies, we believe that these modifications may explain alcohol-induced defects in microtubule-dependent basolateral secretion and in nuclear translocation of a subset of transcription factors. A common feature of these delivery routes is their dependence on microtubule-based motors. Since cytoplasmic dynein/dynactin, a minus-end directed motor and associated protein complex, is known to both mediate nuclear translocation and basolateral to apical transcytosis, we predicted that transcytosis would also be impaired in ethanol-treated cells. To test this possibility, we monitored transcytosis of three classes of newly synthesized apical proteins (lipid-anchored, single spanning membrane proteins and polymeric IgA receptor) in polarized, hepatic WIF-B cells. As predicted, apical delivery of all proteins tested was impaired in ethanol-treated cells. Unlike in control cells, transcytosing proteins were observed in discrete sub-apical puncta en route to the apical surface indicating that delayed delivery was not due to decreased basolateral internalization, but to impaired vesicle translocation. Furthermore, we determined that dynein/dynactin labeling redistributed from a diffuse cytosolic pool to discrete sub-apical puncta in ethanoltreated cells, a distribution also consistent with stalled microtubule-associated vesicles. Because microtubule motors are known to more tightly bind hyperacetylated microtubules, we assayed dynein/dynactin microtubule binding properties in control and ethanol-treated cells. We found that increased levels of dynein (but not dynactin) were recovered in taxol-stabilized pellets from treated cells consistent with enhanced associations. In contrast, no changes in vesicle association were observed for either dynein or dynactin implying vesicle binding is not altered by ethanol treatment. Thus, we propose that enhanced motor binding to microtubules in ethanol-treated cells leads to decreased motor processivity resulting in vesicle stalling and ultimately in impaired delivery. We are currently doing live cell imaging of transcytosis and developing cell-free vesicle motility assays to reveal the mechanism of motor impairment.


The following people have nothing to disclose: Jennifer L. Groebner, David J. Fernandez, Dean J. Tuma, Pamela L. Tuma


The role of neutrophil endotoxin tolerance in the predisposition to sepsis in alcohol-related liver disease

Jennifer M. Ryan1,Godhev K. Manakkat Vijay1, (Robin) Daniel Abeles1, Thomas Tranah1, Lee J. Morkwick2, Laura J. Blockmore1, Antonio Rivo2, Nikhil Vergis3, Nicholas J. Taylor1, Shilpo Choksh2, Yun Mo1, John G. OGrody1, Debbie Showcross1

1 Institute of Liver Studies and Transplantation, King's College London School of Medicine of King's College Hospital, London, United Kingdom; 2Foundofion for Liver Research, London, United Kingdom; 3Hepatology Research Unit, St. Mary's Hospital, Imperial College London, London, United Kingdom

Introduction: Due to the high rates of sepsis it is essential to improve understanding of the immunodeficiency present in cirrhosis. Systemic endotoxemia in patients with advanced cirrhosis, particularly alcohol-related, is widely recognised and has been purported to induce up-regulation of circulating neutrophil Toll-like receptor (TLR) expression. It is unclear, however, whether chronic endotoxin exposure alters the neutrophil response to an acute endotoxin challenge or ‘second hit' which frequently precipitates acute-on-chronic liver failure with high mortality. Aims: We examined circulating neutrophil phenotype and function pre- and post-endotoxin challenge in 29 patients with cirrhosis"[9 actively drinking (AA); 7 abstinent (ABA); 13 with acute alcoholic hepatitis (AH) with a discriminant factor >32] and 10 healthy controls (HC). Methods: Neutrophil phenotype was characterised in peripheral blood using fluorochrome conjugated monoclonal antibodies anti-CD16, -CD11b, -TLR-2 and -TLR-4. Reactive oxygen species (ROS), as a surrogate marker of endotoxemia, were measured by the conversion of dihydrorhodamine to rhodamine and quantified by flow cytometry. Neutrophils were stimulated with 0.2ng/μL lipopolysacchararide (LPS) for 2 hours and response analysed. Results: Median MELD scores for the AA, ABA and AH cohorts were 15, 11 and 26 respectively. 4/9 patients in the AA, 1/7 in the ABA and 4/13 in the AH groups were culture-positive. Only the circulating AH neutrophils had reduced CD16 (p<0.01) and TLR-2 expression (p<0.05) and showed a trend towards reduced TLR-4 expression (p=0.09) at baseline with elevated production of ROS (p<0.05) compared to HC. Neutrophils from HC up-regulated TLR-4 receptor expression in response to challenge with LPS (p=0.02) but the cirrhotic and AH patients failed to do so. A significant increase in TLR2 expression was seen following stimulation with LPS in the HC and ABA group (p<0.05) but not in the AA and AH patients. 6/8 patients who had microbiological proven infection displayed inappropriate down-regulation of TLR-4 post stimulation. Conclusion: Neutrophils from AH patients had reduced activation markers and elevated production of R〇S. Within all patient groups circulating neutrophils failed to appropriately up-regulate TLR-4 expression and in those who went on to develop culture-positive infection TLR-4 was in fact down-regulated upon stimulation. The failure to adequately respond to infection or a ‘second hit' may relate to chronic systemic neutrophil endotoxin exposure and active alcohol intake. Strategies to overcome endotoxin tolerance may be beneficial in reducing life-threatening septic episodes.


John G. O'Grady - Advisory Committees or Review Panels: Astellas, Novartis; Speaking and Teaching: Astellas, Roche

Debbie Shawcross - Advisory Committees or Review Panels: Norgine; Speaking and Teaching: Norgine

The following people have nothing to disclose: Jennifer M. Ryan, Godhev K. Manakkat Vijay, (Robin) Daniel Abeles, Thomas Tranah, Lee J. Markwick, Laura J. Blackmore, Antonio Riva, Nikhil Vergis, Nicholas J. Taylor, Shilpa Chokshi, Yun Ma