Inflammation and fibrosis


145

HIV-infection of Kupffer cells results in a dysregulated response to LPS despite effective Anti-Retroviral Therapy

Arevik Mosoian1, Feng Hong1, Yedidya Saiman1, Adeeb Rahman1, Andrea D. Branch1, Sasan Roayaie1, Sander Florman1, Francesc Cunyat2, Mario Stevenson2, Meena Bansal1

1lcahn School of Medicine at Mount Sinai, New York, NY; 2University of Miami Leonard M. Miller School of Medicine, Miami, FL

HIV infection causes CD4+ lymphocyte depletion in the gut and has been linked to the disruption of gut epithelial integrity and increased microbial translocation. Microbial translocation has been linked to liver disease progression in HIV/HCV coinfected patients and to systemic immune activation in HIVinfected patients. Since Kupffer cells (KCs) are known targets of HIV in vivo, are responsible for the uptake of translocated microbial products entering the portal circulation, and are tolerant to effects of LPS, we hypothesized that HIV infection of KCs may alter their response to LPS and postulated that despite effective systemic anti-retroviral therapy, their response to LPS may be persistently dysregulated Methods: KCs were isolated by density centrifugation from livers of patients with and without HIV infection who underwent resection or transplant (3HIV/HCV and 1-HIV/HBV/HCV). All HIV patients had undetectable plasma HIV for a minimum of one year. Purity of KC preps was assessed by RT-qPCR for CD68, CD14, TLR4, CD3, and CD31.HIV-BaL (R5 tropic) and HIV-IIIB (X4 tropic) were used to infect primary KCs. Cytokine levels in response to LPS were examined by RT-qPCR and ELISA in both the in vitro infected KCs and the KCs derived from HIV-infected patients. Evidence of latent HIV infection in KCs was assessed by RTqPCR for HIV multiply-spliced RNA products and by qPCR for HIV DNA. Results: R5 tropic HIV-Bal productively infected KCs and resulted in large amounts of viral production for over one month in culture suggesting that infection is not cytopathic. KCs challenged with LPS showed a minimal inflammatory response. However, infection of KCs with R5 or X4 tropic HIV resulted in a significantly enhanced production of pro-inflammatory cytokines (IL-8, TNFα, IL-6) and the pro-fibrogenic cytokine (TGFβ1) in response to LPS but not to poly-IC suggesting a specific effect on TLR4 ligands. Most notably, we detected HIV DNA by qPCR and multiply-spliced RNA by RT-qPCR in KCs derived from the livers of all aviremic HIV patients and these cells maintained a dysregulated pro-inflammatory response to LPS. Conclusions: HIV-1 infection of Kupffer cells in vitro results in an augmented pro-inflammatory and pro-fibrogenic response to LPS but not poly-IC. Despite undetectable plasma HIV RNA in patients on ART, KCs contain HIV DNA and multiply-spliced RNA and maintain a dysregulated pro-inflammatory response to LPS. These findings suggest that HIV infection of Kupffer cells may contribute to hepatic inflammation and fibrosis by altering response to translocated microbial products.

Disclosures:

Andrea D. Branch - Grant/Research Support: Kadmon, Gilead, Janssen

The following people have nothing to disclose: Arevik Mosoian, Feng Hong, Yedidya Saiman, Adeeb Rahman, Sasan Roayaie, Sander Florman, Francesc Cunyat, Mario Stevenson, Meena Bansal

146

HCV is taken up by human liver sinusoidal endothelial cells (LSECs) and triggers IFN Type I and III (lambda) production and autocrine/paracrine signaling that inhibits HCV replication

Silvia Giugliano1, Lucy Golden-Mason 1, 2, Evgenia Dobrinskikh1, Amy E. Stone1, 2, Alejandro Soto-Gutierrez6, Michael Gale4, Vijay Shah5, Hugo R. Rosen1, 3;

1Gastroenterology and Hepatology, University of Colorado at Denver, Denver, CO; 2Immunology, National Jewish Hospital, Denver, CO; 3VA Hospital, Denver, CO; 4Immunology, University of Washington, Seattle, WA; 5Hepatology, Mayo Clinic, Rochester, MN; 6Surgery, University of Pittsburgh, Pittsburgh, PA

LSECs have been highly conserved during evolution to clear waste molecules entering the circulation and make up ∼50% of non-parenchymal cells in the liver. LSECs are known to transcytose HCV and facilitate direct contact with hepatocytes, but their roles in HCV RNA sensing and induction of innate antiviral responses remains largely unexplored. Methods/Results: We used both primary human LSECs (from eight subjects) and an immortalized LSEC line. LSECs express high levels of CD31, ICAM-1, DC-SIGN, L-SIGN, stabilin-1, as well as receptors implicated in HCV entry (LDL-R, CD81, SR-BI). Confocal microscopy demonstrated the presence of HCV core and HCV NS5A proteins 24 hrs after addition of full-length HCV JFH-1 (MO 0.1). The uptake of HCV occurred independently of direct hepatocyte contact. This process was mediated by clathrindependent endocytosis. Although LSECs took up HCV, they did not sustain replication at at 24, 48 hrs, and 72 hrs. Next, primary LSECs were transfected with an HCV PAMP (the pU/UC tract in the 3΄ UTR); as a negative control, primary human LSECs cells were transfected with the adjacent highly-conserved X-region. After 8 hrs, qRT-PCR was performed on multiple antiviral genes. IFN-β and IFNL1, IFNL2, IFNL3 were all upregulated > 20-fold following PAMP transfection. In contrast, stimulation with Poly I: C (synthetic TLR3 ligand) failed to induce IFNLs; LPS induced TNF-α and IL-6 but no IFNs. IL-28B (IFNL3) genotyping (rs12979860) demonstrated that favorable CC genotype was associated with significantly higher transcription of IFNLs by LSECs following PAMP stimulation. In order to examine autocrine and paracrine signaling pathways, LSECs were treated with recombinant IFN-α or recombinant Type III IFNs and mRNA for IFNs and ISGs tested at 8 and 24 hrs. Remarkably, IFNL3 was the most consistently-induced IFN. 〇O the ISGs, viperin (virus inhibitory protein, endosplasmic reticulum-associated, IFN-inducible), known to have inhibitory effects on viral replication by physically associating with the HCV NS5A, was the most highly induced (>10, 000 fold) with LSECs. Supernatants from resting LSECs were able to inhibit JFH-1 replication in Huh7.5.1cells by more than 80% and supernatants from IFN-treated LSECs totally eliminated HCV replication. Conclusion: For the first time, we show that human endocytose HCV. HCV RNA triggers robust Type I and III IFNs which in turn induce autocrine IFN production and remarkably high ISGs, in particular viperin within LSECs that may contribute to the restricted replication of HCV. Both resting and IFN-treated LSECs inhibit HCV replication in hepatocytes.

Disclosures:

The following people have nothing to disclose: Silvia Giugliano, Lucy GoldenMason, Evgenia Dobrinskikh, Amy E. Stone, Alejandro Soto-Gutierrez, Michael Gale, Vijay Shah, Hugo R. Rosen

147

Hepatic sdf-1 (CXCL12) acts downstream of VEGF to recruit liver sinusoidal endothelial cell progenitor cells from the bone marrow in liver regeneration

Laurie D. DeLeve1,Xiangdong Wang1, Lei Wang1, William A. Gaarde2

1 Division of Gastrointestinal and Liver Diseases, Usc Keck School of Medicine, Los Angeles',CA; 2 ISIS Pharmaceuticals, Carlsbad, CA

We have previously demonstrated that recruitment of liver sinusoidal endothelial cell progenitor cells (SPC) from the bone marrow is essential for liver regeneration (Wang et al, JCI 122: 1567-1573, 2012). After both partial hepatectomy and toxininduced liver injury, hepatic vascular endothelial growth factor (VEGF) plays a central role in regulating proliferation of sPC in the bone marrow, mobilization of SPC to the circulation, engraftment of SPC in the liver, and differentiation of SPC to liver sinusoidal endothelial cells (Wang et al, Gastroenterology 143: 1555-1563, 2012). However it is not intuitively obvious why a growth factor would regulate homing and engraftment of cells. We therefore examined whether the chemokine sdf-1 (stromal derived factor 1 or CXCL12), which is involved in homing and migration of T-lymphocytes, monocytes, and stem and progenitor cells, might be a downstream regulator of this process. Methods: Studies were performed in the two-thirds partial hepatectomy model in rats. VEGF and sdf-1 were knocked down by 4-weeks of a twice-weekly i. p. injection of their specific antisense oligonucleotides (ASO; ISIS Pharmaceuticals) with scrambled ASOas controls. SPC were identified as CD133+31 +45+ cells in bone marrow (BM SPC) or in the circulation. Engraftment of BM SPC as liver sinusoidal endothelial cells after partial hepatectomy was determined by GFP tracking in wild type Lewis rats transplanted with bone marrow from Lew-Tg(CAGEGFP)ys Lewis rats. Results: (1)Knockdown of VEGF with ASO decreased sdf-1 gene and protein expression by 60% (p<0.001) and 80% (p<0.005), respectively, demonstrating that sdf-1 is downstream of VEGF. (2) After partial hepatectomy, hepatic sdf-1 gene and protein expression increased more than 6-fold (p<0.0005) and 2-fold (p<0.01), respectively. (3) As was seen with VEGF knockdown, sdf-1 knockdown with ASO completely prevented the partial hepatectomy-induced increase in proliferation of SPC in the bone marrow (p<0.01 compared to scrambled AS〇 control) and mobilization of SPC to the circulation (p<0.05 compared to scrambled ASO control). Engraftment of BM SPC after partial hepatectomy was decreased by 50% after sdf-1 ASO(p<0.005). Conclusion: Hepatic sdf-1 acts downstream from VEGF to regulate homing and engraftment of bone marrow SPC after partial hepatectomy.

Disclosures:

Laurie D. DeLeve - Advisory Committees or Review Panels: Pfizer, Takeda, BristolMyers Squibb, Amgen

William A. Gaarde - Employment: ISIS, ISIS, ISIS, ISIS

The following people have nothing to disclose: Xiangdong Wang, Lei Wang

148

Hepatic Stellate Cells Orchestrate Clearance of Dead Hepatocytes from the Liver after Acute Injury in a Hypoxia-inducible Factor-1α-dependent Manner

Bryan Copple1, Aaron Pace1,Akie J. Mochizuki1, Keara Towery2, James P Luyendyk2

1 Pharmacology and Toxicology, Michigan State University, East Lansing, Ml; 2Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI

Hypoxia-inducible factor-1 α (HIF-1 α) is a transcription factor activated in cells by hypoxia, as well as other mediators. We recently demonstrated that HIF-1 a is activated in hepatic stellate cells (HSCs) in diseased human livers. In addition, we demonstrated that HIF-1 α regulates genes in HSCs important for tissue repair, including genes involved in angiogenesis, matrix remodeling, and immunomodulation. This suggested that activation of HIF-1α a in HSCs may be critical for liver regeneration after acute injury. To test this hypothesis, mice were generated with reduced levels of HIF-1 α in HSCs by crossing HIF-1 α floxed mice with mice that express Cre recombinase under control of the glial fibrillary acidic protein (GFAP) promoter (i. e., HIF-1 αGFAP Cre+ mice). These mice and control mice (i. e., HIF-1 αGFAP Cre- mice) were treated with 1 ml/kg of carbon tetrachloride and liver injury and repair were assessed. Liver injury, as measured by serum ALT activity, was not different between the two mouse strains at 24, 48 or 72 hours after treatment. By 72 hours after carbon tetrachloride, necrotic hepatocytes were largely cleared from the livers of HIF-1 α-GFAP Cremice. In striking contrast, large areas of necrosis remained in the livers of HIF-1 α-GFAP Cre+ mice, suggesting a defect in the clearance of necrotic cells from the liver. In these mice, the persistence of necrotic hepatocytes stimulated a fibrotic response characterized by extensive collagen deposition and elevated αsmooth muscle actin levels. To elucidate the mechanism by which activation of HIF-1 α in HSCs mediated clearance of necrotic hepatocytes, we determined the impact of HIF-1 α deletion on recruitment of phagocytic innate immune cells to the liver. Numbers of CD68 positive and F4/80 positive macrophages were not different between HIF-1 α-GFAP Cre+ and HIF-1 α-GFAP Cre- mice, however, fewer neutrophils accumulated in the livers of HIF-1 α-GFAP Cre+ mice after carbon tetrachloride. Consistent with this, levels of several inflammatory cytokines and chemokines, critical for neutrophil recruitment, were lower in HIF-1 α-GFAP Cre+ mice after carbon tetrachloride. This suggests that activation of HIF-1 α in HSCs elicits production of inflammatory cytokines which recruit neutrophils to the liver to remove dead cell debris—a critical aspect of liver remodeling during repair. Collectively, these studies have identified a novel function for HSCs in orchestrating the clearance of necrotic hepatocytes from the liver, and demonstrated a key role for HIF-1 α in modulating the hepatic inflammatory response during acute liver injury.

Disclosures:

The following people have nothing to disclose: Bryan Copple, Aaron Pace, Akie J. Mochizuki, Keara Towery, James P. Luyendyk

149

Macrophage autophagy protects from liver fibrosis

Jasper Ladder1, 2, Marie-Noële Chobert1, 2, Sophie Lotersztajn1, 2, Fatima Teixeira-Clerc1, 2

1U955, INSERM, Créteil, France; 2UMR_S955, Université Paris-Est, Créteil,France

Aim: Macrophages play a central role in the development of liver fibrosis by promoting activation of hepatic fibrogenic cells. Autophagy is a lysosomal degradation pathway of cellular components that plays an essential role in cellular homeostasis. Recent studies have shown that macrophage autophagy displays anti-inflammatory properties in inflammatory conditions such as atherosclerosis, colitis and tuberculosis. The aim of this study was to investigate the role of macrophage autophagy in the pathogenesis of liver fibrosis. Methods: Experiments were performed in mice invalidated for the autophagy gene, ATG5, in the myeloid lineage (ATG5Mye-/- mice) and the ATG5Mye+/+ littermate wild-type (WT) mice. Liver fibrosis was induced in mice by repeated intraperitoneal injection of CCl4.In vitro studies were performed on peritoneal macrophages isolated from WT and ATG5Mye-/- mice and on hepatic myofibroblasts isolated from WT mice. Results: Studies in isolated peritoneal macrophages demonstrate that macrophages invalidated for the ATG5 gene are deficient in autophagy. Moreover, the expression of the pro-inflammatory cytokines, IL-6, IL-1 α, ll1β and TNF-α, was significantly increased in peritoneal macrophages isolated from ATG5Mye-/- mice and treated with LPS, compared to macrophages derived from WT mice and treated with LPS. In addition, the expression of the profibrogenic markers TGF-β1, TIMP-1, MMP2 and MMP9 was increased in myofibroblasts exposed to conditioned medium of LPS-treated ATG5-/- macrophages compared to myofibroblasts incubated with conditioned medium from LPS-treated WT macrophages. ATG5Mye-/- mice showed increased liver fibrosis, as shown by enhanced matrix deposition and increased expression of collagen I mRNA in CCl4-treated ATG5Mye-/- mice as compared to WT mice. Moreover, macrophage autophagy invalidation enhances the expression of the fibrogenic markers TGF-β1, TIMP-1, MMP2 and MMP9 and of the myofibroblast marker, a-SMA. Finally, macrophage autophagy inactivation promoted inflammatory cell recruitment, as reflected by increased expression of Ly6C, CCR2, MARCO Ly6G and MPO. Conclusion: These data demonstrate that macrophage autophagy invalidation promotes liver fibrosis by regulating hepatic inflammation. These results suggest that macrophage autophagy may display anti-inflammatory and anti-fibrogenic properties.

Disclosures:

The following people have nothing to disclose: Jasper Lodder, Marie-Noële Chobert, Sophie Lotersztajn, Fatima Teixeira-Clerc

150

The liver-derived plasma protein histidine-rich glycoprotein promotes chronic liver injury and fibrosis by polarizing hepatic macrophages towards the inflammatory M1 phenotype

Matthias Bartneck1, Viktor Fech1, Josef Ehling2, 3, Xiao Wei1, Klaudia Warzecha1, Kanishko Hittatiya4, Nikolaus Gassler3, Twan Lammers2, 5, Willi Jahnen-Dechent6, Christian Trautwein1, Frank Tacke1

1Medical Clinic III, Medical Faculty, RWTH University, Aachen, Germany; 2Department of Experimental Molecular Imaging, Helmholtz Institute for Biomedical Engineering, Medical Faculty, RWTH University' Aachen, Germany; 3Institute of Pathology, Medical Faculty, RWTH University, Aachen, Germany; 4Department of Pathology, Rheinische Friedrich-Wilhelms-University, Bonn, Germany; 5Department of Targeted Therapeutics, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, Netherlands; 6Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen, Germany, Aachen, Germany

Purpose of the study: Hepatic macrophages critically promote hepatic inflammation and fibrogenesis. However, the role of macrophages polarization for the outcome of liver diseases is not clear. The histidine-rich glycoprotein (HRG) is an abundant plasma protein that mediates the transition of alternatively activated (M2) to pro-inflammmatory (M1) macrophages that have been recently shown to inhibit tumor growth and metastasis. Methods: The functional role of HRG was studied in C57BL6 wildtype and HRG-/- mice in models of carbon tetrachloride (CCl4)-induced acute liver injury, CCl4-induced fibrosis and methionine-choline deficient (MCD) diet-mediated steatohepatitis using biochemistry (ALT, AST, hydroxyproline), histology, immunohistochemistry, micro-computerized tomography (μ-CT), cell cultures of bone marrow derived macrophages (BMM), and flow cytometric immune cell phenotyping. Results summary: In steady state conditions, hepatic macrophages from HRG-/-mice are polarized towards the “anti-inflammatory” M2 subtype, alongside less activated hepatic natural killer (NK) cells as reflected by reduced LAMP-1 expression. Upon chronic liver injury induced by CCl4 or MCD diet, liver injury and fibrosis were attenuated in HRG-/- compared to WT mice. This was accompanied by lower numbers of inflammatory macrophages, Kupffer cells, and fewer degranulated (LAMP-1+) NK cells. In order to dissect whether the soluble factor HRG (and not its expression of macrophages) is truly responsible for altered macrophage polarization, we performed extensive in vitro experiments with WT and HRG-deficient BMM, revealing that HRG-/- macrophages can still be polarized towards functional M1- or M2-macrophages upon specific stimulation by interferon gamma (IFN۷) or interleukin-4 (IL4), respectively. Strikingly, HRG-deficient mice showed significantly enhanced hepatic vascularization by histology and μ-CT-based determination of hepatic blood volume, suggesting proangiogenic activities of hepatic M2-polarized macrophages, independent of fibrosis development. Conclusion: The liver-derived plasma protein HRG is a novel endogenous molecular factor promoting the polarization of hepatic macrophages towards the M1 phenotype, thereby accentuating chronic liver injury and fibrosis progression, but limiting neo-angiogenesis. The HRG protein might therefore represent a promising therapeutic target in chronic liver fibrosis.

Disclosures:

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, Viktor Fech, Josef Ehling, Xiao Wei, Klaudia Warzecha, Kanishka Hittatiya, Nikolaus Gassler, Twan Lammers, Willi Jahnen-Dechent

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