Mechanisms of Injury


A unique subset of CD11c-positive dendritic cells mediate immunological tolerance via TLR9 in Concanavalin A-induced liver injury in mice

Nobuhiro Nakamoto1, Hirotoshi Ebinuma1, Takanori Kanai1, Yuko Wakayama1, Nobuhito Taniki1, Hiroko Murata1, Yohei Mikami1, Po-sung Chu1, Kazuo Sugiyama1, Hidetsugu Saito2,1, Toshifumi Hibi1;

1Department of Internal Medicine, University of Keio, School of Medicine, Tokyo, Japan; 2 Department of Pharmacotherapeutics, Keio Unversity, School of Pharmacy, Tokyo, Japan

BACKGROUND & AIMS: We recently reported that C-C chemokine receptor (CCR) 9+ CD11 b+ macrophages play a critical role in the pathogenesis of acute liver injury by a single injection of Concanavalin A (Con A) in mice (Gastroenterology 2012). On the other hand, it is known that a repetitive injection of Con A results in immunological tolerance under a specific condition. In this study, we tried to elucidate a participation of a unique subset of APCs in mediating liver tolerance, especially focusing on the disease specific toll like receptors (TLRs). METHODS: Male C57BL/6 mice were re-injected with a sublethal dose of Con A 1, 3, or 7 days after an initial Con A administration to induce immunological tolerance. Liver mononuclear cells were separated 12 hours after the final Con A administration, and soluble cytokines production from these cells stimulated with TLR1-9 agonist in vitro was measured by Cytometric Bead Array (CBA) assay. RESULTS: Con A pretreated mice were partially protected from liver injury by Con A rechallenge 7 days after pretreatment, while liver damage was more aggravated by Con A rechallenge as early as 3 days after pretreatment. We next performed a serial analysis of the number and the function of hepatic APCs following Con A administration. The number of CD11 b+ F4/80+ macrophages dramatically increased that peaked at 24 hours and decreased thereafter. In contrast, the number of CD 11c+ DCs decreased that peaked at 24 hours perhaps due to an increased cell death, and returned to the baseline by 7 days. CD11c+ DCs within Con A-treated liver were phenotypically mature with increased expression of CD80, CD86, and MHC class2. In vitro stimulation of whole liver cells with TLR4, 6, and 9 agonist induced the production of pro-inflammatory cytokines such as IL-6 and TNF-α, while the production of IL-10 was only induced by TLR9 agonist stimulation and the Th 1 /Th2 balance via TLR9 shifted to Th2 dominant with time following Con A administration. Hepatic CD11c+ DCs sorted by MACS beads 7 days after Con A administration (CD11c+ DCs-D7) have a maximum potential to produce IL-10 and TGF-β by TLR9 agonist stimulation compared with other APCs subset. These CD11c+ DCs prompted naïve CD4 T cells to differentiate to a regulatory phenotype in the presence of TLR9 agonist in vitro. Finally, Pretreatment with CD11c+ DCs-D7, but not CD11c+ DCs sorted at earlier time point or CD11 b+ macrophages, protected mice from Con A induced acute liver damage in vivo. CONCLUSIONS: In summary, IL-10 producing MHC class2+ CD11c+ DCs play a role in mediating liver tolerance via TLR9 as an important negative regulator of the excessive liver inflammation by Con A in mice.


Toshifumi Hibi - Grant/Research Support: Abbott Japan, Ajinomoto Pharma, Astrazeneca Phramaceuticals, Janssen Pharmaceutical K.K, Tanabe Mitsubishi Pharma

The following people have nothing to disclose: Nobuhiro Nakamoto, Hirotoshi Ebinuma, Takanori Kanai, Yuko Wakayama, Nobuhito Taniki, Hiroko Murata, Yohei Mikami, Po-sung Chu, Kazuo Sugiyama, Hidetsugu Saito


Inflammatory Cytokines Released by Visceral Adipose Tissue (VAT) Is Associated with Development of Non-Alcoholic Fatty Liver Disease (NAFLD)

Aybike Birerdinc1,2, Katherine Doyle2, Lei Wang3, Rohini Mehta1,3, Zahra Younoszai1, Vikas Chandhoke2, Zachary D. Goodman2, Ancha Baranova1,2, Zobair M. Younossi1,3;

1Betty and Guy Beatty Center for Integrated Research, Inova Health System, Falls Church, VA; 2Center for the Study of Chronic Metabolic Diseases, George Mason University, Fairfax, VA; 3Center for Liver Disease, Department of Medicine, Inova Fairfax Hospital, Falls Church, VA

Visceral adipose is now known to be an active endocrine organ. The cytokines released by visceral fat (VAT) are thought to have both local and systemic effects. Our aim was to assess the role of visceral fat TGFb1 gene expression and the associated cytokine-signaling cascade in distinguishing NASH from non-NASH NAFLD. Methods: RNAs were extracted from frozen visceral fat samples of 241 patients with liver biopsy proven NAFLD using Bio-Rad's Aurum Total RNA Fatty and Fibrous Tissue Kit. 1 μg of RNA from each sample was converted to cDNA using SABiosciences' RT2 First Strand Kit. cDNA samples were added to 10 μL q-RTPCR reactions in a 96 well plate format. Serum was tested with the Biorad Bio-Plex Pro TGFb1 and 17-plex cytokine assays. All primers were custom designed and validated. Group differences in expression levels were assessed using Mann-Whitney tests. An ANOVA tests were performed to distinguish the contribution of relevant cytokines to the presence of NASH and non-NASH NAFLD. Results: A total of 241 patients (39.1% NASH, 33.9% NAFLD, 20.8% with type 2 diabetes, age 43.61 +/-11.41 years, BMI 46.39+/-10.91) were included. When control subjects with no disease were compared to those with NAFLD, the differential factors were the ratio of ALT/AST (P < 0.0019) and levels of glucose (P < 0.0016), whereas in the comparison of non-NAFLD diseases and NASH the strongest differentiation factor was MIP-1b (p<0.003). Interestingly, serum levels of cytokines such as TGFb1 (p < 0.006), MIP-1b (p <0.00273), IL-8 (P < 0.0002), and IL-17 (p< 0.002) all have similar differentiating power for the group with no disease/NASH and non-NASH NAFLD/NASH, while the adipose-specific gene expression levels TGFb1 (p<0.002) and serum IL-5 (p<0.004) were capable of differentiate these groups. Additionally, TGFb1 gene expression in VAT and TGFb1 in serum shows strong negative correlations with scored histopathological features such as hepatocyte ballooning (r=-0.2241 p<0.04433), Kupffer cell hypertrophy (r=-0.3687, p<0.0007078), Lymphocyte infiltration (r=-0.3368, p<0.002112) and the presence of polymor-phonucleated cells (r=-0.2836, p<0.0103). Conclusion: Cytokines released by VAT may guide the development of the inflammatory component of liver disease in patients with NASH. The relationship between the expression of TGFb1 gene in VAT and serum levels of inflammatory cytokines warrants further investigations.


Zachary D. Goodman - Grant/Research Support: Gilead Sciences, Fibrogen, Galectin Therapeutics, Merck, Vertex

Zobair M. Younossi - Advisory Committees or Review Panels: Merck, Vertex, Tibotec/J andJ; Consulting: Gilead Sciences

The following people have nothing to disclose: Aybike Birerdinc, Katherine Doyle, Lei Wang, Rohini Mehta, Zahra Younoszai, Vikas Chandhoke, Ancha Baranova


Microbiota modulates susceptibility to T-cell mediated liver injury

Stela Celaj, Michael W. Gleeson, Jie Deng, James D. Gorham;

The Geisel School of Medicine at Dartmouth, Lebanon, NH

Variability in disease progression is common to all liver diseases caused by T cell mediated hepatocellular injury, and is one of the most challenging aspects of the effective management of patients with inflammatory liver disease. Identifying factors that regulate liver inflammation and injury is critical to understanding how and why some patients progress rapidly. We show that resident gut microbiota is a major regulator of T cell mediated liver injury. Hepatic inflammation was induced in BALB/c mice through administration of Con A, which activates NKT cells and T cells, and leads to acute damage through hepatocyte Fas activation. Mice from different vendors (Jackson Labs (JAX); Taconic (TAC)) exhibited different levels of liver damage following Con A: JAX mice were highly sensitive and TAC mice were more resistant. Analysis of 1400 SNPs genome-wide showed that JAX and TAC BALB/c mice were genetically indistinguishable. Assessment of fecal microbiota using 16S deep sequencing showed distinct microbial populations in JAX mice and TAC mice, including differential levels of segmented filamentous bacteria. Importantly, sensitivity to Con A could be transferred between mice following co-housing. Preliminary analysis showed that liver immune cell composition was similar between JAX mice and TAC mice, as were liver cytokines and chemokines released following Con A. Interestingly, JAX mice were much more sensitive than TAC mice to liver damage induced by injection of the Fas activating mAb Jo-2, a maneuver that bypasses the immune system and induces liver injury directly by activating Fas on hepatocytes. Similarly, treatment of JAX mice with oral antibiotics greatly reduced Jo-2 induced liver injury. Thus, the microbiota potently regulates T cell mediated liver injury, and exerts its influence not by modulating the immune system per se, but rather by acting at the level of the hepatocyte, serving as a rheostat to modulate the hepatocellular response to Fas mediated cell death.


The following people have nothing to disclose: Stela Celaj, Michael W. Gleeson, Jie Deng, James D. Gorham


Identification of the Signal Transduction Pathways Critical for the Interaction between Bile Acids and the IL-23/IL-1 7A Axis during Cholestasis

Kate M. O'Brien, Kara Kelly, Bryan Copple; Pharmacology and Toxicology, Michigan State University, East Lansing, MI

Bile acids and the IL-23/IL-17A axis are critical mediators of inflammation in the liver during cholestasis. We recently showed that bile acids and the IL-23/IL-17A axis interact by two separate mechanisms to elicit an inflammatory response. First, the bile acid, taurocholic acid (TCA), stimulates hepatocytes to

produce IL-23, a key cytokine for maintenance of Th17 cells, the major source of IL-17A. Second, IL-17A synergistically enhances production of inflammatory mediators by TCA-treated hepatocytes. Considering the importance of these two pathways to cholestatic liver disease, the present studies aimed to elucidate the signal transduction pathways that mediate these two mechanisms. Studies have shown that IL-17A activates the transcription factor, CCAAT/enhancer binding protein beta (C/EBPβ) in hepatocytes. Accordingly, we hypothesized that IL-17A activates C/EBPβ which synergistically enhances upregu-lation of the proinflammatory cytokine, macrophage inflammatory protein-2 (MIP-2), by TCA. Primary hepatocytes were isolated from C/EBPβ heterozygous mice or wild-type (WT) littermates and treated with 10 ng/mL IL-17A in the presence or absence of 200 μM TCA. MIP-2 mRNA levels were measured by real-time PCR. In WT hepatocytes, IL-17A synergistically enhanced induction of MIP-2 by TCA; whereas, heterozygous deletion of C/EBPβ completely prevented this synergistic interaction. These data suggest that C/EBPβ is critical for the synergistic interaction between IL-1 7A and TCA in hepatocytes. Next, we identified the signal transduction pathways that mediate upregulation of IL-23 by TCA. Treatment of hepatocytes with TCA activated PI3K/AKT, JNK, and p38. Inhibition of PI3K/AKT and JNK attenuated the induction of IL-23 by TCA; whereas, p38 inhibition enhanced TCA-induced IL-23 production. Overall, these studies identified the key signal transduction pathways that mediate the interaction between bile acids and the IL-23/IL-17A axis. Pharmacological targeting of these pathways could alleviate hepatic inflammation and injury in patients with cholestatic liver disease.


The following people have nothing to disclose: Kate M. O'Brien, Kara Kelly, Bryan Copple


Gene expression and immunohistochemical changes in liver tissue from fulminant hepatitis E, versus fulminant hepatitis B and normal liver

Anshu Naik1, Amit Goel1, Vinita Agrawal2, Aditya N. Sarangi1, Nanda Chhavi1, Vineeta Singh3, Shahid Jameel4, Rakesh Aggar-wal1;

1Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India; 2Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India; 3San-dor Proteomics Pvt Ltd, Hyderabad, India; 4Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India

Introduction: It is unclear whether liver injury in acute hepatitis E is due to virus-induced cytolysis or the host immune response. We therefore studied host gene expression and enumerated immune cells in liver tissues from fulminant hepatitis E (FHF-E) patients, in comparison with healthy livers and those from fulminant hepatitis B (FHF-B) patients. Methods: Microarray-based expression profiling was done on post-mortem liver tissue from 5 FHF-E and 6 FHF-B patients, and normal liver tissue from 6 persons. Differential expression was defined as ≥2.0-fold change with Benjamini-Hochberg corrected p-value below 0.05. CD4+, CD8+ and CD56+ cells were counted using immunohistochemistry. Results: Compared to normal, the livers from FHF-E and FHF-B showed differential expression of 3377 (up-regulated 1703, down-regulated 1674) and 2572 (up 1164, down 1408) entities, respectively. This included 2142 (up 896, down 1246) entities that were common between the two sets; most of these belonged to metabolic, hemostatic (intrinsic and extrinsic prothrombin activation), and complement (classical, alternative and lectin-induced) pathways. Analysis of 1235 (up 807, down 428) entities with differential expression in FHF-E but not in FHF-B showed activation of several immune response pathways, particularly those involving cytotoxic T cells (Table). CD8+ T cells were increased in both FHF-E (median 53.4 [range 31.2-99.9]) and FHF-B (49.3 [19.3-51]; p=0.005) compared to controls (6.9 [3.1-14.9]). Conclusion: Liver tissue from FHF-E patients showed increased expression of genes belonging to cytotoxic T cell effector pathways, accompanied by CD8+ T cell infiltration. This suggests a role for CD8+ T cells in the pathogenesis of hepatitis E.

Pathways whose genes were over-represented among entities differentially expressed in fulminant hepatitis E (FHF-E), and in FHF-E but not in fulminant hepatitis B (FHF-B)

Table 1. 
BioCarta pathwayFHF-E vs normalIn FHF-E vs normal but not in FHF-B vs normal
Gene countP valueGene countP value
  1. p values are Benjamini-Hochberg corrected and considered significant if <0.10

T cytotoxic cell surface molecules80.0079370.00053
T helper cell surface molecules70.0344760.00431
Cells and molecules involved in local acute inflammatory response80.04921--
Monocyte and its surface molecules60.08014--
Neutrophil and its surface molecules50.08370--
CTL mediated immune response against target cells--70.00221
Co-stimulatory signal during T-cell activation--70.00850
T cell receptor signaling pathway--100.00488
Lck and Fyn tyrosine kinases in initiation of TCR activation--50.01867
IL-7 signal transduction--50.04457
T cell receptor and CD3 complex--30.05294
IL 17 signaling pathway--50.05615


The following people have nothing to disclose: Anshu Naik, Amit Goel, Vinita Agrawal, Aditya N. Sarangi, Nanda Chhavi, Vineeta Singh, Shahid Jameel, Rakesh Aggarwal


CEACAM1 (Carcinoembryonic antigen-related cell adhesion molecule 1) protects from acute immune-mediated liver injury

Claudia Wegscheid1, Andrea K. Horst2, Gisa Tiegs1;

1 Experimental Immunology and Hepatology, University Medical Centre Ham-burg-Eppendorf, Hamburg, Germany; 2Institute of Clinical Chemistry, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

The T cell mitogenic plant lectin concanavalin A (ConA) induces acute immune-mediated liver injury. Hallmarks of liver injury are increased plasma transaminase activities and the release of pro-inflammatory cytokines. The cellular adhesion molecule CEACAM1 is expressed on epithelia, endothelia and leukocytes. CEACAM1 has been originally identified as an intercellular, homophilic adhesion molecule on hepatocytes. The CEACAM1 isoform with a long cytoplasmic domain contains an immune receptor tyrosine-based inhibition motif (ITIM) that is pivotal in for the negative regulation of leukocyte activation. CEACAM1-long suppresses the activity of NK cells, T cells and myeloid cells, such as granulocytes and monocytes/macrophages. This negative regulation modulates innate immunity in both infection and sterile inflammation. Thus, elucidation of CEACAM1-dependent regulatory mechanisms in the murine ConA model might be useful to evaluate novel therapeutic approaches in human liver disorders. In order to identify CEACAM1-dependent effects in ConA-induced liver injury, we analyzed plasma transaminase activities and pro-inflammatory cytokine expression (8 and 24 hrs after i.v. injection of ConA (5mg/ml) into C57Bl/6 (wt) and Ceacam1-/- mice. Furthermore, the distribution and subtypes of CEACAM1+ and CEACAM1- T cells were analyzed in livers and spleens. Interestingly, we observed exacerbated liver damage in Ceacam1-/- mice, evident by significant elevation of plasma transaminase activities and an exaggerated Th1-cytokine response. More specifically, CEACAM1 expression was markedly increased on CD4+ T cells, CD4+Foxp3+ regulatory T cells (Treg) as well as CD8+ T cells after ConA. Also, we observed higher abundance of CEACAM1-expression on CD4+ T cells, CD4+Foxp3+ Treg and well as CD8+ T cells after ConA treatment. Furthermore, CD4+Foxp3+ Treg were more abundant in livers and spleens of naïve wt mice in contrast to Ceacam1-/- mice. Based on the observation that liver injury is aggravated in Ceacam1-/- mice, we suggest an involvement of CEACAM1 + T cells in the attenuation of immune-mediated liver disease. In the ConA model, CEACAM1 exerts immune modulatory functions by regulating hepatic and splenic CD4+ and CD8+ T cell abundance and polarization. Further studies are under way to characterize the molecular basis for the immune modulatory role of CEACAM1 + Th1 effector cells, CEACAM1+ Tregs, and CEACAM1 +CD8+ T cells in ConA-mediated acute liver injury. Using this model, we will describe the functional role of T cell activation and the immunosuppressive capacity of CEACAM1+ Tregs following a Th1-polarized immune response yielding liver protection.


The following people have nothing to disclose: Claudia Wegscheid, Andrea K. Horst, Gisa Tiegs


Liver May be the Target Organ of SFTSV Infection Injuries

Jun Li, Yaping Han, Longfeng Jiang, Zuhu Huang;

Department of Infectious Diseases, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China

Objective As a new emerging virus in China, fever with throm-bocytopenia syndrome virus (SFTSV) infection can cause severe tissue damage. We aim to study the relationship between liver damage and viral load, lymphocyte subsets during SFTSV-infec-tion process and its influence on prognosis. Methods SFTSV-RNA from serum samples was detected dynamically by real-time PCR. Lymphocyte subsets were tested by FACS. Meanwhile ALT/AST/CK /WBC/PLT were examined to analyze the relationship between viral load and tissue damage. Results SFTSV- RNA of all the 22 patients was positive. SFTSV load <lxlO6 (copies/ml) in 18 patients, 1 patient died, but SFTSV load > 1x107 (copies / ml) in 4 patients, 2 cases died; In the early stage of SFTSV-infection, serum ALT/AST/LDH/CK markedly elevated and WBC and PLT in peripheral blood decreased significantly. The level of ALT/AST/LDH/CK, WBC and PLT in 19 cured patients was gradually backed to normal with the reducing of viral load. The elevated level of serum AST/LDH/CK and SFTSV load positively correlated, PLT and SFTSV load negatively correlated. At the same stage of infection process, ALT/AST/LDH/CK in incurable patients were significantly higher than in cured patients; Compared with healthy population, the number of CD4+ cells was lower in the onset of the first 5 to 9 days; CD8+ cells was higher and NK cells was lower in 9 to 15 days. Lymphocyte subsets of most patients were normal with the viral load undetectable; The number of T cells of incurable patients significantly decreased, while NK cells increased. A variety of detection indicators could not recover with extension of disease course. Conclusion Liver may be one of the target organs of SFTSV-infection injuries. Severity of tissue damage is closely related to serum SFTSV load. High viral load, decreasing of T cells, increasing of NK cells may be the important factors of poor prognosis.


The following people have nothing to disclose: Jun Li, Yaping Han, Longfeng Jiang, Zuhu Huang


Minocycline decreases liver injury following resuscitated hemorrhagic shock

Paul J. Matheson3,2, Jason Smith3, Keith C. Falkner1, Jane Frimodig1, Cynthia Downard3, Craig J. McClain1,4, Richard N. Garrison3,4;

1 Department of Medicine/GI, University of Louisville, Louisville, KY; 2Physiology and Biophysics, University of Louisville, Louisville, KY;3 Surgery, University of Louisville, Louisville, KY;4Rob-ley Rex Veterans Administration Medical Center, Louisville, KY

Background: Conventional resuscitation (CR) from hemorrhagic shock (HS) that restores central hemodynamic function nonetheless results in gut and liver hypoperfusion and hypoxia, organ and cellular edema, and liver injury. This can lead to multi-system organ failure and death. Minocycline stabilizes mitochondrial membrane function inhibiting mitochondrial transition pore opening and cytochrome C release-mediated apoptosis. We hypothesized that minocycline might improve post-resuscitation (post-RES) hepatic function and, thus, prevent hepatic injury following hemorrhagic shock. Methods: Anesthetized male Sprague-Dawley rats were randomized to groups (n=7/group): 1) Sham (no HS); 2) Sham + Minocycline at time of RES (Sham + Min(0″)); 3) Sham + Min at 120″ min post-RES (Min(120″)); 4) HS + CR; 5) HS + CR + Min(0″); and 6) HS + CR + Min(120″). At 4 hours post-RES, we measured: 1) effective hepatic blood flow (EHBF) by galactose clearance; 2) hepatic injury (serum ALT); 3) CMP & CBC; 4) tissue edema (lung, liver, or ileum wet-dry weight ratios); and 5) lung, liver, and ileum histopathology. Results: Histopathology showed lung and liver injury in HS + CR at 4 hours post-RES. Serum ALT levels were increased in HS+CR but not in HS + CR + Min(0″) or HS + CR + Min(120″). During HS, all HS + CR groups had decreased EHBF that was restored by i.v. blood and saline RES. However, at 4 hours post-RES, EHBF was decreased in all HS+CR groups despite minocycline treatment. EHBF was stable in all Sham groups, and all Sham groups had no evidence of liver injury or tissue edema. Discussion: Despite adequate RES after HS to restore central hemodynamic function, liver blood flow was compromised at 4 hours post-RES. Minocycline treatment at the time of RES prevented liver injury (serum ALT) but did not significantly improve liver blood flow. One therapeutic mechanism of action of minocycline might be that minocycline effectively inhibited hepatic apoptosis in the reperfusion period. We postulate that minocycline might provide a beneficial effect to trauma patients undergoing standard of care treatment fluid resuscitation after hemorrhagic shock.


Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche

The following people have nothing to disclose: Paul J. Matheson, Jason Smith, Keith C. Falkner, Jane Frimodig, Cynthia Downard, Richard N. Garrison


MicroRNA122 Levels in Hepatitis-C Patients Associated with Variables Unrelated to Hepatic Injury

Perry H. Dubin1,2, Mamta K. Jain3, William M. Lee1;

1Digestive and Liver Diseases, UT Southwestern Medical Center, Dallas, TX; 2Doris Duke Foundation, New York, NY; 3Division of Infectious Diseases, UT Southwestern Medical Center, Dallas, TX

Background: Serum levels of microRNA-122 (miR-122) are variably elevated in patients with chronic hepatitis C (CHC). To further examine its clinical role, we aimed to identify which demographic or laboratory variables were associated with miR-122. Methods: miR-122 values were determined in sera from 43 CHC patients, measured in triplicate using the mirVana™ PARIS™ kit. Banked sera were pulled from two CHC databases from clinics at the University of Texas Southwestern Medical Center and Parkland Health and Hospital System. The following demographic and clinical data were retrospectively collected from the date of initial serum banking: HIV co-infection, sex, race, HCV genotype, cirrhosis, white blood cell count, hemoglobin, platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, albumin, total and direct bilirubin, and HCV viral load. Using SPSS V21, univariate non-parametric testing was performed, followed by a multivariate linear regression for variables meeting univariate significance of p<0.05. Unavailable data were censored from analysis. Results: In univariate analysis, HIV co-infection was the only categorical variable significantly associated with miR-122, where co-infection was associated with lower miR-122 levels (p=0.016), and significant positive Spearman's correlations were identified for hemoglobin (rho=0.361, p=0.028), ALT (rho=0.602, p<0.001), AST (rho=0.331, p=0.045), and albumin (rho=0.417, p=0.042). Multivariate linear regression including these five variables was significant (p<0.001), with ALT (p<0.001) and albumin (p=0.030) remaining significant in the model. Conclusions: Since miR-122 has been investigated as a marker for hepatic injury, ALT and AST were significant as expected. However, it was surprising to identify associations with variables unrelated to injury: HIV co-infection, hemoglobin, and albumin. In fact, co-infected patients, who encounter increased liver injury and rapid progression of fibrosis, had lower levels of miR-122. The variables hemoglobin and albumin may explain the lower miR-122 levels identified in HIV infection, as HIV is known to cause anemia and has been associated with under-nutrition (HIV co-infected serum had lower median values for both hemoglobin and albumin, though not significantly). Additionally, the absence of an association between miR-122 and cirrhosis suggests that care must be taken when evaluating miR-122, as it may be influenced by factors distinct from liver injury.


Mamta K. Jain - Advisory Committees or Review Panels: Merck, Vertex, Boehringer Ingelheim ; Grant/Research Support: Vertex, Bristol-Myers Squibb, Pfizer, Janssen, Gilead, Boehrnger Ingelheim , Viiv Healthcare, EMD Serono

William M. Lee-Consulting: Eli Lilly, Novartis; Grant/Research Support: Gilead, Roche, Vertex, BI, Anadys, BMS, merck; Speaking and Teaching: Merck

The following people have nothing to disclose: Perry H. Dubin


Berberine ameliorates experimental liver injury limiting activation of NALP3 inflammasome, through an interaction with the P2X7 purinergic receptor

Elisa Vivoli1, Stefano Milani1, Angela Provenzano1, Andrea Cap-pon1, Erica Novo2, Claudia P. Oliveira3, Alessio Masi1, Roberto Narducci1, Guido Mannaioni1, Gloriano Moneti1, Maurizio Parola2, Fabio Marra1;

1University of Florence, Florence, Italy; 2University of Turin, Turin, Italy; 3University of Sao Paulo, Sao Paulo, Brazil

The natural alkaloid berberine (BRB) is commonly used as a supplement in the US, and has been shown to possess a wide range of pharmacological and biochemical effects, including protection against liver injury. In this study, we analyzed the effects of BRB in different murine models of liver injury, and investigated the underlying mechanism of action. BRB (5 mg/kg i.p.) was tested in steatohepatitis induced by administration of a methionine and choline deficient (MCD) diet and in acute acetaminophen (APAP) intoxication. The mechanism of action of BRB was further investigated in LPS-stimulated murine macrophages (RAW 264.7) in vitro. Activation of the P2X7 receptor was assessed by measurement of calcium transients. BRB markedly suppressed ALT elevation and necroinflammation in MCD-fed mice. In addition, intrahepatic gene expression of CD11 b, CCL2, TGF-b, type I procollagen, andTNF were significantly downregulated in MCD-fed, BRB-treated mice. Feeding a MCD diet increased hepatic levels of mature IL-1 β and expression of all components of the NALP3 inflammasome pathway, while these effects were limited by BRB. Surprisingly, administration of BRB to MCD-fed mice caused an excess of mortality, which could not be attributed to increased serum levels of BRB, measured by LC/MS mass spectrometry. In the model of APAP-induced hepatotoxicity, pretreatment with BRB reduced ALT elevation. Moreover, upregulation of the expression of NALP3 inflammasome components induced by APAP was limited by BRB, which also reduced mortality. Inflammasome activation in LPS-stimulated RAW 264.7 was markedly decreased by BRB, demonstrating a direct interference with activation of the inflammasome pathway. BRB did not affect the activation of NF-κB pathway, which provides an initial signal leading to inflammasome activation. However, exposure of RAW 264.7 cells to BRB limited the elevation of intracellular calcium caused by a selective ligand of P2X7, a purinergic receptor involved in the later phases of inflammasome assembly. In conclusion, administration of BRB ameliorates necroinflammation and expression of proinflammatory cytokines in experimental steatohepatitis. In this model, but not in other models, berberine was associated with an excess mortality, which was unrelated to the liver phenotype. We also demonstrate for the first time that BRB interferes with the activation of the inflammasome pathway in vivo and in vitro, through a previously unidentified mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.


Maurizio Parola - Independent Contractor: Shire Pharmaceutical Ltd, Basingstoke, UK

Fabio Marra - Advisory Committees or Review Panels: Abbott; Consulting: Bayer Healthcare, Gilead; Grant/Research Support: ViiV

The following people have nothing to disclose: Elisa Vivoli, Stefano Milani, Angela Provenzano, Andrea Cappon, Erica Novo, Claudia P. Oliveira, Alessio Masi, Roberto Narducci, Guido Mannaioni, Gloriano Moneti


Association of Immune-related Markers in Bile and Serum with Gallbladder Cancer

Jill Koshiol1, Troy J. Kemp2, Felipe A. Castro1, Yu-Tang Gao3, Leticia Nogueira1, Asif Rashid4, Ann W. Hsing5,6, Allan Hildesheim1, Ruth M. Pfeiffer1, Ligia Pinto2;

1Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD; 2HPV Immunology Laboratory, Frederick National Laboratory for Cancer Research, SAIC-Frederick, Inc., Frederick, MD; 3Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China; 4Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX; 5Cancer Prevention Institute of California, Fremont, CA; 6Stanford Cancer Institute, Palo Alto, CA

Background: Gallbladder cancer (GBC) is a highly fatal disease, with a median survival of 4 months. Although gallstones (GS) are the major risk factor, only 1% of GS patients develop GBC. While inflammation is clearly implicated in gallbladder carcinogenesis, its precise mechanisms remain unclear. Elucidation of these mechanisms could help identify a subset of GS patients at risk for developing GBC, which may facilitate targeted prevention efforts. While measurement of circulating inflammatory immune-related markers is appealing, circulating markers may not reflect what happens at the gallbladder since the markers are secreted from a variety of cell types throughout the body. We therefore examined the correlation between bile and serum immune marker levels and explored the association of immune markers with GBC compared to GS. Methods: Using multiplexed assays, we measured 52 immune-related markers in serum and bile from 43 GBC cases and 127 GS controls from a population-based case-control study conducted in Shanghai, China. We evaluated the correlation between bile and serum markers using Spearman correlation coefficients in cases and controls separately and calculated age- and sex-adjusted odds ratios (ORs) for the association with GBC. Results: The correlations between serum and bile immune-related markers varied from -0.23 to 0.47 among GS controls and from -0.23 to 0.65 among GBC cases. Only three markers had correlations >0.4 in GBC and/or GS patients (P≤0.004): CRP, ENA78, and SAA. Despite the lack of strong correlation, many markers were strongly associated with GBC versus GS based on measurements in both serum and bile; comparing the highest marker level group vs. the lowest group, 17 markers (33%) were significantly associated with GBC risk in both serum and bile (adipsin, CCL20/MIP3a, CRP, CXCL6/GCP2, CXCL9/MIG, GRO, IL-12p40, IL-16, IL-8, IP10, lipocalin2, MCP1, MIP1d, resistin, SAA, TNFa, VEGF). The ORs for these 17 markers ranged from 0.32 (95% CI: 0.11-0.94) for adipsin to 96.4 (95% CI: 11.8-785.4) for IL-8 in serum and from 0.17 (95% CI: 0.06-0.54) for MIP1d to 54.8 (95% CI: 6.9-432.5) for IL-8 in bile. Conclusion: Using multiplexed technology, we were able to identify a number of inflammatory and angiogenic markers in serum and bile associated with GBC risk. These results provide biological evidence for the role of inflammatory/immune processes involved in GBC and may offer a first step toward identifying biomarkers that could help identify gallstone patients at high risk of developing gallbladder cancer.


The following people have nothing to disclose: Jill Koshiol, Troy J. Kemp, Felipe A. Castro, Yu-Tang Gao, Leticia Nogueira, Asif Rashid, Ann W. Hsing, Allan Hildesheim, Ruth M. Pfeiffer, Ligia Pinto


IL-12/STAT4 plays a protective role in Con A induced T cell-mediated hepatitis

Yan Wang1,2, Dechun Feng1, Hua Wang1, Ming-Jiang Xu1, Ogyi Park1, Yongmei Li1, Bin Gao1;

1Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD; 2Department of Infectious Diseases, Peking University First Hospital, Beijing, China

Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway has been implicated in a variety of cellular functions, playing important roles in controlling immune-mediated liver injury. The functions of STAT1 and STAT3 in the pathogenesis of liver diseases have been extensively investigated; however, the role of STAT4 remains largely unknown. Here we demonstrated that STAT4 activation was detected in liver immune cells from patients with viral hepatitis, autoimmune hepatitis, nonalcoholic steatohepatitis, and alcoholic liver diseases, as well as in a mouse model of con-canavalin A (Con A)-induced T cell hepatitis. Flow cytometric and immunohistochemistry analyses revealed that STAT4 activation was mainly detected in T and NKT cells in Con A-induced T cell hepatitis model, such activation was diminished in IL-12a-/- and IL-12b-/- mice. As expected, disruption of the STAT4 reduced the production of both Th1 and Th2 cytokines, but surprisingly exacerbated Con A-induced liver injury. In agreement with these findings, ablation of IL-12a or IL-12b also augmented Con A-induced hepatocellular damage. Further studies showed that hepatic NKT cells from Con A-treated STAT4-/- mice had higher levels of FasL expression and cyto-toxicity against hepatocytes than those from Con A-treated WT mice. In vitro studies revealed that blocking FasL by antibodies on NKT cells attenuated hepatic NKT cytotoxicity against hepatocytes. Our results suggest that IL-12 activation of STAT4 up-regulates expression of Th1 and Th2 cytokines but inhibits the expression of FasL on NKT cells. The suppression of FasL expression contributes to the protective effects of IL-12/STAT4 on Con A-induced T cell mediated hepatitis.


The following people have nothing to disclose: Yan Wang, Dechun Feng, Hua Wang, Ming-Jiang Xu, Ogyi Park, Yongmei Li, Bin Gao


A TLR5 agonist inhibits ischemic reperfusion injury in a mouse model

Adrian Keogh1, Rene Fahrner1, Michaela Medova2, Daniel Aeber-sold2, Daniel Candinas1, Andrei Gudkov3, Yitzhak Zimmer2, Deborah Stroka1;

1Clinic of Visceral Surgery and Medicine, University Hospital Bern, Bern, Switzerland;2Department of Radiation Oncology, University Hospital Bern, Bern, Switzerland; 3Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY

Background CBLB502 (aka Entolimod) is a pharmacologically optimized derivative of bacterial protein flagellin, an agonist of toll-like receptor 5 (TLR5). Stimulation of TLR5 with CBLB502 was shown previously to have a radioprotective property in mouse and primate models. Here we investigated its stimulation of innate immune responses in mice and its potential hepato-protective effect in a liver ischemia reperfusion (I/R) model. Methods Expression of TLR5 was determined in isolated liver cell populations and whole liver by RT-qPCR and immunohistochemistry. Alterations in gene expression were determined in mice treated with CBLB502 for 2 and 6 hours by RT-qPCR of liver tissue. Activation of an innate immune response was assessed by a CD62L shedding assay. A mouse model of partial liver I/R was used to assess the hepatoprotective effect of CBLB502 against acute liver injury. Injury was assessed by serum ALT/AST levels, leukocyte infiltrate and myeloperoxidase activity. Results Hepatic expression of TLR5 was found on hepatocytes, biliary cells and infiltrating mononuclear cells. CBLB502 was more a potent monocyte activator than flagellin, LC500.02 vs. 0.68 ng/ml respectively. After 2 hrs, CBLB increased inflammatory (TNF; 22-fold), neutrophil chemoat-tractant (CXCL1; 77-fold, CXCL2; 51-fold), TH2 (IL-10; 25-fold) and cytoprotective (TNFAIP3; 350-fold, HMOX1; 19-fold) gene expression, but not TH1 genes (IFN gamma and IL2; not detectable). Preliminary data show that in mice treated with 0.2mgkg-1, s.c., CBLB502 there is a beneficial influence on clinical symptoms of hepatic ischemia reperfusion injury by reduced serum transaminases (p<0.05) and reduced myeloperoxidase activity reflecting reduced neutrophil infiltration (p<0.0005). Conclusions I/R injury associated with hepatic resections and liver transplantation remains a serious complication in clinical practice. Hepatic damage could potentially be diminished by prior activation of an innate immune response targetingTLR5. Materials were provided by Cleveland BioLabs, Inc. All right, title, and interest in these materials are owned by Cleveland BioLabs, Inc.


Andrei Gudkov- Board Membership: Cleveland BioLabs, Inc.; Consulting: Cleveland BioLabs, Inc.; Grant/Research Support: Cleveland BioLabs, Inc.

The following people have nothing to disclose: Adrian Keogh, Rene Fahrner, Michaela Medova, Daniel Aebersold, Daniel Candinas, Yitzhak Zimmer, Deborah Stroka


Activation of senescence pathways in chronic and acute-on-chronic liver failure rat brain

Marc Oria1,2, Fausto Andreola1, Rita Garcia-Martinez1, Rajiv Jalan1;

1Liver Failure Group-UCL Institute for Liver and Digestive Health-Royal Free Hospital, University College London, London, United Kingdom; 2Damme Sheila Sherlock EASL Post Doctoral Research Fellow, EASL, London, Switzerland

Background: Cirrhotic patients can suffer from a neurodegenerative process related to hepatic encephalopathy (HE). Reduction in brain size is associated with the number of overt HE episodes. Hepatocerebral degeneration is a chronic disorder that may persist after liver transplantation and has important consequences on daily life. The development of this disorder has been associated with the severity of liver failure. We hypothesize that neurodegeneration that is consequent on episodes of HE may be due to hyperammonemia and/or activation of inflammation secondary to infectious complications, which often results in acute-on chronic liver failure (ACLF). Aims: Using a well-characterized animal model for chronic liver failure and ACLF we aimed to characterize molecular signatures and assess the relationship between nitrogen metabolism, inflammation and neurodegeneration. Methods: Brain (cortex and cerebellum) samples were collected from 6 weeks bile duct ligated rats (BDL) (chronic liver failure model), BDL+LPS (Kleb-siella Pneumoniae 0.3 mg/Kg, 3 hours infusion i.v) (ACLF model) and sham-operated rats (control) (n=6/group), respectively. RNA was isolated from the tissues using Qiazol; RNA pellets were partially re-extracted, precipitated, DNAseI-digested and cleaned. Samples were qualitatively and quantitatively analyzed with the Bioanalyser. 1 ug of RNA/sample was retro-transcribed and then run on RT2 PCR array rat cellular senescence plates. Ammonia and TNFα were assessed in plasma. Results: Senescence-associated genes were differentially expressed in each pathological model and in each brain region compared to controls. The genes that were up- or down regulated are summarized in Table 1.BDL rats showed higher ammonia and TNFα levels compared with control rats 7.2±4.5 and 71.5±22.4 pg/mL respectively. In BDL+LPS, ammonia remained stable but TNFα increased to 789.5±212.9 pg/mL. Conclusions: Dysregulation of senescence pathways within brain regions was observed in the chronic liver failure and ACLF models. In BDL rats, oxidative stress (NOX4) and cyclin D (CDKN2B) pathways were affected in the cortex and the cerebellum respectively. When liver injury was exacerbated by LPS (development of ACLF), the dysegulation of further senescence pathways was observed suggesting that precipitating factors and consequent inflammation might induce neurodegeneration in the ACLF.

Table 2. Genes up- and down-regulated in rat brain of chronic and ACLF (.2-fold change, p<0.05)
Chronic (BDL)1 N0X4--1 CDKN2B


Rajiv Jalan - Consulting: Ocera Therapeutics, Conatus

The following people have nothing to disclose: Marc Oria, Fausto Andreola, Rita Garcia-Martinez


The Role of Sphingosine Kinase 1 and Sphingosine-1-Phosphate in Acute Hepatic Failure

Dorit Avni1, Wei-Ching Huang1, Jeremy Allegood1, Arun J. Sanyal2, Sarah Spiegel1;

1Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, School of Medicine, Richmond, VA;2Department of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University, School of Medicine, Richmond, VA

BACKGROUND: There is growing evidence that the bioactive sphingolipid mediatorsphingosine-1-phosphate (S1P) produced by sphingosine kinase 1 (SphK1) is involved in inflammation and cancer. Although most of the actions of S1 P are mediated by activation of specific cell surface receptors, our lab has shown that intracellular S1 P has a direct role in regulating the TNF-alpha signaling pathway (Alvarez et al. Nature. 465:1084, 2010). AIM: To examine the involvement of the SphK1 /S1 P axis in a murine model of acute liver failure. METHODS: Acute liver failure was induced in SphK1-/- and wild type littermate C57BL/6 mice by administration of a low dose of bacterial lipopolysaccharide (LPS) in the presence of the liver-specific transcriptional inhibitor D-(+)-galactosamine (GalN). This results in endotoxic shock and liver injury that is macrophage-dependent and mediated by secreted TNF-alpha. RESULTS: We found that SphK1-/- mice were protected from acute liver failure. Liver and serum TNF-alpha levels as well as markers of liver apoptosis were dramatically decreased compared to wild type littermates. In agreement, TNF-alpha secretion from LPS-induced peritoneal macrophages deficient in SphK1 was also greatly reduced. Furthermore, treatment of wild type mice with the SphK1 specific inhibitor, SKI-1, also prevented fulminant liver damage and secretion of TNF-alpha induced by GalN/LPS. CONCLUSION: Our results reveal that the SphK1-S1P axis has a pivotal role in liver injury and suggests that it deserves consideration as a therapeutic target for acute liver failure.


Arun J. Sanyal - Advisory Committees or Review Panels: Gore, Gilead, Abbott, Ikaria; Consulting: Salix, Immuron, Exhalenz, Bayer-Onyx, Genentech, Norgine, GalMed, Novartis, Echosens, Takeda; Grant/Research Support: Salix, Genentech, Genfit, Intercept, Ikaria, Takeda, Gilead; Independent Contractor: UpToDate

The following people have nothing to disclose: Dorit Avni, Wei-Ching Huang, Jeremy Allegood, Sarah Spiegel


Salidroside Attenuates LPS-stimulated Activation of THP-1 cell-derived macrophages through Down-regulation of MAPK/NF-kB signaling Pathways

Hongwu Wang1, Ting Wu1, Junying Qi1, Yaqi Wang1, Xiaoping Luo2, Qin Ning1;

1Institute and Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Department of Pediatrics, Tongji Hospital, Wuhan, China

Alcoholic liver disease (ALD) is associated with a spectrum of liver injury ranging from steatosis and steatohepatitis to fibrosis and cirrhosis. In response to gut-derived lipopolysaccharide (LPS), activation of Kupffer cells plays a key role in the development and progression of ALD by secreting a variety of proinflammatory cytokines. Consequently, inhibition of macrophage-activation would have therapeutic benefits for alleviating the progression of ALD. Salidroside (Sal), one of main bioactive components isolated from Rhodiola Sachalinensis, has been reported to suppress LPS-induced inflammatory response, but the underlying mechanisms in macrophages remain poorly understood. In this study, we investigate the anti-inflammatory effects of Salidroside and the possible mechanisms in LPS-stimulated phrobol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage models. The results showed that Sal markedly decreased the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX2), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) at both mRNA and protein levels, and there was dose-effect relationship between the three Sal pre-treated groups. Further studies revealed that Sal strongly suppressed NF-κB activation and down-regulated the phosphorylation of ERK, p38 and JNK. Our present study demonstrated that Sal could suppress the production of iNOS, COX2, IL-1 β, IL-6 and TNF-α in LPS-stimulated PMA-differeti-ated THP-1 cells by inhibiting NF-κB activation and MAPK signal pathway phosphorylation.


Qin Ning - Advisory Committees or Review Panels: ROCHE, NOVARTIS, BMS, MSD, GSK; Consulting: ROCHE, NOVARTIS, BMS, MSD, GSK; Grant/Research Support: ROCHE, NOVARTIS, BMS; Speaking and Teaching: ROCHE, NOVARTIS, BMS, MSD, GSK

The following people have nothing to disclose: Hongwu Wang, Ting Wu, Junying Qi, Yaqi Wang, Xiaoping Luo


Liver sinusoids are predominantly populated by M2 not M1 macrophages in alcoholic hepatitis

James Lee1, Barbara A. French1, Timothy R. Morgan2, Samuel W. French1;

1Pathology, Harbor-UCLA Medical Center, Torrance, CA; 2Pathology, VA Long Beach Healthcare System, Long Beach, CA

Background: Liver cell injury in alcoholic hepatitis (AH) is in part, due to macrophage generated proinflammatory cytokines i.e. M1 i, M2a, M2b, and M2c might be involved in ALD. The T cell response to chemokines and cytokines differs not only when M1 and M2 macrophages are compared but even when individual M2 subtypes are profiled. Purpose: In AH, M1 monocytes in the blood show increased sensitivity in the TNF-alpha response in LPS. Immunohistochemistry (IHC) studies showed that the liver sinusoids in ALD are abundantly populated by CD163 expressing type 2 macrophages. In this report, we further subtype these M2 macrophages using IHC. Methods: Using immunofluorescent antibody-labeling, we profiled the proinflammatory markers and chemokines observed in M1 and M2a, M2b, and M2c macrophages in liver biopsiesfrom patients with AH. Results: The increased CD 163 expression was confirmed as well an additional macrophage phenotypic marker CD206 was increased which suggests that ALD pathogenesis is driven mainly by M2a and M2c macrophages. Macrophage expression of the phenotypic markers TLR-2 and TLR-8, as well as the chemokine CCL-18 was found. This suggests that liver pathogenesis in ALD is driven primarily by M2c macrophages. However, IRF-4, which is related to IL-4 production and M2a polarization as well as the cytokines CCL-1, Il-1 Ra and Il-1 Beta and the chemokine CXCL-1 were also observed, suggesting that M2a and M2b also play a role in AH pathogenesis. Notably, cytokines observed in M1 macrophage polarization were absent and the only common cytokine, Il-6, expressed by this macrophage subtype showed only faint expression, Conclusion: In AH, M2c play a more prominent role in liver pathogenesis than other macrophages, especially when compared to the minimal role shown by M1 macrophages.

Table 3. 
SubtypeCytokinesChemokinesPhenotypic markersAdditionally expressed markers
MlPositive: I1-6   
 Negative: I1-1Negative: CXCL9  
M2aPositive: I1-1RaPositive: CCL18Positive: CD163, CD206 
 Negative: I1-10Negative: CCL13  
M2bPositive: I1-1Beta, I1-6Positive: CCL1, CXCL1Positive: CD163 
 Negative: I1-10, TNF-alpha   
M2c Positive: CCL18Positive: CD163, CD206Positive: TLR-8, TLR-2
  Negative: CXCL9  


Timothy R. Morgan - Grant/Research Support: Merck, Vertex, Genentech, Gilead, Bristol Myers Squibb

The following people have nothing to disclose: James Lee, Barbara A. French, Samuel W. French


Elevated acid sphingomyelinase activity and concentration variations of serum sphingolipid metabolites as putative biomarkers in chronic hepatitis C virus infection and non alcoholic fatty liver disease

Georgios Grammatikos1,2, Christiane Mühle3, Nerea Ferreirós4, Dimitra Bogdanou1, Sirkka Schroeter1,2, Stephanie Schwalm2, Gudrun Hintereder5, Johannes Kornhuber3, Stefan Zeuzem1, Christoph Sarrazin1, Josef Pfeilschifter2;

1Medizinische Klinik I, University Hospital Frankfurt, Frankfurt, Germany; 2Pharmazentrum Frankfurt, Institut für Allgemeine Pharmakologie, University Hospital Frankfurt, Frankfurt, Germany; 3Department of Psychiatry and Psychotherapy, Friedrich-Alexander University Hospital, Erlangen-Nürnberg, Germany; 4Pharmazentrum Frankfurt, Institut für klinische Pharmakologie, University Hospital Frankfurt, Frankfurt, Germany; 5Central Laboratory, University Hospital Frankfurt, Frankfurt, Germany

Sphingolipids constitute bioactive molecules with functional implications in the pathogenesis of various diseases. The patho-physiology of liver diseases is tightly associated with several bioactive sphingolipid metabolites, while the acid sphin gomyelinase mediated hydrolysis of sphingomyelin to the antiproliferative ceramide has been shown to modulate significantly hepatocellular apoptosis. However, the role of sphingolipids as possible disease biomarkers in chronic liver disease remains largely unexplored. Methods: In the present study we used mass spectrometry- and spectrofluorometry-methods in order to quantify various sphingolipid metabolites and assess the activity of respective regulating enzymes in the serum of 69 patients with non-alcoholic fatty liver disease and 69 patients with chronic hepatitis C virus infection as compared to72 healthy probands. Results: In our study we observed a significant activation of the acid sphingomyelinase, an enzyme able to hydrolyze sphingomyelin to ceramide, in the serum of patients with chronic liver disease as compared to healthy individuals (p<0.001). Particularly in chronic hepatitis C infection, acid sphingomyelinase correlated significantly with markers of hepatic injury (r=0.312, p=0.009) and showed a high discriminative power as compared to healthy probands. In patients with non-alcoholic fatty liver disease an additional accumulation of various (dihydro-)ceramide species was also identified (p<0.001) and a significant correlation of ceramides to cholesterol levels was observed (r=0.660, p<0.001). Sphin-gosine, a further antiproliferative sphingolipid metabolite was upregulated in chronic liver disease (p<0.001) with no significant variations between patients with non-alcoholic liver disease and chronic hepatitis C virus infection and no significant correlation to markers of hepatic injury. On the contrary the pro-proliferative sphingosine-1-phosphate showed no significant variations in the serum of patients with chronic liver disease as compared to healthy individuals. Conclusion: Chronic hepatitis C virus infection and non-alcoholic fatty liver disease induce a significant deregulation of both the anabolic and the catabolic synthesis of ceramide. Acid sphingomyelinase activity and concentrations of (dihydro-)ceramide species in serum appear as novel biomarkers and as putative therapeutic targets in chronic hepatitis C and non-alcoholic fatty liver disease.


Stefan Zeuzem - Consulting: Abbvie, Achillion Pharmaceuticals, Boehringer Ingelheim GmbH, Bristol-Myers Squibb Co., Gilead, Novartis Pharmaceuticals, Merck & Co., Idenix, Janssen, Roche Pharma AG, Vertex Pharmaceuticals, Presidio, Santaris, Inc

Christoph Sarrazin - Advisory Committees or Review Panels: Boehringer Ingelheim, Vertex, Janssen, Merck/MSD, Gilead, Roche, Boehringer Ingelheim, Achillion, Janssen, Merck/MSD, Gilead, Roche; Consulting: Merck/MSD, Novartis, Merck/MSD, Novartis; Grant/Research Support: Abbott, Intermune, Roche, Merck/MSD, Gilead, Janssen, Abbott, Roche, Merck/MSD, Vertex, Gilead, Janssen; Speaking and Teaching: Bristol-Myers Squibb, Gilead, Novartis, Abbott, Roche, Merck/MSD, Janssen, Siemens, Falk, Boehringer-Ingelheim, Bristol-Myers Squibb, Gilead, Novartis, Abbott, Roche, Merck/MSD, Janssen, Siemens, Falk, Boehringer-Ingelheim

The following people have nothing to disclose: Georgios Grammatikos, Christiane Mühle, Nerea Ferreirós, Dimitra Bogdanou, Sirkka Schroeter, Stephanie Schwalm, Gudrun Hintereder, Johannes Kornhuber, Josef Pfeilschifter


Interference with novel gene KCTD9 inhibits NK cell activation and ameliorates Murine Hepatitis Virus Type 3-induced fulminant hepatitis in BALB/cJ mice

Tao Chen1, Lin Zhu1, Ling Ding1, Li Song1, Xiaoping Zhang1, Aichao Shi1, Xiaoping Luo2, Qin Ning1;

1Institute and Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Department of Pediatrics, Tongji Hospital, Wuhan, China

Background/aims: Our previous reports, both experimental and human studies, have shown the novel gene KCTD9 contributed to liver injury through hepatic NKcell activation in HBV induced acute-on-chronic liver failure. This study aims to elucidate the therapeutic role of KCTD9 in a mice model. Methods: murine hepatitis virus strain 3 (MHV-3) induced fulminant viral hepatitis (MHV-3-FVH) mice model was adopted in the study. The mouse KCTD9 (mKCTD9) expression plasmid and an shRNA plasmid specifically targeting this molecule were constructed and introduced into infected mice by hydrodynamic delivery. The expression of KCTD9 as well as function of hepatic NK cells was detected, respectively. Results: The results showed abundant expression of KCTD9 in hepatic NK cells from mice with MHV-3 infection. The shRNA significantly inhibited KCTD9 expression in hepatic NK cells with decreased CD69 expression, cytotoxicity and ameliorated MHV-3-FVH in these mice demonstrating as an increased survival, improved liver functions and histopathology manifestation. In contrast, delivery of the KCTD9 expression plasmid to MHV-3-infected mice led to a profound progression of liver disease. Conclusion: Our study further elaborated the novel KCTD9 gene contributes to liver injury through NK cell activation in virus-induced liver failure, while interference targeting KCTD9 gene could ameliorated the disease, which provide a potential therapeutic target for virus induced liver failure.


Qin Ning - Advisory Committees or Review Panels: ROCHE, NOVARTIS, BMS, MSD, GSK; Consulting: ROCHE, NOVARTIS, BMS, MSD, GSK; Grant/Research Support: ROCHE, NOVARTIS, BMS; Speaking and Teaching: ROCHE, NOVARTIS, BMS, MSD, GSK

The following people have nothing to disclose: Tao Chen, Lin Zhu, Ling Ding, Li Song, Xiaoping Zhang, Aichao Shi, Xiaoping Luo