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Macrophage Immunoregulatory Galectin-9 Production is increased by Contact with Hepatitis C Virus-infected Hepatocytes and Differentiation

Noah M. Harwood, Lucy Golden-Mason, Hugo R. Rosen, John A. Mengshol
Gastroenterology/Hepatology' Universify of Colorado SOM, Denver VA Medical Center, Denver, CO

Hepatitis C Virus (HCV) infects 3. 2 million people in the United States and leads to cirrhosis in 20% over 20 years. Although therapy has dramatically improved, difficult to treat populations remain. The immunoregulatory protein Galectin-9 (Gal-9) may be partially responsible for the development and maintenance of persistent infection. In HCV patients, Gal-9 is elevated in the sera and liver, and localizes to Kupffer cells. Gal-9 induces apoptosis of HCV antigen-specific T cells and increases inhibitory regulatory T-cells via the receptor Tim-3 (PLoS ONE 2010 5(3): e9504). Our current study focuses on elucidating the signals that increase Gal-9 in Kupffer cells. Methods: しsing quantitative real-time PCR, we analyzed Gal-9 mRNA in co-cultures of the Huh7. 5 cell line infected with JFH-1 HCV and either the THP-1 monocyte cell line or human monocytes from healthy donors. Additionally, we examined Gal-9 levels upon phorbol 12-myristate 13-acetate (PMA)-induced maturation of THP-1 cells to macrophages, and in human monocytes matured to proinflammatory macrophages (M1) with GM-CSF, or alternative (M2) with M-CSF. Flow cytometry confirmed protein expression. Results: THP-1 cells upregulate Gal-9 three to five-fold (p<0.001) when exposed to HCV-infected Huh7. 5s. Induction is likely dependent on contact or proximity, as Gal-9 mRNA levels remain constant when THP-1s and infected Huh7. 5s are cultured on opposite sides of a permeable membrane. Furthermore, medium from infected hepatocytes fails to increase Gal-9 in THP-1s. Gal-9 induction by infected Huh7. 5s is interferon-y independent since a neutralizing antibody has no effect on Gal-9 levels. Gal-9 mRNA is elevated up to 20-fold in co-cultures of infected Huh7. 5s and human monocytes after four days (P=0.02), and increases further with time. To test whether Gal9 levels change with differentiation, we induced macrophage maturation in THP-1 cells with PMA. Basal Gal-9 increases fourfold in mature THP-1 macrophages compared to THP-1 monocytes, with a further Gal-9 increase in THP-1 macrophages exposed to HCV-infected hepatocytes or IFN-y. Human monocytes differentiated into M1s produced 22-fold more Gal-9 than those differentiated into M2s (p<0.02). Conclusions: Gal-9 is produced by monocytes after exposure to HCV-infected Huh7. 5s, and is further heightened during monocyte to macrophage differentiation. HCV-infected cells directly stimulate Gal-9, possibly via contact and uptake by monocytes or macrophages. We are currently investigating whether this occurs via endosomal toll-like receptors. Therapies targeting Gal-9 might provide an immune boost to help eradicate virus in HCV patients.

Disclosures:

The following people have nothing to disclose: Noah M. Harwood, Lucy GoldenMason, Hugo R. Rosen, John A. Mengshol

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Hepatic interferon-stimulated genes are differentially regulated in the liver of chronic hepatitis C patients with different interleukin 28B genotypes

Masao Honda, Takayoshi Shirasaki, Tetsuro Shimakami, Akito Sakai, Rika Horii, Kuniaki Arai, Tatsuya Yamashita, Yoshio Sakai, Taro Yamasnita, Hikari Okada, Mikiko Nakamura, Eishiro Mizukosni, Shuichi Kaneko
Gastroenterology, Kanazawa Univ' Kanazawa, Japan

Background: A previous report showed that pretreatment upregulation of hepatic ISGs had a stronger association with the treatment-resistant IL28B minor genotype (Ml) (TG/GG at rs8099917) than with the treatment-sensitive lL28B major genotype (MA) (Tt at rs8099917) (Gastroenterology, 2010). However, it is unknown how hepatic ISGs are up-regulated in MI patients and why patients with high levels of ISG expression cannot eliminate HCV. Methods: We compared the expression of ISGs in the liver and blood of 146 patients with chronic hepatitis C (CH-C) who received PEGylated-IFN and RBV therapy. Gene expression profiles in the liver and blood of 85 patients were analyzed using Affymetrix GeneChips. Furthermore, ISG expression in liver lobules and portal areas was analyzed using a laser capture microdissection (LCM) method. HCV replication analysis was performed by using an infectious genotype 1a clone, pH77S. 3/Gluc2A that included a Gaussia luciferase reporter gene. Results: ISG expression was correlated between the liver and blood of the MA patients, while no correlation was observed in MI patients. This loss of correlation was due to impaired infiltration of immune cells into the liver lobules of Ml patients, as demonstrated by regional gene expression analysis in liver lobules and portal areas using LCM and immunohistochemical staining. The expression of chemokines, CCL19, CCL21, CCL5 and CXCL13 etc. were significantly repressed in Ml liver. Despite having lower levels of immune cells, hepatic ISGs were up-regulated in MI liver and they were found to be significantly correlated with the expression of IL28 A/B, IFN-4, and WNT5A, while hepatic ISGs in MA liver were not correlated with IFN-λ4 (no expression) and WNT5A. Interestingly, we found WNT5A induced the expression of ISGs in Huh-7 cell line but it increased HCV replication by inducing the expression of the stress granule (SG) protein, G3BP1. In the liver of CH-C, the expression of WNT5A was significantly up-regulated in MI liver and correlated with its receptor, FZD5 and SG protein, G3BP1. Conclusions: In the liver of CH-C patients with IL28B treatment resistant genotype, immune cells were lost and induced the expression of other inflammatory mediators such as WNT5A. WNT5A may support HCV replication and medicate IFN resistance by increasing SG proteins. These changes of signaling pathway might be established in the process of persistent infection of HCV and contribute to the treatment resistant of IL28B Ml genotype.

Disclosures:

Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan

The following people have nothing to disclose: Masao Honda, Takayoshi Shirasaki, Tetsuro Shimakami, Akito Sakai, Rika Horii, Kuniaki Arai, īatsuya Yamashita, Yoshio Sakai, Taro Yamashita, Hikari Okada, Mikiko Nakamura, Eishiro Mizukoshi

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Hepatic IFN lambda receptor 1 expression is induced in chronic hepatitis C and correlates with non-response to IFN alpha and IL28B minor alleles

Francois Duong2, Goio Trincucci2, Tujana Boldanova1,2, Sarah Durand3, 4, Mirjam B. Zeisel3, 4, Thomas F. Baumert5, 6, Markus H. Heim1,2
1Department of Gastroenterology ond Hepatology, University Hospital Basel, Basel, Switzerland; 2Department of Biomedicine, University of Basel, Basel, Switzerland; 3INSERM, U748, Strasbourg, France; 4University of Strasbourg, strasbourg, France; 5Pole des Pathologies Digestives, Hépotiques et Transplantation, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 6Pôle Hépato-digestif, Nouvel Hôpitol Civil, Strasbourg, France

Background: Non-response to pegylated IFNa (pegIFN) and ribavirin in chronic hepatitis C (CHC) is associated with minor alleles of the IFNλ3 (IL28B) genotype and with an activation of the endogenous IFN system in the liver already before treatment. The molecular mechanisms responsible for the constitutive high expression of ISGs and their link to the IFNλ3 genotype are presently unknown. Methods: We measured the expression of IFNλ and of the specific IFNλ receptor chain (IFNλR1) in 122 liver biopsies of patients with CHC and 55 control samples from non-HCV infected patients. Primary human hepatocytes were IFNλ3 (IL28B) genotyped and stimulated with IFNα and the inducible expression of IFNλR1 assessed by qPCR and immunohistochemistry. Huh7 cells were transfected with IFNλR1 and several clones with different expression levels of IFNλR1 were selected and stimulated with IFNλ to investigate the correlation between the IFNλR1 expression and Jak-STAT activation and ISG induction. 20 liver biopsies of patients with CHC were stimulated ex vivo with IFNa and IFNλ to analyse the correlation between IFNλR1 expression and responsiveness to IFNa or IFNλ. Results: We found a very low expression of IFNλR1 in primary human hepatocytes (PHH) and, surprisingly, also in liver biopsies from patients who were not infected with HCV. In PHH, IFNλR1 expression was induced by IFNa, leading to substantially improved responses of the Jak-STAT signalling pathway to IFNλ. The strength of IFNa induced IFNλR1 expression was associated with IFNλ3 genotype. IFNλR1 expression was significantly higher in liver biopsies from patients with CHC compared to uninfected controls. Within the group of patients with CHC, IFNλR1 expression significantly correlated with IFNλ3 genotype. Importantly, ex-vivo treatment of liver biopsies from patients with high-level expression of IFNλR1 showed strong responses to IFNλ and poor responses to IFNα, whereas the contrary was found in patients with lowlevel expression of IFNλR1. Conclusions: These results provide strong evidence that the IFNλ3 genotype determines the extent of IFNα induced IFNλR1 expression in hepatocytes. Minor IFNλ3 alleles are associated with strong IFNλR1 induction. High expression levels of IFNλR1 provide responsiveness to IFNλ and thereby continuous ISG induction, because IFNλ signaling does not become refractory in the liver. CHC patients with constitutive high ISG expression are poor responders to IFNa, but show excellent STAT1 activation in response to IFNλ. Our findings provide a rational for treating pegIFN-a nonresponder patients with peglFN-λ

Disclosures:

The following people have nothing to disclose: Francois Duong, Gaia Trincucci, Tujana Boldanova, Sarah Durand, Mirjam B. Zeisel, Thomas F. Baumert, Markus

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Intrahepatic Changes in the Endogenous Interferon Response are Associated with SVR in Chronic Hepatitis C, Genotype-1 Subjects Treated with Sofosbuvir and Ribavirin

Eric G. Meissner1, Anu Osinusi1,2, Honghui Wang3, John G. McHutchison4, William T. Symonds4, Anthony F. Suffredini3, Michael A. Polis1, Henry Mosur3, Shyam Kottilil1
1Laboratory of Immunoregulation, NIH/NIAID, Bethesda, MD; 2Clinical Research Directorate/CRMP, SAIC-Frederick, Inc, Frederick, MD; 3critical Core Medicine Department, NIH, Bethesda, MD; 4Gilead Sciences, Foster City, CA

Background: To better understand the host response to hepatitis C virus (HCV) during directly acting antiviral (DAA) therapy, we measured endogenous interferon balance over the course of treatment in paired liver biopsies and serum from subjects treated with sofosbuvir and ribavirin and sought associations with treatment outcome. Methods: Sixty treatment naīve subjects with chronic HCV genotype-1 infection were treated with the NS5B RNA polymerase inhibitor sofosbuvir and ribavirin for 24 weeks. Eight subjects had paired pre- and post-treatment liver biopsies available for RNA analysis. Microarrays were performed using the Affymetrix し-219 platform and expression of interferon genes was determined by quantitative RT-PCR. Serum levels of IFNA2 were measured using a Mesoscale Discovery (MSD) single-plex kit. For IFNL3, specific DuoSet antibodies from R&D Systems were adapted for the MSD platform. Results: Of 55 subjects who completed the study, 38 achieved SVR24 and 17 relapsed. In 8 subjects with paired liver biopsies, 7 achieved SVR24 and 1 relapsed. In paired liver biopsies, there were treatment-related decreases in expression of type II and III interferons that correlated with decrease in hepatic expression of interferon stimulated genes. Similarly, receptor expression of type II and III interferons decreased (IFNGR2, IFNLR1) over the course of treatment. In contrast, IFNA2 expression increased in most of the seven subjects who achieved SVR, while expression decreased only in the subject who subsequently relapsed. There was no change in type I receptor expression. IFNA2 was undetectable in patient serum before, during, or at the end of treatment, while IFNL3 and IFNG levels were detectable prior to therapy and decreased by week 12 of therapy irrespective of treatment outcome. Conclusions: Intrahepatic gene expression and quantitative serum protein changes suggest that type II and III interferons drive the intrahepatic interferon gene signature in chronic HCV infection. Elevation of type I interferon gene expression at the end of therapy may be an important determinant of favorable outcome for DAA-based interferon-free therapy.

Disclosures:

John G. McHutchison - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

William ī. Symonds - Employment: Gilead

The following people have nothing to disclose: Eric G. Meissner, Anu Osinusi, Honghui Wang, Anthony F. Suffredini, Michael A. Polis, Henry Masur, Shyam Kot-

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Validation study of the impact of SNPs from GWAS associated with fibrosis progression in chronic hepatitis C (HCV)

Lourdes Rojas1, Javier Ampuero1/, Jose A. Del Compo1, José Raùl Garcίo-Lozano2, Ricard Solá3 , Xavier Forns4, Ricardo MorenoOtero13, Raùl J. Andrade5, Moises Diago6, Javier Solmeron7, Luis Rodrigo8, Jose A Pons9, J. M. Navarro10, Jose L. Collejo11, Javier Gatch-Samaniego12, Manuel Romero-Gomez1
1Digestive, valme Hospital, Seville, Spain; 2Digestive, Hospital Virgen del Rocio, Sevilla, Spain; 3Digestive, Hospital del Mar, Barcelona, Spain; 4Digestive', Hospital Clinic, Barcelona, Spain; 5Digestive, Hospital Virgen de la Victoria, Malaga, Spain; 6Digestivo, Hospital General de Valencia, Valencia, Spain; 7Digestivo, Hospital Son Cecilio, Granada, Spain; 8Hospital Central de Asturias' Oviedo, Spain; 9Hospital Universitario Virgen de Arrixaca, Murcia, Spain; 10Hospital Costa del Sol, Marbella, Spain; 11 Hospital Puerta de Hierro, Madrid, Spain; 12Hospital Carlos III, Madrid, Spain; 13Hosptial La Princesa, Madrid, Spain

Background & Aims: Previous GWAS studies identified several susceptibility loci for HCV-induced liver fibrosis rs4374383 (MERTK), rs16851720 (RNF7), rs361814 (U SP18), rs1626521(UCP3), rs4514994 (MAGI1), rs11943360 (STX18) and rs6725030 (CTNNA2) (Patin et al. Gastro 2012; Del Campo et al. JHepat 2010). To validate the association of these SNPs with fibrosis progression in a cohort of patients with HCV. Methods: We included 810 patients with biopsy-proven HCV. Mean age 45+11 years old; 64% (520/810) males and 36% (290/810) female; HCV-genotype-1 73% (592/810), genotype-2 2% (16/810), genotype-3 16% (131/810) and genotype-4 9% (70/810). IL28B genotype CC was 33% (264/800) and IL28B CT/TT genotype 67% (536/800). Fibrosis F0 was 4% (34/810), F1 52% (418/810), F2 27% (217/810), F3 10% (82/810) and F4 7% (59/810). Advanced fibrosis (F3-F4) was found in 17% (141/810) and mild-moderate fibrosis (F0-F2) 83% (669/810). Steatosis was detected in 37% (303/598). Fibrosis was staged according to Metavir score. The different SNPs were typed using Taqman probes (Applied Byosistems). Results: Genotype CC in UCP3 (28%) and genotype GG in USP18 (18%) were overrepresented in patients with advanced fibrosis in comparison with mild-moderate fibrosis (U. R. 2. 5; 95%CI: 1. 3-4. 8 and 〇. R. 2. 5; 95%CI: 1. 5-4. 2; p<0.001). Multivariate analysis using Backward LR demonstrated UCP3 genotype CC (O. R: 2. 5; 95%CI: 1. 35-4. 58); p<0.001 and FORNS index (O. R: 2; 95%CI: 1. 6-2. 4); p<0.001) as independent variables associated with significant (>F2), advanced fibrosis (>F3) and cirrhosis (F4). Conclusions: UCP3 genotype CC showed a strong association with advanced fibrosis, probably related to the increased lipid-induced oxidative stress in patients with decreased protein function. UCP3 genotype CC and Forns's Index were independently related to advanced fibrosis. New non-invasive methods based on genetic plus standard methods could improve fibrosis prediction.

rsGENEGENOTYPEF0-F2F3-F4O. R (95%)P
rs6725030CTNNA2 (n=472)CC82, 2% (318)17, 8% (69) 0, 067
CT/TT89, 4% (76)10, 6% (9)
rs4514994MAGI1(n= 405)AA/AT87, 7% (142)12, 3% (20) 0, 07
TT80, 7 % (196)19, 3% (47)
rsl1943360STX18(n=482)AA/ AG87, 7% (164)12, 3% (23) 0, 05
GG81, 0% (239)19, 0% (56)
rs 1626521UCP3 (n= 485)CC72, 3% (73)27, 7% (28)2. 5 (1. 5-4. 2)0, 001
CT/TT86, 7% (333)13, 3% (51)
rs361814USP18 (n= 460)GG82% (255)18, 0% (56)2. 5(1. 3-4. 8)0, 003
GT/TT91, 9% (137)8, 1% (12)
rs4374383MERTK (n= 756)AA83, 7% (103)16, 3% (20) 0, 329
AG/GG81, 5% (516)18, 5% (117)
rsl 6851720RNF7 (n= 564)AA/AC83, 2% (452)16, 8% (91) 0, 07
CC100% (21) 0% (0)

Disclosures:

Xavier Forns - Consulting: īibotec/Jansen, MSD, Boheringher Ingelheim; Grant/Research Support: Roche, MSD, Gilead

Moises Diago - Grant/Research Support: BOHERINGER, ROCHE, MSD, GILEAD, BMS, GSK, JANSEN, ABBVIE

Javier Garcίa-Samaniego - Consulting: Boehringer-IngeIheim

Manuel Romero-Gomez - Advisory Committees or Review Panels: Roche Farma, SA., MSD, S. A., Janssen, S. A., Abbott, S. A.; Grant/Research Support: Ferrer, S. A.

The following people have nothing to disclose: Lourdes Rojas, Javier Ampuero, Jose A. Del Campo, Jose Raul Garcla-Lozano, Ricard Sola, Ricardo MorenoOtero, Raul J. Andrade, Javier Salmeron, Luis Rodrigo, Jose A Pons, J. M. Navarro, Jose L. Calleja

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The anti-HCV gene SART1 regulates IFN stimulated genes through nonclassical mechanisms

Wenyu Lin1, Chuanlong Zhu1, Jian Hong1, Lei Zhao1, Qikai Xu2, Foffronucn Chusri1, Nikolaus Jilg1, Dahlene N. Fusco1, Esperance A. Schaefer1, Cynthia Brisoc1, Lee F Peng1, Raymond T Chung1
1Gosfroinfesfinol Unit' Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

Background/Aims: The IFNα's anti-HCV mechanisms are not well understood. In our previous siRNA screen, we identified SART1 which regulated IFN's antiviral effects. SART1 is a splicing factor which involved in RNA splicing and pre-mRNA processing. We hypothesized that SART1 regulates IFN effector genes (IEGs) through mRNA splicing. We performed RNA-Seq to evaluate the possibility that SART1 regulates lEGs at the level of transcription and alternative exon RNA processing. Methods: We performed siRNA knockdown in Huh7. 5. 1 cells. Nonstrand specific RNA sequencing was performed used a largescale, automated variant of the Illumina Tru Seq. Each RNA-Seq sample received at least 75 million reads. We used bedtools with the corresponding GTF files to download GENC〇DE (v12) and ENSEMBL (v68) mapping files respectively for transcriptlevel and exon-level bioinformatics analysis. Selected genes mRNA levels and RNA variants, together with HCV replication were monitored by qPCR and RT-PCR in HCV JFH1 and OR6 replicon cells. Results: We identified 26095 genes from RNASeq transcription level splicing analysis. There were 419 genes with more than 2 fold difference between Neg siRNA and SART1 siRNA whether in the presence or absence of IFN. Ingenuity Pathway Analysis (IPA) identified at least 10 functional pathways, including cell signaling, interferon and antiviral response pathways. Our qPCR data confirmed consistency between RNA-Seq and qPCR analysis. We found that siRNA to SART1 reduced classical ISG mRNA transcription expression including Mx1, IFIH1 (MDA5), OAS3, and DDX58 (RIG-I). However, we found that SART1 did not affect Jak-STAT pathway genes including IFNAR1, STAT1, JAK1, IRF1, and IRF9 mRNA transcription expression. Our bioinformatics alternative exon splicing analysis identified 1589 down-regulated and 1155 up-regulated genes and exons by SART1 or IFNα treatments. Our RT-PCR images have confirmed alternative mRNA splicing for several genes, including N〇M〇3, EIF4G3, MICし1, GORASP2, XP〇1, ZFAND6, and RAB6A. We found that siRNA or overexpression of EIF4G3, GORASP2, or ZFAND6 regulated HCV replication. Conclusions: The spliceosome factor SART1 is not IFN-inducible but is an IFN effector gene. SART1 exerts its anti-HCV action through direct transcriptional downregulation for some ISGs (e. g., IFIH1) and alternative splicing for others, including EIF4G3, G〇RASP2, and ZFAND6. SART1 does not have effects on IFN receptor or components. Taken together, these data imp ulates ISGs in a non-classical manner.

Disclosures:

Raymond ī. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead

The following people have nothing to disclose: Wenyu Lin, Chuanlong Zhu, Jian Hong, Lei Zhao, Qikai Xu, Pattranuch Chusri, Nikolaus Jilg, Dahlene N. Fusco, Esperance A. Schaefer, Cynthia Brisac, Lee F. Peng