- Email: [email protected]
O063 : HCV reinfection cases in phase 3 studies of sofosbuvir
ORAL PRESENTATIONS was identified in 12 patients and 11 of those cases occurred in individuals who had relapsed to injecting drug use. The incidence of reinfection was 1.8/100 PY (95% CI 1.0–3.2) among individuals with injecting drug use prior to treatment, 4.7/100 PY (95% CI 2.7–8.4) among those who had relapsed to injecting behaviour during follow-up and 0.32/100 PY (95% CI 0.04–2.2) among those with no history of injecting drug use. In multivariate regression analysis, low education level (OR 7.3, 95% CI 1.8–29.7; p = 0.006) was the only independent baseline predictor for HCV reinfection. Conclusions: In this seven year follow-up study mainly comprising PWID who had been abstinent from drug use at least six months prior to HCV treatment, we demonstrate frequent relapse to injecting drug use but still low to moderate long-term incidence of HCV reinfection (1.8/100 PY). Our findings are in line with estimates from previous smaller studies and should be addressed when providing HCV care for PWID.
of spontaneous clearance of CHC was 0.36 per 100 person years follow up, occurring after a median 49.5 months of infection. Table 1. Main demographic and clinical patient characteristics Late spontaneous
Chronically infected
clearance (n = 50)
(n = 200)
Male sex [n (%)]
19 (38)
129 (64.5)
0.001
Median age [years (IQR)]
41 (37–48)
43 (38–49)
0.36
Median age at infection [years (IQR)]
28.5 (25–36)
33 (28–38)
0.02
White
48 (96)
194 (97)
Asian
2 (4)
6 (3)
Intravenous drug use
41 (82)
161 (80.5)
Sexual/partner
2 (4)
3 (1.5)
Iatrogenic
2 (4)
6 (3)
Vertical
0 (0)
3 (1.5)
Born abroad
1 (2)
5 (2.5)
Unknown
4 (8)
22 (11)
1
7 (14)
61 (30.5)
2
1 (2)
5 (2.5)
3
9 (18)
52 (26)
Unknown
33 (66)
82 (41)
Positive
2 (4)
3 (1.5)
Negative
36 (72)
98 (49)
Not tested
12 (24)
99 (49.5)
Positive
5 (10)
0 (0)
Negative
43 (86)
99 (49.5)
Not tested
2 (4)
101 (50.5)
Yes
15 (30)
97 (48.5)
No
25 (50)
76 (38)
Unknown
10 (20)
27 (13.5)
Yes
21 (42)
64 (32)
No
24 (48)
109 (54.5)
Unknown
5 (10)
27 (13.5)
Yes
13 (26)
34 (17)
No
25 (50)
104 (52)
Unknown
12 (24)
62 (31)
49.5 (30.5–81.25)
50 (19–101.5)
Median
1000 †
341,142
Interquartile range
1000–375,792
59,496–1,517,864
Ethnic group [n (%)]
0.72
Risk group [n (%)]
0.72
HCV genotype [n (%)]
O062 FACTORS ASSOCIATED WITH SPONTANEOUS CLEARANCE OF CHRONIC HEPATITIS C VIRUS INFECTION: A RETROSPECTIVE CASE CONTROL STUDY N. Bulteel1 , P. Partha Sarathy2 , E. Forrest3 , H. Innes4 , P. Mills5 , H. Valerio4,6 , A. Stanley3 , R.N. Gunson7 , C. Aitken7 , J. Morris8 , S.T. Barclay3 . 1 Centre for Virus Research, MRC – University of Glasgow, 2 Medical School, University of Glasgow, 3 The Walton Liver Clinic, Glasgow Royal Infirmary, 4 School of Health and Life Sciences, Glasgow Caledonian University, 5 Department of Hepatology, Gartnavel General Hospital, 6 Blood-borne Viruses and Sexually Transmitted Infections Section, Health Protection Scotland, 7 West of Scotland Specialist Virology Centre, Glasgow Royal Infirmary, 8 Department of Gastroenterology, Southern General Hospital, Glasgow, United Kingdom E-mail: [email protected]
0.71
Serum HIV IgG [n (%)]
0.52
Serum HBsAg [n (%)]
0.001
Current IDU [n (%)]
0.03
History of alcohol excess/ALD [n (%)]
0.24
Cirrhosis [n (%)]
Background and Aims: Chronic HCV infection (CHC) develops in ~75% of people who acquire HCV, and is defined as infection persisting beyond six months. Spontaneous clearance of CHC is rare and poorly characterised. We conducted a retrospective case control study of patients attending centres in Glasgow to identify factors associated with spontaneous clearance of CHC. Methods: Data were obtained from the West of Scotland specialist virus laboratories on HCV testing between 1994 and 2013. Patients with ≥2 sequential RNA positive samples at least 6 months apart, followed by at least one negative HCV RNA (potential spontaneous clearance cohort), were identified in addition to patients with ≥2 positive samples at least 6 months apart with no subsequent negative samples (chronic infection cohort). Both cohorts were linked to the Scottish Hepatitis C database to exclude treated patients and obtain relevant patient data. A control group was randomly selected from the chronic infection cohort with a frequency of 4 per spontaneous clearer. Case notes were reviewed to verify the clinical course. Categorical variables were compared using c2 . Continuous variables were analysed using the exact Wilcoxon Mann–Whitney test. P values are 2-sided, and values of <0.05 were considered significant. Results: 25113 HCV RNA positive samples, relating to 10318 patients were identified. 116 patients, not linked as treated, had 2 sequential positive results followed by a negative. 66 patients were excluded following case note review leaving 50 patients of interest, contributing 241 person years follow up. 2518 untreated, chronically infected patients were identified, contributing 13766 person years follow up. 200 control patients were randomly selected from this cohort. Table 1 summarises demographic and clinical characteristics of the study populations. Spontaneous clearance of CHC was associated with female gender, HBsAg positivity, younger age at infection and lower HCV RNA. Spontaneous clearance was negatively associated with current IDU. The overall incidence rate S222
Median duration of infection [months (IQR)]
P value
0.24
HCV VL (IU/ml)
0.94 <0.001
† Data on HCV VL only available for 20 patients.
Conclusions: This is the largest cohort of patients with spontaneous clearance of CHC studied to date. Our data suggest that clearance occurs rarely in CHC but can occur after a prolonged duration, and is associated with female sex, HBV co-infection, younger age at infection and low HCV RNA titres. O063 HCV REINFECTION CASES IN PHASE 3 STUDIES OF SOFOSBUVIR E. Svarovskaia1 , R. Martin1 , K. Chodavarapu Chodavarapu1 , C. Hedskog Hedskog1 , D. Brainard1 , M.D. Miller1 , H. Mo1 . 1 Gilead Sciences, Foster City, United States E-mail: [email protected] Background and Aims: Relapse accounted for all virologic failures among treatment-adherent patients in the ledipasvir/sofosbuvir (LDV/SOF) and SOF Phase 3 clinical development program with most of relapse cases occurring 4–12 weeks post-treatment. It has been shown that SVR12 is an appropriate time point for the reliable assessment of a durable treatment response to SOF-containing regimens. However, in very rare cases, patients became positive again for HCV RNA after week 12 post-treatment. In this study, relapse cases that occurred after week 12 post-treatment in the phase 3 SOF studies were investigated for possible HCV reinfection. Methods: Sequencing analysis of the NS5B gene was performed from patients who had relapse HCV after week 12 post-treatment in NEUTRINO, FISSION, POSITRON, FUSION, VALENCE, PHOTON-1, PHOTON-2 and GS-US-334-0118 SOF studies. Maximum likelihood
Journal of Hepatology 2015 vol. 62 | S213–S234
ORAL PRESENTATIONS phylogenetic trees were constructed for baseline and relapse time points to investigate the genetic distance of these HCV samples. Longitudinal samples from patients with viral relapse prior to week 12 and then subsequent follow-up samples were used as a control for viral genetic drift over the same time period. Results: In the 8 SOF studies, of 1154 patients who achieved SVR12, 1142 also had SVR24. However, there were 12 patients who achieved SVR12, but did not achieve SVR24 and had available samples for testing. Of 12 patients, samples from 10 patients were successfully sequenced at baseline and relapse and samples from 2 patients failed amplification at relapse. Phylogenetic analyses that included baseline and relapse samples from these 10 patients as well 30 patients that who relapsed prior to week 12 post-treatment and had longitudinal samples beyond week 12 post-treatment were performed. The genetic distance in NS5B between the baseline and relapse time points in 4 of 10 late virologic failures suggested a reinfection event and not relapse growth of the baseline HCV. In 1 of 4 reinfected patients, there was also a switch in genotype of virus between baseline and reinfection. Longitudinal samples from control patients showed minimal genetic drift over the same time period. Conclusions: About half of the relapse cases in the SOF Phase 3 clinical development program who relapsed after achieving SVR12 were found to have HCV reinfection and not a relapse of the baseline virus. This result further supports SVR12 as an appropriate time point for the reliable assessment of a durable treatment response to SOF regimens.
Fatty liver disease: Experimental O064 GUT–LIVER AXIS DERANGEMENT DUE TO LACK OF INFLAMMASOME ACTIVITY LEADS TO VISCERAL OBESITY AND NASH DEVELOPMENT L. Agostinelli1 , C. Riclicki1 , E. Mingarelli1 , C. Saponaro2 , M. Gaggini2 , E. Buzzigoli2 , I. Pierantonelli1 , S. Saccomanno1 , C. Pinto1 , L. Trozzi1 , A. Benedetti1 , S. De Minicis1 , M. Marzioni1 , A. Gastaldelli2 , G. Svegliati-Baroni1 . 1 Gastroenterology, Polytechnic University of Marche, Ancona, 2 Institute of Clinical Physiology, National Research Council, Pisa, Italy E-mail: [email protected] Background and Aims: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common form of chronic liver disease and can lead to cirrhosis and hepatocellular carcinoma. Gut microflora alterations and bacterial translocation induced by specific dietary habits can elicit a proinflammatory and profibrogenic response. The NLRP3 inflammasome regulates intestinal homeostasis and mediates the release of IL1b and IL18 in response to cellular danger signals. Its role in NAFLD development is controversial. Thus aim of the study was to investigate the role of NLRP3 inflammasome in a Western lifestyle diet model of NAFLD. Methods: Wild-type (WT) C57BL/6 and Nlrp3A350VneoR (Nlrp3−/− ) mice were fed either a chow diet (controls) or a high-fat diet with fructose in drinking water (HFHC) for 12 weeks. Results: Nlrp3−/− HFHC gained more weight (p < 0.01), showed reduced energy expenditure and more fat mass (both measured by doubly labeled water, all p < 0.05) with increased adipose tissue TNFa expression (p < 0.05), and developed more hepatic steatosis measured by triglyceride content (p < 0.01) compared to WT HFHC. HFHC increased intestinal permeability, as showed by reduced zonulin-1 expression in the caecum, that led to higher hepatic expression of TLR4 (p < 0.01) and TLR9 (p < 0.05) in Nlrp3−/− HFHC compared to WT HFHC. In the liver no differences were observed between Nlrp3−/− HFHC and WT HFHC in the expression of downstream SREBP-1c effectors of de novo lipogenesis (ACC, FAS
and SCD-1) that were significantly increased in both groups. On the other hand, compared to WT HFHC, Nlrp3−/− HFHC showed higher expression of the lipogenic transcription factor PPARg2 (p < 0.01) and of its downstream effectors FABP-4 (p < 0.05) and CD36 (p < 0.01) that regulate lipid uptake and storage. In addition Nlrp3−/− HFHC showed increased mitochondrial-oxidation of fatty acid (i.e., higher CPT1A expression) that, associated to the reduced expression of the “master regulator” of the antioxidant response NRF2, led to increased superoxide production (measured by dihydroethidium staining) (all p < 0.01). These series of events were associated to increased macrophage infiltration, type I collagen and MCP1 gene expression (p < 0.05) in Nlrp3−/− HFHC mice only. Conclusions: In the Western lifestyle diet, lack of NLRP3 inflammasome is associated with translocation of bacterial products that leads to severe metabolic alterations in both adipose tissue and the liver, and to NASH development. O065 MECHANISTIC STUDY OF TM6SF2 IN NAFLD PATHOGENESIS: STABLY TRANSFECTED Huh7 CELLS OVER-EXPRESSING THE TM6SF2 E167K VARIANT EXHIBIT GREATER LEVELS OF OXIDATIVE STRESS J. Palmer1 , Y.-L. Liu1 , C.P. Day1 , A. Daly1 , Q.M. Anstee1 . 1 The Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Non-Alcoholic Fatty Liver Disease (NAFLD) is a complex disease trait influenced by genetic modifiers. We, and others, have recently reported that minor (T) allele carriage of a non-synonymous polymorphism in TM6SF2 (rs58542926 C>T, p.E167K), a gene of unknown function, is associated not only with steatosis, but also with NASH and clinically relevant advanced liver fibrosis. Carriage of the C allele is associated with dyslipidaemia and cardiovascular disease. To better understand the mechanisms underlying these associations, we examined the effects of TM6SF2 over-expression in vitro. Methods: Huh7 human hepatoma cell-lines stably transfected to overexpress wild-type (E167) or variant (167K) forms of TM6SF2 were generated. Cells were cultured in standard medium or oleic acid enriched media. Hepatocyte lipid accumulation was assessed by oil red O staining, cellular oxidative stress (ROS) measured by FACS after DCFDA staining and the expression profile of relevant genes involved in lipid metabolism, fatty acid oxidation, inflammation and fibrogenesis were measured by rtPCR. Results: Following stable transfection, TM6SF2 expression was >50fold increased relative to resting Huh7 cells both at mRNA and protein levels. As previously noted, protein expression of the E167 form was approximately twice that of the 167K variant, likely reflecting protein misfolding or accelerated intracellular degradation. Whilst overexpression of either TM6SF2 form was associated with increased hepatic lipid accumulation in oleic acid enriched media, oil red O staining was substantially greater in cells expressing 167K. Consistent with development of NASH, carriage of the 167K variant was also associated with greater levels of ROS. At the transcriptome level, overexpression of TM6SF2 suppressed several lipogenic and pro-inflammatory genes. However, relative to E167, the 167K variant promoted differential expression of key genes including DGAT1, CYP2E1, IL6 and TNFA. Conclusions: These in vitro data suggest that TM6SF2 has profound effects on hepatocyte lipid metabolism. They provide the first evidence that the TM6SF2 p.E167K variant induces increased hepatocyte oxidative stress, possibly by favoring extramitochondrial omega- fatty-acid oxidation, and also promotes hepatocyte inflammatory cytokine expression. Work to further characterize the functional effects of TM6SF2 p.E167K carriage and the mechanisms through which it promotes liver injury and NASH are underway.
Journal of Hepatology 2015 vol. 62 | S213–S234
S223
of spontaneous clearance of CHC was 0.36 per 100 person years follow up, occurring after a median 49.5 months of infection. Table 1. Main demographic and clinical patient characteristics Late spontaneous
Chronically infected
clearance (n = 50)
(n = 200)
Male sex [n (%)]
19 (38)
129 (64.5)
0.001
Median age [years (IQR)]
41 (37–48)
43 (38–49)
0.36
Median age at infection [years (IQR)]
28.5 (25–36)
33 (28–38)
0.02
White
48 (96)
194 (97)
Asian
2 (4)
6 (3)
Intravenous drug use
41 (82)
161 (80.5)
Sexual/partner
2 (4)
3 (1.5)
Iatrogenic
2 (4)
6 (3)
Vertical
0 (0)
3 (1.5)
Born abroad
1 (2)
5 (2.5)
Unknown
4 (8)
22 (11)
1
7 (14)
61 (30.5)
2
1 (2)
5 (2.5)
3
9 (18)
52 (26)
Unknown
33 (66)
82 (41)
Positive
2 (4)
3 (1.5)
Negative
36 (72)
98 (49)
Not tested
12 (24)
99 (49.5)
Positive
5 (10)
0 (0)
Negative
43 (86)
99 (49.5)
Not tested
2 (4)
101 (50.5)
Yes
15 (30)
97 (48.5)
No
25 (50)
76 (38)
Unknown
10 (20)
27 (13.5)
Yes
21 (42)
64 (32)
No
24 (48)
109 (54.5)
Unknown
5 (10)
27 (13.5)
Yes
13 (26)
34 (17)
No
25 (50)
104 (52)
Unknown
12 (24)
62 (31)
49.5 (30.5–81.25)
50 (19–101.5)
Median
1000 †
341,142
Interquartile range
1000–375,792
59,496–1,517,864
Ethnic group [n (%)]
0.72
Risk group [n (%)]
0.72
HCV genotype [n (%)]
O062 FACTORS ASSOCIATED WITH SPONTANEOUS CLEARANCE OF CHRONIC HEPATITIS C VIRUS INFECTION: A RETROSPECTIVE CASE CONTROL STUDY N. Bulteel1 , P. Partha Sarathy2 , E. Forrest3 , H. Innes4 , P. Mills5 , H. Valerio4,6 , A. Stanley3 , R.N. Gunson7 , C. Aitken7 , J. Morris8 , S.T. Barclay3 . 1 Centre for Virus Research, MRC – University of Glasgow, 2 Medical School, University of Glasgow, 3 The Walton Liver Clinic, Glasgow Royal Infirmary, 4 School of Health and Life Sciences, Glasgow Caledonian University, 5 Department of Hepatology, Gartnavel General Hospital, 6 Blood-borne Viruses and Sexually Transmitted Infections Section, Health Protection Scotland, 7 West of Scotland Specialist Virology Centre, Glasgow Royal Infirmary, 8 Department of Gastroenterology, Southern General Hospital, Glasgow, United Kingdom E-mail: [email protected]
0.71
Serum HIV IgG [n (%)]
0.52
Serum HBsAg [n (%)]
0.001
Current IDU [n (%)]
0.03
History of alcohol excess/ALD [n (%)]
0.24
Cirrhosis [n (%)]
Background and Aims: Chronic HCV infection (CHC) develops in ~75% of people who acquire HCV, and is defined as infection persisting beyond six months. Spontaneous clearance of CHC is rare and poorly characterised. We conducted a retrospective case control study of patients attending centres in Glasgow to identify factors associated with spontaneous clearance of CHC. Methods: Data were obtained from the West of Scotland specialist virus laboratories on HCV testing between 1994 and 2013. Patients with ≥2 sequential RNA positive samples at least 6 months apart, followed by at least one negative HCV RNA (potential spontaneous clearance cohort), were identified in addition to patients with ≥2 positive samples at least 6 months apart with no subsequent negative samples (chronic infection cohort). Both cohorts were linked to the Scottish Hepatitis C database to exclude treated patients and obtain relevant patient data. A control group was randomly selected from the chronic infection cohort with a frequency of 4 per spontaneous clearer. Case notes were reviewed to verify the clinical course. Categorical variables were compared using c2 . Continuous variables were analysed using the exact Wilcoxon Mann–Whitney test. P values are 2-sided, and values of <0.05 were considered significant. Results: 25113 HCV RNA positive samples, relating to 10318 patients were identified. 116 patients, not linked as treated, had 2 sequential positive results followed by a negative. 66 patients were excluded following case note review leaving 50 patients of interest, contributing 241 person years follow up. 2518 untreated, chronically infected patients were identified, contributing 13766 person years follow up. 200 control patients were randomly selected from this cohort. Table 1 summarises demographic and clinical characteristics of the study populations. Spontaneous clearance of CHC was associated with female gender, HBsAg positivity, younger age at infection and lower HCV RNA. Spontaneous clearance was negatively associated with current IDU. The overall incidence rate S222
Median duration of infection [months (IQR)]
P value
0.24
HCV VL (IU/ml)
0.94 <0.001
† Data on HCV VL only available for 20 patients.
Conclusions: This is the largest cohort of patients with spontaneous clearance of CHC studied to date. Our data suggest that clearance occurs rarely in CHC but can occur after a prolonged duration, and is associated with female sex, HBV co-infection, younger age at infection and low HCV RNA titres. O063 HCV REINFECTION CASES IN PHASE 3 STUDIES OF SOFOSBUVIR E. Svarovskaia1 , R. Martin1 , K. Chodavarapu Chodavarapu1 , C. Hedskog Hedskog1 , D. Brainard1 , M.D. Miller1 , H. Mo1 . 1 Gilead Sciences, Foster City, United States E-mail: [email protected] Background and Aims: Relapse accounted for all virologic failures among treatment-adherent patients in the ledipasvir/sofosbuvir (LDV/SOF) and SOF Phase 3 clinical development program with most of relapse cases occurring 4–12 weeks post-treatment. It has been shown that SVR12 is an appropriate time point for the reliable assessment of a durable treatment response to SOF-containing regimens. However, in very rare cases, patients became positive again for HCV RNA after week 12 post-treatment. In this study, relapse cases that occurred after week 12 post-treatment in the phase 3 SOF studies were investigated for possible HCV reinfection. Methods: Sequencing analysis of the NS5B gene was performed from patients who had relapse HCV after week 12 post-treatment in NEUTRINO, FISSION, POSITRON, FUSION, VALENCE, PHOTON-1, PHOTON-2 and GS-US-334-0118 SOF studies. Maximum likelihood
Journal of Hepatology 2015 vol. 62 | S213–S234
ORAL PRESENTATIONS phylogenetic trees were constructed for baseline and relapse time points to investigate the genetic distance of these HCV samples. Longitudinal samples from patients with viral relapse prior to week 12 and then subsequent follow-up samples were used as a control for viral genetic drift over the same time period. Results: In the 8 SOF studies, of 1154 patients who achieved SVR12, 1142 also had SVR24. However, there were 12 patients who achieved SVR12, but did not achieve SVR24 and had available samples for testing. Of 12 patients, samples from 10 patients were successfully sequenced at baseline and relapse and samples from 2 patients failed amplification at relapse. Phylogenetic analyses that included baseline and relapse samples from these 10 patients as well 30 patients that who relapsed prior to week 12 post-treatment and had longitudinal samples beyond week 12 post-treatment were performed. The genetic distance in NS5B between the baseline and relapse time points in 4 of 10 late virologic failures suggested a reinfection event and not relapse growth of the baseline HCV. In 1 of 4 reinfected patients, there was also a switch in genotype of virus between baseline and reinfection. Longitudinal samples from control patients showed minimal genetic drift over the same time period. Conclusions: About half of the relapse cases in the SOF Phase 3 clinical development program who relapsed after achieving SVR12 were found to have HCV reinfection and not a relapse of the baseline virus. This result further supports SVR12 as an appropriate time point for the reliable assessment of a durable treatment response to SOF regimens.
Fatty liver disease: Experimental O064 GUT–LIVER AXIS DERANGEMENT DUE TO LACK OF INFLAMMASOME ACTIVITY LEADS TO VISCERAL OBESITY AND NASH DEVELOPMENT L. Agostinelli1 , C. Riclicki1 , E. Mingarelli1 , C. Saponaro2 , M. Gaggini2 , E. Buzzigoli2 , I. Pierantonelli1 , S. Saccomanno1 , C. Pinto1 , L. Trozzi1 , A. Benedetti1 , S. De Minicis1 , M. Marzioni1 , A. Gastaldelli2 , G. Svegliati-Baroni1 . 1 Gastroenterology, Polytechnic University of Marche, Ancona, 2 Institute of Clinical Physiology, National Research Council, Pisa, Italy E-mail: [email protected] Background and Aims: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common form of chronic liver disease and can lead to cirrhosis and hepatocellular carcinoma. Gut microflora alterations and bacterial translocation induced by specific dietary habits can elicit a proinflammatory and profibrogenic response. The NLRP3 inflammasome regulates intestinal homeostasis and mediates the release of IL1b and IL18 in response to cellular danger signals. Its role in NAFLD development is controversial. Thus aim of the study was to investigate the role of NLRP3 inflammasome in a Western lifestyle diet model of NAFLD. Methods: Wild-type (WT) C57BL/6 and Nlrp3A350VneoR (Nlrp3−/− ) mice were fed either a chow diet (controls) or a high-fat diet with fructose in drinking water (HFHC) for 12 weeks. Results: Nlrp3−/− HFHC gained more weight (p < 0.01), showed reduced energy expenditure and more fat mass (both measured by doubly labeled water, all p < 0.05) with increased adipose tissue TNFa expression (p < 0.05), and developed more hepatic steatosis measured by triglyceride content (p < 0.01) compared to WT HFHC. HFHC increased intestinal permeability, as showed by reduced zonulin-1 expression in the caecum, that led to higher hepatic expression of TLR4 (p < 0.01) and TLR9 (p < 0.05) in Nlrp3−/− HFHC compared to WT HFHC. In the liver no differences were observed between Nlrp3−/− HFHC and WT HFHC in the expression of downstream SREBP-1c effectors of de novo lipogenesis (ACC, FAS
and SCD-1) that were significantly increased in both groups. On the other hand, compared to WT HFHC, Nlrp3−/− HFHC showed higher expression of the lipogenic transcription factor PPARg2 (p < 0.01) and of its downstream effectors FABP-4 (p < 0.05) and CD36 (p < 0.01) that regulate lipid uptake and storage. In addition Nlrp3−/− HFHC showed increased mitochondrial-oxidation of fatty acid (i.e., higher CPT1A expression) that, associated to the reduced expression of the “master regulator” of the antioxidant response NRF2, led to increased superoxide production (measured by dihydroethidium staining) (all p < 0.01). These series of events were associated to increased macrophage infiltration, type I collagen and MCP1 gene expression (p < 0.05) in Nlrp3−/− HFHC mice only. Conclusions: In the Western lifestyle diet, lack of NLRP3 inflammasome is associated with translocation of bacterial products that leads to severe metabolic alterations in both adipose tissue and the liver, and to NASH development. O065 MECHANISTIC STUDY OF TM6SF2 IN NAFLD PATHOGENESIS: STABLY TRANSFECTED Huh7 CELLS OVER-EXPRESSING THE TM6SF2 E167K VARIANT EXHIBIT GREATER LEVELS OF OXIDATIVE STRESS J. Palmer1 , Y.-L. Liu1 , C.P. Day1 , A. Daly1 , Q.M. Anstee1 . 1 The Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Non-Alcoholic Fatty Liver Disease (NAFLD) is a complex disease trait influenced by genetic modifiers. We, and others, have recently reported that minor (T) allele carriage of a non-synonymous polymorphism in TM6SF2 (rs58542926 C>T, p.E167K), a gene of unknown function, is associated not only with steatosis, but also with NASH and clinically relevant advanced liver fibrosis. Carriage of the C allele is associated with dyslipidaemia and cardiovascular disease. To better understand the mechanisms underlying these associations, we examined the effects of TM6SF2 over-expression in vitro. Methods: Huh7 human hepatoma cell-lines stably transfected to overexpress wild-type (E167) or variant (167K) forms of TM6SF2 were generated. Cells were cultured in standard medium or oleic acid enriched media. Hepatocyte lipid accumulation was assessed by oil red O staining, cellular oxidative stress (ROS) measured by FACS after DCFDA staining and the expression profile of relevant genes involved in lipid metabolism, fatty acid oxidation, inflammation and fibrogenesis were measured by rtPCR. Results: Following stable transfection, TM6SF2 expression was >50fold increased relative to resting Huh7 cells both at mRNA and protein levels. As previously noted, protein expression of the E167 form was approximately twice that of the 167K variant, likely reflecting protein misfolding or accelerated intracellular degradation. Whilst overexpression of either TM6SF2 form was associated with increased hepatic lipid accumulation in oleic acid enriched media, oil red O staining was substantially greater in cells expressing 167K. Consistent with development of NASH, carriage of the 167K variant was also associated with greater levels of ROS. At the transcriptome level, overexpression of TM6SF2 suppressed several lipogenic and pro-inflammatory genes. However, relative to E167, the 167K variant promoted differential expression of key genes including DGAT1, CYP2E1, IL6 and TNFA. Conclusions: These in vitro data suggest that TM6SF2 has profound effects on hepatocyte lipid metabolism. They provide the first evidence that the TM6SF2 p.E167K variant induces increased hepatocyte oxidative stress, possibly by favoring extramitochondrial omega- fatty-acid oxidation, and also promotes hepatocyte inflammatory cytokine expression. Work to further characterize the functional effects of TM6SF2 p.E167K carriage and the mechanisms through which it promotes liver injury and NASH are underway.
Journal of Hepatology 2015 vol. 62 | S213–S234
S223