- Email: [email protected]
P0940 : Chronic particulate matter exposition represents a second hit in the progression from steatosis to steatohepatitis
POSTERS Table (abstract P0938).
Weight (g) Glycaemia (mg/dl) HOMA-IR Plasmatic IL-6 (pg/ml) Plasmatic TNF-a (pg/ml) Plasmatic IFN-g (pg/ml) Liver weight (g) Total lipid content (%) MUFA hepatic content (g/100 g liver tissue) Hepatic trioleins (g/100 g liver tissue) NAS score Positive Sirius Red cells (%)
Control
HFD-SAF
HFD-EVOO
HFD-O
R
Statistics
42±1.3 107±3 1.7±0.3 8.9±1.8 26.3±3.7 6.1±1.8 1.3±0.2 10±1 38±2 3.2±0.5 0±0 0.2±0.03
52±1.6 192±8 31.7±7.4 22.7±2.6 32.4±4.8 35.4±7.6 2.1±0.6 29±4 53±2 6.7±0.2 4.6±0.3 2.6±0.5
47±1.4 158±6 14.2±4.2 20.9±4.3 29.1±4.0 11.5±4.6 2.3±0.7 21±2 66±2 12.5±0.2 3.6±0.5 1.2±0.6
44±2.6 155±4 11.2±1.2 18.7±2.8 29.5±1.7 4.8±1.9 1.8±0.4 16±3 61±1 12.8±1 2±0 0.4±0.02
43±1.8 115±4 5.1±1.2 11.4±2.9 27.0±3.2 5.6±1.2 1.2±0.15 18±5 ND ND 0±0 0.3±0.02
N = 30/group; *p < 0.05; NS N = 30/group; *p < 0.01; NS N = 30/group; *p < 0.01; NS N = 10/group; NS; NS N = 10/group; NS; NS N = 10/group; *p < 0.01; *p < 0.01 N = 5/group; NS; NS N = 4/group; *p < 0.05; *p < 0.01 N = 5/group; *p < 0.05; NS N = 5/group; *p < 0.01; NS N = 3/group; *p < 0.05; *p < 0.05 N = 3/group; *p < 0.05; *p < 0.01
First statistics are referred to HFD-SAF vs HFD-EVOO and HFD-O; second statistics corresponds to HFD-EVOO vs HFD-O.
Conclusions: Enriched olive oil with HFD decreased NAFLD-induced liver manifestations. Compounds with antioxidant activity partially explains the improvement of some parameters. Insulin resistance decrease and changes in liver lipids content profile, together with the modification of the expression of proteins related with hepatic steatosis and inflammation have a relevant impact in the improvement observed in NAFLD. P0939 FATTY LIVER VS. FIBROTIC LIVER: COMPARISON OF INFECTIONASSOCIATED PORTAL HYPERTENSION J. Schewe1 , M.-C. Makeschin2 , I. Liss1 , A.L. Gerbes1 , C.J. Steib1 . 1 Department of Medicine II, 2 Department of Pathology, LMU Munich, Klinikum Grosshadern, Munich, Germany E-mail: [email protected] Background and Aims: Portal hypertension and alterations of liver structure are of major relevance for chronic liver diseases. We have identified TXA2 as important vasoconstrictor upon infection following TLR 2/6 activation in the liver (e.g. Steib CJ et al., Hepatology 2010). The aim of this study was therefore to investigate, whether the cause of liver disease influences the extent of TXA2 induced portal hypertension. Methods: Bile duct ligation (BDL) in male Sprague-Dawley rats caused fibrosis after 4 weeks (BDL4) and fatty liver was induced by high fat diet. Isolated liver perfusion was performed for 80min. In all three liver models three groups (each n = 5) were investigated: A: control perfusion; B: + Zymosan A (150 mg/ml, 40.–46.min.); C: + U46619 (TXA2 analogue, 0.1mM/ml, 40.–46.min.). Portal perfusion pressure (p) and Thromboxane B2 (TXB2 , stable degradation product) in the perfusate (+ Zym) were measured (mean ± SEM; *p < 0.05). Immunhistochemical evaluation of the livers using CD163 (to determine Kupffer cell density) and a-SMA (to estimate number of activated hepatic stellate cells) is in process. Results: HE staining showed, in comparison to healthy livers, a mild inflammation and moderate to severe fat deposition in fatty livers and a moderate to severe fibrosis with moderate inflammation in BDL4. Portal perfusion pressure and TXB2 production after Zymosan A (group B) and portal perfusion pressure after TXA2 analogue U46619 (group C) are shown in Table 1. Interestingly, pmax was significantly lower after Zymosan A in fatty livers compared to healthy livers and following U46619 pmax was lower in fatty livers compared to fibrotic livers. Conclusions: Fatty livers were less sensitive to TLR 2/6 activation and TXA2 analogue administration than healthy and fibrotic livers. The correlation between portal perfusion pressure and TXB2 decreases from healthy to fatty on fibrotic liver, which might be depend on the increasing damage and alteration of liver architecture because of the liver disease. In summary, this leads to the assumption that fatty livers have a protective effect on infection-associated portal hypertension. Hereby, it offers future
clinical situations the possibility to stratify the risk for patients with fatty livers and prompts further research. This study is supported by DFG STE 1022/2-3 and DFG STE 1022/4-1. Table 1. Portal perfusion pressure (p) and TXB2 production after Zymosan (group B) and portal perfusion pressure after TXA2 analogue U46619 (group C) Group B
Healthy Fatty liver Fibrosis
Group C
pmin vs. pmax (cmH2 O)
TXB2 -min vs. TXB2 -max (pg/mL × g liver)
Correlation coefficient between p and TXB2
pmin vs. pmax (cmH2 O)
4.4±0.4 vs. 17.6±0.9* 4.1±0.2 vs. 11.4±0.6* 8.6±0.3 vs. 15.4±0.6*
41.8±5.8 vs. 1003.1±172* 29.4±2 vs. 532.7±16.2* 87.8±12.7 vs. 1049.2±286.7*
1±0
3.4±0.1 vs. 20.6±0.9* 3.3±0.1 vs. 24±0.9* 8.3±0.2 vs. 30.5±1.9*
0.7±0 0.5±0.1
Mean±SEM; *p < 0.05.
P0940 CHRONIC PARTICULATE MATTER EXPOSITION REPRESENTS A SECOND HIT IN THE PROGRESSION FROM STEATOSIS TO STEATOHEPATITIS M. Tarocchi1 , G. Marroncini1 , S. Polvani1 , S. Tempesti1 , E. Ceni1 , T. Mello1 , M. Peluso2 , A. Galli1 . 1 Department of Experimental and Clinical Biomedical Sciences, University of Florence, 2 Cancer Risk Factor Branch, Cancer Prevention and Research Institute, Florence, Italy E-mail: mirko.tarocchi@unifi.it Background and Aims: Nonalcoholic fatty liver disease (NAFLD), a hepatic manifestation of metabolic syndrome, is the most common chronic liver disease, and the prevalence is rapidly increasing in developed countries. Nonalcoholic steatohepatitis (NASH), the severe form of NAFLD, can also progress to liver cirrhosis and hepatocellular carcinoma. Recent evidences suggest that environmental factors can trigger hepatic inflammation and progression of steatosis to NASH. We evaluate if a western style diet in association with chronic urban particulate matter exposition can modifies the pathogenesis and progression of NASH. Methods: The experimental model was created to reproduce urban lifestyle: C57Bl/6 mice were fed with normal chow (ND) or a western style diet (HFD) rich in fat, cholesterol and sugar, and treated with or without particular matter (PM) collected from the urban area of Florence (Italy). After 4 and 8 weeks were performed the morphologic analysis of liver tissues, the evaluation of inflammatory cell infiltrate and the collagen deposition; we evaluate also the effects of PM on cytokine production and oxidative stress related cellular damage.
Journal of Hepatology 2015 vol. 62 | S263–S864
S697
POSTERS Results: Already after 4 week of treatment were present differences among the HFD group and the HFD-PM group; as expected the HFD groups developed fat accumulation in the liver but, interestingly at 8 weeks, the NASH score was significantly increased in the second group (p < 0.01) based on the histological analysis of the liver, the tissue fat content, the inflammatory cell infiltrate and the collagen deposition. The cytokine profiles indicate an increased production of pro-inflammatory molecules in presence of PM. The levels of DNA adducts were significantly increased in the HFD groups compared to standard chow. Many adducts were identified in liver tissues: one of them was benzo(a)pyrene, that is a well known polycyclic aromatic hydrocarbon with strong mutagenic properties. Interestingly the effects of HFD and PM were synergic in the sense that the levels of DNA adducts were about thirty fold greater in HFD mice in respect to controls treated with the same amounts of air particulate matter extracts mainly due to benzo(a)pyrene increment. Conclusions: Our data suggest that in subject with steatosis due to a western style of life, the association with a chronic exposition to urban particulate matter plays an important role in the pathogenesis of NASH and its evolution to cirrhosis and also to cancer. P0941 CD44 IS AN IMPORTANT ACTOR IN NON ALCOHOLIC STEATOHEPATITIS D. Rousseau1,2 , R. Anty1,2,3 , S. Bonnafous1,2,3 , S. Patouraux1,2,3 , A.-S. Schneck1,2,3 , A. Iannelli1,2,3 , M.-C. Saint-Paul1,2,3 , J. Gugenheim1,2,3 , B. Bailly-Maitre1,2 , A. Tran1,2,3 , P. Gual1,2 . 1 INSERM U1065, Team 8 hepatic complications in obesity, INSERM, 2 University of Nice-Sophia-Antipolis, 3 Centre Hospitalier Universitaire of Nice, Nice, France E-mail: [email protected] Background and Aims: CD44 is expressed on many cell types that contribute to inflammation. CD44 plays an important role in the recruitment of macrophages into adipose tissue with obesity and of leukocyte into liver in response to lithogenic diet. However, its role in hepatic inflammation in obese patients and in mouse models of steatohepatitis has not yet been investigated. Methods: CD44 rs187116 and rs13347 genotypes were determined by allelic discrimination using TaqMan reagents in a cohort of 274 morbidly obese patients with liver biopsy-proven NAFLD. Gene expression of CD44, IL1b, TNFa was evaluated in 30 obese patients (8 without NAFLD, 13 with hepatic steatosis and 9 with NASH). The role of CD44 was evaluated in cd44−/− mice with steatohepatitis induced by methionine- and choline-deficient diet (MCD) (Wt Ctr Diet=8, MCD=14; cd44−/− Ctr Diet=12, MCD=19) by histological, biological, gene expression and flow cytometry analysis. Results: Carriage of CD44 rs187116 minor A allele was associated with presence of hepatic inflammation independently of age, gender, body mass index and the presence of type 2 diabetes in morbidly obese patients (p = 0.0312). Further, CD44 hepatic gene expression was strongly upregulated in NASH patients and correlated with NASH grading (rs =0.668, p < 0.001), NAS (rs =0.562, p = 0.001), ballooning (rs =0.531, p = 0.003) and ALT (rs =0.496, p = 0.005). Experiments in mouse model of steatohepatitis (MCD) further showed the hepatic upregulation of CD44 at the mRNA and protein level. AST and ALT levels were strongly decreased in cd44−/− mice compared with wild-type mice after MCD challenge (−28% and −39%, respectively). Histological analysis of the liver of cd44−/− mice revealed a significantly lower incidence of hepatic steatosis (10±4% vs 48±8%, p < 0.001) and inflammation (2±1 vs 7±2 inflammatory foci/10 fields, p < 0.001). In addition, the hepatic expression level of markers of inflammation (MCP1, TNFa and IL1b) and macrophages (F4/80) was strongly decreased in cd44−/− mice versus wild-type mice after MCD. Flow cytometry analysis also revealed a prevention of F4/80 macrophage infiltration into the liver of cd44−/− mice in response to MCD. S698
Conclusions: Human and experimental data suggest an important role for CD44 in the pathogenesis of NAFLD (steatosis and NASH). P0942 IMMUNE REACTIONS IN STEATOHEPATITIS INITIATION AND PROGRESSION TO FIBROSIS: CRITICAL ROLE OF TH17 AND TH22 LYMPHOCYTES E. Alchera1 , S. Rolla2 , C. Imarisio1 , V. Bardi2 , G. Valente3 , A. Follenzi1 , B. Revanna Chandrashekar1 , F. Novelli2 , R. Carini1 . 1 Health Sciences, University, University of Piemonte Orientale, Novara, 2 Biotechnology and Healthy Sciences, University of Turin, Turin, Italy, Torino, 3 Translational medicine, University of Piemonte Orientale, Novara, Italy E-mail: [email protected] Background and Aims: The mechanisms responsible for steatosis progression to NASH and for NASH evolution to fibrosis are incompletely understood. Recent evidences, indicate that hepatic immune response may also play critical role in NASH. In particular, IL-17 and IL-22 produced by CD4+ T cells can modulate the inflammatory reactions and survival processes. This study investigated Th17 (CD4+ IL-17+ ) and Th22 (CD4+ IL-22+ ) lymphocytes infiltration in mice liver during NASH development and explored the role of IL-17/ IL-22 production in this process. Methods: C57BL/6 WT and IL-17KO mice were fed either a normal diet (ND) or a methionine/choline deficient diet (MCD) diet. Liver T lymphocytes were analyzed by flow cytometry. Primary mouse hepatocyte were exposed to palmitic acid (PA) Results: MCD diet induced progressive hepatic damage as evidenced by ALT increase, progressive micro and macro steatosis, necrosis, lobular inflammation and finally fibrosis. In the liver, Th17 cells significantly increased already at 1 week of MCD diet compared to ND mice, decreased at 2 and 4 weeks, and further increased at 8 weeks. By contrast, Th22 displayed an opposite kinetic. We investigated the role of IL-17 and IL-22 in steatotic liver damage and protection, by employing primary mice hepatocyte exposed to PA. PA alone induced hepatocytes damage and increased JNK phosphorylation, a known marker of hepatocyte lipotoxicity. IL17 treatment further increased these parameters, whereas IL-22 protected them and activated a central mediator of cellular survival processes: Akt. Consistently inhibition of Akt, reversed IL-22-induced prevention of JNK phosphorylation and abolished cytoprotection. To clarify the importance of IL-17 in initiate and sustain liver damage, NASH was induced in IL-17 KO mice. Lower ALT levels and lower NASH and fibrosis markers and an higher percentage of Th22 cells in the liver were observed in IL-17KO mice compared to WT mice. The reduced liver injury and fibrosis in IL-17 KO mice was related to an increased Akt activation and decreased JNK phosphorylation, according to in vitro results. Conclusions: a biphasic increase of Th17 over Th22 is associated to NASH initiation and evolution to fibrosis in mice. Such effects are mediated by a prevalence of the lipotoxic JNK-dependent activity of IL-17 on the Akt-related cytoprotective action of IL-22.
Journal of Hepatology 2015 vol. 62 | S263–S864
Weight (g) Glycaemia (mg/dl) HOMA-IR Plasmatic IL-6 (pg/ml) Plasmatic TNF-a (pg/ml) Plasmatic IFN-g (pg/ml) Liver weight (g) Total lipid content (%) MUFA hepatic content (g/100 g liver tissue) Hepatic trioleins (g/100 g liver tissue) NAS score Positive Sirius Red cells (%)
Control
HFD-SAF
HFD-EVOO
HFD-O
R
Statistics
42±1.3 107±3 1.7±0.3 8.9±1.8 26.3±3.7 6.1±1.8 1.3±0.2 10±1 38±2 3.2±0.5 0±0 0.2±0.03
52±1.6 192±8 31.7±7.4 22.7±2.6 32.4±4.8 35.4±7.6 2.1±0.6 29±4 53±2 6.7±0.2 4.6±0.3 2.6±0.5
47±1.4 158±6 14.2±4.2 20.9±4.3 29.1±4.0 11.5±4.6 2.3±0.7 21±2 66±2 12.5±0.2 3.6±0.5 1.2±0.6
44±2.6 155±4 11.2±1.2 18.7±2.8 29.5±1.7 4.8±1.9 1.8±0.4 16±3 61±1 12.8±1 2±0 0.4±0.02
43±1.8 115±4 5.1±1.2 11.4±2.9 27.0±3.2 5.6±1.2 1.2±0.15 18±5 ND ND 0±0 0.3±0.02
N = 30/group; *p < 0.05; NS N = 30/group; *p < 0.01; NS N = 30/group; *p < 0.01; NS N = 10/group; NS; NS N = 10/group; NS; NS N = 10/group; *p < 0.01; *p < 0.01 N = 5/group; NS; NS N = 4/group; *p < 0.05; *p < 0.01 N = 5/group; *p < 0.05; NS N = 5/group; *p < 0.01; NS N = 3/group; *p < 0.05; *p < 0.05 N = 3/group; *p < 0.05; *p < 0.01
First statistics are referred to HFD-SAF vs HFD-EVOO and HFD-O; second statistics corresponds to HFD-EVOO vs HFD-O.
Conclusions: Enriched olive oil with HFD decreased NAFLD-induced liver manifestations. Compounds with antioxidant activity partially explains the improvement of some parameters. Insulin resistance decrease and changes in liver lipids content profile, together with the modification of the expression of proteins related with hepatic steatosis and inflammation have a relevant impact in the improvement observed in NAFLD. P0939 FATTY LIVER VS. FIBROTIC LIVER: COMPARISON OF INFECTIONASSOCIATED PORTAL HYPERTENSION J. Schewe1 , M.-C. Makeschin2 , I. Liss1 , A.L. Gerbes1 , C.J. Steib1 . 1 Department of Medicine II, 2 Department of Pathology, LMU Munich, Klinikum Grosshadern, Munich, Germany E-mail: [email protected] Background and Aims: Portal hypertension and alterations of liver structure are of major relevance for chronic liver diseases. We have identified TXA2 as important vasoconstrictor upon infection following TLR 2/6 activation in the liver (e.g. Steib CJ et al., Hepatology 2010). The aim of this study was therefore to investigate, whether the cause of liver disease influences the extent of TXA2 induced portal hypertension. Methods: Bile duct ligation (BDL) in male Sprague-Dawley rats caused fibrosis after 4 weeks (BDL4) and fatty liver was induced by high fat diet. Isolated liver perfusion was performed for 80min. In all three liver models three groups (each n = 5) were investigated: A: control perfusion; B: + Zymosan A (150 mg/ml, 40.–46.min.); C: + U46619 (TXA2 analogue, 0.1mM/ml, 40.–46.min.). Portal perfusion pressure (p) and Thromboxane B2 (TXB2 , stable degradation product) in the perfusate (+ Zym) were measured (mean ± SEM; *p < 0.05). Immunhistochemical evaluation of the livers using CD163 (to determine Kupffer cell density) and a-SMA (to estimate number of activated hepatic stellate cells) is in process. Results: HE staining showed, in comparison to healthy livers, a mild inflammation and moderate to severe fat deposition in fatty livers and a moderate to severe fibrosis with moderate inflammation in BDL4. Portal perfusion pressure and TXB2 production after Zymosan A (group B) and portal perfusion pressure after TXA2 analogue U46619 (group C) are shown in Table 1. Interestingly, pmax was significantly lower after Zymosan A in fatty livers compared to healthy livers and following U46619 pmax was lower in fatty livers compared to fibrotic livers. Conclusions: Fatty livers were less sensitive to TLR 2/6 activation and TXA2 analogue administration than healthy and fibrotic livers. The correlation between portal perfusion pressure and TXB2 decreases from healthy to fatty on fibrotic liver, which might be depend on the increasing damage and alteration of liver architecture because of the liver disease. In summary, this leads to the assumption that fatty livers have a protective effect on infection-associated portal hypertension. Hereby, it offers future
clinical situations the possibility to stratify the risk for patients with fatty livers and prompts further research. This study is supported by DFG STE 1022/2-3 and DFG STE 1022/4-1. Table 1. Portal perfusion pressure (p) and TXB2 production after Zymosan (group B) and portal perfusion pressure after TXA2 analogue U46619 (group C) Group B
Healthy Fatty liver Fibrosis
Group C
pmin vs. pmax (cmH2 O)
TXB2 -min vs. TXB2 -max (pg/mL × g liver)
Correlation coefficient between p and TXB2
pmin vs. pmax (cmH2 O)
4.4±0.4 vs. 17.6±0.9* 4.1±0.2 vs. 11.4±0.6* 8.6±0.3 vs. 15.4±0.6*
41.8±5.8 vs. 1003.1±172* 29.4±2 vs. 532.7±16.2* 87.8±12.7 vs. 1049.2±286.7*
1±0
3.4±0.1 vs. 20.6±0.9* 3.3±0.1 vs. 24±0.9* 8.3±0.2 vs. 30.5±1.9*
0.7±0 0.5±0.1
Mean±SEM; *p < 0.05.
P0940 CHRONIC PARTICULATE MATTER EXPOSITION REPRESENTS A SECOND HIT IN THE PROGRESSION FROM STEATOSIS TO STEATOHEPATITIS M. Tarocchi1 , G. Marroncini1 , S. Polvani1 , S. Tempesti1 , E. Ceni1 , T. Mello1 , M. Peluso2 , A. Galli1 . 1 Department of Experimental and Clinical Biomedical Sciences, University of Florence, 2 Cancer Risk Factor Branch, Cancer Prevention and Research Institute, Florence, Italy E-mail: mirko.tarocchi@unifi.it Background and Aims: Nonalcoholic fatty liver disease (NAFLD), a hepatic manifestation of metabolic syndrome, is the most common chronic liver disease, and the prevalence is rapidly increasing in developed countries. Nonalcoholic steatohepatitis (NASH), the severe form of NAFLD, can also progress to liver cirrhosis and hepatocellular carcinoma. Recent evidences suggest that environmental factors can trigger hepatic inflammation and progression of steatosis to NASH. We evaluate if a western style diet in association with chronic urban particulate matter exposition can modifies the pathogenesis and progression of NASH. Methods: The experimental model was created to reproduce urban lifestyle: C57Bl/6 mice were fed with normal chow (ND) or a western style diet (HFD) rich in fat, cholesterol and sugar, and treated with or without particular matter (PM) collected from the urban area of Florence (Italy). After 4 and 8 weeks were performed the morphologic analysis of liver tissues, the evaluation of inflammatory cell infiltrate and the collagen deposition; we evaluate also the effects of PM on cytokine production and oxidative stress related cellular damage.
Journal of Hepatology 2015 vol. 62 | S263–S864
S697
POSTERS Results: Already after 4 week of treatment were present differences among the HFD group and the HFD-PM group; as expected the HFD groups developed fat accumulation in the liver but, interestingly at 8 weeks, the NASH score was significantly increased in the second group (p < 0.01) based on the histological analysis of the liver, the tissue fat content, the inflammatory cell infiltrate and the collagen deposition. The cytokine profiles indicate an increased production of pro-inflammatory molecules in presence of PM. The levels of DNA adducts were significantly increased in the HFD groups compared to standard chow. Many adducts were identified in liver tissues: one of them was benzo(a)pyrene, that is a well known polycyclic aromatic hydrocarbon with strong mutagenic properties. Interestingly the effects of HFD and PM were synergic in the sense that the levels of DNA adducts were about thirty fold greater in HFD mice in respect to controls treated with the same amounts of air particulate matter extracts mainly due to benzo(a)pyrene increment. Conclusions: Our data suggest that in subject with steatosis due to a western style of life, the association with a chronic exposition to urban particulate matter plays an important role in the pathogenesis of NASH and its evolution to cirrhosis and also to cancer. P0941 CD44 IS AN IMPORTANT ACTOR IN NON ALCOHOLIC STEATOHEPATITIS D. Rousseau1,2 , R. Anty1,2,3 , S. Bonnafous1,2,3 , S. Patouraux1,2,3 , A.-S. Schneck1,2,3 , A. Iannelli1,2,3 , M.-C. Saint-Paul1,2,3 , J. Gugenheim1,2,3 , B. Bailly-Maitre1,2 , A. Tran1,2,3 , P. Gual1,2 . 1 INSERM U1065, Team 8 hepatic complications in obesity, INSERM, 2 University of Nice-Sophia-Antipolis, 3 Centre Hospitalier Universitaire of Nice, Nice, France E-mail: [email protected] Background and Aims: CD44 is expressed on many cell types that contribute to inflammation. CD44 plays an important role in the recruitment of macrophages into adipose tissue with obesity and of leukocyte into liver in response to lithogenic diet. However, its role in hepatic inflammation in obese patients and in mouse models of steatohepatitis has not yet been investigated. Methods: CD44 rs187116 and rs13347 genotypes were determined by allelic discrimination using TaqMan reagents in a cohort of 274 morbidly obese patients with liver biopsy-proven NAFLD. Gene expression of CD44, IL1b, TNFa was evaluated in 30 obese patients (8 without NAFLD, 13 with hepatic steatosis and 9 with NASH). The role of CD44 was evaluated in cd44−/− mice with steatohepatitis induced by methionine- and choline-deficient diet (MCD) (Wt Ctr Diet=8, MCD=14; cd44−/− Ctr Diet=12, MCD=19) by histological, biological, gene expression and flow cytometry analysis. Results: Carriage of CD44 rs187116 minor A allele was associated with presence of hepatic inflammation independently of age, gender, body mass index and the presence of type 2 diabetes in morbidly obese patients (p = 0.0312). Further, CD44 hepatic gene expression was strongly upregulated in NASH patients and correlated with NASH grading (rs =0.668, p < 0.001), NAS (rs =0.562, p = 0.001), ballooning (rs =0.531, p = 0.003) and ALT (rs =0.496, p = 0.005). Experiments in mouse model of steatohepatitis (MCD) further showed the hepatic upregulation of CD44 at the mRNA and protein level. AST and ALT levels were strongly decreased in cd44−/− mice compared with wild-type mice after MCD challenge (−28% and −39%, respectively). Histological analysis of the liver of cd44−/− mice revealed a significantly lower incidence of hepatic steatosis (10±4% vs 48±8%, p < 0.001) and inflammation (2±1 vs 7±2 inflammatory foci/10 fields, p < 0.001). In addition, the hepatic expression level of markers of inflammation (MCP1, TNFa and IL1b) and macrophages (F4/80) was strongly decreased in cd44−/− mice versus wild-type mice after MCD. Flow cytometry analysis also revealed a prevention of F4/80 macrophage infiltration into the liver of cd44−/− mice in response to MCD. S698
Conclusions: Human and experimental data suggest an important role for CD44 in the pathogenesis of NAFLD (steatosis and NASH). P0942 IMMUNE REACTIONS IN STEATOHEPATITIS INITIATION AND PROGRESSION TO FIBROSIS: CRITICAL ROLE OF TH17 AND TH22 LYMPHOCYTES E. Alchera1 , S. Rolla2 , C. Imarisio1 , V. Bardi2 , G. Valente3 , A. Follenzi1 , B. Revanna Chandrashekar1 , F. Novelli2 , R. Carini1 . 1 Health Sciences, University, University of Piemonte Orientale, Novara, 2 Biotechnology and Healthy Sciences, University of Turin, Turin, Italy, Torino, 3 Translational medicine, University of Piemonte Orientale, Novara, Italy E-mail: [email protected] Background and Aims: The mechanisms responsible for steatosis progression to NASH and for NASH evolution to fibrosis are incompletely understood. Recent evidences, indicate that hepatic immune response may also play critical role in NASH. In particular, IL-17 and IL-22 produced by CD4+ T cells can modulate the inflammatory reactions and survival processes. This study investigated Th17 (CD4+ IL-17+ ) and Th22 (CD4+ IL-22+ ) lymphocytes infiltration in mice liver during NASH development and explored the role of IL-17/ IL-22 production in this process. Methods: C57BL/6 WT and IL-17KO mice were fed either a normal diet (ND) or a methionine/choline deficient diet (MCD) diet. Liver T lymphocytes were analyzed by flow cytometry. Primary mouse hepatocyte were exposed to palmitic acid (PA) Results: MCD diet induced progressive hepatic damage as evidenced by ALT increase, progressive micro and macro steatosis, necrosis, lobular inflammation and finally fibrosis. In the liver, Th17 cells significantly increased already at 1 week of MCD diet compared to ND mice, decreased at 2 and 4 weeks, and further increased at 8 weeks. By contrast, Th22 displayed an opposite kinetic. We investigated the role of IL-17 and IL-22 in steatotic liver damage and protection, by employing primary mice hepatocyte exposed to PA. PA alone induced hepatocytes damage and increased JNK phosphorylation, a known marker of hepatocyte lipotoxicity. IL17 treatment further increased these parameters, whereas IL-22 protected them and activated a central mediator of cellular survival processes: Akt. Consistently inhibition of Akt, reversed IL-22-induced prevention of JNK phosphorylation and abolished cytoprotection. To clarify the importance of IL-17 in initiate and sustain liver damage, NASH was induced in IL-17 KO mice. Lower ALT levels and lower NASH and fibrosis markers and an higher percentage of Th22 cells in the liver were observed in IL-17KO mice compared to WT mice. The reduced liver injury and fibrosis in IL-17 KO mice was related to an increased Akt activation and decreased JNK phosphorylation, according to in vitro results. Conclusions: a biphasic increase of Th17 over Th22 is associated to NASH initiation and evolution to fibrosis in mice. Such effects are mediated by a prevalence of the lipotoxic JNK-dependent activity of IL-17 on the Akt-related cytoprotective action of IL-22.
Journal of Hepatology 2015 vol. 62 | S263–S864