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OC-8 IL28B and PNPLA3 polymorphisms affect histological liver damage in patients with non-alcoholic fatty liver disease
Abstracts of the A.I.S.F. Annual Meeting 2012 / Digestive and Liver Disease 44S (2012), S1–S48 hepatogenic properties and following systemic administration are able to accelerate the resolution of an acute liver injury without any differentiation and manipulation. These features make UCMSCs strong candidates for future application in regenerative medicine for human acute liver disease.
OC-6 Monocyte/macrophage peribiliary recruitment by Pkhd1-defective cholangiocytes induces expression of αvβ6 integrin on biliary cysts via TGFβ1 and TNFα in congenital hepatic fibrosis L. Locatelli 1 , C. Spirli 2 , M. Cadamuro 3 , S. Lecchi 1 , C.M. Morell 1 , Y. Popov 4 , D. Schuppan 4,5 , M. Strazzabosco 1,2 , L. Fabris 3 1 Department
of Clinical Medicine and Prevention, University of Milan-Bicocca, Milan; 2 Department of Medicine and Liver Center, Yale University, New Haven, CT, USA; 3 Department of Surgical and Gastroenterological Sciences, University of Padova, Padova; 4 Division of Gastroenterology Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 5 Division of Molecular and Translational Medicine, Johannes Gutenberg University, Mainz, Germany Background and Aim: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein expressed in cholangiocyte cilia. CHF is characterized by progressive portal fibrosis and portal hypertension. αvβ6 integrin activates latent TGFβ1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of αvβ6 integrin and its regulation in fibrocystin-defective mice. Methods: In Pkhd1KO mice we studied, at different ages (1-12 months): a)portal fibrosis (Sirius Red); b)αvβ6 mRNA and protein expression (RTPCR, IHC); c)α-SMA, TGFβ1, TGFβ2 mRNA expression (RT-PCR); d)portal inflammatory cells (IHC for CD45[leukocytes] and NIMP-R14[neutrophils], FACS analysis); e)cytokines secretion of primary cholangiocytes (Luminex assay); f)TGFβ1, TGFβ2 and TNFα effects on β6 mRNA expression by cultured cholangiocytes before/after inhibition of the TGFβ receptor type II (TGFβRII). Results: Pkhd1KO mice showed an increase in αvβ6 integrin expression on biliary cyst epithelia. Expression of αvβ6 correlated with portal fibrosis (r=0.94, p<0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r=0.97, p<0.01). Gene expression of TGFβ1/2 showed an age-dependent increase. FACS analysis showed that 81% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Pkhd1KO cholangiocytes secreted IP-10 (48×), LIF (20×) and IL13 (17×), potent monocyte chemoattractants. β6 mRNA by cultured Pkhd1KO cholangiocytes was potently stimulated by TNFα (2.3× vs WT) and TGFβ1 (2×). Inhibition of TGFβRII abolished TGFβ1 but not TNFα effects on β6 mRNA. Microbiological analysis showed that Pkhd1KO livers were sterile. Gut decontamination had no effect on liver fibrosis. Conclusions: Pkhd1KO cholangiocytes secrete cytokines able to recruit mononuclear cells and macrophages into the peribiliary microenvironment. TGFβ1 and TNFα released by macrophages up-regulate αvβ6 integrin that increases the fibrogenic stimulus by activating latent TGFβ1. Notably, TNFα up-regulation of αvβ6 is independent from TGFβ and may be critical in the early phases of the disease.
OC-7 NK profiles of non-responder and relapser patients with cHCV infection treated with SOC B. Oliviero 1 , D. Mele 1 , E. Degasperi 2 , S. Varchetta 1 , A. Aghemo 2 , M.G. Rumi 2 , C. Tinelli 3 , M. Colombo 2 , M.U. Mondelli 1 1 Department
of Infectious Diseases, and; 3 Statistics and Clinical Epidemiology Service, Fondazione IRCCS Policlinico San Matteo and University of Pavia; 2 Division of Gastroenterology, Fondazione IRCCS Ospedale Maggiore Mangiagalli Regina Elena and University of Milan, Italy Background and Aim: Innate immunity is thought to contribute to achieve a sustained virological response (SVR) in patients with cHCV infection treated
S3
with Peg-IFNα/ribavirin (RBV), but a clear immunological discrimination between SVR, non-responder (NR) or relapser (Rel) patients is presently not possible. We analysed changes in NK cell phenotype and function during antiviral therapy to identify immunological biomarker(s) of treatment outcome. Methods: We studied NK cell phenotype and function of 37 patients (25 SVR, 7 NR and 5 Rel, genotypes 1, 2, 4) treated with Peg-IFN/RBV (SOC). Molecules involved in NK cell regulation (CD69, NKp30, NKp46, NKG2D, CD16, NKG2A, KIR3DL1/S1, TRAIL, CD107a, Perforin, Tim-3, Siglec-7) were analysed by flow cytometry at baseline and various time points during treatment and after discontinuation. NK cell function was evaluated as cytokine (IFNγ, TNFα) production by intracellular staining and as cytotoxic activity by CD107a degranulation assay. Results: SOC therapy induced an early increase in expression of CD69, NKp30, NKp46 and decrease of Siglec-7, Tim-3, CD16 and Perforin (PFN). By comparing trends of NK phenotype and function of NR and Rel patients with those of SVR using a linear regression models for repeated measures, a typical NK cell profile emerged in NR patients, featuring a lower PFN content at baseline (p=0.02) and during therapy (p=0.014) and a simultaneously higher expression of CD16 (p=0.017), which were inversely correlated at week 12 (p=0.01, r=0.98). Moreover, there was a trend towards reduced IFNγ production and degranulation. Rel patients showed a distinct profile characterized by higher levels of NKp30 expression before (p=0.01) and during the treatment (p=0.003). Moreover, the percentage of NKp46+ and Tim-3+ NK cells remained consistently higher during therapy (p=0.037 and p=0.03, respectively). Conclusions: HCV-infected patients treated with SOC showed different NK cell phenotypic and functional features which allow distinct profiling of non-response or relapse versus SVR.
OC-8 IL28B and PNPLA3 polymorphisms affect histological liver damage in patients with non-alcoholic fatty liver disease S. Petta 1 , S. Grimaudo 1 , C. Cammà 1 , D. Cabibi 2 , V. Di Marco 1 , G. Licata 1 , R.M. Pipitone 1 , A. Craxì 1 1 Sezione 2
di Gastroenterologia, DiBiMIS, University of Palermo, Italy; Cattedra di Anatomia Patologica, University of Palermo, Italy
Background and Aims: Genetic background may affect the severity of liver damage in patients with non-alcoholic fatty liver disease (NAFLD). We aimed to assess whether IL28B rs12979860 and rs8099917 polymorphisms, together with PNPLA3 rs738409 C>G polymorphism, are associated with histological liver damage in NAFLD patients. Methods: One hundred sixty consecutive NAFLD patients, were assessed by liver biopsy (Kleiner score); anthropometric, and biochemical and metabolic features were included. IL28B rs12979860 C>T, IL28B rs8099917 G>C, and PNPLA3 rs738409 C>G single nucleotide polymorphisms were tested. Results: Fourteen (8.8%) patients had IL28B rs 12979860 TT polymorphism, compared with 72 (45%) and 74 (46.2%) with TC and CC variants, respectively. PNPLA3 rs738409 CC polymorphism was present in 47 (29.4%) patients, compared with 79 (49.4%) and 34 (21.3%) with CC and GG variants, respectively. Multivariate logistic regression analysis showed that older age (OR 1.042,95%CI 1.011–1.075, p=0.008), hyperuricemia (OR 4.904,95%CI 1.769–13.599, p=0.002), IL28B rs 12979860 CC (OR 4.893, 95%CI 2.223– 10.770, p<0.001), and steatosis grade (OR 1.645,95%CI 1.005–2.692, p=0.04) were independently linked to moderate-severe lobular inflammation. By contrast, NAS score ≥5 was independently linked to higher HOMA index (OR 1.179,95%CI 1.023–1.399, p=0.02), hyperuricemia (OR 6.391,95%CI 2.030– 20.122, p=0.002) and PNPLA3 rs738409 GG (OR 3.064,95%CI 1.107–8.480, p=0.03). Patients with both IL28B rs 12979860 CC and PNPLA3 rs738409 GG had a prevalence of moderate-severe lobular inflammation and NAS ≥5 of 84.6% (11/13) and 84.6% (11/13), respectively, significantly higher compared with that of patients without both unfavorable polymorphisms (16/65♦ 24.6%, p<0.001; 33/65 ♦ 50.7%, p=0.02; respectively). Conclusions: In NAFLD patients, IL28B rs 12979860 CC genotype, together with PNPLA3 rs738409 GG, is associated with the severity of liver damage.
OC-6 Monocyte/macrophage peribiliary recruitment by Pkhd1-defective cholangiocytes induces expression of αvβ6 integrin on biliary cysts via TGFβ1 and TNFα in congenital hepatic fibrosis L. Locatelli 1 , C. Spirli 2 , M. Cadamuro 3 , S. Lecchi 1 , C.M. Morell 1 , Y. Popov 4 , D. Schuppan 4,5 , M. Strazzabosco 1,2 , L. Fabris 3 1 Department
of Clinical Medicine and Prevention, University of Milan-Bicocca, Milan; 2 Department of Medicine and Liver Center, Yale University, New Haven, CT, USA; 3 Department of Surgical and Gastroenterological Sciences, University of Padova, Padova; 4 Division of Gastroenterology Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 5 Division of Molecular and Translational Medicine, Johannes Gutenberg University, Mainz, Germany Background and Aim: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein expressed in cholangiocyte cilia. CHF is characterized by progressive portal fibrosis and portal hypertension. αvβ6 integrin activates latent TGFβ1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of αvβ6 integrin and its regulation in fibrocystin-defective mice. Methods: In Pkhd1KO mice we studied, at different ages (1-12 months): a)portal fibrosis (Sirius Red); b)αvβ6 mRNA and protein expression (RTPCR, IHC); c)α-SMA, TGFβ1, TGFβ2 mRNA expression (RT-PCR); d)portal inflammatory cells (IHC for CD45[leukocytes] and NIMP-R14[neutrophils], FACS analysis); e)cytokines secretion of primary cholangiocytes (Luminex assay); f)TGFβ1, TGFβ2 and TNFα effects on β6 mRNA expression by cultured cholangiocytes before/after inhibition of the TGFβ receptor type II (TGFβRII). Results: Pkhd1KO mice showed an increase in αvβ6 integrin expression on biliary cyst epithelia. Expression of αvβ6 correlated with portal fibrosis (r=0.94, p<0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r=0.97, p<0.01). Gene expression of TGFβ1/2 showed an age-dependent increase. FACS analysis showed that 81% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Pkhd1KO cholangiocytes secreted IP-10 (48×), LIF (20×) and IL13 (17×), potent monocyte chemoattractants. β6 mRNA by cultured Pkhd1KO cholangiocytes was potently stimulated by TNFα (2.3× vs WT) and TGFβ1 (2×). Inhibition of TGFβRII abolished TGFβ1 but not TNFα effects on β6 mRNA. Microbiological analysis showed that Pkhd1KO livers were sterile. Gut decontamination had no effect on liver fibrosis. Conclusions: Pkhd1KO cholangiocytes secrete cytokines able to recruit mononuclear cells and macrophages into the peribiliary microenvironment. TGFβ1 and TNFα released by macrophages up-regulate αvβ6 integrin that increases the fibrogenic stimulus by activating latent TGFβ1. Notably, TNFα up-regulation of αvβ6 is independent from TGFβ and may be critical in the early phases of the disease.
OC-7 NK profiles of non-responder and relapser patients with cHCV infection treated with SOC B. Oliviero 1 , D. Mele 1 , E. Degasperi 2 , S. Varchetta 1 , A. Aghemo 2 , M.G. Rumi 2 , C. Tinelli 3 , M. Colombo 2 , M.U. Mondelli 1 1 Department
of Infectious Diseases, and; 3 Statistics and Clinical Epidemiology Service, Fondazione IRCCS Policlinico San Matteo and University of Pavia; 2 Division of Gastroenterology, Fondazione IRCCS Ospedale Maggiore Mangiagalli Regina Elena and University of Milan, Italy Background and Aim: Innate immunity is thought to contribute to achieve a sustained virological response (SVR) in patients with cHCV infection treated
S3
with Peg-IFNα/ribavirin (RBV), but a clear immunological discrimination between SVR, non-responder (NR) or relapser (Rel) patients is presently not possible. We analysed changes in NK cell phenotype and function during antiviral therapy to identify immunological biomarker(s) of treatment outcome. Methods: We studied NK cell phenotype and function of 37 patients (25 SVR, 7 NR and 5 Rel, genotypes 1, 2, 4) treated with Peg-IFN/RBV (SOC). Molecules involved in NK cell regulation (CD69, NKp30, NKp46, NKG2D, CD16, NKG2A, KIR3DL1/S1, TRAIL, CD107a, Perforin, Tim-3, Siglec-7) were analysed by flow cytometry at baseline and various time points during treatment and after discontinuation. NK cell function was evaluated as cytokine (IFNγ, TNFα) production by intracellular staining and as cytotoxic activity by CD107a degranulation assay. Results: SOC therapy induced an early increase in expression of CD69, NKp30, NKp46 and decrease of Siglec-7, Tim-3, CD16 and Perforin (PFN). By comparing trends of NK phenotype and function of NR and Rel patients with those of SVR using a linear regression models for repeated measures, a typical NK cell profile emerged in NR patients, featuring a lower PFN content at baseline (p=0.02) and during therapy (p=0.014) and a simultaneously higher expression of CD16 (p=0.017), which were inversely correlated at week 12 (p=0.01, r=0.98). Moreover, there was a trend towards reduced IFNγ production and degranulation. Rel patients showed a distinct profile characterized by higher levels of NKp30 expression before (p=0.01) and during the treatment (p=0.003). Moreover, the percentage of NKp46+ and Tim-3+ NK cells remained consistently higher during therapy (p=0.037 and p=0.03, respectively). Conclusions: HCV-infected patients treated with SOC showed different NK cell phenotypic and functional features which allow distinct profiling of non-response or relapse versus SVR.
OC-8 IL28B and PNPLA3 polymorphisms affect histological liver damage in patients with non-alcoholic fatty liver disease S. Petta 1 , S. Grimaudo 1 , C. Cammà 1 , D. Cabibi 2 , V. Di Marco 1 , G. Licata 1 , R.M. Pipitone 1 , A. Craxì 1 1 Sezione 2
di Gastroenterologia, DiBiMIS, University of Palermo, Italy; Cattedra di Anatomia Patologica, University of Palermo, Italy
Background and Aims: Genetic background may affect the severity of liver damage in patients with non-alcoholic fatty liver disease (NAFLD). We aimed to assess whether IL28B rs12979860 and rs8099917 polymorphisms, together with PNPLA3 rs738409 C>G polymorphism, are associated with histological liver damage in NAFLD patients. Methods: One hundred sixty consecutive NAFLD patients, were assessed by liver biopsy (Kleiner score); anthropometric, and biochemical and metabolic features were included. IL28B rs12979860 C>T, IL28B rs8099917 G>C, and PNPLA3 rs738409 C>G single nucleotide polymorphisms were tested. Results: Fourteen (8.8%) patients had IL28B rs 12979860 TT polymorphism, compared with 72 (45%) and 74 (46.2%) with TC and CC variants, respectively. PNPLA3 rs738409 CC polymorphism was present in 47 (29.4%) patients, compared with 79 (49.4%) and 34 (21.3%) with CC and GG variants, respectively. Multivariate logistic regression analysis showed that older age (OR 1.042,95%CI 1.011–1.075, p=0.008), hyperuricemia (OR 4.904,95%CI 1.769–13.599, p=0.002), IL28B rs 12979860 CC (OR 4.893, 95%CI 2.223– 10.770, p<0.001), and steatosis grade (OR 1.645,95%CI 1.005–2.692, p=0.04) were independently linked to moderate-severe lobular inflammation. By contrast, NAS score ≥5 was independently linked to higher HOMA index (OR 1.179,95%CI 1.023–1.399, p=0.02), hyperuricemia (OR 6.391,95%CI 2.030– 20.122, p=0.002) and PNPLA3 rs738409 GG (OR 3.064,95%CI 1.107–8.480, p=0.03). Patients with both IL28B rs 12979860 CC and PNPLA3 rs738409 GG had a prevalence of moderate-severe lobular inflammation and NAS ≥5 of 84.6% (11/13) and 84.6% (11/13), respectively, significantly higher compared with that of patients without both unfavorable polymorphisms (16/65♦ 24.6%, p<0.001; 33/65 ♦ 50.7%, p=0.02; respectively). Conclusions: In NAFLD patients, IL28B rs 12979860 CC genotype, together with PNPLA3 rs738409 GG, is associated with the severity of liver damage.