Modelling human liver fibrosis in the context of non-alcoholic steatohepatitis using a microphysiological system

Non-alcoholic steatohepatitis (NASH) is a common form of chronic liver disease characterised by lipid accumulation, infiltration of immune cells, hepatocellular ballooning, collagen deposition and liver fibrosis. There is a high unmet need to develop treatments for NASH. We have investigated how liver fibrosis and features of advanced clinical disease can be modelled using an in vitro microphysiological system (MPS). The NASH MPS model comprises a co-culture of primary human liver cells, which were cultured in a variety of conditions including+/− excess sugar, fat, exogenous TGFβ or LPS. The transcriptomic, inflammatory and fibrotic phenotype of the model was characterised and compared using a system biology approach to identify conditions that mimic more advanced clinical disease. The transcriptomic profile of the model was shown to closely correlate with the profile of patient samples and the model displayed a quantifiable fibrotic phenotype. The effects of Obeticholic acid and Elafibranor, were evaluated in the model, as wells as the effects of dietary intervention, with all able to significantly reduce inflammatory and fibrosis markers. Overall, we demonstrate how the MPS NASH model can be used to model different aspects of clinical NASH but importantly demonstrate its ability to model advanced disease with a quantifiable fibrosis phenotype.

Potential benefits of antihypertensive therapy in NAFLD: results from an experimental model

Background and introduction Non-alcoholic fatty liver disease (NAFLD) is considered as the most frequent cause of chronic hepatic disease in adults. It is strictly correlated with insulin resistance. The renin-angiotensin system (RAS) has been correlated to the whole basic physiopathogenic mechanism of NAFLD in experimental models. Systemic arterial hypertension has been suggested to be associated with NAFLD in approximately 40% of the cases, and NAFLD has been independently associated with an increased risk of arterial hypertension in observational studies. Therefore, we can infer that treating arterial hypertension in NAFLD carriers will often be necessary and that the potential beneficial effects of the antihypertensive might, in this context, influence the choice of the respective drug. Purpose We aimed to evaluate the effects of the renin-angiotensin system blockade with angiotensin-converting enzyme inhibitor ramipril and angiotensin 2 type 1 receptor antagonist olmesartan, both used preventively, in NAFLD induced in rabbits fed a hypercholesterolemic diet and compared the results between the groups. Methods Forty-one rabbits were divided into four groups (normal, control, olmesartan and ramipril). The control, olmesartan and ramipril group were fed a hypercholesterolemic diet. Animals from olmesartan group were treated with olmesartan 1mg/kg/day and animals from ramipril group with ramipril 0.35 mg/kg/day. At the end of the 8th week, all rabbits underwent segmental hepatic resection and were euthanised. Blood samples were collected to determine glucose, creatinine, total cholesterol, triglycerides, high-density lipoprotein cholesterol, and aminotransferase levels at baseline and euthanasia. Haematoxylin and eosin and Gomori trichrome stained slides were analysed based on the histological scoring system for NAFLD. Results The comparison between two groups (olmesartan with placebo and ramipril with placebo) showed that olmesartan and ramipril significantly diminished the development of steatosis (p=0.015, p=0.032), lobular inflammation (p<0.001, p=0.006), hepatocellular ballooning (p<0.001, p=0.023) and fibrosis (p=0.001, p=0.02). Based on NAFLD activity score, olmesartan and ramipril significantly reduced the development of nonalcoholic steatohepatitis (p<0.001, p=0.003). The comparison between olmesartan and ramipril showed that results were similar in all histological parameters evaluated (p=1, p=0.454, p=0.454, p=0.195, p=0.078). Conclusion(s) The preventive use of olmesartan and ramipril attenuates similarly, the development of hepatic steatosis, lobular inflammation, hepatocellular ballooning and fibrosis in hypercholesterolemic rabbits and based on NAFLD activity score both significantly reduced the development of nonalcoholic steatohepatitis. Funding Acknowledgement Type of funding source: None

Circulating Neutrophils of Nonalcoholic Steatohepatitis Patients Show an Activated Phenotype and Suppress T Lymphocytes Activity

Neutrophils or PolyMorphonuclear Neutrophils (PMNs) are key effector cells of the innate immune system and thanks to their remarkable plasticity, establish a cross talk with T cells modulating their survival and effector functions. During Nonalcoholic Steatohepatitis (NASH), the advanced form of hepatic steatosis or NAFL, PMNs infiltrate liver tissue, becoming a histological feature of NASH. Our aim was to evaluate the frequency of PMNs in NAFL and NASH patients in order to understand how they modulate the activity of circulating CD4+ and CD8+ T cells. In our cohort of patients, NASH patients displayed a higher frequency of circulating PMNs that was strongly correlated to liver enzymes, grade of steatosis, inflammation and fibrosis, the hepatocellular ballooning, and NAFLD Activity Score (NAS). Furthermore, even if ex vivo, in both groups of patients, PMNs shared the same phenotype of resting cells, after 24 hours of coculture with autologous CD4+ and CD8+ T cells, PMNs of NASH patients acquired a more active phenotype, becoming able to strongly inhibit proliferation and activation of CD4+ and CD8+ T cells. The higher ability of PMNs of NASH patients in suppressing CD4+ and CD8+ T cells, over time, might contribute in reducing the immunological defense of liver tissue against damages thus taking part in the progression of the NAFL disease toward NASH.

FT3 to FT4 Ratio is Associated with Non-Alcoholic Steatohepatitis and Significant Fibrosis in Euthyroid Subjects with NAFLD

Background & Aims: Studies on the relationship between thyroid function and non-alcoholic fatty liver disease (NAFLD) among euthyroid subjects had shown inconsistent results. Objective of the present study was to exploring the independent relationship between thyroid function parameters and nonalcoholic steatohepatitis (NASH), significant fibrosis (SF) respectively after adjusting other well-identified risk factors.Method: This study enrolled 307 patients with biopsy-proven NAFLD. Thyroid dysfunction defined as serum thyroid-stimulating hormone > 4.5 mIU/l or < 0.5 mIU/l and/ or free thyroxine > 14.41 pmol/l or < 7.86 pmol/l.Results: Stepwise regression analysis showed that the fT3/fT4 ratio, an optimal thyroid function parameter, was associated with NASH and SF in euthyroid subjects with NAFLD. After multivariable analysis, the fT3/fT4 ratio (per 0.1 change) showed significant correlation with NASH (OR 2.03(1.33, 3.11), P = 0.001) and SF (OR 2.11(1.28, 3.46), P = 0.003). When ratio was stratified by quartiles (Q1-Q4) as a categorical variable, the results still significant (Q4 versus Q1 (OR for NASH 4.29(1.68, 10.91), P = 0.002; for SF 4.63(1.49, 14.43), P = 0.008, all P for linear trend < 0.05). The prevalence of NASH and SF rose significantly with increasing in quartiles. Furthermore, ratio was positively correlated with the grade of steatosis, lobular inflammation, hepatocellular ballooning and liver fibrosis stage (all P < 0.05). Subgroup analysis showed that hypertension was a possible effect modification.Conclusion: The findings of the present study confirmed an association between fT3/fT4 ratio and NAFLD in euthyroid subjects in a dose-dependent manner, particularly in non-hypertension adults.There is no trial registration number.

Selenoprotein P in Patients with Nonalcoholic Fatty Liver Disease

Objective Main aim of this study was to evaluate circulating selenoprotein P (SEPP) levels in patients with simple steatosis (SS) and nonalcoholic steatohepatitis (NASH) compared with healthy controls. Methods Thirty-one patients with biopsy-proven NAFLD (15 with SS, 10 with borderline NASH, 6 with definite NASH) and 27 matched controls without NAFLD were enrolled. Serum SEPP levels and liver function tests plus biochemical parameters were measured with ELISA and standard methods, respectively. Homeostatic model of assessment - insulin resistance (HOMA-IR) was calculated. Results SEPP levels were statistically different between groups (p-value for trend=0.043). In pairwise comparisons, SEPP was lower in definite NASH compared with controls (p=0.029), but not SS (p=0.18) or borderline NASH (p=0.35). SEPP was not different between controls, SS and borderline NASH. The unadjusted trend between the controls, SS and NASH patients remained essentially unchanged after adjustment for age, sex, log(ALT) and waist circumference, but it marginally lost significance when log(HOMA-IR) entered into the model. SEPP levels were not different between groups of different severity of steatosis, fibrosis, hepatocellular ballooning, lobular and portal inflammation. Conclusions Lower SEPP levels were observed in patients with definite NASH compared with controls, a finding warranting larger studies.

Iron overload results in hepatic oxidative stress, immune cell activation, and hepatocellular ballooning injury, leading to nonalcoholic steatohepatitis in genetically obese mice

The aim of this study was to determine the effect of iron overload in the development of nonalcoholic steatohepatitis (NASH) in a genetically obese mouse model (Lepr db/db). Leptin receptor-deficient mice were fed a normal or an iron-supplemented chow for 8 wk and switched to normal chow for 8 wk. All dietary iron (DI)-fed mice developed hepatic iron overload predominantly in the reticuloendothelial system. Hepatocellular ballooning injury was observed in the livers of 85% of DI mice, relative to 20% of chow-fed Lepr db/db. Hepatic malonyldialdehyde levels and mRNA levels of antioxidant genes ( Nrf2, Gpx1, and Hmox1) were significantly increased in the DI mice. Hepatic mRNA levels of mitochondrial biogenesis regulators Pgc1α, Tfam, Cox4, and Nrf1 were diminished in the DI mice. In addition, gene expression levels of cytokines ( Il6, Tnfα) and several innate and adaptive immune cell markers such as Tlr4, Inos, CD11c, CD4, CD8, and Ifnγ were significantly increased in livers of the DI group. Strikingly, Nlrp3, a component of the inflammasome and Il18, a cytokine elicited by inflammasome activation, were significantly upregulated in the livers of DI mice. In addition, RAW 264.7 macrophages loaded with exogenous iron showed significantly higher levels of inflammatory markers ( Inos, Tnfα, Mcp1, Tlr4). Thus dietary iron excess leads to hepatic oxidative stress, inflammasome activation, induction of inflammatory and immune mediators, hepatocellular ballooning injury, and therefore NASH in this model. Taken together, these studies indicate a multifactorial role for iron overload in the pathogenesis of NASH in the setting of obesity and metabolic syndrome.