An improved mouse model that rapidly develops fibrosis in non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine‐/choline‐deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long‐term feeding with a high‐fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1–14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Masson's trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.     

Vitronectin deficiency attenuates hepatic fibrosis in a non‐alcoholic steatohepatitis‐induced mouse model

Vitronectin (VN), an extracellular matrix protein, is a promising immune biomarker of non‐alcoholic steatohepatitis (NASH); however, its precise function remains unclear. This study investigated how VN deficiency contributes to the development of NASH. Towards this aim, wild‐type (WT) and VN?/? mice were fed with a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) for 6 and 10 weeks to induce NASH, and the livers were isolated. In WT mice fed with CDAHFD for 6 and 10 weeks, the expression of Vn mRNA and protein was up‐regulated compared with that in mice fed with the MF control diet, indicating that VN is regulated in NASH condition. VN?/? mice showed decreased picrosirius red staining in the liver area and Col1a2 mRNA expression levels, compared with WT mice, indicating that the severity of hepatic fibrosis is attenuated in the CDAHFD‐fed VN?/? mice. In addition, VN deficiency did not affect the area of lipid droplets in haematoxylin‐eosin staining and the mRNA expression levels of fatty acid synthases, Srebp, Acc and Fas in the CDAHFD‐fed mice. Moreover, VN deficiency decreased the inflammation score and the mRNA expression levels of Cd11b and F4/80, macrophage markers, as well as Tnf‐α and Il‐1β, inflammatory cytokines in the CDAHFD‐fed mice. Furthermore, VN deficiency decreased the protein and mRNA expression levels of α‐smooth muscle actin in the CDAHFD‐fed mice, suggesting that VN deficiency inhibits the activation of hepatic stellate cells (HSCs). Our findings indicate that VN contributes to the development of fibrosis in the NASH model mice via modulation of the inflammatory reaction and activation of HSCs.     

Non‐alcoholic fatty liver disease – histological scoring systems: a large cohort single‐center,evaluation study

Non‐alcoholic fatty liver disease (NAFLD) is an increasingly common cause of chronic liver disease. Till date, liver biopsy remains the gold standard for identification and quantification of the wide histological spectra of NAFLD. Histological scorings are very useful and widely applied for the diagnosis and management in clinical trials and follow‐up studies of non‐alcoholic steatohepatitis (NASH). However, in view of scarce published literature, there is a need to evaluate them in large cohort of NAFLD. This study was aimed to evaluate the two histological scoring systems (NAS‐CRN, SAF) in the diagnosis of NAFLD and to assess the role of histological characteristics as injury markers in NAFLD. Retrospective histological study of liver biopsies of 1000 patients diagnosed as NAFLD, between 2010 and 2016, was conducted. Histopathologic evaluation and semiquantiative scoring based on NAS‐CRN and SAF algorithm and their correlation with serum aminotransferase and fibrosis were performed. Liver biopsies were classified according to the NAS‐CRN scoring, as NAS <3 (not NASH) in 72 (7.2%), NAS 3–4 (borderline NASH) in 310 (31%), and NAS ≥5 (definite NASH) in 618 (61.8%), and SAF classified 117 (11.7%) not NASH and 883 (88.3%) definite NASH. There was excellent concordance for definite NASH and not NASH; however, 88.06% of borderline NASH was classified as NASH by SAF. 76.39% by NAS and 78.63% by SAF algorithm who were diagnosed as not NASH showed the presence of fibrosis; however, higher stages of fibrosis were significantly more prevalent in definite NASH, excluding burnt‐out cirrhosis. Serum ALT was significantly associated with increasing stages of fibrosis (p < 0.001) and the three categories (not NASH, borderline NASH, and definite NASH) when classified as with/without fibrosis (p < 0.001). Steatosis of higher grades, more ballooned cells, and more foci of Lobular Inflammation were found in significantly higher proportion of patients with NASH (p < 0.001), with higher fibrosis stages (p < 0.001) and higher serum ALT levels (p < 0.001). NAFLD classifications based on histological scoring NAS‐CRN and SAF algorithm are concordant for the category of definite NASH and not NASH, while borderline NASH shows discrepant interpretation. There was highly significant correlation between the NAS and SAF categories with high grades of histological characteristics, with serum ALT and with higher stages of fibrosis. Exclusion of fibrosis is a limitation with both scores.     

Serum lipocalin-2 is a potential biomarker for the clinical diagnosis of nonalcoholic steatohepatitis

Background/AimsNonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis. We aimed to investigate the usefulness of a key biomarker, lipocalin-2 (LCN2), for the detection of NASH progression.MethodsA mouse NASH model was established using a high-fat diet and a high-sugar drinking water. Gene expression profile of the NASH model was analyzed using RNA sequencing. Moreover, 360 NAFLD patients (steatosis, 83; NASH, 277), 40 healthy individuals, and 87 patients with alcoholic fatty liver disease were recruited.ResultsInflammatory infiltration, focal necrosis in the leaflets, steatosis, and fibrosis were documented in the mouse liver. In total, 504 genes were differentially expressed in the livers of NASH mice, and showed significant functional enrichment in the inflammation-related category. Upregulated liver LCN2 was found to be significantly interactive with various interleukins and toll-like receptors. Serum LCN2 levels were significantly increased in NAFLD patients. Serum LCN2 levels were correlated with steatosis, intralobular inflammation, semiquantitative fibrosis score, and nonalcoholic fatty liver disease activity score. The area under the curve of serum LCN2 was 0.987 with a specificity of 100% and a sensitivity of 93.5% for NASH diagnosis, and 0.977 with almost the same specificity and sensitivity for steatosis.ConclusionsLCN2 might be involved in the transition from NAFL to NASH by mediating inflammation. Serum LCN2 levels might be a novel biomarker for the diagnosis of NASH.     

Effect of Lipopolysaccharide on the Progression of Non‐Alcoholic Fatty Liver Disease in High Caloric Diet‐Fed Mice

The incidence of non‐alcoholic steatohepatitis (NASH) is increasing. Because gut microbiota have been highlighted as one of the key factors in the pathogenesis of metabolic syndrome, we investigated the involvement of the bacterial component in the progression of non‐alcoholic fatty liver (NAFL) to NASH. C57BL/6 mice were fed with maintenance food (MF, groups A and B) or a high caloric diet (HCD, groups C and D) for 1 month. Mice were then divided into four groups: Groups A and C were inoculated with PBS, while groups B and D were inoculated with lipopolysaccharide (LPS) plus complete Freund's adjuvant (CFA). The inoculations were performed a total of 3 times over 3 months. At 6 months, while hepatic steatosis was observed in groups C and D, cellular infiltration and fibrosis were less evident in group C than in group D. Inflammatory cytokines were upregulated in groups B and D. 16S rRNA pyrosequencing of whole colon homogenates containing faeces showed that certain bacterial groups, such as Bacteroidaceae, Peptostreptococcaceae and Erysipelotrichaceae, were increased in groups C and D. Although loading of bacterial components (LPS) resulted in hepatic inflammation in both MF‐ and HCD‐fed mice, HCD feeding was more crucial in the progression of NAFL during the triggering phase.     

Clinicopathological correlations in a series of adult patients with non‐alcoholic fatty liver disease

Possible correlations among clinical data, serum aminotransferase levels and histological features were assessed in a series of 37 adult patients with non‐alcoholic fatty liver disease (NAFLD), consisting of nine patients with fatty liver (FL) and 28 with non‐alcoholic steatohepatitis (NASH). In each liver biopsy, the NAFLD activity score (NAS) and the stage of fibrosis were determined. Additionally, the number of Kupffer cell aggregates (microgranulomas) per centimeter of biopsy length (MG/cm ratio) was assessed on immunohistochemical stains for CD68 antigen. Definite NASH (NAS ≥ 5) was strongly correlated with serum aspartate aminotransferase (AST) level (P= 0.003), stage of fibrosis (P= 0.003) and age (P= 0.014). On multivariate analysis, age >46 years and AST level above normal values were found to be independent clinical predictors of established NASH. The MG/cm ratio increased from control liver to FL to NASH (P < 0.001), and was correlated with the NAS (P= 0.003) and with the stage of fibrosis (P= 0.004), but not with the serum aminotransferase levels. In conclusion, persistent AST elevation in patients with suspected NAFLD should be an indication for liver biopsy, in order to determine the severity of necroinflammatory activity and the stage of fibrosis. Microgranuloma counting may represent a useful complementary marker of necroinflammatory activity in patients with NAFLD.     

Evaluation of high‐fat high‐fructose diet treatment in factor VIII (coagulation factor)‐deficient mouse model

Non‐alcoholic fatty liver disease (NAFLD)‐like conditions enhance the production and action of clotting factors in humans. However, studies examining the effect of NAFLD due to high‐fat high‐fructose (HFHF) diet in factor VIII‐deficient (haemophilia A) animals or patients have not been reported previously. In this study, we investigated the individual role of factor VIII in the progression of diet‐induced NAFLD in the factor 8^?/? (F8^?/?) mouse model system and its consequences on the haemophilic status of the mice. The F8^?/? mice were fed with HFHF diet for 14 weeks. Physiological, biochemical, haematological, molecular, pathological, and immune histochemical analyses were performed to evaluate the effect of this diet. The F8^?/? mice developed hepatic steatosis after 14 weeks HFHF diet and displayed lower energy metabolism, higher myeloid cell infiltration in the liver, decreased platelet count, upregulated de novo fatty acid synthesis, lipid accumulation, and collagen deposition. This study helps to understand the role of factor VIII in NAFLD pathogenesis and to analyse the severity and consequences of steatosis in haemophilic patients as compared to normal population. This study suggests that haemophilic animals (F8^?/? mice) are highly prone to hepatic steatosis and thrombocytopenia.     

Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis

Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the "two hit" hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptor-deficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies.     

Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high-calorie diet

Non‐alcoholic fatty liver disease (NAFLD) is one of the commonest liver diseases in Western countries. Although leptin deficient ob/ob and db/db mice are frequently used as murine models of NAFLD, an exhaustive characterization of their hepatic lesions has not been reported to date, particularly under calorie overconsumption. Thus, liver lesions were characterized in 78 ob/ob and db/db mice fed either a standard or high‐calorie (HC) diet, for one or three months. Steatosis, necroinflammation, apoptosis and fibrosis were assessed and the NAFLD activity score (NAS) was calculated. Steatosis was milder in db/db mice compared to ob/ob mice and was more frequently microvesicular. Although necroinflammation was usually mild in both genotypes, it was aggravated in db/db mice after one month of calorie overconsumption. Apoptosis was observed in db/db mice whereas it was only detected in ob/ob mice after HC feeding. Increased apoptosis was frequently associated with microvesicular steatosis. In db/db mice fed the HC diet for three months, fibrosis was aggravated while steatosis, necroinflammation and apoptosis tended to alleviate. This was associated with increased plasma β‐hydroxybutyrate suggesting an adaptive stimulation of hepatic mitochondrial fatty acid oxidation (FAO). Nevertheless, one‐third of these db/db mice had steatohepatitis (NAS ≥ 5), whereas none of the ob/ob mice developed non‐alcoholic steatohepatitis under the same conditions. Steatosis, necroinflammation, apoptosis and fibrosis are modulated by calorie overconsumption in the context of leptin deficiency. Association between apoptosis and microvesicular steatosis in obese mice suggests common mitochondrial abnormalities. Enhanced hepatic FAO in db/db mice is associated with fibrosis aggravation.     

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.     

Japanese herbal medicines shosaikoto,inchinkoto, and juzentaihoto inhibit high‐fat diet‐induced nonalcoholic steatohepatitis in db/db mice

Few studies have investigated the effects of Japanese herbal medicines on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). To the best of our knowledge, only one study has examined whether high‐fat (HF) diet‐fed db/db mice are appropriate animal models of NASH. We investigated the effects of four types of Japanese herbal medicines (shosaikoto (TJ‐9), inchinkoto (TJ‐135), juzentaihoto (TJ‐48), and keishibukuryogan (TJ‐25)) on hepatic lesions of HF diet‐fed db/db mice. Db/db mice were divided into six groups: control diet (control); HF diet (HF); and HF diet supplemented with TJ‐9, TJ‐135, TJ‐48, or TJ‐25 (TJ‐9, TJ‐135, TJ‐48, and TJ‐25, respectively). Mice were killed after 6 weeks of treatment, and biochemical and pathological analyses were performed. Mice in the HF group consistently developed histopathological features consistent with definite NASH, and marked necroinflammation occurred. Serum alanine aminotransferase levels in the TJ‐9, TJ‐135, and TJ‐48 groups were significantly improved compared with those in the HF group. With regard to liver histology, TJ‐9 and TJ‐48 significantly improved lobular inflammation, and TJ‐135 significantly improved ballooning degeneration. We have shown that HF diet‐fed db/db mice are animal models that correctly recapitulate the histopathology of human NASH and that TJ‐9, TJ‐135, and TJ‐48 inhibit necroinflammatory activity in this model.     

Differential TM4SF5‐mediated SIRT1 modulation and metabolic signaling in nonalcoholic steatohepatitis progression

Nonalcoholic fatty liver disease is a chronic condition involving steatosis, steatohepatitis and fibrosis, and its progression remains unclear. Although the tetraspanin transmembrane 4 L six family member 5 (TM4SF5) is involved in hepatic fibrosis and cancer, its role in nonalcoholic steatohepatitis (NASH) progression is unknown. We investigated the contribution of TM4SF5 to liver pathology using transgenic and KO mice, diet‐ or drug‐treated mice, in vitro primary cells, and in human tissue. TM4SF5‐overexpressing mice exhibited nonalcoholic steatosis and NASH in an age‐dependent manner. Initially, TM4SF5‐positive hepatocytes and liver tissue exhibited lipid accumulation, decreased Sirtuin 1 (SIRT1), increased sterol regulatory‐element binding proteins (SREBPs) and inactive STAT3 via suppressor of cytokine signaling (SOCS)1/3 upregulation. In older mice, TM4SF5 promoted inflammatory factor induction, SIRT1 expression and STAT3 activity, but did not change SOCS or SREBP levels, leading to active STAT3‐mediated ECM production for NASH progression. A TM4SF5‐associated increase in chemokines promoted SIRT1 expression and progression to NASH with fibrosis. Suppression of the chemokine CCL20 reduced immune cell infiltration and ECM production. Liver tissue from high‐fat diet‐ or CCl₄‐treated mice and human patients exhibited TM4SF5‐dependent steatotic or steatohepatitic livers with links between TM4SF5‐mediated SIRT1 modulation and SREBP or SOCS/STAT3 signaling axes. TM4SF5‐mediated STAT3 activation in fibrotic NASH livers increased collagen I and laminin γ2. Both collagen I α1 and laminin γ2 suppression resulted in reduced SIRT1 and active STAT3, but no change in SREBP1 or SOCS, and abolished CCl₄‐mediated mouse liver damage. TM4SF5‐mediated signaling pathways that involve SIRT1, SREBPs and SOCS/STAT3 promoted progression to NASH. Therefore, TM4SF5 and its downstream effectors may be promising therapeutic targets to treat nonalcoholic fatty liver disease. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Manganese superoxide dismutase is reduced in the liver of male but not female humans and rodents with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is among the most common liver diseases. Oxidative stress is one of the pathogenic mechanisms contributing to the progression of simple fatty liver to non-alcoholic steatohepatitis (NASH). Manganese superoxide dismutase (MnSOD) is a mitochondrial antioxidative enzyme and here its expression in rodent and human NAFLD has been analyzed. MnSOD is found reduced in the liver of male mice fed a high fat diet and male ob/ob mice. Female mice fed an atherogenic diet to induce NASH have MnSOD protein levels comparable to controls. In a cohort of 30 controls, 41 patients with fatty liver and 39 NASH patients, MnSOD mRNA is significantly lower in the steatotic and NASH liver. When analyzed in both genders separately reduction of MnSOD expression is only found in males. Here, MnSOD mRNA negatively correlates with steatosis grade but not with extent of fibrosis or inflammation. MnSOD is, however, not reduced in primary human hepatocytes (PHH) treated with palmitate or oleate to increase cellular triglycerides. Lipopolysaccharide, TNF, IL-6, TGFβ and leptin which are all raised in NAFLD do not affect MnSOD in PHH. Adiponectin which attenuates oxidative stress partly by increasing MnSOD in macrophages does not induce MnSOD in PHH. In summary, current data show that hepatic MnSOD is reduced in male but not female humans and rodents with NAFLD.     

Effect of chronic psychosocial stress on nonalcoholic steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which may progress towards inflammation (nonalcoholic steatohepatitis (NASH)). NAFLD is regarded as a consequence of a sedentary, food-abundant lifestyle which, in the modern world, often coincides with chronically high levels of perceived psychosocial stress. Here, we aimed to characterize the effect of chronic psychosocial stress on the development of NAFLD/NASH in male mice either fed with standard chow or NASH-inducing high fat diet. Chronic psychosocial stress was induced by chronic subordinate colony housing (CSC), a pre-clinically validated paradigm relevant for human affective and somatic disorders. Single housed (SHC) mice served as controls. Under standard chow conditions CSC mice revealed lower hepatic triglyceride levels but higher hepatic TNFα, MCP-1 and HMOX mRNA expression, while serum transaminase levels did not significantly differ from SHC mice. Under the NASH-inducing high-fat diet CSC and SHC mice showed similar body weight-gain and serum levels of glucose and adiponectin. Moreover, liver histology as well as TNFα, MCP-1 and HMOX expression were similar in CSC and SHC mice fed with HFD. Surprisingly, CSC showed even significantly lower transaminase levels than SHC mice fed with the same NASH-inducing diet. Together, these data indicate that under normal dietary conditions the CSC model induces noticeable hepatic oxidative stress and inflammation without causing manifest hepatocellular injury. In contrast, CSC exhibited a protective effect on hepatocellular injury in a dietary NASH-model. Identification of the underlying mechanisms of this phenomenon may lead to novel therapeutic strategies to prevent progression of NAFLD.     

Animal models of non‐alcoholic fatty liver disease: current perspectives and recent advances

Non‐alcoholic fatty liver disease (NAFLD) is a continuous spectrum of diseases characterized by excessive lipid accumulation in hepatocytes. NAFLD progresses from simple liver steatosis to non‐alcoholic steatohepatitis and, in more severe cases, to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Because of its growing worldwide prevalence, various animal models that mirror both the histopathology and the pathophysiology of each stage of human NAFLD have been developed. The selection of appropriate animal models continues to be one of the key questions faced in this field. This review presents a critical analysis of the histopathology and pathogenesis of NAFLD, the most frequently used and recently developed animal models for each stage of NAFLD and NAFLD‐induced HCC, the main mechanisms involved in the experimental pathogenesis of NAFLD in different animal models, and a brief summary of recent therapeutic targets found by the use of animal models. Integrating the data from human disease with those from animal studies indicates that, although current animal models provide critical guidance in understanding specific stages of NAFLD pathogenesis and progression, further research is necessary to develop more accurate models that better mimic the disease spectrum, in order to provide both increased mechanistic understanding and identification/testing of novel therapeutic approaches. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Hepatic steatosis causes induction of the chemokine RANTES in the absence of significant hepatic inflammation

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to cirrhosis. Hepatocellular lipid accumulation is a hallmark of both nonalcoholic steatosis and steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine CCL5/RANTES plays an important role in the progression of hepatic inflammation and fibrosis. We here aimed to investigate its expression in NAFLD. Incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation, and corresponding dose-dependent RANTES induction in vitro. Furthermore, we observed significantly elevated hepatic RANTES expression in a dietary model of NAFLD, in which mice were fed a high-fat diet for 12 weeks. This diet induced significant hepatic steatosis but only minimal inflammation. In contrast to the liver, RANTES expression was not induced in visceral adipose tissue of the group fed with high-fat diet. Finally, RANTES serum levels were elevated in patients with ultrasound-diagnosed NAFLD. In conclusion, our data indicate hepatocytes as cellular source of elevated hepatic as well as circulating RANTES levels in response to hepatic steatosis. Noteworthy, upregulation of RANTES in response to lipid accumulation occurs in the absence of relevant inflammation, which further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.     

Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease

Mechanisms associated with the progression of simple steatosis to non-alcoholic fatty liver disease (NAFLD) remain undefined. Regulatory T cells (T(regs)) play a critical role in regulating inflammatory processes in non-alcoholic steatohepatitis (NASH) and because T helper type 17 (Th17) functionally oppose T(reg)-mediated responses, this study focused on characterizing the role of Th17 cells using a NAFLD mouse model. C57BL/6 mice were fed either a normal diet (ND) or high fat (HF) diet for 8 weeks. Mice in the HF group had a significantly higher frequency of liver Th17 cells compared to ND-fed mice. Neutralization of interleukin (IL)-17 in HF mice ameliorated lipopolysaccharide (LPS)-induced liver injury reflected by decreased serum alanine aminotransferase (ALT) levels and reduced inflammatory cell infiltrates in the liver. In vitro, HepG2 cells cultured in the presence of free fatty acids (FFA; oleic acid and palmitic acid) for 24 h and IL-17 developed steatosis via insulin-signalling pathway interference. IL-17 and FFAs synergized to induce IL-6 production by HepG2 cells and murine primary hepatocytes which, in combination with transforming growth factor (TGF-β), expanded Th17 cells. It is likely that a similar process occurs in NASH patients, as there were significant levels of IL-17(+) cell infiltrates in NASH patient livers. The hepatic expression of Th17 cell-related genes [retinoid-related orphan receptor gamma (ROR)γt, IL-17, IL-21 and IL-23] was also increased significantly in NASH patients compared to healthy controls. Th17 cells and IL-17 were associated with hepatic steatosis and proinflammatory response in NAFLD and facilitated the transition from simple steatosis to steatohepatitis. Strategies designed to alter the balance between Th17 cells and T(regs) should be explored as a means of preventing progression to NASH and advanced liver diseases in NAFLD patients.     

Immunotherapy with oral administration of humanized anti‐CD3 monoclonal antibody: a novel gut‐immune system‐based therapy for metaflammation and NASH

The immune system plays a role in the pathogenesis of non‐alcoholic steatohepatitis (NASH) underlying hepatocyte injury and fibrosis progression at all disease stages. Oral administration of anti‐CD3 monoclonal antibody (mAb) has been shown in preclinical studies to be an effective method for systemic immune modulation and alleviates immune‐mediated disorders without T cell depletion. In the present review, we summarize the concept of the oral administration of humanized anti‐CD3 mAb in patients with NASH and discuss the potential of this treatment to address the current requirements of treatments for NASH. Recently published preclinical and clinical data on oral administration of anti CD3 are discussed. Human trials have shown that the oral administration of anti‐CD3 in healthy volunteers, patients with chronic hepatitis C virus (HCV) infection and patients with NASH and type 2 diabetes is safe and well tolerated, as well as biologically active. Oral anti‐CD3 induces regulatory T cells, suppresses the chronic inflammatory state associated with NASH and exerts a beneficial effect on clinically relevant parameters. Foralumab is a fully human anti‐CD3 mAb that has recently been shown to exert a potent anti‐inflammatory effect in humanized mice. It is being developed for treatment of NASH and primary biliary cholangitis (PBC). Oral administration of anti CD3 may provide an effective therapy for patients with NASH.     

Inhibition of Glutaminyl Cyclases alleviates CCL2‐mediated inflammation of non‐alcoholic fatty liver disease in mice

Inflammation is an integral part of non‐alcoholic fatty liver disease (NAFLD), the most prevalent form of hepatic pathology found in the general population. In this context, recently we have examined the potential role of Glutaminyl Cyclases (QC and isoQC), and their inhibitors, in the maturation of chemokines, for example, monocyte chemoattractant protein 1 (MCP‐1, CCL2), to generate their bioactive conformation. Catalysis by isoQC leads to the formation of an N‐terminal pyroglutamate residue protecting CCL2 against degradation by aminopeptidases. This is of importance because truncated forms possess a reduced potential to attract immune cells. Since liver inflammation is characterized by the up‐regulation of different chemokine pathways, and within this CCL2 is known to be a prominent example, we hypothesised that application of QC/isoQC inhibitors may alleviate liver inflammation by destabilizing CCL2. Therefore, we investigated the role of QC/isoQC inhibition, in comparison with the angiotensin receptor blocker Telmisartan, during development of pathology in a mouse model of non‐alcoholic fatty liver disease. Application of a QC/isoQC inhibitor led to a significant reduction in circulating alanine aminotransferase and NAFLD activity score accompanied by an inhibitory effect on hepatocyte ballooning. Further analysis revealed a specific reduction of inflammation by decreasing the number of F4/80‐positive macrophages, which is in agreement with the proposed CCL2‐related mechanism of action of QC/isoQC inhibitors. Finally, QC/isoQC inhibitor application attenuated liver fibrosis as characterized by reduced collagen deposition in the liver parenchyma. Thus in conclusion, QC/isoQC inhibitors are a promising novel class of anti‐non‐alcoholic steatohepatitis drugs which have a comparable disease‐modifying effect to that of Telmisartan, which is probably mediated via specific interference with a comparable monocyte/macrophage infiltration that occurs under inflammatory conditions.     

Novel non‐alcoholic steatohepatitis model with histopathological and insulin‐resistant features

Although several non‐alcoholic steatohepatitis (NASH) models have been reported to date, few of these models fully reflect the histopathology and pathophysiology of human NASH. The aim of this study was to establish a novel NASH model by feeding a high‐fat (HF) diet and administering both carbon tetrachloride (CCl₄) and the Liver X receptor agonist T0901317. Male C57BL/6J mice were divided into four groups (each n = 5): HF, HF + CCl₄, HF + T0901317, and the novel NASH model (HF + CCl₄ + T0901317). CCl₄ (0.1 mL/kg) and T0901317 (2.5 mg/kg) were intraperitoneally administered four times and five times, respectively. The livers of the novel NASH model group presented a whitish colour. The serum levels of TNF‐α and IL‐6 were significantly increased in the novel NASH model group, and mice in this group exhibited histopathological features and insulin resistance reflective of NASH, i.e., macrovesicular hepatic steatosis, ballooning hepatocytes, Mallory‐Denk bodies, lobular inflammation and fibrosis. The novel NASH model group presented significantly upregulated expression levels of mRNAs related to lipogenesis, oxidative stress, fibrosis and steatosis and significantly downregulated expression levels of mRNAs related to triglyceride export. We successfully established a novel experimental NASH model that exhibits similar histopathology and pathophysiology to human NASH.