Murine Nonalcoholic Steatohepatitis Research Articles

Metformin treatment rescues CD8+ T-cell response to immune checkpoint inhibitor therapy in mice with NAFLD.

Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. Invivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which hastherapeutic implications in patients with NASH-related liver cancer. Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.

Depletion of hepatic stellate cells inhibits hepatic steatosis in mice.

Hepatic stellate cells (HSCs), the main source of extracellular matrix in hepatic fibrogenesis, produce various cytokines, growth factors, and morphogenetic proteins. Among these, several factors are known to promote hepatocyte lipid accumulation, suggesting that HSCs can be efficient therapeutic targets for non-alcoholic steatohepatitis (NASH). This study aimed to investigate the effects of HSC depletion on the development of hepatic steatosis and fibrosis in a murine NASH model. C57BL/6 mice were treated with gliotoxin (GTX), an apoptosis inducer of activated HSCs under the feeding of a choline-deficient l-amino acid-defined high-fat diet for 4weeks. For in vitro study, Hc3716 cells, immortalized human hepatocytes, were treated with fatty acids in the presence or absence of LX2, immortalized HSCs. Choline-deficient l-amino acid-defined high-fat diet increased pronounced hepatic steatosis, which was attenuated by GTX treatment, together with a reduction in the number of activated HSCs. This change was associated with the downregulation of the peroxisome proliferator-activated receptor gamma (PPARγ) and its downstream genes, including adipocyte protein 2, cluster of differentiation 36 (CD36), and fatty acid transport protein 1, all of which increase the fatty acid uptake into hepatocytes. As expected, GTX treatment improved hepatic fibrosis. Co-culture of hepatocytes with HSCs enhanced intracellular lipid accumulation, together with the upregulation of PPARγ and CD36 protein expressions. In addition to the improvement in hepatic fibrogenesis, depletion of HSCs had a favorable effect on hepatic lipid metabolism in a mouse NASH model, suggesting that HSCs are potentially efficient targets for the treatment of NASH.

From the Editor’s Desk…

From the Editor’s Desk…

Butyrolactone I attenuates inflammation in murine NASH by inhibiting the NF-κB signaling pathway

Nonalcoholic steatohepatitis (NASH) is the development of non-alcoholic fatty liver disease (NAFLD) and a key element in the exacerbation of NAFLD. Since there are currently no drugs approved by the U.S. Food and Drug Administration to treat this disease, the search for treatments that can be translated into clinical use is urgent. Butyrolactone I (BLI), isolated from Aspergillus terreus, is an active compound possessing multiple biological activities. However, the effects of BLI on NASH have never been reported. In this study, RAW264.7 cells stimulated by lipopolysaccharide (LPS) were applied to study the anti-inflammatory effect and the underlying mechanisms of BLI in vitro. Following this, mice fed with high-fat and -fructose diet (HFFD) were used to explore the alleviation of NASH by BLIin vivo. We found that BLI attenuated inflammation in LPS-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway and downregulating the expression of iNOS and COX-2. Moreover, results of experiments in vivo demonstrated that BLI reduced serum transaminase levels, decreased hepatic fat accumulation, inhibited inflammation, suppressed oxidative stress, and ameliorated liver fibrosis. For the first time, we investigated the role of BLI in the treatment of murine NASH. We found that BLI alleviates NASH partly by inhibiting the NF-κB pathway of signaling. Given its hepatoprotective effects and non-toxic properties, BLI can be a novel and effective drug for NASH patients.

Alisol B Alleviates Hepatocyte Lipid Accumulation and Lipotoxicity via Regulating RARα-PPARγ-CD36 Cascade and Attenuates Non-Alcoholic Steatohepatitis in Mice.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease worldwide, with no effective therapies available. Discovering lead compounds from herb medicine might be a valuable strategy for the treatment of NASH. Here, we discovered Alisol B, a natural compound isolated from Alisma orientalis (Sam.), that attenuated hepatic steatosis, inflammation, and fibrosis in high-fat diet plus carbon tetrachloride (DIO+CCl4)-induced and choline-deficient and amino acid-defined (CDA)-diet-induced NASH mice. RNA-seq showed Alisol B significantly suppressed CD36 expression and regulated retinol metabolism in NASH mice. In mouse primary hepatocytes, Alisol B decreased palmitate-induced lipid accumulation and lipotoxicity, which were dependent on CD36 suppression. Further study revealed that Alisol B enhanced the gene expression of RARα with no direct RARα agonistic activity. The upregulation of RARα by Alisol B reduced HNF4α and PPARγ expression and further decreased CD36 expression. This effect was fully abrogated after RARα knockdown, suggesting Alisol B suppressed CD36 via regulating RARα-HNF4α-PPARγ cascade. Moreover, the hepatic gene expression of RARα was obviously decreased in murine NASH models, whereas Alisol B significantly increased RARα expression and decreased CD36 expression, along with the downregulation of HNF4α and PPARγ. Therefore, this study showed the unrecognized therapeutic effects of Alisol B against NASH with a novel mechanism by regulating RARα-PPARγ-CD36 cascade and highlighted Alisol B as a promising lead compound for the treatment of NASH.

High-fat diet-induced nonalcoholic steatohepatitis is accelerated by low carnitine and impaired glucose tolerance in novel murine models

AbstractCarnitine deficiency and impaired glucose tolerance (IGT) exacerbate liver steatosis. Given the current lack of ideal murine nonalcoholic steatohepatitis (NASH) models, we investigated new NASH models using jvs/+ mice with low carnitine and wild-type mice with low-dose alloxan-induced IGT. The jvs/+ and wild-type mice were divided into jvs/+ mice fed a high-fat diet (HFD) from 3 weeks of age (HF hetero group), wild-type mice with low-dose alloxan treatment fed HFD (AL + HF wild group), wild-type mice fed HFD (HF wild group), and two types of mice fed a normal diet—jvs/+ and wild-type (intact group). All mice were sacrificed at 20 or 40 weeks of age. All male HFD-fed mice showed obesity, IGT, high blood insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR), high liver enzyme levels, and high cholesterol levels. The degree of IGT was the worst in the AL + HF wild group, and blood insulin levels and HOMA-IR score were remarkably increased from 20 to 40 weeks of age. Almost all HFD-fed mice showed steatosis, fibrosis, and lobular inflammation in the centrilobular zone. These changes were accompanied by hepatocyte ballooning and were enhanced at 40 weeks of age. Furthermore, the incidence rate of nodular hyperplasia and adenoma in both the HF hetero and AL + HF wild groups was nearly 30%. We successfully established two novel murine models of NASH using male jvs/+ mice with low carnitine and male wild-type mice with IGT that eventually developed obesity, fatty liver, insulin resistance, liver fibrosis, and tumorigenesis. These results suggest that low carnitine levels and early-stage induction of IGT are important factors in the progression of NASH to tumorigenesis, similar to human NASH.

1351-P: Molecular Pathogenesis of NASH through the Dysregulation of Metabolic Organ Network in the NASH-HCC Model Mouse Treated with Streptozotocin, High-Fat Diet

Introduction: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease, often associated with type II diabetes, which sometimes progresses to hepatocellular carcinoma (HCC) . NASH has become a health problem worldwide, but therapeutic agents for NASH have not yet been approved. Although the mechanism of NASH progression has not been fully elucidated, the multiple parallel hits hypothesis suggests abnormalities in adipocytokines, endotoxins, etc could contribute to the development of NASH. The STAM™ model is a clinically-correlated murine NASH model which shows the same pathological progression as human patients. Diabetes is a well-known comorbidity of NASH, and it is evident in the STAM™ model but analysis has focused mostly on the liver. Considering organ crosstalk interactions between the liver, and the gut and adipose tissue in the model should be clarified. Methods: NASH was induced by injection of 200 µg streptozotocin solution 2 days after birth and feeding with HFD after 4 weeks of age. The mice were sacrificed at NASH stage. Colon samples were snap frozen in liquid nitrogen and stored at -80°C for tight junction-related protein analysis. Adipose tissue was prepared into paraffin blocks for HE staining. Blood adiponectin was analyzed to confirm changes in adipocytokine profile. Results: Tight junction-related proteins in the intestine of STAM™ showed that expression of ZO-1 decreased with the progression of disease. Increased expression of endotoxin in the blood and decreased expression of adiponectin were also observed. HE staining of adipose tissue revealed hypertrophy of adipocytes. Conclusion: The data suggested the occurrence of leaky gut, and abnormalities in adipocytokine secretion. Together with the liver, phenotypes in these organs are highly similar to human NASH patients, and might be involved in the pathogenesis of NASH. Disclosure L.P.Bui: None. Y.Sakakibara: None. R.Tanaka: None. E.H.Pigney: None. T.Hashiguchi: None.

Receptor-interacting protein kinase-1 ablation in liver parenchymal cells promotes liver fibrosis in murine NASH without affecting other symptoms.

Non-alcoholic steatohepatitis (NASH), a chronic liver disease that emerged in industrialized countries, can further progress into liver fibrosis, cirrhosis, and hepatocellular carcinoma. In the next decade, NASH is predicted to become the leading cause of liver transplantation, the only current interventional therapeutic option. Hepatocyte death, triggered by different death ligands, plays key role in its progression. Previously, we showed that the receptor-interacting protein kinase-1 (RIPK1) in hepatocytes exhibits a protective role in ligand-induced death. Now, to decipher the role of RIPK1 in NASH, Ripk1LPC-KO mice, deficient for RIPK1 only in liver parenchymal cells, and their wild-type littermates (Ripk1fl/fl) were fed for 3, 5, or 12weeks with high-fat high-cholesterol diet (HFHCD). The main clinical signs of NASH were analyzed to compare the pathophysiological state established in mice. Most of the symptoms evolved similarly whatever the genotype, whether it was the increase in liver to body weight ratio, the steatosis grade or the worsening of liver damage revealed by serum transaminase levels. In parallel, inflammation markers followed the same kinetics with significant equivalent inductions of cytokines (hepatic mRNA levels and blood cytokine concentrations) and a main peak of hepatic infiltration of immune cells at 3weeks of HFHCD. Despite this identical inflammatory response, more hepatic fibrosis was significantly evidenced at week 12 in Ripk1LPC-KO mice. This coincided with over-induced rates of transcripts of genes implied in fibrosis development (Tgfb1, Tgfbi, Timp1, and Timp2) in Ripk1LPC-KO animals. In conclusion, our results show that RIPK1 in hepatocyte limits the progression of liver fibrosis during NASH.

Hepatocyte-specific activity of TSC22D4 triggers progressive NAFLD by impairing mitochondrial function

ObjectiveFibrotic organ responses have recently been identified as long-term complications in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date. MethodsBased on our previous discovery of transforming growth factor beta-like stimulated clone (TSC)22D4 as a key driver of insulin resistance and glucose intolerance in obesity and type 2 diabetes, we generated a TSC22D4-hepatocyte specific knockout line (TSC22D4-HepaKO) and exposed mice to control or NASH diet models. Mechanistic insights were generated by metabolic phenotyping and single-nuclei RNA sequencing. ResultsHepatic TSC22D4 expression was significantly correlated with markers of liver disease progression and fibrosis in both murine and human livers. Indeed, hepatic TSC22D4 levels were elevated in human NASH patients as well as in several murine NASH models. Specific genetic deletion of TSC22D4 in hepatocytes led to reduced liver lipid accumulation, improvements in steatosis and inflammation scores and decreased apoptosis in mice fed a lipogenic MCD diet. Single-nuclei RNA sequencing revealed a distinct TSC22D4-dependent gene signature identifying an upregulation of mitochondrial-related processes in hepatocytes upon loss of TSC22D4. An enrichment of genes involved in the TCA cycle, mitochondrial organization, and triglyceride metabolism underscored the hepatocyte-protective phenotype and overall decreased liver damage as seen in mouse models of hepatocyte-selective TSC22D4 loss-of-function. ConclusionsTogether, our data uncover a new connection between targeted depletion of TSC22D4 and intrinsic metabolic processes in progressive liver disease. Hepatocyte-specific reduction of TSC22D4 improves hepatic steatosis and promotes hepatocyte survival via mitochondrial-related mechanisms thus paving the way for targeted therapies.

Role of AKR1B10 and AKR1B8 in the pathogenesis of non-alcoholic steatohepatitis (NASH) in mouse.

Non-alcoholic steatohepatitis (NASH) is a clinically important spectrum of non-alcoholic fatty liver disease (NAFLD) in humans. NASH is a stage of NAFLD progression wherein liver steatosis accompanies inflammation and pro-fibrotic events. Presently, there are no approved drugs for NASH, which has become a leading cause of liver transplant worldwide. To discover novel drug targets for NASH, we analyzed a human transcriptomic NASH dataset and found Aldo-keto reductase family 1 member B10 (AKR1B10) as a significantly upregulated gene in livers of human NASH patients. Similarly murine Akr1b10 and Aldo-keto reductase family 1 member B8 (Akr1b8) gene, which is a murine ortholog of human AKR1B10, were also found to be upregulated in a mouse model of diet-induced NASH. Furthermore, pharmacological inhibitors of AKR1B10 significantly reduced the pathological features of NASH such as steatosis, inflammation and fibrosis in mouse. In addition, genetic silencing of both mouse Akr1b10 and Akr1b8 significantly reduced the expression of proinflammatory cytokines from hepatocytes. These results, thus, underscore the involvement of murine AKR1B10 and AKR1B8 in the pathogenesis of murine NASH and raise an intriguing possibility of a similar role of AKR1B10 in human NASH.

Deep proteomic profiling unveils arylsulfatase A as a non-alcoholic steatohepatitis inducible hepatokine and regulator of glycemic control

Non-alcoholic steatohepatitis (NASH) and type 2 diabetes are closely linked, yet the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. Using proteomic approaches, we interrogate hepatocyte protein secretion in two models of murine NASH to understand how liver-derived factors modulate lipid metabolism and insulin sensitivity in peripheral tissues. We reveal striking hepatokine remodelling that is associated with insulin resistance and maladaptive lipid metabolism, and identify arylsulfatase A (ARSA) as a hepatokine that is upregulated in NASH and type 2 diabetes. Mechanistically, hepatic ARSA reduces sulfatide content and increases lysophosphatidylcholine (LPC) accumulation within lipid rafts and suppresses LPC secretion from the liver, thereby lowering circulating LPC and lysophosphatidic acid (LPA) levels. Reduced LPA is linked to improvements in skeletal muscle insulin sensitivity and systemic glycemic control. Hepatic silencing of Arsa or inactivation of ARSA’s enzymatic activity reverses these effects. Together, this study provides a unique resource describing global changes in hepatokine secretion in NASH, and identifies ARSA as a regulator of liver to muscle communication and as a potential therapeutic target for type 2 diabetes.

Chronic exposure to low-dose cadmium facilitated nonalcoholic steatohepatitis in mice by suppressing fatty acid desaturation

Exposure to cadmium (Cd), a toxic metal, is epidemiologically linked to nonalcoholic steatohepatitis (NASH) in humans. However, the role of Cd in NASH remains to be fully elucidated. This study employed a novel murine NASH model to investigate the effects of chronic low-dose Cd on hepatic pathology and its underlying mechanisms. NASH is characterized by lipid accumulation, extensive cell death, and persistent inflammation in the liver. We found that treatment with Cd in drinking water (10 mg/L) for 6 or 12 weeks significantly boosted hepatic fat deposition, increased hepatocyte destruction, and amplified inflammatory responses in mice, confirming that low-dose Cd can facilitate NASH development in vivo. Mechanistically, chronic Cd exposure reshaped the hepatic transcriptional landscape, with PPAR-mediated fatty acid metabolic pathways being the most significantly altered. In particular, Cd repressed fatty acid desaturation, leading to the accumulation of saturated fatty acids whose lipotoxicity exacerbated cell death and, consequently, inflammatory activation. In summary, we validated the causal effects of chronic low-dose Cd on NASH in vivo and identified the fatty acid desaturation program as a novel target for Cd to instigate hepatopathological alterations.

Beneficial Activities of Alisma orientale Extract in a Western Diet-Induced Murine Non-Alcoholic Steatohepatitis and Related Fibrosis Model via Regulation of the Hepatic Adiponectin and Farnesoid X Receptor Pathways.

The hepatic adiponectin and farnesoid X receptor (FXR) signaling pathways play multiple roles in modulating lipid and glucose metabolism, reducing hepatic inflammation and fibrosis, and altering various metabolic targets for the management of non-alcoholic fatty liver disease (NAFLD). Alisma orientale (AO, Ze xie in Chinese and Taeksa in Korean) is an herbal plant whose tubers are enriched with triterpenoids, which have been reported to exhibit various bioactive properties associated with NAFLD. Here, the present study provides a preclinical evaluation of the biological functions and related signaling pathways of AO extract for the treatment of NAFLD in a Western diet (WD)-induced mouse model. The findings showed that AO extract significantly reversed serum markers (liver function, lipid profile, and glucose) and improved histological features in the liver sections of mice fed WD for 52 weeks. In addition, it also reduced hepatic expression of fibrogenic markers in liver tissue and decreased the extent of collagen-positive areas, as well as inhibited F4/80 macrophage aggregation and inflammatory cytokine secretion. The activation of adiponectin and FXR expression in hepatic tissue may be a major mechanistic signaling cascade supporting the promising role of AO in NAFLD pharmacotherapy. Collectively, our results demonstrated that AO extract improves non-alcoholic steatohepatitis (NASH) resolution, particularly with respect to NASH-related fibrosis, along with the regulation of liver enzymes, postprandial hyperglycemia, hyperlipidemia, and weight loss, probably through the modulation of the hepatic adiponectin and FXR pathways.

Effects of pemafibrate and bezafibrate on CDAHFD-induced murine nonalcoholic steatohepatitis (NASH) model

Effects of pemafibrate and bezafibrate on CDAHFD-induced murine nonalcoholic steatohepatitis (NASH) model

Dual role of neutrophils in modulating liver injury and fibrosis during development and resolution of diet-induced murine steatohepatitis

Inflammatory changes in the liver represent a key feature of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD). Innate immune activation including hepatic neutrophilic infiltration acts as an important inflammatory trigger as well as a potential mediator of inflammation resolution. In this study, we dissected the effects of neutrophil depletion via anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies administration during ongoing high fat-fructose-cholesterol (FFC) diet-induced murine NASH and during inflammation resolution by switching into a low-fat control diet. During NASH progression, protective effects were shown as HSC activation, cell infiltration and activation of pro-inflammatory macrophages were ameliorated. Furthermore, these changes were contrasted with the effects observed when neutrophil depletion was performed during the resolution phase. Impaired resolving mechanisms, such as a failure to balance the pro and anti-inflammatory cytokines ratio, deficient macrophage phenotypic switch into a pro-restorative profile, and defective repair and remodeling processes were observed when neutrophils were depleted in this scenario. This study described phase-dependent contrasting roles of neutrophils as triggers and pro-resolutive mediators of liver injury and fibrosis associated with diet-induced NASH in mice. These findings have important translational implications at the time of designing NASH therapeutic strategies.

Pcsk9 Deletion Promotes Murine Nonalcoholic Steatohepatitis and Hepatic Carcinogenesis: Role of Cholesterol.

Proprotein convertase subtilisin/kexin type 9 (Pcsk9) binds to hepatic low‐density lipoprotein receptor (LDLR) and induces its internalization and degradation. Pcsk9 inhibition increases LDLR expression by hepatocytes, which causes increased uptake of circulating LDL, thereby reducing plasma LDL‐cholesterol. However, by increasing the uptake of LDL by the liver, Pcsk9 inhibition increases the exposure of the liver to cholesterol, which may result in higher risk of steatohepatitis and ever carcinogenesis. We compared Pcsk9‐/‐ knockout (KO) mice and appropriate wild‐type (WT) controls of the same strain assigned to a high‐fat (15%, wt/wt) diet for 9 months supplemented with 0.25%, 0.5%, or 0.75% dietary cholesterol. Pcsk9 KO mice on a high‐fat, high‐cholesterol diet exhibited higher levels of hepatic free cholesterol loading and hepatic cholesterol crystallization than their WT counterparts. Pcsk9 KO mice developed crown‐like structures of macrophages surrounding cholesterol crystal‐containing lipid droplets and hepatocytes, exhibited higher levels of apoptosis, and developed significantly more hepatic inflammation and fibrosis consistent with fibrosing steatohepatitis, including 5‐fold and 11‐fold more fibrosis at 0.5% and 0.75% dietary cholesterol, respectively. When injected with diethylnitrosamine, a hepatic carcinogen, early‐in‐life Pcsk9 KO mice were more likely to develop liver cancer than WT mice. Conclusion: Pcsk9 KO mice on high‐cholesterol diets developed increased hepatic free cholesterol and cholesterol crystals and fibrosing steatohepatitis with a higher predisposition to liver cancer compared with WT mice. Future studies should evaluate whether patients on long‐term treatment with anti‐PSCK9 monoclonal antibodies are at increased risk of hepatic steatosis, steatohepatitis or liver cancer, while accounting for concurrent use of statins.

Abstract 10784: Circulating Obesity Associated Pro-Fibrotic Protein Promotes Fibrosis in Liver and Heart

Introduction: Non-alcoholic steatohepatitis (NASH), driven by the obesity epidemic, has become the most common form of liver disease. Obesity contributes inflamed visceral adipose tissue secretes pro-inflammatory adipokines that is causal for systemic metabolic disorders. Despite its importance, the mechanism of NASH in the presence of morbid obesity related adipokines from BAT remains unclear. Hypothesis: Various kinds of white adipokines are known in obesity. However, brown adipokines still remain to be explored. Methods: To identify the function of obesity associated pro-fibrotic protein (OAFP), we generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice, and murine systemic OAFP knockout (KO) model. We also conducted functional in vitro studies with differentiated brown adipocytes. Results: Analyzing two sets of DNA microarray data with bioinformatics, we identified a secreted form OAFP expressed in dysfunctional brown adipose tissue (BAT) in mice. Testing our biobank samples, we found this protein increased in plasma of NASH patients or aged individuals. We generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice, and found that OAFP is produced predominantly by BAT. In this model, we also found that OAFP increased in plasma. For this reason, we generated a murine systemic OAFP knockout (KO) model and found that liver fibrosis ameliorated in OAFP-KO model. In addition, vaccination therapy also inhibited liver fibrosis. In contrast, liver fibrosis augmented in OAFP gain of function (GOF) model, and these results suggested that OAFP has causal role for the progression of fibrotic response in liver. In the obese NASH model, we found that cardiac fibrosis also developed and this ameliorated in OAFP-KO model, indicating that OAFP may have pathological roles for heart failure with preserved ejection fraction (HFpEF) related with age-related disorders. In vitro studies with differentiated brown adipocytes showed that OAFP was upregulated through by PERK/JNK/c-fos/AP1 pathways. Conclusions: Our results suggest that OAFP contributes for the progression of fibrotic responses in obese or aged models. Inhibition of OAFP may become a therapy for NASH or HFpEF.

Conditioned medium from stem cells derived from human exfoliated deciduous teeth ameliorates NASH via the Gut-Liver axis

Non-alcoholic steatohepatitis (NASH) occurrence has been increasing and is becoming a major cause of liver cirrhosis and liver cancer. However, effective treatments for NASH are still lacking. We examined the benefits of serum-free conditioned medium from stem cells derived from human exfoliated deciduous teeth (SHED-CM) on a murine non-alcoholic steatohepatitis (NASH) model induced by a combination of Western diet (WD) and repeated administration of low doses of carbon tetrachloride intraperitoneally, focusing on the gut-liver axis. We showed that repeated intravenous administration of SHED-CM significantly ameliorated histological liver fibrosis and inflammation in a murine NASH model. SHED-CM inhibited parenchymal cell apoptosis and reduced the activation of inflammatory macrophages. Gene expression of pro-inflammatory and pro-fibrotic mediators (such as Tnf-α, Tgf-β, and Ccl-2) in the liver was reduced in mice treated with SHED-CM. Furthermore, SHED-CM protected intestinal tight junctions and maintained intestinal barrier function, while suppressing gene expression of the receptor for endotoxin, Toll-like receptor 4, in the liver. SHED-CM promoted the recovery of Caco-2 monolayer dysfunction induced by IFN-γ and TNF-α in vitro. Our findings suggest that SHED-CM may inhibit NASH fibrosis via the gut-liver axis, in addition to its protective effect on hepatocytes and the induction of macrophages with unique anti-inflammatory phenotypes.

The Detrimental Role of Regulatory T Cells in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is induced by steatosis and metabolic inflammation. While involvement of the innate immune response has been shown, the role of the adaptive immune response in NASH remains controversial. Likewise, the role of regulatory T cells (Treg) in NASH remains unclear although initial clinical trials aim to target these regulatory responses. High‐fat high‐carbohydrate (HF‐HC) diet feeding of NASH‐resistant BALB/c mice as well as the corresponding recombination activating 1 (Rag)‐deficient strain was used to induce NASH and to study the role of the adaptive immune response. HF‐HC diet feeding induced strong activation of intrahepatic T cells in BALB/c mice, suggesting an antigen‐driven effect. In contrast, the effects of the absence of the adaptive immune response was notable. NASH in BALB/c Rag1−/− mice was substantially worsened and accompanied by a sharp increase of M1‐like macrophage numbers. Furthermore, we found an increase in intrahepatic Treg numbers in NASH, but either adoptive Treg transfer or anti‐cluster of differentiation (CD)3 therapy unexpectedly increased steatosis and the alanine aminotransferase level without otherwise affecting NASH. Conclusion: Although intrahepatic T cells were activated and marginally clonally expanded in NASH, these effects were counterbalanced by increased Treg numbers. The ablation of adaptive immunity in murine NASH led to marked aggravation of NASH, suggesting that Tregs are not regulators of metabolic inflammation but rather enhance it.

The Specific NLRP3 Antagonist IFM-514 Decreases Fibrosis and Inflammation in Experimental Murine Non-Alcoholic Steatohepatitis.

Background and Aims: Activation of the inflammasome NLRP3 (NOD-, LRR- and pyrin domain containing 3) contributes to the development of non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH). Therefore, this study explored the therapeutic effects of a novel and selective NLRP3 antagonist in a murine dietary model of NASH.Methods: Groups of 12-week-old ApoE -/- mice were fed ad lib for 7 weeks with a methionine/choline deficient (MCD) and western diet (WD). After 3 weeks of diet-induced injury, mice were injected i. p. with the NLRP3 antagonist IFM-514 (100 mg/kg body weight) or vehicle (0.5% carmellose) every day, 5 days/week for a further 4 weeks. Several markers of inflammation, fibrosis and steatosis were evaluated. Whole transcriptome sequencing and panel RNA expression analysis (NanoString) were performed.Results: IFM-514 inhibited IL-1β production in mice challenged with 20 mg/kg lipopolysaccharide, and in mouse and human inflammatory cells in vitro. IFM-514 inhibited hepatic inflammation in the in vivo non-alcoholic steatohepatitis model assessed by H&E staining and in the hepatic gene expression of inflammasome-related proinflammatory cytokines. This effect was associated with significant reduction in caspase-1 activation. Similarly, IFM-514 was efficacious in vivo in MDC-fed ApoE -/- mice, markedly reducing portal pressure, Sirius red staining and 4-hydroxyproline content compared to vehicle-treated mice. Moreover, IFM-514 significantly reduced hepatic steatosis in MCD-fed ApoE -/- mice, as evidenced by NAFLD scores, oil red O staining, hepatic triglycerides and gene expression. In WD treated animals, similar trends in inflammation and fibrosis were observed, although not sufficient IFM-514 levels were reached.Conclusion: Overall, IFM-514 reduced liver inflammation and fibrosis, with mild effects on liver steatosis in experimental murine NASH. Blocking of NLRP3 may be an attractive therapeutic approach for NASH patients.