TLR4 Deficiency Protects against Hepatic Fibrosis and Diethylnitrosamine-Induced Pre-Carcinogenic Liver Injury in Fibrotic Liver

Background The development of hepatocellular carcinoma (HCC) is a common consequence of advanced liver fibrosis but the interactions between fibrogenesis and carcinogenesis are still poorly understood. Recently it has been shown that HCC promotion depends on Toll-like receptor (TLR) 4. Pre-cancerogenous events can be modelled in mice by the administration of a single dose of diethylnitrosamine (DEN), with HCC formation depending amongst others on interleukin (IL) 6 production. Mice lacking the hepatocanalicular phosphatidylcholine transporter ABCB4 develop liver fibrosis spontaneously, resemble patients with sclerosing cholangitis due to mutations of the orthologous human gene, and represent a valid model to study tumour formation in pre-injured cholestatic liver. The aim of this study was to investigate DEN-induced liver injury in TLR4-deficient mice with biliary fibrosis. Methods ABCB4-deficient mice on the FVB/NJ genetic background were crossed to two distinct genetic backgrounds (TLR4-sufficient C3H/HeN and TLR4-deficient C3H/HeJ) for more than 10 generations. The two congenic knockout and the two corresponding wild-type mouse lines were treated with a single dose of DEN for 48 hours. Phenotypic differences were assessed by measuring hepatic collagen contents, inflammatory markers (ALT, CRP, IL6) as well as hepatic apoptosis (TUNEL) and proliferation (Ki67) rates. Results Hepatic collagen accumulation is significantly reduced in ABCB4-/-:TLR4-/-double-deficient mice. After DEN challenge, apoptosis, proliferation and inflammatory markers are decreased in TLR4-deficient in comparison to TLR4-sufficient mice. When combining ABCB4 and TLR4 deficiency with DEN treatment, hepatic IL6 expression and proliferation rates are lowest in fibrotic livers from the double-deficient line. Consistent with these effects, selective digestive tract decontamination in ABCB4-/- mice also led to reduced tumor size and number after DEN. Conclusion This study demonstrates that liver injury upon DEN challenge depends on pre-existing fibrosis and genetic background. The generation of ABCB4-/: TLR4-/- double-deficient mice illustrates that TLR4-deficiency protects against hepatic injury in a preclinical mouse model of chronic liver disease

CCL20 mediates lipopolysaccharide induced liver injury and is a potential driver of inflammation and fibrosis in alcoholic hepatitis

OBJECTIVE: Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury. DESIGN: CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo. RESULTS: CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs. CONCLUSIONS: Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH

Promotion of Hepatocellular Carcinoma by the Intestinal Microbiota and TLR4

Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota, and TLR4 activation in non-bone marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease

CCL20 mediates lipopolysaccharide induced liver injury and is a potential driver of inflammation and fibrosis in alcoholic hepatitis

Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury

ESRRG and PERM1 Govern Mitochondrial Conversion in Brite/Beige Adipocyte Formation

When exposed to cold temperatures, mice increase their thermogenic capacity by an expansion of brown adipose tissue mass and the formation of brite/beige adipocytes in white adipose tissue depots. However, the process of the transcriptional changes underlying the conversion of a phenotypic white to brite/beige adipocytes is only poorly understood. By analyzing transcriptome profiles of inguinal adipocytes during cold exposure and in mouse models with a different propensity to form brite/beige adipocytes, we identified ESRRG and PERM1 as modulators of this process. The production of heat by mitochondrial uncoupled respiration is a key feature of brite/beige compared to white adipocytes and we show here that both candidates are involved in PGC1α transcriptional network to positively regulate mitochondrial capacity. Moreover, we show that an increased expression of ESRRG or PERM1 supports the formation of brown or brite/beige adipocytes in vitro and in vivo. These results reveal that ESRRG and PERM1 are early induced in and important regulators of brite/beige adipocyte formation.ISSN:1664-239

A Genetic Model to Study the Contribution of Brown and Brite Adipocytes to Metabolism

UCP1-dependent thermogenesis is studied to define new strategies to ameliorate obesity and type 2 diabetes; however, animal models are mostly limited to germline mutations of UCP1, which can effect adaptive changes in UCP1-independent pathways. We develop an inducible mouse model for the sequential ablation of UCP1+ brown and brite/beige adipocytes in adult mice. We demonstrate that activated brown adipocytes can increase systemic energy expenditure (EE) by 30%, while the contribution of brite/beige UCP1+ cells is <5%. Notably, UCP1+ adipocytes do not contribute to circulating FGF21 levels, either at room temperature or after cold exposure. We demonstrate that the FGF21-mediated effects on EE and glucose homeostasis are partially dependent on the presence of UCP1+ cells, while the effect on weight loss is not. In conclusion, acute UCP1+ cell deletion may be a useful model to study the impact of brown and brite/beige adipocytes on metabolism.ISSN:2666-3864ISSN:2211-124

Fibrosis progression depends on TLR4 status.

<p>ABCB4-deficient, ABCB4/TLR4-double-deficient and the corresponding control mice were compared with respect to hepatic damage and fibrosis at 16 weeks of age when fibrosis is already established (n = 6 per line; 3 males, 3 females). (A) Hepatic collagen contents, measured as μg hydroxyproline (HYP) per g liver. (B) Relative hepatic <i>Col1a1</i> mRNA expression. ABCB4^+/+:TLR4^+/+ mice were set as 1. (C) Relative hepatic <i>a-Sma</i> mRNA expression. ABCB4^+/+:TLR4^+/+ mice were set as 1. (D) Relative hepatic <i>Crp</i> mRNA expression. ABCB4^+/+:TLR4^+/+ mice were set as 1. (E) Relative hepatic <i>Il6</i> mRNA expression. ABCB4^+/+:TLR4^+/+ mice were set as 1. (F) Plasma alanine aminotransferase (ALT) activities, measured in units per liter (U/l). (G) Hepatocellular apoptosis rates. (H) Hepatocellular proliferation rates. *p < 0.05.</p

TLR4 deficiency protects against DEN-induced liver injury in fibrotic liver.

<p>ABCB4-deficient and ABCB4/TLR4-double-deficient mice were subjected to DEN at 16 weeks of age (n = 12 per line; 6 males, 6 females). (A) Plasma ALT activities, measured in U/l. (B) Relative hepatic <i>Col1a1</i> mRNA expression. ABCB4^-/-:TLR4^+/+ mice were set as 1. (C) Relative hepatic <i>a-SMA</i> mRNA expression. ABCB4^-/-:TLR4^+/+ mice were set as 1. (D) Relative hepatic <i>Il6</i> expression. ABCB4^-/-: TLR4^+/+ mice were set as 1. (E) Relative hepatic <i>Crp</i> expression. ABCB4^-/-:TLR4^+/+ mice were set as 1. (D) Hepatocellular apoptosis rate. (E) Hepatocellular proliferation rate. *p<0.05; **p<0.01.</p

TLR4 deficiency increases apoptosis and proliferation at early fibrotic stages.

<p>ABCB4-deficient and ABCB4/TLR4-double-deficient mice were subjected to DEN at 7 weeks of age (n = 12 per line; 6 males, 6 females). (A) Plasma ALT activities, measured in U/l. (B) Relative hepatic <i>Col1a1</i> mRNA expression. ABCB4^-/-:TLR4^+/+ mice were set as 1. (C) Relative hepatic <i>a-SMA</i> mRNA expression. ABCB4^-/-:TLR4^+/+ mice were set as 1. (D) Relative hepatic <i>Il6</i> expression. ABCB4^-/-:TLR4^+/+ mice were set as 1. (E) Hepatocellular apoptosis rate. (F) Hepatocellular proliferation rate. *p < 0.05.</p

Gut sterilization leads to reduced tumor growth.

<p>ABCB4-deficient mice underwent DEN treatment and received a combination of four antibiotics (Abx) in drinking water until sacrifice (SAC). (A) Experimental design. (B) Macroscopic appearance of livers from ABCB4-deficient mice with or without Abx treatment. (C) Tumor sizes and numbers in the two experimental groups (-ABx: n = 10; +ABx: n = 6). *p<0.05.</p