Anne-Marie Cassard-Doulcier

Intestinal microbiota determines development of non-alcoholic fatty liver disease in mice

Objective Non-alcoholic fatty liver disease (NAFLD) is prevalent among obese people and is considered the hepatic manifestation of metabolic syndrome. However, not all obese individuals develop NAFLD. Our objective was to demonstrate the role of the gut microbiota in NAFLD development using transplantation experiments in mice. Design Two donor C57BL/6J mice were selected on the basis of their responses to a high-fat diet (HFD). Although both mice displayed similar body weight gain, one mouse, called the ‘responder’, developed hyperglycaemia and had a high plasma concentration of pro-inflammatory cytokines. The other, called a ‘non-responder’, was normoglycaemic and had a lower level of systemic inflammation. Germ-free mice were colonised with intestinal microbiota from either the responder or the non-responder and then fed the same HFD. Results Mice that received microbiota from different donors developed comparable obesity on the HFD. The responder-receiver (RR) group developed fasting hyperglycaemia and insulinaemia, whereas the non-responder-receiver (NRR) group remained normoglycaemic. In contrast to NRR mice, RR mice developed hepatic macrovesicular steatosis, which was confirmed by a higher liver concentration of triglycerides and increased expression of genes involved in de-novo lipogenesis. Pyrosequencing of the 16S ribosomal RNA genes revealed that RR and NRR mice had distinct gut microbiota including differences at the phylum, genera and species levels. Conclusions Differences in microbiota composition can determine response to a HFD in mice. These results further demonstrate that the gut microbiota contributes to the development of NAFLD independently of obesity.

Nonalcoholic fatty liver disease: from pathogenesis to patient care

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. It encompasses a wide spectrum of liver lesions, from pure steatosis to end-stage liver disease with cirrhosis and hepatocellular carcinoma. Nonalcoholic steatohepatitis corresponds only to one stage of NAFLD. As NAFLD can be considered a liver manifestation of the metabolic syndrome, its prevalence is high in obese people and in patients who have type 2 diabetes—insulin resistance is one of the key elements of the pathogenesis of NAFLD. This disease is often asymptomatic in the absence of decompensated cirrhosis, but should be suspected in patients with elevated aminotransferase levels or radiological evidence of a fatty liver or hepatomegaly. Liver fibrosis is associated with age over 50 years, obesity, diabetes and high triglyceride levels. Liver biopsy is the only way to assess the histologic features of necrotic inflammation and fibrosis that define nonalcoholic steatohepatitis and to determine its probable prognosis. The prognosis is good for pure steatosis, whereas the presence of necrotic inflammation is associated with a significant risk of progression to cirrhosis and, possibly, hepatocellular carcinoma. Lifestyle changes, such as dietary modifications and exercise, are recommended. To date, there have been very few randomized, placebo-controlled trials of drug treatments for NAFLD.

Harmful effect of adipose tissue on liver lesions in patients with alcoholic liver disease.

BACKGROUND & AIMS Adipose tissue is an important source of cytokines. Excess weight is an independent risk factor for steatosis, acute alcoholic hepatitis (AAH), and cirrhosis in patients with alcoholic liver disease (ALD). In this study, we investigated the role of adipose tissue in human ALD. PATIENTS AND METHODS Fifty patients with ALD underwent liver and abdominal subcutaneous adipose tissue biopsies and supplied blood samples for the investigation of cytokine gene expression and secretion, as well as liver histology. RESULTS The levels of TNF-alpha and IL-10 in adipose tissue were higher in patients with AAH. IL-10 level in adipose tissue was also correlated with fibrosis score. TNF-alpha gene expression in adipose tissue was correlated with Maddrey score, blood C-reactive protein (CRP) concentration and liver IL-6 concentration. IL-6 production levels in the liver were higher in patients with AAH and correlated with AAH score, liver histological lesions, liver TNF-alpha concentration, Maddrey score, and blood CRP concentration. Plasma concentrations of soluble forms of TNF-receptor were correlated with inflammatory lesions in the liver, Maddrey score and fibrosis score. CONCLUSION In patients with ALD, inflammation occurs not only in the liver, but also in the adipose tissue. Adipose tissue inflammation is correlated with the severity of pathological features in the liver. Our findings may account for the harmful interactions between body mass index, AAH, fibrosis, and cirrhosis in alcoholic patients.

Housekeeping gene variability in the liver of alcoholic patients.

BACKGROUND Quantification of gene expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) requires normalization to an endogenous reference gene termed housekeeping gene (HKG). Many of the commonly used HKGs are regulated and vary under experimental conditions and disease stages. Alcoholic liver disease (ALD) is associated with several different liver histological lesions that may modulate HKG expression. We investigated the variability of commonly used HGKs (18S, β-actin, glyceraldehyde-3-phosphate [GAPDH], and arginine/serine-rich splicing factor [SFRS4]) in the liver of patients with ALD. METHODS Fifty consecutive patients at different stages of ALD underwent liver biopsy. The stability of HKG was assessed according to liver histological lesions. RESULTS β-actin had the highest coefficient of dispersion (COD) (23.9). β-actin tended to decrease with steatosis and to increase with alcoholic hepatitis; β-actin also increased in patients with both alcoholic hepatitis and cirrhosis. GAPDH and SFRS4 COD were 2.8 and 2.1, respectively. GAPDH was decreased with steatosis and increased with alcoholic hepatitis and fibrosis. 18S had the lowest COD (1.4). Both 18S and SFRS4 levels were not significantly modified with respect to all alcohol-induced liver histological lesions. CONCLUSIONS In patients with ALD, the most constantly expressed HKGs are 18S and SFRS4. These genes are appropriate reference genes for normalization of RT-qPCR in the liver of patients with ALD. The use of other HKGs such as β-actin or GAPDH would lead to misinterpretation of the results.

Glucocorticoid-induced leucine zipper: A key protein in the sensitization of monocytes to lipopolysaccharide in alcoholic hepatitis.

Glucocorticoid‐induced leucine zipper (GILZ), a recently identified protein induced by glucocorticoids (GCs), inhibits the nuclear factor κB pathway and the activation of monocytes/macrophages by lipopolysaccharides (LPS). This study aimed to elucidate the contribution of GILZ to the pathogenesis of alcoholic hepatitis (AH): we (1) assessed GILZ expression in the livers of patients with AH and (2) treated patients with severe AH with GCs (prednisolone 40 mg/day) and studied the effect of GILZ modulation on circulating monocyte function. We quantified GILZ expression in the livers of 42 consecutive alcoholic patients (21 with and 21 without AH). GILZ messenger RNA (mRNA) levels were lower in the livers of patients with AH versus those without AH (P < 0.05). We collected circulating monocytes from patients with severe AH before and 48 hours after GC treatment to quantify GILZ expression and cytokine secretion. GC treatment induced significantly higher levels of GILZ mRNA than that observed before treatment and impaired LPS‐induced tumor necrosis factor‐α (TNF‐α) and regulated upon activation, normal T cell–expressed secretion (RANTES) by these monocytes. We transfected circulating monocytes with GILZ small interfering RNA (siRNA), specifically blocking GILZ expression, to demonstrate the role of GILZ in mediating GC effect. GILZ siRNA abrogated the effect of GC treatment on LPS‐induced TNF‐α and RANTES secretion. Conclusion: Low expression of GILZ may contribute to liver inflammation in AH. GCs enhance GILZ expression, abrogating macrophage sensitivity to LPS and proinflammatory cytokine secretion. These findings may explain the beneficial effect of GC treatment in patients with severe AH. (HEPATOLOGY 2007;46:1986–1992.)

LIM-Only Protein FHL2 Activates NF-κB Signaling in the Control of Liver Regeneration and Hepatocarcinogenesis

ABSTRACT Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor–NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2−/− macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2−/− mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

Independent and opposite associations of trunk fat and leg fat with liver enzyme levels

Background: In contrast to trunk fat mass (TFM), which is associated with cardiovascular risk markers, leg fat mass (LFM) displays independent protective effects against atherosclerosis. Little is known about the respective influence of central and peripheral adiposity on liver enzyme levels.

Alcohol withdrawal alleviates adipose tissue inflammation in patients with alcoholic liver disease

Patients with alcoholic liver disease (ALD) display inflammation of the subcutaneous adipose tissue (SAT) which correlates with liver lesions. We examined macrophage markers and polarization in the SAT of alcoholic patients and adipokine expression according to liver inflammation; we studied the consequences of alcohol withdrawal.

CXCR4 dysfunction in non-alcoholic steatohepatitis in mice and patients.

Homing of inflammatory cells to the liver is key in the progression of non-alcoholic steatohepatitis (NASH). An abnormal response of CD4+ T-cells from obese mice to the chemotactic effect of CXCL12 has been reported but the mechanism involved in this process and relevance in patients are unknown. We aimed to explore the mechanism involved in the abnormal chemotaxis of CXC chemokine ligand 12 (CXCL12) in several mouse models of NASH and the relevance in the context of human non-alcoholic fatty liver disease (NAFLD). We assessed chemotactic responsiveness of CD4+ T-cells to CXCL12, the effect of AMD3100, a CXC chemokine receptor 4 (CXCR4) antagonist, in mice and lymphocytes from patients with NAFLD, and the affinity of CXCL12 for CXCR4. CXCL12-promoted migration of CD4+ T-cells from three different mouse models of NASH was increased and dependent of CXCR4. CD4+ T-cells from patients with NASH, but not from patients with pure steatosis, responded more strongly to the chemotactic effect of CXCL12, and this response was inhibited by AMD3100. Treatment with AMD3100 decreased the number of CD4+ T-cells to the liver in ob/ob mice. CXCL12 expression in the liver, CXCR4 and CXCR7 expression in CD4+ T-cells were not increased in three different mouse models of NASH. However, the affinity of CXCL12 for CXCR4 was increased in CD4+ T-cells of ob/ob mice. In conclusion, the CXCL12/CXCR4 pathway contributes in both mice and patients to the enhanced recruitment of CD4+ T-cells in NASH. An increased affinity of CXCL12 to CXCR4 rather than a higher expression of the chemokine or its receptors is involved in this process.

555 ALCOHOL WITHDRAWAL ALLEVIATES SUBCUTANEOUS ADIPOSE TISSUE INFLAMMATION IN PATIENTS WITH ALCOHOLIC LIVER DISEASE

555 ALCOHOL WITHDRAWAL ALLEVIATES SUBCUTANEOUS ADIPOSE TISSUE INFLAMMATION IN PATIENTS WITH ALCOHOLIC LIVER DISEASE C.S. Voican, M. Njike-Nakseu, H. Boujedidi, N. Barri-Ova, L. Bouchet-Delbos, H. Agostini, S. Maitre, S. Prevot, A.-M. Cassard-Doulcier, S. Naveau, G. Perlemuter. Hopital Antoine Beclere, INSERM, U 996, IPSIT, Clamart, Unite de Recherche Clinique Paris-Sud, Bicetre, France E-mail: cosmin_voican@yahoo.com