Correction of all-trans retinoic acid deficiency in alcoholic cirrhosis lessens the excessive inflammatory monocyte response: a translational study.

BACKGROUND & AIMS: Patients with alcoholic liver disease (ALD) have vitamin A (VA) deficiency and an enhanced immune response associated with disease severity. All-trans retinoic acid (ATRA), a VA-active metabolite, has anti-inflammatory effects and its deficiency could contribute to the exacerbated proinflammatory reaction. The aim of this study was to investigate the effects of ATRA/VA deficiency and supplementation on the monocyte response in ALD. METHODS: Vitamin A and ATRA plasma levels were quantified in ALD patients and healthy subjects (HS). The in vitro effect of ATRA on lipopolysaccharide (LPS)-induced TNF-α production by human peripheral blood mononuclear cells (PBMC) was assessed by ELISA and RT-PCR. The activation pattern of peritoneal macrophages (PerMΦ) and circulating monocytes isolated from VA-deficient mice and ALD patients, respectively, was evaluated by flow cytometry, quantification of TNF-α and NO2 production. RESULTS: Alcoholic liver disease patients (n = 85) showed plasmatic VA deficiency that was correlated with scores of severity and with the hepatic venous pressure gradient. ATRA levels correlated significantly with VA levels. In vitro, ATRA pretreatment decreased the overproduction of TNF-α by LPS-stimulated PBMC of ALD patients. In vivo, VA deficiency in mice was associated with increased activation of PerMΦ, while oral ATRA supplementation normalized it. CONCLUSION: For the first time, we show that VA/ATRA deficiencies in ALD patients are associated with disease severity. Furthermore, our data strongly suggest that the VA deficiency observed in ALD patients might participate in the pathophysiology of the disease by priming immune cells, and that ATRA supplementation could downregulate the deleterious proinflammatory state in cirrhosis and might thus be of therapeutic use.

Marked 25-hydroxyvitamin D deficiency is associated with poor prognosis in patients with alcoholic liver disease.

BACKGROUND & AIMS: Vitamin D deficiency has been frequently reported in advanced liver disease. However, its influence on alcoholic liver disease (ALD) has been poorly elucidated. We investigated the association of vitamin D with clinical, biological, and histological parameters and survival in ALD patients. Furthermore, we explored the effect of vitamin D treatment on ALD patient peripheral blood mononuclear cells (PBMCs), and in a murine experimental model of ALD. METHODS: Serum levels of 25-hydroxyvitamin D [25(OH)D] were determined in 324 Caucasian ALD patients and 201 healthy controls. In vitro experiments on vitamin D pre-treated PBMCs evaluated TNFα production by ELISA in culture supernatants. Mice were submitted to an ethanol-fed diet and some of them were orally supplemented three times per week with 1,25(OH)2D. RESULTS: Severe deficiency in 25(OH)D (<10 ng/ml) was significantly associated with higher aspartate aminotransferase levels (p=1.00 × 10(-3)), increased hepatic venous pressure gradient (p=5.80 × 10(-6)), MELD (p=2.50 × 10(-4)), and Child-Pugh scores (p=8.50 × 10(-7)). Furthermore, in multivariable analysis, a low 25(OH)D concentration was associated with cirrhosis (OR=2.13, 95% CI=1.18-3.84, p=0.013) and mortality (HR=4.33, 95% CI=1.47-12.78, p=7.94 × 10(-3)) at one year. In addition, in vitro, 1,25(OH)2D pretreatment decreased TNFα production by stimulated PBMCs of ALD patients (p=3.00 × 10(-3)), while in vivo, it decreased hepatic TNFα expression in ethanol-fed mice (p=0.04). CONCLUSIONS: Low 25(OH)D levels are associated with increased liver damage and mortality in ALD. Our results suggest that vitamin D might be both a biomarker of severity and a potential therapeutic target in ALD.

Glycogen synthase kinase 3 involvement in the excessive proinflammatory response to LPS in patients with decompensated cirrhosis.

BACKGROUND & AIMS: In decompensated cirrhosis, the early innate immune response to the Toll-like receptor 4 (TLR4) agonist, lipopolysaccharides (LPS), is characterized by a hyper-production of pro-inflammatory cytokines and hypo-production of the anti-inflammatory cytokine IL-10. In LPS-stimulated non-cirrhotic immune cells, the constitutively active glycogen synthase kinase (GSK) 3 favors pro- vs. anti-inflammatory cytokines, by acting on gene induction. However, in these cells, TLR4 dampens its own pro-inflammatory response by inducing early (within minutes) AKT-mediated phosphorylation of GSK3ß (one of two GSK3 isoforms) on Ser9. Phosphorylation of GSK3ß (Ser9) inhibits its activity, decreases pro-inflammatory cytokines, and increases IL-10. Thus, we investigated the role of GSK3 in LPS-induced cytokine production by peripheral blood mononuclear cells (PBMCs) or monocytes from patients with advanced cirrhosis and normal subjects. METHODS: Cells were pre-incubated with or without GSK3 inhibitor (SB216763 or lithium chloride) for 1h and then stimulated with LPS. Cytokine production was assessed at mRNA and secreted proteins levels, by real-time RT-PCR at 1h and ELISA at 20 h, respectively. GSK3ß phosphorylation was assessed using Western blotting. RESULTS: In cirrhotic and normal PBMCs pretreated with GSK3 inhibitors, LPS-induced production of pro-inflammatory proteins TNF-α and IL-12p40 was significantly decreased while that of IL-10 was increased. LPS-induced, AKT-mediated phosphorylation of GSK3ß on Ser9 found in normal monocytes, was abolished in cirrhotic cells. CONCLUSIONS: GSK3 is involved in the early TLR4-mediated pro-inflammatory response in patients with decompensated cirrhosis. This was associated with a defect in AKT-mediated GSK3ß phosphorylation resulting in unrestricted 'pro-inflammatory' activity of the enzyme.