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International audience; Il‐17A is considered to guide liver inflammation and fibrosis. Using human liver slice culture, we aim to study whether IL‐17A is capable of influencing the fibrogenesis, but also, to look into lymphatic immune cell composition, secreting IL‐17A. Using human liver samples (F0‐F4) and blood samples, collected after partial hepatectomy due to different pathologies, we analyzed IL‐17A secretion and immune cell profile. Liver tissue was used for primary culture of human liver slices, followed by subsequent cytokine stimulation and analysis by Elisa of fibrotic markers. The Huh7.5.1 cell line was used for SeaHorse and WB analysis. IL‐17A concentration in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells are the main sources of IL‐17A in both compartments, liver and blood. Moreover, the presence of liver Th17IL‐ 17A+INFγ+ cells were detected. IL‐17A stimulation of human liver slices increases the expression of profibrotic markers. Stimulation by IL‐17A+TGF‐β1 removes the reserve respiratory capacity in Huh7.5.1 cells, while RA addition is capable to restore it and to reverse the expression of LC3II/LC3I ratio. IL‐17A, secreted by Th17 and MAIT cells, induce the expression of pro‐fibrotic markers. However, the level of hepatic IL‐17A secretion is probably more related to the underlying pathologies that cause fibrosis rather than to the mechanism of fibrosis itself. There is also a terrain of communication between IL‐17A and retinoic acid in the liver, that should be investigated.

International audience; Background: Liver fibrosis can result in end-stage liver failure and death.Aim: To examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model.Methods: Fibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected from patients after liver resection. Human liver slices (HLS) were cultivated for up to 21 days. Hepatitis C virus (HCV) infection, alcohol (ethanol stimulation) and steatosis (palmitate stimulation) were examined in fibrotic (F2 to F4) liver slices infected (or not) with HCV. F0-F1 HLS were used as controls. At day 0, either ursodeoxycholic acid (choleretic and hepatoprotective properties) and/or α-tocopherol (antioxidant properties) were added to standard of care on HLS and fibrotic liver slices, infected (or not) with HCV. Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay.Results: The cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs. Expression of the biomarkers of fibrosis and the progression to steatosis (estimated by triglycerides production) was increased with the addition of HCV and /or ethanol or palmitate. From day 15 of the follow-up studies, a significant decrease of both transforming growth factor β-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.Conclusion: These results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver (viral, alcoholic, nonalcoholic steatohepatitis liver diseases) and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination.

International audience

International audience; Background: To study the first steps of liver molecular fibrogenesis in our ex vivo model of human liver slices culture. Methods: Human fibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected after liver resection. Human liver slices of 350 μm (2.7x106cells per slice) were cultivated for up to 21 days. Fibrotic liver slices F2 to F4 (FLS) were infected (or not) with HCVcc supernatant infection [Con1/C3 (genotype1b)] (MOI = 0.1) (INF FLS). F0-F1 HLS (HLS) were used as controls. At day 0, either ursodeoxycholic acid (UDCA, only choleretic and hepatoprotective properties) and/or a-tocopherol (Toco, anti-oxydant properties which could reduce fibrosis progression) were added to standard of care concentrations on HLS and FLS. The following fibrosis markers expression were assayed in HLS, in FLS and in INF FLS, [tumor growth factor b (TGFβ), Heat shock protein 47 (Hsp47), Alpha smooth muscle actin (α-Sma), Procollagen1 A1 (Procl1A1), Matrix metalloproteinases 2, 9 (MMP-2, 9), Vascular Endothelial Growth Factor (VEGF)] and checked by RT-qPCR and the triglyceride production by ELISA assays. Results: The TGFβ, Hsp47,α-Sma, Procl1A1, MMP-2, MMP-9, VEGF expression and the triglyceride production increased significantly in both HLS and FLS but to a higher extent in FLS as compared to HLS, with a triglyceride production higher x 3.2] in FLS. HCV infection increased significantly the fibrotic markers expression and triglyceride production in non- infected (NI) FLS and INF FLS to a higher extent in INF FLS as compared to controls. The induced molecular fibrogenesis significantly increased, particularly true for the advanced stage of fibrosis, including cirrhosis. Under a-tocopherol exposure, a significant reduction of TGF-β RNA expression was observed in NINF and INF FLS but only for the stage F4. UDCA did not impact TGFβ RNA expression in NINF F0 to F4 liver slices, but significantly reduces its expression in F2F3F4 infected liver slices. Combination of UDCA and Toco treatments did not result in an additive effect and only reduced the TGFβ RNA expression in F2-F4 INF HLS but not in F2-F4 NI HLS. Conclusion: Our ex vivo model of human liver slices culture, supporting hepatocyte-specific gene expression for 21 days, provides an easy tool for studying human molecular liver fibrogenesis which could accelerate the evaluation of the potency of new anti-fibrotic therapies, alone or in combination.

International audience; BACKGROUNDLiver fibrosis can result in end-stage liver failure and death.AIMTo examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model.METHODSFibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected from patients after liver resection. Human liver slices (HLS) were cultivated for up to 21 days. Hepatitis C virus (HCV) infection, alcohol (ethanol stimulation) and steatosis (palmitate stimulation) were examined in fibrotic (F2 to F4) liver slices infected (or not) with HCV. F0-F1 HLS were used as controls. At day 0, either ursodeoxycholic acid (choleretic and hepatoprotective properties) and/or α-tocopherol (antioxidant properties) were added to standard of care on HLS and fibrotic liver slices, infected (or not) with HCV. Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay.RESULTSThe cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs. Expression of the biomarkers of fibrosis and the progression to steatosis (estimated by triglycerides production) was increased with the addition of HCV and /or ethanol or palmitate. From day 15 of the follow-up studies, a significant decrease of both transforming growth factor β-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.CONCLUSIONThese results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver (viral, alcoholic, nonalcoholic steatohepatitis liver diseases) and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination

Despite all the medical advances mortality due to cirrhosis and hepatocellular carcinoma, the end stages of fibrosis, continuously increases. Recent data suggest that liver fibrosis is guided by type 3 inflammation with IL-17A at the top of the line. The storage of vitamin A and its active metabolites, as well as genetics, can influence the development and progression of liver fibrosis and inflammation. Retinoic acid (active metabolite of vitamin A) is able to regulate the differentiation of IL-17A+/IL-22–producing cells as well as the expression of profibrotic markers. IL-17A and its pro-fibrotic role in the liver is the most studied, while the interaction and communication between IL-17A, IL-22, and vitamin A–active metabolites has not been investigated. We aim to update what is known about IL-17A, IL-22, and retinoic acid in the pathobiology of liver diseases.

The liver is the central metabolic organ in the human body, and also plays an essential role in innate and adaptive immunity. While mouse models offer significant insights into immune-inflammatory liver disease, human immunology differs in important respects. It is not easy to address those differences experimentally. Therefore, to improve the understanding of human liver immunobiology and pathology, we have established precision-cut human liver slices to study innate immunity in human tissue. Human liver slices collected from resected livers could be maintained in ex vivo culture over a two-week period. Although an acute inflammatory response accompanied by signs of tissue repair was observed in liver tissue following slicing, the expression of many immune genes stabilized after day 4 and remained stable until day 15. Remarkably, histological evidence of pre-existing liver diseases was preserved in the slices for up to 7 days. Following 7 days of culture, exposure of liver slices to the toll-like receptor (TLR) ligands, TLR3 ligand Poly-I:C and TLR4 ligand LPS, resulted in a robust activation of acute inflammation and cytokine genes. Moreover, Poly-I:C treatment induced a marked antiviral response including increases of interferons IFNB, IL-28B and a group of interferon-stimulated genes. Therefore, precision-cut liver slices emerge as a valuable tool to study human innate immunity.