Chantal Combe

Adrenalectomy enhances pro-inflammatory cytokines gene expression, in the spleen, pituitary and brain of mice in response to lipopolysaccharide.

To assess the possible influence of endogenous glucocorticoids on cytokine expression in the brain, adrenalectomized mice and sham operated mice were injected with saline or lipopolysaccharide (LPS, 10 micrograms/mouse, subcutaneously) and the levels of transcripts for IL-1 alpha, IL-1 beta, IL-1ra, IL-6 and tumor necrosis factor-alpha (TNF alpha) were determined 2 h after treatment in the spleen, pituitary, hypothalamus, hippocampus and striatum, using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Levels of IL-1 beta were measured by ELISA in plasma and tissues of mice sacrificed after the administration of LPS or saline. LPS induced the expression of pro-inflammatory cytokines at the mRNA level in all tissues under investigation, except for TNF alpha in the hippocampus. This effect was potentiated by adrenalectomy in the spleen for IL-1 alpha and IL-1ra, the pituitary for cytokines other than IL-1ra, the hypothalamus for all cytokines, the hippocampus for cytokines other than TNF alpha, and the striatum for IL-1 alpha and IL-6. In saline-treated mice, adrenalectomy increased IL-1 alpha and IL-1 beta gene expression in the hypothalamus and IL-1 alpha gene expression in the hippocampus and striatum. LPS increased plasma and tissue levels of IL-1 beta, as determined by ELISA, and this effect was potentiated by adrenalectomy in plasma and tissues other than the spleen. These results can be interpreted to suggest that endogenous glucocorticoids regulate the neural components of the host response to infection and inflammation by inhibiting cytokine expression in peripheral organs and the brain.

A functional screening identifies five microRNAs controlling glypican-3: role of miR-1271 down-regulation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the major primary liver cancer. Glypican‐3 (GPC3), one of the most abnormally expressed genes in HCC, participates in liver carcinogenesis. Based on data showing that GPC3 expression is posttranscriptionally altered in HCC cells compared to primary hepatocytes, we investigated the implication of microRNAs (miRNAs) in GPC3 overexpression and HCC. To identify GPC3‐regulating miRNAs, we developed a dual‐fluorescence FunREG (functional, integrated, and quantitative method to measure posttranscriptional regulations) system that allowed us to screen a library of 876 individual miRNAs. Expression of candidate miRNAs and that of GPC3 messenger RNA (mRNA) was measured in 21 nontumoral liver and 112 HCC samples. We then characterized the phenotypic consequences of modulating expression of one candidate miRNA in HuH7 cells and deciphered the molecular mechanism by which this miRNA controls the posttranscriptional regulation of GPC3. We identified five miRNAs targeting GPC3 3′‐untranslated region (UTR) and regulating its expression about the 876 tested. Whereas miR‐96 and its paralog miR‐1271 repressed GPC3 expression, miR‐129‐1‐3p, miR‐1291, and miR‐1303 had an inducible effect. We report that miR‐1271 expression is down‐regulated in HCC tumor samples and inversely correlates with GPC3 mRNA expression in a particular subgroup of HCC. We also report that miR‐1271 inhibits the growth of HCC cells in a GPC3‐dependent manner and induces cell death. Conclusion: Using a functional screen, we found that miR‐96, miR‐129‐1‐3p, miR‐1271, miR‐1291, and miR‐1303 differentially control GPC3 expression in HCC cells. In a subgroup of HCC, the up‐regulation of GPC3 was associated with a concomitant down‐regulation of its repressor miR‐1271. Therefore, we propose that GPC3 overexpression and its associated oncogenic effects are linked to the down‐regulation of miR‐1271 in HCC. (HEPATOLOGY 2013)

Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices

During cholestasis, bile acids accumulate in the liver, and induce cellular alterations. Cholestasis is a major cause of liver fibrosis. We have used precision-cut liver slices (PCLS) in culture to investigate the effects of bile acids on hepatic cells. Rat PCLS were placed on an insert in a vial containing culture medium, and gently agitated on a roller platform. PCLS were treated with 100 μM taurolithocholate (TLC), taurodeoxycholate (TDC) or taurocholate (TC) for 24 or 48 h. PCLS viability was measured, and immunohistochemistry was performed with antibodies against active caspase 3, platelet-derived growth factor (PDGF) receptor-β and ED-A fibronectin. TDC and TLC, two hydrophobic bile acids, induced hepatocyte necrosis and apoptosis, whereas TC, an hydrophilic bile acid, improved slice viability as compared with controls. Both TDC and TC induced biliary epithelial cell proliferation, together with portal fibroblast proliferation and activation, as shown by PDGF receptor-β and ED-A fibronectin expression. TLC induced biliary epithelial cell apoptosis. Our results indicate that individual bile acids induce cell type-specific effects in a complex liver microenvironment. The fact that PCLS support biliary epithelial cell and portal fibroblast proliferation will make this model very useful for the study of the mechanisms involved in portal fibrosis.

ATP binding cassette transporter ABC1 is required for the release of interleukin‐1β by P2X7‐stimulated and lipopolysaccharide‐primed mouse Schwann cells

Schwann cells are best known as myelinating glial cells of the peripheral nervous system, but they also participate actively in the sphere of immunity by producing pro‐inflammatory cytokines, such as interleukin‐1β (IL‐1β). In a previous study, we demonstrated that posttranslational processing of IL‐1β by immune‐challenged Schwann cells required the P2X7 receptor. Remarkably, the release of IL‐1β was not associated with cell death, indicating the involvement of an active mechanism. ATP binding cassette (ABC) transporters are known to transport leaderless secretory proteins, such as IL‐1β; therefore, we investigated whether such transporters were at work in Schwann cells. Mouse Schwann cells expressed ABC1 transporter mRNA and displayed the functional protein. Glybenclamide and diisothiocyanato‐stilbene‐disulfonic acid (DIDS), two blockers of chloride fluxes that drive the export activity of ABC1 transporters, inhibited IL‐1β release without altering its intracellular processing. Enhancing chloride efflux potentiated the release of IL‐1β, while decreasing it led to a strong reduction in its release. Because the stimulation of the P2X7 receptor also activates a chloride conductance, we investigated the possibility of a sole anionic pathway mobilized by the P2X7 receptor and ABC1. Glybenclamide and DIDS had no significant effects on the P2X7‐activated chloride current suggesting therefore the existence of two different pathways. In summary, ABC1 transporters are required for the release of IL‐1β by mouse Schwann cells. Being associated together with chloride conductance, P2X7 receptors and ABC1 transporters delineate a subtle and complex regulation of IL‐1β production in mammalian Schwann cells. Furthermore, ABC1 transporters could be a target of therapeutic interest for regulating IL‐1β activity in neuroinflammation disorders. GLIA, 2004.

Effects of lipopolysaccharide and glucocorticoids on expression of interleukin-1β converting enzyme in the pituitary and brain of mice

The present study was carried out to determine whether those factors which regulate the expression of IL-1 beta in immune and non-immune tissues are also able to regulate the expression of ICE. In a first experiment, mice were injected with LPS (10 micrograms/mouse, i.p.) and killed before, 1, 3 or 6 h after the injection. Total RNAs were extracted from the spleen, pituitary and brain (hippocampus and hypothalamus) and submitted to RT-PCR to determine the levels of ICE mRNA as compared to beta 2 microglobulin mRNA. ICE mRNAs were more abundant in the spleen and hippocampus than in the pituitary and hypothalamus but they were not significantly altered by LPS treatment. In a second experiment mice were submitted to adrenalectomy or a 15 min restraint stress and injected with saline or LPS (10 micrograms/mouse. sc). They were killed 1-2 h later and total RNA was extracted from the same tissues as in experiment 1. Adrenalectomized mice had significantly higher ICE mRNA levels whereas stressed mice had significantly lower ICE mRNA levels than their respective controls. These results are discussed with respect to the possible regulatory influence of glucocorticoids on the expression of ICE.

Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices

Background: The debate concerning the potential remodelling and/or reversibility of cirrhotic lesions and biliary fibrosis is still open.

Specific activation of the different fibrogenic cells in rat cultured liver slices mimicking in vivo situations

Due to the loss of cell–cell and cell–matrix interactions, cell culture models poorly mimic the in vivo situation. Therefore, we tested the applicability of precision-cut liver slices (PCLS) to study the early activation of the two main liver fibrogenic cell subpopulations: hepatic stellate cells (HSC) and portal fibroblasts (PF). PCLS were treated with thioacetamide or acetaminophen to induce HSC activation. In PCLS culture, both were able to trigger centrolobular lesion and HSC activation as observed in vivo. However, thioacetamide also presented a toxic effect on portal tract cells. In this PCLS model of centrolobular lesion, the antioxidant N-acetylcysteine was able to prevent acetaminophen-induced injury. To induce a specific activation of PF, PCLS were treated with epidermal growth factor or β-oestradiol. As in vivo, epidermal growth factor and β-oestradiol induced bile duct epithelial cell proliferation accompanied by PF activation; however, β-oestradiol also triggers sinusoidal cell proliferation. We demonstrated that treatments usually used in vivo to induce liver fibrosis allow, in cultured PCLS, the specific activation of the two main liver fibrogenic cell subpopulations, making this model very useful to study the mechanisms involved in early fibrogenic cell activation.

Role of interleukin-1β and tumour necrosis factor-α in lipopolysaccharide-induced sickness behaviour: a study with interleukin-1 type I receptor-deficient mice: Brain IL-1 and TNF-α in sickness behaviour

Interleukin‐1 (IL‐1) mediates symptoms of sickness during the host response to infection. IL‐1 exerts its effects via several subtypes of receptors. To assess the role of IL‐1 receptor type I (IL‐1RI) in the sickness‐inducing effects of IL‐1, IL‐1β and the cytokine inducer lipopolysaccharide were administered to IL‐1RI‐deficient mice (IL‐1RI–/–). Sickness was assessed by depression of social exploration, anorexia, immobility and body weight loss. IL‐1RI–/– mice were resistant to the sickness‐inducing effects of IL‐1β administered intraperitoneally (2 µg/mouse) and intracerebroventricularly (2 ng/mouse), but still fully responsive to lipopolysaccharide administered intraperitoneally (2.5 µg/mouse) and intracerebroventricularly (3 ng/mouse). The sensitivity of IL‐1RI–/– mice to lipopolysaccharide was not due to a higher brain expression of proinflammatory cytokines other than IL‐1, since lipopolysaccharide‐induced expression of brain IL‐1 β, tumour necrosis factor‐α (TNF‐α) and IL‐6 transcripts were identical in IL‐1RI–/– and control mice when measured by semiquantitative reverse‐transcriptase polymerase chain reaction 1 h after treatment. Blockade of TNF‐α action in the brain by intracerebroventricular administration of a fragment of the soluble TNF receptor, TNF binding protein (3.6 µg/mouse), attenuated the depressive effects of intraperitoneal injection of lipopolysaccharide (1 µg/mouse) on behaviour in IL‐1RI–/– but not in control mice. Since IL‐1RI–/– mice were not more sensitive to intracerebroventricularly TNF‐α (50 ng) than control mice, these results indicate that IL‐1RI mediates the sickness effect of IL‐1 and that TNF‐α simply replaces IL‐1 when this last cytokine is deficient.

Smoothelin, a new marker to determine the origin of liver fibrogenic cells.

AIM To explore this hypothesis that smooth muscle cells may be capable of acquiring a myofibroblastic phenotype, we have studied the expression of smoothelin in fibrotic conditions. METHODS Normal liver tissue (n = 3) was obtained from macroscopically normal parts of hepatectomy, taken at a distance from hemangiomas. Pathological specimens included post-burn cutaneous hypertrophic scars (n = 3), fibrotic liver tissue (n = 5), cirrhotic tissue (viral and alcoholic hepatitis) (n = 5), and hepatocellular carcinomas (n = 5). Tissue samples were fixed in 10% formalin and embedded in paraffin for immunohistochemistry or were immediately frozen in liquid nitrogen-cooled isopentane for confocal microscopy analysis. Sections were stained with antibodies against smoothelin, which is expressed exclusively by smooth muscle cells, and α-smooth muscle actin, which is expressed by both smooth muscle cells and myofibroblasts. RESULTS In hypertrophic scars, α-smooth muscle actin was detected in vascular smooth muscle cells and in numerous myofibroblasts present in and around nodules, whereas smoothelin was exclusively expressed in vascular smooth muscle cells. In the normal liver, vascular smooth muscle cells were the only cells that express α-smooth muscle actin and smoothelin. In fibrotic areas of the liver, myofibroblasts expressing α-smooth muscle actin were detected. Myofibroblasts co-expressing α-smooth muscle actin and smoothelin were observed, and their number was slightly increased in parallel with the degree of fibrosis (absent in liver with mild or moderate fibrosis; 5% to 10% positive in liver showing severe fibrosis). In cirrhotic septa, numerous myofibroblasts co-expressed α-smooth muscle actin and smoothelin (more than 50%). In hepatocellular carcinomas, the same pattern of expression for α-smooth muscle actin and smoothelin was observed in the stroma reaction surrounding the tumor and around tumoral cell plates. In all pathological liver samples, α-smooth muscle actin and smoothelin were co-expressed in vascular smooth muscle cells. CONCLUSION During development of advanced liver fibrosis, a subpopulation of myofibroblasts expressing smoothelin may be derived from vascular smooth muscle cells, illustrating the different cellular origins of myofibroblasts.