Thomas Gicquel

Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1β or TNF-α Release from Human Hepatic Stellate Cells

Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.

Quantitative Analysis of Acetaminophen and its Primary Metabolites in Small Plasma Volumes by Liquid Chromatography–Tandem Mass Spectrometry

A fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the simultaneous determination of acetaminophen (APAP) and its glucuronide and sulfate metabolites (APAP-GLU and APAP-SUL) in small plasma volumes. This method included a simple step of sample preparation and a chromatographic separation on an LC-MS-MS system equipped with an electrospray ionization source and a tandem triple quadrupole mass spectrometer in multiple reaction monitoring mode. The analytes and internal standard, APAP deuterated analog, were separated on a C18 column (3.0 µm, 2.1 × 100 mm), using aqueous 1% formic acid and methanol (80:20, v/v) as the mobile phase. The LC-MS-MS method was validated for accuracy, precision, linearity, extraction efficiency, process efficiency and matrix effect. Calibration curves were obtained by fortifying drug-free plasma and ranges of linearity were set between 0.25-20 mg/L. The mean correlation coefficients, r², were >0.99 for APAP and its metabolites. The inter-day and intra-day precision values were less than 11.75 and 13.03%, respectively, at the lower limit of quantification concentration. The usability of the method was demonstrated by studying APAP metabolism in C57BL/6J wild-type and obese ob/ob female mice, in which only small plasma volumes were available. The results showed that APAP glucuronidation was enhanced in obese mice, suggesting that changes in APAP metabolism could modify its toxicity in obesity and related fatty liver disease.

Involvement of matrix metalloproteinases (MMPs) and inflammasome pathway in molecular mechanisms of fibrosis.

Fibrosis is a basic connective tissue lesion defined by the increase in the fibrillar extracellular matrix (ECM) components in tissue or organ. Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turn-over of ECM and so they are suggested to be important in tissue remodelling observed during fibrogenic process associated with chronic inflammation. Tissue remodelling is the result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of ECM components markedly controlled by the MMPs/TIMP imbalance. We previously showed an association of the differences in collagen deposition in the lungs of bleomycin-treated mice with a reduced molar pro-MMP-9/TIMP-1 ratio. Using the carbon tetrachloride (CCl4) preclinical model of liver fibrosis in mice, we observed a significant increase in collagen deposition with increased expression and release of tissue inhibitors of metalloproteinase (TIMP)-1 both at 24 h and 3 weeks later. This suggests an early altered regulation of matrix turnover involved in the development of fibrosis. We also demonstrated an activation of NLRP3-inflammasome pathway associated with the IL-1R/MyD88 signalling in the development of experimental fibrosis both in lung and liver. This was also associated with an increased expression of purinergic receptors mainly P2X7. Finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of tissue remodelling and fibrosis.

A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen

Obesity and nonalcoholic fatty liver disease (NAFLD) can increase susceptibility to hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are poorly understood. For acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during NAFLD. The first aim of our study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, human HepaRG cells were incubated for one week with stearic acid or oleic acid, in the presence of different concentrations of insulin. Although cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids, CYP2E1 activity was significantly increased only by stearic acid. CYP2E1 activity was reduced by insulin and this effect was reproduced in cultured primary human hepatocytes. Next, APAP cytotoxicity was assessed in HepaRG cells with or without lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations showed that the loss of ATP and glutathione was almost always greater in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole, recovery of ATP was significantly higher in the presence of stearate with low (2.5mM) or high (20mM) concentrations of APAP. Levels of APAP-glucuronide were significantly enhanced by insulin. Hence, HepaRG cells can be used as a valuable model of NAFLD to unveil important metabolic and hormonal factors which can increase susceptibility to drug-induced hepatotoxicity.

Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages

Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD‐like receptor‐family protein 3 (NLRP3)‐inflammasome leading to the pro‐inflammatory cytokine, interleukin (IL)‐1β, release in the lung. The NLRP3‐inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5′‐[γ‐thio]triphosphate tetralithium salt (ATPγS) and 2′,3′‐O‐(4‐benzoylbenzoyl) adenosine 5′‐triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL‐1β from human monocyte‐derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL‐1α and IL‐6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7R). NLRP3 and IL‐1β mRNA expression were induced from LPS‐primed macrophages, but also after 5‐h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7R were not induced by BzATP. We observed that P2X7R antagonists, A‐438079 and A‐740003, were able to reduce the release of IL‐1β, but not of IL‐1α and IL‐6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7R‐NLRP3 inflammasome pathway in the secretion of IL‐1β from ATP‐stimulated human macrophages, and suggest that P2X7R were not involved in IL‐1α and IL‐6 release. This study also points out that repression of the P2X7R represents a novel potential therapeutic approach to control fibrosis in lung injury.

Amatoxins (α- and β-Amanitin) and Phallotoxin (Phalloidin) Analyses in Urines Using High-Resolution Accurate Mass LC–MS Technology

Mycotoxin intoxications can result from the consumption of amatoxins like α- and β-amanitin or of phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatoxins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography-high-resolution-mass spectrometry, LC-HR-MS), Q Exactive™ (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 × 2.1 mm, 2.6 µm) using a gradient of 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for α-amanitin, β-amanitin, phalloidin and IS of 1.9, 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC-HR-MS system was performed by extracting the exact mass value of each protonated species using a 5-p.p.m. mass window, which was 919.3614, 920.3455, 789.3257 and 388.1586 for α-amanitin, β-amanitin, phalloidin and IS, respectively. Calibration curves were obtained by spiking drug-free urine at 1-100 ng/mL. Mean correlation coefficients, r(2), were above 0.99 for each amatoxins and phalloidin. According to currently accepted validation procedures, the method was tested for selectivity, calibration, accuracy, matrix effect, precision and recovery. Authentic urine samples from 43 patients suffering from a suspected intoxication with mushrooms were analyzed by LC-HR-MS, and the results were compared with ELISA competitive immunoassay. The LC-HR-MS presented large benefits over immunoassay of being specific, faster and more sensitive, making it suitable for daily emergency toxicological analysis.

Roflumilast n-oxide associated with PGE2 prevents the neutrophil elastase-induced production of chemokines by epithelial cells.

Neutrophil chemotaxis is involved in the lung inflammatory process in conditions such as chronic obstructive pulmonary disease (COPD). Neutrophil elastase (NE), one of the main proteases produced by neutrophils, has an important role in the inflammatory process via the release of chemokines from airway epithelial cells. It was recently shown that roflumilast N-oxide has therapeutic potential in COPD. The aim of the present study was to investigate roflumilast N-oxides effect on NE-induced chemokine production and signaling pathways in A549 epithelial cells. A549 cells were incubated with NE for 30min, washed with PBS and then cultured for 2h (for measurement of mRNA expression) and 24h (for chemokine release) or for 5 to 30min (for protein phosphorylation assays). Prior to the addition of NE, cells were also pre-incubated with prostaglandin E2 (PGE2), alone and in combination with roflumilast N-oxide. Addition of NE was associated with elevated chemokine production by A549 cells and induction of the p38α pathway. In contrast when combined with PGE2, the roflumilast N-oxide had an additive effect on the inhibition of NE-induced chemokine release and p38α and other kinases activation. In conclusion, we demonstrated that NE is able to increase the release of chemokines from epithelial cells via the activation of p38α MAP-kinase and that roflumilast N-oxide when combined with PGE2 lowers NE-induced kinase activation and chemokine production.

In vitro characterization of NPS metabolites produced by human liver microsomes and the HepaRG cell line using liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis: application to furanyl fentanyl

BACKGROUNDnIdentification of metabolites is of importance in the challenge of new psychoactive substances (NPS) as it could improve the detection window in biological matrices in clinical and forensic cases of intoxication. Considering the numerous and diverse NPS reported each year, producers increasingly appear today to be targeting non-controlled synthetic opioids, involving fentanyl derivatives such as furanyl fentanyl (Fu-F).nnnOBJECTIVEnThis work aims to investigate and compare metabolites of Fu-F using two in vitro experimental approaches.nnnMETHODSnCYP- and UGT-dependent metabolites of Fu-F were investigated by means of analyses of both human liver microsome (HLM) and hepatic (HepaRG) cell line incubates using liquid chromatography with high-resolution mass detection and, subsequently, compared and confronted to recently published data.nnnRESULTSnSeventeen Fu-F metabolites were produced and several metabolic pathways can be postulated. HLMs and HepaRG cultures appear to be complementary: HepaRG cells produced 9 additional metabolites, but which appear to be minor in vivo metabolites. Specific* and/or abundant Fu-F metabolites are dihydrodiol-Fu-F*, norFu-F* and despropionylfentanyl. However, norFu-F seems to be inconstantly observed in in vivo cases. Furthermore, a sulfate metabolite presents at significant rate in urine obtained from FU-F users was not identified here, as in another in vitro study.nnnCONCLUSIONnHLMs represent an acceptable first choice tool for a single NPS metabolism study in forensic laboratories. Dihydrodiol-Fu-F and despropionylfentanyl could be proposed as reliable metabolites to be recorded in HRMS libraries in order to improve detection of Fu-F users. Nevertheless, additional verifications of in vivo data remain necessary to confirm relevant blood and urinary metabolites of Fu-F.

The release of pro-inflammatory cytokines is mediated via mitogen-activated protein kinases rather than by the inflammasome signalling pathway in keratinocytes

Toll‐like receptors (TLRs) are expressed in the skin and airway epithelial tissues, which are the most important sites of host–pathogen interactions. TLRs recognize the 3‐D structures of pathogen‐associated molecules and are therefore useful markers of the innate immune response. Here, we investigated the role of lipopolysaccharides and monosodium urate (MSU) crystals in the activation of the TLR and NOD‐like receptor (NLR) pathways in human keratinocytes. Analysis of the inflammasome compounds revealed that NOD‐like receptor P3 and TLR4, both of which are components of inflammasome complexes involved in the activation of interleukin (IL)‐1β, were not expressed in keratinocytes. Transcriptomic analysis showed that the combination of MSU and lipopolysaccharide priming did not elicit significant results compared to MSU treatment, which induced the expression of TLR2, IL‐6 and IL‐8/chemokine (C‐X‐C motif) ligand 8 CXCL8 in the keratinocyte cell line HaCaT. Furthermore, MSU promoted the phosphorylation of extracellular signal‐regulated kinase 1/2 and MAPK14/p38α mitogen‐activated protein kinases. We concluded that MSU stimulates a pro‐inflammatory response in keratinocytes via mitogen‐activated protein kinase pathway to induce production of IL‐8/CXCL8 chemokine (C‐X‐C motif) ligand 8 and TLR2.

Opsonisation of nanoparticles prepared from poly(β-hydroxybutyrate) and poly(trimethylene carbonate)-b-poly(malic acid) amphiphilic diblock copolymers: Impact on the in vitro cell uptake by primary human macrophages and HepaRG hepatoma cells.

The present work reports the investigation of the biocompatibility, opsonisation and cell uptake by human primary macrophages and HepaRG cells of nanoparticles (NPs) formulated from poly(β-malic acid)-b-poly(β-hydroxybutyrate) (PMLA-b-PHB) and poly(β-malic acid)-b-poly(trimethylene carbonate) (PMLA-b-PTMC) diblock copolymers, namely PMLA800-b-PHB7300, PMLA4500-b-PHB4400, PMLA2500-b-PTMC2800 and PMLA4300-b-PTMC1400. NPs derived from PMLA-b-PHB and PMLA-b-PTMC do not trigger lactate dehydrogenase release and do not activate the secretion of pro-inflammatory cytokines demonstrating the excellent biocompatibility of these copolymers derived nano-objects. Using a protein adsorption assay, we demonstrate that the binding of plasma proteins is very low for PMLA-b-PHB-based nano-objects, and higher for those prepared from PMLA-b-PTMC copolymers. Moreover, a more efficient uptake by macrophages and HepaRG cells is observed for NPs formulated from PMLA-b-PHB copolymers compared to that of PMLA-b-PTMC-based NPs. Interestingly, the uptake in HepaRG cells of NPs formulated from PMLA800-b-PHB7300 is much higher than that of NPs based on PMLA4500-b-PHB4400. In addition, the cell internalization of PMLA800-b-PHB7300 based-NPs, probably through endocytosis, is strongly increased by serum pre-coating in HepaRG cells but not in macrophages. Together, these data strongly suggest that the binding of a specific subset of plasmatic proteins onto the PMLA800-b-PHB7300-based NPs favors the HepaRG cell uptake while reducing that of macrophages.