Effects of the nerve growth factor and its carrier protein on the inflammatory response from human monocytes.

BACKGROUND: The nerve growth factor (NGF) has been previously shown to be involved in cellular proliferation, differentiation, survival, or wound healing. This factor displays a variety of biological effects that yet remain to be explored. Previous data on cell lines show a pro-inflammatory role of NGF on monocytes. OBJECTIVES: The objective of the study was to investigate the pro-inflammatory effect of NGF, using a model of fresh human monocytes. METHODS: Monocytes obtained from PBMC were exposed to NGF at various concentrations. Alternatively, monocytes were exposed to BSA, the NGF carrier protein without the NGF. Gene expression and cytokine release in the supernatant were monitored. RESULTS: We found that NGF increased the expression of pro-inflammatory, chemotactic, and remodeling genes such as interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and C-X-C motif ligand (CXCL)8. The protein levels of CXCL8 and matrix metalloproteinase (MMP)-9 were also increased in the cell supernatants following NGF exposure. BSA alone was found to drive part of this response, bringing nuance to the inflammatory potential of the NGF. CONCLUSION: These data suggest that NGF is able to enhance monocyte inflammatory responses once cells are stimulated with another signal but is possibly not able to directly activate it. This could have implications for example in patients with bacterial infections, where NGF could worsen the local inflammation by over-activating immune cells.

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Impact of JAK/STAT inhibitors on human monocyte-derived-macrophages stimulated by cigarette smoke extract and lipopolysaccharide.

The main risk factor for chronic obstructive pulmonary disease (COPD) is cigarette smoke (CS). It can alter many immune cells functions such as phagocytosis, efferocytosis and cytokine production. Cytokines play a role in the orchestration of inflammation in COPD. The JAK/STAT pathways are among the most important signalling components of cytokines. The objective of this work was to investigate the role of the JAK/STAT pathway with regard to cytokine release and microsphere uptake capacity (to minimize the non-specific scavenging) in human monocyte-derived-macrophages (MDMs). The MDMs were stimulated by cigarette smoke extract (CSE) alone or in combination with lipopolysaccharide (LPS). CSE alone was not associated with significant changes in the cytokine, with the exception of IL-8/CXCL8 production. However, CSE disturbed cytokine production in LPS-stimulated MDMs. CSE increase CXCL-8 and CCL2 release in LPS-stimulated monocyte-derived macrophages and suppressed the production of IL-6 and CXCL1 in these cells. CSE also decreased microsphere uptake capacity by MDMs. Then, CSE + LPS-stimulated MDMs were treated with two different JAK inhibitors. AG490 (specific inhibitor of JAK2) and ruxolitinib (inhibitor of JAK1 and JAK2). JAK/STAT inhibitors, particularly ruxolitinib, attenuated in cytokine production without completely inhibiting when compared with dexamethasone. On the other hand, the cells exposed to dexamethasone are nearly unable to capture the microspheres, while both JAK inhibitors do not affect the uptake capacity. In summary, our results showed the versatility of ruxolitinib which might bring a better balance disturbance of cytokine release and uptake capacity. The information regarding the distinctive effect of JAK/STAT inhibitors may be useful in the development of novel treatments for COPD.

Circumsporozoite Protein of Plasmodium berghei- and George Baker Virus A-Derived Peptides Trigger Efficient Cell Internalization of Bioconjugates and Functionalized Poly(ethylene glycol)-b-poly(benzyl malate)-Based Nanoparticles in Human Hepatoma Cells.

In order to identify the peptides, selected from the literature, that exhibit the strongest tropism towards human hepatoma cells, cell uptake assays were performed using biotinylated synthetic peptides bound to fluorescent streptavidin or engrafted onto nanoparticles (NPs), prepared from biotin-poly(ethylene glycol)-block-poly(benzyl malate) (Biot-PEG-b-PMLABe) via streptavidin bridging. Two peptides, derived from the circumsporozoite protein of Plasmodium berghei- (CPB) and George Baker (GB) Virus A (GBVA10-9), strongly enhanced the endocytosis of both streptavidin conjugates and NPs in hepatoma cells, compared to primary human hepatocytes and non-hepatic cells. Unexpectedly, the uptake of CPB- and GBVA10-9 functionalized PEG-b-PMLABe-based NPs by hepatoma cells involved, at least in part, the peptide binding to apolipoproteins, which would promote NP's interactions with cell membrane receptors of HDL particles. In addition, CPB and GBVA10-9 peptide-streptavidin conjugates favored the uptake by hepatoma cells over that of the human macrophages, known to strongly internalize nanoparticles by phagocytosis. These two peptides are promising candidate ligands for targeting hepatocellular carcinomas.

Non-oxidative ethanol metabolism in human hepatic cells in vitro: Involvement of uridine diphospho-glucuronosyltransferase 1A9 in ethylglucuronide production.

Ethanol is the most frequently psychoactive substance used in the world, leading to major public health problems with several millions of deaths attributed to alcohol consumption each year. Metabolism of ethanol occurs mainly in the liver via the predominant oxidative metabolism pathway involving phase I enzymes including alcohol dehydrogenases (ADH), cytochrome P450 (CYP) 2E1 and catalase. In a lesser extent, an alternative non-oxidative pathway also contributes to the metabolism of ethanol, which involves the uridine diphospho-glucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II enzymes. Using liquid chromatography-high resolution mass spectrometry, ethylglucuronide (EtG) and ethylsulfate (EtS) produced respectively by UGT and SULT conjugation and detected in various biological samples are direct markers of alcohol consumption. We report herein the efficient non-oxidative metabolic pathway of ethanol in human differentiated HepaRG cells compared to primary human hepatocytes (HH). We showed dose- and time-dependent production of EtS and EtG after ethanol (25 or 50 mM) treatment in culture media of differentiated HepaRG cells and HH and a significant induction of CYP2E1 mRNA expression upon acute ethanol exposure in HepaRG cells. These differentiated hepatoma cells thus represent a suitable in vitro human liver cell model to explore ethanol metabolism and more particularly EtG and EtS production. In addition, using recombinant HepG2 cells expressing different UGT1A genes, we found that UGT1A9 was the major UGT involved in ethanol glucuronidation.

Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity.

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.