Benedetta Riccardi

Human macrophage differentiation induces OCTN2–mediated L-carnitine transport through stimulation of mTOR–STAT3 axis

l‐Carnitine, in addition to playing a fundamental role in the β‐oxidation of fatty acids, has been recently identified as a modulator of immune function, although the mechanisms that underlie this role remain to be clarified. In this study, we addressed the modulation of l‐carnitine transport and expression of related transporters during differentiation of human monocytes to macrophages. Whereas monocytes display a modest uptake of l‐carnitine, GM‐CSF–induced differentiation massively increased the saturable Na+‐dependent uptake of l‐carnitine. Kinetic and inhibition analyses demonstrate that in macrophage l‐carnitine transport is mediated by a high‐affinity component (Km ∼4 µM) that is identifiable with the operation of OCTN2 transporter and a low‐affinity component (Km > 10 mM) that is identifiable with system A for neutral amino acids. Consistently, both SLC22A5/OCTN2 and SLC38A2/SNAT2 are induced during the differentiation of monocytes to macrophages at gene and protein levels. Elucidation of GM‐CSF signaling demonstrates that the cytokine causes the activation of mTOR kinase, leading to the phosphorylation and activation of STAT3, which, in turn, is responsible for OCTN2 transcription. SLC22A5/OCTN2 therefore emerges as a novel member of the set of genes markers of macrophage differentiation.

Functional activity of L-carnitine transporters in human airway epithelial cells

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects.

In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitor.

Abstract 1. The metabolism of CHF 6001, a novel PDE4 inhibitor, was determined in vitro in mouse, rat, dog, monkey and human microsomes and hepatocytes and in vivo in plasma, urine, feces and bile of rats after intravenous and intratracheal administration. 2. The behavior of CHF 6001 in microsomes and hepatocytes changed across species. CYP3A4/5 isoenzymes were identified to be the primary enzymes responsible for the metabolism of CHF 6001 in human liver microsomes. 3. In the rat, CHF 6001 was found extensively metabolized in urine, feces and bile, but not in plasma, where CHF 6001 was the main compound present. The metabolite profiles were different in the four biological matrices from both qualitative and quantitative point of view. 4. CHF 6001 was metabolized through hydrolysis with the formation of the alcohol CHF 5956, loss of a chlorine atom, loss of the N-oxide, hydroxylation, loss of the cyclopropylmethyl group in the alcohol moiety, conjugation with glucuronic acid, glutathione and cysteine–glycine. 5. The major metabolite present in the bile was isolated and characterized by nuclear magnetic resonance analysis. It derived from CHF 6001 through contraction of the pyridine-N-oxide ring to N-hydroxy pyrrole and conjugation with glucuronic acid.

High Throughput Screening of β-Amyloid Secretion Inhibitors Using Homogenous Time-Resolved Fluorescence

A cell-based assay using homogeneous time-resolved fluorescence has been developed for high throughput screening of putative β-amyloid (Aβ)production inhibitors. In this assay, total Aβ is detected by simply adding two commercially available antibody complexes. The first was a biotinylated monoclonal antibody (4G8), specifically recognizing an epitope comprising the residues 17-24 of the Aβ peptide, complexed with europium cryptate-streptavidin conjugate. The second was a polyclonal antibody (BioS-N), raised against the N-terminus of the Aβ peptide, complexed with an allophycocyanin-anti rabbit antibody conjugate. Binding of the two complexes to the Aβ peptide brought europium cryptate (fluorescence donor) and allophycocyanin (fluorescence acceptor) into close proximity, consequently a fluorescent resonance energy transfer signal was produced upon excitation at 337 nm. The resulting fluorescence signal (665 nm) was then detected using a Discovery™ or a ViewLux™ reader. Detection of Aβ by the proposed method is possible at concentrations of approximately 1 nM. The method was employed for the detection of Aβ secreted from a stable transfected human neuroglioma cell line (H4) overexpressing a mutated form of the human amyloid precursor protein (APP695NL) and developed for robotic automation. At optimized conditions, signal-to-background ratios exceeding 5 and Z factors around 0.7 were achieved in a 384-well format. High throughput screening of 56,913 potential Aβ production inhibitors led to identification of new non-cytotoxic and cell permeable compounds with potencies in the submicromolar range.

Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1β and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.