Corinne Martin-Chouly

Inorganic arsenic alters expression of immune and stress response genes in activated primary human T lymphocytes

Inorganic arsenic, a carcinogenic environmental contaminant, exerts immunosuppressive effects on human T lymphocytes. In particular, interleukin-2 (IL2) secretion and T cell proliferation are reduced when peripheral blood mononuclear cells (PBMC) from individuals chronically exposed to arsenic are stimulated ex vivo with lectins such as phytohemaglutinin (PHA). However, it is not clear whether the metalloid directly acts on T cells or blocks monocyte-dependent accessory signals activated by PHA. We report that in vitro pre-treatment of PBMC with sodium arsenite (NaAs) reduces IL2 secretion and T cell proliferation induced by PHA, but does not prevent expression of monocyte-derived cytokines (IL1, IL6, TNFα) functioning as lymphocyte-activating factors. In addition, we found that NaAs delays induction of IL2 and IL2 receptor α chain (IL2RA) mRNA levels in human primary isolated T cells activated by PHA. Kinetic analysis showed that NaAs pre-treatment first inhibits, but thereafter markedly increases, induction of IL2 and IL2RA mRNA when T cells are stimulated with PHA for 8 h and 72 h, respectively. We conducted whole genome microarray-based analysis of gene expression in primary T cell cultures derived from independent donors. NaAs systematically and significantly up-regulated a set of 35 genes, including several immune and stress genes, such as IL13, granulocyte-macrophage colony stimulating factor, lymphotoxin α and heme oxygenase-1 (HO-1). Up-regulation of HO-1, a stress and immunosuppressive protein, was rapidly detectable, both in T cells and in PBMC treated with NaAs. Inhibition of the immunosuppressive activity of HO-1 in PBMC however failed to prevent NaAs-dependent inhibition of T cell proliferation induced by PHA. Our findings demonstrate that, at least in vitro, inorganic arsenic acts directly on human T cells and impairs their activity, probably independently of HO-1 expression and monocyte-related accessory signals.

Global effects of inorganic arsenic on gene expression profile in human macrophages

Inorganic arsenic, a major environmental contaminant, exerts immunosuppressive effects towards human cells. We previously demonstrated that relevant environmental concentrations of inorganic arsenic altered morphology and functions of human primary macrophages, suggesting interference with macrophage differentiation program. The goal of this study was to determine global effect of low concentrations of arsenic trioxide (As(2)O(3)) on gene expression profile in human primary macrophages, in order to identify molecular targets of inorganic arsenic, especially those relevant of macrophage differentiation process. Using a pan-genomic microarray, we demonstrate that exposure of human blood monocyte-derived macrophages to 1microM As(2)O(3) for 72h, a non-cytototoxic concentration, results in up-regulation of 32 genes and repression of 91 genes. Among these genes, 26 are specifically related to differentiation program of human macrophages. Particularly, we validated that As(2)O(3) strongly alters expression of MMP9, MMP12, CCL22, SPON2 and CXCL2 genes, which contribute to major macrophagic functions. Most of these metalloid effects were reversed when As(2)O(3)-treated macrophages were next cultured in arsenic-free medium. We also show that As(2)O(3) similarly regulates expression of this macrophagic gene subset in human alveolar macrophages, the phenotype of which closely resembles that of blood monocyte-derived macrophage. In conclusion, our study demonstrates that environmentally relevant concentrations of As(2)O(3) impair expression of macrophage-specific genes, which fully supports interference of metalloid with differentiation program of human macrophages.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstructive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.

Anti-Inflammatory Effect of Fluvastatin on IL-8 Production Induced by Pseudomonas aeruginosa and Aspergillus fumigatus Antigens in Cystic Fibrosis

Background Early in life, patients with cystic fibrosis (CF) are infected with microorganisms including bacteria and fungi, particularly Pseudomonas aeruginosa and Aspergillus fumigatus. Since recent research has identified the anti-inflammatory properties of statins (besides their lipid-lowering effects), we investigated the effect of fluvastatin on the production of the potent neutrophil chemoattractant chemokine, IL-8, in whole blood from CF patients, stimulated by Pseudomonas aeruginosa (LPS) and Aspergillus fumigatus (AFA) antigens. Results Whole blood from adult patients with CF and from healthy volunteers was collected at the Rennes University Hospital (France). Blood was pretreated for 1 h with fluvastatin (0–300 µM) and incubated for 24 h with LPS (10 µg/mL) and/or AFA (diluted 1/200). IL-8 protein levels, quantified by ELISA, were increased in a concentration-dependent manner when cells were stimulated by LPS or AFA. Fluvastatin strongly decreased the levels of IL-8, in a concentration-dependent manner, in whole blood from CF patients. However, its inhibitory effect was decreased or absent in whole blood from healthy subjects. Furthermore, the inhibition induced by fluvastatin in CF whole blood was reversed in the presence of intermediates within the cholesterol biosynthesis pathway, mevalonate, farnesyl pyprophosphate or geranylgeranyl pyrophosphate that activate small GTPases by isoprenylation. Conclusions For the first time, the inhibitory effects of fluvastatin on CF systemic inflammation may reveal the important therapeutic potential of statins in pathological conditions associated with the over-production of pro-inflammatory cytokines and chemokines as observed during the manifestation of CF. The anti-inflammatory effect could be related to the modulation of the prenylation of signalling proteins.

Increased extracellular matrix metalloproteinase inducer (EMMPRIN) expression in pulmonary fibrosis.

Extracellular matrix metalloproteinase inducer (EMMPRIN) was examined on bronchoalveolar lavage fluids (BALFs) and lung tissue from patients with fibrosis (usual interstitial pneumonia–idiopathic pulmonary fibrosis [UIP-IPF], n = 15; diffuse parenchymal lung diseases without IPF characteristics on computerized tomography scan, n = 8) and without fibrosis (n = 6). In UIP-IPF, EMMPRIN staining was increased in areas of fibrosis, mainly in macrophages and in epithelial cells. EMMPRIN was also found in the extracellular medium with significant levels in patients with lung fibrosis compared to subjects without fibrosis. Moreover, macrophages from patients with lung fibrosis spontaneously produce EMMPRIN. These findings show that EMMPRIN is increased in lung fibrosis.

Synthesis and Potential Anti-Inflammatory Activity of Some Tetrahydrophthalazinones

A solid-phase route for the preparation of 4a,5,8,8a-tetrahydrophthalazinon-1-ones employing the Diels-Alder reaction has been developed. Some of the new compounds have been tested for inhibition of LPS-stimulated TNF-α production in human whole blood from patients with chronic obstructive pulmonary disease (COPD). This evaluation revealed two compounds 17 and 18 of interest, incorporating an arylpiperazine moiety, which were found to inhibit LPS- induced TNF-α release like the well known anti-inflammatory PDE4 inhibitors, rolipram and roflumilast.

Solid-phase synthesis and evaluation of libraries of substituted 4,5-dihydropyridazinones as vasodilator agents

The solid‐phase parallel preparation of a library of 4,5‐dihydropyridazin‐3(2H)‐one derivatives substituted at position 6 with piperazinylmethyl or tetrahydroquinolinylmethyl groups and analogues (3) is reported. Polymer‐supported γ‐keto‐δ‐aminoesters prepared from Wang resin reacted with hydrazine or methylhydrazine to afford pyridazinones in good yields after a cyclization cleavage approach. We have evaluated these novel analogues and several compounds of other series (1, 2) for their vasorelaxant effect. Among the products tested, 3I and 3d proved to be efficacious and potent relaxant agents of the isolated rat aorta. Inhibitors of phosphodiesterase (PDE3), responsible for the breakdown of cyclic AMP in the vascular smooth muscle, are currently developed for cardiac heart failure because of their inotropic effect and coronary vasodilatation. We had expected that the vasodilatation induced by 3l, as efficient as reference PDE3 inhibitors, milrinone or CI‐930, to be due to PDE3 inhibition. However 3I and 3d exhibited a low inhibitory effect against PDE3 isoenzyme activity. These compounds induced a significant vasorelaxation, which could be of therapeutic interest even if their mechanism of action remains to be determined.

Regulation of MMP/TIMP Balance as Therapeutic Target in Pulmonary Diseases

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases, regulated by endogenous tissue inhibitors (TIMPs) and inducer (EMMPRIN). The demonstration of a functional role of MMPs in pulmonary diseases raises the possibility of therapeutic intervention targeting MMP/TIMP balance to prevent pathological processes.

026 Augmentation des taux d’EMMPRIN et de MT1-MMP dans le plasma et le parenchyme pulmonaire des patients atteints de mucoviscidose

Introduction Les maladies respiratoires chroniques, dont la mucoviscidose, sont associees a une inflammation et un remodelage tissulaire. Ce processus fait intervenir un desequilibre dans la balance proteases-antiproteases impliquant les metalloproteinases de la matrice (MMPs) et conduit a une degradation ou un exces de matrice extra-cellulaire. Le role des MMPs dans le developpement physiopathologique de la mucoviscidose reste mal connu. Methodes 14 plasmas et 27 expectorations de patients adultes stables ont ete recoltees. Les gelatinases ont ete analysees par zymo-graphie puis quantifiees par methode ELISA. L’EMMPRIN et la MT1-MMP ont ete mis en evidence puis quantifies par western blot. Les TIMPs ont ete quantifies par methode ELISA. Dans les 20 parenchymes pulmonaires (pieces operatoires de transplantation), la localisation de l’EMMPRIN a ete determinee par immuno-histochimie. La severite de la mucoviscidose a ete jugee sur 5 criteres : VEMS et CVF, l’indice de masse corporelle (IMC), le score de Shwachman (clinico-radiologique) et le score de Brasfield (radiologique). Resultats Dans le plasma, les taux de pro-MMP-2 sont inversement correles avec tous les criteres de severite (i.e. VEMS : r 2 = 0,75, p = 0,003), alors que les taux d’EMMPRIN et de MT1-MMP sont positivement correles (i.e. VEMS respectivement : r 2 = 0,77, p = 0,004 et r 2 = 0,58, p = 0,002). En revanche, nous n’avons pas observe de correlation pour la pro-MMP-9, TIMP-1 et TIMP-2 dans le plasma. Dans les expectorations, nous n’avons pas mis en evidence de correlation entre les criteres de severite et les taux de pro-MMP-2, pro-MMP-9, TIMP-1, TIMP-2 et EMMPRIN. Sur les coupes de parenchymes pulmonaires, nous avons pu montrer que les principales localisations de l’EMMPRIN sont les macrophages alveolaires et les pneumocytes de type 2. Conclusion Lorsque la severite de la mucoviscidose des patients adultes stables augmente, les taux de pro-MMP-2 diminuent alors que ceux d’EMMPRIN et de MT1-MMP augmentent. Une activa-tion de la pro-MMP-2 par la MT1-MMP dont les productions sont stimulees par l’EMMPRIN peut expliquer la diminution de la pro-MMP-2 observee dans notre etude. Nos travaux suggerent aussi que les macrophages alveolaires et les pneumocytes de type 2 sont impliques dans la production d’EMMPRIN.