Amandine Vincent

Marion Thauvin

Elodie Quévrain

Emilie Mathieu

Sarah Layani-Moreno

Philippe Layani

Ines Seksik

Sophie Batinic-Haberle

Clotilde Vriz

Nicolas Policar

Delsuc

Sarah Layani

Philippe Seksik

Ines Batinic-Haberle

Sophie Vriz

Clotilde Policar

Nicolas Delsuc
email: nicolas.delsuc@ens.psl.eu

Evaluation of the compounds commonly known as superoxide dismutase and catalase mimics in cellular models

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Introduction

Oxidative stress is associated with many physiological pathologies such as diabetes, inflammatory bowel diseases, cancers, and neurodegenerative diseases [START_REF] Halliwell | Free radicals in biology and medicine, Fifth[END_REF]. It results from an imbalance between the concentration of reactive species (RS) responsible for deleterious damages, and antioxidant defenses [START_REF] Auten | Oxygen toxicity and reactive oxygen species: the devil is in the details[END_REF]. Among naturally occurring antioxidants, the metalloenzymes superoxide dismutases (SOD), catalase (CAT) and glutathione peroxidases (GPxs) play crucial roles in redox homeostasis. SODs catalyze the dismutation of the superoxide anion O O or alcohols. These enzymes are thus an extraordinary source of inspiration for the design of low molecular weight complexes possessing antioxidant properties that could be used as catalytic drugs to supplement these enzymes under pathological situations [START_REF] Soldevila-Barreda | Approaches to the design of catalytic metallodrugs[END_REF]. Complexes mimicking SOD have attracted much interest since superoxide is the first reactive species in the O 2 reduction cascade produced as byproduct during respiration in living aerobic organisms [START_REF] Iranzo | Manganese complexes displaying superoxide dismutase activity: A balance between different factors[END_REF][START_REF] Batinic-Haberle | SOD Therapeutics: Latest Insights into Their Structure-Activity Relationships and Impact on the Cellular Redox-Based Signaling Pathways[END_REF][START_REF] Policar | Mimicking SODs, Why and How: Bio-Inspired Manganese Complexes as SOD Mimics[END_REF][START_REF] Batinić-Haberle | Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential[END_REF][START_REF] Miriyala | Manganese superoxide dismutase, MnSOD and its mimics[END_REF][START_REF] Mathieu | A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases[END_REF].

Among the metals used to develop SOD mimics, manganese is the most studied. If released, manganese does not induce Fenton chemistry which would have otherwise led to increased oxidative stress [START_REF] Policar | Mimicking SODs, Why and How: Bio-Inspired Manganese Complexes as SOD Mimics[END_REF][START_REF] Charrier | On dithiothreitol (DTT) as a measure of oxidative potential for ambient particles: evidence for the importance of soluble transition metals[END_REF]. Indeed, to date, a huge diversity of manganese complexes has been reported for their ability to react catalytically with superoxide. They involved ligands such as salen derivatives [START_REF] Baudry | Salenmanganese complexes are superoxide dismutase-mimics[END_REF][START_REF] Puglisi | Bioconjugates of cyclodextrins of manganese salentype ligand with superoxide dismutase activity[END_REF][START_REF] Park | Effect of the oligo(ethylene glycol) group on the antioxidant activity of manganese salen complexes[END_REF], cyclic polyamine [START_REF] Salvemini | Superoxide Dismutase Mimetics[END_REF][START_REF] Rush | The superoxide dismutase activities of two higher-valent manganese complexes, MnIV desferrioxamine and MnIII cyclam[END_REF][START_REF] Riley | Manganese macrocyclic ligand complexes as mimics of superoxide dismutase[END_REF][START_REF] Riley | Computer-Aided Design (CAD) of Synzymes: Use of Molecular Mechanics (MM) for the Rational Design of Superoxide Dismutase Mimics[END_REF][START_REF] Salvemini | A Nonpeptidyl Mimic of Superoxide Dismutase with Therapeutic Activity in Rats[END_REF][START_REF] Dees | Water Exchange on Seven-Coordinate Mn(II) Complexes with Macrocyclic Pentadentate Ligands: Insight in the Mechanism of Mn(II) SOD Mimetics[END_REF], tri-or dipodal nitrogen-centered ligands [START_REF] Kitajima | Monomeric (benzoato)manganese(II) complexes as manganese superoxide dismutase mimics[END_REF][START_REF] Deroche | A Seven-Coordinate Manganese(II) Complex Formed with a Single Tripodal Heptadentate Ligand as a New Superoxide Scavenger[END_REF][START_REF] Yamato | Superoxide Dismutase Mimetic Complex of Mn(II) / N,N-Bis(2pyridylmethyl)-(S)-Histidine[END_REF][START_REF] Policar | New MnII Complexes with an N/O Coordination Sphere from Tripodal N-Centered Ligands -Characterization from Solid State to Solution and Reaction with Superoxide in Non-Aqueous and Aqueous Media[END_REF][START_REF] Lewis | A manganese superoxide dismutase mimic based on cis,cis-1,3,5-triaminocyclohexane[END_REF], 1,2-ethanediaminecentered ligands [START_REF] Liao | Synthesis, characterization and SOD-like activities of manganese-containing complexes with N,N,N′,N′-tetrakis(2′-benzimidazolyl methyl)-1,2-ethanediamine (EDTB)[END_REF][START_REF] Groni | Reactivity of MnII with Superoxide. Evidence for a [MnIIIOO]+ Unit by Low-Temperature Spectroscopies[END_REF][START_REF] Cisnetti | Synthesis and superoxide dismutase-like activity of new manganese(III) complexes based on tridentate N2O ligands derived from histamine[END_REF], desferrioxamine derivatives [START_REF] Rush | The superoxide dismutase activities of two higher-valent manganese complexes, MnIV desferrioxamine and MnIII cyclam[END_REF][START_REF] Beyer | Characterization of a superoxide dismutase mimic prepared from desferrioxamine and MnO2[END_REF][START_REF] Faulkner | Characterization of Mn(III) Complexes of Linear and Cyclic Desferrioxamines as Mimics of Superoxide Dismutase Activity[END_REF], polyaminocarboxylato- [START_REF] Lati | Oxidation of first-row bivalent transition-metal complexes containing ethylenediaminetetra-acetate and nitrilotriacetate ligands by free radicals: a pulse-radiolysis study[END_REF][START_REF] Stein | Superoxide and manganese(III). Reactions of manganese-EDTA and manganese-CyDTA complexes with molecular oxygen. X-ray structure of potassium manganese-EDTA.2 water[END_REF][START_REF] Koppenol | Catalysis of superoxide dismutation by manganese aminopolycarboxylate complexes[END_REF] or polycarboxylato ligands [START_REF] Archibald | The scavenging of superoxide radical by manganous complexes: In vitro[END_REF], peptides [START_REF] Bailey | A novel anti-oxidant and anti-cancer strategy: a peptoid anti-inflammatory drug conjugate with SOD mimic activity[END_REF][START_REF] Fisher | Metal ion chelating peptides with superoxide dismutase activity[END_REF][START_REF] Piacham | Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase[END_REF], porphyrins [START_REF] Valentine | Reaction of superoxide with the manganese(III) tetraphenylporphine cation[END_REF][START_REF] Faulkner | Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo[END_REF][START_REF] Batinić-Haberle | A Potent Superoxide Dismutase Mimic: Manganese β-Octabromo-meso-tetrakis-(N-methylpyridinium-4-yl) Porphyrin[END_REF][START_REF] Batinic-Haberle | The Ortho Effect Makes Manganese(III) Meso -Tetrakis( N -Methylpyridinium-2-yl)Porphyrin a Powerful and Potentially Useful Superoxide Dismutase Mimic[END_REF][START_REF] Gauuan | Superoxide dismutase mimetics: synthesis and structure-activity relationship study of MnTBAP analogues[END_REF][START_REF] Batinić-Haberle | New PEG-ylated Mn( III ) porphyrins approaching catalytic activity of SOD enzyme[END_REF], phthalocyanines [START_REF] Matemadombo | Evaluation of the Performance of Manganese Phthalocyanines as Superoxide Dismutase Mimics[END_REF], texaphyrins [START_REF] Brewster | Metallotexaphyrins as MRI-Active Catalytic Antioxidants for Neurodegenerative Disease: A Study on Alzheimer's Disease[END_REF][START_REF] Arambula | Texaphyrins: Tumor Localizing Redox Active Expanded Porphyrins[END_REF], corroles [START_REF] Gomes | A catalytic antioxidant for limiting amyloid-beta peptide aggregation and reactive oxygen species generation[END_REF][START_REF] Okun | Cellular Uptake and Organ Accumulation of Amphipolar Metallocorroles with Cytoprotective and Cytotoxic Properties[END_REF][START_REF] Haber | Catalytic antioxidant therapy by metallodrugs: lessons from metallocorroles[END_REF] or biliverdin and its derivatives [START_REF] Spasojević | Manganese(III) Biliverdin IX Dimethyl Ester: A Powerful Catalytic Scavenger of Superoxide Employing the Mn(III)/Mn(IV) Redox Couple[END_REF]. Some of these complexes have thus far been assayed on cellular [START_REF] Spasojević | Manganese(III) Biliverdin IX Dimethyl Ester: A Powerful Catalytic Scavenger of Superoxide Employing the Mn(III)/Mn(IV) Redox Couple[END_REF][START_REF] Patel | Metalloporphyrins improve the survival of Sod2-deficient neurons[END_REF][START_REF] Munroe | Only one of a wide assortment of manganese-containing SOD mimicking compounds rescues the slow aerobic growth phenotypes of both Escherichia coli and Saccharomyces cerevisiae strains lacking superoxide dismutase enzymes[END_REF][START_REF] Pollard | Radioprotective effects of manganese-containing superoxide dismutase mimics on ataxia-telangiectasia cells[END_REF][START_REF] Alexandre | Improvement of the Therapeutic Index of Anticancer Drugs by the Superoxide Dismutase Mimic Mangafodipir[END_REF][START_REF] Filipović | Striking Inflammation from Both Sides: Manganese(II) Pentaazamacrocyclic SOD Mimics Act Also as Nitric Oxide Dismutases: A Single-Cell Study[END_REF][START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF] and in vivo models [START_REF] Mathieu | A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases[END_REF][START_REF] Alexandre | Improvement of the Therapeutic Index of Anticancer Drugs by the Superoxide Dismutase Mimic Mangafodipir[END_REF][START_REF] Gauter-Fleckenstein | Comparison of two Mn porphyrin-based mimics of superoxide dismutase (SOD) in pulmonary radioprotection[END_REF][START_REF] Gauter-Fleckenstein | Early and late administration of MnTE-2-PyP5+ in mitigation and treatment of radiation-induced lung damage[END_REF][START_REF] Doctrow | A Synthetic Superoxide Dismutase/Catalase Mimetic EUK-207 Mitigates Radiation Dermatitis and Promotes Wound Healing in Irradiated Rat Skin[END_REF][START_REF] Doctrow | Salen-Manganese Complexes as Catalytic Scavengers of Hydrogen Peroxide and Cytoprotective Agents: Structure-Activity Relationship Studies[END_REF][START_REF] Sheng | Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia[END_REF][START_REF] Masini | Protective effects of M40403, a selective superoxide dismutase mimetic, in myocardial ischaemia and reperfusion injury in vivo[END_REF][START_REF] Rong | EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainateinduced neuropathology[END_REF][START_REF] Mcdonald | A superoxide dismutase mimetic with catalase activity (EUK-8) reduces the organ injury in endotoxic shock[END_REF][START_REF] Masini | Reduction of antigen-induced respiratory abnormalities and airway inflammation in sensitized guinea pigs by a superoxide dismutase mimetic[END_REF][START_REF] Guillaumot | Oxaliplatin-induced neuropathy: the preventive effect of a new super-oxide dismutase modulator[END_REF]. Interestingly, Mn cyclic polyamine Mn(II) dichloro[(4aR,13aR,17aR,21aR)-1,2,3,4,4a,5,6,12,13,13a, 14,15,16,17,17a,18,19,20,21,21aeicosahydro-11,7-nitrilo-7Hdibenzo[b,h] [START_REF] Halliwell | Free radicals in biology and medicine, Fifth[END_REF][START_REF] Iranzo | Manganese complexes displaying superoxide dismutase activity: A balance between different factors[END_REF][START_REF] Batinić-Haberle | Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential[END_REF][START_REF] Charrier | On dithiothreitol (DTT) as a measure of oxidative potential for ambient particles: evidence for the importance of soluble transition metals[END_REF] tetraazacycloheptadecine-κN5,κN13,κN18,κN21, κN22] (Imisopasem manganese, M40403, CG4419) and Mn porphyrins (Mn(III) meso-tetrakis(Nethylpyridinium-2-yl)porphyrin called MnTE-2-PyP 5+ and Mn(III) meso-tetrakis(N-(2′nbutoxyethyl)pyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP 5+ ) called MnTnBuOE-2-PyP 5+ have advanced to several clinical trials [START_REF] Batinic-Haberle | Thiol regulation by Mn porphyrins, commonly known as SOD mimics[END_REF].

The efficiency of the complexes was linked to their intrinsic SOD activity but also to parameters such as cellular uptake, localization inside cells and cellular fragments and their stability. It has been shown that SOD mimics can also exhibit catalase activity and their dual activities have been recently reviewed by Batinic-Haberle et al. and Signorella et al. [START_REF] Batinic-Haberle | SOD Therapeutics: Latest Insights into Their Structure-Activity Relationships and Impact on the Cellular Redox-Based Signaling Pathways[END_REF][START_REF] Batinic-Haberle | Thiol regulation by Mn porphyrins, commonly known as SOD mimics[END_REF][START_REF] Tovmasyan | A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics[END_REF][START_REF] Tovmasyan | Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate[END_REF][START_REF] Signorella | Rationally designed mimics of antioxidant manganoenzymes: Role of structural features in the quest for catalysts with catalase and superoxide dismutase activity[END_REF]. We aimed here to undergo comparative study of several compounds which are frequently tested in cellular and animal models where they showed efficacy ultimately reducing oxidative stress. Small molecules such as those used here do not have protein tertiary structure that would allow specificity for certain reactive species, and thus react with variety of reactive species, both oxidizing or reducing them [START_REF] Batinić-Haberle | Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential[END_REF][START_REF] Batinic-Haberle | SOD Therapeutics: Latest Insights into Their Structure-Activity Relationships and Impact on the Cellular Redox-Based Signaling Pathways[END_REF][START_REF] Tovmasyan | Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate[END_REF][START_REF] Batinic-Haberle | Thiol regulation by Mn porphyrins, commonly known as SOD mimics[END_REF]. In particular, studies on Mn porphyrins have demonstrated that these compounds upregulate MnSOD, catalase, glutaredoxins (Grx), peroxiredoxins (Prxs) and other antioxidants via oxidizing cysteine of Kelch-like enoyl-coenzyme A hydratase-associated protein 1 (Keap1) of Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor [START_REF] Tovmasyan | Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate[END_REF][START_REF] Batinic-Haberle | Thiol regulation by Mn porphyrins, commonly known as SOD mimics[END_REF][START_REF] Batinic-Haberle | Mn Porphyrin-Based Redox-Active Drugs: Differential Effects as Cancer Therapeutics and Protectors of Normal Tissue Against Oxidative Injury[END_REF]. In that way, rather than acting as SOD and catalase mimics, they indirectly affect levels of O 2 -and H 2 O 2 .

We used here two assays that are specific for O 2 -and H 2 O 2 to see if we can correlate the SOD and CAT like activities of those compounds to their efficacy. For these assays, we have intentionally set up experiments lasting less than 1h to focus on the mimics reactivity and to avoid upregulation of antioxidant enzymes. Then, we looked at their efficacy in suppressing the inflammation in HT29-MD2 cells submitted to bacterial lipopolysaccharide (LPS) challenge. The three cellular assays have been combined with inductively coupled plasma mass spectrometry (ICP-MS) quantification of the Mn center of compounds to get insights into their cell penetration.

Experimental

1.1. Reagents and instruments LPS (Escherichia coli O55:B5), bovine CuZnSOD, Nicotinamide adenine dinucleotide phosphate (NADH), ferricytochrome c and pyruvic acid were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France). Interleukine-8 (IL-8) detection by enzyme-linked immunosorbent assay (ELISA) was performed using a kit (Duoset) provided by R&D Systems (Minneapolis, Minnesota, USA). Bicinchoninic acid assay (BCA) and bovine serum albumin (BSA) were from Uptima-Interchim (Montluçon, France). Detection Enhanced Chemiluminescence (ECL) system and nitrocellulose membranes were from Amersham Biosciences (Piscataway, New Jersey, USA). Dulbecco's modified Eagle medium (DMEM), DMEM without NaHCO 3 (10 g.L -1 ) and blasticidin, were from Invitrogen (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Fetal bovine serum (FBS) was from GE Healthcare Life Sciences (South Logan, Utah, USA). (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution (1M), Ethylenediaminetetraacetic acid (EDTA) solution (0.5 M), and Dulbecco's phosphate Buffered Saline (10X, DPBS) were from Gibco (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The protease inhibitor cocktail was from Roche Diagnostics (Meylan, France). Mn Porphyrins were obtained from I. Batinic-Haberle and prepared as detailed in: Rajic Z. )methyl]-ethane-1,2-diamine) and complex were synthesized according to previously described protocols [START_REF] Cisnetti | A New Pentadentate Ligand Forms Both a Di-and a Mononuclear MnII Complex: Electrochemical, Spectroscopic and Superoxide Dismutase Activity Studies[END_REF]. UV-visible spectra were recorded on a Varian Cary 300 Bio spectrophotometer and ICP-MS analysis were performed on an Agilent 7700 X.

Methods

Stock solutions

Mito-Tempol, M40403 and EUK-134. 5 mM stock solutions in H 2 O were prepared and the compounds were then added to reach the expected incubation concentrations. Mn1. A 10 mM stock solution of the 1:1 complex was prepared in HEPES (50 mM, pH 7.5). The complex was then added to reach the expected incubation concentrations. Mn Porphyrins. stock solutions in water were purchased and the compounds were directly added to reach the expected incubation concentrations. [MnTE-2-PyP 5+ ] = 4.5 mM, [MnTnBuOE-2-PyP 5+ ] = 5.23 mM and [MnTBAP 3-] = 4.47 mM.

Assay on macrophages

Cell culture. Murine macrophage RAW 264.7 (American Type Culture Collection) cell line was cultured at 37°C under a 5% CO 2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing 1.0 g. L -1 D-glucose and sodium pyruvate (Invitrogen). The medium was supplemented with 5% fetal bovine serum (Invitrogen) and 20 µg. mL -1 gentamicin (Sigma). Viability assay. Cell growth and viability of murine macrophages (RAW 264.7) was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as previously reported [START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF]. Superoxide measurement. Ferricytochrome c reduction was used to assess the superoxide ion production in RAW 264.7 cells. Cells were stimulated with culture medium containing 1 U mL -1 Interferon gamma (IFN-) and 1 ng mL -1 LPS for 24 h at 37°C (except for the negative control). They were then incubated 1h at 37°C in a home-made medium with 100 μM ferricytochrome c (Sigma Aldrich, prepared without using trichloroacid (TCA)), with or without antioxidant and with 800 nM phorbol 12-myristate 13-acetate (PMA). A home-made medium is necessary to get rid of the colored pH indicator present in DMEM that absorbs in the same range of wavelength as ferricytochrome c. The medium is composed of 14.6 mM glucose, 358 mM NaCl, 12.7 mM KCl, 3.1 mM KH 2 PO 4 , 6.1 mM MgSO 4 , 3.1 mM CaCl 2 , 13 mM NaHCO 3 and 53 mM HEPES buffer. The absorbance of supernatants was measured at 550 nm, where ferrocytochrome c displays a peak. Contribution of ferricytochrome c at 100 µM was subtracted from the absorbance of each sample. This protocol leads to an integration over 1h of the production of superoxide in the extracellular medium. Note that this assay was shown to provide a positive result with antioxidants not able to enter cells, such as purified SOD in the extracellular medium [START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF], with its overall negative charge, as found again here in Figure 2. For that reason, it was labeled "extracellular activity assay" [START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF], but it most probably provide an activity measurement composite between extracellular and the amount that penetrate in cells during the 1-h incubation.

Assay on HeLa HyPer cells

Cell culture. Stable cell line HeLa HyPer1 was prepared using the HeLa Flp-In cell line, which was kindly provided by Stephen Taylor [START_REF] Tighe | Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores[END_REF], and cultured at 37°C under a 5% CO 2 atmosphere in DMEM containing 4.5 g.L -1 D-glucose (Invitrogen) supplemented with 10% of heat-inactivated FBS (Invitrogen). HyPer1 expression in this stable cell line was controlled by doxycycline, added 24 h after seeding. The cells were cultured for an additional 24 h before being processed for analysis. 1h before imaging, the medium was replaced by DMEM without NaHCO 3 containing 1.0 g.L -1 D-glucose and 30 mM HEPES (pH 7.5). This cell is of interest in this context as they endogenously produce H 2 O 2 a way to evaluate the catalase activity of antioxidants. Pharmacological treatments. Cells were incubated with or without 100 µM of antioxidant during 1 hr at 37°C under a 5% CO 2 atmosphere. Imaging. Imaging was performed with a CSU-W1 Yokogawa spinning disk coupled to a Zeiss Axio Observer Z1 inverted microscope equipped with a sCMOS Hamamatsu camera and a 63× objective (63×/1.4 oil WD: 0.17 mm) oil objective. DPSS 100 mW 405 nm and 150 mW 491 nm lasers and a 525/50 bandpass emission filter were used. H 2 O 2 levels quantification and statistical analysis. Images were processed with the Fiji software, to obtain the HyPer1 ratio of the emission at 530 nm (491/530)/(405/530). HyPer ratio was then measured for several cells, and normalized to the ratio value of the control condition. Data were analyzed using Kalaeidagraph and expressed as the mean ± standard error of the mean (SEM). Statistical significance was calculated using an ordinary one-way ANOVA followed by Tukey's multiple comparison test. Note that this assay was shown to provide a positive result with antioxidants not able to enter cells, such as purified catalase in the extracellular medium [START_REF] Amblard | H 2 O 2 and Engrailed 2 paracrine activity synergize to shape the zebrafish optic tectum[END_REF]. Indeed, H 2 O 2 is able to cross membranes and its consumption in the extracellular medium shifts the equilibrium and, in fine, consumes also H 2 O 2 inside cells, where it is measured by the Hyper probe. In this set-up, with a short incubation, the measure is most probably dominated by the extracellular effect, although a contribution of the amount of antioxidants that may have penetrated in cells in 1h is not excluded.

Assay on HT29-MD2 cells

Cell culture. HT29 human colon adenocarcinoma were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Wiltshire, UK) and stably transfected to over-express MD2 as previously described. [START_REF] Lenoir | MD-2 controls bacterial lipopolysaccharide hyporesponsiveness in human intestinal epithelial cells[END_REF] Cells were cultured in DMEM supplemented with 10% of heat-inactivated FBS, and 0.1% of blasticidin (10 µg mL -1 ) at 37 °C in a 5% CO 2 /air atmosphere. Cytotoxicity assay. Cytotoxicity of tested compounds and controls, with LPS, was assessed using lactate dehydrogenase (LDH) release assay, by following the release of the cytosolic lactate dehydrogenase (LDH) into the supernatant, indicating membrane damages [START_REF] Grondin | Regulation of colon cancer cell proliferation and migration by MD-2 activity[END_REF]. Cytotoxicity was considered when LDH release was more than 10%. Concentration of LDH in supernatant: 800 µL of a pyruvate/NADH solution (see below) was added into a 1 mL plastic cuvette, as well as 200 µL of supernatant, and the decrease in absorbance at 340 nm was immediately monitored for 1 minute. The slope is proportional to LDH concentration in supernatant. Concentration of LDH in cell lysate: 800 µL of a pyruvate/NADH solution (see below) was added into a 1 mL plastic cuvette, as well as 190 µL of 0.1 M PBS and 10 µL of cell lysate, and the decrease in absorbance at 340 nm was immediately measured for 1 minute. The slope is proportional to LDH concentration in cell lysate. The percentage of LDH released in the supernatant was calculated as follows: Solution of pyruvate/NADH: 4.1 mg of pyruvic acid (0.62 mM), and 7.7 mg of NADH (0.18 mM) in 60 mL of 0.1 M PBS (pH 7.4). Cell activation with LPS and incubation with the compounds. HT29-MD2 cells were seeded in 12 or 24 well-plates at 200 000 cells/well to reach 90% confluence after 3 or 4 days. Cells were incubated with media only, or tested compounds at the desired concentration for 6 h in DMEM supplemented with 10% of heat-inactivated FBS and with LPS (0.1 µg/mL). Supernatants were collected, and stored at -20 °C before ELISA and LDH assay. Determination of proteins concentrations in cell lysates. In order to normalize the IL-8 concentrations measured, protein content of each cell lysate was measured. They were determined using BCA protein assay reagents and BSA as standard according to the manufacturer's instructions. Briefly, a solution (98% BCA, 2% CuSO 4 ) was added to the protein solution in 96-wells plate. After 30 min at 37°C, the absorbance was monitored at 560 nm in a SpectraMax M5e microplate reader from Molecular Devices. Absorbance was linked to protein mass thanks to a calibration curve with BSA. IL8 quantification. Levels of the pro-inflammatory cytokine IL8 produced by cells were determined in cell supernatants using a commercially available ELISA kit according to the instructions of the manufacturer. IL8 levels were normalized by the protein content determined in the corresponding cell lysates.

Quantification of manganese in cell lysates by ICP-MS

Cells were cultured in a 75 cm 2 plastic flask to reach 90% confluency. They were incubated with medium only, or tested compounds at the desired concentration for one or twenty-four hours, at 37 °C. Cells were then washed twice with 0.9% NaCl. 700 µL of HEPES 0.1 M was then added to scratch cells. For HT29-MD2 cells, 50 µL of the cell suspension was added to an aqueous solution of HNO 3 2% (950 µL). The resulting solution was heated at 90°C overnight to ensure decoordination of Mn from the porphyrins (Figure S5a, SI). The solution was diluted with a solution of HNO 3 2% (1 mL), filtrated on 0.2 µm sized filters to get rid of cellular debris before analysis. A calibration curve was established using anhydrous MnCl 2 . A range of concentration going from zero to 100 ppb was generally used for calibration.

For RAW cells, the same above-mentioned procedure was used but using 100 µL of the cell suspension in 0.9 mL of an aqueous solution of HNO 3 2%. 1.2.6. Quantification of porphyrins in HT29-MD2 cell lysates by UV-vis spectrometry Cells were cultured in a 75 cm 2 flask to reach 90% confluency. They were incubated with medium only, or tested compounds at the desired concentration for one or twenty-four hours, at 37 °C. Cells were then washed twice with 0.9% NaCl. 700 µL of HEPES 0.1 M was then added to scratch cells. Cells were lysed by sonication. UV-vis spectra of the resulting solutions were recorded and the spectrum of a solution of non-incubated cell was subtracted to get rid of the cell absorption. The absorbance of the Soret band was compared with those of solutions (5 µM) of the pure porphyrins in water. To compare the concentrations found by UV-vis with the concentrations measured by ICP-MS, the concentrations in µM were converted into ppb considering the dilution used for samples preparation in ICP-MS experiments (dilution  40).

Results and discussion

A nitroxide radical (Mito-TEMPOL) and several MnSOD mimics (Figure 1) belonging to the above-mentioned SOD mimics families, covering a large range of SOD activity and/or catalase activity were selected (Table 1). Mito-TEMPOL was included for comparison, since it does not involve a metal cation to perform the redox catalysis, does not exhibit catalase activity but shows a weak SOD activity only in acidic region [START_REF] Miriyala | Manganese superoxide dismutase, MnSOD and its mimics[END_REF][START_REF] Tovmasyan | A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics[END_REF]. The catalysis of superoxide dismutation occurs via redox couple RNO  /RNO + [START_REF] Goldstein | The Role of Oxoammonium Cation in the SOD-Mimic Activity of Cyclic Nitroxides[END_REF]. In addition, Mito-TEMPOL, possesses a triphenylphosphonium group to increase its accumulation into mitochondria where reactive oxygen species (ROS) are mainly produced [START_REF] Antonenko | Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: Synthesis and in vitro studies[END_REF]. Among the Mn porphyrins studied, two positively charged and differently lipophilic (MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ ) and one negatively charged (MnTBAP 3-) have been selected. The positively charged Mn porphyrins were shown to enter the cell, where they prefer nucleus and mitochondria being driven there by anionic phosphate groups and mitochondrial negative potential [START_REF] Batinic-Haberle | SOD Therapeutics: Latest Insights into Their Structure-Activity Relationships and Impact on the Cellular Redox-Based Signaling Pathways[END_REF][START_REF] Batinic-Haberle | Mn Porphyrin-Based Redox-Active Drugs: Differential Effects as Cancer Therapeutics and Protectors of Normal Tissue Against Oxidative Injury[END_REF][START_REF] Spasojević | Mn porphyrin-based superoxide dismutase (SOD) mimic, MnIIITE-2-PyP5+, targets mouse heart mitochondria[END_REF][START_REF] Smith | Delivery of bioactive molecules to mitochondria in vivo[END_REF]. It is expected that the negatively charged porphyrin will be less reactive towards the negatively charged superoxide and less cell penetrant. While not reported as not being a catalytic SOD mimic [START_REF] Rebouças | Pure manganese(III) 5,10,15,20tetrakis(4-benzoic acid)porphyrin (MnTBAP) is not a superoxide dismutase mimic in aqueous systems: a case of structure-activity relationship as a watchdog mechanism in experimental therapeutics and biology[END_REF], MnTBAP 3-can be interesting as it reacts with peroxynitrite and has reportedly shown efficacy in vitro and in vivo [START_REF] Miriyala | Manganese superoxide dismutase, MnSOD and its mimics[END_REF][START_REF] Batinić-Haberle | Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury, an SODspecific Escherichia coli model and carrageenan-induced pleurisy[END_REF][START_REF] Rocha | Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer[END_REF]. We refer herein as intrinsic activities to the kinetics of the reaction with superoxide or hydrogen peroxide outside of any cellular context, whether it is catalytic or not, typically reported in Table 1 [START_REF] Policar | Mimicking SODs, Why and How: Bio-Inspired Manganese Complexes as SOD Mimics[END_REF].

In order to compare the antioxidants selected for their SOD activity in cells, we have first used an assay involving murine macrophages RAW 264.7 [START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF]. On this cell line, the production of RS including superoxide can be induced upon stimulation with bacterial lipopolysaccharide (LPS) and interferon  (IFN-). The release of these RS in the surrounding medium is then triggered using phorbol 12-myristate 13-acetate (PMA) [START_REF] Filipović | Striking Inflammation from Both Sides: Manganese(II) Pentaazamacrocyclic SOD Mimics Act Also as Nitric Oxide Dismutases: A Single-Cell Study[END_REF][START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF][START_REF] Amatore | Electrochemical Monitoring of Single Cell Secretion: Vesicular Exocytosis and Oxidative Stress[END_REF]. Quantification of superoxide concentration is made possible by using ferricytochrome c as superoxide UV-vis marker. As ferricytochrome c is not able to penetrate cells, only the superoxide released extracellularly is measured. CuZnSOD was incubated at 100 U/mL and all of the other antioxidants were incubated, in presence of ferricytochrome c, for 1h at 5 µM. At this concentration, no toxicity was observed (MTT assay, Figure S1, SI). It is worth mentioning that 1h-inccubation may be too short to lead to antioxidant proteins upregulation [START_REF] Harris | Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1[END_REF]. In presence of superoxide, the ferricytochrome c is reduced into ferrocytochrome c. A SOD mimic competes with ferricytochrome c for reaction with superoxide and leads to a lowering of the amount in ferrocytochrome c. The ferrocytochrome c concentrations measured in the cell supernatants were compared and the concentration found after activation and in absence of any antioxidant was set at 100%. Of note, even if the superoxide concentration is measured outside the cells, an antioxidant that enters cells and decreases superoxide concentration inside cells would also result in lowering ferricytochrome c reduction [START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF]. As shown in Figure 2, three main groups emerged: very efficient catalysts (% ferricytochrome c < 20 % activated), efficient catalysts (20 % < % ferricytochrome c < 80 % activated), and inactive catalysts (% ferricytochrome c > 80 % activated). In the first group, the recombinant CuZnSOD is very efficient at reducing the extracellular superoxide concentration and was even able to result in superoxide levels lower than that obtained for nonactivated cells. In the second group, Mn1, M40403, EUK-134 and the two positively charged porphyrins MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ were able to decrease the extracellular superoxide concentrations to levels close to the non-activated situation. With exception of EUK-134, such data reflect their similar SOD-like activities. In the third group, Mito-TEMPOL and MnTBAP 3- were found inactive in the tested conditions. As expected, the obtained results are in line with the lack of their SOD activities reported in Table 1 (k McCF or k cat ). Both compounds have SOD-like activities lower than the rate for O 2 -selfdismutation of ~5 x 10 5 M -1 s -1 and are thus not true catalytic SOD mimics [START_REF] Batinić-Haberle | Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential[END_REF].

In a second assay, we wanted to compare the antioxidants according to their catalase activity. We have used HeLa cells expressing intracellularly the ratiometric fluorescent sensor of H 2 O 2 called HyPer [START_REF] Belousov | Genetically encoded fluorescent indicator for intracellular hydrogen peroxide[END_REF]. HyPer is a circularly permuted yellow fluorescent protein (cpYFP) emitting at 530 nm integrated into the regulatory domain of the bacterial H 2 O 2 sensing protein OxyR (OxyR-RD). Upon oxidation of HyPer thiols by H 2 O 2 , disulfide bridges formation induces a modification of the spectral properties of the YFP. This leads to a ratiometric modification of the excitation spectrum of HyPer that possesses two excitation maxima at 420 and 488 nm. Thus, by measuring the normalized ratio of the intensity I (491/530) /I (405/530) , it is possible to monitor specifically H 2 O 2 concentration in cells. It is worth mentioning that since H 2 O 2 is able to passively diffuse across cytoplasmic membrane, the observed effect of the antioxidants may be composite: it may be due to dismutation of H 2 O 2 inside or outside the cells. For instance, incubation of HeLa HyPer with the recombinant heme catalase, has shown to reduce H 2 O 2 concentration inside cells whereas it does not cross cells membrane [START_REF] Amblard | H 2 O 2 and Engrailed 2 paracrine activity synergize to shape the zebrafish optic tectum[END_REF].

HeLa HyPer cells were incubated with the antioxidants for 1h at 100 µM in HEPES buffer and imaged at 530 nm by fluorescence microscopy upon excitation at two wavelengths 405 and 491 nm respectively (Figures S2 andS3) [START_REF] Mishina | Chapter Three -Visualization of Intracellular Hydrogen Peroxide with HyPer, a Genetically Encoded Fluorescent Probe[END_REF]. This incubation time is long enough to detect an effect on H 2 O 2 concentration and short enough to exclude a possible feedback of the cell expressing antioxidant enzymes [START_REF] Harris | Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1[END_REF]. Hence, this assay is meant to evidence a direct effect of the compounds to reduce the level of H 2 O 2 . The ratios of the intensity I (491/530) /I (405/530) normalized against the control (NFR for normalized fluorescence ratio) was determined for several cells (typically 50-150 cells) and were reported in the Figure 3. In this assay, cells were not stressed, and, consequently, the effects reported are those on basal H 2 O 2 levels.

As shown in Figure 3, again, three groups of antioxidants were observed: efficacious catalyst (0.5 < NFR < 0.75), moderately efficacious catalysts (0.75 < NFR < 1) and prooxidant compounds (NFR > 1). In the first group, the two more efficacious CAT mimics from aqueous chemistry studies were MnTE-2-PyP 5+ (k cat H 2 O 2 = 63.3 M -1 s -1 ) [START_REF] Tovmasyan | A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics[END_REF] and MnTnBuOE-2-PyP 5+ (k cat H 2 O 2 = 88.5 M -1 s -1 ) [START_REF] Tovmasyan | A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics[END_REF] and were indeed found with NFR of 0.68 and 0.60 respectively. The effect of MnTnBuOE-2-Pyp 5+ may be higher as it distributes to a higher level to cells than does MnTE-2-PyP 5+ [START_REF] Tovmasyan | Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate[END_REF]. In the second group, Mn1 and EUK-134 reduced similarly the H 2 O 2 concentration in HeLa cells which correlates with their similar moderate intrinsic CAT activities (2.3 and 13 M -1 s -1 respectively) [START_REF] Bernard | Evaluation of the anti-oxidant properties of a SODmimic Mn-complex in activated macrophages[END_REF][START_REF] Tovmasyan | A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics[END_REF][START_REF] Melov | Lifespan Extension and Rescue of Spongiform Encephalopathy in Superoxide Dismutase 2 Nullizygous Mice Treated with Superoxide Dismutase-Catalase Mimetics[END_REF]. Surprisingly, Mito-TEMPOL was found active in this assay whereas no CAT activity is reported for this compound. In contrast, although M40403 exhibits a CAT activity (8.2 M -1 s -1 ) similar to Mn1 and EUK-134 in aqueous studies, in cells, M40403 induced increased levels of H 2 O 2 [START_REF] Tovmasyan | A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics[END_REF]. In eukaryotic organisms, the steady state concentration of superoxide and H 2 O 2 were estimated to be 10 -10 and 50 10 -10 M respectively, attesting the higher tolerance of eukaryotic cells for H 2 O 2 than for O 2 - [START_REF] Cadenas | Guest Editors[END_REF]. Owing to this ratio, it is very unlikely that by catalyzing superoxide dismutation the antioxidants lead to a measurable increase of H 2 O 2 concentration and thus to an increased fluorescence ratio. The increased level of H 2 O 2 observed with M40403 may instead be due to a low stability of the complexes which may partly dissociate and lead to manganese release as reported recently [START_REF] Weekley | Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide Dismutase (SOD) Mimetics: Fluorescently Labeled MnSOD Mimetics, X-ray Absorption Spectroscopy, and X-ray Fluorescence Microscopy Studies[END_REF][START_REF] Mathieu | Intracellular location matters: rationalization of the anti-inflammatory activity of a manganese(II) superoxide dismutase mimic complex[END_REF]; there may be a ligand chemistry or toxicity we are still not aware of and would require further studies. Increased levels of H 2 O 2 were also observed after incubation with the redox silent analogue of Mn1 called Zn1 and the manganese salt MnCl 2 .The negatively charged porphyrin MnTBAP 3-did not show a significant effect on H 2 O 2 concentration as it has no CAT-like activity. Overall, as H 2 O 2 is mainly produced at the plasma membrane and can easily diffuse, and as the results are in line with the catalytic constants measured in test tubes with the positively charged porphyrins being the most efficacious [START_REF] Winterbourn | Biological Production, Detection, and Fate of Hydrogen Peroxide[END_REF], these results suggest that the measured activities are mainly due to the portion of antioxidants that remain outside the cells. To go further, we have assayed the selected antioxidants on HT29-MD2 cells. These intestine epithelial cells have been stably transfected to overexpress MD2 protein that promotes an enhanced inflammatory response in presence of bacterial lipopolysaccharide [START_REF] Lenoir | MD-2 controls bacterial lipopolysaccharide hyporesponsiveness in human intestinal epithelial cells[END_REF]. Besides, it has been shown that such inflammatory reaction is mediated by oxidative stress [START_REF] Valentine | Acute-phase induction of manganese superoxide dismutase in intestinal epithelial cell lines[END_REF][START_REF] Denis | Apple Peel Polyphenols and Their Beneficial Actions on Oxidative Stress and Inflammation[END_REF]. HT29-MD2 cells were thus incubated in presence of LPS and the antioxidants at non-toxic concentrations: 10 and 100 µM (Figure S4, SI) [START_REF] Mathieu | A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases[END_REF][START_REF] Mathieu | Intracellular location matters: rationalization of the anti-inflammatory activity of a manganese(II) superoxide dismutase mimic complex[END_REF][START_REF] Mathieu | Anti-inflammatory activity of superoxide dismutase mimics functionalized with cell-penetrating peptides[END_REF]. In this assay, 6h incubations were required to observe a significant inflammatory response. Consequently, the observed anti-inflammatory effect of the antioxidants may be related to either their direct scavenging of O 2 -or H 2 O 2 , or mediated metabolic processes that would result in reducing levels of those species and oxidative stress in general. The concentration of an inflammatory marker, the interleukin 8 (IL8) has been measured by ELISA to compare the effects of the antioxidants. As expected, upon LPS challenge, the IL8 concentration of non-incubated cells increased significantly. Thus, the decreased level of IL8 in the presence of an antioxidant was indicative of antiinflammatory activity (Figure 4). After incubation of the antioxidants at 10 µM, only M40403, and MnTnBuOE-2-PyP 5+ to a lesser extent, showed a moderate anti-inflammatory effect. The other compounds were found inactive or even pro-inflammatory in the case of Mn1. Taking into account Mn1 dissociation constant in solution that has been estimated to be 6.5 ± 1.7 10 -7 M [START_REF] Ching | Bioinspired superoxide-dismutase mimics: The effects of functionalization with cationic polyarginine peptides[END_REF], at a concentration of 10 µM, it is expected that at least 23% of the ligand is not coordinated to Mn and this value may increase in a competitive ligand biological environment. Because the ligand is toxic for the cells [START_REF] Mathieu | A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases[END_REF], the pro-inflammatory response observed may be due to the toxicity of the non-coordinated ligand. Note that in this model, MnCl 2 was previously assayed and found to be not active or proinflammatory [START_REF] Mathieu | A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases[END_REF][START_REF] Mathieu | Intracellular location matters: rationalization of the anti-inflammatory activity of a manganese(II) superoxide dismutase mimic complex[END_REF][START_REF] Mathieu | Anti-inflammatory activity of superoxide dismutase mimics functionalized with cell-penetrating peptides[END_REF]. After incubation at a higher concentration (100 µM), as expected, M40403 and MnTnBuOE-2-PyP 5+ were found more active than at 10 µM. Mn1 also exhibited high antiinflammatory effect as already reported [START_REF] Mathieu | A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases[END_REF]. Surprisingly, MnTE-2-PyP 5+ did not lead to a significant decrease of IL8 level whereas it possesses similar catalytic constants for superoxide and H 2 O 2 dismutation as MnTnBuOE-2-PyP 5+ [START_REF] Tovmasyan | Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate[END_REF].

The bioavailability of cationic Mn-porphyrins in cellular fragments, cells and tissue has been comprehensively studied and reported [START_REF] Batinic-Haberle | Mn Porphyrin-Based Redox-Active Drugs: Differential Effects as Cancer Therapeutics and Protectors of Normal Tissue Against Oxidative Injury[END_REF]. In this cellular assay, we observed that both the more hydrophilic MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ penetrate similarly, as shown by quantification of the Mn content by ICP-MS in cell lysates incubated with these porphyrins that lead to similar values (Figure S6, SI). A similar trend was observed when the porphyrins were quantified in cell lysates using their specific UV-Vis signature (Figure S5 b-c and Figure S6, SI). To note, very surprisingly, quantification experiments showed that the negatively charged MnTBAP 3-was more internalized than the positively charged porphyrins (Figure S5 b-c and Figure S6, SI), which was not expected. It should however be mentioned that MnTBAP 3-was suggested to display an intracellular and intramitochondrial bio-activity [START_REF] Li | Dependence of excitotoxic neurodegeneration on mitochondrial aconitase inactivation[END_REF]. Although it penetrates in this model, no activity was observed for this negatively charged porphyrin. Interestingly too, this study suggests that M40403 and EUK134 penetrate into cells and confirmed the previous results reported for Mn1 [START_REF] Mathieu | A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases[END_REF][START_REF] Mathieu | Intracellular location matters: rationalization of the anti-inflammatory activity of a manganese(II) superoxide dismutase mimic complex[END_REF][START_REF] Mathieu | Anti-inflammatory activity of superoxide dismutase mimics functionalized with cell-penetrating peptides[END_REF]. The different activities observed here may be related to different cellular localizations. In this assay, antioxidants that are poor SOD mimics (MITO-TEMPOL, EUK-134, MnTBAP 3-) are not efficient whatever their catalase-like activity, suggesting that superoxide and/or its progeny may be the main reactive species that had to be controlled in this model and played an important role in the inflammatory responses. While inferior at lower 10 µM concentration, due to its low metal-ligand stability and/or inertia, at 100 µM, though, Mn1 showed the best anti-inflammatory activity in this cellular model, likely due to its high SOD activity and high bioavailability.

Conclusion

In conclusion, we have shown that the assays on macrophages (for SOD activity) and on HeLa HyPer cells (for CAT activity) are useful to validate the activity of antioxidants in cellular contexts. They allow verifying that the compounds are efficient under biological conditions which data justify their further evaluation under more complex conditions. The assay on HT29-MD2 cells affords more relevant information, in particular for SOD mimics, since in this assay, among the ROS produced, it seems that superoxide or its progeny plays an important role in the inflammatory response of the cells. The bioactivities measured were related not only to the intrinsic catalytic activities of the antioxidants but also with their penetration inside the cells. Evidences suggested that their cellular localization is also an important factor; this assay gained an insight into their bioavailabilities. This three-cell-model assay thus provides a comparison of different families of SOD mimics on cellular models relevant for the development of such compounds, and a broader overview of the potential of antioxidants for their use as catalytic drugs.

  et al. for MnTnBuOE-2-PyP [71], Reboucas J.S.R. et al. for Manganese(III) 5,10,15,20-tetrakis(4benzoic acid)porphyrin (MnTBAP) [72], Batinic-Haberle I. et al. for MnTE-2-PyP [73,74]. 2,2,6,6-Tetramethyl-4-[[5-(triphenylphosphonio)pentyl]oxy]-1-piperidinyloxy bromide (Mito-Tempol) was purchased from Sigma Aldrich, the enantiomer CG4419 of M40403 from Astatech and Mn(III) 2,2′-[1,2-Ethanediylbis(nitrilomethylidyne)]bis[6-methoxy-phenol) (EUK134) from Clinisciences. Mn1 ligand (N-(hydrobenzyl)-N,N'-bis[2-(N-methyl-imidazolyl

Figure 1 .

 1 Figure 1. Structure of the selected antioxidants studied in this article.

Figure 2 .

 2 Figure 2. Evaluation of antioxidants on murine macrophages RAW 264.7. Reported values are related to the values observed for activated cells and arbitrarily set at 100%. Macrophages were incubated with IFN- (1 U mL -1 ) and LPS (1 ng mL -1 ) for 24 h at 37°C in DMEM followed by an incubation with ferricytochrome c (100 µM), PMA (800 nM) and antioxidants (100 µM) at 37°C for 1h in a homemade medium (see experimental section). Data are mean ± SEM of three independent experiments with (**) p < 0.01 and (*) p < 0.05 versus activated cells.

Figure 3 .

 3 Figure 3. H 2 O 2 levels measured in HyPer HeLa cells. The cells were incubated 1h at 37°C in HEPES (30 mM pH 7.5) under 5% CO 2 without (control) or with antioxidants at 100 µM. Fluorescence images were recorded after excitation at 405 nm and 491 nm and the ratio of the emission at 530 nm I (491/530) /I (405/530) was measured for several cells. The ratio was arbitrarily set at 1 for control cells i.e. cells incubated with HEPES only. Data are

MnTnBuOE- 2 -

 2 PyP5 + , that is now in clinical trial, shows efficacy on all of the models. Its effects may be due to direct scavenging of H 2 O 2 and O 2 -(for the two first cellular models, with short incubation times) and/or to the Mn porphyrin-driven upregulation of MnSOD and catalase that would then take care of H 2 O 2 and O 2 -(inflammation model requiring a longer 6h incubation, compatible with protein up-regulation). These data support its progress into clinical[START_REF] Batinic-Haberle | Thiol regulation by Mn porphyrins, commonly known as SOD mimics[END_REF][START_REF] Zhao | A novel redox regulator, MnTnBuOE-2-PyP5+, enhances normal hematopoietic stem/progenitor cell function[END_REF].

Figure 4 .

 4 Figure 4. Quantification of the inflammatory marker IL8 by ELISA after incubation of HT29-MD2 cells with the antioxidants at 10 or 100 µM. Antioxidants were incubated for 6 h at 37°C in supplemented DMEM in presence of LPS (0.1 µg mL -1 ) except for the control where no antioxidants and LPS were added. The absorbance measured for activated cells was arbitrary set at 100%. Data represent mean  SEM for two independent experiments for MnTnBuOE-2-PyP 5+ and M40403 and three independent experiments for Mito-TEMPOL, Mn1, EUK-134, MnTE-2-PyP 5+ and MnTBAP 3-with **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 vs. activated cells.

  2 -into H 2 O 2 and O 2 , CAT catalyzes H 2 O 2 dismutation into H 2 O and O 2 and GPxs catalyze the reduction of H 2 O 2 and others hydroperoxides into H 2

Table 1 .

 1 Antioxidants intrinsic dismutation constants. k McCF refers to the rate constant calculated from the McCord and Fridovich assay.

	6 M -1 s -1 )	k cat H 2 O 2 (M -1 s -1 ) f	references

a Ferricytochrome c, phosphate buffer (50 mM, pH 8). b Ferricytochrome c, phosphate buffer (50 mM, pH 7.8). c HEPES (60 mM, pH 7.4). d Phosphate buffer (50 mM, pH 7.4). e Phosphate buffer (50 mM, pH 7.8). f Unless specified, catalytic constants were measured by polarography using a Clarktype electrode in tris(hydroxymethyl)aminomethane (Tris) buffer (50 mM, pH 7.8). g HEPES (100 mM, pH 7.4). h Value found for EUK-8 instead of EUK-134 that differs only by the two methoxy substituents. SF means stopped-flow and PR pulse radiolysis.

Acknowledgments

We thank Ecole Normale Supérieure Paris Saclay for A. Vincent's PhD fellowship, TGE RENARD (FR 3443, CNRS) and UMR8601 and S. Lajnef for access to the EPR spectrometer for the EPR experiments. We also thank B. Goetz and P. Legeay for help with ICP-MS. ANR (ANR-15-CE07-0027), Association François Aupetit and the Fondation pour la Recherche Biomédicale (project DIE20151234413) are acknowledged for financial supports.


domain: spi phys chim math info

text:
C Ghouila-Houri

Ieee Member

M Kazar-Mendes

T Arnoult

R Viard

Q Gallas

E Garnier

A Merlen

A Talbi

P Pernod

Aerodynamic wall pressure measurement using a high temperature gradient Pirani micro-sensor

Keywords: Pirani sensor, Pressure sensor, MEMS, Aerodynamic wall pressure. I

This paper presents and discusses the first demonstration of aerodynamic wall pressure measurement using a thermal Pirani effect based micro-sensor. The thermal micro-sensor is designed as a suspended micro-hot-wire separated from the substrate by a nanoscale gap, and mechanically supported by perpendicular micro-bridges. The micro-sensor was calibrated for pressure measurement from 10 kPa to 800 kPa. It was then used as aerodynamic wall pressure sensor in a turbulent boundary layer wind tunnel and successfully measured the 1.1 kPa depression occurring in the wind tunnel when the flow velocity goes up to 40 m/s. (Abstract)

INTRODUCTION

Aerodynamic turbulent flows are characterized by a complex behavior with multiple vortices of varying size, intensity and frequency. The complexity of turbulence lies in the Navier-Stockes equations that can only be solved in simple geometries and low turbulent flows (low Reynolds number flows) [START_REF] Löfdahl | MEMS applications in turbulence and flow control[END_REF]. For complex geometries and highly turbulent flows (high Reynolds number flows), computer simulations require experimental data. More precisely, two quantities are of great interest in turbulence equations: the wall shear stress and the wall pressure. Therefore, engineers and scientists have worked on the development of wall shear stress and wall pressure sensors for aerodynamic applications [START_REF] Löfdahl | MEMS-based pressure and shear stress sensors for turbulent flows[END_REF], [START_REF] Ghouila-Houri | Unsteady flows measurements using a calorimetric wall shear stress micro-sensor[END_REF].

One particularity of turbulent flows is that spatial and temporal scales of turbulence structures decrease when the Reynolds number increases. The need is thereby of small and fast sensors and MEMS (Micro Electro Mechanical Systems) technologies logically appeared as a potential solution to address this issue. The measurement of wall shear stress using MEMS devices was, and is still, vastly investigated, with technologies using various physical principles: from floating element based capacitive sensors to thermal sensors [4]. Our group in IEMN (Institute of Electronics, Microelectronics and Nanotechnology) designed, fabricated, characterized and calibrated bidirectional wall shear stress thermal micro-sensors ( [START_REF] Ghouila-Houri | High temperature gradient micro-sensor for wall shear stress and flow direction measurements[END_REF], [START_REF] Ghouila-Houri | Unsteady flows measurements using a calorimetric wall shear stress micro-sensor[END_REF]), based on a suspended hotwire microstructure.

For the measurement of wall pressure fluctuations, most technologies are based on a membrane design. The membrane is deformed due to the pressure fluctuations and the transducers either detects the stress using piezoresistive effect as in [START_REF] Berns | AeroMEMS sensor array for high-resolution wall pressure measurements[END_REF] or the deflection using capacitive measurement [START_REF] Zagnoni | A non-invasive capacitive sensor strip for aerodynamic pressure measurement[END_REF]. In each case, the measurement of pressure involves a movable mechanical part, sensitive to structure vibrations.

On the contrary, the Pirani effect exploits thermal transfers to measure the absolute pressure. Indeed, the gas thermal conductivity is pressure dependent. Consequently, Pirani sensors are composed of a hot-wire and a heat sink, and they measure the thermal transfer from the hot-wire to the substrate through the gas present in the gap between them [START_REF] Santagata | An analytical model and verification for MEMS Pirani gauges[END_REF]. When the pressure is high, the heat transfer, which is realized by the collision of gas molecules, is fully efficient. When pressure lowers, the mean free path of gas molecules increases. At the so-called pressure of transition, the mean free path of gas molecules becomes similar to the gap. Consequently, the heat transfer by collision becomes less efficient and the thermal conductivity decreases until the hot-wire is thermally isolated from the substrate [START_REF] Ghouila-Houri | High temperature gradient nanogap-Pirani micro-sensor with maximum sensitivity around atmospheric pressure[END_REF]. Thus, no movable part is involved in the working principle of such sensors. However, Pirani sensors are usually used for vacuum pressure applications [START_REF] Grau | Optimized MEMS Pirani sensor with increased pressure measurement sensitivity in the fine and rough vacuum regimes[END_REF] because for the measurement of pressure near the atmospheric pressure, the gap needs to be at nanoscales [START_REF] Khosraviani | The nanogap Pirani-a pressure sensor with superior linearity in an atmospheric pressure range[END_REF]. Our group developed a MEMS Pirani sensor with a nanoscale gap demonstrating a maximum of sensitivity around atmospheric pressure [START_REF] Ghouila-Houri | High temperature gradient nanogap-Pirani micro-sensor with maximum sensitivity around atmospheric pressure[END_REF].

In the present work, we investigated the utilization of this Pirani micro-sensor for aerodynamic wall pressure measurement in a turbulent boundary layer wind tunnel. This paper will be divided into two main parts. Firstly, we will introduce the micro-sensor particular design achieving a high temperature gradient, a large heat exchange area and a nanoscale gap. We will also present the calibration of the Pirani sensor for a wide range of pressure going from 10 kPa to 800 kPa. Secondly, we will present the integration of the Pirani micro-sensor in wind tunnel facilities. In this part, we will describe and discuss the aerodynamic wall pressure measurement performed in the wind tunnel.

II. THE PIRANI MICRO-SENSOR DESIGN AND CALIBRATION

A. The design

As mentioned before, Pirani sensors are usually composed of a hot-wire suspended over the substrate with a micro-bridge design anchored to the substrate at each extremity [START_REF] Santagata | An analytical model and verification for MEMS Pirani gauges[END_REF]. In our work, the design is slightly different. The micro-sensor is designed as a 1 mm long hot-wire based microstructure which is suspended over the substrate and mechanically supported by perpendicular silicon dioxide micro-bridges (Fig. 1 (a)). With such design, the present micro-sensor acts as multiple micro-Pirani gauges, which are about 10 µm long, anchored to two micro-bridges on extremities, and placed in series to reach 1 mm long. The gap between the hot-wire and the substrate is height-controlled and can be reduced up to 100 nm. Consequently, the microsensor reach both a nanoscale gap and a large heat exchange area that improves the signal to noise ratio and the dynamic range of the measure.

The micro-sensor was fabricated in the IEMN cleanroom facilities, using conventional surface micromachining techniques as described in [START_REF] Ghouila-Houri | High temperature gradient nanogap-Pirani micro-sensor with maximum sensitivity around atmospheric pressure[END_REF]. Figure 1 (b) and(c) display Scanning Electron Microscopy (SEM) pictures of the fabricated micro-sensor. In Figure 1 (b), one can see the series of 10 µm long micro-Pirani gauges shaping the 1 mm long hot-wire. In Figure 1 (c), the nanogap is visible: both the wire and the micro-bridges are suspended over the substrate.

The microstructure allowed then a nanoscale gap but with a 1 mm long hot-wire which allows to have a large heat exchange area and a high temperature gradient (2°C/ mW [START_REF] Ghouila-Houri | High temperature gradient nanogap-Pirani micro-sensor with maximum sensitivity around atmospheric pressure[END_REF]) for high signal to noise ratio, compared to nanowires.

B. Calibration of the device

The micro-sensor was calibrated in a pressure-controlled chamber (Fig. 2 (a)). The pressure was controlled using a PPC4 Fluke Pressure calibrator.

The micro-sensor works in Constant Temperature (CT) mode: the heating current is electronically regulated to maintain the wire temperature constant. We calibrated the micro-sensor for pressure going from 10 kPa to 800 kPa. The dynamic range considered here is far larger than the needed range in aerodynamics. Nonetheless, as shown in Figure 2 (b), the micro-sensor has not reached its saturation limits, neither at high pressure nor at low pressure. The maximum of sensitivity is reached for 110 kPa [START_REF] Ghouila-Houri | High temperature gradient nanogap-Pirani micro-sensor with maximum sensitivity around atmospheric pressure[END_REF]. The direction of current variation can be noted: it increases with over pressure and decreases with depression.

III. AERODYNAMIC WALL PRESSURE MEASUREMENTS

A. Integration in wind tunnel

Wind tunnel experiments were performed in the ONERA Lille Turbulent Boundary Layer wind tunnel (Fig. 3 (a)). The wind tunnel test section is 30 cm by 30 cm and the mainstream flow velocity reaches a maximum of 40 m/s. The Pirani micro-sensor was packaged on a support and contacts were recovered using wire bonding (Fig. 3 (b)).

The integration of the micro-sensor in the wind tunnel wall needed however the addition of a rigid cover with a hole, allowing pressure measurement while avoiding convective heat transfer. Indeed, the micro-sensor is based on thermal principle as thermal flow sensors and is therefore sensitive to wall shear stress by convective heat transfer as described in [START_REF] Ghouila-Houri | Robust thermal microstructure for designing flow sensors and pressure sensors[END_REF]. However, it is simple to know if the microsensor measures pressure or wall shear stress. Indeed, with the increasing flow velocity, the wall pressure decreases whereas the wall shear stress increases. As regards the micro-sensor operation, the heating current increases when the wall shear stress increases [START_REF] Ghouila-Houri | Robust thermal microstructure for designing flow sensors and pressure sensors[END_REF] whereas it decreases when measuring a depression.

B. Results

We realized two experiments: first we measured the variation of heating current of the Pirani micro-sensor with the flow velocity. Then we replaced the Pirani micro-sensor by a membrane based commercial piezo-resistive pressure sensor and realized the measurements again as reference measurements. Thereby we could compare the two results. In Figure 4, we displayed the measured variation of pressure versus the mainstream flow velocity U∞ for both the reference sensor and the Pirani micro-sensor. For the latter, we used the calibration curve obtained in Figure 2 (b) to convert the variation of heating current in pressure variation.

The results presented in Figure 4 show that both sensors measure almost the same diminution of pressure with the flow velocity, with up to -1.1 kPa for 40 m/s. This is the first demonstration of aerodynamic wall pressure measurement using a thermal Pirani sensor. Further work will investigate the measurement of the dynamic wall pressure fluctuations using the thermal Pirani micro-sensor.

IV. CONCLUSION

In this paper, we presented the first demonstration of aerodynamic wall pressure measurement using a high temperature gradient Pirani based micro-sensor. The microsensor was designed with a nanoscale gap and a large heat exchange area for obtaining both maximum sensitivity at atmospheric pressure and high signal to noise ratio. It was fabricated in the IEMN cleanroom using CMOS compatible micromachining techniques, allowing future integration in microelectronic chip. The micro-sensor was integrated in a wind tunnel wall and we measured the diminution of pressure with the increasing mainstream flow velocity. The comparison of the obtained result to a reference sensor demonstrated the ability of the Pirani micro-sensor to measure aerodynamic wall pressure. Future work will investigate the dynamic calibration of the micro-sensor and the use of the micro-sensor to measure the wall pressure fluctuations in various fluidic configurations (flat plate, separated flow…).

Fig 1 :

 1 Fig 1: (a) Schematic of the Pirani micro-sensor (not to scale) (b) SEM picture of the fabricated micro-sensor (c) Zoom on the gap (SEM

Fig. 2 :

 2 Fig. 2: (a) Experimental set up for pressure calibration (b) Calibration of the Pirani micro-sensor [9].

Fig3:

  Fig3: (a) ONERA Lille wind tunnel (b) Micro-Pirani Sensor wire bonded on a support (c) Integration in the wind tunnel wall with a rigid cover avoiding convective heat transfer

ACKNOWLEDGMENT

This work was funded by the French National Research Agency (ANR) in the framework of the ANR ASTRID "CAMELOTT" Project. It is supported by the regional platform CONTRAERO in the framework of the CPER ELSAT 2020 Project. The authors also thank RENATECH, the French national nanofabrication network, and FEDER.