S Ployon

A Attina

J Vialaret

Anne Sophie Walker

Christophe Hirtz

Cédric Saucier
email: cedric.saucier@umontpellier.fr

& C Saucier

Laccases 2 & 3 as biomarkers of Botrytis cinerea infection in sweet white wines

Keywords: Botrytized white wines, Botrytis cinerea, noble rot, laccases

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Botrytized wines are sweet wines which are made with overripe berries from Vitis vinifera infected by the fungus Botrytis cinerea which are rich in aroma compounds [START_REF] Tosi | Effects of noble rot on must composition and aroma profile of Amarone wine produced by the traditional grape withering protocol[END_REF]. They are produced in different countries in the world such as Hungary (Tokaji Aszu), Germany (Trockenbeerenauslese and Beerenauslese wines) and France (Sauternes) [START_REF] Magyar | Botrytized Wines[END_REF][START_REF] Teissedre | Botrytized Wines : Sauternes, German Wines[END_REF]. Recently, the production of botrytized wines has increased in Australia, New Zealand, South Africa, and California [START_REF] Magyar | Botrytized Wines[END_REF].

Botrytis cinerea (Botryotinia fuckeliana) is a filamentous and phytopathogenic fungus with a wide host range since it can infect more than 220 eudicot plants [START_REF] Fournier | The 'Dr Jekyll and Mr Hyde fungus' : Noble rot versus gray mold symptoms of Botrytis cinerea on grapes[END_REF]. B. cinerea is notably the main pathogen infecting grapevines. Contamination of grape berries by B. cinerea was well compared to "Dr Jekyll and Mr Hyde" since its infection may take two very distinctive pathways, namely "noble rot" or "bunch rot", closely depending on weather conditions [START_REF] Fournier | The 'Dr Jekyll and Mr Hyde fungus' : Noble rot versus gray mold symptoms of Botrytis cinerea on grapes[END_REF]. The development of B. cinerea under mild climatic conditions (i.e. dry days followed by moisty nights) and a late grape harvest promotes noble rot development which induces favourable changes in berry composition. During the natural noble rot process, water evaporation through grape skin leads to a decrease of berry water content. Besides this process, many metabolic and physicochemical changes occur in the berries: an increase of sugar content due to berry dehydration, changes in sugar profile, changes in acid concentrations [START_REF] Ribéreau-Gayon | Handbook of enology[END_REF]. [START_REF] Blanco-Ulate | Developmental and metabolic plasticity of white-skinned grape berries in response to Botrytis cinerea during noble rot[END_REF] reported an activation of the stilbene, flavonoid and anthocyanin biosynthesis in noble rot berries [START_REF] Blanco-Ulate | Developmental and metabolic plasticity of white-skinned grape berries in response to Botrytis cinerea during noble rot[END_REF]. The noble rot process impacts the berry aroma composition [START_REF] Sarrazin | Characterization of key-aroma compounds of botrytized wines, influence of grape botrytization[END_REF] and especially its volatile thiol content [START_REF] Thibon | Aromatic potential of botrytized white wine grapes : Identification and quantification of new cysteine-S-conjugate flavor precursors[END_REF][START_REF] Tominaga | Stereoisomeric Distribution of 3-Mercaptohexan-1-ol and 3-Mercaptohexyl Acetate in Dry and Sweet White Wines Made from Vitis vinifera (Var. Sauvignon Blanc and Semillon)[END_REF], providing botrytized wines with a distinctive sensory profile with more grapefruit and passion fruit notes.

By contrast, a cool weather associated with heavy rainfalls favours B.cinerea development as bunch or grey rot in grape clusters. This leads to unwanted organoleptic properties in musts and wines such as the appearance of earthy mushroom aromas and other off-flavor compounds [START_REF] Lopez Pinar | Effects of Botrytis cinerea and Erysiphe necator fungi on the aroma character of grape must : A comparative approach[END_REF][START_REF] Steel | Grapevine Bunch Rots : Impacts on Wine Composition, Quality, and Potential Procedures for the Removal of Wine Faults[END_REF]. In addition, the oxidation of phenolic compounds by B. cinerea laccase enzymes leads to a decline of must and wine colour stability [START_REF] Claus | Laccases of Botrytis cinerea[END_REF][START_REF] Ribéreau-Gayon | Handbook of enology[END_REF][START_REF] Steel | Grapevine Bunch Rots : Impacts on Wine Composition, Quality, and Potential Procedures for the Removal of Wine Faults[END_REF].

B. cinerea also impacts the grapes and wine proteomes as it secretes some enzymes and exhibits a high proteolytic activity leading to grape protein degradation [START_REF] Cilindre | Influence of Botrytis cinerea infection on Champagne wine proteins (characterized by two-dimensional electrophoresis/immunodetection) and wine foaming properties[END_REF][START_REF] Kupfer | Comparative protein profile analysis of wines made from Botrytis cinerea infected and healthy grapes reveals a novel biomarker for gushing in sparkling wine[END_REF][START_REF] Marchal | Effects of Botrytis cinerea Infection on the Must Protein Electrophoretic Characteristics[END_REF].

This alteration of grape proteins affects wine properties and quality such as gushing [START_REF] Kupfer | Comparative protein profile analysis of wines made from Botrytis cinerea infected and healthy grapes reveals a novel biomarker for gushing in sparkling wine[END_REF] and foaming properties [START_REF] Cilindre | Influence of Botrytis cinerea infection on Champagne wine proteins (characterized by two-dimensional electrophoresis/immunodetection) and wine foaming properties[END_REF][START_REF] Marchal | Evidence for Protein Degradation by Botrytis cinerea and Relationships with Alteration of Synthetic Wine Foaming Properties[END_REF]. Infection is also characterised by production of B. cinerea proteins that can be found in berries and resulting wines, such as ATP synthase, hydroxysteroid dehydrogenases [START_REF] Lorenzini | Identification of potential protein markers of noble rot infected grapes[END_REF], glycoside hydrolase, beta-1,3-gucanase [START_REF] Perutka | High-proline proteins in experimental hazy white wine produced from partially botrytized grapes : High-Proline Proteins in Hazy White Wine[END_REF] and laccases [START_REF] Kupfer | Comparative protein profile analysis of wines made from Botrytis cinerea infected and healthy grapes reveals a novel biomarker for gushing in sparkling wine[END_REF][START_REF] Perutka | High-proline proteins in experimental hazy white wine produced from partially botrytized grapes : High-Proline Proteins in Hazy White Wine[END_REF]. Laccases (EC 1.10.3.2) are well-known and the most studied proteins of B. cinerea since they are responsible for major organoleptic problems and alteration of wine stability. They are highly glycosylated extracellular enzymes that belong to the family of polyphenol oxidase (PPO) [START_REF] Claus | Laccases : Structure, reactions, distribution[END_REF]. These multi-cooper oxidases are responsible for oxidative reactions in plants, using oxygen to oxidise a wide range of aromatic and non-aromatic substrates by a radical-catalysed reaction mechanism [START_REF] Claus | Laccases : Structure, reactions, distribution[END_REF]. In the case of grey mould contamination, the oxidation of phenolic compounds into highly reactive quinones that polymerize into brownish polymers responsible for wines colour alteration [START_REF] Ribéreau-Gayon | Handbook of enology[END_REF]. The effect of laccase activity on organoleptic properties of musts and wines during noble rot infection is less clear. Even though laccase activity was detected in noble rot berries, its implication in the gold colour of botrytized wines was not elucidated and might be caused by glucose-oxidase activity more than laccase activity [START_REF] Vivas | Occurrence and specificity of glucose oxidase (E.C: 1.1.3.4) in botrytized sweet white wine. Comparison with laccase (E.C: 1.10.3.2[END_REF]. A recent study reported the presence of a single laccase in hazy white wines produced with partially botrytized grapes [START_REF] Perutka | High-proline proteins in experimental hazy white wine produced from partially botrytized grapes : High-Proline Proteins in Hazy White Wine[END_REF]. However, two laccases were identified by our research group from in vitro cultures of B. cinerea for three different strains [START_REF] Quijada-Morin | Strain effect on extracellular laccase activities from Botrytis cinerea : Strain effect on laccases from Botrytis cinerea[END_REF]. To our knowledge, there was no study on laccases produced by B. cinerea in sweet white wines. The aim of our study is then to identify laccases present in botrytized white wines. First, a bottom-up qualitative proteomic approach was used to identify laccases present in

Protein Precipitation

TCA/Acetone

SDS-PAGE Electrophoresis

NanoFlow LC -Q-TOF-MS

Laccase identification

LC -MRM

Laccase

Semi-Quantification 6

Material & Methods

Wine samples

Three commercial non-botrytized sweet white wines (SWs) and 3 commercial botrytized wines (BWs) were used in this study. Samples are listed in table 1. After bottles opening, wines were divided into 15 ml tube aliquots and kept at -80°C.

Chemicals

Acetone (analytic grade), trifluroroacetic acid (TFA) and dithiothreitol (DTT) were purchased from Sigma (Saint Quentin Fallavier, France). Trichloroacetic acid (TCA) was purchased from Acros Organics (Thermo Fisher). The reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Invitrogen. Urea was purchased from Merck KGaA (Darmstadt, Germany) and ammonium bicarbonate (ABC) was purchase from Fluka Analytical (Bucharest, Romania). Tris(hydroxymethyl)aminomethane (Tris) and iodoacetic acid (IAA) were purchased from VWR (Fontenay-sous-Bois, France). Water ULC-MS, formic acid ULC-MS (FA), acetonitrile ULC-MS (ACN), methanol ULC-MS (MeOH) were all from Biosolve (Dieuze, France).

Trypsin Gold, Mass Spectrometry Grade, was purchased from Promega (Charbonnières-les-Bains, France).

Trichloroacetic acid/acetone wine proteins extraction

Total proteins from 10 ml of wines were precipitated overnight by adding 40 ml of 10% TCA/chilled acetone (-20°C). After precipitation, samples were centrifuged at 10 000 g for 20 min at 4°C. Proteins pellets were washed twice with 1 ml of cold acetone and centrifuged at 10 000 g for 20 min at 4°C.

Pellets were stored at -80°C until use for Q-TOf and MRM analysis. Blue Coomassie Instant Blue (Expedeon, San Diego, USA). For each wine sample, 8 bands were excised from the gel on a molecular weight range from 30 to 190 kDa. In gel digestion of bands was performed as following. Bands were washed successively twice with 150 μL of 50% ethanol in 50mmol/L ABC (pH 8.4) and were dried using a Speedvac (Labconco, Kansas City, MO, USA). Then, the bands were successively reduced (10 mmol/L of DTT, 56°C;1 h), alkylated (55 mmol/L of IAA, 37°C, 45 min), dehydrated (50% ethanol in 50 mmol/L ABC (pH 8.4)) and dried using a Speedvac.

Bands were digested overnight at 37°C in 12.5 mg/mL of tryspin solution in 25 mmol/L ammonium carbonate. The digestion was stopped by adding 15 μL of pure FA (pH < 4). The tryptic peptides were desalted on BRAVO AssayMap (Agilent Technologies, Les Ulis, France) with C18 Tips conditioned with 50 μL of 70% ACN/0.1% FA and equilibrated with 50 μL of 0.1% FA. Loaded samples were washed twice with 50 μL of 0.1% FA and eluted with 50 μL of 70% ACN/0.1% FA. Samples were dried using a Speedvac (Labconco, Kansas City, MO, USA) and resuspended in 10 μL of phase A (0.1% formic acid, 2% ACN in water) for LC-MS/MS injections.

Sample preparation for MRM analyses

Protein pellets were obtained as described above. All the following steps were performed on Amicon Ultra 4, 30kDa centrifugal filter units (Merck Millipore, Molsheim, France). Protein pellets were suspended and washed twice by 8 M urea in Tris-HCl (pH 8.5) and successively reduced in 10 mM of DTT in Tris-HCl (pH 8.5), alkylated in IAA in 0.1 M in Tris-HCl (pH 8.5), washed twice with 50 mM ABC buffer and then digested overnight (37°C) with trypsin at 1 µg/ml in 50 mM ABC buffer. Tryptic peptides were dried using a Speedvac (Thermo Fisher Scientific, Villebon sur Yvette, France) and resuspended in 30 µl ACN with 0.1% FA.

Mass Analyses

Nano-Q-TOF-MS analyses for protein identification

Seven microliters of the samples were injected in an HPLC NanoElute (Bruker, Billerica, USA) system coupled to a Q-TOF-MS instrument (Impact II, Bruker Daltonics, Champs sur Marne, France).

LC-MRM analyses for laccase targeted detection

The two following peptides, YDSSSTVDPTSVGVTPR (laccase 2) and YTTNTITAGK (laccase 3)

were purchased from Eurogentec (Angers, France). Linearity of the two peptides was assessed.

Synthetic standards were mixed and diluted in 0.1% FA (Phase A) at 1.4, 0.14 and 0.014 pmol/µL for YDSSSTVDPTSVGVTPR and 0.14, 0.014 and 0.0014 pmol/µL for YTTNTITAGK. monitored to detect the laccase-3-BcLCC3. MS signal was integrated, manually checked, and exported to an Excel file with Skyline 4.1 (University of Washington). Analyses were performed on the same day in order to avoid inter-day variability on 3 biological replicates.

Statistical analysis

For MRM analyses, mean values of peak area are reported with their standard deviation. For each laccase, peptide peak area of the two groups BWs and SWs were compared by a Student t-test (α = 0.05) using XLSTAT 2018.4 (Addinsoft, Paris, France).

Results & Discussion

Identification of laccases present in sweet wines by nano-LC-Q-TOF-MS

Total proteins of a botrytized wine sample (BW1) and a non-botrytized wine sample (SW1) were isolated by TCA/acetone precipitation and separated by SDS-PAGE electrophoresis. Tryptic digests were analysed by nano-LC-MS and peptides were compared against two distinct databases with a focus to identify laccases. In the molecular weight range studied (i.e. 30 to 190 kDa) fifteen proteins were identified in the UniprotKB/SwissProt global protein database, mainly yeast protein from Saccharomyces cerevisiae and enzymes from B. cinerea (Supp. Material S1).

A short homemade laccase database was established using laccase genes data collected by [START_REF] Simon | Cross-reference table for Botrytis cinerea B0510 and T4 gene ids[END_REF] to specifically identify laccases present in BW1 and SW1. Results are listed in Table 2. Five different laccases were detected in the samples. Laccase-2-BcLCC2, laccase-3-BcLCC7 and laccase-8-BcLCC8 were identified in both SW1 and BW1 samples. BcLCC12 was detected only in SW1 while laccase-1-BcLCC1 was detected only in BW1. Laccases were identified as isoenzymes in more than 115 fungus species [START_REF] Baldrian | Fungal laccases -occurrence and properties[END_REF].

From a genetic point of view, 13 genes (BcLcc1 to BcLcc13) encoding for laccases were identified in B. cinerea genome [START_REF] Simon | Cross-reference table for Botrytis cinerea B0510 and T4 gene ids[END_REF].

Laccase-2-BcLCC2 is a 581 amino acids protein encoded by the BcLcc2 gene. The molecular weight calculated for the enzyme is 63.4. A higher apparent molecular weight around 94 kDa was observed by SDS-PAGE electrophoresis likely due to glycosylation of these enzymes [START_REF] Quijada-Morin | Strain effect on extracellular laccase activities from Botrytis cinerea : Strain effect on laccases from Botrytis cinerea[END_REF]. Laccase-2-BcLCC2 is the main laccase identified in B. cinerea cultures independently of the strain since studies reported its secretion by B0510 [START_REF] González-Fernández | Proteomic analysis of mycelium and secretome of different Botrytis cinerea wildtype strains[END_REF][START_REF] Quijada-Morin | Strain effect on extracellular laccase activities from Botrytis cinerea : Strain effect on laccases from Botrytis cinerea[END_REF][START_REF] Schouten | Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase[END_REF]), RM344 and VA612 (Quijada-Morin et al., 2017) and T4 (González-Fernández et al., 2014) strains. Laccase-2-BcLCC2 was recently detected in hazy white wine [START_REF] Perutka | High-proline proteins in experimental hazy white wine produced from partially botrytized grapes : High-Proline Proteins in Hazy White Wine[END_REF] and gushing sparkling white wine [START_REF] Kupfer | Comparative protein profile analysis of wines made from Botrytis cinerea infected and healthy grapes reveals a novel biomarker for gushing in sparkling wine[END_REF] produced with partially infected grapes. Laccase-2-BcLCC2 could be involved in detoxification of compounds contained in the plant, that are responsible for fungus development inhibition such as resveratrol [START_REF] Schouten | Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase[END_REF]. Its ability to degrade the phenolic antibiotic 2,4-diacetylphloroglucinol was also demonstrated in vitro [START_REF] Schouten | Involvement of the ABC transporter BcAtrB and the laccase BcLCC2 in defence of Botrytis cinerea against the broad-spectrum antibiotic 2,4-diacetylphloroglucinol[END_REF].

Laccase-3-BcLCC7 is a 663 amino acids protein of 71.1 kDa encoded by the gene BcLcc7 [START_REF] Simon | Cross-reference table for Botrytis cinerea B0510 and T4 gene ids[END_REF]. The expression of this laccase was reported for the first time by Quijada-Morin et al (2017) for three different B. cinerea strains, with slight differences in the amino acid sequence for the strains RM344 and VA612 compared to the reference strain B0510 (Quijada-Morin et al., 2017). In both cases, a lower number of peptides and a lower sequence coverage was observed compared to laccase-2-BcLCC2. To our knowledge, it is the first time that the laccase-3-BcLCC3 was identified in a wine matrix.

As far as we know, there are no proteomic study that report the presence of other three laccases in B. cinerea secretome. BcLcc1 and BcLcc8 genes expression was investigated by transcriptomic studies. BcLcc1 transcriptome was not detected in B. cinerea cultures, even after induction by phenolic compounds [START_REF] Schouten | Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase[END_REF]. Regarding laccase-8-BcLCC8, an upregulation of BcLcc8 gene during inoculation of tomatoes by B. cinerea was reported, but no protein expression was evidenced [START_REF] Petrasch | Infection Strategies Deployed by Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer as a Function of Tomato Fruit Ripening Stage[END_REF].

The abundance of MS fragments suggests that the amount of laccase-2-BcLCC2 is higher than other laccases in both wines. However, the Q-TOF results did not allow to discriminate both wines.

Thus, a second approach was developed to provide a semi quantitative measurement of laccases in sweet white wines. Considering the high M. scores obtained for the identification of laccase-2-BcLCC2 and laccase-3-BcLCC7 (i.e. up to 400 for laccase-3-BcLCC7 and up to 2000 for laccase-2-BcLCC2 in BW, compared to values under 100 for laccase-1-BcLCC1, laccase-8-BcLCC8 and laccase-12-BcLCC12), we chose to focus on laccases-2-BcLCC2 and -3-BcLCC7 for the MRM semiquantitative study.

Multiple reaction monitoring (MRM) analysis for targeted detection of laccases-2-BcLCC2 & laccase-3-BcLCC7

The relative abundance of both laccase-2-BcLCC2 and laccase-3-BcLCC7 was investigated in three botrytized and three non-botrytized wines. Two peptides with a specific sequence for each enzyme were chosen: YDSSSTVDPTSVGVTPR for laccase-2-BcLCC2 and YTTNTITAGK for laccase-3-BcLCC7. These peptides were then monitored by LC-MRM after tryptic digestion of the protein extracts.

Linearity of the MS signal of the two peptides was confirmed (R² > 0.99) by using analytical standards in ACN. The peptide linearity ranges from 0.141 to 28.16 pmol for YDSSSTVDPTSVGVTPR (Figure 2A) and from 0.0028 to 2.816 pmol for YTTNTITAGK (Figure 2B). For YDSSSTVDPTSVGVTPR (laccase 2), the LOD and LOQ calculated were 0.014 pmol/µL and 0.14 pmol/µL, respectively. For YTTNTITAGK (laccase 3), the LOD and LOQ calculated were 0.0014 pmol/µL and 0.0028 pmol/µL, respectively. The relative abundance of each peptide suggests that laccase-2-BcLCC2 was the major laccase present in all the wines. Generally, the composition of the growing environment of the fungus influences laccase production and some compounds may act as inducers of laccase secretion that can impact the laccase substrate specificity, molecular weight, glycosylation level and sugar pattern [START_REF] Marbach | Inducer and culture medium dependent properties of extracellular laccase from Botrytis cinerea[END_REF]. A previous study reported that laccase-2-BcLCC2 production was induced by gallic acid [START_REF] Quijada-Morin | Strain effect on extracellular laccase activities from Botrytis cinerea : Strain effect on laccases from Botrytis cinerea[END_REF]. An increase of BcLcc2 gene transcriptome was also evidenced in B. cinerea culture in presence of resveratrol and tannic acid [START_REF] Schouten | Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase[END_REF], that belong to the stilbenes and the tannins families, respectively, and in presence of cooper (Udugama Vithanage, Savocchia, Strappe, Schmidtke, [START_REF] Udugama Vithanage | Copper induction of laccases in the plant pathogenic fungus Botrytis cinerea[END_REF]. In accordance with our results, laccase-3-BcLCC7 seemed also to be minor in culture media in presence of gallic acid, suggesting the phenolic acid is not the main inducer of this laccase [START_REF] Quijada-Morin | Strain effect on extracellular laccase activities from Botrytis cinerea : Strain effect on laccases from Botrytis cinerea[END_REF].

Conclusion

Using an untargeted proteomic approach, five different laccases (laccase-1-BcLCC1, laccase-2-BcLCC2, laccase-3-BcLCC7, laccase-8-BcLCC8 and laccase-12-BcLCC12) were identified in sweet and botrytized wines. To our knowledge, this is the first time that the presence of laccase-1-BcLCC1, laccase-3-BcLCC7, laccase-8-BcLCC8 and laccase-12-BcLCC12 is reported in grape or wine.

Two laccases, namely laccase-2-BcLCC2 and laccase-3-BcLCC7 were semi-quantified in sweet and botrytized wines. Laccase-2-BcLCC2, which was also detected in bunch rot wines [START_REF] Perutka | High-proline proteins in experimental hazy white wine produced from partially botrytized grapes : High-Proline Proteins in Hazy White Wine[END_REF] and culture media [START_REF] Quijada-Morin | Strain effect on extracellular laccase activities from Botrytis cinerea : Strain effect on laccases from Botrytis cinerea[END_REF][START_REF] Schouten | Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase[END_REF] seemed to be the main laccase produced by the fungus during grape infection. The second targeted laccase, laccase-3-BcLCC7, was detected in much lower amount.

This study revealed that laccase-2-BcLCC2 and laccase-3-BcLCC7 are still detectable in wines made with noble rot infected berries after several years of bottle ageing. Targeted detection by LC-MRM of the two laccases allowed the discrimination of botrytized from non-botrytized sweet white wines. These proteins are then very good quantitative biomarkers of Botrytis infection and are suitable for high throughput analysis of white wine quality in future studies.

FIGURE CAPTIONS

FIGURE

  FIGURE 1: Workflow

  and in-gel protein digestion for Q-TOf protein identification Wine protein pellets were separated by SDS-PAGE electrophoresis using à 4-12 % Tris-Acetate Protein Gels (Invitrogen, Illkirch, France). Briefly, pellets were suspended in 50 µl of milliQ water, 26 µl of the samples were mixed with 10 µl Novex NuPAGE LDS (4X) buffer (Invitrogen, Illkirch, France) and 4 µl of reducting agent NuPAGE10X (Invitrogen, Illkirch, France). Denaturation was performed 5 min at 90°C. Finally, samples were centrifuged to remove insoluble particles and loaded in SDS-PAGE gel with ready-to-use molecular-weight marker (SeeBlue® Plus 2 Prestained Standard 1X (Invitrogen, Illkirch, France). Electrophoresis was carried out at 200 mA. Gel was stained with

  Analyses were operated in nanoflow mode (400nL/min) NanoFlow LC. Samples were desalted and pre-concentrated on a PepMap u-precolumn (300 μm × 5 mm, C18 PepMap 100, 5 μm, 100 Å) (Thermo Fisher Scientific, Illkirch, France). The capillary pump was set at 20 μL/min and the mobile phase was 0.05% TFA, 2% ACN in water. Peptides were separated on a C18 analytical column (75 μm × 500 mm; Acclaim Pepmap RSLC, C18, 2 μm, 100 Å). The peptides were eluted with a gradient consisting of 5-26% B for 45 min and 90% B for 4 min (A, 0.1% formic acid, 2% ACN in water; B, 0.1% formic acid in ACN) at 400 nL/min. The temperature was set at 50°C. Peptides were identified by data-dependent acquisition (DDA). A lock-mass [m/z 1222, Hexakis (1H, 1H, 4Hhexafluorobutyloxy) phosphazine] was used as the internal calibrator. Instant Expertise software (Bruker Daltonics) selected as many as possible of the most intense ions per cycle of 3 seconds and active exclusion was performed after one spectrum for 2 minutes only if the precursor ion exhibited an intensity three times greater than the previous scan. MS/MS spectra were searched against a SwissProt_2017_07_28 database and against a home-made laccase short database adapted from Simon and Viaud (2018)[START_REF] Simon | Cross-reference table for Botrytis cinerea B0510 and T4 gene ids[END_REF]. Mascot v 2.4.1 algorithm (Matrix Science, http://www.matrixscience.com/) was used with the following settings: enzyme: trypsin; variable modifications: oxidation (M) and deamidated (N,Q); fixed modifications: carbamidomethyl (C); missed cleavages: 1; instrument type CID: ESI-QUAD-TOF; peptide tolerance: 10.0 ppm; MS/MS tolerance: 0.05 Da; peptide charge: 1+, 2+ and 3+; mass: monoisotopic; C13: 1; minimum peptide length: 5; peptide decoy: ON; adjust FDR (%): 2.5; percolator: ON ; ions score cut-off: 12; ions score threshold for significant peptide IDs: 12.

FIGURE 2 :

 2 FIGURE 2: Linearity of signals of the peptides (A) YDSSSTVDPTSVGVTPR (transition 884.42 -> 913.51) and (B) YTTNTITAGK (transition 535.28 -> 805.44).The abundance of laccase-2-BcLCC2 and laccase-3-BcLCC7 peptides in three botrytized and three non-botrytized wines are presented in figure3 (A) and (B) respectively. Laccase-2-BcLCC2 was detected in all samples BWs and SWs. The peak areas range from 3.30.10 5 AU to 7.70.10 5 AU in the BWs and from 0.16.10 3 AU to 1.04.10 3 AU in the 3 SWs, indicating a significant greater laccase 2 concentration (p-value < 0.0001) in the 3 BWs compared to the 3 SWs.Similarly, laccase-3-BcLCC7 was detected in the six wines samples. A significant difference (p-value < 0.0001) was observed between BWs and SWs for their laccase 3 content. Peak areas were significantly higher in BWs with value up to 1.36.10 3 AU than in SWs, were peak area did not exceed 0.05.10 3 AU. That represents more than a factor 27 for the intensities of the signals. Nevertheless, the presence of both laccases in non-botrytized SWs could indicate a very minor contamination of grapes used for their winemaking.

FIGURE

  FIGURE 1: Workflow

TABLE 1 : List of wine samples used in the study, years, wine origin Sample name Year Appellation Non-Botrytized Sweet Wins (SWs)

 1

		SW1	2016	Jurançon
		SW2	2015	Alsace
		SW3	2014	Beaumes de Venise
		BW1	2013	Sauternes
	Botrytized Wins (BWs)	BW2	2015	Sauternes
		BW3	2015	Sauternes

TABLE 2 : MS identification table of laccases in sweet botrytized (BW1) or non-botrytized (SW1) wines using Laccase short database (adapted from Simon & Viaud, 2018). Gene name, protein description, theoretical molecular weight (Th. MW), Isoelectric point (pI), Mascot score (M. score), number of identified peptides, and sequence coverage (SC) are reported.

 2

	Sample	B0510-BROAD gene name	Usual gene name	Protein	Th. MW [kDa]	pI	M. Scores	#Peptides SC [%]
		BC1G_08553.1	BcLcc2	Laccase-2-BcLCC2	63.4	4.5	1101.5	18	26.7
	SW1	BC1G_10329.1 BC1G_14349.1	BcLcc7 BcLcc8	Laccase-3-BcLCC7 Laccase-8-BcLCC8	71.1 76.4	5.7 5.4	64.6 23.6	1 1	1.5 1.3
		BC1G_15514.1 BcLcc12 Laccase-12-BcLCC12	67.1	5.2	15.5	1	2.6
		BC1G_08553.1	BcLcc2	Laccase-2-BcLCC2	63.4	4.5	2074.0	23	26.7
	BW1	BC1G_10329.1 BC1G_14349.1	BcLcc7 BcLcc8	Laccase-3-BcLCC7 Laccase-8-BcLCC8	71.1 76.4	5.7 5.4	407.6 51.6	9 1	16 1.3
		BC1G_00138.1	BcLcc1	Laccase-1-BcLCC1	60.8	5.9	30.8	1	1.8

Semi-quantification of laccase-2-BcLCC2 peptide (transition 884.42 -> 913.51) (A) and laccase- 3-BcLCC7 peptide (transition 535.28 -> 805.44) (B) in botrytized sweet white wines (BWs) and non-botrytized sweet white wines (SWs). Significant difference between the two groups evaluated by Student t-test *** p- value < 0.0001 (Values are given in Supp. Material S2).

	A FIGURE 3:						B
		1.E+06	y = 31000x -16204					4.E+05	y = 102715x -2561
	Peak Area	2.E+05 4.E+05 6.E+05 8.E+05	R² = 0.9981				Peak Area	1.E+05 2.E+05 3.E+05		R² = 0.9985
		0.E+00							0.E+00
		0	5	10	15	20	25	30		0	0.5	1	1.5	2	2.5	3
					pmol								pmol

TABLE 1 : List of wine samples used in the study, years, wine origin TABLE 2: MS identification table of laccases in sweet botrytized (BW1) or non-botrytized (SW1) wines using Laccase short database (adapted from Simon & Viaud, 2018). Gene name, protein description, theoretical molecular weight (Th. MW), Isoelectric point (pI), Mascot score (M. score), number of identified peptides, and sequence coverage (SC) are reported. SUPPLEMENTARY MATERIAL S1: Total protein identification table SUPPLEMENTARY MATERIAL S2: Transition results peptides YDSSSTVDPTSVGVTPR (transition 884.42 -> 913.51) and YTTNTITAGK (transition 535.28 -> 805.44).

 1

Acknowledgements

We thank Mr Laurent Tiers for technical assistance. We thank the Biolaffort Company for funding.

Conflict of interest statement

The authors declare no conflict of interest.


Fernanda C Moraes
email: fernanda.moraes@inserm.fr

Joana C Antunes
email: joana.antunes@2c2t.uminho.pt

Laura Marcela

Forero Ramirez
email: lmforeror@gmail.com

Paola Aprile
email: paola.aprile@inserm.fr

Gregory Franck
email: gregory.franck@inserm.fr

Cédric Chauvierre
email: cedric.chauvierre@inserm.fr

Frédéric Chaubet
email: fchaubet@gmail.com

Didier Letourneur
email: didier.letourneur@inserm.fr

Synthesis of cationic quaternized pullulan derivatives for miRNA delivery

Keywords: pullulan, cationic derivatives, polyplexes, microRNA, gene therapy miRNA, MicroRNA, ncRNAs, small Non Coding RNAs, pDNA, Plasmid DNA, PEI, Polyethyleneimine, PAMAM Polyamidoamine, siRNA, small interfering RNA, QA-pullulan, Quaternized ammonium-pullulan, Mw, Weight average molecular weight, Mn, Number average molecular weight, GTMAC, Glycidyltrimethylammonium chloride, 6-FAM, 6-Fluorescein amidite, SEC-MALLS, Size exclusion chromatography-multiangle laser light scattering, FTIR, Fourier-transform infrared spectroscopy, 1 H-NMR, Proton nuclear magnetic resonance, TBE, Tris-Borate-EDTA, PdI, Polydispersity Index, HUVECs, Human umbilical vein endothelial cells, PBS, Phosphate buffered saline, DS, Degree of substitution, AGU, Anhydroglucose unit, DAPI, 4

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Gene therapy uses nucleic acids (DNA or RNA) for the treatment, cure or prevention of human disorders by the insertion of new genes into target cells [START_REF] Cao | Recent Advances in Chitosan-Based Carriers for Gene Delivery[END_REF]. The use of nucleic acids as drugs has been described for two decades [START_REF] Wolff | Direct gene transfer into mouse muscle in vivo[END_REF], being mainly based on viral vectors and plasmid DNA technology [START_REF] Xiong | Biomedical applications of mRNA nanomedicine[END_REF][START_REF] Zarghampoor | Improved translation efficiency of therapeutic mRNA[END_REF]. More recently, small non coding RNAs (ncRNAs), namely microRNAs (miRNAs), have emerged as key regulators of gene expression as a promising alternative to conventional plasmid DNA (pDNA) due to its multiple unique features and advantages when compared to DNA-based therapy [START_REF] Bernardo | miRNA therapeutics: a new class of drugs with potential therapeutic applications in the heart[END_REF][START_REF] Xiong | Biomedical applications of mRNA nanomedicine[END_REF]. miRNAs are small endogenous single stranded non-coding RNA molecules that play important roles in many cellular processes such as cell differentiation, proliferation and survival acting on post-transcriptional level by binding to complementary target mRNAs, resulting in mRNA translational inhibition or degradation [START_REF] Rupaimoole | MicroRNA therapeutics: towards a new era for the management of cancer and other diseases[END_REF]. miRNAs have been proposed and investigated as therapeutics and biomarkers for various diseases such as cancer, diabetes, cardiovascular diseases and other pathologies whose etiology is related to aberrant gene expression [START_REF] Candido | The analysis of miRNA expression profiling datasets reveals inverse microRNA patterns in glioblastoma and Alzheimer's disease[END_REF][START_REF] Falzone | Identification of Novel MicroRNAs and Their Diagnostic and Prognostic Significance in Oral Cancer[END_REF][START_REF] Gaur | Chitosan nanoparticle-mediated delivery of miRNA-34a decreases prostate tumor growth in the bone and its expression induces non-canonical autophagy[END_REF][START_REF] Hou | The let-7 microRNA enhances heme oxygenase-1 by suppressing Bach1 and attenuates oxidant injury in human hepatocytes[END_REF][START_REF] Poddar | miR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells[END_REF]Zhu et al., 2019). At the target, miRNAs can generate strong, sustained and comprehensive biological effects, through endogenous gene silencing with very low concentrations (at least 0.5 nM) [START_REF] Devaux | for the Biomarker subcommittee of the TTM trial (Target Temperature Management After Cardiac Arrest, NCT01020916)[END_REF][START_REF] Qiagen | Guidelines for miRNA mimic and miRNA inhibitor experiments[END_REF]. Additionally, miRNAs are able to affect multiple pathways/cellular processes rather than specific targets [START_REF] Van Rooij | Toward MicroRNA-Based Therapeutics for Heart Disease: The Sense in Antisense[END_REF]. The success of miRNA-based therapies depends on the development of suitable vehicles able to specifically target cells for the efficient delivery of the nucleic acids, with minimum toxicity [START_REF] Chakraborty | Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine[END_REF].

Compared to DNA-based gene therapy, the delivery of miRNA is more challenging due to important differences in terms of size and structure. Most of the polymeric delivery strategies originally developed for plasmid DNA (pDNA) in gene therapy cannot be used for miRNA delivery without formulation optimization since miRNA are much smaller molecules (21-23 bp) than DNA (several kilo bp). Thereby, small RNAs are more difficult to be packaged and condensed into stable systems for protection against early clearance and degradation [START_REF] Nuhn | Size-Dependent Knockdown Potential of siRNA-Loaded Cationic Nanohydrogel Particles[END_REF][START_REF] Scholz | Therapeutic plasmid DNA versus siRNA delivery: Common and different tasks for synthetic carriers[END_REF]. Moreover, the vulnerability of RNA to enzymatic degradation (hydrolyses) by serum nucleases due to an extra hydroxyl group in the 2' position of pentose ring in ribose structure, instead of a hydrogen atom present in DNA structure, induced additional challenges to RNA delivery [START_REF] Gary | Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery[END_REF][START_REF] Mao | Chitosan-based formulations for delivery of DNA and siRNA[END_REF].

Several types of biomaterials have been used as platforms for gene delivery, and in particular positively-charged polymers represent a high proportion. They can bind nucleic acids via electrostatic interactions to form particulate structures named polyplexes [START_REF] Shen | Polymer-Nucleic Acid Interactions[END_REF]. Polyplexes are formed in solution by the direct mixing of two oppositely charged polyelectrolytes and the main driving force that guides polyplex formation is an increase in the entropy of the system due to the release of low-molecular weight counterions which is able to produce nanoparticles containing the nucleic acid payload in a compacted and protected structure [START_REF] Lächelt | Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50 Years (and Beyond)[END_REF][START_REF] Lounis | Prediction of Polyelectrolyte Complex Stoichiometry for Highly Hydrophilic Polyelectrolytes[END_REF]. The easy manipulation and versatility of physicochemical properties are some of the most important advantages of polymeric gene carriers [START_REF] Patil | DNA-based therapeutics and DNA delivery systems: A comprehensive review[END_REF]. Cationic polymers that have been used for the delivery of miRNAs include polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimers and chitosan [START_REF] Mao | Chitosan-based formulations for delivery of DNA and siRNA[END_REF][START_REF] Scholz | Therapeutic plasmid DNA versus siRNA delivery: Common and different tasks for synthetic carriers[END_REF]. Polymers derived from natural sources could be preferable in gene therapy applications because they are naturally biocompatible, biodegradable and nontoxic and can overcome the cytotoxicity associate with the use of several synthetic polymers which does not satisfy the requirements for clinical applications. However, cationic chitosan, one of the most studied polymers of the list, has its own drawbacks such as low water solubility at physiological pH because of partial protonation of primary amino groups which can affects nucleic acid interactions and stability of complexes in bloodstream [START_REF] Gary | Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery[END_REF][START_REF] Ragelle | Chitosan nanoparticles for siRNA delivery: Optimizing formulation to increase stability and efficiency[END_REF]. Thus, the design of biocompatible, stable and efficient nanocarriers is needed [START_REF] Shi | Challenges in DNA Delivery and Recent Advances in Multifunctional Polymeric DNA Delivery Systems[END_REF]. In contrast to the large amount of synthetic non-viral gene delivery systems, there is only a small number of polycations of natural origin [START_REF] Thanou | Quaternized chitosan oligomers as novel gene delivery vectors in epithelial cell lines[END_REF].

Pullulan is a natural polysaccharide derived from Aureobasidium pullulans that consists of maltotriose units and due to its non-toxic, non-immunogenic and biodegradable properties, is considered as a promising material for biomedical applications (K.R. and V., 2017). It is admitted safe in the United States for a wide range of applications, such as an excipient in pharmaceutical tablets and listed in the Japanese Standards as accepted adjuvant for drug delivery [START_REF] Prajapati | Pullulan: An exopolysaccharide and its various applications[END_REF][START_REF] Rekha | Blood compatibility and in vitro transfection studies on cationically modified pullulan for liver cell targeted gene delivery[END_REF]. Cationic pullulan derivatives grafted with primary or tertiary amine groups were previously demonstrated to be able to form complexes with plasmid DNA as well as siRNA in cationized pullulan-based hydrogels [START_REF] San Juan | Development of a Functionalized Polymer for Stent Coating in the Arterial Delivery of Small Interfering RNA[END_REF], 2007). However, the ionic interaction between the macromolecules closely depend on the pH. Hydroxyl groups of pullulan can be easily substituted with quaternized ammonium groups (QA) to improve the interaction with negatively charged nucleic acids [START_REF] San Juan | Tubular cationized pullulan hydrogels as local reservoirs for plasmid DNA[END_REF][START_REF] Shao | Elucidating the role of free polycationic chains in polycation gene carriers by free chains of polyethylenimine or N,N,N-trimethyl chitosan plus a certain polyplex[END_REF]. QA derivatives are an interesting strategy for gene therapy due to their permanent positive charges independent of the pH [START_REF] Ahmed | Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery[END_REF]. In this work, polycations have been obtained by grafting QA using glycidyltrimethylammonium chloride at different feeding ratios into the pullulan backbone. Then, QA-pullulan formulations were used to form nanosized polyplexes with miRNAs opening the way to their use as a platform for gene delivery.

Materials and Methods

Materials

Pullulan (Mw 200 kg/mol) was purchased from Hayashibara Company (Okayama, Japan).

Glycidyltrimethylammonium chloride (GTMAC) (Mw 151.63 g/mol) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dialysis membranes of 20 kDa was purchased from Spectrum Laboratories (New Brunswick, NJ, USA). Single-stranded miRNA and 6-Fluorescein AMidite (6-FAM)-miRNA scramble oligonucleotides with 23 bp of length (RNA sequence: 5'-UUA AUG CUA AUC GUG AUA GGG GU-3') used to mimic the natural human mature microRNA miR-155-5p were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Sequence was reconstituted in RNase-free water to achieve a final concentration of 20 μM and stock solutions were stored at -20°C. Ultrapure water was prepared by ion-exchange of running tap water (Millipore, France).

Synthesis of cationized pullulan

Alkylation with GTMAC

Pullulan was synthetized based on a protocol previously described by San Juan et al. to graft primary and tertiary amine groups into pullulan backbone [START_REF] San Juan | Tubular cationized pullulan hydrogels as local reservoirs for plasmid DNA[END_REF]. Briefly, pullulan (500 mg) was dissolved in 5 mL of an aqueous solution of 10 M NaOH. Then, the solution was placed at 4°C under magnetic stirring until pullulan was completely solubilized. Glycidyltrimethylammonium chloride (GTMAC) was added to polysaccharide solution at different feeding ratios (1 eq = 3.1 mmol; 2 eq = 6.2 mmol, and 3 eq = 9.3 mmol) drop by drop and under high magnetic stirring at 4°C. The solution was placed under magnetic stirring at 55°C for 4 hours. After cooling down to room temperature, the pH was adjusted to 7 with concentrated hydrochloric acid solution. The mixture was exhaustively dialyzed (MWCO of 20 kDa) for 48 hours with ultrapure water and the derivatives were obtained as fluffy white materials after freeze-drying.

Chemical characterization of modified pullulan

Elemental analysis

Elemental analysis (C, H, N, O) was performed by the Microanalysis service of Centre National de la Recherche Scientifique (CNRS) (Gif/Yvette, France) (Table 1 andTable A.1). Degree of substitution was estimated using N content values.

Molecular weight determination

Weight-average molecular weight (Mw) and number-average molecular weight (Mn) were determined by size exclusion chromatography (SEC) coupled to a multiangle laser light scattering (MALLS) detector (DAWN-F, Wyatt Technology, Santa Barbara, CA, USA). The SEC line consisted of a TSK-Gel PWXL Guardcolumn and TSK-Gel GMPWXL in series (Tosoh Bioscience, Stuttgart, Germany). The eluent was 0.2 M NaNO3 containing 0.02% (w/v) NaN3 at 0.5 mL/min flow rate. The samples were dissolved in the eluent at a concentration of 10 mg/mL. The dn/dc value used for the molecular weight distribution calculations was 0.14. The collected data were analyzed using the software Astra V-6.1.2 (Santa Barbara, CA).

FTIR spectroscopy

FTIR spectroscopy was carried out with KBr pellets on a Thermo-Nicolet-AVATAR 370 FTIR (Thermo Electron Corporation, Waltham, MA). A first pellet with 200 mg of KBr was prepared. Then, pellets were made using 200 mg KBr and 2 mg of polymer. A 32-scan interferogram was collected in transmittance mode. Scans were taken from 400 cm -1 to 4000 cm -1 at a resolution of 2 cm -1 . The spectrum of KBr pellet was used as background.

NMR spectroscopy

1 H-NMR spectra was recorded with a Bruker-400 Ultrashield (Bruker, Germany) operating at 400 MHz. Polymers samples were prepared in D2O at a concentration of 20 mg/mL.

Synthesis of QA-pullulan/miRNA Polyplexes

Briefly, cationic pullulan and anionic miRNA were dissolved in ultrapure water to obtain stock solutions at 0.08 mg/mL and 0.04 mg/mL, respectively. All polysaccharide solutions were filtered before use (0.2 µm). Nanosized polyplexes were produced by dropping 1 mL of miRNA solution over 1 mL of quaternized pullulan solution under high magnetic stirring at room temperature in aqueous solution.

Samples were placed at 4ºC during 20 min for stabilization before use. and Electrophoretic Light Scattering (ELS) using a Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK) respectively. Size and zeta potential measurements were performed at 25°C using a 173° and 13° scattering angle for size and zeta potential measurements, respectively, equipped with a 4 mW HeNe laser beam with a wavelength of 633 nm. Size and ζ potential values were automatically calculated through the software DTS Nano v.6.30 (Malvern, UK) using the Stokes-Einstein equation and the Henry equation with the Smoluchowski approximation, respectively. Size measurements were performed in water using disposable polystyrene cuvettes and zeta potential measurements were performed in 1 mM KCl solution using a DTS1070-folded capillary cell (ELS, Malvern, UK). Data were acquired in triplicate.

Morphology

The morphology of optimized polyplexes was assessed using a Transmission Electron Microscope (TEM) Tecnai 12 (80 kV; FEI; Hillsboro, OR, USA) with a 1K x 1K KeenView camera (OSiS). Nanosized polyplexes were produced as previously described. Then, 3 μL of polyplexes suspension was placed onto a 400 μL mesh copper grid coated with carbon (EMS) without any sample dilution. Uranyl acetate 1% (w/v) was used for negative staining. TEM pictures was performed at the platform ImagoSeine of the Institute Jacques Monod (Paris, France).

miRNA presence into polyplexes

The presence of the miRNA was monitored by agarose gel electrophoresis (AGE) and UV spectrophotometry after miRNA desorption. Agarose gel electrophoresis was perfomed using 5% agarose gels containing ethidium bromide in TBE (Tris-Borate-EDTA, Thermo Fisher Scientific, Vilnius, Lithuania) buffer. An Ultra-Low Range GeneRuler DNA Ladder (10-300 bp; Thermo Fisher Scientific, Bleiswijk, Netherlands) was used to monitor miRNA presence. Samples included solution of free polymer (QA-pullulan), plus additional controls from different steps of the miRNA loading: miRNA alone (at the concentration used for loading), QA-pullulan polysaccharide solution and QA-pullulan/miRNA polyplexes. Samples were run by electrophoresis (50 V, 120 min) and detected through a UV transilluminator (Vilber Lourmat smart imaging system, Collegien, France). Additional miRNA quantification was performed by UV spectrophotometry using NanoDrop™ 2000/2000c Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

Stability of QA-pullulan/miRNA Polyplexes

The stability of complexes was evaluated over time while stored at 4°C in aqueous media. Size and PdI (Polydispersity Index) evolutions of polyplexes suspensions were monitored by DLS (n= 3) at each time point (0h, 6h, 12h, 18h, 24h). 2.5.5 Cytotoxicity of QA-pullulan and QA-pullulan/miRNA Polyplexes Cytotoxicity was evaluated in Human Umbilical Vein Endothelial Cells (HUVECs, ATCC CRL-1730, Manassas, VA, USA) by resazurin conversion assay (Sigma-Aldrich, Saint Louis, MO, USA). Cells were seeded at a concentration of 10,000 cells/well onto 96-well plates in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 10% (v/v) of fetal bovine serum, 1% (v/v) of penicillin and 1% (v/v) of streptomycin in a humidified atmosphere with 5% CO2 at 37°C. At a subconfluent to confluent stage, cells were detached with 0.05% trypsin/EDTA (Gibco) and re-seeded until the required number of cells for the subsequent experiments was obtained. Cell metabolic activity was assessed after different culture conditions by resazurin conversion. Cells were incubated with medium containing 10% resazurin for 2 hours at 37°C. Fluorescence intensity (530 nm/590 nm) of the cell culture supernatants was measured with an Infinite® 200 PRO microplate reader (TECAN Group Ltd., Männedorf, Switzerland). A blank control comprising only medium was also included. Bright field images (EVOS™ XL Core Cell Imaging System, Thermo Fisher Scientific, Carlsbad, CA, USA) were taken during culture to monitor cell morphology.

miRNA delivery

Human Umbilical Vein Endothelial Cells (HUVECs, ATCC CRL-1730, Manassas, VA, USA) cells were seeded at a concentration of 8,000 cells/well onto Nunc™ Lab-Tek™ chamber slides (Thermo Fisher Scientific) and grown for 48 hours. Polyplexes of QA-pullulan and scramble FAM-miRNA were prepared as previously described on the same day of treatment and incubated for 20 min at 4°C before adding to the cells. After 30 min of incubation using serum-free supplemented cell culture medium, cells were washed with PBS and fixed with fresh 4% paraformaldehyde. Cell nucleus was stained with 4',6-DiAmidino-2-PhenylIndole (DAPI) (Sigma Aldrich, St. Louis, MO, USA). Coverslips were mounted using Ibidi Mounting Medium (IMM) for fluorescence microscopy (ibidi GmbH, Germany). Fluorescence images were taken to evaluate miRNA delivery (Axiovert Observer Z1 inverted microscope, Zeiss, Germany).

Statistical analysis

The data were expressed as mean ± SD (standard deviation). Statistical analysis was performed using GraphPad Prism (version: 5.0a, San Diego, CA, USA). Since data followed a non-parametric distribution, Wilcoxon matched-pairs test was performed to compare each paired group and p smaller than 0.05 was considered to be statistically significant.

Results and discussion

Chemical modification of pullulan and characterization

Pullulan was grafted with glycidyltrimethylammonium chloride to give QA groups (Figure 1) with molar feeding ratios of 1, 2 and 3 to get QA-pullulan. Cationic derivatives were characterized to confirm the success of incorporation of trimethylammonium groups into the pullulan backbone.

Nitrogen elemental analysis confirmed the substitution reaction with N content ranging from 1.8 to 3.3% (w/w) corresponding to 1.3 to 2.3 mmol of QA groups per g of polymer. SEC-MALLS analysis evidenced monomodal molecular weight distributions (PdI ranging from 1.1 to 1.4) (Table 1 andTable A.1) The extent of the derivatisation reaction was given by the degree of substitution (DS) defined as the number of substituted hydroxyls groups per AGU (AnhydroGlucose Unit) [START_REF] Cumpstey | Chemical Modification of Polysaccharides[END_REF]. DS can be calculated using N content from elemental analysis results. Derivatives with a degree of substitution in QA groups around 0.3-0.5 were obtained in one step and in a shorter time (4 hours) compared to others protocols used to obtain quaternized polymers with reaction time of 8, 14 and 24 hours [START_REF] Dionísio | Charged pullulan derivatives for the development of nanocarriers by polyelectrolyte complexation[END_REF]Liu et al., 2015;[START_REF] Stefan | New cationically modified pullulan attenuates atherogenesis and influences lipid metabolism in apoE-knockout mice[END_REF]. Moreover, the protocol of Liu et al. performed in 8h used a mixture of water and isopropanol as reaction medium contrary to our protocol performed only in water without organic solvent (Liu et al., 2015).

IR spectra of pullulan and its cationic derivative presented the general pattern characteristic of polysaccharides [START_REF] Stuart | Infrared Spectroscopy: Fundamentals and Applications[END_REF]. The substitution with QA groups was confirmed by a signal at 1470 cm -1 (Figure 2) due to C-H scissoring in methyl groups [START_REF] Li | Synthesis of water soluble chitosan derivatives with halogeno-1,2,3-triazole and their antifungal activity[END_REF][START_REF] Stefan | New cationically modified pullulan attenuates atherogenesis and influences lipid metabolism in apoE-knockout mice[END_REF]. This was further confirmed by 1 H-NMR spectra (Figure 3) with signals at 3.2 ppm and 4.4 ppm [START_REF] Dionísio | Charged pullulan derivatives for the development of nanocarriers by polyelectrolyte complexation[END_REF][START_REF] Stefan | New cationically modified pullulan attenuates atherogenesis and influences lipid metabolism in apoE-knockout mice[END_REF].

Highly charged cationic polymers can have non-specific interactions with polyanionic biomolecules in the bloodstream leading to the inhibition of many crucial cellular processes and causing severe inflammatory reactions [START_REF] Rekha | Blood compatibility and in vitro transfection studies on cationically modified pullulan for liver cell targeted gene delivery[END_REF][START_REF] Shi | Challenges in DNA Delivery and Recent Advances in Multifunctional Polymeric DNA Delivery Systems[END_REF]. When compared to non-modified pullulan, cationic pullulan did not show toxicity up to 72h of incubation with increasing concentrations of QA-pullulan until 1,000 µg/mL (Figure 4) and supports results of literature that pullulan modified with glycidyltrimethylammonium chloride is a nontoxic polymer [START_REF] Stefan | New cationically modified pullulan attenuates atherogenesis and influences lipid metabolism in apoE-knockout mice[END_REF].

Synthesis and characterization of QA-pullulan/miRNA Polyplexes

Polyplexes were prepared by mixing miRNA and QA-pullulan at different concentrations under strong magnetic stirring (Figure 5). They were incubated at 4°C for 20 minutes for stabilization of complexes before use. Once QA-pullulan modified with only 1.83% of N showed to be able to form complexes with miRNA by electrostatic interactions, this derivative was selected for all further optimizations. To optimize the production of stable polyplexes, a fixed concentration of miRNA of 5 nmol (0.04 mg/mL) was selected based on previous works performed by [START_REF] Antunes | Core-Shell Polymer-Based Nanoparticles To Deliver MiR-155-5p To Endothelial Cells[END_REF][START_REF] Antunes | Core-Shell Polymer-Based Nanoparticles To Deliver MiR-155-5p To Endothelial Cells[END_REF]. Different N/P ratios (0.5-15) were tested by increasing the concentration of QA-pullulan. All physicochemical characterizations are summarized in Figure 6. Samples were characterized by DLS/ELS regarding size, PdI and zeta potential values (Figure 6C). Complexes were formed at an optimized N/P molar ratio of 0.8 producing polyplexes in the nanosized range with a diameter around 130 ± 30 nm and narrow distribution as shown by the polydispersity index (0.07 ± 0.04). More negative zeta potential values of QA-pullulan/miRNA polyplexes (-12 ± 5 mV) as compared to zeta potential of cationic pullulan solution alone (31 ± 6 mV), suggested the presence of additional negative charges at the surface due to miRNA presence in the complex. The morphology of complexes was characterized by TEM (Figure 6B) evidencing the formation of homogeneous spherical particles. The observed sizes were well-correlated with those determined by DLS. miRNA presence was confirmed by agarose gel electrophoresis by the presence of a band in the expected region of 23 bp after miRNA desorption from QA-pullulan (Figure 7A). miRNA was quantified using the software ImageJ by the intensity of the band of polyplexes in the agarose gel normalized by the control naked miRNA and the loading efficiency was around 80%. There was no miRNA degradation or fragmentation after loading as suggested by the absence of smeared bands in AGE indicating that QA-pullulan could effectively protected miRNA. Further miRNA quantification by UV spectrophotometry (Figure 7B) displayed a similar trend as in AGE results.

The N/P ratio, which corresponds to the ratio between QA groups of pullulan derivatives to the phosphate groups of nucleic acid, plays an important role in the capacity of complexation, particle diameter, stability and cytotoxicity of carriers [START_REF] Tekie | Chitosan polyplex nanoparticle vector for miR-145 expression in MCF-7: Optimization by design of experiment[END_REF][START_REF] Vauthier | Chitosan-based nanoparticles for in vivo delivery of interfering agents including siRNA[END_REF][START_REF] Zhao | N/P Ratio Significantly Influences the Transfection Efficiency and Cytotoxicity of a Polyethylenimine/Chitosan/DNA Complex[END_REF]. Generally, polyplexes can be formed between polycations and nucleic acids at relatively low N/P ratios [START_REF] Shao | Elucidating the role of free polycationic chains in polycation gene carriers by free chains of polyethylenimine or N,N,N-trimethyl chitosan plus a certain polyplex[END_REF]. In our case, this optimum was found at N/P of 0.8. Furthermore, it was described that an increase in the average molecular weight (Mn) of polymer dramatically improved oligonucleotides encapsulation at low N/P ratios [START_REF] Ahmed | Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery[END_REF][START_REF] De | Synthesis and evaluation of diethylethylamine-chitosan for gene delivery: composition effects on the in vitro transfection efficiency[END_REF][START_REF] Vauthier | Chitosan-based nanoparticles for in vivo delivery of interfering agents including siRNA[END_REF]. In a study of Liu and co-workers chitosan with chains having 5 to 10 times the length of siRNA (i.e. with a molecular weight ranging from 65 to 170 kDa)

were most suitable to form stable complexes [START_REF] Liu | The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing[END_REF]. Further studies could investigate the effect of the molecular weight of pullulan and its consequence in terms of both physicochemical characteristics of the polyplexes and in vivo biodistribution/biodegradation.

Stability of complexes in aqueous media was evaluated here under storage at 4°C over 1 day without forming aggregates (Figure 8). In addition, it is noteworthy that our method of complex formation was developed without the use of any chemical crosslinking agent.

The increase of the ionic strength has a significant impact on colloidal stability with aggregate formation, and higher PdI values correlated with the heterogeneity of complexes in this medium.

Alameh et al. described that the colloidal instability in 150 mM NaCl increased with the increase of molecular weight when comparing chitosan with low (10 kDa) and high (120 kDa) Mn [START_REF] Alameh | siRNA Delivery with Chitosan: Influence of Chitosan Molecular Weight, Degree of Deacetylation, and Amine to Phosphate Ratio on in Vitro Silencing Efficiency, Hemocompatibility, Biodistribution, and in Vivo Efficacy[END_REF]. Keeping a good stability in physiological conditions is one of the major challenges when designing vectors intended for intravenous administration [START_REF] Ragelle | Chitosan nanoparticles for siRNA delivery: Optimizing formulation to increase stability and efficiency[END_REF]. To achieve this goal, several strategies were thought to overcome this limitation and improve stability in biological fluids [START_REF] Mao | Chitosan-based formulations for delivery of DNA and siRNA[END_REF]. A classical strategy uses cross-linkers such as sodium tripolyphosphate (TPP) for the preparation of chitosan NPs with nucleic acids [START_REF] Karimi | Evaluation of Chitosan-Tripolyphosphate Nanoparticles as a p-shRNA Delivery Vector: Formulation, Optimization and Cellular Uptake Study[END_REF][START_REF] Wang | Degradable Hyaluronic Acid/Protamine Sulfate Interpolyelectrolyte Complexes as miRNA-Delivery Nanocapsules for Triple-Negative Breast Cancer Therapy[END_REF]. However, TPP is a chemical agent with limited sites of interaction for ionotropic gelation [START_REF] Avadi | Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method[END_REF].

Hence, the colloidal stability of polyplexes nanosystems in physiological conditions was far from being fully addressed in the previous investigations [START_REF] Wu | Stabilization of chitosan/hyaluronan colloidal polyelectrolyte complexes in physiological conditions[END_REF].

To assess if the developed polyplexes could be used in biomedical applications, namely as vectors for gene therapy, the cytocompatibility towards HUVECs was evaluated here through an in vitro toxicological assay based on resazurin conversion. In vitro tests did not evidence any toxic effect of the QA-pullulan/miRNA complexes after 1 day of incubation up to the maximum tested dose of 200 µg/mL of polyplexes (n=3) (Figure 9A). We did not observe any alteration in cell morphology (Figure 9B). This important result is a key aspect to further investigate the potential use of the new biocompatible systems.

Finally, a comparative study of free miRNA, neutral pullulan/miRNA and polyplexes of QA-pullulan/miRNA, was done using a fluorescently labeled miRNA to evaluate their cellular uptake.

As it can be seen from Figures 10B and10C, no miRNA could be detected inside cells in case of administration of free miRNA or unmodified pullulan and miRNA indicating that free miRNA could hardly be internalized by cells. miRNA from polyplexes, in contrast, was clearly incorporated by cells (Figure 10D) by forming complexes with QA-pullulan that is able to promote their internalization usually via endocytic pathways being 150 nm the accepted size limit for cellular uptake by non-specific endocytosis [START_REF] Ganas | Biodegradable capsules as non-viral vectors for in vitro delivery of PEI/siRNA polyplexes for efficient gene silencing[END_REF][START_REF] Gary | Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery[END_REF][START_REF] Goyal | Layer-by-Layer Assembled Gold Nanoshells for the Intracellular Delivery of miR-34a[END_REF].

Conclusions

This work demonstrated that nanosized polyplexes are formed and are stable in aqueous medium by simply mixing miRNA with pullulan substituted with quaternized ammonium groups. Such pullulan derivatives have been previously used by André and co-workers to modify the surface of nanoparticles based on sorbitan monooleate (Span ® 80) in order to increase the interaction of this system with siRNA for cellular therapy [START_REF] André | Characterization and comparison of two novel nanosystems associated with siRNA for cellular therapy[END_REF]. To the best of our knowledge, cationized pullulan have been used for the first time to build miRNA delivery nanosystems. The obtained nanoparticles proved to be non-cytotoxic on HUVECs cells after 1 day incubation. Neither toxic reagent nor organic solvent were used. With further experiments proving the efficacy of the systems in vivo, we expect to contribute for the development of new safe pullulan-based delivery systems for miRNA therapeutics.

Appendices:

2. 5

 5 Characterization of QA-pullulan/miRNA Polyplexes 2.5.1 Size and zeta potential measurements Particle size and zeta potential of polyplexes were measured by Dynamic Light Scattering (DLS)

a

  Feeding: molamine/molhydroxyl groups, b Measured by SEC-MALLS, c SD ± 5-10%, d From nitrogen elemental analysis, e Degree of substitution of QA-pullulan calculated per anydroglucose unit.

Figure 1 .

 1 Figure 1. Schematic representation of synthesis route for alkylation of pullulan using glycidyltrimethylammonium chloride (GTMAC).

Figure 2 .

 2 Figure 2. FTIR spectra of (a) non-modified pullulan and (b) QA-pullulan.

Figure 3 .

 3 Figure 3. 1 H-NMR spectra of (a) non-modified pullulan and (b) QA-pullulan in D2O. 1A, 1B, 1C signals depicted anomeric H from maltotriose base unit of pullulan. Numbers 2-6 corresponded to H on anydroglucose unit. H from GTMAC substituent (b) were depicted with numbers from 7 to 10.

Figure 4 .

 4 Figure 4. Cytocompatibility of (a) non-modified pullulan and (b) QA-pullulan towards HUVECs at 200, 500 and 1,000 µg/mL after 24, 48 and 72h of incubation. Results are shown as mean ± SD from 3 independent experiments with no statistical differences detected using the Wilcoxon matched-pairs signed rank test.

Figure 5 .

 5 Figure 5. Schematic representation of preparation of QA-pullulan/miRNA polyplexes by electrostatic interactions.

Figure 6 .

 6 Figure 6. Physico-chemical characterization of QA-pullulan/miRNA polyplexes: (a) Size distribution from Light Scattering (LS) expressed by intensity and number percentages; (b) Morphology of polyplexes obtained by TEM microscopy; (c) Size (nm), PdI, attenuator and count rate values measured by LS and zeta potential (mV) measured by Doppler laser electrophoresis. Results are shown as mean ± standard deviation of 3 different samples.

Figure 7 .

 7 Figure 7. (a) miRNA presence onto polyplexes after miRNA desorption confirmed by agarose gel electrophoresis using an Ultra-Low Range GeneRuler DNA Ladder (10-300 bp) as control and RNA amount (ng) quantified by the intensity of the band when compared to miRNA alone (0.04 mg/mL, 9 µL) using ImageJ software; (b) RNA amount (ng/µL) quantified by UV absorbance (n=3).

Figure 8 .

 8 Figure 8. Stability of QA-pullulan/miRNA polyplexes evaluated by size (left) and PdI evolution (right) measured by Light Scattering as function of time of incubation in water at 4°C (mean ± SD, n=3).

Figure 9 .

 9 Figure 9. (a) Cytocompatibility of complexes towards HUVECs exposed to increasing concentrations of QA-pullulan/miRNA polyplexes measured by resazurin conversion after 1 day of incubation. Results are shown as mean ± SD from 3 independent experiments with no statistical differences detected using the Wilcoxon matched-pairs signed rank test. (b) Optical images of confluent cells without polyplexes and with 100 µg/mL and

Figure 10 .

 10 Figure 10. Fluorescence microscopy images of HUVECs evidencing miRNA delivery. Left column: cells' nuclei stained with DAPI (depicted in blue); middle column: FAM labelled miRNA (depicted in green); right column: merged images. (A) without treatment, (B) cells treated with FAM-labelled miRNA, (C) cells treated with FAM-labelled miRNA in presence of unmodified pullulan; (D) cells treated with polyplexes formed by

Table 1 .

 1 . Characterization of pullulan and cationic pullulan derivatives.

Table A . 1 .

 A1 Elemental analysis of pullulan and cationic pullulan derivatives.

a Feeding: molamine/molhydroxyl groups

  Table A.1: Elemental analysis of pullulan and cationic pullulan derivatives. and miR-5187-5p as biomarkers for the diagnosis of unprotected left main coronary artery disease. J.

	Thorac. Dis. 11, 1799-1808. https://doi.org/10.21037/jtd.2019.05.24

Funding: This work was supported by INSERM, Université de Paris and Université Paris 13. The PhD fellowship was funded by CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO (CNPq) from Brazil grant number 201245/2017-5. Acknowledgments: The authors are grateful to PhD fellowship for Fernanda C. Moraes from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Thanks also to Camille Le Guilcher and Soraya Lanouar for assistance in data analysis.

Conflicts of Interest:

The authors declare no conflict of interest.


Hafid Ait-Oufella
email: hafid.aitoufella@inserm.fr

Peter Libby

Alain Tedgui

Antibody-based immunotherapy targeting cytokines and atherothrombotic cardiovascular diseases: A review for future perspectives

Keywords: Cytokines, Immunotherapy, Monoclonal antibody, Atherosclerosis

Introduction

During the past three decades, animal experiments have immeasurably aided understanding the mechanisms of atherosclerosis development and subsequent complications [START_REF] Libby | Inflammation and immunity in diseases of the arterial tree: players and layers[END_REF]. In particular, the use of genetically engineered atherosclerosis-prone mice affirmed a major role for both innate and adaptive immunity in response to elevated plasma lipid concentrations. The immune/inflammatory cells recruited into the artery wall can secrete cytokines, including tumour necrosis factor (TNF)-α, interleukin (IL)-1β or IL-6, that may promote plaque growth and properties related to their propensity to provoke clinical complications in humans [START_REF] Ait-Oufella | Recent advances on the role of cytokines in atherosclerosis[END_REF]. Yet, the clinical relevance of these experimental findings has long remained unclear. The successful development of monoclonal antibody-based immunotherapies that target specific cytokines in the treatment of chronic immune-inflammatory diseases associated with increased cardiovascular risk, including psoriasis, rheumatoid arthritis and ankylosing spondylitis, provides interesting opportunities to address this knowledge gap [START_REF] Ait-Oufella | Anticytokine Immune Therapy and Atherothrombotic Cardiovascular Risk[END_REF] (Table 1).

TNF-α blockade

Human atherosclerotic plaques contain TNF-α, and TNF-α plasma concentrations associate with recurrence of major adverse cardiovascular events (MACE) in patients with acute coronary syndromes (ACS), even after adjustment for conventional cardiovascular risk factors [START_REF] Ridker | Elevation of tumor necrosis factor-alpha and increased risk of recurrent coronary events after myocardial infarction[END_REF]. Despite some conflicting results, experiments in mice established the atherogenicity of TNF-α, and Tnf-α gene deletion protects against experimental atherosclerosis. In patients with psoriatic arthritis, neutralizing anti-TNF-α monoclonal antibodies significantly reduced the progression of carotid atherosclerotic plaques, measured by ultrasound in non-randomized observations when compared to disease-modifying antirheumatic drugs (DMARDs) and non-selective immunomodulators including sulfasalazine, methotrexate and cyclosporin [START_REF] Minno | Carotid intimamedia thickness in psoriatic arthritis: differences between tumor necrosis factor-alpha blockers and traditional disease-modifying antirheumatic drugs[END_REF]. Positive vascular effects of anti-TNF-α antibodies associated with improved clinical outcomes in patients with rheumatoid arthritis or psoriasis, but the conclusions from these observational studies must be analysed with caution due to potential confounding factors. Nevertheless, based on experimental and clinical data, European experts recommended the use of either methotrexate or a TNF-α-blocking agents for treatment of patients with severe psoriasis at high cardiovascular risk. Anti-TNF-α therapy should not be used in patients with heart failure. This limitation emerged from the ATTACH study, a multicentre, randomized controlled study that showed that high doses of anti-TNF-α increased the risk of death and/or rehospitalization in patients with moderate to severe heart failure [START_REF] Chung | Randomized, double-blind, placebo-controlled, pilot trial of infliximab, a chimeric monoclonal antibody to tumor necrosis factor-alpha, in patients with moderate-to-severe heart failure: results of the anti-TNF Therapy Against Congestive Heart Failure (ATTACH) trial[END_REF]. The mechanisms behind these deleterious effects of TNF-α inhibitors remain uncertain. It merits mention that anti-TNF-α drugs in patients with lupus, obesity, metabolic syndrome or type 2 diabetes yielded inconsistent results, perhaps depending on patient characteristics.

IL-17 blockade

IL-17A, produce by CD4 + , natural killer (NK) and NKT cells, as well as vascular cells, participates in host defence against bacterial and fungal infections. The role of IL-17 in atherosclerosis remains unsettled.

Several studies reported a pathogenic effect of IL-17 in atherosclerotic mice, orchestrating the recruitment and the activation of myeloid cells within the intima, promoting the release of chemokines and proinflammatory cytokines [START_REF] Taleb | IL-17 and Th17 cells in atherosclerosis: subtle and contextual roles[END_REF]. Other studies, however, have shown protective effects of IL-17A by use of loss-or gain-of-function experiments attributed to decreased endothelial expression of VCAM-1 and increased collagen synthesis by vascular smooth muscle cells. In patients with ACS, plasma IL-17 can predict the recurrence of MACE, after adjustment for cardiovascular risk factors, treatments and Creactive protein (CRP) [START_REF] Simon | Circulating levels of interleukin-17 and cardiovascular outcomes in patients with acute myocardial infarction[END_REF]. The role of IL-17 in atherosclerosis may thus depend on context. Patients with high cardiovascular risk treated with inhibitors of the IL-17 pathway for dermatologic or rheumatologic diseases may therefore warrant intensive preventive efforts.

Currently, anti-IL-17 immunotherapy is used to treat patients with arthritis or psoriasis. Two recent trials in patients with psoriatic arthritis and ankylosing spondylitis, treated with an anti-IL-17A monoclonal antibody or placebo, reported a trend for increased MACE in groups treated with anti-IL-17A antibody [START_REF] Mease | Secukinumab Inhibition of Interleukin-17A in Patients with Psoriatic Arthritis[END_REF].

Even though the difference in adverse cardiovascular outcomes was not statistically significant in these studies, they sound a note of caution, especially in light of conflicting preclinical data. The ongoing postmarketing registry results should soon shed light on the long-term cardiovascular effects of anti-IL-17 immunotherapies, and provide more definitive information regarding cardiovascular safety than the smaller studies that may be underpowered for this assessment. Monoclonal antibodies that target IL-12p40/IL-23p19 have undergone evaluation in the treatment of severe forms of psoriasis. Briakinumab, a fully human anti-p40 monoclonal antibody, was withdrawn from the market due to frequent MACE during phase I and II studies. A meta-analysis found an increased risk of MACE under anti-IL-23 immunotherapy [START_REF] Tzellos | Association of anti-IL-12/23 biologic agents ustekinumab and briakinumab with major adverse cardiovascular events[END_REF]. A recent study presented at the French dermatology meeting on the SNIRAM (Système National d'Information Inter-régimes de l'Assurance Maladie) database confirmed this increase of MACE in individuals receiving anti-IL-23 therapy, mainly in patients with high cardiovascular risk. Such studies contrast with a pooled safety analysis of four trials reporting a similar overall rate of MACE in ustekinumab-treated patients and those receiving anti-TNF agents for moderateto-severe psoriasis [START_REF] Papp | Efficacy and safety of secukinumab in the treatment of moderate-to-severe plaque psoriasis: a randomized, double-blind, placebocontrolled phase II dose-ranging study[END_REF]. Three monoclonal antibodies that selectively inhibit the IL-23p19 subunit are in development for the treatment of moderate-to-severe psoriasis. Some cardiovascular safety data may become available soon.

IL-23 blockade

IL-6 blockade

IL-6, produced by both vascular and immune cells, mediates acute and chronic inflammatory responses [START_REF] Libby | All roads lead to IL-6: A central hub of cardiometabolic signaling[END_REF]. The vascular effects of IL-6 vary according to experimental conditions and atherosclerosis stages in mice. In humans, high IL-6 plasma levels associate with worse outcomes in people with or without manifest cardiovascular diseases. A large-scale collaborative genetic association analysis found that the IL6R rs7529229 variant, which associates with high plasma levels of soluble IL-6R and low CRP levels, also associated with reduced coronary artery disease events [START_REF] Swerdlow | The interleukin-6 receptor as a target for prevention of coronary heart disease: a mendelian randomisation analysis[END_REF].

Tocilizumab, an IL-6 receptor-blocking monoclonal antibody, has become available to treat rheumatoid arthritis. In different database studies, IL-6 blockade does not increase cardiovascular risk and possibly even protects against MACE occurrence, although it consistently raises plasma triglyceride concentrations [START_REF] Kim | Cardiovascular Safety of Tocilizumab Versus Tumor Necrosis Factor Inhibitors in Patients With Rheumatoid Arthritis: A Multi-Database Cohort Study[END_REF]. An ongoing phase II trial named ASSAIL-MI will evaluate the ability of a single administration of tocilizumab to reduce myocardial injury damage in patients with acute coronary syndrome.

IL-1β blockade

IL-1β production increases in response to a number of mediators including TNF-α and cholesterol crystals. In animal experiments, IL-1β exerts proatherogenic effects. Il-1β gene inactivation reduced atherosclerosis in mice whereas periarterial IL-1β administration accelerated vascular disease in pigs by increasing the activation of both immune and endothelial cells.

A human neutralizing anti-IL-1β monoclonal antibody, canakinumab, has approval for the treatment of rare inflammasome gain-of-function diseases, as well as juvenile chronic arthritis or familial Mediterranean fever. In a randomized trial including patients with diabetes at high cardiovascular risk, anti-IL-1β treatment reduced CRP and IL-6 plasma levels in a dose-dependent manner. More recently, in the international randomized CANTOS trial (n=10,061) with a median follow-up of 3.7 years, treatment with canakinumab reduced MACE in patients with stable coronary artery disease and high-sensitivity CRP >2 mg/L despite optimal medical treatment. In an on-treatment analysis, the most benefit from canakinumab accrued to participants who achieved high-sensitivity CRP concentrations less than 2 mg/L or IL-6 levels below the study median value of 1.65 ng/L after 3 months [START_REF] Ridker | Modulation of the interleukin-6 signalling pathway and incidence rates of atherosclerotic events and all-cause mortality: analyses from the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS)[END_REF]. CANTOS provided the first proof in humans that targeting inflammation can reduce cardiovascular events independently of lipid lowering. Nonetheless, because of pharmacoeconomic issues, canakinumab is not commercialized for cardiovascular indications. An exploratory analysis of CANTOS showed a striking reduction in cancer incidence and mortality, an observation that spurred a number of ongoing clinical trials in the oncology arena.

Perspectives

In this review, we focus on antibody-based immunotherapy targeting cytokines, but other immunomodulatory strategies directed at enhancing immune tolerance are currently being explored, including low-dose IL-2-induced expansion of CD4 + regulatory T cells or vaccination against low-density lipoprotein-derived peptides.

The development of immunomodulatory therapies in chronic inflammatory diseases and cancers provides an exciting look to the future of treatment for patients with cardiovascular disease. After years of accumulating experimental evidence for a role of the immune system in atherosclerosis, we have now crossed the threshold of clinical translation. With regard to the use of anticytokine immunotherapy, the task remains to balance the risk of impaired host defences and tumour surveillance against antiinflammatory efficacy. The information reviewed above indicates that we will soon have many opportunities to harness immunomodulatory strategies to lower cardiovascular risk. Yet, only large prospective randomized trials will move the field forward from concept, laboratory investigation and small clinical studies to the many patients who remain at cardiovascular risk despite the best of current care.

  IL-23 consists of the two subunits, IL-12p40 and IL-23p19. It maintains IL-17 production and promotes the survival of Th17 cells. Studies in atherosclerotic mice have yielded conflicting findings. IL-23 receptor deficiency in Ldlr -/-mice had no effect on atherosclerosis, whereas haematopoietic Il-23 gene inactivation accelerated atherosclerosis by inducing gut dysbiosis and increasing intestinal barrier permeability.

Table 1

 1 Summary of the atherothrombotic cardiovascular effects of antibody-based immunotherapytargeting cytokines. CAD: coronary artery disease; IL: interleukin; RA: rheumatoid arthritis; TNF: tumour necrosis factor.

Funding

This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM) (HAO and AT). Peter Libby receives funding support from the National Heart, Lung, and Blood Institute (R01HL080472), and the RRM Charitable Fund.

Disclosures

Hafid Ait-Oufella declares having received fees for medical trainings from the laboratories Abbvie, Lilly, UCB Pharma, Pfizer.

Alain Tedgui declares having received fees for scientific training from Novartis Pharma.

Peter Libby is an unpaid consultant to, or involved in clinical trials for, Amgen, AstraZeneca, Esperion Therapeutics, Ionis Pharmaceuticals, Kowa Pharmaceuticals, Novartis, Pfizer, Sanofi-Regeneron, and XBiotech, Inc. Peter Libby is a member of scientific advisory board for Amgen, Corvidia Therapeutics, DalCor Pharmaceuticals, IFM Therapeutics, Kowa Pharmaceuticals, Olatec Therapeutics, Medimmune, Novartis, and XBiotech, Inc. Dr. Libby's laboratory has received research funding in the last 2 years from Novartis.